Pentavalent Bismuth-Mediated Glycosylation Methods to Activate Sialic and Uronic Acids and the Incorporation of Sialic Acids Into Insulin

NASA Astrophysics Data System (ADS)

Kabotso, Daniel Elorm Kwame

The negative charge at physiological pH of carboxylic acid-containing monosaccharides modulate the properties of many natural biomolecules such as oligosaccharides and glycoconjugates. Unfortunately, these altered electronic properties also make the incorporation of such acidic sugars more challenging as compared to the more commonly studied neutral sugars. Herein are reported the first demonstration of glycosylation reactions mediated by triphenylbis(1,1,1-trifluoromethanesulfonato)-bismuth with thioglycosides containing carboxylic acid substituents protected as esters. Unlike with many neutral sugar substrates, the addition of 1-propanethiol to the reactions proved critical to obtaining good yields of the desired glycosylation products using sialic acid, galacturonic acid, and glucuronic acid. The protocol was demonstrated to be amenable to automation using a liquid-handling platform. The consequences of artificially incorporating carboxylic-acid-containing sugars into proteins were tested by the design of a linker containing 1 to 4 sialic acids--a sugar found in many human proteins and brain tissues--that was attached via reductive amination of trityl thiopropylaldehyde at the phenyl alanine terminal end of the protein insulin produced through solid-phase peptide synthesis. Removal of the trityl group with neat trifluoroacetic acid furnished the thiol-free modified insulin that was ligated via a disulfide bond to the peptide scaffold bearing acetyl protected sialic acids. A 14-15% ammonium hydroxide solution was found to be effective in deprotecting the acetyl groups without degradation of the disulfide bond. In addition to maintaining the potency and bioactivity of insulin, the sialic acid-containing linker rendered insulin more resistant to aggregation due to heat and mechanical agitation compared to the unmodified protein.

Plasmonics-Based Detection of Virus Using Sialic Acid Functionalized Gold Nanoparticles.

PubMed

Lee, Changwon; Wang, Peng; Gaston, Marsha A; Weiss, Alison A; Zhang, Peng

2017-01-01

Biosensor for the detection of virus was developed by utilizing plasmonic peak shift phenomenon of the gold nanoparticles and viral infection mechanism of hemagglutinin on virus and sialic acid on animal cells. The plasmonic peak of the colloidal gold nanoparticles changes with the aggregation of the particles due to the plasmonic interaction between nearby particles and the color of the colloidal nanoparticle solution changes from wine red to purple. Sialic acid reduced and stabilized colloidal gold nanoparticle aggregation is induced by the addition of viral particles in the solution due to the hemagglutinin-sialic acid interaction. In this work, sialic acid reduced and stabilized gold nanoparticles (d = 20.1 ± 1.8 nm) were synthesized by a simple one-pot, green method without chemically modifying sialic acid. The gold nanoparticles showed target-specific aggregation with viral particles via hemagglutinin-sialic acid binding. A linear correlation was observed between the change in optical density and dilution of chemically inactivated influenza B virus species. The detection limit of the virus dilution (hemagglutinination assay titer, 512) was shown to be 0.156 vol% and the upper limit of the linearity can be extended with the use of more sialic acid-gold nanoparticles.

Effect of sialic acid loss on dendritic cell maturation

PubMed Central

Crespo, Hélio J; Guadalupe Cabral, M; Teixeira, Alexandra V; Lau, Joseph T Y; Trindade, Hélder; Videira, Paula A

2009-01-01

Sialic acids are key structural determinants and contribute to the functionality of a number of immune cell receptors. Previously, we demonstrated that differentiation of human dendritic cells (DCs) is accompanied by an increased expression of sialylated cell surface structures, putatively through the activity of the ST3Gal.I and ST6Gal.I sialyltransferases. Furthermore, DC endocytosis was reduced upon removal of the cell surface sialic acid residues by neuraminidase. In the present work, we evaluate the contribution of the sialic acid modifications in DC maturation. We demonstrate that neuraminidase-treated human DCs have increased expression of major histocompatibility complex (MHC) and costimulatory molecules, increased gene expression of specific cytokines and induce a higher proliferative response of T lymphocytes. Together, the data suggest that clearance of cell surface sialic acids contributes to the development of a T helper type 1 proinflammatory response. This postulate is supported by mouse models, where elevated MHC class II and increased maturation of specific DC subsets were observed in DCs harvested from ST3Gal.I−/− and ST6Gal.I−/− mice. Moreover, important qualitative differences, particularly in the extent of reduced endocytosis and in the peripheral distribution of DC subsets, existed between the ST3Gal.I−/− and ST6Gal.I−/− strains. Together, the data strongly suggest not only a role of cell surface sialic acid modifications in maturation and functionality of DCs, but also that the sialic acid linkages created by different sialyltransferases are functionally distinct. Consequently, with particular relevance to DC-based therapies, cell surface sialylation, mediated by individual sialyltransferases, can influence the immunogenicity of DCs upon antigen loading. PMID:19740323

[Roles of sialic acids in sperm maturation and capacitation and sperm-egg recognition].

PubMed

Feng, Ying; Wang, Lin; Wu, Yi-Lun; Liu, Hong-Hua; Ma, Fang

2016-10-01

Sialic acids are a subset of nine-carbon alpha-keto aldonic acids involved in various biological functions. Sialic acid on the sperm surface is closely related to sperm maturation and capacitation and sperm-egg recognition, which makes sperm negatively charged to avoid accumulation and covers some antigenic determinants there to increase the survival rate of sperm in the female reproductive tract. The loss of sialic acids is an important factor mediating sperm capacitation. Moreover, the sialic acid at the extremity of the protein polymer is involved in signal identification in sperm-egg recognition. Here, we review the current understanding of sialic acids in sperm maturation and capacitation and sperm-egg recognition.

Studies on the defect underlying the lysosomal storage of sialic acid in Salla disease. Lysosomal accumulation of sialic acid formed from N-acetyl-mannosamine or derived from low density lipoprotein in cultured mutant fibroblasts.

PubMed Central

Renlund, M; Kovanen, P T; Raivio, K O; Aula, P; Gahmberg, C G; Ehnholm, C

1986-01-01

Salla disease is a lysosomal storage disorder characterized by mental retardation and disturbed sialic acid metabolism. To study endogenous synthesis and breakdown of sialic acid, fibroblasts were incubated for 5 d in the presence and then in the absence of N-[3H]acetylmannosamine. Labeling of free sialic acid was 5-10 times higher in mutant than in normal cells. Radioactivity decreased in 4 d by 75% in normal but only by 30% in mutant fibroblasts. The labeling pattern was not normalized upon coculture of mutant and normal cells. To study the metabolism of extracellular sialic acid, low-density lipoprotein (LDL) was labeled in the sialic acid moiety (periodate-NaB3H4) or in the protein moiety (125I). Binding, internalization, lysosomal degradation, and exit of products of protein catabolism were similar in normal and mutant fibroblasts. Upon incubation with LDL labeled in the sialic acid moiety, mutant cells accumulated 2-3 times more free sialic acid radioactivity than normal fibroblasts, mostly in the lysosomal fraction. After a 24-h chase incubation, radioactivity in free sialic acid decreased by 70-80% in normal but only by 10-30% in mutant cells. In mutant fibroblasts, 40% of the radioactivity remained in lysosomes, whereas no labeled free sialic acid was detected in lysosomes from normal fibroblasts. We conclude that in Salla disease, fibroblast endogenous synthesis of sialic acid and lysosomal cleavage of exogenous glycoconjugates is normal, but free sialic acid cannot leave the lysosome. These findings suggest that the basic defect in Salla disease is deficient transport of free sialic acid through the lysosomal membrane. PMID:3944269

Sialic acid-to-urea ratio as a measure of airway surface hydration

PubMed Central

Hill, David B.; Button, Brian; Shi, Shuai; Jania, Corey; Duncan, Elizabeth A.; Doerschuk, Claire M.; Chen, Gang; Ranganathan, Sarath; Stick, Stephen M.; Boucher, Richard C.

2017-01-01

Although airway mucus dehydration is key to pathophysiology of cystic fibrosis (CF) and other airways diseases, measuring mucus hydration is challenging. We explored a robust method to estimate mucus hydration using sialic acid as a marker for mucin content. Terminal sialic acid residues from mucins were cleaved by acid hydrolysis from airway samples, and concentrations of sialic acid, urea, and other biomarkers were analyzed by mass spectrometry. In mucins purified from human airway epithelial (HAE), sialic acid concentrations after acid hydrolysis correlated with mucin concentrations (r2 = 0.92). Sialic acid-to-urea ratios measured from filters applied to the apical surface of cultured HAE correlated to percent solids and were elevated in samples from CF HAEs relative to controls (2.2 ± 1.1 vs. 0.93 ± 1.8, P < 0.01). Sialic acid-to-urea ratios were elevated in bronchoalveolar lavage fluid (BALF) from β-epithelial sodium channel (ENaC) transgenic mice, known to have reduced mucus hydration, and mice sensitized to house dust mite allergen. In a translational application, elevated sialic acid-to-urea ratios were measured in BALF from young children with CF who had airway infection relative to those who did not (5.5 ± 3.7 vs. 1.9 ± 1.4, P < 0.02) and could be assessed simultaneously with established biomarkers of inflammation. The sialic acid-to-urea ratio performed similarly to percent solids, the gold standard measure of mucus hydration. The method proved robust and has potential to serve as flexible techniques to assess mucin hydration, particularly in samples like BALF in which established methods such as percent solids cannot be utilized. PMID:28062483

Metabolism of Sialic Acid by Bifidobacterium breve UCC2003

PubMed Central

Egan, Muireann; O'Connell Motherway, Mary; Ventura, Marco

2014-01-01

Bifidobacteria constitute a specific group of commensal bacteria that inhabit the gastrointestinal tracts of humans and other mammals. Bifidobacterium breve UCC2003 has previously been shown to utilize several plant-derived carbohydrates that include cellodextrins, starch, and galactan. In the present study, we investigated the ability of this strain to utilize the mucin- and human milk oligosaccharide (HMO)-derived carbohydrate sialic acid. Using a combination of transcriptomic and functional genomic approaches, we identified a gene cluster dedicated to the uptake and metabolism of sialic acid. Furthermore, we demonstrate that B. breve UCC2003 can cross feed on sialic acid derived from the metabolism of 3′-sialyllactose, an abundant HMO, by another infant gut bifidobacterial strain, Bifidobacterium bifidum PRL2010. PMID:24814790

Sialic acids as link to Japanese scientistsDedicated to Prof. Dr. Tamio Yamakawa.

PubMed Central

SCHAUER, Roland

2016-01-01

This manuscript is dedicated to Prof. Tamio Yamakawa and describes my cooperations on sialic acid-related topics with Japanese scientists during the last 40 years. We studied sialic acids and their O-acetylated derivatives in the sea urchin Pseudocentrotus depressus, in Halocynthia species, and in human and bovine milk. In seafood we mainly searched for N-glycolylneuraminic acid. With synthetic substrates it was shown that sialic acid O-acetylation at C-4 hinders the activity of sialidases, with the exception of viral enzymes. The biosynthesis of Neu5Gc was discussed and the distribution of this sialic acid in dogs followed in modern literature and reviewed regarding their migration. An excellent source of sialic acids is edible bird nest substance (Collocalia mucin) which was used for the synthesis of sialylation inhibitors. PMID:27063181

Metabolism of sialic acid by Bifidobacterium breve UCC2003.

PubMed

Egan, Muireann; O'Connell Motherway, Mary; Ventura, Marco; van Sinderen, Douwe

2014-07-01

Bifidobacteria constitute a specific group of commensal bacteria that inhabit the gastrointestinal tracts of humans and other mammals. Bifidobacterium breve UCC2003 has previously been shown to utilize several plant-derived carbohydrates that include cellodextrins, starch, and galactan. In the present study, we investigated the ability of this strain to utilize the mucin- and human milk oligosaccharide (HMO)-derived carbohydrate sialic acid. Using a combination of transcriptomic and functional genomic approaches, we identified a gene cluster dedicated to the uptake and metabolism of sialic acid. Furthermore, we demonstrate that B. breve UCC2003 can cross feed on sialic acid derived from the metabolism of 3'-sialyllactose, an abundant HMO, by another infant gut bifidobacterial strain, Bifidobacterium bifidum PRL2010. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Sialic acid catabolism and transport gene clusters are lineage specific in Vibrio vulnificus.

PubMed

Lubin, Jean-Bernard; Kingston, Joseph J; Chowdhury, Nityananda; Boyd, E Fidelma

2012-05-01

Sialic or nonulosonic acids are nine-carbon alpha ketosugars that are present in all vertebrate mucous membranes. Among bacteria, the ability to catabolize sialic acid as a carbon source is present mainly in pathogenic and commensal species of animals. Previously, it was shown that several Vibrio species carry homologues of the genes required for sialic acid transport and catabolism, which are genetically linked. In Vibrio cholerae on chromosome I, these genes are carried on the Vibrio pathogenicity island-2 region, which is confined to pathogenic isolates. We found that among the three sequenced Vibrio vulnificus clinical strains, these genes are present on chromosome II and are not associated with a pathogenicity island. To determine whether the sialic acid transport (SAT) and catabolism (SAC) region is universally present within V. vulnificus, we examined 67 natural isolates whose phylogenetic relationships are known. We found that the region was present predominantly among lineage I of V. vulnificus, which is comprised mainly of clinical isolates. We demonstrate that the isolates that contain this region can catabolize sialic acid as a sole carbon source. Two putative transporters are genetically linked to the region in V. vulnificus, the tripartite ATP-independent periplasmic (TRAP) transporter SiaPQM and a component of an ATP-binding cassette (ABC) transporter. We constructed an in-frame deletion mutation in siaM, a component of the TRAP transporter, and demonstrate that this transporter is essential for sialic acid uptake in this species. Expression analysis of the SAT and SAC genes indicates that sialic acid is an inducer of expression. Overall, our study demonstrates that the ability to catabolize and transport sialic acid is predominately lineage specific in V. vulnificus and that the TRAP transporter is essential for sialic acid uptake.

Erythrocyte sialic acid content during aging in humans: correlation with markers of oxidative stress.

PubMed

Mehdi, Mohammad Murtaza; Singh, Prabhakar; Rizvi, Syed Ibrahim

2012-01-01

Sialic acids are substituted neuraminic acid derivatives which are typically found at the outermost end of glycan chains on the membrane in all cell types. The role of erythrocyte membrane sialic acids during aging has been established however the relationship between sialic acid and oxidative stress is not fully understood. The present work was undertaken to analyze the relationship between erythrocyte membrane sialic acid with its plasma level, membrane and plasma lipid hydroperoxide levels and plasma total antioxidant capacity. Results show that sialic acid content decreases significantly (P< 0.001) in RBC membrane (r= -0.901) and increases in plasma (r=0.860) as a function of age in humans. Lipid peroxidation measured in the form of hydroperoxides increases significantly (P<0.001) in plasma (r=0.830) and RBC membranes (r=0.875) with age in humans. The Trolox Equivalent Total Antioxidant Capacity (TETAC) of plasma was found to be significantly decreased (P< 0.001, r=-0.844). We observe significant correlations between decrease of erythrocyte membrane sialic acid and plasma lipid hydroperoxide and TETAC. Based on the observed correlations, we hypothesize that increase in oxidative stress during aging may influence the sialic acid decomposition from membrane thereby altering the membrane configuration affecting many enzymatic and transporter activities. Considering the importance of plasma sialic acid as a diagnostic parameter, it is important to establish age-dependent reference.

Erythrocyte Sialic Acid Content during Aging in Humans: Correlation with Markers of Oxidative Stress

PubMed Central

Mehdi, Mohammad Murtaza; Singh, Prabhakar; Rizvi, Syed Ibrahim

2012-01-01

Sialic acids are substituted neuraminic acid derivatives which are typically found at the outermost end of glycan chains on the membrane in all cell types. The role of erythrocyte membrane sialic acids during aging has been established however the relationship between sialic acid and oxidative stress is not fully understood. The present work was undertaken to analyze the relationship between erythrocyte membrane sialic acid with its plasma level, membrane and plasma lipid hydroperoxide levels and plasma total antioxidant capacity. Results show that sialic acid content decreases significantly (P < 0.001) in RBC membrane (r = −0.901) and increases in plasma (r = 0.860) as a function of age in humans. Lipid peroxidation measured in the form of hydroperoxides increases significantly (P < 0.001) in plasma (r = 0.830) and RBC membranes (r = 0.875) with age in humans. The Trolox Equivalent Total Antioxidant Capacity (TETAC) of plasma was found to be significantly decreased (P < 0.001, r = −0.844). We observe significant correlations between decrease of erythrocyte membrane sialic acid and plasma lipid hydroperoxide and TETAC. Based on the observed correlations, we hypothesize that increase in oxidative stress during aging may influence the sialic acid decomposition from membrane thereby altering the membrane configuration affecting many enzymatic and transporter activities. Considering the importance of plasma sialic acid as a diagnostic parameter, it is important to establish age-dependent reference. PMID:22377734

Exogenous sialic acid transport contributes to group B streptococcus infection of mucosal surfaces.

PubMed

Pezzicoli, Alfredo; Ruggiero, Paolo; Amerighi, Fulvia; Telford, John L; Soriani, Marco

2012-09-15

By sequence analysis of available group B streptococcus (GBS) genomes, we discovered a conserved putative operon involved in the catabolism of sialic acid, containing a tripartite transporter formed by two integral membrane components and a sugar-binding unit, named SAL0039. Expression analysis in the presence of different substrates revealed that SAL0039 was specifically upregulated by the presence of sialic acid and downregulated when bacteria were grown in human blood or in the presence of a high concentration of glucose. The role of SAL0039 in sugar transport was supported by the inability of the sal0039 deletion mutant strain to import exogenous sialic acid and to grow in semidefined medium supplemented with this sugar. Furthermore, in vivo evidence showed that the presence of exogenous sialic acid significantly increased the capacity of GBS to infect mice at the mucosal level. These findings suggest that transport of sialic acid may also contribute to GBS infections.

Sialic acid-triggered macroscopic properties switching on a smart polymer surface

NASA Astrophysics Data System (ADS)

Xiong, Yuting; Li, Minmin; Wang, Hongxi; Qing, Guangyan; Sun, Taolei

2018-01-01

Constructing smart surfaces with responsive polymers capable of dynamically and reversibly changing their chemical and physical properties by responding to the recognition of biomolecules remains a challenging task. And, the key to achieving this purpose relies on the design of polymers to precisely interact with the target molecule and successfully transform the interaction signal into tunable macroscopic properties, further achieve special bio-functions. Herein, inspired by carbohydrate-carbohydrate interaction (CCI) in life system, we developed a three-component copolymer poly(NIPAAm-co-PT-co-Glc) bearing a binding unit glucose (Glc) capable of recognizing sialic acid, a type of important molecular targets for cancer diagnosis and therapy, and reported the sialic acid triggered macroscopic properties switching on this smart polymer surface. Detailed mechanism studies indicated that multiple hydrogen bonding interactions between Glc unit and Neu5Ac destroyed the initial hydrogen bond network of the copolymer, leading to a reversible "contraction-to-swelling" conformational transition of the copolymer chains, accompanied with distinct macroscopic property switching (i.e., surface wettability, morphology, stiffness) of the copolymer film. And these features enabled this copolymer to selectively capture sialic acid-containing glycopeptides from complex protein samples. This work provides an inspiration for the design of novel smart polymeric materials with sensitive responsiveness to sialic acid, which would promote the development of sialic acid-specific bio-devices and drug delivery systems.

Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

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

Gangi Setty, Thanuja; Cho, Christine; Govindappa, Sowmya

2014-07-01

Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which oftenmore » takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states.« less

Terminal sialic acid linkages determine different cell infectivities of human parainfluenza virus type 1 and type 3.

PubMed

Fukushima, Keijo; Takahashi, Tadanobu; Ito, Seigo; Takaguchi, Masahiro; Takano, Maiko; Kurebayashi, Yuuki; Oishi, Kenta; Minami, Akira; Kato, Tatsuya; Park, Enoch Y; Nishimura, Hidekazu; Takimoto, Toru; Suzuki, Takashi

2014-09-01

Human parainfluenza virus type 1 (hPIV1) and type 3 (hPIV3) initiate infection by sialic acid binding. Here, we investigated sialic acid linkage specificities for binding and infection of hPIV1 and hPIV3 by using sialic acid linkage-modified cells treated with sialidases or sialyltransferases. The hPIV1 is bound to only α2,3-linked sialic acid residues, whereas hPIV3 is bound to α2,6-linked sialic acid residues in addition to α2,3-linked sialic acid residues in human red blood cells. α2,3 linkage-specific sialidase treatment of LLC-MK2 cells and A549 cells decreased the infectivity of hPIV1 but not that of hPIV3. Treatment of A549 cells with α2,3 linkage-specific sialyltransferase increased infectivities of both hPIV1 and hPIV3, whereas α2,6 linkage-specific sialyltransferase treatment increased only hPIV3 infectivity. Clinical isolates also showed similar sialic acid linkage specificities. We concluded that hPIV1 utilizes only α2,3 sialic acid linkages and that hPIV3 makes use of α2,6 sialic acid linkages in addition to α2,3 sialic acid linkages as viral receptors. Copyright © 2014. Published by Elsevier Inc.

Sialic Acid Catabolism Confers a Competitive Advantage to Pathogenic Vibrio cholerae in the Mouse Intestine▿

PubMed Central

Almagro-Moreno, Salvador; Boyd, E. Fidelma

2009-01-01

Sialic acids comprise a family of nine-carbon ketosugars that are ubiquitous on mammalian mucous membranes. However, sialic acids have a limited distribution among Bacteria and are confined mainly to pathogenic and commensal species. Vibrio pathogenicity island 2 (VPI-2), a 57-kb region found exclusively among pathogenic strains of Vibrio cholerae, contains a cluster of genes (nan-nag) putatively involved in the scavenging (nanH), transport (dctPQM), and catabolism (nanA, nanE, nanK, and nagA) of sialic acid. The capacity to utilize sialic acid as a carbon and energy source might confer an advantage to V. cholerae in the mucus-rich environment of the gut, where sialic acid availability is extensive. In this study, we show that V. cholerae can utilize sialic acid as a sole carbon source. We demonstrate that the genes involved in the utilization of sialic acid are located within the nan-nag region of VPI-2 by complementation of Escherichia coli mutants and gene knockouts in V. cholerae N16961. We show that nanH, dctP, nanA, and nanK are highly expressed in V. cholerae grown on sialic acid. By using the infant mouse model of infection, we show that V. cholerae ΔnanA strain SAM1776 is defective in early intestinal colonization stages. In addition, SAM1776 shows a decrease in the competitive index in colonization-competition assays comparing the mutant strain with both O1 El Tor and classical strains. Our data indicate an important relationship between the catabolism of sialic acid and bacterial pathogenesis, stressing the relevance of the utilization of the resources found in the host's environment. PMID:19564383

Sialic acid catabolism confers a competitive advantage to pathogenic vibrio cholerae in the mouse intestine.

PubMed

Almagro-Moreno, Salvador; Boyd, E Fidelma

2009-09-01

Sialic acids comprise a family of nine-carbon ketosugars that are ubiquitous on mammalian mucous membranes. However, sialic acids have a limited distribution among Bacteria and are confined mainly to pathogenic and commensal species. Vibrio pathogenicity island 2 (VPI-2), a 57-kb region found exclusively among pathogenic strains of Vibrio cholerae, contains a cluster of genes (nan-nag) putatively involved in the scavenging (nanH), transport (dctPQM), and catabolism (nanA, nanE, nanK, and nagA) of sialic acid. The capacity to utilize sialic acid as a carbon and energy source might confer an advantage to V. cholerae in the mucus-rich environment of the gut, where sialic acid availability is extensive. In this study, we show that V. cholerae can utilize sialic acid as a sole carbon source. We demonstrate that the genes involved in the utilization of sialic acid are located within the nan-nag region of VPI-2 by complementation of Escherichia coli mutants and gene knockouts in V. cholerae N16961. We show that nanH, dctP, nanA, and nanK are highly expressed in V. cholerae grown on sialic acid. By using the infant mouse model of infection, we show that V. cholerae DeltananA strain SAM1776 is defective in early intestinal colonization stages. In addition, SAM1776 shows a decrease in the competitive index in colonization-competition assays comparing the mutant strain with both O1 El Tor and classical strains. Our data indicate an important relationship between the catabolism of sialic acid and bacterial pathogenesis, stressing the relevance of the utilization of the resources found in the host's environment.

Sialic acid-dependent cell entry of human enterovirus D68

DOE PAGES

Liu, Yue; Sheng, Ju; Baggen, Jim; ...

2015-11-13

Human enterovirus D68 (EV-D68) is a causative agent of childhood respiratory diseases and has now emerged as a global public health threat. Nevertheless, knowledge of the tissue tropism and pathogenesis of EV-D68 has been hindered by a lack of studies on the receptor-mediated EV-D68 entry into host cells. Here we demonstrate that cell surface sialic acid is essential for EV-D68 to bind to and infect susceptible cells. Crystal structures of EV-D68 in complex with sialylated glycan receptor analogues show that they bind into the ‘canyon’ on the virus surface. The sialic acid receptor induces a cascade of conformational changes inmore » the virus to eject a fatty-acid-like molecule that regulates the stability of the virus. Furthermore, virus binding to a sialic acid receptor and to immunoglobulin-like receptors used by most other enteroviruses share a conserved mechanism for priming viral uncoating and facilitating cell entry.« less

Sialic acid-dependent cell entry of human enterovirus D68

PubMed Central

Liu, Yue; Sheng, Ju; Baggen, Jim; Meng, Geng; Xiao, Chuan; Thibaut, Hendrik J.; van Kuppeveld, Frank J. M.; Rossmann, Michael G.

2015-01-01

Human enterovirus D68 (EV-D68) is a causative agent of childhood respiratory diseases and has now emerged as a global public health threat. Nevertheless, knowledge of the tissue tropism and pathogenesis of EV-D68 has been hindered by a lack of studies on the receptor-mediated EV-D68 entry into host cells. Here we demonstrate that cell surface sialic acid is essential for EV-D68 to bind to and infect susceptible cells. Crystal structures of EV-D68 in complex with sialylated glycan receptor analogues show that they bind into the ‘canyon' on the virus surface. The sialic acid receptor induces a cascade of conformational changes in the virus to eject a fatty-acid-like molecule that regulates the stability of the virus. Thus, virus binding to a sialic acid receptor and to immunoglobulin-like receptors used by most other enteroviruses share a conserved mechanism for priming viral uncoating and facilitating cell entry. PMID:26563423

Examination of Signatures of Recent Positive Selection on Genes Involved in Human Sialic Acid Biology.

PubMed

Moon, Jiyun M; Aronoff, David M; Capra, John A; Abbot, Patrick; Rokas, Antonis

2018-03-28

Sialic acids are nine carbon sugars ubiquitously found on the surfaces of vertebrate cells and are involved in various immune response-related processes. In humans, at least 58 genes spanning diverse functions, from biosynthesis and activation to recycling and degradation, are involved in sialic acid biology. Because of their role in immunity, sialic acid biology genes have been hypothesized to exhibit elevated rates of evolutionary change. Consistent with this hypothesis, several genes involved in sialic acid biology have experienced higher rates of non-synonymous substitutions in the human lineage than their counterparts in other great apes, perhaps in response to ancient pathogens that infected hominins millions of years ago (paleopathogens). To test whether sialic acid biology genes have also experienced more recent positive selection during the evolution of the modern human lineage, reflecting adaptation to contemporary cosmopolitan or geographically-restricted pathogens, we examined whether their protein-coding regions showed evidence of recent hard and soft selective sweeps. This examination involved the calculation of four measures that quantify changes in allele frequency spectra, extent of population differentiation, and haplotype homozygosity caused by recent hard and soft selective sweeps for 55 sialic acid biology genes using publicly available whole genome sequencing data from 1,668 humans from three ethnic groups. To disentangle evidence for selection from confounding demographic effects, we compared the observed patterns in sialic acid biology genes to simulated sequences of the same length under a model of neutral evolution that takes into account human demographic history. We found that the patterns of genetic variation of most sialic acid biology genes did not significantly deviate from neutral expectations and were not significantly different among genes belonging to different functional categories. Those few sialic acid biology genes that

Synthesis and Biological Evaluation of Non-Hydrolizable 1,2,3-Triazole Linked Sialic Acid Derivatives as Neuraminidase Inhibitors

PubMed Central

Weïwer, Michel; Chen, Chi-Chang; Kemp, Melissa M.; Linhardt, Robert J.

2013-01-01

α-Sialic acid azide 1 has been used as a substrate for the efficient preparation of 1,2,3-triazole derivatives of sialic acid using the copper-catalyzed azide-alkyne Huisgen cycloaddition (“click chemistry”). Our approach is to generate non-natural N-glycosides of sialic acid that are resistant to neuraminidase catalyzed hydrolysis as opposed to the natural O-glycosides. These N-glycosides would act as neuraminidase inhibitors to prevent the release of new virions. As a preliminary study, a small library of 1,2,3-triazole-linked sialic acid derivatives has been synthesized in 71-89% yield. A disaccharide mimic of sialic acid has also been prepared using the α-sialic acid azide 1 and a C-8 propargyl sialic acid acceptor in 68% yield. A model sialic acid coated dendrimer was also synthesized from a per-propargylated pentaerythritol acceptor. These novel sialic acid derivatives were then evaluated as potential neuraminidase inhibitors using a 96-well plate fluorescence assay; micromolar IC50 values were observed, comparable to the known sialidase inhibitor Neu5Ac2en. PMID:24223493

The evaluation of serum total sialic acid and lipid-bound sialic acid levels in chronically exposed rats to 7,12-dimethylbenz(a)anthracene and fluoride

NASA Astrophysics Data System (ADS)

Oto, Gokhan; Ekin, Suat; Uyar, Hasan; Ozdemir, Hulya; Yıldız, Damla; Karakuş, Yagmur

2017-04-01

In this study, changes in serum total sialic acid (TSA) and lipid-bound sialic acid (LSA) levels were examined in chronically exposed rats to 7,12-dimethylbenz(a)anthracene (DMBA) and fluoride. This study demonstrated that the TSA, LSA levels increased more in DMBA-treated groups compared to the fluoride treated groups. The result obtained has shown that the harmful effect of DMBA which is also causing more cell membrane damage on human and animal health should be taken into consideration.

Fetal ascites and oligohydramnios: prenatal diagnosis of a sialic acid storage disease (index case).

PubMed

Poulain, P; Odent, S; Maire, I; Milon, J; Proudhon, J F; Jouan, H; Le Marec, B

1995-09-01

In a 20-year-old primiparous patient, a routine ultrasound scan performed at 28 weeks revealed fetal ascites, bilateral talipes, and oligohydramnios. This woman, married to possibly her first cousin, was at risk for an autosomal recessive disease, a metabolic disorder. At 29 weeks, an amniotic fluid biochemical study revealed the presence of an abnormal band of free sialic acid, leading to a diagnosis of a congenital form of sialic acid storage disease. Termination of pregnancy was performed at 30 weeks. Measurement of free sialic acid in cultured fetal skin fibroblasts confirmed the diagnosis.

Evaluation of serum sialic acid, fucose levels and their ratio in oral squamous cell carcinoma.

PubMed

Chinnannavar, Sangamesh Ningappa; Ashok, Lingappa; Vidya, Kodige Chandrashekhar; Setty, Sunil Mysore Kantharaja; Narasimha, Guru Eraiah; Garg, Ranjana

2015-01-01

Detection of cancer at the early stage is of utmost importance to decrease the morbidity and mortality of the disease. Apart from the conventional biopsy, minimally invasive methods like serum evaluation are used for screening large populations. Thus, this study aimed to estimate serum levels of sialic acid and fucose and their ratio in oral cancer patients and in healthy control group to evaluate their role in diagnosis. Serum samples were collected from 52 healthy controls (group I) and 52 squamous cell carcinoma patients (group II). Estimation of serum levels of sialic acid and fucose and their ratio was performed. This was correlated histopathologically with the grades of carcinoma. Statistical analysis was done by using analysis of variance (ANOVA) test and unpaired "t" test. Results showed that serum levels of sialic acid and fucose were significantly higher in oral cancer patients compared to normal healthy controls (P < 0.001). The sialic acid to fucose ratio was significantly lower in cancer patients than in normal controls (P < 0.01). However, comparison with histological grading, habits, gender, and age group did not show any significant result. The mean serum sialic acid and fucose levels showed an increasing trend from controls to malignant group and their corresponding ratio showed decreasing trend from controls to malignant group. The ratio of sialic acid to fucose can be a useful diagnostic aid for oral cancer patients.

Modulation of substrate specificities of D-sialic acid aldolase through single mutations of Val-251.

PubMed

Chou, Chien-Yu; Ko, Tzu-Ping; Wu, Kuan-Jung; Huang, Kai-Fa; Lin, Chun-Hung; Wong, Chi-Huey; Wang, Andrew H-J

2011-04-22

In a recent directed-evolution study, Escherichia coli D-sialic acid aldolase was converted by introducing eight point mutations into a new enzyme with relaxed specificity, denoted RS-aldolase (also known formerly as L-3-deoxy-manno-2-octulosonic acid (L-KDO) aldolase), which showed a preferred selectivity toward L-KDO. To investigate the underlying molecular basis, we determined the crystal structures of D-sialic acid aldolase and RS-aldolase. All mutations are away from the catalytic center, except for V251I, which is near the opening of the (α/β)(8)-barrel and proximal to the Schiff base-forming Lys-165. The change of specificity from D-sialic acid to RS-aldolase can be attributed mainly to the V251I substitution, which creates a narrower sugar-binding pocket, but without altering the chirality in the reaction center. The crystal structures of D-sialic acid aldolase·l-arabinose and RS-aldolase·hydroxypyruvate complexes and five mutants (V251I, V251L, V251R, V251W, and V251I/V265I) of the D-sialic acid aldolase were also determined, revealing the location of substrate molecules and how the contour of the active site pocket was shaped. Interestingly, by mutating Val251 alone, the enzyme can accept substrates of varying size in the aldolase reactions and still retain stereoselectivity. The engineered D-sialic acid aldolase may find applications in synthesizing unnatural sugars of C(6) to C(10) for the design of antagonists and inhibitors of glycoenzymes.

Identification of Sialic Acid Linkages on Intact Glycopeptides via Differential Chemical Modification Using IntactGIG-HILIC

NASA Astrophysics Data System (ADS)

Yang, Shuang; Wu, Wells W.; Shen, Rong-Fong; Bern, Marshall; Cipollo, John

2018-04-01

Mass spectrometric analysis of intact glycopeptides can reveal detailed information about glycosite, glycan structural features, and their heterogeneity. Sialyl glycopeptides can be positively, negatively, or neutrally charged depending on pH of their buffer solution and ionization conditions. To detect sialoglycopeptides, a negative-ion mode mass spectrometry may be applied with a minimal loss of sialic acids, although the positively charged or neutral glycopeptides may be excluded. Alternatively, the sialyl glycopeptides can be identified using positive-ion mode analysis by doping a high concentration of sodium salts to the analytes. Although manipulation of unmodified sialoglycopeptides can be useful for analysis of samples, less than optimal ionization, facile loss of sialyl and unfavorable ionization of accompanying non-sialyl peptides make such strategies suboptimal. Currently available chemical derivatization methods, while stabilizing for sialic acid, mask sialic acid linkage configuration. Here, we report the development of a novel approach to neutralize sialic acids via sequentially chemical modification that also reveals their linkage configuration, often an important determinant in biological function. This method utilizes several components to facilitate glycopeptide identification. These include the following: solid phase derivatization, enhanced ionization of sialoglycopeptides, differentiation of sialic acid linkage, and enrichment of the modified glycopeptides by hydrophilic interaction liquid chromatography. This technology can be used as a tool for quantitative analysis of protein sialylation in diseases with determination of sialic acid linkage configuration. [Figure not available: see fulltext.

Structure of the LPS O-chain from Fusobacterium nucleatum strain 10953, containing sialic acid

PubMed Central

Vinogradov, Evgeny; St. Michael, Frank; Homma, Kiyonobu; Sharma, Ashu; Cox, Andrew D.

2017-01-01

Fusobacterium nucleatum is an anaerobic bacterium found in the human mouth where it causes periodontitis. Recently, it has been gaining attention as a potential causative agent for colorectal cancer and is strongly linked with pregnancy complications including pre-term and still births. Little is known about virulence factors of this organism and thus we have initiated studies to examine the bacterial surface glycochemistry. Consistent with a recent paper suggesting that F. nucleatum strain 10593 can synthesize sialic acid, a staining technique identified sialic acid on the bacterial surface. We isolated lipopolysaccharide from this F. nucleatum strain and performed structural analysis on the O-antigen. Our studies identified a trisaccharide repeating unit of the O-antigen with the following structure: -[→4)-α-Neup5Ac-(2→4)-β-D-Galp-(1→3)-α-D-FucpNAc4NAc-(1-]-where Ac indicates 4-N-acetylation of ∼30% FucNAc4N residues. The presence of sialic acid as a constituent of the O-antigen is consistent with recent data identifying de novo sialic acid synthesis in this strain. PMID:28199859

Serum sialic acid levels in patients with sympathetic ophthalmitis.

PubMed

Lamba, P A; Pandey, P K; Sarin, G S; Mathur, M D

1993-12-01

Serum sialic acid levels were measured in 16 patients with sympathetic ophthalmitis, 36 with neglected traumatic uveitis following penetrating injury and 40 healthy subjects. There was no significant alteration of its level in patients with traumatic uveitis. However, its level was significantly elevated in patients with sympathetic ophthalmitis. It was high even in the early stage of the disease. It decreased significantly at the remission stage. It is proposed that measurement of sialic acid level in serum can be used as a diagnostic aid when the diagnosis of sympathetic ophthalmitis remains doubtful on clinical grounds. The extent of rise in its level may be considered a good parameter of the degree of severity of sympathetic ophthalmitis. It may also act as a useful tool to evaluate the drug efficacy in this disease.

Sialic acid content in human saliva and anti-influenza activity against human and avian influenza viruses.

PubMed

Limsuwat, Nattavatchara; Suptawiwat, Ornpreya; Boonarkart, Chompunuch; Puthavathana, Pilaipan; Wiriyarat, Witthawat; Auewarakul, Prasert

2016-03-01

It was shown previously that human saliva has higher antiviral activity against human influenza viruses than against H5N1 highly pathogenic avian influenza viruses, and that the major anti-influenza activity was associated with sialic-acid-containing molecules. To further characterize the differential susceptibility to saliva among influenza viruses, seasonal influenza A and B virus, pandemic H1N1 virus, and 15 subtypes of avian influenza virus were tested for their susceptibility to human and chicken saliva. Human saliva showed higher hemagglutination inhibition (HI) and neutralization (NT) titers against seasonal influenza A virus and the pandemic H1N1 viruses than against influenza B virus and most avian influenza viruses, except for H9N2 and H12N9 avian influenza viruses, which showed high HI and NT titers. To understand the nature of sialic-acid-containing anti-influenza factors in human saliva, α2,3- and α2,6-linked sialic acid was measured in human saliva samples using a lectin binding and dot blot assay. α2,6-linked sialic acid was found to be more abundant than α2,3-linked sialic acid, and a seasonal H1N1 influenza virus bound more efficiently to human saliva than an H5N1 virus in a dot blot analysis. These data indicated that human saliva contains the sialic acid type corresponding to the binding preference of seasonal influenza viruses.

A sialic acid assay in isolation and purification of bovine k-casein glycomacropeptide: a review.

PubMed

Nakano, Takuo; Ozimek, Lech

2014-01-01

Sialic acid is a carbohydrate moiety of k-casein glycomacropeptide (GMP), which is a 64 amino acid residue C-terminal sialylated phosphorylated glycopeptide released from k-casein by the action of chymosin during cheese making. GMP lacks aromatic amino acids including phenylalanine, tyrosine, and tryptophan. Because of its unique amino acid composition and various biological activities, GMP is thought to be a potential ingredient for dietetic foods (e.g., a food for PKU patients) and pharmaceuticals. Thus, increased attention has been given to the development of techniques to purify GMP. In this review, techniques of GMP purification described in patents and scientific research papers were introduced. A sialic acid assay is the important method to track GMP isolation and purification processes, for which the thiobarbituric acid reaction with 1-propanol as a chromophore extracting solvent is an inexpensive, practical and specific technique. Sephacryl S-200 gel filtration chromatography, cellulose acetate electrophoresis, and sodium dodecyl sulfate polyacrylamide gel electrophoresis are the major techniques to identify sialic acid specific to GMP. Sephacryl S-200 chromatography and cellulose acetate electrophoresis are also used to detect GMP sialic acid in whey pearmeate and whey added commercial margarine samples. Future research includes development of an economical industrial scale method to produce high purity GMP.

Localization of sialic acid in kidney glomeruli: regionalization in the podocyte plasma membrane and loss in experimental nephrosis.

PubMed

Charest, P M; Roth, J

1985-12-01

Sialic acid residues were localized by electron microscopy in renal glomeruli of normal and puromycin-treated rats with a cytochemical technique that utilized the Limax flavus lectin. In Lowicryl K4M thin sections from normal rats, sialic acid residues were found along the plasma membrane of the various glomerular cell types and in the glomerular basement membrane as well as the mesangial matrix. In NaDodSO4/PAGE, sialic acid residues of normal glomeruli were mainly confined to a 140-kDa protein previously identified as podocalyxin. The distribution of sialic acid residues in the podocyte plasma membrane was found to be remarkably regionalized. Based on the differential labeling intensity, three plasma membrane domains could be defined: the foot process base, the foot process region above the slit diaphragm, and the body of podocytes. Cytochemical and biochemical analysis of glomeruli from puromycin-treated rats showed a loss of sialic acid residues from glomerular sialoglycoconjugates indicating a perturbated glycosylation.

Host-Derived Sialic Acids Are an Important Nutrient Source Required for Optimal Bacterial Fitness In Vivo.

PubMed

McDonald, Nathan D; Lubin, Jean-Bernard; Chowdhury, Nityananda; Boyd, E Fidelma

2016-04-12

A major challenge facing bacterial intestinal pathogens is competition for nutrient sources with the host microbiota.Vibrio cholerae is an intestinal pathogen that causes cholera, which affects millions each year; however, our knowledge of its nutritional requirements in the intestinal milieu is limited. In this study, we demonstrated that V. cholerae can grow efficiently on intestinal mucus and its component sialic acids and that a tripartite ATP-independent periplasmic SiaPQM strain, transporter-deficient mutant NC1777, was attenuated for colonization using a streptomycin-pretreated adult mouse model. In in vivo competition assays, NC1777 was significantly outcompeted for up to 3 days postinfection. NC1777 was also significantly outcompeted in in vitro competition assays in M9 minimal medium supplemented with intestinal mucus, indicating that sialic acid uptake is essential for fitness. Phylogenetic analyses demonstrated that the ability to utilize sialic acid was distributed among 452 bacterial species from eight phyla. The majority of species belonged to four phyla, Actinobacteria (members of Actinobacillus, Corynebacterium, Mycoplasma, and Streptomyces), Bacteroidetes (mainly Bacteroides, Capnocytophaga, and Prevotella), Firmicutes (members of Streptococcus, Staphylococcus, Clostridium, and Lactobacillus), and Proteobacteria (including Escherichia, Shigella, Salmonella, Citrobacter, Haemophilus, Klebsiella, Pasteurella, Photobacterium, Vibrio, and Yersinia species), mostly commensals and/or pathogens. Overall, our data demonstrate that the ability to take up host-derived sugars and sialic acid specifically allows V. cholerae a competitive advantage in intestinal colonization and that this is a trait that is sporadic in its occurrence and phylogenetic distribution and ancestral in some genera but horizontally acquired in others. Sialic acids are nine carbon amino sugars that are abundant on all mucous surfaces. The deadly human pathogen Vibrio cholerae contains

Effect of fluoride on salivary immunoglobulins and sialic acid.

PubMed

Güzel, Kadriye Görkem Ulu; Kirzioğlu, Zuhal; Adiloğlu, Ali Kudret; Ertürk, Münciye Semra Özay

2017-04-01

The aim of our study was to evaluate the effect of fluoride on salivary immunoglobulin and sialic acid levels in children with dental fluorosis and healthy teeth who live in places with high fluoride concentration in drinking water. Fifty-one (51) healthy children between 6 and 12 years old with no caries were randomly selected from primary schools enrolled in the dental-care program operated by the Department of Pediatric Dentistry. The children were divided into two groups: group I comprised 26 children with dental fluorosis [Thylstrup-Fejerskov Dental Fluorosis Index (TFI) = 4] who lived in Isparta (2.7-2.8 ppm), and group II consisted of 25 children without dental fluorosis who were born in low-fluoride areas and had lived in Isparta for only the previous two years. Stimulated and unstimulated saliva were collected and analyzed for fluoride, salivary immunoglobulins and sialic acid levels. Sialic acid level was correlated negatively with age. Levels of secretory immunoglobulin A (sIgA) and secretory immunoglobulin G (sIgG) were higher in children with dental fluorosis compared with those in group II, although these differences were not significant. Increased sIgA and sIgG levels may arrest the progression of caries in subjects with dental fluorosis. Given the risks of dental fluorosis, further studies of the effects of different fluoride levels in drinking water on salivary composition of children with mixed dentition are needed to confirm the results of our study and to provide data for comparison.

Contribution of Sialic Acid to the Voltage Dependence of Sodium Channel Gating

PubMed Central

Bennett, Eric; Urcan, Mary S.; Tinkle, Sally S.; Koszowski, Adam G.; Levinson, Simon R.

1997-01-01

A potential role for sialic acid in the voltage-dependent gating of rat skeletal muscle sodium channels (rSkM1) was investigated using Chinese hamster ovary (CHO) cells stably transfected with rSkM1. Changes in the voltage dependence of channel gating were observed after enzymatic (neuraminidase) removal of sialic acid from cells expressing rSkM1 and through the expression of rSkM1 in a sialylation-deficient cell line (lec2). The steady-state half-activation voltages (Va) of channels under each condition of reduced sialylation were ∼10 mV more depolarized than control channels. The voltage dependence of the time constants of channel activation and inactivation were also shifted in the same direction and by a similar magnitude. In addition, recombinant deletion of likely glycosylation sites from the rSkM1 sequence resulted in mutant channels that gated at voltages up to 10 mV more positive than wild-type channels. Thus three independent means of reducing channel sialylation show very similar effects on the voltage dependence of channel gating. Finally, steady-state activation voltages for channels subjected to reduced sialylation conditions were much less sensitive to the effects of external calcium than those measured under control conditions, indicating that sialic acid directly contributes to the negative surface potential. These results are consistent with an electrostatic mechanism by which external, negatively charged sialic acid residues on rSkM1 alter the electric field sensed by channel gating elements. PMID:9089440

Leishmania donovani Utilize Sialic Acids for Binding and Phagocytosis in the Macrophages through Selective Utilization of Siglecs and Impair the Innate Immune Arm.

PubMed

Roy, Saptarshi; Mandal, Chitra

2016-08-01

Leishmania donovani, belonging to a unicellular protozoan parasite, display the differential level of linkage-specific sialic acids on their surface. Sialic acids binding immunoglobulin-like lectins (siglecs) are a class of membrane-bound receptors present in the haematopoetic cell lineages interact with the linkage-specific sialic acids. Here we aimed to explore the utilization of sialic acids by Leishmania donovani for siglec-mediated binding, phagocytosis, modulation of innate immune response and signaling pathways for establishment of successful infection in the host. We have found enhanced binding of high sialic acids containing virulent strains (AG83+Sias) with siglec-1 and siglec-5 present on macrophages compared to sialidase treated AG83+Sias (AG83-Sias) and low sialic acids-containing avirulent strain (UR6) by flow cytometry. This specific receptor-ligand interaction between sialic acids and siglecs were further confirmed by confocal microscopy. Sialic acids-siglec-1-mediated interaction of AG83+Sias with macrophages induced enhanced phagocytosis. Additionally, sialic acids-siglec-5 interaction demonstrated reduced ROS, NO generation and Th2 dominant cytokine response upon infection with AG83+Sias in contrast to AG83-Sias and UR6. Sialic acids-siglecs binding also facilitated multiplication of intracellular amastigotes. Moreover, AG83+Sias induced sialic acids-siglec-5-mediated upregulation of host phosphatase SHP-1. Such sialic acids-siglec interaction was responsible for further downregulation of MAPKs (p38, ERK and JNK) and PI3K/Akt pathways followed by the reduced translocation of p65 subunit of NF-κβ to the nucleus from cytosol in the downstream signaling pathways. This sequence of events was reversed in AG83-Sias and UR6-infected macrophages. Besides, siglec-knockdown macrophages also showed the reversal of AG83+Sias infection-induced effector functions and downstream signaling events. Taken together, this study demonstrated that virulent parasite

3DSDSCAR--a three dimensional structural database for sialic acid-containing carbohydrates through molecular dynamics simulation.

PubMed

Veluraja, Kasinadar; Selvin, Jeyasigamani F A; Venkateshwari, Selvakumar; Priyadarzini, Thanu R K

2010-09-23

The inherent flexibility and lack of strong intramolecular interactions of oligosaccharides demand the use of theoretical methods for their structural elucidation. In spite of the developments of theoretical methods, not much research on glycoinformatics is done so far when compared to bioinformatics research on proteins and nucleic acids. We have developed three dimensional structural database for a sialic acid-containing carbohydrates (3DSDSCAR). This is an open-access database that provides 3D structural models of a given sialic acid-containing carbohydrate. At present, 3DSDSCAR contains 60 conformational models, belonging to 14 different sialic acid-containing carbohydrates, deduced through 10 ns molecular dynamics (MD) simulations. The database is available at the URL: http://www.3dsdscar.org. Copyright 2010 Elsevier Ltd. All rights reserved.

Total sialic acid profile in regressing and remodelling organs during the metamorphosis of marsh frog (Pelophylax ridibundus Pallas 1771).

PubMed

Kaptan, Engin; Bas, Serap Sancar; Inceli, Meliha Sengezer

2013-03-01

This study aimed to investigate the functional relationship of sialic acid in regressing and remodelling organs such as the tail, small intestine and liver during the metamorphosis of Pelophylax ridibundus. For this purpose, four groups were composed according to developmental periods by considering Gosner's criteria (1964). Our findings showed that the sialic acid content of the larval tail has an opposite profile to cell death process. Although the sialic acid content of the small intestine and liver did not change evidently during metamorphosis, it increased after the completion of metamorphosis. Frog tail extensively exhibited cell death process and decreased proliferative activity and underwent complete degeneration during metamorphic climax. In spite of increased apoptotic index, a decreased sialic acid level in the tail tissues during climax can be the indication of a death cell removal process. However, the intestine and the liver included both cell death and proliferative process and remodelling in their adult forms. Thus, their sialic acid profiles during metamorphosis were different from the tail's profile. These data show that sialic acid may be an indicator of the presence of some cellular events during metamorphosis and that it can have different roles in the developmental process depending on the organ's fate throughout metamorphosis. Copyright © 2012 John Wiley & Sons, Ltd.

Prototype amperometric biosensor for sialic acid determination.

PubMed

Marzouk, Sayed A M; Ashraf, S S; Tayyari, Khawla A Al

2007-02-15

This paper describes the first report on the development, characterization, and applications of a prototype amperometric biosensor for free sialic acid (SA). The sensor was constructed by the coimmobilization of two enzymes, i.e., N-acetylneuraminic acid aldolase and pyruvate oxidase, on a polyester microporous membrane, which was then mounted on top of a platinum disk electrode. The SA biosensor operation was based on the sequential action of the two enzymes to ultimately produce hydrogen peroxide, which was then detected by anodic amperometry at the platinum electrode. The surface of the platinum electrode was coated with an electropolymeric layer to enhance the biosensor selectivity in the presence of interfering oxidizable species. Optimization of the enzyme layer composition resulted in a fast and steady current response in phosphate buffer pH 7.2 at 37 degrees C. The limit of detection was 10 microM, and the response was linear to 3.5 mM (r = 0.9987). The prepared SA biosensors retained approximately 85% of their initial sensitivity after 8 days and showed excellent response reproducibility (CV = 2.3%). Utilization of a third enzyme, sialidase, expanded the scope of the present SA biosensor to determine bound sialic acid as well. The merits of the described biosensor allowed its successful application in determining SA in biological and pharmaceutical samples. The obtained results indicated that the presented SA biosensor should be a useful bioanalytical tool in several biological and clinical applications such as screening of SA as a nonspecific tumor marker as well as monitoring of tumor therapy.

Affinity chromatography matrices for depletion and purification of casein glycomacropeptide from bovine whey.

PubMed

Baieli, María F; Urtasun, Nicolás; Martinez, María J; Hirsch, Daniela B; Pilosof, Ana M R; Miranda, María V; Cascone, Osvaldo; Wolman, Federico J

2017-01-01

Casein glycomacropeptide (CMP) is a 64- amino acid peptide found in cheese whey, which is released after κ-casein specific cleavage by chymosin. CMP lacks aromatic amino acids, a characteristic that makes it usable as a nutritional supplement for people with phenylketonuria. CMP consists of two nonglycosylated isoforms (aCMP A and aCMP B) and its different glycosylated forms (gCMP A and gCMP B). The most predominant carbohydrate of gCMP is N-acetylneuraminic acid (sialic acid). Here, we developed a CMP purification process based on the affinity of sialic acid for wheat germ agglutinin (WGA). After formation of chitosan beads and adsorption of WGA, the agglutinin was covalently attached with glutaraldehyde. Two matrices with different WGA density were assayed for CMP adsorption. Maximum adsorption capacities were calculated according to the Langmuir model from adsorption isotherms developed at pH 7.0, being 137.0 mg/g for the matrix with the best performance. In CMP reduction from whey, maximum removal percentage was 79% (specifically 33.7% of gCMP A and B, 75.8% of aCMP A, and 93.9% of aCMP B). The CMP was recovered as an aggregate with an overall yield of 64%. Therefore, the matrices developed are promising for CMP purification from cheese whey. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:171-180, 2017. © 2016 American Institute of Chemical Engineers.

Measles virus fusion machinery activated by sialic acid binding globular domain.

PubMed

Talekar, Aparna; Moscona, Anne; Porotto, Matteo

2013-12-01

Paramyxoviruses, including the human pathogen measles virus (MV) and the avian Newcastle disease virus (NDV), enter host cells through fusion of the viral envelope with the target cell membrane. This fusion is driven by the concerted action of two viral envelope glycoproteins: the receptor binding protein and the fusion protein (F). The MV receptor binding protein (hemagglutinin [H]) attaches to proteinaceous receptors on host cells, while the receptor binding protein of NDV (hemagglutinin-neuraminidase [HN]) interacts with sialic acid-containing receptors. The receptor-bound HN/H triggers F to undergo conformational changes that render it competent to mediate fusion of the viral and cellular membranes. The mechanism of fusion activation has been proposed to be different for sialic acid-binding viruses and proteinaceous receptor-binding viruses. We report that a chimeric protein containing the NDV HN receptor binding region and the MV H stalk domain can activate MV F to fuse, suggesting that the signal to the stalk of a protein-binding receptor binding molecule can be transmitted from a sialic acid binding domain. By engineering the NDV HN globular domain to interact with a proteinaceous receptor, the fusion activation signal was preserved. Our findings are consistent with a unified mechanism of fusion activation, at least for the Paramyxovirinae subfamily, in which the receptor binding domains of the receptor binding proteins are interchangeable and the stalk determines the specificity of F activation.

Glycoproteomics enabled by tagging sialic acid- or galactose-terminated glycans

PubMed Central

Ramya, T N C; Weerapana, Eranthie; Cravatt, Benjamin F; Paulson, James C

2013-01-01

In this paper, we present two complementary strategies for enrichment of glycoproteins on living cells that combine the desirable attributes of “robust enrichment” afforded by covalent-labeling techniques and “specificity for glycoproteins” typically provided by lectin or antibody affinity reagents. Our strategy involves the selective introduction of aldehydes either into sialic acids by periodate oxidation (periodate oxidation and aniline-catalyzed oxime ligation (PAL)) or into terminal galactose and N-acetylgalactosamine residues by galactose oxidase (galactose oxidase and aniline-catalyzed oxime ligation (GAL)), followed by aniline-catalyzed oxime ligation with aminooxy-biotin to biotinylate the glycans of glycoprotein subpopulations with high efficiency and cell viability. As expected, the two methods exhibit reciprocal tagging efficiencies when applied to fully sialylated cells compared with sialic acid-deficient cells. To assess the utility of these labeling methods for glycoproteomics, we enriched the PAL- and GAL-labeled (biotinylated) glycoproteome by adsorption onto immobilized streptavidin. Glycoprotein identities (IDs) and N-glycosylation site information were then obtained by liquid chromatography-tandem mass spectrometry on total tryptic peptides and on peptides subsequently released from N-glycans still bound to the beads using peptide N-glycosidase F. A total of 175 unique N-glycosylation sites were identified, belonging to 108 nonredundant glycoproteins. Of the 108 glycoproteins, 48 were identified by both methods of labeling and the remainder was identified using PAL on sialylated cells (40) or GAL on sialic acid-deficient cells (20). Our results demonstrate that PAL and GAL can be employed as complementary methods of chemical tagging for targeted proteomics of glycoprotein subpopulations and identification of glycosylation sites of proteins on cells with an altered sialylation status. PMID:23070960

Linkage-specific sialic acid derivatization for MALDI-TOF-MS profiling of IgG glycopeptides.

PubMed

de Haan, Noortje; Reiding, Karli R; Haberger, Markus; Reusch, Dietmar; Falck, David; Wuhrer, Manfred

2015-08-18

Glycosylation is a common co- and post-translational protein modification, having a large influence on protein properties like conformation and solubility. Furthermore, glycosylation is an important determinant of efficacy and clearance of biopharmaceuticals such as immunoglobulin G (IgG). Matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF)-mass spectrometry (MS) shows potential for the site-specific glycosylation analysis of IgG at the glycopeptide level. With this approach, however, important information about glycopeptide sialylation is not duly covered because of in-source and metastable decay of the sialylated species. Here, we present a highly repeatable sialic acid derivatization method to allow subclass-specific MALDI-TOF-MS analysis of tryptic IgG glycopeptides. The method, employing dimethylamidation with the carboxylic acid activator 1-ethyl-3-(3-dimethylamino)propyl)carbodiimide (EDC) and the catalyst 1-hydroxybenzotriazole (HOBt), results in different masses for the functionally divergent α2,3- and α2,6-linked sialic acids. Respective lactonization and dimethylamidation leads to their direct discrimination in MS and importantly, both glycan and peptide moieties reacted in a controlled manner. In addition, stabilization allowed the acquisition of fragmentation spectra informative with respect to glycosylation and peptide sequence. This was in contrast to fragmentation spectra of underivatized samples, which were dominated by sialic acid loss. The method allowed the facile discrimination and relative quantitation of IgG Fc sialylation in therapeutic IgG samples. The method has considerable potential for future site- and sialic acid linkage-specific glycosylation profiling of therapeutic antibodies, as well as for subclass-specific biomarker discovery in clinical IgG samples derived from plasma.

Exploring the mechanism of βamyloid toxicity attenuation by multivalent sialic acid polymers through the use of mathematical models

PubMed Central

Cowan, Christopher B.; Patel, Dhara A.; Good, Theresa A.

2009-01-01

β-Amyloid peptide (Aβ), the primary protein component in senile plaques associated with Alzheimer’s disease (AD), has been implicated in neurotoxicity associated with AD. Previous studies have shown that the Aβ-neuronal membrane interaction plays a role in the mechanism of Aβ toxicity. More specifically, it is thought that Aβ interacts with ganglioside rich and sialic acid rich regions of cell surfaces. In light of such evidence, we have used a number of different sialic acid compounds of different valency or number of sialic acid moieties per molecule to attenuate Aβ toxicity in a cell culture model. In this work, we proposed various mathematical models of Aβ interaction with both the cell membrane and with the multivalent sialic acid compounds, designed to act as membrane mimics. These models allow us to explore the mechanism of action of this class of sialic acid membrane mimics in attenuating the toxicity of Aβ. The mathematical models, when compared with experimental data, facilitate the discrimination between different modes of action of these materials. Understanding the mechanism of action of Aβ toxicity inhibitors should provide insight into the design of the next generation of molecules that could be used to prevent Aβ toxicity associated with Alzheimer’s disease. PMID:19217912

[Analysis of sialic acid in infant formulas using ultra performance liquid chromatography-triple quadrupole mass spectrometry].

PubMed

Xie, Honglei; Li, Chun; Liu, Ning

2013-08-01

The method for analysing sialic acid in infant formulas by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) has been established. Sialic acid in milk was released via acid hydrolysis, and purified by an HLB solid phase extraction cartridge. The UPLC separation was performed on an ACQUITY UPLC BEH HILIC column (50 mm x 2.1 mm, 1.7 microm) utilizing a gradient elution program of acetonitrile and water (containing 0.1% formic acid) as the mobile phases at a flow rate of 0.25 mL/min. Injection volume and column temperature were set at 5 microL and 30 degrees C, respectively. The identification and quantification were achieved by using electrospray ionisation (ESI)-MS/MS in positive ion mode and multiple reaction monitoring (MRM) mode. The linear range was from 0.05 to 5.0 mg/L for sialic acid and the correlation coefficient (R(2)) was greater than 0.99. The average recoveries spiked at the four concentration levels of 0.1, 0.5, 2.5 and 5.0 mg/L ranged between 84.3% and 98.9% with the relative standard deviations from 4.9% to 8.2%. The limit of detection was 0.01 mg/L. Therefore, this method has the characteristics of simple operation, high reproducibility and sensitivity. It can be widely applied to determine the total contents of sialic acid in infant formula, cow milk and human milk.

Sialic Acids in the Brain: Gangliosides and Polysialic Acid in Nervous System Development, Stability, Disease, and Regeneration

PubMed Central

Gerardy-Schahn, Rita; Hildebrandt, Herbert

2014-01-01

Every cell in nature carries a rich surface coat of glycans, its glycocalyx, which constitutes the cell's interface with its environment. In eukaryotes, the glycocalyx is composed of glycolipids, glycoproteins, and proteoglycans, the compositions of which vary among different tissues and cell types. Many of the linear and branched glycans on cell surface glycoproteins and glycolipids of vertebrates are terminated with sialic acids, nine-carbon sugars with a carboxylic acid, a glycerol side-chain, and an N-acyl group that, along with their display at the outmost end of cell surface glycans, provide for varied molecular interactions. Among their functions, sialic acids regulate cell-cell interactions, modulate the activities of their glycoprotein and glycolipid scaffolds as well as other cell surface molecules, and are receptors for pathogens and toxins. In the brain, two families of sialoglycans are of particular interest: gangliosides and polysialic acid. Gangliosides, sialylated glycosphingolipids, are the most abundant sialoglycans of nerve cells. Mouse genetic studies and human disorders of ganglioside metabolism implicate gangliosides in axon-myelin interactions, axon stability, axon regeneration, and the modulation of nerve cell excitability. Polysialic acid is a unique homopolymer that reaches >90 sialic acid residues attached to select glycoproteins, especially the neural cell adhesion molecule in the brain. Molecular, cellular, and genetic studies implicate polysialic acid in the control of cell-cell and cell-matrix interactions, intermolecular interactions at cell surfaces, and interactions with other molecules in the cellular environment. Polysialic acid is essential for appropriate brain development, and polymorphisms in the human genes responsible for polysialic acid biosynthesis are associated with psychiatric disorders including schizophrenia, autism, and bipolar disorder. Polysialic acid also appears to play a role in adult brain plasticity

Chemoselective synthesis of sialic acid 1,7-lactones.

PubMed

Allevi, Pietro; Rota, Paola; Scaringi, Raffaella; Colombo, Raffaele; Anastasia, Mario

2010-08-20

The chemoselective synthesis of the 1,7-lactones of N-acetylneuraminic acid, N-glycolylneuraminic acid, and 3-deoxy-d-glycero-d-galacto-nononic acid is accomplished in two steps: a simple treatment of the corresponding free sialic acid with benzyloxycarbonyl chloride and a successive hydrogenolysis of the formed 2-benzyloxycarbonyl 1,7-lactone. The instability of the 1,7-lactones to protic solvents has been also evidenced together with the rationalization of the mechanism of their formation under acylation conditions. The results permit to dispose of authentic 1,7-sialolactones to be used as reference standards and of a procedure useful for the preparation of their isotopologues to be used as inner standards in improved analytical procedures for the gas liquid chromatography-mass spectrometry (GLC-MS) analysis of 1,7-sialolactones in biological media.

Development of magnetic resonance imaging based detection methods for beta amyloids via sialic acid-functionalized magnetic nanoparticles

NASA Astrophysics Data System (ADS)

Kouyoumdjian, Hovig

The development of a non-invasive method for the detection of Alzheimer's disease is of high current interest, which can be critical in early diagnosis and in guiding preventive treatment of the disease. The aggregates of beta amyloids are a pathological hallmark of Alzheimer's disease. Carbohydrates such as sialic acid terminated gangliosides have been shown to play significant roles in initiation of amyloid aggregation. Herein, we report a biomimetic approach using sialic acid coated iron oxide superparamagnetic nanoparticles for in vitro detection in addition to the assessment of the in vivo mouse-BBB (Blood brain barrier) crossing of the BSA (bovine serum albumin)-modified ones. The sialic acid functionalized dextran nanoparticles were shown to bind with beta amyloids through several techniques including ELISA (enzyme linked immunosorbent assay), MRI (magnetic resonance imaging), TEM (transmission electron microscopy), gel electrophoresis and tyrosine fluorescence assay. The superparamagnetic nature of the nanoparticles allowed easy detection of the beta amyloids in mouse brains in both in vitro and ex vivo model by magnetic resonance imaging. Furthermore, the sialic acid nanoparticles greatly reduced beta amyloid induced cytotoxicity to SH-SY5Y neuroblastoma cells, highlighting the potential of the glyconanoparticles for detection and imaging of beta amyloids. Sialic acid functionalized BSA (bovine serum albumin) nanoparticles also showed significant binding to beta amyloids, through ELISA and ex vivo mouse brain MRI experiments. Alternatively, the BBB crossing was demonstrated by several techniques such as confocal microscopy, endocytosis, exocytosis assays and were affirmed by nanoparticles transcytosis assays through bEnd.3 endothelial cells. Finally, the BBB crossing was confirmed by analyzing the MRI signal of nanoparticle-injected CD-1 mice.

A GntR-type transcriptional repressor controls sialic acid utilization in Bifidobacterium breve UCC2003.

PubMed

Egan, Muireann; O'Connell Motherway, Mary; van Sinderen, Douwe

2015-02-01

Bifidobacterium breve strains are numerically prevalent among the gut microbiota of healthy, breast-fed infants. The metabolism of sialic acid, a ubiquitous monosaccharide in the infant and adult gut, by B. breve UCC2003 is dependent on a large gene cluster, designated the nan/nag cluster. This study describes the transcriptional regulation of the nan/nag cluster and thus sialic acid metabolism in B. breve UCC2003. Insertion mutagenesis and transcriptome analysis revealed that the nan/nag cluster is regulated by a GntR family transcriptional repressor, designated NanR. Crude cell extract of Escherichia coli EC101 in which the nanR gene had been cloned and overexpressed was shown to bind to two promoter regions within this cluster, each of which containing an imperfect inverted repeat that is believed to act as the NanR operator sequence. Formation of the DNA-NanR complex is prevented in the presence of sialic acid, which we had previously shown to induce transcription of this gene cluster. © FEMS 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

A Small Molecule Inhibits Virion Attachment to Heparan Sulfate- or Sialic Acid-Containing Glycans

PubMed Central

Colpitts, Che C.

2014-01-01

ABSTRACT Primary attachment to cellular glycans is a critical entry step for most human viruses. Some viruses, such as herpes simplex virus type 1 (HSV-1) and hepatitis C virus (HCV), bind to heparan sulfate, whereas others, such as influenza A virus (IAV), bind to sialic acid. Receptor mimetics that interfere with these interactions are active against viruses that bind to either heparan sulfate or to sialic acid. However, no molecule that inhibits the attachment of viruses in both groups has yet been identified. Epigallocatechin gallate (EGCG), a green tea catechin, is active against many unrelated viruses, including several that bind to heparan sulfate or to sialic acid. We sought to identify the basis for the broad-spectrum activity of EGCG. Here, we show that EGCG inhibits the infectivity of a diverse group of enveloped and nonenveloped human viruses. EGCG acts directly on the virions, without affecting the fluidity or integrity of the virion envelopes. Instead, EGCG interacts with virion surface proteins to inhibit the attachment of HSV-1, HCV, IAV, vaccinia virus, adenovirus, reovirus, and vesicular stomatitis virus (VSV) virions. We further show that EGCG competes with heparan sulfate for binding of HSV-1 and HCV virions and with sialic acid for binding of IAV virions. Therefore, EGCG inhibits unrelated viruses by a common mechanism. Most importantly, we have identified EGCG as the first broad-spectrum attachment inhibitor. Our results open the possibility for the development of small molecule broad-spectrum antivirals targeting virion attachment. IMPORTANCE This study shows that it is possible to develop a small molecule antiviral or microbicide active against the two largest groups of human viruses: those that bind to glycosaminoglycans and those that bind to sialoglycans. This group includes the vast majority of human viruses, including herpes simplex viruses, cytomegalovirus, influenza virus, poxvirus, hepatitis C virus, HIV, and many others. PMID

Evaluation of the antitumor effect of dexamethasone palmitate and doxorubicin co-loaded liposomes modified with a sialic acid-octadecylamine conjugate.

PubMed

Sun, Jing; Song, Yanzhi; Lu, Mei; Lin, Xiangyun; Liu, Yang; Zhou, Songlei; Su, Yuqing; Deng, Yihui

2016-10-10

Dexamethasone palmitate has the potential to inhibit the activity of tumor-associated macrophages, which promote cancer proliferation, invasion, and metastasis; however, only very high and frequent doses are capable of inducing antitumor effects. With the aim to reduce the anticancer dose and decrease the nonspecific toxicity, we designed a liposomal system to co-deliver dexamethasone palmitate and doxorubicin. Furthermore, a ligand conjugate sialic acid-octadecylamine, with enhanced affinity towards the membrane receptors over-expressed in tumors, was anchored on the surface of the liposomes to increase drug distribution to the tumor tissue. Co-loaded liposomes were developed using lipid film hydration method to load dexamethasone palmitate and remote loading technology to load doxorubicin. The co-loaded liposomes modified with sialic acid-octadecylamine represented comparable physicochemical properties and blood plasma profiles with conventional co-loaded liposomes, but the biodistribution proved that sialic acid-octadecylamine modified liposomes accumulated more in tumor. The co-loaded liposomes showed higher tumor growth suppression than the single-drug loaded liposomes, while showing no additional drug toxicity in S180-bearing Kunming mice. The co-loaded liposomes modified with sialic acid-octadecylamine achieved a significantly better antitumor effect, and induced "shedding" of cancerous tissue in the mice. These finding suggested that co-loaded liposomes modified with sialic acid-octadecylamine provided a safe therapeutic strategy with outstanding anticancer activity. Copyright © 2016 Elsevier B.V. All rights reserved.

Preparation of a molecularly imprinted sensor based on quartz crystal microbalance for specific recognition of sialic acid in human urine.

PubMed

Qiu, Xiuzhen; Xu, Xian-Yan; Chen, Xuncai; Wu, Yiyong; Guo, Huishi

2018-05-08

A novel molecularly imprinted quartz crystal microbalance (QCM) sensor was successfully prepared for selective determination of sialic acid (SA) in human urine samples. To obtain the QCM sensor, we first modified the gold surface of the QCM chip by self-assembling of allylmercaptane to introduce polymerizable double bonds on the chip surface. Then, SA molecularly imprinted polymer (MIP) nanofilm was attached to the modified QCM chip surface. For comparison, we have also characterized the nonmodified and improved surfaces of the QCM sensor by using atomic force microscopy (AFM) and Fourier transform infrared (FTIR) spectroscopy. We then tested the selectivity and detection limit of the imprinted QCM sensor via a series of adsorption experiments. The results show a linear response in the range of 0.025-0.50 μmol L -1 for sialic acid. Moreover, the limit of detection (LOD) of the prepared imprinted QCM sensor was found to be 1.0 nmol L -1 for sialic acid, and high recovery values range from 87.6 to 108.5% with RSD < 8.7 (n = 5) for the spiked urine sample obtained. Overall, this work presents how a novel QCM sensor was developed and used to detect sialic acid in human urine samples. Graphical abstract Specific recognition of sialic acid by the MIP-QCM sensor system.

Host-Derived Sialic Acids Are an Important Nutrient Source Required for Optimal Bacterial Fitness In Vivo

PubMed Central

McDonald, Nathan D.; Lubin, Jean-Bernard; Chowdhury, Nityananda

2016-01-01

ABSTRACT A major challenge facing bacterial intestinal pathogens is competition for nutrient sources with the host microbiota. Vibrio cholerae is an intestinal pathogen that causes cholera, which affects millions each year; however, our knowledge of its nutritional requirements in the intestinal milieu is limited. In this study, we demonstrated that V. cholerae can grow efficiently on intestinal mucus and its component sialic acids and that a tripartite ATP-independent periplasmic SiaPQM strain, transporter-deficient mutant NC1777, was attenuated for colonization using a streptomycin-pretreated adult mouse model. In in vivo competition assays, NC1777 was significantly outcompeted for up to 3 days postinfection. NC1777 was also significantly outcompeted in in vitro competition assays in M9 minimal medium supplemented with intestinal mucus, indicating that sialic acid uptake is essential for fitness. Phylogenetic analyses demonstrated that the ability to utilize sialic acid was distributed among 452 bacterial species from eight phyla. The majority of species belonged to four phyla, Actinobacteria (members of Actinobacillus, Corynebacterium, Mycoplasma, and Streptomyces), Bacteroidetes (mainly Bacteroides, Capnocytophaga, and Prevotella), Firmicutes (members of Streptococcus, Staphylococcus, Clostridium, and Lactobacillus), and Proteobacteria (including Escherichia, Shigella, Salmonella, Citrobacter, Haemophilus, Klebsiella, Pasteurella, Photobacterium, Vibrio, and Yersinia species), mostly commensals and/or pathogens. Overall, our data demonstrate that the ability to take up host-derived sugars and sialic acid specifically allows V. cholerae a competitive advantage in intestinal colonization and that this is a trait that is sporadic in its occurrence and phylogenetic distribution and ancestral in some genera but horizontally acquired in others. PMID:27073099

Acetylated sialic acid residues and blood group antigens localise within the epithelium in microvillous atrophy indicating internal accumulation of the glycocalyx

PubMed Central

Phillips, A D; Brown, A; Hicks, S; Schüller, S; Murch, S H; Walker-Smith, J A; Swallow, D M

2004-01-01

Background: Microvillous atrophy, a disorder of intractable diarrhoea in infancy, is characterised by the intestinal epithelial cell abnormalities of abnormal accumulation of periodic acid-Schiff (PAS) positive secretory granules within the apical cytoplasm and the presence of microvillous inclusions. The identity of the PAS positive material is not known, and the aim of this paper was to further investigate its composition. Methods: Formaldehyde fixed sections were stained with alcian blue/PAS to identify the acidic or neutral nature of the material, phenylhydrazine blocking was employed to stain specifically for sialic acid, and saponification determined the presence of sialic acid acetylation. The specificity of sialic acid staining was tested by digestion with mild sulphuric acid. Expression of blood group related antigens was tested immunochemically. Results: Alcian blue/PAS staining identified a closely apposed layer of acidic material on the otherwise neutral (PAS positive) brush border in controls. In microvillous atrophy, a triple layer was seen with an outer acidic layer, an unstained brush border region, and accumulation within the epithelium of a neutral glycosubstance that contained acetylated sialic acid. Blood group antigens were detected on the brush border, in mucus, and within goblet cells in controls. In microvillous atrophy they were additionally expressed within the apical cytoplasm of epithelial cells mirroring the PAS abnormality. Immuno electron microscopy localised expression to secretory granules. Conclusions: A neutral, blood group antigen positive, glycosubstance that contains acetylated sialic acid accumulates in the epithelium in microvillous atrophy. Previous studies have demonstrated that the direct and indirect constitutive pathways are intact in this disorder and it is speculated that the abnormal staining pattern reflects accumulation of glycocalyx related material. PMID:15542511

Abiotic stress of ambient cold temperature regulates the host receptivity to pathogens by cell surfaced sialic acids.

PubMed

Moon, Seong-Cheol; Joo, Su-Yeon; Chung, Tae-Wook; Choi, Hee-Jung; Park, Mi-Ju; Choi, Hee-Jin; Bae, Sung-Jin; Kim, Keuk-Jun; Kim, Cheorl-Ho; Joo, Myungsoo; Ha, Ki-Tae

2016-07-29

Ambient cold temperature, as an abiotic stress, regulates the survival, stability, transmission, and infection of pathogens. However, the effect of cold temperature on the host receptivity to the pathogens has not been fully studied. In this study, the expression of terminal α-2,3- and α-2,6-sialic acids were increased in murine lung tissues, especially bronchial epithelium, by exposure to cold condition. The expression of several sialyltransferases were also increased by exposure to cold temperature. Furthermore, in human bronchial epithelial BEAS-2B cells, the expressions of α-2,3- and α-2,6-sialic acids, and mRNA levels of sialyltransferases were increased in the low temperature condition at 33 °C. On the other hand, the treatment of Lith-Gly, a sialyltransferase inhibitor, blocked the cold-induced expression of sialic acids on surface of BEAS-2B cells. The binding of influenza H1N1 hemagglutinin (HA) toward BEAS-2B cells cultured at low temperature condition was increased, compared to 37 °C. In contrast, the cold-increased HA binding was blocked by treatment of lithocholicglycine and sialyl-N-acetyl-D-lactosamines harboring α-2,3- and α-2,6-sialyl motive. These results suggest that the host receptivity to virus at cold temperature results from the expressions of α-2,3- and α-2,6-sialic acids through the regulation of sialyltransferase expression. Copyright © 2016 Elsevier Inc. All rights reserved.

When contemporary aminoacyl-tRNA synthetases invent their cognate amino acid metabolism

PubMed Central

Roy, Hervé; Becker, Hubert Dominique; Reinbolt, Joseph; Kern, Daniel

2003-01-01

Faithful protein synthesis relies on a family of essential enzymes called aminoacyl-tRNA synthetases, assembled in a piecewise fashion. Analysis of the completed archaeal genomes reveals that all archaea that possess asparaginyl-tRNA synthetase (AsnRS) also display a second ORF encoding an AsnRS truncated from its anticodon binding-domain (AsnRS2). We show herein that Pyrococcus abyssi AsnRS2, in contrast to AsnRS, does not sustain asparaginyl-tRNAAsn synthesis but is instead capable of converting aspartic acid into asparagine. Functional analysis and complementation of an Escherichia coli asparagine auxotrophic strain show that AsnRS2 constitutes the archaeal homologue of the bacterial ammonia-dependent asparagine synthetase A (AS-A), therefore named archaeal asparagine synthetase A (AS-AR). Primary sequence- and 3D-based phylogeny shows that an archaeal AspRS ancestor originated AS-AR, which was subsequently transferred into bacteria by lateral gene transfer in which it underwent structural changes producing AS-A. This study provides evidence that a contemporary aminoacyl-tRNA synthetase can be recruited to sustain amino acid metabolism. PMID:12874385

Properties and substrate specificities of the phenylalanyl-transfer-ribonucleic acid synthetases of Aesculus species

PubMed Central

Anderson, J. W.; Fowden, L.

1970-01-01

1. Phenylalanyl-tRNA synthetases have been partially purified from cotyledons of seeds of Aesculus californica, which contains 2-amino-4-methylhex-4-enoic acid, and from four other species of Aesculus that do not contain this amino acid. The A. californica preparation was free from other aminoacyl-tRNA synthetases, and the contaminating synthetase activity in preparations from A. hippocastanum was decreased to acceptable limits by conducting assays of pyrophosphate exchange activity in 0.5m-potassium chloride. 2. The phenylalanyl-tRNA synthetase from each species activated 2-amino-4-methylhex-4-enoic acid with Km 30–40 times that for phenylalanine. The maximum velocity for 2-amino-4-methylhex-4-enoic acid was only 30% of that for phenylalanine with the A. californica enzyme, but the maximum velocities for the two substrates were identical for the other four species. 3. 2-Amino-4-methylhex-4-enoic acid was not found in the protein of A. californica, so discrimination against this amino acid probably occurs in the step of transfer to tRNA, though subcellular localization, or subsequent steps of protein synthesis could be involved. 4. Crotylglycine, methallylglycine, ethallylglycine, 2-aminohex-4,5-dienoic acid, 2-amino-5-methylhex-4-enoic acid, 2-amino-4-methylhex-4-enoic acid, β-(thien-2-yl)alanine, β-(pyrazol-1-yl)alanine, phenylserine and m-fluorophenylalanine were substrates for pyrophosphate exchange catalysed by the phenylalanyl-tRNA synthetases of A. californica or A. hippocastanum. Allylglycine, phenylglycine and 2-amino-4-phenylbutyric acid were inactive. PMID:5493504

Saccharomyces boulardii expresses neuraminidase activity selective for α2,3-linked sialic acid that decreases Helicobacter pylori adhesion to host cells.

PubMed

Sakarya, Serhan; Gunay, Necati

2014-10-01

Helicobacter pylori is a major causative agent of gastritis and peptic ulcer disease and is an established risk factor for gastric malignancy. Antibiotic combination therapy can eradicate H. pylori. As these same regimens can evoke adverse effects and resistance, new alternative therapies or adjunctive treatments are needed. A probiotic approach may provide a novel strategy for H. pylori treatment. In the current study, two probiotic bacteria, Lactobacillus acidophilus and Lactobacillus reuteri, and a probiotic yeast, Saccharomyces boulardii, were evaluated for their ability to influence H. pylori viability, adherence to gastric and duodenal cells, as well as the effect of S. boulardii on cell surface expression of sialic acid. Our results indicate that S. boulardii contains neuraminidase activity selective for α(2-3)-linked sialic acid. This neuraminidase activity removes surface α(2-3)-linked sialic acid, the ligand for the sialic acid-binding H. pylori adhesin, which in turn, inhibits H. pylori adherence to duodenal epithelial cells. © 2014 APMIS. Published by John Wiley & Sons Ltd.

Mutations in type 3 reovirus that determine binding to sialic acid are contained in the fibrous tail domain of viral attachment protein sigma1.

PubMed

Chappell, J D; Gunn, V L; Wetzel, J D; Baer, G S; Dermody, T S

1997-03-01

The reovirus attachment protein, sigma1, determines numerous aspects of reovirus-induced disease, including viral virulence, pathways of spread, and tropism for certain types of cells in the central nervous system. The sigma1 protein projects from the virion surface and consists of two distinct morphologic domains, a virion-distal globular domain known as the head and an elongated fibrous domain, termed the tail, which is anchored into the virion capsid. To better understand structure-function relationships of sigma1 protein, we conducted experiments to identify sequences in sigma1 important for viral binding to sialic acid, a component of the receptor for type 3 reovirus. Three serotype 3 reovirus strains incapable of binding sialylated receptors were adapted to growth in murine erythroleukemia (MEL) cells, in which sialic acid is essential for reovirus infectivity. MEL-adapted (MA) mutant viruses isolated by serial passage in MEL cells acquired the capacity to bind sialic acid-containing receptors and demonstrated a dependence on sialic acid for infection of MEL cells. Analysis of reassortant viruses isolated from crosses of an MA mutant virus and a reovirus strain that does not bind sialic acid indicated that the sigma1 protein is solely responsible for efficient growth of MA mutant viruses in MEL cells. The deduced sigma1 amino acid sequences of the MA mutant viruses revealed that each strain contains a substitution within a short region of sequence in the sigma1 tail predicted to form beta-sheet. These studies identify specific sequences that determine the capacity of reovirus to bind sialylated receptors and suggest a location for a sialic acid-binding domain. Furthermore, the results support a model in which type 3 sigma1 protein contains discrete receptor binding domains, one in the head and another in the tail that binds sialic acid.

The Structure of a Cyanobacterial Bicarbonate Transport Protein, CmpA

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

Koropatkin, Nicole M.; Koppenaal, David W.; Pakrasi, Himadri B.

2007-01-26

Cyanobacteria, blue-green algae, are the most abundant autotrophs in aquatic environments and form the base of the food chain by fixing carbon and nitrogen into cellular biomass. To compensate for the low selectivity of Rubisco for CO₂ over O₂, Cyanobacteria have developed highly efficient CO₂concentrating machinery of which the ABC transport system CmpABCD from Synechocystis PCC 6803 is one component. Here we describe the structure of the bicarbonate binding protein, CmpA, in the absence and presence of bicarbonate and carbonic acid. CmpA is highly homologous to the nitrate transport protein, NrtA. CmpA binds carbonic acid at the entrance to themore » ligand-binding pocket whereas bicarbonate binds in nearly an identical location compared to nitrate binding to NrtA. Unexpectedly, bicarbonate binding is accompanied by a metal ion, identified as Ca²⁺ via inductively coupled plasma optical emission spectrometry. The binding of bicarbonate and metal is highly cooperative and suggests that CmpA co-transports bicarbonate and calcium.« less

HA Antibody-Mediated FcγRIIIa Activity Is Both Dependent on FcR Engagement and Interactions between HA and Sialic Acids.

PubMed

Cox, Freek; Kwaks, Ted; Brandenburg, Boerries; Koldijk, Martin H; Klaren, Vincent; Smal, Bastiaan; Korse, Hans J W M; Geelen, Eric; Tettero, Lisanne; Zuijdgeest, David; Stoop, Esther J M; Saeland, Eirikur; Vogels, Ronald; Friesen, Robert H E; Koudstaal, Wouter; Goudsmit, Jaap

2016-01-01

Interactions with receptors for the Fc region of IgG (FcγRs) have been shown to contribute to the in vivo protection against influenza A viruses provided by broadly neutralizing antibodies (bnAbs) that bind to the viral hemagglutinin (HA) stem. In particular, Fc-mediated antibody-dependent cellular cytotoxicity (ADCC) has been shown to contribute to protection by stem-binding bnAbs. Fc-mediated effector functions appear not to contribute to protection provided by strain-specific HA head-binding antibodies. We used a panel of anti-stem and anti-head influenza A and B monoclonal antibodies with identical human IgG1 Fc domains and investigated their ability to mediate ADCC-associated FcγRIIIa activation. Antibodies which do not interfere with sialic acid binding of HA can mediate FcγRIIIa activation. However, the FcγRIIIa activation was inhibited when a mutant HA, unable to bind sialic acids, was used. Antibodies which block sialic acid receptor interactions of HA interfered with FcγRIIIa activation. The inhibition of FcγRIIIa activation by HA head-binding and sialic acid receptor-blocking antibodies was confirmed in plasma samples of H5N1 vaccinated human subjects. Together, these results suggest that in addition to Fc-FcγR binding, interactions between HA and sialic acids on immune cells are required for optimal Fc-mediated effector functions by anti-HA antibodies.

Changes in human parotid salivary protein and sialic acid levels during pregnancy.

PubMed

D'Alessandro, S; Curbelo, H M; Tumilasci, O R; Tessler, J A; Houssay, A B

1989-01-01

Saliva was collected with a Carlson-Crittenden device, under citric acid stimulation, in 107 pregnant women, 9 puerperal and 7 non-pregnant controls. No significant changes were found in salivary flow rate, pH and amylase levels. The total protein levels were decreased during pregnancy and the puerperium. The sialic acid levels decreased gradually but markedly during pregnancy, returning to normal levels in the puerperium. These changes in parotid saliva may be related to the hormonal changes of pregnancy.

Liquid chromatography-tandem mass spectrometry assay for the quantification of free and total sialic acid in human cerebrospinal fluid.

PubMed

van der Ham, Maria; de Koning, Tom J; Lefeber, Dirk; Fleer, André; Prinsen, Berthil H C M T; de Sain-van der Velden, Monique G M

2010-05-01

Analysis of sialic acid (SA) metabolites in cerebrospinal fluid (CSF) is important for clinical diagnosis. In the present study, a high-performance liquid chromatography-tandem mass spectrometry (HPLC/MS/MS) method for free sialic acid (FSA) and total sialic acid (TSA) in human CSF was validated. The method utilized a simple sample-preparation procedure of protein precipitation for FSA and acid hydrolysis for TSA. Negative electrospray ionisation was used to monitor the transitions m/z 308.2-->87.0 (SA) and m/z 311.2--> 90.0 ((13)C(3)-SA). Conjugated sialic acid (CSA) was calculated by subtracting FSA from TSA. We established reference intervals for FSA, TSA and CSA in CSF in 217 control subjects. The method has been applied to patients' samples with known differences in SA metabolites like meningitis (n=6), brain tumour (n=2), leukaemia (n=5), and Salla disease (n=1). Limit of detection (LOD) was 0.54 microM for FSA and 0.45 mM for TSA. Intra- and inter-assay variation for FSA (21.8 microM) were 4.8% (n=10) and 10.4% (n=40) respectively. Intra- and inter-assay variation for TSA (35.6 microM) were 9.7% (n=10) and 12.8% (n=40) respectively. Tested patients showed values of TSA above established reference value. The validated method allows sensitive and specific measurement of SA metabolites in CSF and can be applied for clinical diagnoses. 2010 Elsevier B.V. All rights reserved.

The phagocytic capacity and immunological potency of human dendritic cells is improved by α2,6‐sialic acid deficiency

PubMed Central

Cabral, M. Guadalupe; Silva, Zélia; Ligeiro, Dário; Seixas, Elsa; Crespo, Hélio; Carrascal, Mylène A.; Silva, Mariana; Piteira, Ana R.; Paixão, Paulo; Lau, Joseph T.; Videira, Paula A.

2013-01-01

Summary Dendritic cells (DCs) play an essential role in immunity against bacteria by phagocytosis and by eliciting adaptive immune responses. Previously, we demonstrated that human monocyte‐derived DCs (MDDCs) express a high content of cell surface α2,6‐sialylated glycans. However, the relative role of these sialylated structures in phagocytosis of bacteria has not been reported. Here, we show that treatment with a sialidase significantly improved the capacity of both immature and mature MDDCs to phagocytose Escherichia coli. Desialylated MDDCs had a significantly more mature phenotype, with higher expression of MHC molecules and interleukin (IL)‐12, tumour necrosis factor‐α, IL‐6 and IL‐10 cytokines, and nuclear factor‐κB activation. T lymphocytes primed by desialylated MDDCs expressed more interferon‐γ when compared with priming by sialylated MDDCs. Improved phagocytosis required E. coli sialic acids, indicating a mechanism of host–pathogen interaction dependent on sialic acid moieties. The DCs harvested from mice deficient in the ST6Gal.1 sialyltransferase showed improved phagocytosis capacity, demonstrating that the observed sialidase effect was a result of the removal of α2,6‐sialic acid. The phagocytosis of different pathogenic E. coli isolates was also enhanced by sialidase, which suggests that modifications on MDDC sialic acids may be considered in the development of MDDC‐based antibacterial therapies. Physiologically, our findings shed new light on mechanisms that modulate the function of both immature and mature MDDCs, in the context of host–bacteria interaction. Hence, with particular relevance to DC‐based therapies, the engineering of α2,6‐sialic acid cell surface is a novel possibility to fine tune DC phagocytosis and immunological potency. PMID:23113614

CD22 and Siglec-G regulate inhibition of B-cell signaling by sialic acid ligand binding and control B-cell tolerance.

PubMed

Nitschke, Lars

2014-09-01

CD22 and Siglec-G are two B-cell expressed members of the Siglec (sialic acid-binding immunoglobulin (Ig)-like lectin) family and are potent inhibitors of B-cell signaling. Genetic approaches have provided evidence that this inhibition of B-cell antigen receptor (BCR) signaling by Siglecs is dependent on ligand binding to sialic acids in specific linkages. The cis-ligand-binding activity of CD22 leads to homo-oligomer formation, which are to a large extent found in membrane domains that are distinct from those containing the BCR. In contrast, Siglec-G is recruited via sialic acid binding to the BCR. This interaction of Siglec-G with mIgM leads to an inhibitory function that seems to be specific for B-1 cells. Both CD22 and Siglec-G control B-cell tolerance and loss of these proteins, its ligands or its inhibitory pathways can increase the susceptibility for autoimmune diseases. CD22 is a target protein both in B-cell leukemias and lymphomas, as well as in B-cell mediated autoimmune diseases. Both antibodies and synthetic chemically modified sialic acids are currently tested to target Siglecs on B cells. © The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

[Role of sialic acid loss in the myocardium in depressing the contractile function of the heart muscle during stress].

PubMed

Meerson, F Z; Saulia, A I; Gudumak, V S

1985-01-01

Under conditions of stress a time-dependent decrease in content of sialic acids was found in adult rats; within 9 hrs of the animal immobilization the sialic acid content was decreased by 40% as compared with controls. At the same time, activities of trypsin and LDHI were increased in blood serum. The data obtained suggest that activation of proteases occurring during the stress led to increased hydrolysis of base components of glycocalyx and to impairment of the cardiomyocyte sarcolemma. These phenomena appear to be responsible for the post-stress deterioration of heart muscle contractile functions.

Microscale Measurements of Michaelis-Menten Constants of Neuraminidase with Nanogel Capillary Electrophoresis for the Determination of the Sialic Acid Linkage.

PubMed

Gattu, Srikanth; Crihfield, Cassandra L; Holland, Lisa A

2017-01-03

Phospholipid nanogels enhance the stability and performance of the exoglycosidase enzyme neuraminidase and are used to create a fixed zone of enzyme within a capillary. With nanogels, there is no need to covalently immobilize the enzyme, as it is physically constrained. This enables rapid quantification of Michaelis-Menten constants (K M ) for different substrates and ultimately provides a means to quantify the linkage (i.e., 2-3 versus 2-6) of sialic acids. The fixed zone of enzyme is inexpensive and easily positioned in the capillary to support electrophoresis mediated microanalysis using neuraminidase to analyze sialic acid linkages. To circumvent the limitations of diffusion during static incubation, the incubation period is reproducibly achieved by varying the number of forward and reverse passes the substrate makes through the stationary fixed zone using in-capillary electrophoretic mixing. A K M value of 3.3 ± 0.8 mM (V max , 2100 ± 200 μM/min) was obtained for 3'-sialyllactose labeled with 2-aminobenzoic acid using neuraminidase from Clostridium perfringens that cleaves sialic acid monomers with an α2-3,6,8,9 linkage, which is similar to values reported in the literature that required benchtop analyses. The enzyme cleaves the 2-3 linkage faster than the 2-6, and a K M of 2 ± 1 mM (V max , 400 ± 100 μM/min) was obtained for the 6'-sialyllactose substrate. An alternative neuraminidase selective for 2-3 sialic acid linkages generated a K M value of 3 ± 2 mM (V max , 900 ± 300 μM/min) for 3'-sialyllactose. With a knowledge of V max , the method was applied to a mixture of 2-3 and 2-6 sialyllactose as well as 2-3 and 2-6 sialylated triantennary glycan. Nanogel electrophoresis is an inexpensive, rapid, and simple alternative to current technologies used to distinguish the composition of 3' and 6' sialic acid linkages.

Microscale Measurements of Michaelis–Menten Constants of Neuraminidase with Nanogel Capillary Electrophoresis for the Determination of the Sialic Acid Linkage

PubMed Central

2016-01-01

Phospholipid nanogels enhance the stability and performance of the exoglycosidase enzyme neuraminidase and are used to create a fixed zone of enzyme within a capillary. With nanogels, there is no need to covalently immobilize the enzyme, as it is physically constrained. This enables rapid quantification of Michaelis–Menten constants (KM) for different substrates and ultimately provides a means to quantify the linkage (i.e., 2-3 versus 2-6) of sialic acids. The fixed zone of enzyme is inexpensive and easily positioned in the capillary to support electrophoresis mediated microanalysis using neuraminidase to analyze sialic acid linkages. To circumvent the limitations of diffusion during static incubation, the incubation period is reproducibly achieved by varying the number of forward and reverse passes the substrate makes through the stationary fixed zone using in-capillary electrophoretic mixing. A KM value of 3.3 ± 0.8 mM (Vmax, 2100 ± 200 μM/min) was obtained for 3′-sialyllactose labeled with 2-aminobenzoic acid using neuraminidase from Clostridium perfringens that cleaves sialic acid monomers with an α2-3,6,8,9 linkage, which is similar to values reported in the literature that required benchtop analyses. The enzyme cleaves the 2-3 linkage faster than the 2-6, and a KM of 2 ± 1 mM (Vmax, 400 ± 100 μM/min) was obtained for the 6′-sialyllactose substrate. An alternative neuraminidase selective for 2-3 sialic acid linkages generated a KM value of 3 ± 2 mM (Vmax, 900 ± 300 μM/min) for 3′-sialyllactose. With a knowledge of Vmax, the method was applied to a mixture of 2-3 and 2-6 sialyllactose as well as 2-3 and 2-6 sialylated triantennary glycan. Nanogel electrophoresis is an inexpensive, rapid, and simple alternative to current technologies used to distinguish the composition of 3′ and 6′ sialic acid linkages. PMID:27936604

Evaluation of the relationship between passive smoking and salivary electrolytes, protein, secretory IgA, sialic acid and amylase in young children.

PubMed

Avşar, Aysun; Darka, Ozge; Bodrumlu, Ebru Hazar; Bek, Yüksel

2009-05-01

To evaluate the relationship between passive smoking as determined by salivary cotinine levels and salivary electrolytes, protein, secretory IgA, sialic acid and amylase in children. Saliva was collected from 90 passive smoker (PS) subjects (the study group) and 90 healthy age-matched children (the control group). The study group was divided into three subgroups according the number of cigarettes smoked. Socio-economic status, dental and dietary habits were recorded by questionnaire. Stimulated salivary calcium (Ca), phosphate (P), sodium (Na), potassium (P), total protein, amylase activity, sialic acid level, secretory IgA concentration and cotinine level were analysed. All data were analysed using SPSS, version 13.0. Socio-economic status, dental and dietary habits were similar between the two groups. The salivary electrolytes concentrations did not reveal significant difference between the two groups (p>0.05). The mean cotinine levels of PS children were 1.58+/-4.3 ng/mL. The salivary concentrations of protein were similar between the two groups (p>0.05). The salivary secretory IgA concentration was significantly lower in the PS group than controls. The sialic acid level and amylase activity in PS group were found significantly higher compared with the controls (p<0.05). No difference was observed for all these parameters with sex (p>0.05). When saliva samples were analysed for output, the sialic acid level and amylase activity increased significantly in PS subjects (p<0.05). Further, the output of secretory IgA concentration was found significantly lower compared with the controls (p<0.05). In conclusion, we show that passive smoking was associated with a decrease in secretory IgA concentration, whereas with increase in amylase activity and sialic acid level of stimulated whole saliva in young children.

Double mimicry evades tRNA synthetase editing by toxic vegetable-sourced non-proteinogenic amino acid.

PubMed

Song, Youngzee; Zhou, Huihao; Vo, My-Nuong; Shi, Yi; Nawaz, Mir Hussain; Vargas-Rodriguez, Oscar; Diedrich, Jolene K; Yates, John R; Kishi, Shuji; Musier-Forsyth, Karin; Schimmel, Paul

2017-12-22

Hundreds of non-proteinogenic (np) amino acids (AA) are found in plants and can in principle enter human protein synthesis through foods. While aminoacyl-tRNA synthetase (AARS) editing potentially provides a mechanism to reject np AAs, some have pathological associations. Co-crystal structures show that vegetable-sourced azetidine-2-carboxylic acid (Aze), a dual mimic of proline and alanine, is activated by both human prolyl- and alanyl-tRNA synthetases. However, it inserts into proteins as proline, with toxic consequences in vivo. Thus, dual mimicry increases odds for mistranslation through evasion of one but not both tRNA synthetase editing systems.

Comparative Analyses of the Lipooligosaccharides from Nontypeable Haemophilus influenzae and Haemophilus haemolyticus Show Differences in Sialic Acid and Phosphorylcholine Modifications

PubMed Central

Post, Deborah M. B.; Ketterer, Margaret R.; Coffin, Jeremy E.; Reinders, Lorri M.; Munson, Robert S.; Bair, Thomas; Murphy, Timothy F.; Foster, Eric D.; Gibson, Bradford W.

2016-01-01

Haemophilus haemolyticus and nontypeable Haemophilus influenzae (NTHi) are closely related upper airway commensal bacteria that are difficult to distinguish phenotypically. NTHi causes upper and lower airway tract infections in individuals with compromised airways, while H. haemolyticus rarely causes such infections. The lipooligosaccharide (LOS) is an outer membrane component of both species and plays a role in NTHi pathogenesis. In this study, comparative analyses of the LOS structures and corresponding biosynthesis genes were performed. Mass spectrometric and immunochemical analyses showed that NTHi LOS contained terminal sialic acid more frequently and to a higher extent than H. haemolyticus LOS did. Genomic analyses of 10 strains demonstrated that H. haemolyticus lacked the sialyltransferase genes lic3A and lic3B (9/10) and siaA (10/10), but all strains contained the sialic acid uptake genes siaP and siaT (10/10). However, isothermal titration calorimetry analyses of SiaP from two H. haemolyticus strains showed a 3.4- to 7.3-fold lower affinity for sialic acid compared to that of NTHi SiaP. Additionally, mass spectrometric and immunochemical analyses showed that the LOS from H. haemolyticus contained phosphorylcholine (ChoP) less frequently than the LOS from NTHi strains. These differences observed in the levels of sialic acid and ChoP incorporation in the LOS structures from H. haemolyticus and NTHi may explain some of the differences in their propensities to cause disease. PMID:26729761

The remarkable stability of chimeric, sialic acid-derived alpha/delta-peptides in human blood plasma.

PubMed

Saludes, Jonel P; Natarajan, Arutselvan; DeNardo, Sally J; Gervay-Hague, Jacquelyn

2010-05-01

Peptides are labile toward proteolytic enzymes, and structural modifications are often required to prolong their metabolic half-life and increase resistance. One modification is the incorporation of non-alpha-amino acids into the peptide to deter recognition by hydrolytic enzymes. We previously reported the synthesis of chimeric alpha/delta-peptides from glutamic acids (Glu) and the sialic acid derivative Neu2en. Conformational analyses revealed these constructs adopt secondary structures in water and may serve as conformational surrogates of polysialic acid. Polysialic acid is a tumor-associated polysaccharide and is correlated with cancer metastasis. Soluble polysialic acid is rapidly cleared from the blood limiting its potential for vaccine development. One motivation in developing structural surrogates of polysialic acid was to create constructs with increased bioavailability. Here, we report plasma stability profiles of Glu/Neu2en alpha/delta-peptides. DOTA was conjugated at the peptide N-termini by solid phase peptide synthesis, radiolabeled with (111)In, incubated in human blood plasma at 37 degrees C, and their degradation patterns monitored by cellulose acetate electrophoresis and radioactivity counting. Results indicate that these peptides exhibit a long half-life that is two- to three-orders of magnitude higher than natural alpha-peptides. These findings provide a viable platform for the synthesis of plasma stable, sialic acid-derived peptides that may find pharmaceutical application.

Identification, characterization and leucocyte expression of Siglec-10, a novel human sialic acid-binding receptor.

PubMed Central

Munday, J; Kerr, S; Ni, J; Cornish, A L; Zhang, J Q; Nicoll, G; Floyd, H; Mattei, M G; Moore, P; Liu, D; Crocker, P R

2001-01-01

Here we characterize Siglec-10 as a new member of the Siglec family of sialic acid-binding Ig-like lectins. A full-length cDNA was isolated from a human spleen library and the corresponding gene identified. Siglec-10 is predicted to contain five extracellular Ig-like domains and a cytoplasmic tail containing three putative tyrosine-based signalling motifs. Siglec-10 exhibited a high degree of sequence similarity to CD33-related Siglecs and mapped to the same region, on chromosome 19q13.3. The expressed protein was able to mediate sialic acid-dependent binding to human erythrocytes and soluble sialoglycoconjugates. Using specific antibodies, Siglec-10 was detected on subsets of human leucocytes including eosinophils, monocytes and a minor population of natural killer-like cells. The molecular properties and expression pattern suggest that Siglec-10 may function as an inhibitory receptor within the innate immune system. PMID:11284738

Polysaccharide capsule and sialic acid-mediated regulation promote biofilm-like intracellular bacterial communities during cystitis.

PubMed

Anderson, Gregory G; Goller, Carlos C; Justice, Sheryl; Hultgren, Scott J; Seed, Patrick C

2010-03-01

Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infections (UTIs). A murine UTI model has revealed an infection cascade whereby UPEC undergoes cycles of invasion of the bladder epithelium, intracellular proliferation in polysaccharide-containing biofilm-like masses called intracellular bacterial communities (IBC), and then dispersal into the bladder lumen to initiate further rounds of epithelial colonization and invasion. We predicted that the UPEC K1 polysaccharide capsule is a key constituent of the IBC matrix. Compared to prototypic E. coli K1 strain UTI89, a capsule assembly mutant had a fitness defect in functionally TLR4(+) and TLR4(-) mice, suggesting a protective role of capsule in inflamed and noninflamed hosts. K1 capsule assembly and synthesis mutants had dramatically reduced IBC formation, demonstrating the common requirement for K1 polysaccharide in IBC development. The capsule assembly mutant appeared dispersed in the cytoplasm of the bladder epithelial cells and failed to undergo high-density intracellular replication during later stages of infection, when the wild-type strain continued to form serial generations of IBC. Deletion of the sialic acid regulator gene nanR partially restored IBC formation in the capsule assembly mutant. These data suggest that capsule is necessary for efficient IBC formation and that aberrant sialic acid accumulation, resulting from disruption of K1 capsule assembly, produces a NanR-mediated defect in intracellular proliferation and IBC development. Together, these data demonstrate the complex but important roles of UPEC polysaccharide encapsulation and sialic acid signaling in multiple stages of UTI pathogenesis.

Sialic Acid-Responsive Polymeric Interface Material: From Molecular Recognition to Macroscopic Property Switching

NASA Astrophysics Data System (ADS)

Xiong, Yuting; Jiang, Ge; Li, Minmin; Qing, Guangyan; Li, Xiuling; Liang, Xinmiao; Sun, Taolei

2017-01-01

Biological systems that utilize multiple weak non-covalent interactions and hierarchical assemblies to achieve various bio-functions bring much inspiration for the design of artificial biomaterials. However, it remains a big challenge to correlate underlying biomolecule interactions with macroscopic level of materials, for example, recognizing such weak interaction, further transforming it into regulating material’s macroscopic property and contributing to some new bio-applications. Here we designed a novel smart polymer based on polyacrylamide (PAM) grafted with lactose units (PAM-g-lactose0.11), and reported carbohydrate-carbohydrate interaction (CCI)-promoted macroscopic properties switching on this smart polymer surface. Detailed investigations indicated that the binding of sialic acid molecules with the grafted lactose units via the CCIs induced conformational transformation of the polymer chains, further resulted in remarkable and reversible switching in surface topography, wettability and stiffness. With these excellent recognition and response capacities towards sialic acid, the PAM-g-lactose0.11 further facilitated good selectivity, strong anti-interference and high adsorption capacity in the capture of sialylated glycopeptides (important biomarkers for cancers). This work provides some enlightenment for the development of biointerface materials with tunable property, as well as high-performance glycopeptide enrichment materials.

Direct detection of ligand binding to Sepharose-immobilised protein using saturation transfer double difference (STDD) NMR spectroscopy

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

Haselhorst, Thomas; Muenster-Kuehnel, Anja K.; Oschlies, Melanie

2007-08-10

We report an easy and direct application of 'Saturation Transfer Double Difference' (STDD) NMR spectroscopy to identify ligands that bind to a Sepharose-immobilised target protein. The model protein, cytidine 5'-monophosphate sialic acid (CMP-Sia) synthetase, was expressed as a Strep-Tag II fusion protein and immobilised on Strep-Tactin Sepharose. STD NMR experiments of the protein-enriched Sepharose matrix in the presence of a binding ligand (cytidine 5'-triphosphate, CTP) and a non-binding ligand ({alpha}/{beta}-glucose) clearly show that CTP binds to the immobilised enzyme, whereas glucose has no affinity. This approach has three major advantages: (a) only low quantities of protein are required, (b) nomore » specialised NMR technology or the application of additional data analysis by non-routine methods is required, and (c) easy multiple use of the immobilised protein is available.« less

Molecular interaction of Siglecs (sialic acid-binding Ig-like lectins) with sialylated ligands on Trypanosoma cruzi.

PubMed

Jacobs, Thomas; Erdmann, Hanna; Fleischer, Bernhard

2010-01-01

The protozoan parasite Trypanosoma cruzi (T. cruzi) is transmitted by blood-sucking insect vectors. After transmission, parasites circulate in the blood as trypomastigotes and invade a variety of cells to multiply intracellularly as amastigotes. The acute phase triggers an immune response that restricts the dissemination and proliferation of parasites. However, parasites are able to persist in different tissues for decades causing the pathology of Chagas' disease. T. cruzi expresses a trans-sialidase (TS). This unique enzyme transfers sialic acid from host glycoconjugates to mucin-like molecules on the parasite and is supposed to be a major virulence factor. TS and sialylated structures were implicated in the persistence of parasites. We discuss here the recent findings on the function of sialylated structures on the surface of T. cruzi with a special emphasis on their property to interact with sialic acid-binding Ig-like lectins, which may allow the parasite to modulate the immune system of the host. Copyright (c) 2009 Elsevier GmbH. All rights reserved.

Unravelling the specificity and mechanism of sialic acid recognition by the gut symbiont Ruminococcus gnavus.

PubMed

Owen, C David; Tailford, Louise E; Monaco, Serena; Šuligoj, Tanja; Vaux, Laura; Lallement, Romane; Khedri, Zahra; Yu, Hai; Lecointe, Karine; Walshaw, John; Tribolo, Sandra; Horrex, Marc; Bell, Andrew; Chen, Xi; Taylor, Gary L; Varki, Ajit; Angulo, Jesus; Juge, Nathalie

2017-12-19

Ruminococcus gnavus is a human gut symbiont wherein the ability to degrade mucins is mediated by an intramolecular trans-sialidase (RgNanH). RgNanH comprises a GH33 catalytic domain and a sialic acid-binding carbohydrate-binding module (CBM40). Here we used glycan arrays, STD NMR, X-ray crystallography, mutagenesis and binding assays to determine the structure and function of RgNanH_CBM40 (RgCBM40). RgCBM40 displays the canonical CBM40 β-sandwich fold and broad specificity towards sialoglycans with millimolar binding affinity towards α2,3- or α2,6-sialyllactose. RgCBM40 binds to mucus produced by goblet cells and to purified mucins, providing direct evidence for a CBM40 as a novel bacterial mucus adhesin. Bioinformatics data show that RgCBM40 canonical type domains are widespread among Firmicutes. Furthermore, binding of R. gnavus ATCC 29149 to intestinal mucus is sialic acid mediated. Together, this study reveals novel features of CBMs which may contribute to the biogeography of symbiotic bacteria in the gut.

Colloidal and electrochemical aspects of copper-CMP

NASA Astrophysics Data System (ADS)

Sun, Yuxia

Copper based interconnects with low dielectric constant layers are currently used to increase interconnect densities and reduce interconnect time delays in integrated circuits. The technology used to develop copper interconnects involves Chemical Mechanical Planarization (CMP) of copper films deposited on low-k layers (silica or silica based films), which is carried out using slurries containing abrasive particles. One issue using such a structure is copper contamination over dielectric layers (SiO2 film), if not reduced, this contamination will cause current leakage. In this study, the conditions conducive to copper contamination onto SiO2 films during Cu-CMP process were studied, and a post-CMP cleaning technique was discussed based on experimental results. It was found that the adsorption of copper onto a silica surface is kinetically fast (<0.5 minute). The amount of copper absorbed is pH and concentration dependent and affected by presence of H2O2, complexing agents, and copper corrosion inhibitor Benzotrazole. Based on de-sorption results, DI water alone was unable to reduce adsorbed copper to an acceptable level, especially for adsorption that takes place at a higher pH condition. The addition of complex agent, citric acid, proved effective in suppressing copper adsorption onto oxide silica during polishing or post-CMP cleaning by forming stable copper-CA complexes. Surface Complexation Modeling was used to simulate copper adsorption isotherms and predict the copper contamination levels on SiO2 surfaces. Another issue with the application of copper CMP is its environmental impact. CMP is a costly process due to its huge consumption of pure water and slurry. Additionally, Cu-CMP processing generates a waste stream containing certain amounts of copper and abrasive slurry particles. In this study, the separation technique electrocoagulation was investigated to remove both copper and abrasive slurry particles simultaneously. For effluent containing ˜40 ppm

Sialic acids regulate microvessel permeability, revealed by novel in vivo studies of endothelial glycocalyx structure and function

PubMed Central

Betteridge, Kai B.; Arkill, Kenton P.; Neal, Christopher R.; Harper, Steven J.; Foster, Rebecca R.; Satchell, Simon C.; Bates, David O.

2017-01-01

Key points We have developed novel techniques for paired, direct, real‐time in vivo quantification of endothelial glycocalyx structure and associated microvessel permeability.Commonly used imaging and analysis techniques yield measurements of endothelial glycocalyx depth that vary by over an order of magnitude within the same vessel.The anatomical distance between maximal glycocalyx label and maximal endothelial cell plasma membrane label provides the most sensitive and reliable measure of endothelial glycocalyx depth.Sialic acid residues of the endothelial glycocalyx regulate glycocalyx structure and microvessel permeability to both water and albumin. Abstract The endothelial glycocalyx forms a continuous coat over the luminal surface of all vessels, and regulates multiple vascular functions. The contribution of individual components of the endothelial glycocalyx to one critical vascular function, microvascular permeability, remains unclear. We developed novel, real‐time, paired methodologies to study the contribution of sialic acids within the endothelial glycocalyx to the structural and functional permeability properties of the same microvessel in vivo. Single perfused rat mesenteric microvessels were perfused with fluorescent endothelial cell membrane and glycocalyx labels, and imaged with confocal microscopy. A broad range of glycocalyx depth measurements (0.17–3.02 μm) were obtained with different labels, imaging techniques and analysis methods. The distance between peak cell membrane and peak glycocalyx label provided the most reliable measure of endothelial glycocalyx anatomy, correlating with paired, numerically smaller values of endothelial glycocalyx depth (0.078 ± 0.016 μm) from electron micrographs of the same portion of the same vessel. Disruption of sialic acid residues within the endothelial glycocalyx using neuraminidase perfusion decreased endothelial glycocalyx depth and increased apparent solute permeability to albumin in the same

Evaluation of the antitumor effects of vitamin K2 (menaquinone-7) nanoemulsions modified with sialic acid-cholesterol conjugate.

PubMed

Shi, Jia; Zhou, Songlei; Kang, Le; Ling, Hu; Chen, Jiepeng; Duan, Lili; Song, Yanzhi; Deng, Yihui

2018-02-01

Numerous studies have recently shown that vitamin K 2 (VK 2 ) has antitumor effects in a variety of tumor cells, but there are few reports demonstrating antitumor effects of VK 2 in vivo. The antitumor effects of VK 2 in nanoemulsions are currently not known. Therefore, we sought to characterize the antitumor potential of VK 2 nanoemulsions in S180 tumor cells in the present study. Furthermore, a ligand conjugate sialic acid-cholesterol, with enhanced affinity towards the membrane receptors overexpressed in tumors, was anchored on the surface of the nanoemulsions to increase VK 2 distribution to the tumor tissue. VK 2 was encapsulated in oil-in-water nanoemulsions, and the physical and chemical stability of the nanoemulsions were characterized during storage at 25 °C. At 25 °C, all nanoemulsions remained physically and chemically stable with little change in particle size. An in vivo study using syngeneic mice with subcutaneously established S180 tumors demonstrated that intravenous or intragastric administration of VK 2 nanoemulsions significantly suppressed the tumor growth. The VK 2 nanoemulsions modified with sialic acid-cholesterol conjugate showed higher tumor growth suppression than the VK 2 nanoemulsions, while neither of them exhibited signs of drug toxicity. In summary, VK 2 exerted effective antitumor effects in vivo, and VK 2 nanoemulsions modified with sialic acid-cholesterol conjugate enhanced the antitumor activity, suggesting that these VK 2 may be promising agents for the prevention or treatment of tumor in patients.

Quantum Mechanics/Molecular Mechanics Study of the Sialyltransferase Reaction Mechanism.

PubMed

Hamada, Yojiro; Kanematsu, Yusuke; Tachikawa, Masanori

2016-10-11

The sialyltransferase is an enzyme that transfers the sialic acid moiety from cytidine 5'-monophospho-N-acetyl-neuraminic acid (CMP-NeuAc) to the terminal position of glycans. To elucidate the catalytic mechanism of sialyltransferase, we explored the potential energy surface along the sialic acid transfer reaction coordinates by the hybrid quantum mechanics/molecular mechanics method on the basis of the crystal structure of sialyltransferase CstII. Our calculation demonstrated that CstII employed an S N 1-like reaction mechanism via the formation of a short-lived oxocarbenium ion intermediate. The computational barrier height was 19.5 kcal/mol, which reasonably corresponded with the experimental reaction rate. We also found that two tyrosine residues (Tyr156 and Tyr162) played a vital role in stabilizing the intermediate and the transition states by quantum mechanical interaction with CMP.

Metabolism of vertebrate amino sugars with N-glycolyl groups: mechanisms underlying gastrointestinal incorporation of the non-human sialic acid xeno-autoantigen N-glycolylneuraminic acid.

PubMed

Banda, Kalyan; Gregg, Christopher J; Chow, Renee; Varki, Nissi M; Varki, Ajit

2012-08-17

Although N-acetyl groups are common in nature, N-glycolyl groups are rare. Mammals express two major sialic acids, N-acetylneuraminic acid and N-glycolylneuraminic acid (Neu5Gc). Although humans cannot produce Neu5Gc, it is detected in the epithelial lining of hollow organs, endothelial lining of the vasculature, fetal tissues, and carcinomas. This unexpected expression is hypothesized to result via metabolic incorporation of Neu5Gc from mammalian foods. This accumulation has relevance for diseases associated with such nutrients, via interaction with Neu5Gc-specific antibodies. Little is known about how ingested sialic acids in general and Neu5Gc in particular are metabolized in the gastrointestinal tract. We studied the gastrointestinal and systemic fate of Neu5Gc-containing glycoproteins (Neu5Gc-glycoproteins) or free Neu5Gc in the Neu5Gc-free Cmah(-/-) mouse model. Ingested free Neu5Gc showed rapid absorption into the circulation and urinary excretion. In contrast, ingestion of Neu5Gc-glycoproteins led to Neu5Gc incorporation into the small intestinal wall, appearance in circulation at a steady-state level for several hours, and metabolic incorporation into multiple peripheral tissue glycoproteins and glycolipids, thus conclusively proving that Neu5Gc can be metabolically incorporated from food. Feeding Neu5Gc-glycoproteins but not free Neu5Gc mimics the human condition, causing tissue incorporation into human-like sites in Cmah(-/-) fetal and adult tissues, as well as developing tumors. Thus, glycoproteins containing glycosidically linked Neu5Gc are the likely dietary source for human tissue accumulation, and not the free monosaccharide. This human-like model can be used to elucidate specific mechanisms of Neu5Gc delivery from the gut to tissues, as well as general mechanisms of metabolism of ingested sialic acids.

TRYPTOPHANASE-TRYPTOPHAN SYNTHETASE SYSTEMS IN ESCHERICHIA COLI I.

PubMed Central

Freundlich, Martin; Lichstein, Herman C.

1962-01-01

Freundlich, Martin (University of Minnesota, Minneapolis) and Herman C. Lichstein. Tryptophanase-tryptophan synthetase systems in Escherichia coli. I. Effect of tryptophan and related compounds. J. Bacteriol. 84:979–987. 1962.—The effect of tryptophan and related compounds on tryptophanase and tryptophan synthetase formation in Escherichia coli was determined. Several of these compounds stimulated the formation of tryptophanase while concomitantly decreasing the production of synthetase. A number of tryptophan analogues were found to inhibit growth. The possible mode of action of these substances was examined further. 5-Hydroxytryptophan greatly inhibited the formation of synthetase and also reduced growth. Its inhibitory action on growth was attributed, at least partially, to the false feedback inhibition of anthranilic acid formation. Tryptamine was found to be a potent inhibitor of the activity of synthetase, as well as of the enzyme(s) involved in the synthesis of anthranilic acid from shikimic acid. However, growth reduction was only partially reversed by tryptophan. Indole-3-acetic acid and indole-3-propionic acid decreased growth and increased the formation of synthetase six- to eightfold. The action of these compounds was ascribed to their ability to block the endogenous formation of tryptophan. PMID:13959621

CMP‑N‑acetylneuraminic acid synthetase interacts with fragile X related protein 1.

PubMed

Ma, Yun; Tian, Shuai; Wang, Zongbao; Wang, Changbo; Chen, Xiaowei; Li, Wei; Yang, Yang; He, Shuya

2016-08-01

Fragile X mental retardation protein (FMRP), fragile X related 1 protein (FXR1P) and FXR2P are the members of the FMR protein family. These proteins contain two KH domains and a RGG box, which are characteristic of RNA binding proteins. The absence of FMRP, causes fragile X syndrome (FXS), the leading cause of hereditary mental retardation. FXR1P is expressed throughout the body and important for normal muscle development, and its absence causes cardiac abnormality. To investigate the functions of FXR1P, a screen was performed to identify FXR1P‑interacting proteins and determine the biological effect of the interaction. The current study identified CMP‑N‑acetylneuraminic acid synthetase (CMAS) as an interacting protein using the yeast two‑hybrid system, and the interaction between FXR1P and CMAS was validated in yeast using a β‑galactosidase assay and growth studies with selective media. Furthermore, co‑immunoprecipitation was used to analyze the FXR1P/CMAS association and immunofluorescence microscopy was performed to detect expression and intracellular localization of the proteins. The results of the current study indicated that FXR1P and CMAS interact, and colocalize in the cytoplasm and the nucleus of HEK293T and HeLa cells. Accordingly, a fragile X related 1 (FXR1) gene overexpression vector was constructed to investigate the effect of FXR1 overexpression on the level of monosialotetrahexosylganglioside 1 (GM1). The results of the current study suggested that FXR1P is a tissue‑specific regulator of GM1 levels in SH‑SY5Y cells, but not in HEK293T cells. Taken together, the results initially indicate that FXR1P interacts with CMAS, and that FXR1P may enhance the activation of sialic acid via interaction with CMAS, and increase GM1 levels to affect the development of the nervous system, thus providing evidence for further research into the pathogenesis of FXS.

Serum sialic acid and oxidative stress parameters changes in cattle with leptospirosis.

PubMed

Erdogan, H M; Karapehlivan, M; Citil, M; Atakisi, O; Uzlu, E; Unver, A

2008-04-01

This study was designed to disclose some indicators of oxidative stress and inflammation in natural cases of bovine leptospirosis. For this purpose, 12 bulls exhibiting clinical signs of leptospirosis and 10 healthy bulls were used. Animals were subjected to thorough clinical examination and the clinical signs were recorded. All animals were blood sampled in order to determine serum total sialic acid (TSA), lipid bound sialic acid (LBSA), malondialdehyde (MDA), reduced glutathione (GSH), nitric oxide (NO), uric acid (UA), total protein (TP), albumin and glucose. Urine samples were collected from each animal and examined under dark-field microscope to observe spirochetes. Diseased animals exhibited clinical signs suggesting leptospirosis and the diagnosis was supported by positive dark-field microscope examination. Mean TSA (mmol/L), LBSA (mmol/L), TP (g/dl), albumin (g/dl), glucose (mg/dl), MDA (micromol/L), GSH (mg/dl), NO (nmol/ml), and UA (mg/L) levels were 1.63 +/- 0.02, 0.40 +/- 0.10, 7.18 +/- 0.24, 3.23 +/- 0.5, 64.96 +/- 1.88, 5.71 +/- 0.11, 78.68 +/- 0.72, 7.94 +/- 0.34, and 8.75 +/- 0.41 in healthy bulls, and 2.50 +/- 0.05, 0.70 +/- 0.2, 9.27 +/- 0.17, 2.55 +/- 0.62, 107.93 +/- 2.52, 8.82 +/- 0.14, 47.85 +/- 1.85, 14.57 +/- 0.63 and 15.85 +/- 0.80 in leptospirosis cases, respectively. The differences between the two groups were statistically significant (P < 0.001). Increased TSA, LBSA, MDA, NO, UA, TP, glucose and decreased GSH and albumin concentrations were suggestive of inflammation and oxidative stress in diseased bulls. The results obtained may suggest that oxidative damage along with other mechanisms might have taken part in the pathogenesis of bovine leptospirosis and further detailed studies are needed to fully understand the mechanism(s) of the disease.

N-Glycolylneuraminic acid deficiency worsens cardiac and skeletal muscle pathophysiology in α-sarcoglycan-deficient mice

PubMed Central

Martin, Paul T; Camboni, Marybeth; Xu, Rui; Golden, Bethannie; Chandrasekharan, Kumaran; Wang, Chiou-Miin; Varki, Ajit; Janssen, Paul M L

2013-01-01

Roughly 3 million years ago, an inactivating deletion occurred in CMAH, the human gene encoding CMP-Neu5Ac (cytidine-5′-monophospho-N-acetylneuraminic acid) hydroxylase (Chou HH, Takematsu H, Diaz S, Iber J, Nickerson E, Wright KL, Muchmore EA, Nelson DL, Warren ST, Varki A. 1998. A mutation in human CMP-sialic acid hydroxylase occurred after the Homo-Pan divergence. Proc Natl Acad Sci USA. 95:11751–11756). This inactivating deletion is now homozygous in all humans, causing the loss of N-glycolylneuraminic acid (Neu5Gc) biosynthesis in all human cells and tissues. The CMAH enzyme is active in other mammals, including mice, where Neu5Gc is an abundant form of sialic acid on cellular membranes, including those in cardiac and skeletal muscle. We recently demonstrated that the deletion of mouse Cmah worsened the severity of pathophysiology measures related to muscular dystrophy in mdx mice, a model for Duchenne muscular dystrophy (Chandrasekharan K, Yoon JH, Xu Y, deVries S, Camboni M, Janssen PM, Varki A, Martin PT. 2010. A human-specific deletion in mouse Cmah increases disease severity in the mdx model of Duchenne muscular dystrophy. Sci Transl Med. 2:42–54). Here, we demonstrate similar changes in cardiac and skeletal muscle pathology and physiology resulting from Cmah deletion in α-sarcoglycan-deficient (Sgca−/−) mice, a model for limb girdle muscular dystrophy 2D. These experiments demonstrate that loss of mouse Cmah can worsen disease severity in more than one form of muscular dystrophy and suggest that Cmah may be a general genetic modifier of muscle disease. PMID:23514716

Chlamydia trachomatis Scavenges Host Fatty Acids for Phospholipid Synthesis via an Acyl-Acyl Carrier Protein Synthetase*

PubMed Central

Yao, Jiangwei; Dodson, V. Joshua; Frank, Matthew W.; Rock, Charles O.

2015-01-01

The obligate intracellular parasite Chlamydia trachomatis has a reduced genome but relies on de novo fatty acid and phospholipid biosynthesis to produce its membrane phospholipids. Lipidomic analyses showed that 8% of the phospholipid molecular species synthesized by C. trachomatis contained oleic acid, an abundant host fatty acid that cannot be made by the bacterium. Mass tracing experiments showed that isotopically labeled palmitic, myristic, and lauric acids added to the medium were incorporated into C. trachomatis-derived phospholipid molecular species. HeLa cells did not elongate lauric acid, but infected HeLa cell cultures elongated laurate to myristate and palmitate. The elongated fatty acids were incorporated exclusively into C. trachomatis-produced phospholipid molecular species. C. trachomatis has adjacent genes encoding the separate domains of the bifunctional acyl-acyl carrier protein (ACP) synthetase/2-acylglycerolphosphoethanolamine acyltransferase gene (aas) of Escherichia coli. The CT775 gene encodes an acyltransferase (LpaT) that selectively transfers fatty acids from acyl-ACP to the 1-position of 2-acyl-glycerophospholipids. The CT776 gene encodes an acyl-ACP synthetase (AasC) with a substrate preference for palmitic compared with oleic acid in vitro. Exogenous fatty acids were elongated and incorporated into phospholipids by Escherichia coli-expressing AasC, illustrating its function as an acyl-ACP synthetase in vivo. These data point to an AasC-dependent pathway in C. trachomatis that selectively scavenges host saturated fatty acids to be used for the de novo synthesis of its membrane constituents. PMID:26195634

Host-Selected Amino Acid Changes at the Sialic Acid Binding Pocket of the Parvovirus Capsid Modulate Cell Binding Affinity and Determine Virulence

PubMed Central

López-Bueno, Alberto; Rubio, Mari-Paz; Bryant, Nathan; McKenna, Robert; Agbandje-McKenna, Mavis; Almendral, José M.

2006-01-01

The role of receptor recognition in the emergence of virulent viruses was investigated in the infection of severe combined immunodeficient (SCID) mice by the apathogenic prototype strain of the parvovirus minute virus of mice (MVMp). Genetic analysis of isolated MVMp viral clones (n = 48) emerging in mice, including lethal variants, showed only one of three single changes (V325M, I362S, or K368R) in the common sequence of the two capsid proteins. As was found for the parental isolates, the constructed recombinant viruses harboring the I362S or the K368R single substitutions in the capsid sequence, or mutations at both sites, showed a large-plaque phenotype and lower avidity than the wild type for cells in the cytotoxic interaction with two permissive fibroblast cell lines in vitro and caused a lethal disease in SCID mice when inoculated by the natural oronasal route. Significantly, the productive adsorption of MVMp variants carrying any of the three mutations selected through parallel evolution in mice showed higher sensitivity to the treatment of cells by neuraminidase than that of the wild type, indicating a lower affinity of the viral particle for the sialic acid component of the receptor. Consistent with this, the X-ray crystal structure of the MVMp capsids soaked with sialic acid (N-acetyl neuraminic acid) showed the sugar allocated in the depression at the twofold axis of symmetry (termed the dimple), immediately adjacent to residues I362 and K368, which are located on the wall of the dimple, and approximately 22 Å away from V325 in a threefold-related monomer. This is the first reported crystal structure identifying an infectious receptor attachment site on a parvovirus capsid. We conclude that the affinity of the interactions of sialic-acid-containing receptors with residues at or surrounding the dimple can evolutionarily regulate parvovirus pathogenicity and adaptation to new hosts. PMID:16415031

Characterization of chemical interactions during chemical mechanical polishing (CMP) of copper

NASA Astrophysics Data System (ADS)

Lee, Seung-Mahn

2003-10-01

Chemical mechanical polishing (CMP) has received much attention as an unique technique to provide a wafer level planarization in semiconductor manufacturing. However, despite the extensive use of CMP, it still remains one of the least understood areas in semiconductor processing. The lack of the fundamental understanding is a significant barrier to further advancements in CMP technology. One critical aspect of metal CMP is the formation of a thin surface layer on the metal surface. The formation and removal of this layer controls all the aspects of the CMP process, including removal rate, surface finish, etc. In this dissertation, we focus on the characterization of the formation and removal of the thin surface layer on the copper surface. The formation dynamics was investigated using static and dynamic electrochemical techniques, including potentiodynamic scans and chronoamperometry. The results were validated using XPS measurements. The mechanical properties of the surface layer were investigated using nanoindentation measurements. The electrochemical investigation showed that the thickness of the surface layer is controlled by the chemicals such as an oxidizer (hydrogen peroxide), a corrosion inhibitor (benzotriazole), a complexing agent (citric acid), and their concentrations. The dynamic electrochemical measurements indicated that the initial layer formation kinetics is unaffected by the corrosion inhibitors. The passivation due to the corrosion inhibitor becomes important only on large time scales (>200 millisecond). The porosity and the density of the chemically modified surface layer can be affected by additives of other chemicals such as citric acid. An optimum density of the surface layer is required for high polishing rate while at the same time maintaining a high degree of surface finish. Nanoindentation measurements indicated that the mechanical properties of the surface layer are strongly dependent on the chemical additives in the slurry. The CMP

Synthesis of novel ganglioside GM4 analogues containing N-deacetylated and lactamized sialic acid: probes for searching new ligand structures for human L-selectin.

PubMed

Otsubo, N; Ishida, H; Kiso, M

2001-01-15

Novel ganglioside GM4 analogues, which contain N-deacetylated or lactamized sialic acid instead of usual N-acetylneuraminic acid, were synthesized in a highly efficient manner. (Methyl 4,7,8,9-tetra-O-acetyl-3,5-dideoxy-5-trifluoroacetamido-D-glycero-alpha-D-galacto-2-nonulopyranosylonate)-(2-->3)-4,6-di-O-acetyl-2-O-benzoyl-D-galactopyranosyl trichloroacetimidate was coupled with 2-(tetradecyl)hexadecanol to give the desired beta-glycoside in high yield. Successive O- and N-deacylation, and saponification of the methyl ester group afforded the N-deacetylated sialyl derivative that was converted by treatment with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in Me2SO into the lactamized sialic acid-containing ganglioside GM4 analogue.

4-O-Acetyl-sialic acid (Neu4,5Ac2) in acidic milk oligosaccharides of the platypus (Ornithorhynchus anatinus) and its evolutionary significance.

PubMed

Urashima, Tadasu; Inamori, Hiroaki; Fukuda, Kenji; Saito, Tadao; Messer, Michael; Oftedal, Olav T

2015-06-01

Monotremes (echidnas and platypus) retain an ancestral form of reproduction: egg-laying followed by secretion of milk onto skin and hair in a mammary patch, in the absence of nipples. Offspring are highly immature at hatching and depend on oligosaccharide-rich milk for many months. The primary saccharide in long-beaked echidna milk is an acidic trisaccharide Neu4,5Ac2(α2-3)Gal(β1-4)Glc (4-O-acetyl 3'-sialyllactose), but acidic oligosaccharides have not been characterized in platypus milk. In this study, acidic oligosaccharides purified from the carbohydrate fraction of platypus milk were characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and (1)H-nuclear magnetic resonance spectroscopy. All identified structures, except Neu5Ac(α2-3)Gal(β1-4)Glc (3'-sialyllactose) contained Neu4,5Ac2 (4-O-acetyl-sialic acid). These include the trisaccharide 4-O-acetyl 3'-sialyllactose, the pentasaccharide Neu4,5Ac2(α2-3)Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)Glc (4-O-acetyl-3'-sialyllacto-N-tetraose d) and the hexasaccharide Neu4,5Ac2(α2-3)Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-3)Gal(β1-4)Glc (4-O-acetyl-3'-sialyllacto-N-fucopentaose III). At least seven different octa- to deca-oligosaccharides each contained a lacto-N-neohexaose core (LNnH) and one or two Neu4,5Ac2 and one to three fucose residues. We conclude that platypus milk contains a diverse (≥ 20) array of neutral and acidic oligosaccharides based primarily on lactose, lacto-N-neotetraose (LNnT) and LNnH structural cores and shares with echidna milk the unique feature that all identified acidic oligosaccharides (other than 3'-sialyllactose) contain the 4-O-acetyl-sialic acid moiety. We propose that 4-O-acetylation of sialic acid moieties protects acidic milk oligosaccharides secreted onto integumental surfaces from bacterial hydrolysis via steric interference with bacterial sialidases. This may be of evolutionary significance since taxa ancestral to monotremes and other mammals are

Arabidopsis thaliana GH3.5 acyl acid amido synthetase mediates metabolic crosstalk in auxin and salicylic acid homeostasis

PubMed Central

Westfall, Corey S.; Sherp, Ashley M.; Zubieta, Chloe; Alvarez, Sophie; Schraft, Evelyn; Marcellin, Romain; Ramirez, Loren; Jez, Joseph M.

2016-01-01

In Arabidopsis thaliana, the acyl acid amido synthetase Gretchen Hagen 3.5 (AtGH3.5) conjugates both indole-3-acetic acid (IAA) and salicylic acid (SA) to modulate auxin and pathogen response pathways. To understand the molecular basis for the activity of AtGH3.5, we determined the X-ray crystal structure of the enzyme in complex with IAA and AMP. Biochemical analysis demonstrates that the substrate preference of AtGH3.5 is wider than originally described and includes the natural auxin phenylacetic acid (PAA) and the potential SA precursor benzoic acid (BA). Residues that determine IAA versus BA substrate preference were identified. The dual functionality of AtGH3.5 is unique to this enzyme although multiple IAA-conjugating GH3 proteins share nearly identical acyl acid binding sites. In planta analysis of IAA, PAA, SA, and BA and their respective aspartyl conjugates were determined in wild-type and overexpressing lines of A. thaliana. This study suggests that AtGH3.5 conjugates auxins (i.e., IAA and PAA) and benzoates (i.e., SA and BA) to mediate crosstalk between different metabolic pathways, broadening the potential roles for GH3 acyl acid amido synthetases in plants. PMID:27849615

Neurologic syndrome associated with homozygous mutation at MAG sialic acid binding site.

PubMed

Roda, Ricardo H; FitzGibbon, Edmond J; Boucekkine, Houda; Schindler, Alice B; Blackstone, Craig

2016-08-01

The MAG gene encodes myelin-associated glycoprotein (MAG), an abundant protein involved in axon-glial interactions and myelination during nerve regeneration. Several members of a consanguineous family with a clinical syndrome reminiscent of Pelizaeus-Merzbacher disease and demyelinating leukodystrophy on brain MRI were recently found to harbor a homozygous missense p.Ser133Arg MAG mutation. Here, we report two brothers from a nonconsanguineous family afflicted with progressive cognitive impairment, neuropathy, ataxia, nystagmus, and gait disorder. Exome sequencing revealed the homozygous missense mutation p.Arg118His in MAG. This Arg118 residue in immunoglobulin domain 1 is critical for sialic acid binding, providing a compelling mechanistic basis for disease pathogenesis.

HALE STAIN FOR SIALIC ACID-CONTAINING MUCINS. ADAPTATION TO ELECTRON MICROSCOPY.

PubMed

GASIC, G; BERWICK, L

1963-10-01

The feasibility of using the Hale stain to identify cellular sialic acid-containing mucins by electron microscopy was investigated. Three kinds of mouse ascites tumor cells were fixed in neutral buffered formalin, exposed to fresh colloidal ferric oxide, treated with potassium ferrocyanide, imbedded in Selectron, and sectioned for electron microscopy. Additional staining with uranyl acetate and potassium permanganate was done after sectioning in order to increase contrast. Those cells known to be coated with sialomucin showed deposits of electron-opaque ferric ferrocyanide crystals in the areas where sialomucin concentrations were expected. When these cells were treated with neuraminidase beforehand, these deposits did not appear. It was concluded that, with the precautions and modifications described, the Hale stain can be successfully combined with electron microscopy to identify sialomucin.

The Aminoacyl-tRNA Synthetase Complex.

PubMed

Mirande, Marc

2017-01-01

Aminoacyl-tRNA synthetases (AARSs) are essential enzymes that specifically aminoacylate one tRNA molecule by the cognate amino acid. They are a family of twenty enzymes, one for each amino acid. By coupling an amino acid to a specific RNA triplet, the anticodon, they are responsible for interpretation of the genetic code. In addition to this translational, canonical role, several aminoacyl-tRNA synthetases also fulfill nontranslational, moonlighting functions. In mammals, nine synthetases, those specific for amino acids Arg, Asp, Gln, Glu, Ile, Leu, Lys, Met and Pro, associate into a multi-aminoacyl-tRNA synthetase complex, an association which is believed to play a key role in the cellular organization of translation, but also in the regulation of the translational and nontranslational functions of these enzymes. Because the balance between their alternative functions rests on the assembly and disassembly of this supramolecular entity, it is essential to get precise insight into the structural organization of this complex. The high-resolution 3D-structure of the native particle, with a molecular weight of about 1.5 MDa, is not yet known. Low-resolution structures of the multi-aminoacyl-tRNA synthetase complex, as determined by cryo-EM or SAXS, have been reported. High-resolution data have been reported for individual enzymes of the complex, or for small subcomplexes. This review aims to present a critical view of our present knowledge of the aminoacyl-tRNA synthetase complex in 3D. These preliminary data shed some light on the mechanisms responsible for the balance between the translational and nontranslational functions of some of its components.

Crystallization and preliminary X-ray diffraction analysis of the sialic acid-binding domain (VP8*) of porcine rotavirus strain CRW-8

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

Scott, Stacy A.; Holloway, Gavan; Coulson, Barbara S.

2005-06-01

The sialic acid-binding domain (VP8*) component of the porcine CRW-8 rotavirus spike protein has been overexpressed in E. coli, purified and co-crystallized with an N-acetylneuraminic acid derivative. X-ray diffraction data have been collected to 2.3 Å, which has enabled determination of the structure by molecular replacement. Rotavirus recognition and attachment to host cells involves interaction with the spike protein VP4 that projects outwards from the surface of the virus particle. An integral component of these spikes is the VP8* domain, which is implicated in the direct recognition and binding of sialic acid-containing cell-surface carbohydrates and facilitates subsequent invasion by themore » virus. The expression, purification, crystallization and preliminary X-ray diffraction analysis of VP8* from porcine CRW-8 rotavirus is reported. Diffraction data have been collected to 2.3 Å resolution, enabling the determination of the VP8* structure by molecular replacement.« less

Establishment and characterization of an Epstein-Barr virus-transformed B cell line, KM/C8, from a patient with infantile sialic acid storage disease.

PubMed

Nagatsuka, Y; Nakano, C; Nemoto, N; Jike, T; Ono, Y; Hirabayashi, Y

1998-07-23

Nakano et al. have recently reported a Japanese case of infantile sialic acid storage disease [C. Nakano, Y. Hirabayashi, K. Ohno, T. Yano, T. Mito, M. Sakurai, Brain Dev., 18 (1996) 153-156]. For further etiological analysis of this disease, we prepared the Epstein-Barr virus (EBV)-transformed cell line (LCL) from the peripheral lymphocytes of this patient and performed initial characterization of the cells. Electron microscopy of the cells showed that the cells contained many vacuoles and swelled lysosomes. Cytochemical staining with sialic acid-specific lectin, Limax flavus agglutinin (LFA), showed strong staining on membranes and subcellular organelles on the patient-derived cells, whereas LCL from a normal person was only weakly stained. The cells from the patient contained 5.5-7.3 nmol/107 cells of free N-acetyl neuraminic acid, whereas three strains of LCLs derived from normal persons contained 1 nmol/107 cells. The culture supernatant of LCL from the patient contained 144 nmol/ml of free N-acetyl neuraminic acid, whereas the LCL culture supernatant from normal persons contained 57-73 nmol/ml of free sialic acid, which was the same or only at a slightly higher level than the fresh medium. In addition, cellular acidic sialidase measured as 4-methylumbelliferyl sialidase was elevated (107 nmol 4-methylumbelliferon released/mg cellular protein/60 min). The EBV-LCL from an ISSD patient is considered to remain as the abnormality of the cell donor. Copyright 1998 Elsevier Science B.V. All rights reserved.

The trans-sialidase from Trypanosoma cruzi induces thrombocytopenia during acute Chagas' disease by reducing the platelet sialic acid contents.

PubMed

Tribulatti, María Virginia; Mucci, Juan; Van Rooijen, Nico; Leguizamón, María Susana; Campetella, Oscar

2005-01-01

Strong thrombocytopenia is observed during acute infection with Trypanosoma cruzi, the parasitic protozoan agent of American trypanosomiasis or Chagas' disease. The parasite sheds trans-sialidase, an enzyme able to mobilize the sialyl residues on cell surfaces, which is distributed in blood and is a virulence factor. Since the sialic acid content on the platelet surface is crucial for determining the half-life of platelets in blood, we examined the possible involvement of the parasite-derived enzyme in thrombocytopenia induction. We found that a single intravenous injection of trans-sialidase into naive mice reduced the platelet count by 50%, a transient effect that lasted as long as the enzyme remained in the blood. CD43(-/-) mice were affected to a similar extent. When green fluorescent protein-expressing platelets were treated in vitro with trans-sialidase, their sialic acid content was reduced together with their life span, as determined after transfusion into naive animals. No apparent deleterious effect on the bone marrow was observed. A central role for Kupffer cells in the clearance of trans-sialidase-altered platelets was revealed after phagocyte depletion by administration of clodronate-containing liposomes and splenectomy. Consistent with this, parasite strains known to exhibit more trans-sialidase activity induced heavier thrombocytopenia. Finally, the passive transfer of a trans-sialidase-neutralizing monoclonal antibody to infected animals prevented the clearance of transfused platelets. Results reported here strongly support the hypothesis that the trans-sialidase is the virulence factor that, after depleting the sialic acid content of platelets, induces the accelerated clearance of the platelets that leads to the thrombocytopenia observed during acute Chagas' disease.

SPOT4 Operational Control Center (CMP)

NASA Technical Reports Server (NTRS)

Zaouche, G.

1993-01-01

CNES(F) is responsible for the development of a new generation of Operational Control Center (CMP) which will operate the new heliosynchronous remote sensing satellite (SPOT4). This Operational Control Center takes large benefit from the experience of the first generation of control center and from the recent advances in computer technology and standards. The CMP is designed for operating two satellites all the same time with a reduced pool of controllers. The architecture of this CMP is simple, robust, and flexible, since it is based on powerful distributed workstations interconnected through an Ethernet LAN. The application software uses modern and formal software engineering methods, in order to improve quality and reliability, and facilitate maintenance. This software is table driven so it can be easily adapted to other operational needs. Operation tasks are automated to the maximum extent, so that it could be possible to operate the CMP automatically with very limited human interference for supervision and decision making. This paper provides an overview of the SPOTS mission and associated ground segment. It also details the CMP, its functions, and its software and hardware architecture.

Steric and thermodynamic limits of design for the incorporation of large unnatural amino acids in aminoacyl-tRNA synthetase enzymes.

PubMed

Armen, Roger S; Schiller, Stefan M; Brooks, Charles L

2010-06-01

Orthogonal aminoacyl-tRNA synthetase/tRNA pairs from archaea have been evolved to facilitate site specific in vivo incorporation of unnatural amino acids into proteins in Escherichia coli. Using this approach, unnatural amino acids have been successfully incorporated with high translational efficiency and fidelity. In this study, CHARMM-based molecular docking and free energy calculations were used to evaluate rational design of specific protein-ligand interactions for aminoacyl-tRNA synthetases. A series of novel unnatural amino acid ligands were docked into the p-benzoyl-L-phenylalanine tRNA synthetase, which revealed that the binding pocket of the enzyme does not provide sufficient space for significantly larger ligands. Specific binding site residues were mutated to alanine to create additional space to accommodate larger target ligands, and then mutations were introduced to improve binding free energy. This approach was used to redesign binding sites for several different target ligands, which were then tested against the standard 20 amino acids to verify target specificity. Only the synthetase designed to bind Man-alpha-O-Tyr was predicted to be sufficiently selective for the target ligand and also thermodynamically stable. Our study suggests that extensive redesign of the tRNA synthatase binding pocket for large bulky ligands may be quite thermodynamically unfavorable.

Overexpression of acetyl-CoA synthetase in Saccharomyces cerevisiae increases acetic acid tolerance

PubMed Central

Ding, Jun; Holzwarth, Garrett; Penner, Michael H.; Patton-Vogt, Jana; Bakalinsky, Alan T.

2015-01-01

Acetic acid-mediated inhibition of the fermentation of lignocellulose-derived sugars impedes development of plant biomass as a source of renewable ethanol. In order to overcome this inhibition, the capacity of Saccharomyces cerevisiae to synthesize acetyl-CoA from acetic acid was increased by overexpressing ACS2 encoding acetyl-coenzyme A synthetase. Overexpression of ACS2 resulted in higher resistance to acetic acid as measured by an increased growth rate and shorter lag phase relative to a wild-type control strain, suggesting that Acs2-mediated consumption of acetic acid during fermentation contributes to acetic acid detoxification. PMID:25673654

Mutual promotion of electrochemical-localized surface plasmon resonance on nanochip for sensitive sialic acid detection.

PubMed

Li, Shuang; Liu, Jinglong; Lu, Yanli; Zhu, Long; Li, Candong; Hu, Lijiang; Li, Jun; Jiang, Jing; Low, Szeshin; Liu, Qingjun

2018-06-01

Localized surface plasmon resonance (LSPR) induced charge separation were concentrated on the metal nanoparticles surface, which made it sensitive to the surface refractive index changes during optical sensing. Similarly, electrochemical detection was based on the electron transformation on the electrode surface. Herein, we fabricated a nanochip by decorating a nanocone-array substrate with gold nanoparticles and silver nanoparticles for dynamic electro-optical spectroscopy. Mercaptophenyl boronic acid (MPBA) was immobilized firmly on the nanochip by the metal-S bond for sensitive sialic acid sensing. Owing to the high stability of gold nanoparticles and the high sensitivity of silver nanoparticles, the nanochip showed good performance in LSPR detection with rich and high responses. Besides, the nanochip also showed sensitive electrical signals during electrochemical detection due to the excitation of the energetic charges from the nanoparticles surface to the reaction system. The dynamic electro-optical spectroscopy was based on a unique combination of LSPR and linear sweep voltammetry (LSV). On the one hand, electrochemical signals activated the electrons on the nanochip to promote the propagation and resonance of surface plasmon. On the other hand, LSPR concentrated the electrons on the nanochip surface, which made the electrons easily driven to enhance the current in electrochemical detection. Results showed that mutual promotion of electrochemical-LSPR on nanochip covered a linear dynamic range from 0.05 mM to 5 mM on selective sialic acid detection with a low detection limit of 17 μM. The synchronous amplification of the electro-optical response during electrochemical-LSPR, opened up a new perspective for efficient and sensitive biochemical detection. Copyright © 2018 Elsevier B.V. All rights reserved.

Salmonella O48 Serum Resistance is Connected with the Elongation of the Lipopolysaccharide O-Antigen Containing Sialic Acid

PubMed Central

Pawlak, Aleksandra; Rybka, Jacek; Dudek, Bartłomiej; Krzyżewska, Eva; Rybka, Wojciech; Kędziora, Anna; Klausa, Elżbieta; Bugla-Płoskońska, Gabriela

2017-01-01

Complement is one of the most important parts of the innate immune system. Some bacteria can gain resistance against the bactericidal action of complement by decorating their outer cell surface with lipopolysaccharides (LPSs) containing a very long O-antigen or with specific outer membrane proteins. Additionally, the presence of sialic acid in the LPS molecules can provide a level of protection for bacteria, likening them to human cells, a phenomenon known as molecular mimicry. Salmonella O48, which contains sialic acid in the O-antigen, is the major cause of reptile-associated salmonellosis, a worldwide public health problem. In this study, we tested the effect of prolonged exposure to human serum on strains from Salmonella serogroup O48, specifically on the O-antigen length. After multiple passages in serum, three out of four tested strains became resistant to serum action. The gas-liquid chromatography/tandem mass spectrometry analysis showed that, for most of the strains, the average length of the LPS O-antigen increased. Thus, we have discovered a link between the resistance of bacterial cells to serum and the elongation of the LPS O-antigen. PMID:28934165

Salmonella O48 Serum Resistance is Connected with the Elongation of the Lipopolysaccharide O-Antigen Containing Sialic Acid.

PubMed

Pawlak, Aleksandra; Rybka, Jacek; Dudek, Bartłomiej; Krzyżewska, Eva; Rybka, Wojciech; Kędziora, Anna; Klausa, Elżbieta; Bugla-Płoskońska, Gabriela

2017-09-21

Complement is one of the most important parts of the innate immune system. Some bacteria can gain resistance against the bactericidal action of complement by decorating their outer cell surface with lipopolysaccharides (LPSs) containing a very long O-antigen or with specific outer membrane proteins. Additionally, the presence of sialic acid in the LPS molecules can provide a level of protection for bacteria, likening them to human cells, a phenomenon known as molecular mimicry. Salmonella O48, which contains sialic acid in the O-antigen, is the major cause of reptile-associated salmonellosis, a worldwide public health problem. In this study, we tested the effect of prolonged exposure to human serum on strains from Salmonella serogroup O48, specifically on the O-antigen length. After multiple passages in serum, three out of four tested strains became resistant to serum action. The gas-liquid chromatography/tandem mass spectrometry analysis showed that, for most of the strains, the average length of the LPS O-antigen increased. Thus, we have discovered a link between the resistance of bacterial cells to serum and the elongation of the LPS O-antigen.

Density functional theory based probe of the affinity interaction of saccharide ligands with extra-cellular sialic acid residues.

PubMed

Patel, Anjali; Tiwari, Sanjay; Jha, Prafulla K

2018-05-10

Changes in glycosylation pattern leads to malignant transformations among the cells. In combination with upregulated actions of sialyltransferases, it ultimately leads to differential expression of sialic acid (SA) at cell surface. Given its negative charge and localization to extracellular domain, SA has been exploited for the development of targeted theranostics using approaches, such as, cationization and appending recognition saccharides on carrier surface. In this study, we have performed quantum mechanical calculations based on density functional theory (DFT) to study the interaction of saccharides with extracellular SA. Gradient-corrected DFT with the three parameter function (B3) was utilized for the calculation of Lee-Yang-Parr (LYP) correlation function. Atomic charge, vibrational frequencies and energy of the optimized structures were calculated through B3LYP. Our calculations demonstrate a stronger galactose-sialic acid interaction at tumour-relevant low pH and hyperthermic condition. These results support the application of pH responsive delivery vehicles and targeted hyperthermic chemotherapy for eradicating solid tumour deposits. These studies, conducted a priori, can guide the formulation scientists over appropriate choice of ligands and their applications in the design of 'smart' theranostic tools.

Steric and Thermodynamic Limits of Design for the Incorporation of Large UnNatural Amino Acids in Aminoacyl-tRNA Synthetase Enzymes

PubMed Central

Armen, Roger S.; Schiller, Stefan M.; Brooks, Charles L.

2015-01-01

Orthogonal aminoacyl-tRNA synthetase/tRNA pairs from archaea have been evolved to facilitate site specific in vivo incorporation of unnatural amino acids into proteins in Escherichia coli. Using this approach, unnatural amino acids have been successfully incorporated with high translational efficiency and fidelity. In this study, CHARMM-based molecular docking and free energy calculations were used to evaluate rational design of specific protein-ligand interactions for aminoacyl-tRNA synthetases. A series of novel unnatural amino acid ligands were docked into the p-benzoyl-L-phenylalanine tRNA synthetase, which revealed that the binding pocket of the enzyme does not provide sufficient space for significantly larger ligands. Specific binding site residues were mutated to alanine to create additional space to accommodate larger target ligands, and then mutations were introduced to improve binding free energy. This approach was used to redesign binding sites for several different target ligands, which were then tested against the standard 20 amino acids to verify target specificity. Only the synthetase designed to bind Man-α-O-Tyr was predicted to be sufficiently selective for the target ligand and also thermodynamically stable. Our study suggests that extensive redesign of the tRNA synthatase binding pocket for large bulky ligands may be quite thermodynamically unfavorable. PMID:20310065

Human erythrocyte band 3 functions as a receptor for the sialic acid-independent invasion of Plasmodium falciparum. Role of the RhopH3-MSP1 complex.

PubMed

Baldwin, Michael; Yamodo, Innocent; Ranjan, Ravi; Li, Xuerong; Mines, Gregory; Marinkovic, Marina; Hanada, Toshihiko; Oh, Steven S; Chishti, Athar H

2014-12-01

Plasmodium falciparum takes advantage of two broadly defined alternate invasion pathways when infecting human erythrocytes: one that depends on and the other that is independent of host sialic acid residues on the erythrocyte surface. Within the sialic acid-dependent (SAD) and sialic acid-independent (SAID) invasion pathways, several alternate host receptors are used by P. falciparum based on its particular invasion phenotype. Earlier, we reported that two putative extracellular regions of human erythrocyte band 3 termed 5C and 6A function as host invasion receptor segments binding parasite proteins MSP1 and MSP9 via a SAID mechanism. In this study, we developed two mono-specific anti-peptide chicken IgY antibodies to demonstrate that the 5C and 6A regions of band 3 are exposed on the surface of human erythrocytes. These antibodies inhibited erythrocyte invasion by the P. falciparum 3D7 and 7G8 strains (SAID invasion phenotype), and the blocking effect was enhanced in sialic acid-depleted erythrocytes. In contrast, the IgY antibodies had only a marginal inhibitory effect on FCR3 and Dd2 strains (SAD invasion phenotype). A direct biochemical interaction between erythrocyte band 3 epitopes and parasite RhopH3, identified by the yeast two-hybrid screen, was established. RhopH3 formed a complex with MSP119 and the 5ABC region of band 3, and a recombinant segment of RhopH3 inhibited parasite invasion in human erythrocytes. Together, these findings provide evidence that erythrocyte band 3 functions as a major host invasion receptor in the SAID invasion pathway by assembling a multi-protein complex composed of parasite ligands RhopH3 and MSP1. Copyright © 2014 Elsevier B.V. All rights reserved.

Overexpression of acetyl-CoA synthetase in Saccharomyces cerevisiae increases acetic acid tolerance.

PubMed

Ding, Jun; Holzwarth, Garrett; Penner, Michael H; Patton-Vogt, Jana; Bakalinsky, Alan T

2015-01-01

Acetic acid-mediated inhibition of the fermentation of lignocellulose-derived sugars impedes development of plant biomass as a source of renewable ethanol. In order to overcome this inhibition, the capacity of Saccharomyces cerevisiae to synthesize acetyl-CoA from acetic acid was increased by overexpressing ACS2 encoding acetyl-coenzyme A synthetase. Overexpression of ACS2 resulted in higher resistance to acetic acid as measured by an increased growth rate and shorter lag phase relative to a wild-type control strain, suggesting that Acs2-mediated consumption of acetic acid during fermentation contributes to acetic acid detoxification. © FEMS 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Differences in sialic acid residues among bone alkaline phosphatase isoforms: a physical, biochemical, and immunological characterization.

PubMed

Magnusson, P; Farley, J R

2002-12-01

High-performance liquid chromatography (HPLC) separates three human bone alkaline phosphatase (BALP) isoforms in serum; two major BALP isoforms, B1 and B2, and a minor fraction, B/I, which is composed on average of 70% bone and 30% intestinal ALP. The current studies were intended to identify an in vitro source of the BALP isoforms for physical, biochemical, and immunological characterizations. The three BALP isoforms were identified in extracts of human osteosarcoma (SaOS-2) cells, by HPLC, after separation by anion-exchange chromatography. All three BALP isoforms were similar with respect to freeze-thaw stability, solubility, heat inactivation, and inhibition by L-phenylalanine, L-homoarginine, and levamisole. The isoforms were also kinetically similar (i.e., maximal velocity and KM at pH 8.8 and pH 10.0). The isoforms differed, however, with respect to sensitivity to precipitation with wheat germ agglutinin (WGA), P < 0.001, but not Concanavalin A. At 3.0 mg/ml, WGA precipitated approximately 25% of B/I but more than 80% of B1 and B2. Molecular weights were estimated by native gradient gel electrophoresis: B/I, 126 kDa; B1, 136 kDa; and B2, 141 kDa. Desialylation with neuraminidase reduced the apparent sizes of B1 and B2 to 127 kDa (i.e., approximately to that of B/I). The total carbohydrate content was calculated to be 18 kDa, 28 kDa, and 33 kDa (i.e., 14%, 21%, and 23%) for the BALP isofonns, B/I, B1, and B2, respectively. The number of sialic acid residues was estimated to be 29 and 45, for each B1 and B2 homodimer, respectively. Apparent discrepancies between these estimates of molecular weight and estimates based on gel filtration chromatography were attributed to nonspecific interactions between carbohydrate residues and the gel filtration beads. All three BALP isoforms showed similar dose-dependent linearity in the commercial Alkphase-B and Tandem-MP Ostase immunoassays, r = 0.944 and r = 0.985, respectively (P < 0.001). In summary, our data indicate that

Rational design of aminoacyl-tRNA synthetase specific for p-acetyl-L-phenylalanine.

PubMed

Sun, Renhua; Zheng, Heng; Fang, Zhengzhi; Yao, Wenbing

2010-01-01

The Methanococcus jannaschii tRNA(Tyr)/tyrosyl-tRNA synthetase pair has been engineered to incorporate unnatural amino acids into proteins in Escherichia coli site-specifically. In order to add other unnatural amino acids into proteins by this approach, the amino acid binding site of M. jannaschii tyrosyl-tRNA synthetase need to be mutated. The crystal structures of M. jannaschii tyrosyl-tRNA synthetase and its mutations were determined, which provided an opportunity to design aminoacyl-tRNA synthetases specific for other unnatural amino acids. In our study, we attempted to design aminoacyl-tRNA synthetases being able to deliver p-acetyl-L-phenylalanine into proteins. p-Acetyl-L-phenylalanine was superimposed on tyrosyl in M. jannaschii tyrosyl-tRNA synthetase-tyrosine complex. Tyr32 needed to be changed to non-polar amino acid with shorter side chain, Val, Leu, Ile, Gly or Ala, in order to reduce steric clash and provide hydrophobic environment to acetyl on p-acetyl-L-phenylalanine. Asp158 and Ile159 would be changed to specific amino acids for the same reason. So we designed 60 aminoacyl-tRNA synthetases. Binding of these aminoacyl-tRNA synthetases with p-acetyl-L-phenylalanine indicated that only 15 of them turned out to be able to bind p-acetyl-L-phenylalanine with reasonable poses. Binding affinity computation proved that the mutation of Tyr32Leu and Asp158Gly benefited p-acetyl-L-phenylalanine binding. And two of the designed aminoacyl-tRNA synthetases had considerable binding affinities. They seemed to be very promising to be able to incorporate p-acetyl-L-phenylalanine into proteins in E. coli. The results show that the combination of homology modeling and molecular docking is a feasible method to filter inappropriate mutations in molecular design and point out beneficial mutations. Copyright 2009 Elsevier Inc. All rights reserved.

A Chemical Biology Solution to Problems with Studying Biologically Important but Unstable 9-O-Acetyl Sialic Acids.

PubMed

Khedri, Zahra; Xiao, An; Yu, Hai; Landig, Corinna Susanne; Li, Wanqing; Diaz, Sandra; Wasik, Brian R; Parrish, Colin R; Wang, Lee-Ping; Varki, Ajit; Chen, Xi

2017-01-20

9-O-Acetylation is a common natural modification on sialic acids (Sias) that terminate many vertebrate glycan chains. This ester group has striking effects on many biological phenomena, including microbe-host interactions, complement action, regulation of immune responses, sialidase action, cellular apoptosis, and tumor immunology. Despite such findings, 9-O-acetyl sialoglycoconjugates have remained largely understudied, primarily because of marked lability of the 9-O-acetyl group to even small pH variations and/or the action of mammalian or microbial esterases. Our current studies involving 9-O-acetylated sialoglycans on glycan microarrays revealed that even the most careful precautions cannot ensure complete stability of the 9-O-acetyl group. We now demonstrate a simple chemical biology solution to many of these problems by substituting the oxygen atom in the ester with a nitrogen atom, resulting in sialic acids with a chemically and biologically stable 9-N-acetyl group. We present an efficient one-pot multienzyme method to synthesize a sialoglycan containing 9-acetamido-9-deoxy-N-acetylneuraminic acid (Neu5Ac9NAc) and compare it to the one with naturally occurring 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac 2 ). Conformational resemblance of the two molecules was confirmed by computational molecular dynamics simulations. Microarray studies showed that the Neu5Ac9NAc-sialoglycan is a ligand for viruses naturally recognizing Neu5,9Ac 2 , with a similar affinity but with much improved stability in handling and study. Feeding of Neu5Ac9NAc or Neu5,9Ac 2 to mammalian cells resulted in comparable incorporation and surface expression as well as binding to 9-O-acetyl-Sia-specific viruses. However, cells fed with Neu5Ac9NAc remained resistant to viral esterases and showed a slower turnover. This simple approach opens numerous research opportunities that have heretofore proved intractable.

Characterization of shallow trench isolation CMP process and its application

NASA Astrophysics Data System (ADS)

Li, Helen; Zhang, ChunLei; Liu, JinBing; Liu, ZhengFang; Chen, Kuang Han; Gbondo-Tugbawa, Tamba; Ding, Hua; Li, Flora; Lee, Brian; Gower-Hall, Aaron; Chiu, Yang-Chih

2016-03-01

Chemical mechanical polishing (CMP) has been a critical enabling technology in shallow trench isolation (STI), which is used in current integrated circuit fabrication process to accomplish device isolation. Excessive dishing and erosion in STI CMP processes, however, create device yield concerns. This paper proposes characterization and modeling techniques to address a variety of concerns in STI CMP. In the past, majority of CMP publications have been addressed on interconnect layers in backend- of-line (BEOL) process. However, the number of CMP steps in front-end-of-line (FEOL) has been increasing in more advanced process techniques like 3D-FinFET and replacement metal gate, as a results incoming topography induced by FEOL CMP steps can no longer be ignored as the topography accumulates and stacks up across multiple CMP steps and eventually propagating to BEOL layers. In this paper, we first discuss how to characterize and model STI CMP process. Once STI CMP model is developed, it can be used for screening design and detect possible manufacturing weak spots. We also work with process engineering team to establish hotspot criteria in terms of oxide dishing and nitride loss. As process technologies move from planar transistor to 3D transistor like FinFet and multi-gate, it is important to accurately predict topography in FEOL CMP processes. These incoming topographies when stacked up can have huge impact in BEOL copper processes, where copper pooling becomes catastrophic yield loss. A calibration methodology to characterize STI CMP step is developed as shown in Figure 1; moreover, this STI CMP model is validated from silicon data collected from product chips not used in calibration stage. Additionally, wafer experimental setup and metrology plan are instrumental to an accurate model with high predictive power. After a model is generated, spec limits and threshold to establish hotspots criteria can be defined. Such definition requires working closely with foundry

The endolysins of bacteriophages CMP1 and CN77 are specific for the lysis of Clavibacter michiganensis strains.

PubMed

Wittmann, Johannes; Eichenlaub, Rudolf; Dreiseikelmann, Brigitte

2010-08-01

Putative endolysin genes of bacteriophages CMP1 and CN77, which infect Clavibacter michiganensis subsp. michiganensis and C. michiganensis subsp. nebraskensis, respectively, were cloned and expressed in Escherichia coli. The His-tagged endolysin of CMP1 consists of 306 amino acids and has a calculated molecular mass of 34.8 kDa, while the His-tagged endolysin of CN77 has 290 amino acids with a molecular mass of 31.9 kDa. The proteins were purified and their bacteriolytic activity was demonstrated. The bacteriolytic activity of both enzymes showed a host range which was limited to the respective C. michiganensis subspecies and did not affect other bacteria, even those closely related to Clavibacter. Due to the high specificity of the CMP1 and CN77 endolysins they may be useful tools for biocontrol of plant-pathogenic C. michiganensis without affecting other bacteria in the soil.

Carboxymethyl pachyman (CMP) reduces intestinal mucositis and regulates the intestinal microflora in 5-fluorouracil-treated CT26 tumour-bearing mice.

PubMed

Wang, Canhong; Yang, Shuxian; Gao, Li; Wang, Lili; Cao, Li

2018-05-23

The compound 5-fluorouracil (5-FU) is the first choice chemotherapeutic agent for the treatment of colorectal cancer (CRC), but intestinal mucositis is a primary limiting factor in anticancer therapy. There is currently no broadly effective targeted treatment to cure this side effect. Carboxymethylated pachyman (CMP) is a polysaccharide that is modified from the structure of pachyman isolated from Poria cocos (Chinese name: Fu Ling). Meanwhile, recent studies have shown that CMP exhibits immune regulatory, anti-inflammatory and antioxidant activities. Therefore, the purpose of this study was to evaluate the intestinal protective effect of CMP in 5-FU-treated CT26 tumour-bearing mice and to further explore its underlying mechanism(s) of action. Initially, a CT26 colon carcinoma xenograft mice model was established. The colon length, colon tissue injury, intestinal flora, short-chain fatty acids (SCFAs) and indicators linked to inflammation, antioxidation and apoptosis were then measured. Our results showed that CMP in combination with 5-FU reversed intestinal shortening (p < 0.01) and alleviated 5-FU-induced colon injury (p < 0.001) via suppression of ROS production; increasing the levels of CAT, GSH-Px and GSH; decreasing expression of NF-κB, p-p38 and Bax; and elevating the levels of Nrf2 and Bcl-2. More importantly, CMP had a significant impact and counteracted the intestinal microflora disorders produced by 5-FU by increasing the proportion of Bacteroidetes, lactobacilli, and butyric acid-producing and acetic acid-producing bacteria and restoring the intestinal flora diversity. Overall, this work suggested that CMP could regulate the ecological balance of the intestinal flora and reduce colon injuries induced by 5-FU in CT26 tumour-bearing mice, and the mechanism involved may be associated with the regulation of the NF-κB, Nrf2-ARE and MAPK/P38 pathways.

Regulation of B cell functions by the sialic acid-binding receptors siglec-G and CD22.

PubMed

Jellusova, Julia; Nitschke, Lars

2011-01-01

B cell antigen receptor (BCR) engagement can lead to many different physiologic outcomes. To achieve an appropriate response, the BCR signal is interpreted in the context of other stimuli and several additional receptors on the B cell surface participate in the modulation of the signal. Two members of the Siglec (sialic acid-binding immunoglobulin-like lectin) family, CD22 and Siglec-G have been shown to inhibit the BCR signal. Recent findings indicate that the ability of these two receptors to bind sialic acids might be important to induce tolerance to self-antigens. Sialylated glycans are usually absent on microbes but abundant in higher vertebrates and might therefore provide an important tolerogenic signal. Since the expression of the specific ligands for Siglec-G and CD22 is tightly regulated and since Siglecs are not only able to bind their ligands in trans but also on the same cell surface this might provide additional mechanisms to control the BCR signal. Although both Siglec-G and CD22 are expressed on B cells and are able to inhibit BCR mediated signaling, they also show unique biological functions. While CD22 is the dominant regulator of calcium signaling on conventional B2 cells and also seems to play a role on marginal zone B cells, Siglec-G exerts its function mainly on B1 cells and influences their lifespan and antibody production. Both Siglec-G and CD22 have also recently been linked to toll-like receptor signaling and may provide a link in the regulation of the adaptive and innate immune response of B cells.

Oligosaccharide composition of the neurotoxin responsive Na/sup +/ channel and the requirement of sialic acid for activity

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

Negishi, M.; Shaw, G.W.; Glick, M.C.

1986-05-01

The neurotoxin responsive Na/sup +/ channel was purified to homogeneity in an 18% yield from a clonal cell line of mouse neuroblastoma, N-18, metabolically labeled with L-(/sup 3/H)fucose. The Na/sup +/ channel, a glycoprotein, M/sub r/=200,000 (gradient 7-14% PAGE) was digested with Pronase and the glycopeptides were characterized by serial lectin affinity chromatography. greater than 90% of the oligosaccharides contained sialic acid and 18% were biantennary, 39% were triantennary and 30% tetraantennary. The glycoprotein was reconstituted into artificial phospholipid vesicles and /sup 86/Rb flux was stimulated (65%) by 200 ..mu..M veratridine and 1.2 ..mu..g of scorpion venom and was inhibitedmore » (95%) by 5 ..mu..M tetrodotoxin. The requirement of sialic acid for Na/sup +/ channel activity was demonstrated since neuraminidase (0.01 U) treatment of the reconstituted glycoprotein eliminated the response of /sup 86/Rb flux to the stimulating neurotoxins. In other experiments, treatment of N-18 cells with 10 ..mu..M swainsonine, an inhibitor of glycoprotein processing, altered the oligosaccharide composition of the Na/sup +/ channel. When the abnormally glycosylated Na/sup +/ channel was reconstituted into artificial phospholipid vesicles, /sup 86/Rb flux in response to neurotoxins was impaired. Thus, glycosylation of the polypeptide with oligosaccharides of specific composition and structure is essential for expression of the biological activity of the neurotoxin responsive Na/sup +/ channel.« less

Coding of Class I and II aminoacyl-tRNA synthetases

PubMed Central

Carter, Charles W.

2018-01-01

SUMMARY The aminoacyl-tRNA synthetases and their cognate transfer RNAs translate the universal genetic code. The twenty canonical amino acids are sufficiently diverse to create a selective advantage for dividing amino acid activation between two distinct, apparently unrelated superfamilies of synthetases, Class I amino acids being generally larger and less polar, Class II amino acids smaller and more polar. Biochemical, bioinformatic, and protein engineering experiments support the hypothesis that the two Classes descended from opposite strands of the same ancestral gene. Parallel experimental deconstructions of Class I and II synthetases reveal parallel losses in catalytic proficiency at two novel modular levels—protozymes and Urzymes—associated with the evolution of catalytic activity. Bi-directional coding supports an important unification of the proteome; affords a genetic relatedness metric—middle base-pairing frequencies in sense/antisense alignments—that probes more deeply into the evolutionary history of translation than do single multiple sequence alignments; and has facilitated the analysis of hitherto unknown coding relationships in tRNA sequences. Reconstruction of native synthetases by modular thermodynamic cycles facilitated by domain engineering emphasizes the subtlety associated with achieving high specificity, shedding new light on allosteric relationships in contemporary synthetases. Synthetase Urzyme structural biology suggests that they are catalytically active molten globules, broadening the potential manifold of polypeptide catalysts accessible to primitive genetic coding and motivating revisions of the origins of catalysis. Finally, bi-directional genetic coding of some of the oldest genes in the proteome places major limitations on the likelihood that any RNA World preceded the origins of coded proteins. PMID:28828732

Influence of sialic acids on the galactose-recognizing receptor of rat peritoneal macrophages.

PubMed

Lee, H Y; Kelm, S; Michalski, J C; Schauer, R

1990-04-01

The interaction of the galactose-recognizing receptor from rat peritoneal macrophages with ligands containing terminal galactose residues, such as asialoorosomucoid, desialylated erythrocytes or lymphocytes, can be inhibited by free N-acetylneuraminic acid (Neu5Ac) and oligosaccharides or glycoproteins containing this sugar in terminal position. This effect of Neu5Ac on the receptor is specific. The other naturally occurring or most of synthetic neuraminic acid derivatives tested do not exhibit an equivalent inhibitory potency as Neu5Ac. Although free Neu5Ac inhibits 5-fold stronger (K50 = 0.2mM) than free galactose, clustering of Neu5Ac in oligosaccharides and glycoproteins does not lead to stronger inhibition, which is in contrast to galactose-containing ligands. A more branched (triantennary) sialooligosaccharide inhibits less than biantennary and unbranched sialooligosaccharides. This may be the reason, why complex sialic acid-containing ligands like native orosomucoid or blood cells are not bound and internalized by the macrophages. The dissociation of asialoorosomucoid from the receptor is slow under the influence of Neu5Ac and requires relatively high concentrations of this sugar, whereas the dissociation mediated by galactose is rapid and requires lower concentrations. An allosteric influence of Neu5Ac on the binding of galactose by the receptor is discussed.

Continuous directed evolution of aminoacyl-tRNA synthetases

PubMed Central

Bryson, David I.; Fan, Chenguang; Guo, Li-Tao; Miller, Corwin; Söll, Dieter; Liu, David R.

2017-01-01

Directed evolution of orthogonal aminoacyl-tRNA synthetases (AARSs) enables site-specific installation of non-canonical amino acids (ncAAs) into proteins. Traditional evolution techniques typically produce AARSs with greatly reduced activity and selectivity compared to their wild-type counterparts. We designed phage-assisted continuous evolution (PACE) selections to rapidly produce highly active and selective orthogonal AARSs through hundreds of generations of evolution. PACE of a chimeric Methanosarcina spp. pyrrolysyl-tRNA synthetase (PylRS) improved its enzymatic efficiency (kcat/KMtRNA) 45-fold compared to the parent enzyme. Transplantation of the evolved mutations into other PylRS-derived synthetases improved yields of proteins containing non-canonical residues up to 9.7-fold. Simultaneous positive and negative selection PACE over 48 h greatly improved the selectivity of a promiscuous Methanocaldococcus jannaschii tyrosyl-tRNA synthetase variant for site-specific incorporation of p-iodo-L-phenylalanine. These findings offer new AARSs that increase the utility of orthogonal translation systems and establish the capability of PACE to efficiently evolve orthogonal AARSs with high activity and amino acid specificity. PMID:29035361

Non-standard amino acid recognition by Escherichia coli leucyl-tRNA synthetase

NASA Technical Reports Server (NTRS)

Martinis, S. A.; Fox, G. E.

1997-01-01

Recombinant E. coli leucyl-tRNA synthetase was screened for amino acid-dependent pyrophosphate exchange activity using noncognate aliphatic amino acids including norvaline, homocysteine, norleucine, methionine, and homoserine. [32P]-labeled reaction products were separated by thin layer chromatography using a novel solvent system and then quantified by phosphorimaging. Norvaline which differs from leucine by only one methyl group stimulated pyrophosphate exchange activity as did both homocysteine and norleucine to a lesser extent. The KM parameters for leucine and norvaline were measured to be 10 micromoles and 1.5 mM, respectively. Experiments are in progress to determine if norvaline is transferred to tRNA(Leu) and/or edited by a pre- or post-transfer mechanism.

Regulation of B Cell Functions by the Sialic Acid-Binding Receptors Siglec-G and CD22

PubMed Central

Jellusova, Julia; Nitschke, Lars

2011-01-01

B cell antigen receptor (BCR) engagement can lead to many different physiologic outcomes. To achieve an appropriate response, the BCR signal is interpreted in the context of other stimuli and several additional receptors on the B cell surface participate in the modulation of the signal. Two members of the Siglec (sialic acid-binding immunoglobulin-like lectin) family, CD22 and Siglec-G have been shown to inhibit the BCR signal. Recent findings indicate that the ability of these two receptors to bind sialic acids might be important to induce tolerance to self-antigens. Sialylated glycans are usually absent on microbes but abundant in higher vertebrates and might therefore provide an important tolerogenic signal. Since the expression of the specific ligands for Siglec-G and CD22 is tightly regulated and since Siglecs are not only able to bind their ligands in trans but also on the same cell surface this might provide additional mechanisms to control the BCR signal. Although both Siglec-G and CD22 are expressed on B cells and are able to inhibit BCR mediated signaling, they also show unique biological functions. While CD22 is the dominant regulator of calcium signaling on conventional B2 cells and also seems to play a role on marginal zone B cells, Siglec-G exerts its function mainly on B1 cells and influences their lifespan and antibody production. Both Siglec-G and CD22 have also recently been linked to toll-like receptor signaling and may provide a link in the regulation of the adaptive and innate immune response of B cells. PMID:22566885

Ribosomal incorporation of backbone modified amino acids via an editing-deficient aminoacyl-tRNA synthetase.

PubMed

Iqbal, Emil S; Dods, Kara K; Hartman, Matthew C T

2018-02-14

The ability to incorporate non-canonical amino acids (ncAA) using translation offers researchers the ability to extend the functionality of proteins and peptides for many applications including synthetic biology, biophysical and structural studies, and discovery of novel ligands. Here we describe the high promiscuity of an editing-deficient valine-tRNA synthetase (ValRS T222P). Using this enzyme, we demonstrate ribosomal translation of 11 ncAAs including those with novel side chains, α,α-disubstitutions, and cyclic β-amino acids.

E-selectin-targeted Sialic Acid-PEG-dexamethasone Micelles for Enhanced Anti-Inflammatory Efficacy for Acute Kidney Injury.

PubMed

Hu, Jing-Bo; Kang, Xu-Qi; Liang, Jing; Wang, Xiao-Juan; Xu, Xiao-Ling; Yang, Ping; Ying, Xiao-Ying; Jiang, Sai-Ping; Du, Yong-Zhong

2017-01-01

The effective treatment for acute kidney injury (AKI) is currently limited, and care is primarily supportive. Sialic acid (SA) is main component of Sialyl Lewis x antigen on the mammalian cell surface, which participates in E-selectin binding. Therefore, dexamethasone(DXM)-loaded E-selectin-targeting sialic acid-polyethylene glycol-dexamethasone (SA-PEG-DXM/DXM) conjugate micelles are designed for ameliorating AKI. The conjugates are synthesized via the esterification reaction between PEG and SA or DXM, and can spontaneously form micelles in an aqueous solution with a 65.6 µg/mL critical micelle concentration. Free DXM is incorporated into the micelles with 6.28 ± 0.21% drug loading content. In vitro DXM release from SA-PEG-DXM/DXM micelles can be prolonged to 48h. Much more SA-PEG-DXM micelles can be internalized by lipopolysaccharide (LPS)-activated human umbilical vein endothelial cells (HUVECs) in comparison to PEG-DXM micelles due to specific interaction between SA and E-selectin expressed on HUVECs, and consequently more SA-PEG-DXM micelles are accumulated in the kidney of AKI murine model. Furthermore, SA in SA-PEG-DXM conjugates can significantly ameliorate LPS-induced production of pro-inflammatory cytokines via suppressing LPS-activated Beclin-1/Atg5-Atg12-mediated autophagy to attenuate toxicity. Compared with free DXM and PEG-DXM/DXM micelles, SA-PEG-DXM/DXM micelles show better therapeutical effects, as reflected by the improved renal function, histopathological changes, pro-inflammatory cytokines, oxidative stress and expression of apoptotic related proteins.

Targeted delivery of epirubicin to tumor-associated macrophages by sialic acid-cholesterol conjugate modified liposomes with improved antitumor activity.

PubMed

Zhou, Songlei; Zhang, Ting; Peng, Bo; Luo, Xiang; Liu, Xinrong; Hu, Ling; Liu, Yang; Di, Donghua; Song, Yanzhi; Deng, Yihui

2017-05-15

With the knowledge that the receptors of sialic acid are overexpressed on the surface of tumor-associated macrophages (TAMs), which play a crucial role in the tumor's progression and metastasis, a sialic acid-cholesterol conjugate (SA-CH) was synthesized and modified on the surface of epirubicin (EPI)-loaded liposomes (EPI-SAL) to improve the delivery of EPI to the TAMs. The liposomes were developed using remote loading technology via a pH gradient. The liposomes were evaluated for particle size, encapsulation efficiency, in vitro release, stability, in vitro cytotoxicity and pharmacokinetics. And the in vitro and in vivo cellular uptake studies demonstrated EPI-SAL achieved enhanced accumulation of EPI into TAMs. The antitumor studies indicated that EPI-SAL provided the strongest antitumor activity compared with the other formulations (EPI-S, EPI-CL and EPI-PL represent EPI solution, conventional liposomal EPI, PEGylated liposomal EPI, respectively), and the survival percent of tumor-bearing mice was 83.3%. The superior antitumor efficacy was probably attributed to the killing of TAMs by EPI-SAL, and modulating the tumor microenvironment with the depletion of TAMs. These findings suggested that SA-CH decorated EPI-loaded liposomes may present an effective strategy to eradicate TAMs, which may be a promising approach for cancer therapy. Copyright © 2017 Elsevier B.V. All rights reserved.

42 CFR 417.407 - Requirements for a Competitive Medical Plan (CMP).

Code of Federal Regulations, 2010 CFR

2010-10-01

... 42 Public Health 3 2010-10-01 2010-10-01 false Requirements for a Competitive Medical Plan (CMP... AND HUMAN SERVICES (CONTINUED) MEDICARE PROGRAM HEALTH MAINTENANCE ORGANIZATIONS, COMPETITIVE MEDICAL... Requirements for a Competitive Medical Plan (CMP). (a) General rule. To qualify as a CMP, an entity must be...

42 CFR 417.407 - Requirements for a Competitive Medical Plan (CMP).

Code of Federal Regulations, 2011 CFR

2011-10-01

... 42 Public Health 3 2011-10-01 2011-10-01 false Requirements for a Competitive Medical Plan (CMP... AND HUMAN SERVICES (CONTINUED) MEDICARE PROGRAM HEALTH MAINTENANCE ORGANIZATIONS, COMPETITIVE MEDICAL... Requirements for a Competitive Medical Plan (CMP). (a) General rule. To qualify as a CMP, an entity must be...

Polyspecific pyrrolysyl-tRNA synthetases from directed evolution.

PubMed

Guo, Li-Tao; Wang, Yane-Shih; Nakamura, Akiyoshi; Eiler, Daniel; Kavran, Jennifer M; Wong, Margaret; Kiessling, Laura L; Steitz, Thomas A; O'Donoghue, Patrick; Söll, Dieter

2014-11-25

Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA(Pyl) have emerged as ideal translation components for genetic code innovation. Variants of the enzyme facilitate the incorporation >100 noncanonical amino acids (ncAAs) into proteins. PylRS variants were previously selected to acylate N(ε)-acetyl-Lys (AcK) onto tRNA(Pyl). Here, we examine an N(ε)-acetyl-lysyl-tRNA synthetase (AcKRS), which is polyspecific (i.e., active with a broad range of ncAAs) and 30-fold more efficient with Phe derivatives than it is with AcK. Structural and biochemical data reveal the molecular basis of polyspecificity in AcKRS and in a PylRS variant [iodo-phenylalanyl-tRNA synthetase (IFRS)] that displays both enhanced activity and substrate promiscuity over a chemical library of 313 ncAAs. IFRS, a product of directed evolution, has distinct binding modes for different ncAAs. These data indicate that in vivo selections do not produce optimally specific tRNA synthetases and suggest that translation fidelity will become an increasingly dominant factor in expanding the genetic code far beyond 20 amino acids.

Polyspecific pyrrolysyl-tRNA synthetases from directed evolution

PubMed Central

Guo, Li-Tao; Wang, Yane-Shih; Nakamura, Akiyoshi; Eiler, Daniel; Kavran, Jennifer M.; Wong, Margaret; Kiessling, Laura L.; Steitz, Thomas A.; O’Donoghue, Patrick; Söll, Dieter

2014-01-01

Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNAPyl have emerged as ideal translation components for genetic code innovation. Variants of the enzyme facilitate the incorporation >100 noncanonical amino acids (ncAAs) into proteins. PylRS variants were previously selected to acylate Nε-acetyl-Lys (AcK) onto tRNAPyl. Here, we examine an Nε-acetyl-lysyl-tRNA synthetase (AcKRS), which is polyspecific (i.e., active with a broad range of ncAAs) and 30-fold more efficient with Phe derivatives than it is with AcK. Structural and biochemical data reveal the molecular basis of polyspecificity in AcKRS and in a PylRS variant [iodo-phenylalanyl-tRNA synthetase (IFRS)] that displays both enhanced activity and substrate promiscuity over a chemical library of 313 ncAAs. IFRS, a product of directed evolution, has distinct binding modes for different ncAAs. These data indicate that in vivo selections do not produce optimally specific tRNA synthetases and suggest that translation fidelity will become an increasingly dominant factor in expanding the genetic code far beyond 20 amino acids. PMID:25385624

Mechanism of inhibition of mammalian tumor and other thymidylate synthases by N sup 4 -hydroxy-dCMP, N sup 4 -hydroxy-5-fluoro-dCMP, and related analogues

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

Rode, W.; Zielinski, Z.; Dzik, J.M.

1990-12-01

N{sup 4}-Hydroxy-dCMP (N{sup 4}-OH-dCMP), N{sup 4}-methoxy-dCMP (N{sup 4}-OMe-dCMP), and their 5-fluoro congeners were all slow-binding inhibitors of Ehrlich carcinoma thymidylate synthase (TS), competitive with respect to dUMP, and had differing kinetic constants describing interactions with the two TS binding sites. N{sup 4}-OH-dCMP was not a substrate and its inactivation of TS was methylenetetrahydrofolate-dependent, hence mechanism-based. K{sub i} values for N{sup 4}-OH-dCMP and its 5-fluoro analogue were in the range 10{sup {minus}7}-10{sup {minus}8} M, 2-3 orders of magnitude higher for the corresponding N{sup 4}-OMe analogues. The 5-methyl analogue of N{sup 4}-OHdCMP was 10{sup 4}-fold less potent, pointing to the anti rotamermore » of the imino form of exocyclic N{sup 4}-OH, relative to the ring N(3), as the active species. This is consistent with weaker slow-binding inhibition of the altered enzyme from 5-FdUrd-resistant, relative to parent, L1210 cells by both FdUMP and N{sup 4}-OH-dCMP, suggesting interaction of both N{sup 4}-OH and C(5)-F groups with the same region of the active center. Kinetic studies with purified enzyme from five sources, viz., Ehrlich carcinoma, L1210 parental, and 5-FdUrd-resistant cells, regenerating rat liver, and the tapeworm Hymenolepis diminuta, demonstrated that addition of a 5-fluoro substituent to N{sup 4}-OH-dCMP increased its affinity from 2- to 20-fold for the enzyme from different sources. With the Ehrlich and tapeworm enzymes, N{sup 4}-OH-FdCMP and FdUMP were almost equally effective inhibitors.« less

Colloquium paper: uniquely human evolution of sialic acid genetics and biology.

PubMed

Varki, Ajit

2010-05-11

Darwinian evolution of humans from our common ancestors with nonhuman primates involved many gene-environment interactions at the population level, and the resulting human-specific genetic changes must contribute to the "Human Condition." Recent data indicate that the biology of sialic acids (which directly involves less than 60 genes) shows more than 10 uniquely human genetic changes in comparison with our closest evolutionary relatives. Known outcomes are tissue-specific changes in abundant cell-surface glycans, changes in specificity and/or expression of multiple proteins that recognize these glycans, and novel pathogen regimes. Specific events include Alu-mediated inactivation of the CMAH gene, resulting in loss of synthesis of the Sia N-glycolylneuraminic acid (Neu5Gc) and increase in expression of the precursor N-acetylneuraminic acid (Neu5Ac); increased expression of alpha2-6-linked Sias (likely because of changed expression of ST6GALI); and multiple changes in SIGLEC genes encoding Sia-recognizing Ig-like lectins (Siglecs). The last includes binding specificity changes (in Siglecs -5, -7, -9, -11, and -12); expression pattern changes (in Siglecs -1, -5, -6, and -11); gene conversion (SIGLEC11); and deletion or pseudogenization (SIGLEC13, SIGLEC14, and SIGLEC16). A nongenetic outcome of the CMAH mutation is human metabolic incorporation of foreign dietary Neu5Gc, in the face of circulating anti-Neu5Gc antibodies, generating a novel "xeno-auto-antigen" situation. Taken together, these data suggest that both the genes associated with Sia biology and the related impacts of the environment comprise a relative "hot spot" of genetic and physiological changes in human evolution, with implications for uniquely human features both in health and disease.

Expression of Human CTP Synthetase in Saccharomyces cerevisiae Reveals Phosphorylation by Protein Kinase A*

PubMed Central

Han, Gil-Soo; Sreenivas, Avula; Choi, Mal-Gi; Chang, Yu-Fang; Martin, Shelley S.; Baldwin, Enoch P.; Carman, George M.

2005-01-01

CTP synthetase (EC 6.3.4.2, UTP: ammonia ligase (ADP-forming)) is an essential enzyme in all organisms; it generates the CTP required for the synthesis of nucleic acids and membrane phospholipids. In this work we showed that the human CTP synthetase genes, CTPS1 and CTPS2, were functional in Saccharomyces cerevisiae and complemented the lethal phenotype of the ura7Δ ura8Δ mutant lacking CTP synthetase activity. The expression of the CTPS1-and CTPS2-encoded human CTP synthetase enzymes in the ura7Δ ura8Δ mutant was shown by immunoblot analysis of CTP synthetase proteins, the measurement of CTP synthetase activity, and the synthesis of CTP in vivo. Phosphoamino acid and phosphopeptide mapping analyses of human CTP synthetase 1 isolated from 32Pi-labeled cells revealed that the enzyme was phosphorylated on multiple serine residues in vivo. Activation of protein kinase A activity in yeast resulted in transient increases (2-fold) in the phosphorylation of human CTP synthetase 1 and the cellular level of CTP. Human CTP synthetase 1 was also phosphorylated by mammalian protein kinase A in vitro. Using human CTP synthetase 1 purified from Escherichia coli as a substrate, protein kinase A activity was dose- and time-dependent, and dependent on the concentrations of CTP synthetase1 and ATP. These studies showed that S. cerevisiae was useful for the analysis of human CTP synthetase phosphorylation. PMID:16179339

Increased production of free fatty acids in Aspergillus oryzae by disruption of a predicted acyl-CoA synthetase gene.

PubMed

Tamano, Koichi; Bruno, Kenneth S; Koike, Hideaki; Ishii, Tomoko; Miura, Ai; Umemura, Myco; Culley, David E; Baker, Scott E; Machida, Masayuki

2015-04-01

Fatty acids are attractive molecules as source materials for the production of biodiesel fuel. Previously, we attained a 2.4-fold increase in fatty acid production by increasing the expression of fatty acid synthesis-related genes in Aspergillus oryzae. In this study, we achieved an additional increase in the production of fatty acids by disrupting a predicted acyl-CoA synthetase gene in A. oryzae. The A. oryzae genome is predicted to encode six acyl-CoA synthetase genes and disruption of AO090011000642, one of the six genes, resulted in a 9.2-fold higher accumulation (corresponding to an increased production of 0.23 mmol/g dry cell weight) of intracellular fatty acid in comparison to the wild-type strain. Furthermore, by introducing a niaD marker from Aspergillus nidulans to the disruptant, as well as changing the concentration of nitrogen in the culture medium from 10 to 350 mM, fatty acid productivity reached 0.54 mmol/g dry cell weight. Analysis of the relative composition of the major intracellular free fatty acids caused by disruption of AO090011000642 in comparison to the wild-type strain showed an increase in stearic acid (7 to 26 %), decrease in linoleic acid (50 to 27 %), and no significant changes in palmitic or oleic acid (each around 20-25 %).

The importance of cytosolic glutamine synthetase in nitrogen assimilation and recycling

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

Bernard, S.M.; Habash, D.Z.

2009-07-02

Glutamine synthetase assimilates ammonium into amino acids, thus it is a key enzyme for nitrogen metabolism. The cytosolic isoenzymes of glutamine synthetase assimilate ammonium derived from primary nitrogen uptake and from various internal nitrogen recycling pathways. In this way, cytosolic glutamine synthetase is crucial for the remobilization of protein-derived nitrogen. Cytosolic glutamine synthetase is encoded by a small family of genes that are well conserved across plant species. Members of the cytosolic glutamine synthetase gene family are regulated in response to plant nitrogen status, as well as to environmental cues, such as nitrogen availability and biotic/abiotic stresses. The complex regulationmore » of cytosolic glutamine synthetase at the transcriptional to post-translational levels is key to the establishment of a specific physiological role for each isoenzyme. The diverse physiological roles of cytosolic glutamine synthetase isoenzymes are important in relation to current agricultural and ecological issues.« less

Four Trypanosoma brucei fatty acyl-CoA synthetases: fatty acid specificity of the recombinant proteins.

PubMed Central

Jiang, D W; Englund, P T

2001-01-01

As part of our investigation of fatty acid metabolism in Trypanosoma brucei, we have expressed four acyl-CoA synthetase (TbACS) genes in Esherichia coli. The recombinant proteins, with His-tags on their C-termini, were purified to near homogeneity using nickel-chelate affinity chromatography. Although these enzymes are highly homologous, they have distinct specificities for fatty acid chain length. TbACS1 prefers saturated fatty acids in the range C(11:0) to C(14:0) and TbACS2 prefers shorter fatty acids, mainly C(10:0). TbACS3 and 4, which have 95% sequence identity, have similar specificities, favouring fatty acids between C(14:0) and C(17:0). In addition, TbACS1, 3 and 4 function well with a variety of unsaturated fatty acids. PMID:11535136

The microsomal dicarboxylyl-CoA synthetase.

PubMed Central

Vamecq, J; de Hoffmann, E; Van Hoof, F

1985-01-01

Dicarboxylic acids are products of the omega-oxidation of monocarboxylic acids. We demonstrate that in rat liver dicarboxylic acids (C5-C16) can be converted into their CoA esters by a dicarboxylyl-CoA synthetase. During this activation ATP, which cannot be replaced by GTP, is converted into AMP and PPi, both acting as feedback inhibitors of the reaction. Thermolabile at 37 degrees C, and optimally active at pH 6.5, dicarboxylyl-CoA synthetase displays the highest activity on dodecanedioic acid (2 micromol/min per g of liver). Cell-fractionation studies indicate that this enzyme belongs to the hepatic microsomal fraction. Investigations about the fate of dicarboxylyl-CoA esters disclosed the existence of an oxidase, which could be measured by monitoring the production of H2O2. In our assay conditions this H2O2 production is dependent on and closely follows the CoA consumption. It appears that the chain-length specificity of the handling of dicarboxylic acids by this catabolic pathway (activation to acyl-CoA and oxidation with H2O2 production) parallels the pattern of the degradation of exogenous dicarboxylic acids in vivo. PMID:4062873

Human Coronavirus HKU1 Spike Protein Uses O-Acetylated Sialic Acid as an Attachment Receptor Determinant and Employs Hemagglutinin-Esterase Protein as a Receptor-Destroying Enzyme.

PubMed

Huang, Xingchuan; Dong, Wenjuan; Milewska, Aleksandra; Golda, Anna; Qi, Yonghe; Zhu, Quan K; Marasco, Wayne A; Baric, Ralph S; Sims, Amy C; Pyrc, Krzysztof; Li, Wenhui; Sui, Jianhua

2015-07-01

Human coronavirus (hCoV) HKU1 is one of six hCoVs identified to date and the only one with an unidentified cellular receptor. hCoV-HKU1 encodes a hemagglutinin-esterase (HE) protein that is unique to the group a betacoronaviruses (group 2a). The function of HKU1-HE remains largely undetermined. In this study, we examined binding of the S1 domain of hCoV-HKU1 spike to a panel of cells and found that the S1 could specifically bind on the cell surface of a human rhabdomyosarcoma cell line, RD. Pretreatment of RD cells with neuraminidase (NA) and trypsin greatly reduced the binding, suggesting that the binding was mediated by sialic acids on glycoproteins. However, unlike other group 2a CoVs, e.g., hCoV-OC43, for which 9-O-acetylated sialic acid (9-O-Ac-Sia) serves as a receptor determinant, HKU1-S1 bound with neither 9-O-Ac-Sia-containing glycoprotein(s) nor rat and mouse erythrocytes. Nonetheless, the HKU1-HE was similar to OC43-HE, also possessed sialate-O-acetylesterase activity, and acted as a receptor-destroying enzyme (RDE) capable of eliminating the binding of HKU1-S1 to RD cells, whereas the O-acetylesterase-inactive HKU1-HE mutant lost this capacity. Using primary human ciliated airway epithelial (HAE) cell cultures, the only in vitro replication model for hCoV-HKU1 infection, we confirmed that pretreatment of HAE cells with HE but not the enzymatically inactive mutant blocked hCoV-HKU1 infection. These results demonstrate that hCoV-HKU1 exploits O-Ac-Sia as a cellular attachment receptor determinant to initiate the infection of host cells and that its HE protein possesses the corresponding sialate-O-acetylesterase RDE activity. Human coronaviruses (hCoV) are important human respiratory pathogens. Among the six hCoVs identified to date, only hCoV-HKU1 has no defined cellular receptor. It is also unclear whether hemagglutinin-esterase (HE) protein plays a role in viral entry. In this study, we found that, similarly to other members of the group 2a CoVs, sialic

Sialylation of lipooligosaccharides is dispensable for the virulence of Haemophilus ducreyi in humans.

PubMed

Spinola, Stanley M; Li, Wei; Fortney, Kate R; Janowicz, Diane M; Zwickl, Beth; Katz, Barry P; Munson, Robert S

2012-02-01

Sialylated glycoconjugates on the surfaces of mammalian cells play important roles in intercellular communication and self-recognition. The sialic acid preferentially expressed in human tissues is N-acetylneuraminic acid (Neu5Ac). In a process called molecular mimicry, many bacterial pathogens decorate their cell surface glycolipids with Neu5Ac. Incorporation of Neu5Ac into bacterial glycolipids promotes bacterial interactions with host cell receptors called Siglecs. These interactions affect bacterial adherence, resistance to serum killing and phagocytosis, and innate immune responses. Haemophilus ducreyi, the etiologic agent of chancroid, expresses lipooligosaccharides (LOS) that are highly sialylated. However, an H. ducreyi sialyltransferase (lst) mutant, whose LOS contain reduced levels of Neu5Ac, is fully virulent in human volunteers. Recently, a second sialyltransferase gene (Hd0053) was discovered in H. ducreyi, raising the possibility that Hd0053 compensated for the loss of lst during human infection. CMP-Neu5Ac is the obligate nucleotide sugar donor for all bacterial sialyltransferases; LOS derived from an H. ducreyi CMP-Neu5Ac synthetase (neuA) mutant has no detectable Neu5Ac. Here, we compared an H. ducreyi neuA mutant to its wild-type parent in several models of pathogenesis. In human inoculation experiments, the neuA mutant formed papules and pustules at rates that were no different than those of its parent. When grown in media with and without Neu5Ac supplementation, the neuA mutant and its parent had similar phenotypes in bactericidal, macrophage uptake, and dendritic cell activation assays. Although we cannot preclude a contribution of LOS sialylation to ulcerative disease, these data strongly suggest that sialylation of LOS is dispensable for H. ducreyi pathogenesis in humans.

Lipoic acid synthetase deficiency causes neonatal-onset epilepsy, defective mitochondrial energy metabolism, and glycine elevation.

PubMed

Mayr, Johannes A; Zimmermann, Franz A; Fauth, Christine; Bergheim, Christa; Meierhofer, David; Radmayr, Doris; Zschocke, Johannes; Koch, Johannes; Sperl, Wolfgang

2011-12-09

Lipoic acid is an essential prosthetic group of four mitochondrial enzymes involved in the oxidative decarboxylation of pyruvate, α-ketoglutarate, and branched chain amino acids and in the glycine cleavage. Lipoic acid is synthesized stepwise within mitochondria through a process that includes lipoic acid synthetase. We identified the homozygous mutation c.746G>A (p.Arg249His) in LIAS in an individual with neonatal-onset epilepsy, muscular hypotonia, lactic acidosis, and elevated glycine concentration in plasma and urine. Investigation of the mitochondrial energy metabolism showed reduced oxidation of pyruvate and decreased pyruvate dehydrogenase complex activity. A pronounced reduction of the prosthetic group lipoamide was found in lipoylated proteins. Copyright © 2011 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Orthogonal use of a human tRNA synthetase active site to achieve multifunctionality.

PubMed

Zhou, Quansheng; Kapoor, Mili; Guo, Min; Belani, Rajesh; Xu, Xiaoling; Kiosses, William B; Hanan, Melanie; Park, Chulho; Armour, Eva; Do, Minh-Ha; Nangle, Leslie A; Schimmel, Paul; Yang, Xiang-Lei

2010-01-01

Protein multifunctionality is an emerging explanation for the complexity of higher organisms. In this regard, aminoacyl tRNA synthetases catalyze amino acid activation for protein synthesis, but some also act in pathways for inflammation, angiogenesis and apoptosis. It is unclear how these multiple functions evolved and how they relate to the active site. Here structural modeling analysis, mutagenesis and cell-based functional studies show that the potent angiostatic, natural fragment of human tryptophanyl-tRNA synthetase (TrpRS) associates via tryptophan side chains that protrude from its cognate cellular receptor vascular endothelial cadherin (VE-cadherin). VE-cadherin's tryptophan side chains fit into the tryptophan-specific active site of the synthetase. Thus, specific side chains of the receptor mimic amino acid substrates and expand the functionality of the active site of the synthetase. We propose that orthogonal use of the same active site may be a general way to develop multifunctionality of human tRNA synthetases and other proteins.

Distribution of O-Acetylated Sialic Acids among Target Host Tissues for Influenza Virus

PubMed Central

Barnard, Karen N.; Ossiboff, Robert J.; Khedri, Zahra; Feng, Kurtis H.; Yu, Hai; Chen, Xi; Varki, Ajit

2017-01-01

ABSTRACT Sialic acids (Sias) are important glycans displayed on the cells and tissues of many different animals and are frequent targets for binding and modification by pathogens, including influenza viruses. Influenza virus hemagglutinins bind Sias during the infection of their normal hosts, while the encoded neuraminidases and/or esterases remove or modify the Sia to allow virion release or to prevent rebinding. Sias naturally occur in a variety of modified forms, and modified Sias can alter influenza virus host tropisms through their altered interactions with the viral glycoproteins. However, the distribution of modified Sia forms and their effects on pathogen-host interactions are still poorly understood. Here we used probes developed from viral Sia-binding proteins to detect O-acetylated (4-O-acetyl, 9-O-acetyl, and 7,9-O-acetyl) Sias displayed on the tissues of some natural or experimental hosts for influenza viruses. These modified Sias showed highly variable displays between the hosts and tissues examined. The 9-O-acetyl (and 7,9-) modified Sia forms were found on cells and tissues of many hosts, including mice, humans, ferrets, guinea pigs, pigs, horses, dogs, as well as in those of ducks and embryonated chicken egg tissues and membranes, although in variable amounts. The 4-O-acetyl Sias were found in the respiratory tissues of fewer animals, being primarily displayed in the horse and guinea pig, but were not detected in humans or pigs. The results suggest that these Sia variants may influence virus tropisms by altering and selecting their cell interactions. IMPORTANCE Sialic acids (Sias) are key glycans that control or modulate many normal cell and tissue functions while also interacting with a variety of pathogens, including many different viruses. Sias are naturally displayed in a variety of different forms, with modifications at several positions that can alter their functional interactions with pathogens. In addition, Sias are often modified or

Post-Training Intrahippocampal Injection of Synthetic Poly-Alpha-2,8-Sialic Acid-Neural Cell Adhesion Molecule Mimetic Peptide Improves Spatial Long-Term Performance in Mice

ERIC Educational Resources Information Center

Florian, Cedrick; Foltz, Jane; Norreel, Jean-Chretien; Rougon, Genevieve; Roullet, Pascal

2006-01-01

Several data have shown that the neural cell adhesion molecule (NCAM) is necessary for long-term memory formation and might play a role in the structural reorganization of synapses. The NCAM, encoded by a single gene, is represented by several isoforms that differ with regard to their content of alpha-2,8-linked sialic acid residues (PSA) on their…

Functional expansion of human tRNA synthetases achieved by structural inventions

PubMed Central

Guo, Min; Schimmel, Paul; Yang, Xiang-Lei

2010-01-01

Known as an essential component of the translational apparatus, the aminoacyl-tRNA synthetase family catalyzes the first step reaction in protein synthesis, that is, to specifically attach each amino acid to its cognate tRNA. While preserving this essential role, tRNA synthetases developed other roles during evolution. Human tRNA synthetases, in particular, have diverse functions in different pathways involving angiogenesis, inflammation and apoptosis. The functional diversity is further illustrated in the association with various diseases through genetic mutations that do not affect aminoacylation or protein synthesis. Here we review the accumulated knowledge on how human tRNA synthetases used structural inventions to achieve functional expansions. PMID:19932696

Harnessing cancer cell metabolism for theranostic applications using metabolic glycoengineering of sialic acid in breast cancer as a pioneering example

PubMed Central

Badr, Haitham A.; AlSadek, Dina M.M.; El-Houseini, Motawa E.; Saeui, Christopher T.; Mathew, Mohit P.; Yarema, Kevin J.; Ahmed, Hafiz

2016-01-01

Abnormal cell surface display of sialic acids – a family of unusual 9-carbon sugars - is widely recognized as distinguishing feature of many types of cancer. Sialoglycans, however, typically cannot be identified with sufficiently high reproducibility and sensitivity to serve as clinically accepted biomarkers and similarly, almost all efforts to exploit cancer-specific differences in sialylation signatures for therapy remain in early stage development. In this report we provide an overview of important facets of glycosylation that contribute to cancer in general with a focus on breast cancer as an example of malignant disease characterized by aberrant sialylation. We then describe how cancer cells experience nutrient deprivation during oncogenesis and discuss how the resulting metabolic reprogramming, which endows breast cancer cells with the ability to obtain nutrients during scarcity, constitutes an “Achilles’ heel” that we believe can be exploited by metabolic glycoengineering (MGE) strategies to develop new diagnostic methods and therapeutic approaches. In particular, we hypothesize that adaptations made by breast cancer cells that allow them to efficiently scavenge sialic acid during times of nutrient deprivation renders them vulnerable to MGE, which refers to the use of exogenously-supplied, non-natural monosaccharide analogues to modulate targeted aspects of glycosylation in living cells and animals. In specific, once non-natural sialosides are incorporated into the cancer “sialome” they can be exploited as epitopes for immunotherapy or as chemical tags for targeted delivery of imaging or therapeutic agents selectively to tumors. PMID:27926828

Functional and structural analysis of the sialic acid-binding domain of rotaviruses.

PubMed Central

Isa, P; López, S; Segovia, L; Arias, C F

1997-01-01

The infectivity of most animal rotaviruses is dependent on the interaction of the virus spike protein VP4 with a sialic acid (SA)-containing cell receptor, and the SA-binding domain of this protein has been mapped between amino acids 93 and 208 of its trypsin cleavage fragment VP8. To identify which residues in this region are essential for the SA-binding activity, we performed alanine mutagenesis of the rotavirus RRV VP8 expressed in bacteria as a fusion polypeptide with glutathione S-transferase. Tyrosines were primarily targeted since tyrosine has been involved in the interaction of other viral hemagglutinins with SA. Of the 15 substitutions carried out, 10 abolished the SA-dependent hemagglutination activity of the protein, as well as its ability to bind to glycophorin A in a solid-phase assay. However, only alanine substitutions for tyrosines 155 and 188 and for serine 190 did not affect the overall conformation of the protein, as judged by their interaction with a panel of conformationally sensitive neutralizing VP8 monoclonal antibodies (MAbs). These findings suggest that these three amino acids play an essential role in the SA-binding activity of the protein, presumably by interacting directly with the SA molecule. The predicted secondary structure of VP8 suggests that it is organized as 11 beta-strands separated by loops; in this model, Tyr-155 maps to loop 7 while Tyr-188 and Ser-190 map to loop 9. The close proximity of these two loops is also supported by previous results from competition experiments with neutralizing MAbs directed at RRV VP8. PMID:9261399

SEMICONDUCTOR TECHNOLOGY Dummy fill effect on CMP planarity

NASA Astrophysics Data System (ADS)

Junxiong, Zhou; Lan, Chen; Wenbiao, Ruan; Zhigang, Li; Weixiang, Shen; Tianchun, Ye

2010-10-01

With the use of a chemical-mechanical polishing (CMP) simulator verified by testing data from a foundry, the effect of dummy fill characteristics, such as fill size, fill density and fill shape, on CMP planarity is analyzed. The results indicate that dummy density has a significant impact on oxide erosion, and copper dishing is in proportion to dummy size. We also demonstrate that cross shape dummy fill can have the best dishing performance at the same density.

Avian and human influenza virus compatible sialic acid receptors in little brown bats.

PubMed

Chothe, Shubhada K; Bhushan, Gitanjali; Nissly, Ruth H; Yeh, Yin-Ting; Brown, Justin; Turner, Gregory; Fisher, Jenny; Sewall, Brent J; Reeder, DeeAnn M; Terrones, Mauricio; Jayarao, Bhushan M; Kuchipudi, Suresh V

2017-04-06

Influenza A viruses (IAVs) continue to threaten animal and human health globally. Bats are asymptomatic reservoirs for many zoonotic viruses. Recent reports of two novel IAVs in fruit bats and serological evidence of avian influenza virus (AIV) H9 infection in frugivorous bats raise questions about the role of bats in IAV epidemiology. IAVs bind to sialic acid (SA) receptors on host cells, and it is widely believed that hosts expressing both SA α2,3-Gal and SA α2,6-Gal receptors could facilitate genetic reassortment of avian and human IAVs. We found abundant co-expression of both avian (SA α2,3-Gal) and human (SA α2,6-Gal) type SA receptors in little brown bats (LBBs) that were compatible with avian and human IAV binding. This first ever study of IAV receptors in a bat species suggest that LBBs, a widely-distributed bat species in North America, could potentially be co-infected with avian and human IAVs, facilitating the emergence of zoonotic strains.

Sialic Acid Metabolic Engineering: A Potential Strategy for the Neuroblastoma Therapy

PubMed Central

Gnanapragassam, Vinayaga S.; Bork, Kaya; Galuska, Christina E.; Galuska, Sebastian P.; Glanz, Dagobert; Nagasundaram, Manimozhi; Bache, Matthias; Vordermark, Dirk; Kohla, Guido; Kannicht, Christoph; Schauer, Roland; Horstkorte, Rüdiger

2014-01-01

Background Sialic acids (Sia) represent negative-charged terminal sugars on most glycoproteins and glycolipids on the cell surface of vertebrates. Aberrant expression of tumor associated sialylated carbohydrate epitopes significantly increases during onset of cancer. Since Sia contribute towards cell migration ( =  metastasis) and to chemo- and radiation resistance. Modulation of cellular Sia concentration and composition poses a challenge especially for neuroblastoma therapy, due to the high heterogeneity and therapeutic resistance of these cells. Here we propose that Metabolic Sia Engineering (MSE) is an effective strategy to reduce neuroblastoma progression and metastasis. Methods Human neuroblastoma SH-SY5Y cells were treated with synthetic Sia precursors N-propanoyl mannosamine (ManNProp) or N-pentanoyl mannosamine (ManNPent). Total and Polysialic acids (PolySia) were investigated by high performance liquid chromatography. Cell surface polySia were examined by flow-cytometry. Sia precursors treated cells were examined for the migration, invasion and sensitivity towards anticancer drugs and radiation treatment. Results Treatment of SH-SY5Y cells with ManNProp or ManNPent (referred as MSE) reduced their cell surface sialylation significantly. We found complete absence of polysialylation after treatment of SH-SY5Y cells with ManNPent. Loss of polysialylation results in a reduction of migration and invasion ability of these cells. Furthermore, radiation of Sia-engineered cells completely abolished their migration. In addition, MSE increases the cytotoxicity of anti-cancer drugs, such as 5-fluorouracil or cisplatin. Conclusions Metabolic Sia Engineering (MSE) of neuroblastoma cells using modified Sia precursors reduces their sialylation, metastatic potential and increases their sensitivity towards radiation or chemotherapeutics. Therefore, MSE may serve as an effective method to treat neuroblastoma. PMID:25148252

A novel approach for quantitation of nonderivatized sialic acid in protein therapeutics using hydrophilic interaction chromatographic separation and nano quantity analyte detection.

PubMed

Chemmalil, Letha; Suravajjala, Sreekanth; See, Kate; Jordan, Eric; Furtado, Marsha; Sun, Chong; Hosselet, Stephen

2015-01-01

This paper describes a novel approach for the quantitation of nonderivatized sialic acid in glycoproteins, separated by hydrophilic interaction chromatography, and detection by Nano Quantity Analyte Detector (NQAD). The detection technique of NQAD is based on measuring change in the size of dry aerosol and converting the particle count rate into chromatographic output signal. NQAD detector is suitable for the detection of sialic acid, which lacks sufficiently active chromophore or fluorophore. The water condensation particle counting technology allows the analyte to be enlarged using water vapor to provide highest sensitivity. Derivatization-free analysis of glycoproteins using HPLC/NQAD method with PolyGLYCOPLEX™ amide column is well correlated with HPLC method with precolumn derivatization using 1, 2-diamino-4, 5-methylenedioxybenzene (DMB) as well as the Dionex-based high-pH anion-exchange chromatography (or ion chromatography) with pulsed amperometric detection (HPAEC-PAD). With the elimination of derivatization step, HPLC/NQAD method is more efficient than HPLC/DMB method. HPLC/NQAD method is more reproducible than HPAEC-PAD method as HPAEC-PAD method suffers high variability because of electrode fouling during analysis. Overall, HPLC/NQAD method offers broad linear dynamic range as well as excellent precision, accuracy, repeatability, reliability, and ease of use, with acceptable comparability to the commonly used HPAEC-PAD and HPLC/DMB methods. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

TreeCmp: Comparison of Trees in Polynomial Time

PubMed Central

Bogdanowicz, Damian; Giaro, Krzysztof; Wróbel, Borys

2012-01-01

When a phylogenetic reconstruction does not result in one tree but in several, tree metrics permit finding out how far the reconstructed trees are from one another. They also permit to assess the accuracy of a reconstruction if a true tree is known. TreeCmp implements eight metrics that can be calculated in polynomial time for arbitrary (not only bifurcating) trees: four for unrooted (Matching Split metric, which we have recently proposed, Robinson-Foulds, Path Difference, Quartet) and four for rooted trees (Matching Cluster, Robinson-Foulds cluster, Nodal Splitted and Triple). TreeCmp is the first implementation of Matching Split/Cluster metrics and the first efficient and convenient implementation of Nodal Splitted. It allows to compare relatively large trees. We provide an example of the application of TreeCmp to compare the accuracy of ten approaches to phylogenetic reconstruction with trees up to 5000 external nodes, using a measure of accuracy based on normalized similarity between trees.

Peptide Synthetase Gene in Trichoderma virens

PubMed Central

Wilhite, S. E.; Lumsden, R. D.; Straney, D. C.

2001-01-01

Trichoderma virens (synonym, Gliocladium virens), a deuteromycete fungus, suppresses soilborne plant diseases caused by a number of fungi and is used as a biocontrol agent. Several traits that may contribute to the antagonistic interactions of T. virens with disease-causing fungi involve the production of peptide metabolites (e.g., the antibiotic gliotoxin and siderophores used for iron acquisition). We cloned a 5,056-bp partial cDNA encoding a putative peptide synthetase (Psy1) from T. virens using conserved motifs found within the adenylate domain of peptide synthetases. Sequence similarities with conserved motifs of the adenylation domain, acyl transfer, and two condensation domains support identification of the Psy1 gene as a gene that encodes a peptide synthetase. Disruption of the native Psy1 gene through gene replacement was used to identify the function of this gene. Psy1 disruptants produced normal amounts of gliotoxin but grew poorly under low-iron conditions, suggesting that Psy1 plays a role in siderophore production. Psy1 disruptants cannot produce the major T. virens siderophore dimerum acid, a dipetide of acylated Nδ-hydroxyornithine. Biocontrol activity against damping-off diseases caused by Pythium ultimum and Rhizoctonia solani was not reduced by the Psy1 disruption, suggesting that iron competition through dimerum acid production does not contribute significantly to disease suppression activity under the conditions used. PMID:11679326

Pyrrolysyl-tRNA Synthetase, an Aminoacyl-tRNA Synthetase for Genetic Code Expansion

DOE PAGES

Crnkovic, Ana; Suzuki, Tateki; Soll, Dieter; ...

2016-06-14

Genetic code expansion (GCE) has become a central topic of synthetic biology. GCE relies on engineered aminoacyl-tRNA synthetases (aaRSs) and a cognate tRNA species to allow codon reassignment by co-translational insertion of non-canonical amino acids (ncAAs) into proteins. Introduction of such amino acids increases the chemical diversity of recombinant proteins endowing them with novel properties. Such proteins serve in sophisticated biochemical and biophysical studies both in vitro and in vivo, they may become unique biomaterials or therapeutic agents, and they afford metabolic dependence of genetically modified organisms for biocontainment purposes. In the Methanosarcinaceae the incorporation of the 22nd genetically encodedmore » amino acid, pyrrolysine (Pyl), is facilitated by pyrrolysyl-tRNA synthetase (PylRS) and the cognate UAG-recognizing tRNAPyl. This unique aaRS•tRNA pair functions as an orthogonal translation system (OTS) in most model organisms. The facile directed evolution of the large PylRS active site to accommodate many ncAAs, and the enzyme’s anticodon-blind specific recognition of the cognate tRNAPyl make this system highly amenable for GCE purposes. The remarkable polyspecificity of PylRS has been exploited to incorporate >100 different ncAAs into proteins. Here we review the Pyl-OT system and selected GCE applications to examine the properties of an effective OTS.« less

Tandem mass spectrometry of isomeric aniline-labeled N-glycans separated on porous graphitic carbon: Revealing the attachment position of terminal sialic acids and structures of neutral glycans.

PubMed

Michael, Claudia; Rizzi, Andreas M

2015-07-15

Quantitative monitoring of changes in the N-glycome upon disease has gained significance in the context of biomarker discovery. Separation and quantification of isobaric glycan isomers can be attained by using high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS). Collision-induced dissociation (CID)-based fragmentation of separated isobaric glycans is evaluated in respect to its potential of providing fragment ions specific for the linkage positions of terminal sialic acids and the presence of intersecting GlcNAc moieties, respectively. N-Glycans were labeled via reductive amination using (12)C6-aniline and (13)C6-aniline as isotope-coded labeling reagents. The differently labeled glycans were merged and separated into various species using a porous graphitic carbon (PGC) stationary phase. Identification of structural features of separated isobaric isomers was performed by CID-based tandem mass spectrometry (MS/MS) carried out in a quadrupole time-of-flight (QqTOF) or a quadrupole ion-trap (IT) mass spectrometer. Working in the negative ion mode, new diagnostic CID fragment ions could be found that are indicative for the α2,6-type linkage of sialic acids. Other diagnostic ions, identified before as being indicative for the substitution of the 6-antenna, could be confirmed as being of relevance also in the case of aniline labeling. In the positive ion mode, CID fragment ions indicative for the structure of short neutral N-glycans were identified. One new diagnostic ion specific for the linkage position of the terminal sialic acids and one for the presence of bisecting GlcNAc in N-glycans were identified. The aniline label introduced for improved relative quantitation in MS(1) was found not to significantly alter the CID fragmentation patterns that were reported previously by other authors for unlabeled/reduced glycans or for glycans with more polar labels. Copyright © 2015 John Wiley & Sons, Ltd.

Physiological Exploration of the Long Term Evolutionary Selection against Expression of N-Glycolylneuraminic Acid in the Brain*♦

PubMed Central

Naito-Matsui, Yuko; Davies, Leela R. L.; Takematsu, Hiromu; Chou, Hsun-Hua; Tangvoranuntakul, Pam; Carlin, Aaron F.; Verhagen, Andrea; Heyser, Charles J.; Yoo, Seung-Wan; Choudhury, Biswa; Paton, James C.; Paton, Adrienne W.; Varki, Nissi M.; Schnaar, Ronald L.; Varki, Ajit

2017-01-01

All vertebrate cell surfaces display a dense glycan layer often terminated with sialic acids, which have multiple functions due to their location and diverse modifications. The major sialic acids in most mammalian tissues are N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc), the latter being derived from Neu5Ac via addition of one oxygen atom at the sugar nucleotide level by CMP-Neu5Ac hydroxylase (Cmah). Contrasting with other organs that express various ratios of Neu5Ac and Neu5Gc depending on the variable expression of Cmah, Neu5Gc expression in the brain is extremely low in all vertebrates studied to date, suggesting that neural expression is detrimental to animals. However, physiological exploration of the reasons for this long term evolutionary selection has been lacking. To explore the consequences of forced expression of Neu5Gc in the brain, we have established brain-specific Cmah transgenic mice. Such Neu5Gc overexpression in the brain resulted in abnormal locomotor activity, impaired object recognition memory, and abnormal axon myelination. Brain-specific Cmah transgenic mice were also lethally sensitive to a Neu5Gc-preferring bacterial toxin, even though Neu5Gc was overexpressed only in the brain and other organs maintained endogenous Neu5Gc expression, as in wild-type mice. Therefore, the unusually strict evolutionary suppression of Neu5Gc expression in the vertebrate brain may be explained by evasion of negative effects on neural functions and by selection against pathogens. PMID:28049733

Psathyrella velutina Mushroom Lectin Exhibits High Affinity toward Sialoglycoproteins Possessing Terminal N-Acetylneuraminic Acid alpha 2,3-Linked to Penultimate Galactose Residues of Trisialyl N-Glycans. Comparison with other sialic acid-specific lectins.

PubMed

Ueda, Haruko; Matsumoto, Hanako; Takahashi, Noriko; Ogawa, Haruko

2002-07-12

A lectin from the fruiting body of the Psathyrella velutina mushroom (PVL) was found to bind specifically to N-acetylneuraminic acid, as well as to GlcNAc (Ueda, H., Kojima, K., Saitoh, T., and Ogawa, H. (1999) FEBS Lett. 448, 75-80). In this study, the glycan sequences that PVL recognizes with high affinity on sialoglycoproteins were revealed. Among sialic acid-specific lectins only PVL could reveal the sialylated N-acetyllactosamine structure of glycoproteins in blotting studies, based on the dual specificity. The affinity of PVL to fetuin was measured by surface plasmon resonance to be 10(7) m(-1), which is an order of magnitude higher than those of Sambucus nigra agglutinin and Maackia amurensis mitogen, whereas affinity to asialofetuin was approximately 0 and to asialo-agalactofetuin was 10(8) m(-1), suggesting that PVL exhibits remarkably high affinities toward glycoproteins possessing trisialo- or GlcNAc-exposed glycans. Transferrin was separated into fractions that correspond to the sialylation states on an immobilized PVL column. Transferrin-possessing trisialoglycans containing alpha2,3-linked N-acetylneuraminic acid on the beta1,4-linked GlcNAc branch bound to the PVL column and eluted with GlcNAc; those containing only alpha2,6-linked sialic acids were retarded, whereas other transferrin fractions passed through the column. These results indicate that PVL is a lectin with potential for separation and detection of sialoglycoproteins because of its dual specificity toward sialoglycans and GlcNAc exposed glycans.

The study on the effect of pattern density distribution on the STI CMP process

NASA Astrophysics Data System (ADS)

Sub, Yoon Myung; Hian, Bernard Yap Tzen; Fong, Lee It; Anak, Philip Menit; Minhar, Ariffin Bin; Wui, Tan Kim; Kim, Melvin Phua Twang; Jin, Looi Hui; Min, Foo Thai

2017-08-01

The effects of pattern density on CMP characteristics were investigated using specially designed wafer for the characterization of pattern-dependencies in STI CMP [1]. The purpose of this study is to investigate the planarization behavior based on a direct STI CMP used in cerium (CeO2) based slurry system in terms of pattern density variation. The minimal design rule (DR) of 180nm generation technology node was adopted for the mask layout. The mask was successfully applied for evaluation of a cerium (CeO2) abrasive based direct STI CMP process. In this study, we described a planarization behavior of the loading-effects of pattern density variation which were characterized with layout pattern density and pitch variations using masks mentioned above. Furthermore, the characterizing pattern dependent on the variations of the dimensions and spacing features, in thickness remaining after CMP, were analyzed and evaluated. The goal was to establish a concept of library method which will be used to generate design rules reducing the probability of CMP-related failures. Details of the characterization were measured in various layouts showing different pattern density ranges and the effects of pattern density on STI CMP has been discussed in this paper.

The group B streptococcal sialic acid O-acetyltransferase is encoded by neuD, a conserved component of bacterial sialic acid biosynthetic gene clusters.

PubMed

Lewis, Amanda L; Hensler, Mary E; Varki, Ajit; Nizet, Victor

2006-04-21

Nearly two dozen microbial pathogens have surface polysaccharides or lipo-oligosaccharides that contain sialic acid (Sia), and several Sia-dependent virulence mechanisms are known to enhance bacterial survival or result in host tissue injury. Some pathogens are also known to O-acetylate their Sias, although the role of this modification in pathogenesis remains unclear. We report that neuD, a gene located within the Group B Streptococcus (GBS) Sia biosynthetic gene cluster, encodes a Sia O-acetyltransferase that is itself required for capsular polysaccharide (CPS) sialylation. Homology modeling and site-directed mutagenesis identified Lys-123 as a critical residue for Sia O-acetyltransferase activity. Moreover, a single nucleotide polymorphism in neuD can determine whether GBS displays a "high" or "low" Sia O-acetylation phenotype. Complementation analysis revealed that Escherichia coli K1 NeuD also functions as a Sia O-acetyltransferase in GBS. In fact, NeuD homologs are commonly found within Sia biosynthetic gene clusters. A bioinformatic approach identified 18 bacterial species with a Sia biosynthetic gene cluster that included neuD. Included in this list are the sialylated human pathogens Legionella pneumophila, Vibrio parahemeolyticus, Pseudomonas aeruginosa, and Campylobacter jejuni, as well as an additional 12 bacterial species never before analyzed for Sia expression. Phylogenetic analysis shows that NeuD homologs of sialylated pathogens share a common evolutionary lineage distinct from the poly-Sia O-acetyltransferase of E. coli K1. These studies define a molecular genetic approach for the selective elimination of GBS Sia O-acetylation without concurrent loss of sialylation, a key to further studies addressing the role(s) of this modification in bacterial virulence.

Pseudouridylate Synthetase of Escherichia coli: a Catabolite-Repressible Enzyme

PubMed Central

Solomon, L. R.; Breitman, T. R.

1971-01-01

The growth on pseudouridine of two pyrimidine auxotrophs of Escherichia coli (Bu− and W63-86) was markedly enhanced when glycerol replaced glucose as a carbon source or when adenosine 3′:5′-cyclic monophosphoric acid was added to medium containing glucose. These results indicated that an enzyme catalyzing a reaction in the pathway of pseudouridine conversion to uracil was sensitive to catabolite repression. The following pathway is proposed for pseudouridine utilization: [Formula: see text] [Formula: see text] Pseudouridylate synthetase was sensitive to catabolite repression in strains Bu− and W63-86. In contrast, strains B5RU and W5RU, mutants of Bu− and W63-86 which were selected for their ability to grow rapidly on pseudouridine in the presence of glucose, had high levels of pseudouridylate synthetase in the presence of glucose. In the case of B5RU but not W5RU, synthetase activity was greater in cells grown on glycerol or on glucose plus adenosine 3′:5-cyclic monophosphoric acid than on glucose. PMID:4329733

Crystal structure of human cytosolic aspartyl-tRNA synthetase, a component of multi-tRNA synthetase complex

PubMed Central

Kim, Kyung Rok; Park, Sang Ho; Kim, Hyoun Sook; Rhee, Kyung Hee; Kim, Byung-Gyu; Kim, Dae Gyu; Park, Mi Seul; Kim, Hyun-Jung; Kim, Sunghoon; Han, Byung Woo

2013-01-01

Human cytosolic aspartyl-tRNA synthetase (DRS) catalyzes the attachment of the amino acid aspartic acid to its cognate tRNA and it is a component of the multi-tRNA synthetase complex (MSC) which has been known to be involved in unexpected signaling pathways. Here, we report the crystal structure of DRS at a resolution of 2.25 Å. DRS is a homodimer with a dimer interface of 3750.5 Å2 which comprises 16.6% of the monomeric surface area. Our structure reveals the C-terminal end of the N-helix which is considered as a unique addition in DRS, and its conformation further supports the switching model of the N-helix for the transfer of tRNAAsp to elongation factor 1α. From our analyses of the crystal structure and post-translational modification of DRS, we suggest that the phosphorylation of Ser146 provokes the separation of DRS from the MSC and provides the binding site for an interaction partner with unforeseen functions. PMID:23609930

Crystal structure of human cytosolic aspartyl-tRNA synthetase, a component of multi-tRNA synthetase complex.

PubMed

Kim, Kyung Rok; Park, Sang Ho; Kim, Hyoun Sook; Rhee, Kyung Hee; Kim, Byung-Gyu; Kim, Dae Gyu; Park, Mi Seul; Kim, Hyun-Jung; Kim, Sunghoon; Han, Byung Woo

2013-10-01

Human cytosolic aspartyl-tRNA synthetase (DRS) catalyzes the attachment of the amino acid aspartic acid to its cognate tRNA and it is a component of the multi-tRNA synthetase complex (MSC) which has been known to be involved in unexpected signaling pathways. Here, we report the crystal structure of DRS at a resolution of 2.25 Å. DRS is a homodimer with a dimer interface of 3750.5 Å(2) which comprises 16.6% of the monomeric surface area. Our structure reveals the C-terminal end of the N-helix which is considered as a unique addition in DRS, and its conformation further supports the switching model of the N-helix for the transfer of tRNA(Asp) to elongation factor 1α. From our analyses of the crystal structure and post-translational modification of DRS, we suggest that the phosphorylation of Ser146 provokes the separation of DRS from the MSC and provides the binding site for an interaction partner with unforeseen functions. Copyright © 2013 Wiley Periodicals, Inc.

Medicinal Chemistry of the Noncanonical Cyclic Nucleotides cCMP and cUMP.

PubMed

Schwede, Frank; Rentsch, Andreas; Genieser, Hans-Gottfried

2017-01-01

After decades of intensive research on adenosine-3',5'-cyclic monophosphate (cAMP)- and guanosine-3',5'-cyclic monophosphate (cGMP)-related second messenger systems, also the noncanonical congeners cyclic cytidine-3',5'-monophosphate (cCMP) and cyclic uridine-3',5'-monophosphate (cUMP) gained more and more interest. Until the late 1980s, only a small number of cCMP and cUMP analogs with sometimes undefined purities had been described. Moreover, most of these compounds had been rather synthesized as precursors of antitumor and antiviral nucleoside-5'-monophosphates and hence had not been tested for any second messenger activity. Along with the recurring interest in cCMP- and cUMP-related signaling in the early 2000s, it became evident that well-characterized small molecule analogs with reliable purities would serve as highly valuable tools for the evaluation of a putative second messenger role of cyclic pyrimidine nucleotides. Meanwhile, for this purpose new cCMP and cUMP derivatives have been developed, and already known analogs have been resynthesized and highly purified. This chapter summarizes early medicinal chemistry work on cCMP and cUMP and analogs thereof, followed by a description of recent synthetic developments and an outlook on potential future directions.

Dynamic NMR Study of Model CMP Slurry Containing Silica Particles as Abrasives

NASA Astrophysics Data System (ADS)

Odeh, F.; Al-Bawab, A.; Li, Y.

2018-02-01

Chemical mechanical planarization (CMP) should provide a good surface planarity with minimal surface defectivity. Since CMP slurries are multi-component systems, it is very important to understand the various processes and interactions taking place in such slurries. Several techniques have been employed for such task, however, most of them lack the molecular recognition to investigate molecular interactions without adding probes which in turn increase complexity and might alter the microenvironment of the slurry. Nuclear magnetic resonance (NMR) is a powerful technique that can be employed in such study. The longitudinal relaxation times (T1) of the different components of CMP slurries were measured using Spin Echo-NMR (SE-NMR) at a constant temperature. The fact that NMR is non-invasive and gives information on the molecular level gives more advantage to the technique. The model CMP slurry was prepared in D2O to enable monitoring of T1 for the various components' protons. SE-NMR provide a very powerful tool to study the various interactions and adsorption processes that take place in a model CMP silica based slurry which contains BTA and/or glycine and/or Cu+2 ions. It was found that BTA is very competitive towards complexation with Cu+2 ions and BTA-Cu complex adsorbs on silica surface.

Recognition of fibronectin by Penicillium marneffei conidia via a sialic acid-dependent process and its relationship to the interaction between conidia and laminin.

PubMed

Hamilton, A J; Jeavons, L; Youngchim, S; Vanittanakom, N

1999-10-01

Adhesion of Penicillium marneffei conidia to the extracellular matrix protein laminin via a sialic acid-dependent process has previously been demonstrated. This study describes the interaction of P. marneffei conidia with fibronectin and examines the relationship of this process to the recognition of laminin via conidia. Immunofluorescence microscopy demonstrated that fibronectin bound to the surface of conidia and to phialides, but not to hyphae, in a pattern similar to that reported for laminin. Conidia were able to bind to fibronectin immobilized on microtiter plates in a concentration-dependent manner. However, binding to fibronectin (at any given concentration of protein and conidia) was less than that to laminin under equivalent conditions. Soluble fibronectin and antifibronectin antibody inhibited adherence of conidia to fibronectin in the plate adherence assay; soluble laminin also caused pronounced inhibition. Various monosaccharides and several peptides had no effect on adherence to fibronectin. However, N-acetylneuraminic acid abolished adherence to fibronectin, indicating that the interaction was mediated through a sialic acid-dependent process; the latter parallels observations of laminin binding by conidia. Fibronectin binding (and binding of laminin) was considerably reduced by prolonged preincubation of conidia with chymotrypsin, suggesting the protein nature of the binding site. Conidia from older cultures were more adherent to both immobilized fibronectin and laminin than conidia from younger cultures. Ligand affinity binding demonstrated the presence of a 20-kDa protein with the ability to bind both fibronectin and laminin. There would therefore appear to be a common receptor for the binding of fibronectin and laminin on the surface of P. marneffei, and the interaction described here maybe important in mediating attachment of the fungus to host tissue.

Enzymatic Production of Glutathione by Bifunctional γ-Glutamylcysteine Synthetase/Glutathione Synthetase Coupled with In Vitro Acetate Kinase-Based ATP Generation.

PubMed

Jiang, Yu; Tao, Rongsheng; Shen, Zhengquan; Sun, Liangdong; Zhu, Fuyun; Yang, Sheng

2016-12-01

Glutathione (γ-glutamyl-L-cysteinylglycine, GSH) is a pharmaceutical compound often used in food additives and the cosmetics industry. GSH can be produced biologically from L-glutamic acid, L-cysteine, and glycine through an enzymatic process traditionally involving two sequential adenosine triphosphate (ATP)-dependent reactions catalyzed by γ-glutamylcysteine synthetase (γ-GCS or GSHI, EC 6.3.2.2) and GSH synthetase (GS or GSHII, EC 6.3.2.3). Here, we report the enzymatic production of GSH by recombinant cell-free bifunctional γ-glutamylcysteine synthetase/glutathione synthetase (γ-GCS-GS or GshF) coupled with in vitro acetate kinase-based ATP generation. GSH production by an acetate kinase-integrated Escherichia coli Rosetta(DE3) mutant expressing Streptococcus thermophilus GshF reached 18.3 ± 0.1 g l -1 (59.5 ± 0.3 mM) within 3 h, with a molar yield of 0.75 ± 0.00 mol mol -1 added cysteine and a productivity of 6.1 ± 0.0 g l -1  h -1 . This is the highest GSH titer reported to date. This newly developed biocatalytic process offers a promising approach for meeting the industrial requirements for GSH production.

Methods and composition for the production of orthogonal tRNA-aminoacyltRNA synthetase pairs

DOEpatents

Schultz, Peter G.; Wang, Lei; Anderson, John Christopher; Chin, Jason; Liu, David R.; Magliery, Thomas J.; Meggers, Eric L.; Mehl, Ryan Aaron; Pastrnak, Miro; Santoro, Stephen William; Zhang, Zhiwen

2010-05-11

This invention provides compositions and methods for generating components of protein biosynthetic machinery including orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases. Methods for identifying orthogonal pairs are also provided. These components can be used to incorporate unnatural amino acids into proteins in vivo.

Methods and composition for the production of orthogonal tRNA-aminoacyltRNA synthetase pairs

DOEpatents

Schultz, Peter G [La Jolla, CA; Wang, Lei [San Diego, CA; Anderson, John Christopher [San Diego, CA; Chin, Jason [Cambridge, GB; Liu, David R [Lexington, MA; Magliery, Thomas J [North Haven, CT; Meggers, Eric L [Philadelphia, PA; Mehl, Ryan Aaron [Lancaster, PA; Pastrnak, Miro [San Diego, CA; Santoro, Steven William [Cambridge, MA; Zhang, Zhiwen [San Diego, CA

2012-05-22

This invention provides compositions and methods for generating components of protein biosynthetic machinery including orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases. Methods for identifying orthogonal pairs are also provided. These components can be used to incorporate unnatural amino acids into proteins in vivo.

Methods and composition for the production of orthogonal tRNA-aminoacyltRNA synthetase pairs

DOEpatents

Schultz, Peter G [La Jolla, CA; Wang, Lei [San Diego, CA; Anderson, John Christopher [San Diego, CA; Chin, Jason [Cambridge, GB; Liu, David R [Lexington, MA; Magliery, Thomas J [North Haven, CT; Meggers, Eric L [Philadelphia, PA; Mehl, Ryan Aaron [Lancaster, PA; Pastrnak, Miro [San Diego, CA; Santoro, Steven William [Cambridge, MA; Zhang, Zhiwen [San Diego, CA

2008-04-08

This invention provides compositions and methods for generating components of protein biosynthetic machinery including orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases. Methods for identifying orthogonal pairs are also provided. These components can be used to incorporate unnatural amino acids into proteins in vivo.

Proximity labeling of cis-ligands of CD22/Siglec-2 reveals stepwise α2,6 sialic acid-dependent and -independent interactions.

PubMed

Alborzian Deh Sheikh, Amin; Akatsu, Chizuru; Imamura, Akihiro; Abdu-Allah, Hajjaj H M; Takematsu, Hiromu; Ando, Hiromune; Ishida, Hideharu; Tsubata, Takeshi

2018-01-01

Lectins expressed on the cell surface are often bound and regulated by the membrane molecules containing the glycan ligands on the same cell (cis-ligands). However, molecular nature and function of cis-ligands are generally poorly understood partly because of weak interaction between lectins and glycan ligands. Cis-ligands are most extensively studied in CD22 (also known as Siglec-2), an inhibitory B lymphocyte receptor specifically recognizing α2,6 sialic acids. CD22, CD45 and IgM are suggested to be ligands of CD22. Here we labeled molecules in the proximity of CD22 in situ on B cell surface using biotin-tyramide. Molecules including CD22, CD45 and IgM were labeled in wild-type but not ST6GalI -/- B cells that lack α2,6 sialic acids, indicating that these molecules associate with CD22 by lectin-glycan interaction, and are therefore cis-ligands. In ST6GalI -/- B cells, these cis-ligands are located in a slightly more distance from CD22. Thus, the lectin-glycan interaction recruits cis-ligands already located in the relative proximity of CD22 through non-lectin-glycan interaction to the close proximity. Moreover, cis-ligands are labeled in Cmah -/- B cells that lack Neu5Gc preferred by mouse CD22 as efficiently as in wild-type B cells, indicating that very low affinity lectin-glycan interaction is sufficient for recruiting cis-ligands, and can be detected by proximity labeling. Thus, proximity labeling with tyramide appears to be a useful method to identify cis-ligands and to analyze their interaction with the lectins. Copyright © 2017 Elsevier Inc. All rights reserved.

Modulation of phenytoin teratogenicity and embryonic covalent binding by acetylsalicylic acid, caffeic acid, and alpha-phenyl-N-t-butylnitrone: implications for bioactivation by prostaglandin synthetase

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

Wells, P.G.; Zubovits, J.T.; Wong, S.T.

1989-02-01

Teratogenicity of the anticonvulsant drug phenytoin is thought to involve its bioactivation by cytochromes P-450 to a reactive arene oxide intermediate. We hypothesized that phenytoin also may be bioactivated to a teratogenic free radical intermediate by another enzymatic system, prostaglandin synthetase. To evaluate the teratogenic contribution of this latter pathway, an irreversible inhibitor of prostaglandin synthetase, acetylsalicylic acid (ASA), 10 mg/kg intraperitoneally (ip), was administered to pregnant CD-1 mice at 9:00 AM on Gestational Days 12 and 13, 2 hr before phenytoin, 65 mg/kg ip. Other groups were pretreated 2 hr prior to phenytoin administration with either the antioxidant caffeicmore » acid or the free radical spin trapping agent alpha-phenyl-N-t-butylnitrone (PBN). Caffeic acid and PBN were given ip in doses that respectively were up to 1.0 to 0.05 molar equivalents to the dose of phenytoin. Dams were killed on Day 19 and the fetuses were assessed for teratologic anomalies. A similar study evaluated the effect of ASA on the in vivo covalent binding of radiolabeled phenytoin administered on Day 12, in which case dams were killed 24 hr later on Day 13. ASA pretreatment produced a 50% reduction in the incidence of fetal cleft palates induced by phenytoin (p less than 0.05), without significantly altering the incidence of resorptions or mean fetal body weight. Pretreatment with either caffeic acid or PBN resulted in dose-related decreases in the incidence of fetal cleft palates produced by phenytoin, with maximal respective reductions of 71 and 82% at the highest doses of caffeic acid and PBN (p less than 0.05).« less

Thiol synthetases of legumes: immunogold localization and differential gene regulation by phytohormones.

PubMed

Clemente, Maria R; Bustos-Sanmamed, Pilar; Loscos, Jorge; James, Euan K; Pérez-Rontomé, Carmen; Navascués, Joaquín; Gay, Marina; Becana, Manuel

2012-06-01

In plants and other organisms, glutathione (GSH) biosynthesis is catalysed sequentially by γ-glutamylcysteine synthetase (γECS) and glutathione synthetase (GSHS). In legumes, homoglutathione (hGSH) can replace GSH and is synthesized by γECS and a specific homoglutathione synthetase (hGSHS). The subcellular localization of the enzymes was examined by electron microscopy in several legumes and gene expression was analysed in Lotus japonicus plants treated for 1-48 h with 50 μM of hormones. Immunogold localization studies revealed that γECS is confined to chloroplasts and plastids, whereas hGSHS is also in the cytosol. Addition of hormones caused differential expression of thiol synthetases in roots. After 24-48 h, abscisic and salicylic acids downregulated GSHS whereas jasmonic acid upregulated it. Cytokinins and polyamines activated GSHS but not γECS or hGSHS. Jasmonic acid elicited a coordinated response of the three genes and auxin induced both hGSHS expression and activity. Results show that the thiol biosynthetic pathway is compartmentalized in legumes. Moreover, the similar response profiles of the GSH and hGSH contents in roots of non-nodulated and nodulated plants to the various hormonal treatments indicate that thiol homeostasis is independent of the nitrogen source of the plants. The differential regulation of the three mRNA levels, hGSHS activity, and thiol contents by hormones indicates a fine control of thiol biosynthesis at multiple levels and strongly suggests that GSH and hGSH play distinct roles in plant development and stress responses.

Two non-redundant fragments in the N-terminal peptide of human cytosolic methionyl-tRNA synthetase were indispensable for the multi-synthetase complex incorporation and enzyme activity.

PubMed

He, Ran; Zu, Li-Dong; Yao, Peng; Chen, Xin; Wang, En-Duo

2009-02-01

In human cytoplasm, nine aminoacyl-tRNA synthetases (aaRSs) and three protein factors form a multi-synthetase complex (MSC). Human cytosolic methionyl-tRNA synthetase (hcMetRS) is a component of the MSC. Sequence alignment revealed that hcMetRS has an N-terminal extension of 267 amino acid residues. This extension can be divided into three sub-domains: GST-like, GN, and GC sub-domains. The effect of each sub-domain in the N-terminal extension of hcMetRS on enzymatic activity and incorporation into the MSC was studied. The results of cellular assay showed that the GST-like sub-domain was responsible for the incorporation of hcMetRS into the MSC. The entire N-terminal extension of hcMetRS is indispensable for the enzymatic activity. Deletion mutagenesis revealed that a seven-amino acid motif within the sub-domain GC was important for the activity of amino acid activation. A conserved proline residue within the seven-amino acid motif was crucial, while the other six residues were moderately important for the amino acid activation activity. Thus, the last 15 residues of previously defined N-terminal extension of hcMetRS was a part of the catalytic domain; whereas the first 252 residues of hcMetRS constitute the N-terminal extended domain of hcMetRS. The formerly defined N-terminal extension of hcMetRS possesses two functions of two different domains.

Metabolism of vertebrate amino sugars with N-glycolyl groups: resistance of α2-8-linked N-glycolylneuraminic acid to enzymatic cleavage.

PubMed

Davies, Leela R L; Pearce, Oliver M T; Tessier, Matthew B; Assar, Siavash; Smutova, Victoria; Pajunen, Maria; Sumida, Mizuki; Sato, Chihiro; Kitajima, Ken; Finne, Jukka; Gagneux, Pascal; Pshezhetsky, Alexey; Woods, Robert; Varki, Ajit

2012-08-17

The sialic acid (Sia) N-acetylneuraminic acid (Neu5Ac) and its hydroxylated derivative N-glycolylneuraminic acid (Neu5Gc) differ by one oxygen atom. CMP-Neu5Gc is synthesized from CMP-Neu5Ac, with Neu5Gc representing a highly variable fraction of total Sias in various tissues and among different species. The exception may be the brain, where Neu5Ac is abundant and Neu5Gc is reported to be rare. Here, we confirm this unusual pattern and its evolutionary conservation in additional samples from various species, concluding that brain Neu5Gc expression has been maintained at extremely low levels over hundreds of millions of years of vertebrate evolution. Most explanations for this pattern do not require maintaining neural Neu5Gc at such low levels. We hypothesized that resistance of α2-8-linked Neu5Gc to vertebrate sialidases is the detrimental effect requiring the relative absence of Neu5Gc from brain. This linkage is prominent in polysialic acid (polySia), a molecule with critical roles in vertebrate neural development. We show that Neu5Gc is incorporated into neural polySia and does not cause in vitro toxicity. Synthetic polymers of Neu5Ac and Neu5Gc showed that mammalian and bacterial sialidases are much less able to hydrolyze α2-8-linked Neu5Gc at the nonreducing terminus. Notably, this difference was not seen with acid-catalyzed hydrolysis of polySias. Molecular dynamics modeling indicates that differences in the three-dimensional conformation of terminal saccharides may partly explain reduced enzymatic activity. In keeping with this, polymers of N-propionylneuraminic acid are sensitive to sialidases. Resistance of Neu5Gc-containing polySia to sialidases provides a potential explanation for the rarity of Neu5Gc in the vertebrate brain.

Orthogonal use of a human tRNA synthetase active site to achieve multi-functionality

PubMed Central

Zhou, Quansheng; Kapoor, Mili; Guo, Min; Belani, Rajesh; Xu, Xiaoling; Kiosses, William B.; Hanan, Melanie; Park, Chulho; Armour, Eva; Do, Minh-Ha; Nangle, Leslie A.; Schimmel, Paul; Yang, Xiang-Lei

2011-01-01

Protein multi-functionality is an emerging explanation for the complexity of higher organisms. In this regard, while aminoacyl tRNA synthetases catalyze amino acid activation for protein synthesis, some also act in pathways for inflammation, angiogenesis, and apoptosis. How multiple functions evolved and their relationship to the active site is not clear. Here structural modeling analysis, mutagenesis, and cell-based functional studies show that the potent angiostatic, natural fragment of human TrpRS associates via Trp side chains that protrude from the cognate cellular receptor VE-cadherin. Modeling indicates that (I prefer the way it was because the conclusion was reached not only by modeling, but more so by experimental studies.)VE-cadherin Trp side chains fit into the Trp-specific active site of the synthetase. Thus, specific side chains of the receptor mimic (?) amino acid substrates and expand the functionality of the active site of the synthetase. We propose that orthogonal use of the same active site may be a general way to develop multi-functionality of human tRNA synthetases and other proteins. PMID:20010843

SEMICONDUCTOR TECHNOLOGY A signal processing method for the friction-based endpoint detection system of a CMP process

NASA Astrophysics Data System (ADS)

Chi, Xu; Dongming, Guo; Zhuji, Jin; Renke, Kang

2010-12-01

A signal processing method for the friction-based endpoint detection system of a chemical mechanical polishing (CMP) process is presented. The signal process method uses the wavelet threshold denoising method to reduce the noise contained in the measured original signal, extracts the Kalman filter innovation from the denoised signal as the feature signal, and judges the CMP endpoint based on the feature of the Kalman filter innovation sequence during the CMP process. Applying the signal processing method, the endpoint detection experiments of the Cu CMP process were carried out. The results show that the signal processing method can judge the endpoint of the Cu CMP process.

Inhibition of DNA replication in Saccharomyces cerevisiae by araCMP.

PubMed

McIntosh, E M; Kunz, B A; Haynes, R H

1986-01-01

Cytosine arabinoside (araC), a potent inhibitor of DNA replication in mammalian cells, was found to be completely ineffective in Saccharomyces cerevisiae. The 5' monophosphate derivative, araCMP, is toxic and effectively inhibits both nuclear and mitochondrial DNA synthesis in this organism. Although wild-type strains can be inhibited by araCMP, dTMP permeable (tup-) strains were found to be much more sensitive to the analogue. In vivo labelling experiments indicate that araC enters yeast cells; however, it is extensively catabolized by deamination and breakage of the glycosidic bond. In addition, the analogue is not efficiently phosphorylated in S. cerevisiae owing to an apparent lack of deoxynucleoside kinase activity. These results provide further evidence that deoxyribonucleotides can be synthesized only through de novo pathways in this organism. Finally, araCMP was found to be recombinagenic in S. cerevisiae which suggests, together with other previous studies, that, in general, inhibition of DNA synthesis in yeast promotes mitotic recombination events.

Structure and function of nucleotide sugar transporters: Current progress.

PubMed

Hadley, Barbara; Maggioni, Andrea; Ashikov, Angel; Day, Christopher J; Haselhorst, Thomas; Tiralongo, Joe

2014-06-01

The proteomes of eukaryotes, bacteria and archaea are highly diverse due, in part, to the complex post-translational modification of protein glycosylation. The diversity of glycosylation in eukaryotes is reliant on nucleotide sugar transporters to translocate specific nucleotide sugars that are synthesised in the cytosol and nucleus, into the endoplasmic reticulum and Golgi apparatus where glycosylation reactions occur. Thirty years of research utilising multidisciplinary approaches has contributed to our current understanding of NST function and structure. In this review, the structure and function, with reference to various disease states, of several NSTs including the UDP-galactose, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine, GDP-fucose, UDP-N-acetylglucosamine/UDP-glucose/GDP-mannose and CMP-sialic acid transporters will be described. Little is known regarding the exact structure of NSTs due to difficulties associated with crystallising membrane proteins. To date, no three-dimensional structure of any NST has been elucidated. What is known is based on computer predictions, mutagenesis experiments, epitope-tagging studies, in-vitro assays and phylogenetic analysis. In this regard the best-characterised NST to date is the CMP-sialic acid transporter (CST). Therefore in this review we will provide the current state-of-play with respect to the structure-function relationship of the (CST). In particular we have summarised work performed by a number groups detailing the affect of various mutations on CST transport activity, efficiency, and substrate specificity.

Multiple Click-Selective tRNA Synthetases Expand Mammalian Cell-Specific Proteomics.

PubMed

Yang, Andrew C; du Bois, Haley; Olsson, Niclas; Gate, David; Lehallier, Benoit; Berdnik, Daniela; Brewer, Kyle D; Bertozzi, Carolyn R; Elias, Joshua E; Wyss-Coray, Tony

2018-06-13

Bioorthogonal tools enable cell-type-specific proteomics, a prerequisite to understanding biological processes in multicellular organisms. Here we report two engineered aminoacyl-tRNA synthetases for mammalian bioorthogonal labeling: a tyrosyl ( ScTyr Y43G ) and a phenylalanyl ( MmPhe T413G ) tRNA synthetase that incorporate azide-bearing noncanonical amino acids specifically into the nascent proteomes of host cells. Azide-labeled proteins are chemoselectively tagged via azide-alkyne cycloadditions with fluorophores for imaging or affinity resins for mass spectrometric characterization. Both mutant synthetases label human, hamster, and mouse cell line proteins and selectively activate their azido-bearing amino acids over 10-fold above the canonical. ScTyr Y43G and MmPhe T413G label overlapping but distinct proteomes in human cell lines, with broader proteome coverage upon their coexpression. In mice, ScTyr Y43G and MmPhe T413G label the melanoma tumor proteome and plasma secretome. This work furnishes new tools for mammalian residue-specific bioorthogonal chemistry, and enables more robust and comprehensive cell-type-specific proteomics in live mammals.

Distinct Signaling Roles of cIMP, cCMP, and cUMP.

PubMed

Seifert, Roland

2016-10-04

The cyclic purine nucleotide cIMP and the cyclic pyrimidine nucleotides cCMP and cUMP are emerging second messengers. These cNMPs show different biological effects, but the molecular mechanisms remain elusive. In this issue of Structure, Ng et al. (2016) provide structural evidence for distinct interactions of cIMP, cCMP, and cUMP with ion channels. Copyright © 2016 Elsevier Ltd. All rights reserved.

Affinity purification of the voltage-sensitive sodium channel from electroplax with resins selective for sialic acid

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

James, W.M.; Emerick, M.C.; Agnew, W.S.

1989-07-11

The voltage-sensitive sodium channel present in the eel (Electrophorus electricus) has an unusually high content of sialic acid, including {alpha}-(2{yields}8)-linked polysialic acid, not found in other electroplax membrane glycopeptides. Lectins from Limax flavus (LFA) and wheat germ (WGA) proved the most effective of 11 lectin resins tried. The most selective resin was prepared from IgM antibodies against Neisseria meningitidis {alpha}-(2{yields}8)-polysialic acid which were affinity purified and coupled to Sepharose 4B. The sodium channel was found to bind to WGA, LFA, and IgM resins and was readily eluted with the appropriate soluble carbohydrates. Experiments with LFA and IgM resins demonstrated bindingmore » and unbinding rates and displacement kinetics, which suggest highly specific binding at multiple sites on the sodium channel protein. In preparative-scale purification of protein previously fractionated by anion-exchange chromatography, without stabilizing TTX, high yields were reproducibly obtained. Further, when detergent extracts were prepared from electroplax membranes fractionated by low-speed sedimentation, a single step over the IgM resin provided a 70-fold purification, yielding specific activities of 3,200 pmol of ({sup 3}H)TTX-binding sites/mg of protein and a single polypeptide of {approximately}285,000 Da on SDS-acrylamide gels. No small peptides were observed after this 5-h isolation. The authors describe a cation-dependent stabilization with millimolar levels of monovalent and micromolar levels of divalent species.« less

TRYPTOPHAN SYNTHETASE LEVELS IN ESCHERICHIA COLI, SHIGELLA DYSENTERIAE, AND TRANSDUCTION HYBRIDS

PubMed Central

Eisenstein, Richard B.; Yanofsky, Charles

1962-01-01

Eisenstein, Richard B. (Western Reserve University, Cleveland, Ohio) and Charles Yanofsky. Tryptophan synthetase levels in Escherichia coli, Shigella dysenteriae, and transduction hybrids. J. Bacteriol. 83:193–204. 1962—Shigella dysenteriae and Escherichia coli, strains K-12 and B, were found to produce low levels of tryptophan synthetase, although some hybrids, formed by the introduction of the gene cluster concerned with tryptophan synthesis from S. dysenteriae into E. coli, produced high levels of this enzyme system. A revertant obtained from a tryptophan-requiring mutant also formed high levels of tryptophan synthetase. The gene or genes responsible for high enzyme production in these strains was shown to be linked to the cluster of genes concerned with tryptophan synthesis. The cause of high enzyme production was investigated. Various lines of evidence, including stimulation of growth by tryptophan precursors, sensitivity to inhibition by 5-methyltryptophan, absence of accumulation of tryptophan, and repression of enzyme formation by anthranilic acid and tryptophan, suggested that high enzyme production in the strains examined results from a partial block in the tryptophan pathway and not from resistance to repression by tryptophan. The conversion of shikimic acid-5-phosphate to anthranilic acid appears to be the partially blocked reaction in the strains studied. PMID:13889700

Methods and compositions for the production of orthogonal tRNA-aminoacyl tRNA synthetase pairs

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

Schultz, Peter G.; Wang, Lei; Anderson, John Christopher

2015-10-20

This invention provides compositions and methods for generating components of protein biosynthetic machinery including orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases. Methods for identifying orthogonal pairs are also provided. These components can be used to incorporate unnatural amino acids into proteins in vivo.

Methods and compositions for the production of orthogonal tRNA-aminoacyl tRNA synthetase pairs

DOEpatents

Schultz, Peter; Wang, Lei; Anderson, John Christopher; Chin, Jason; Liu, David R.; Magliery, Thomas J.; Meggers, Eric L.; Mehl, Ryan Aaron; Pastrnak, Miro; Santoro, Stephen William; Zhang, Zhiwen

2006-08-01

This invention provides compositions and methods for generating components of protein biosynthetic machinery including orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases. Methods for identifying orthogonal pairs are also provided. These components can be used to incorporate unnatural amino acids into proteins in vivo.

Methods and composition for the production of orthogonal tRNA-aminoacyl tRNA synthetase pairs

DOEpatents

Schultz, Peter G [La Jolla, CA; Wang, Lei [San Diego, CA; Anderson, John Christopher [San Diego, CA; Chin, Jason W [San Diego, CA; Liu, David R [Lexington, MA; Magliery, Thomas J [North Haven, CT; Meggers, Eric L [Philadelphia, PA; Mehl, Ryan Aaron [San Diego, CA; Pastrnak, Miro [San Diego, CA; Santoro, Stephen William [San Diego, CA; Zhang, Zhiwen [San Diego, CA

2012-05-08

This invention provides compositions and methods for generating components of protein biosynthetic machinery including orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases. Methods for identifying orthogonal pairs are also provided. These components can be used to incorporate unnatural amino acids into proteins in vivo.

Methods and compositions for the production of orthogonal tRNA-aminoacyl-tRNA synthetase pairs

DOEpatents

Schultz, Peter G [La Jolla, CA; Wang, Lei [San Diego, CA; Anderson, John Christopher [San Diego, CA; Chin, Jason W [San Diego, CA; Liu, David R [Lexington, MA; Magliery, Thomas J [North Haven, CT; Meggers, Eric L [Philadelphia, PA; Mehl, Ryan Aaron [San Diego, CA; Pastrnak, Miro [San Diego, CA; Santoro, Stephen William [San Diego, CA; Zhang, Zhiwen [San Diego, CA

2011-09-06

This invention provides compositions and methods for generating components of protein biosynthetic machinery including orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases. Methods for identifying orthogonal pairs are also provided. These components can be used to incorporate unnatural amino acids into proteins in vivo.

42 CFR 417.460 - Disenrollment of beneficiaries by an HMO or CMP.

Code of Federal Regulations, 2011 CFR

2011-10-01

... or CMP's geographic area does not expand that area to encompass the location of the enrollee's new... to the HMO's or CMP's geographic area within 1 year from the date he or she left that area, the HMO... date the enrollee left that area in accordance with paragraph (f)(1) of this section. (g) Failure to...

42 CFR 417.460 - Disenrollment of beneficiaries by an HMO or CMP.

Code of Federal Regulations, 2010 CFR

2010-10-01

... or CMP's geographic area does not expand that area to encompass the location of the enrollee's new... to the HMO's or CMP's geographic area within 1 year from the date he or she left that area, the HMO... date the enrollee left that area in accordance with paragraph (f)(1) of this section. (g) Failure to...

Tumor Targeting via Sialic Acid: [68Ga]DOTA-en-pba as a New Tool for Molecular Imaging of Cancer with PET.

PubMed

Tsoukalas, Charalambos; Geninatti-Crich, Simonetta; Gaitanis, Anastasios; Tsotakos, Theodoros; Paravatou-Petsotas, Maria; Aime, Silvio; Jiménez-Juárez, Rogelio; Anagnostopoulos, Constantinos D; Djanashvili, Kristina; Bouziotis, Penelope

2018-02-20

The aim of this study was to demonstrate the potential of Ga-68-labeled macrocycle (DOTA-en-pba) conjugated with phenylboronic vector for tumor recognition by positron emission tomography (PET), based on targeting of the overexpressed sialic acid (Sia). The imaging reporter DOTA-en-pba was synthesized and labeled with Ga-68 at high efficiency. Cell binding assay on Mel-C and B16-F10 melanoma cells was used to evaluate melanin production and Sia overexpression to determine the best model for demonstrating the capability of [ 68 Ga]DOTA-en-pba to recognize tumors. The in vivo PET imaging was done with B16-F10 tumor-bearing SCID mice injected with [ 68 Ga]DOTA-en-pba intravenously. Tumor, blood, and urine metabolites were assessed to evaluate the presence of a targeting agent. The affinity of [ 68 Ga]DOTA-en-pba to Sia was demonstrated on B16-F10 melanoma cells, after the production of melanin as well as Sia overexpression was proved to be up to four times higher in this cell line compared to that in Mel-C cells. Biodistribution studies in B16-F10 tumor-bearing SCID mice showed blood clearance at the time points studied, while uptake in the tumor peaked at 60 min post-injection (6.36 ± 2.41 % ID/g). The acquired PET images were in accordance with the ex vivo biodistribution results. Metabolite assessment on tumor, blood, and urine samples showed that [ 68 Ga]DOTA-en-pba remains unmetabolized up to at least 60 min post-injection. Our work is the first attempt for in vivo imaging of cancer by targeting overexpression of sialic acid on cancer cells with a radiotracer in PET.

Surface topography analysis and performance on post-CMP images (Conference Presentation)

NASA Astrophysics Data System (ADS)

Lee, Jusang; Bello, Abner F.; Kakita, Shinichiro; Pieniazek, Nicholas; Johnson, Timothy A.

2017-03-01

Surface topography on post-CMP processing can be measured with white light interference microscopy to determine the planarity. Results are used to avoid under or over polishing and to decrease dishing. The numerical output of the surface topography is the RMS (root-mean-square) of the height. Beyond RMS, the topography image is visually examined and not further quantified. Subjective comparisons of the height maps are used to determine optimum CMP process conditions. While visual comparison of height maps can determine excursions, it's only through manual inspection of the images. In this work we describe methods of quantifying post-CMP surface topography characteristics that are used in other technical fields such as geography and facial-recognition. The topography image is divided into small surface patches of 7x7 pixels. Each surface patch is fitted to an analytic surface equation, in this case a third order polynomial, from which the gradient, directional derivatives, and other characteristics are calculated. Based on the characteristics, the surface patch is labeled as peak, ridge, flat, saddle, ravine, pit or hillside. The number of each label and thus the associated histogram is then used as a quantified characteristic of the surface topography, and could be used as a parameter for SPC (statistical process control) charting. In addition, the gradient for each surface patch is calculated, so the average, maximum, and other characteristics of the gradient distribution can be used for SPC. Repeatability measurements indicate high confidence where individual labels can be lower than 2% relative standard deviation. When the histogram is considered, an associated chi-squared value can be defined from which to compare other measurements. The chi-squared value of the histogram is a very sensitive and quantifiable parameter to determine the within wafer and wafer-to-wafer topography non-uniformity. As for the gradient histogram distribution, the chi-squared could

Cm-p5: an antifungal hydrophilic peptide derived from the coastal mollusk Cenchritis muricatus (Gastropoda: Littorinidae).

PubMed

López-Abarrategui, Carlos; McBeth, Christine; Mandal, Santi M; Sun, Zhenyu J; Heffron, Gregory; Alba-Menéndez, Annia; Migliolo, Ludovico; Reyes-Acosta, Osvaldo; García-Villarino, Mónica; Nolasco, Diego O; Falcão, Rosana; Cherobim, Mariana D; Dias, Simoni C; Brandt, Wolfgang; Wessjohann, Ludger; Starnbach, Michael; Franco, Octavio L; Otero-González, Anselmo J

2015-08-01

Antimicrobial peptides form part of the first line of defense against pathogens for many organisms. Current treatments for fungal infections are limited by drug toxicity and pathogen resistance. Cm-p5 (SRSELIVHQRLF), a peptide derived from the marine mollusk Cenchritis muricatus peptide Cm-p1, has a significantly increased fungistatic activity against pathogenic Candida albicans (minimal inhibitory concentration, 10 µg/ml; EC50, 1.146 µg/ml) while exhibiting low toxic effects against a cultured mammalian cell line. Cm-p5 as characterized by circular dichroism and nuclear magnetic resonance revealed an α-helical structure in membrane-mimetic conditions and a tendency to random coil folding in aqueous solutions. Additional studies modeling Cm-p5 binding to a phosphatidylserine bilayer in silico and isothermal titration calorimetry using lipid monophases demonstrated that Cm-p5 has a high affinity for the phospholipids of fungal membranes (phosphatidylserine and phosphatidylethanolamine), only moderate interactions with a mammalian membrane phospholipid, low interaction with ergosterol, and no interaction with chitin. Adhesion of Cm-p5 to living C. albicans cells was confirmed by fluorescence microscopy with FITC-labeled peptide. In a systemic candidiasis model in mice, intraperitoneal administration of Cm-p5 was unable to control the fungal kidney burden, although its low amphiphaticity could be modified to generate new derivatives with improved fungicidal activity and stability. © FASEB.

Thiol synthetases of legumes: immunogold localization and differential gene regulation by phytohormones

PubMed Central

Clemente, Maria R.; Bustos-Sanmamed, Pilar; Loscos, Jorge; James, Euan K.; Pérez-Rontomé, Carmen; Navascués, Joaquín; Gay, Marina; Becana, Manuel

2012-01-01

In plants and other organisms, glutathione (GSH) biosynthesis is catalysed sequentially by γ-glutamylcysteine synthetase (γECS) and glutathione synthetase (GSHS). In legumes, homoglutathione (hGSH) can replace GSH and is synthesized by γECS and a specific homoglutathione synthetase (hGSHS). The subcellular localization of the enzymes was examined by electron microscopy in several legumes and gene expression was analysed in Lotus japonicus plants treated for 1–48 h with 50 μM of hormones. Immunogold localization studies revealed that γECS is confined to chloroplasts and plastids, whereas hGSHS is also in the cytosol. Addition of hormones caused differential expression of thiol synthetases in roots. After 24–48 h, abscisic and salicylic acids downregulated GSHS whereas jasmonic acid upregulated it. Cytokinins and polyamines activated GSHS but not γECS or hGSHS. Jasmonic acid elicited a coordinated response of the three genes and auxin induced both hGSHS expression and activity. Results show that the thiol biosynthetic pathway is compartmentalized in legumes. Moreover, the similar response profiles of the GSH and hGSH contents in roots of non-nodulated and nodulated plants to the various hormonal treatments indicate that thiol homeostasis is independent of the nitrogen source of the plants. The differential regulation of the three mRNA levels, hGSHS activity, and thiol contents by hormones indicates a fine control of thiol biosynthesis at multiple levels and strongly suggests that GSH and hGSH play distinct roles in plant development and stress responses. PMID:22442424

Structure of Escherichia coli Arginyl-tRNA Synthetase in Complex with tRNAArg: Pivotal Role of the D-loop.

PubMed

Stephen, Preyesh; Ye, Sheng; Zhou, Ming; Song, Jian; Zhang, Rongguang; Wang, En-Duo; Giegé, Richard; Lin, Sheng-Xiang

2018-05-25

Aminoacyl-tRNA synthetases are essential components in protein biosynthesis. Arginyl-tRNA synthetase (ArgRS) belongs to the small group of aminoacyl-tRNA synthetases requiring cognate tRNA for amino acid activation. The crystal structure of Escherichia coli (Eco) ArgRS has been solved in complex with tRNA Arg at 3.0-Å resolution. With this first bacterial tRNA complex, we are attempting to bridge the gap existing in structure-function understanding in prokaryotic tRNA Arg recognition. The structure shows a tight binding of tRNA on the synthetase through the identity determinant A20 from the D-loop, a tRNA recognition snapshot never elucidated structurally. This interaction of A20 involves 5 amino acids from the synthetase. Additional contacts via U20a and U16 from the D-loop reinforce the interaction. The importance of D-loop recognition in EcoArgRS functioning is supported by a mutagenesis analysis of critical amino acids that anchor tRNA Arg on the synthetase; in particular, mutations at amino acids interacting with A20 affect binding affinity to the tRNA and specificity of arginylation. Altogether the structural and functional data indicate that the unprecedented ArgRS crystal structure represents a snapshot during functioning and suggest that the recognition of the D-loop by ArgRS is an important trigger that anchors tRNA Arg on the synthetase. In this process, A20 plays a major role, together with prominent conformational changes in several ArgRS domains that may eventually lead to the mature ArgRS:tRNA complex and the arginine activation. Functional implications that could be idiosyncratic to the arginine identity of bacterial ArgRSs are discussed. Copyright © 2018 Elsevier Ltd. All rights reserved.

Glutamate-Dependent Translational Control of Glutamine Synthetase in Bergmann Glia Cells.

PubMed

Tiburcio-Félix, Reynaldo; Escalante-López, Miguel; López-Bayghen, Bruno; Martínez, Daniel; Hernández-Kelly, Luisa C; Zinker, Samuel; Hernández-Melchor, Dinorah; López-Bayghen, Esther; Olivares-Bañuelos, Tatiana N; Ortega, Arturo

2018-06-01

Glutamate is the major excitatory transmitter of the vertebrate brain. It exerts its actions through the activation of specific plasma membrane receptors expressed both in neurons and in glial cells. Recent evidence has shown that glutamate uptake systems, particularly enriched in glia cells, trigger biochemical cascades in a similar fashion as receptors. A tight regulation of glutamate extracellular levels prevents neuronal overstimulation and cell death, and it is critically involved in glutamate turnover. Glial glutamate transporters are responsible of the majority of the brain glutamate uptake activity. Once internalized, this excitatory amino acid is rapidly metabolized to glutamine via the astrocyte-enriched enzyme glutamine synthetase. A coupling between glutamate uptake and glutamine synthesis and release has been commonly known as the glutamate/glutamine shuttle. Taking advantage of the established model of cultured Bergmann glia cells, in this contribution, we explored the gene expression regulation of glutamine synthetase. A time- and dose-dependent regulation of glutamine synthetase protein and activity levels was found. Moreover, glutamate exposure resulted in the transient shift of glutamine synthetase mRNA from the monosomal to the polysomal fraction. These results demonstrate a novel mode of glutamate-dependent glutamine synthetase regulation and strengthen the notion of an exquisite glia neuronal interaction in glutamatergic synapses.

Transfer of D-phenylalanine from gramicidin S synthetase 1 to gramicidin S synthetase 2 in gramicidin S synthesis.

PubMed

Hori, K; Kanda, M; Miura, S; Yamada, Y; Saito, Y

1983-01-01

The transfer of phenylalanine from gramicidin S synthetase 1 (GS 1) to gramicidin S synthetase 2 (GS 2) was studied by the use of combinations of wild-type GS 1 with various GS 2s from a wild strain and gramicidin S non-producing mutant strains of Bacillus brevis Nagano. The combinations of mutant GS 2s lacking 4'-phosphopantetheine (from BI-4, C-3, E-1, and E-2) did not transfer D-phenylalanine from GS 1, although they could activate all the constituent amino acids. Other mutant GS 2s containing 4'-phosphopantetheine, except GS 2 from BII-3 (proline-activation lacking) accepted D-phenylalanine from intact GS 1. To ascertain more directly whether 4'-phosphopantetheine is involved in the transfer of D-phenylalanine from GS 1 to GS 2, pepsin digests of GS 2 that accepted [14C]phenylalanine were analyzed by Sephadex G-50 column chromatography and thin-layer chromatography (TLC). Radioactivity of [14C]phenylalanine was always associated with a peptide containing 4'-phosphopantetheine. Furthermore, the position of radioactivity was distinct from the position of 4'-phosphopantetheine on TLC after alkaline treatment or performic acid oxidation of the digests.

Benzotriazole removal on post-Cu CMP cleaning

NASA Astrophysics Data System (ADS)

Jiying, Tang; Yuling, Liu; Ming, Sun; Shiyan, Fan; Yan, Li

2015-06-01

This work investigates systematically the effect of FA/O II chelating agent and FA/O I surfactant in alkaline cleaning solutions on benzotriazole (BTA) removal during post-Cu CMP cleaning in GLSI under the condition of static etching. The best detergent formulation for BTA removal can be determined by optimization of the experiments of single factor and compound cleaning solution, which has been further confirmed experimentally by contact angle (CA) measurements. The resulting solution with the best formulation has been measured for the actual production line, and the results demonstrate that the obtained cleaning solution can effectively and efficiently remove BTA, CuO and abrasive SiO2 without basically causing interfacial corrosion. This work demonstrates the possibility of developing a simple, low-cost and environmentally-friendly cleaning solution to effectively solve the issues of BTA removal on post-Cu CMP cleaning in a multi-layered copper wafer. Project supported by the Major National Science and Technology Special Projects (No. 2009ZX02308).

A non-canonical peptide synthetase adenylates 3-methyl-2-oxovaleric acid for auriculamide biosynthesis.

PubMed

Braga, Daniel; Hoffmeister, Dirk; Nett, Markus

2016-01-01

Auriculamide is the first natural product known from the predatory bacterium Herpetosiphon aurantiacus. It is composed of three unusual building blocks, including the non-proteinogenic amino acid 3-chloro-L-tyrosine, the α-hydroxy acid L-isoleucic acid, and a methylmalonyl-CoA-derived ethane unit. A candidate genetic locus for auriculamide biosynthesis was identified and encodes four enzymes. Among them, the non-canonical 199 kDa four-domain nonribosomal peptide synthetase, AulA, is extraordinary in that it features two consecutive adenylation domains. Here, we describe the functional characterization of the recombinantly produced AulA. The observed activation of 3-methyl-2-oxovaleric acid by the enzyme supports the hypothesis that it participates in the biosynthesis of auriculamide. An artificially truncated version of AulA that lacks the first adenylation domain activated this substrate like the full-length enzyme which shows that the first adenylation domain is dispensable. Additionally, we provide evidence that the enzyme tolerates structural variation of the substrate. α-Carbon substituents significantly affected the substrate turnover. While all tested aliphatic α-keto acids were accepted by the enzyme and minor differences in chain size and branches did not interfere with the enzymatic activity, molecules with methylene α-carbons led to low turnover. Such enzymatic plasticity is an important attribute to help in the perpetual search for novel molecules and to access a greater structural diversity by mutasynthesis.

Investigation of aluminum gate CMP in a novel alkaline solution

NASA Astrophysics Data System (ADS)

Cuiyue, Feng; Yuling, Liu; Ming, Sun; Wenqian, Zhang; Jin, Zhang; Shuai, Wang

2016-01-01

Beyond 45 nm, due to the superior CMP performance requirements with the metal gate of aluminum in the advanced CMOS process, a novel alkaline slurry for an aluminum gate CMP with poly-amine alkali slurry is investigated. The aluminum gate CMP under alkaline conditions has two steps: stock polishing and fine polishing. A controllable removal rate, the uniformity of aluminum gate and low corrosion are the key challenges for the alkaline polishing slurry of the aluminum gate CMP. This work utilizes the complexation-soluble function of FA/O II and the preference adsorption mechanism of FA/O I nonionic surfactant to improve the uniformity of the surface chemistry function with the electrochemical corrosion research, such as OCP-TIME curves, Tafel curves and AC impedance. The result is that the stock polishing slurry (with SiO2 abrasive) contains 1 wt.% H2O2,0.5 wt.% FA/O II and 1.0 wt.% FA/O I nonionic surfactant. For a fine polishing process, 1.5 wt.% H2O2, 0.4 wt.% FA/O II and 2.0 wt.% FA/O I nonionic surfactant are added. The polishing experiments show that the removal rates are 3000 ± 50 Å/min and 1600 ± 60 Å/min, respectively. The surface roughnesses are 2.05 ± 0.128 nm and 1.59 ± 0.081 nm, respectively. A combination of the functions of FA/O II and FA/O I nonionic surfactant obtains a controllable removal rate and a better surface roughness in alkaline solution.

Clostridium botulinum serotype D neurotoxin and toxin complex bind to bovine aortic endothelial cells via sialic acid.

PubMed

Yoneyama, Tohru; Miyata, Keita; Chikai, Tomoyuki; Mikami, Akifumi; Suzuki, Tomonori; Hasegawa, Kimiko; Ikeda, Toshihiko; Watanabe, Toshihiro; Ohyama, Tohru; Niwa, Koichi

2008-12-01

Botulinum neurotoxin (BoNT) is produced as a large toxin complex (L-TC) associated with nontoxic nonhemagglutinin (NTNHA) and three hemagglutinin subcomponents (HA-70, -33 and -17). The binding properties of BoNT to neurons and L-TC to intestinal epithelial cells are well documented, while those to other tissues are largely unknown. Here, to obtain novel insights into the pathogenesis of foodborne botulism, we examine whether botulinum toxins bind to vascular endothelial cells. BoNT and 750 kDa L-TC (a complex of BoNT, NTNHA and HAs) of Clostridium botulinum serotype D were incubated with bovine aortic endothelial cells (BAECs), and binding to the cells was assessed using sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot. Both BoNT and L-TC bound to BAECs, with L-TC showing stronger binding. Binding of BoNT and L-TC to BAECs was significantly inhibited by N-acetyl neuraminic acid in the cell culture medium or by treatment of the cells with neuraminidase. However, galactose, lactose or N-acetyl galactosamine did not significantly inhibit toxin binding to the cells. This is the first report demonstrating that BoNT and L-TC bind to BAECs via sialic acid, and this mechanism may be important in the trafficking pathway of BoNT in foodborne botulism.

Elucidation of several neglected reactions in the GC-MS identification of sialic acids as heptafluorobutyrates calls for an urgent reassessment of previous claims.

PubMed

Rota, Paola; Anastasia, Luigi; Allevi, Pietro

2015-05-07

The current analytical protocol used for the GC-MS determination of free or 1,7-lactonized natural sialic acids (Sias), as heptafluorobutyrates, overlooks several transformations. Using authentic reference standards and by combining GC-MS and NMR analyses, flaws in the analytical protocol were pinpointed and elucidated, thus establishing the scope and limitations of the method. It was demonstrated that (a) Sias 1,7-lactones, even if present in biological samples, decompose under the acidic hydrolysis conditions used for their release; (b) Sias 1,7-lactones are unpredicted artifacts, accidentally generated from their parent acids; (c) the N-acetyl group is quantitatively exchanged with that of the derivatizing perfluorinated anhydride; (d) the partial or complete failure of the Sias esterification-step with diazomethane leads to the incorrect quantification and structure attribution of all free Sias. While these findings prompt an urgent correction and improvement of the current analytical protocol, they could be instrumental for a critical revision of many incorrect claims reported in the literature.

Properties and substrate specificity of the leucyl-, the threonyl- and the valyl-transfer-ribonucleic acid synthetases from Aesculus species

PubMed Central

Anderson, J. W.; Fowden, L.

1970-01-01

1. Leucyl- and threonyl-tRNA synthetases were partially purified up to 100-fold and 30-fold respectively from cotyledons of Aesculus hippocastanum and were largely separated from the other aminoacyl-tRNA synthetases. Valyl-tRNA synthetase was purified 25-fold from cotyledons of Aesculus californica. 2. Some properties are reported for the three enzymes when assayed by the [32P]pyrophosphate-ATP exchange technique. 3. β-(Methylenecyclopropyl)alanine, isoleucine, azaleucine, norleucine and γ-hydroxynorvaline acted as alternative substrates for the leucyl-tRNA synthetase; the enzyme's affinity for β-(methylenecyclopropyl)-alanine and for isoleucine was about 80-fold less than that exhibited for leucine. 4. α-Cyclopropylglycine and α-cyclobutylglycine acted as alternative substrates for the valyl-tRNA synthetase. PMID:5493505

Cm-p5: an antifungal hydrophilic peptide derived from the coastal mollusk Cenchritis muricatus (Gastropoda: Littorinidae)

PubMed Central

López-Abarrategui, Carlos; McBeth, Christine; Mandal, Santi M.; Sun, Zhenyu J.; Heffron, Gregory; Alba-Menéndez, Annia; Migliolo, Ludovico; Reyes-Acosta, Osvaldo; García-Villarino, Mónica; Nolasco, Diego O.; Falcão, Rosana; Cherobim, Mariana D.; Dias, Simoni C.; Brandt, Wolfgang; Wessjohann, Ludger; Starnbach, Michael; Franco, Octavio L.; Otero-González, Anselmo J.

2015-01-01

Antimicrobial peptides form part of the first line of defense against pathogens for many organisms. Current treatments for fungal infections are limited by drug toxicity and pathogen resistance. Cm-p5 (SRSELIVHQRLF), a peptide derived from the marine mollusk Cenchritis muricatus peptide Cm-p1, has a significantly increased fungistatic activity against pathogenic Candida albicans (minimal inhibitory concentration, 10 µg/ml; EC50, 1.146 µg/ml) while exhibiting low toxic effects against a cultured mammalian cell line. Cm-p5 as characterized by circular dichroism and nuclear magnetic resonance revealed an α-helical structure in membrane-mimetic conditions and a tendency to random coil folding in aqueous solutions. Additional studies modeling Cm-p5 binding to a phosphatidylserine bilayer in silico and isothermal titration calorimetry using lipid monophases demonstrated that Cm-p5 has a high affinity for the phospholipids of fungal membranes (phosphatidylserine and phosphatidylethanolamine), only moderate interactions with a mammalian membrane phospholipid, low interaction with ergosterol, and no interaction with chitin. Adhesion of Cm-p5 to living C. albicans cells was confirmed by fluorescence microscopy with FITC-labeled peptide. In a systemic candidiasis model in mice, intraperitoneal administration of Cm-p5 was unable to control the fungal kidney burden, although its low amphiphaticity could be modified to generate new derivatives with improved fungicidal activity and stability.—López-Abarrategui, C., McBeth, C., Mandal, S. M., Sun, Z. J., Heffron, G., Alba-Menéndez, A., Migliolo, L., Reyes-Acosta, O., García-Villarino, M., Nolasco, D. O., Falcão, R., Cherobim, M. D., Dias, S. C., Brandt, W., Wessjohann, L., Starnbach, M., Franco, O. L., Otero-González, A. J. Cm-p5: an antifungal hydrophilic peptide derived from the coastal mollusk Cenchritis muricatus (Gastropoda: Littorinidae). PMID:25921828

Low incidence of N-glycolylneuraminic acid in birds and reptiles and its absence in the platypus.

PubMed

Schauer, Roland; Srinivasan, G Vinayaga; Coddeville, Bernadette; Zanetta, Jean-Pierre; Guérardel, Yann

2009-08-17

The sialic acids of the platypus, birds, and reptiles were investigated with regard to the occurrence of N-glycolylneuraminic (Neu5Gc) acid. They were released from tissues, eggs, or salivary mucin samples by acid hydrolysis, and purified and analyzed by thin-layer chromatography, high-performance liquid chromatography, and mass spectrometry. In muscle and liver of the platypus only N-acetylneuraminic (Neu5Ac) acid was found. The nine bird species studied also did not express N-glycolylneuraminic acid with the exception of an egg, but not tissues, from the budgerigar and traces in poultry. Among nine reptiles, including one turtle, N-glycolylneuraminic acid was only found in the egg and an adult basilisk, but not in a freshly hatched animal. BLAST analysis of the genomes of the platypus, the chicken, and zebra finch against the CMP-N-acetylneuraminic acid hydroxylase did not reveal the existence of a similar protein structure. Apparently monotremes (platypus) and sauropsids (birds and reptiles) cannot synthesize Neu5Gc. The few animals where Neu5Gc was found, especially in eggs, may have acquired this from the diet or by an alternative pathway. Since Neu5Gc is antigenic to man, the observation that this monosaccharide does not or at least only rarely occur in birds and reptiles, may be of nutritional and clinical significance.

Aminoacyl-tRNA synthetases: versatile players in the changing theater of translation.

PubMed Central

Francklyn, Christopher; Perona, John J; Puetz, Joern; Hou, Ya-Ming

2002-01-01

Aminoacyl-tRNA synthetases attach amino acids to the 3' termini of cognate tRNAs to establish the specificity of protein synthesis. A recent Asilomar conference (California, January 13-18, 2002) discussed new research into the structure-function relationship of these crucial enzymes, as well as a multitude of novel functions, including participation in amino acid biosynthesis, cell cycle control, RNA splicing, and export of tRNAs from nucleus to cytoplasm in eukaryotic cells. Together with the discovery of their role in the cellular synthesis of proteins to incorporate selenocysteine and pyrrolysine, these diverse functions of aminoacyl-tRNA synthetases underscore the flexibility and adaptability of these ancient enzymes and stimulate the development of new concepts and methods for expanding the genetic code. PMID:12458790

Development of Formulations for a-SiC and Manganese CMP and Post-CMP Cleaning of Cobalt

NASA Astrophysics Data System (ADS)

Lagudu, Uma Rames Krishna

We have investigated the chemical mechanical polishing (CMP) of amorphous SiC (a-SiC) and Mn and Post CMP cleaning of cobalt for various device applications. During the manufacture of copper interconnects using the damascene process the polishing of copper is followed by the polishing of the barrier material (Co, Mn, Ru and their alloys) and its post CMP cleaning. This is followed by the a-SiC hard mask CMP. Silicon carbide thin films, though of widespread use in microelectronic engineering, are difficult to process by CMP because of their hardness and chemical inertness. The earlier part of the SiC work discusses the development of slurries based on silica abrasives that resulted in high a-SiC removal rates (RRs). The ionic strength of the silica dispersion was found to play a significant role in enhancing material removal rate, while also providing very good post-polish surface-smoothness. For example, the addition of 50 mM potassium nitrate to a pH 8 aqueous slurry consisting of 10 wt % of silica abrasives and 1.47 M hydrogen peroxide increased the RR from about 150 nm/h to about 2100 nm/h. The role of ionic strength in obtaining such high RRs was investigated using surface zeta-potentials measurements and X-ray photoelectron spectroscopy (XPS). Evidently, hydrogen peroxide promoted the oxidation of Si and C to form weakly adhered species that were subsequently removed by the abrasive action of the silica particles. The effect of potassium nitrate in increasing material removal is attributed to the reduction in the electrostatic repulsion between the abrasive particles and the SiC surface because of screening of surface charges by the added electrolyte. We also show that transition metal compounds when used as additives to silica dispersions enhance a-SiC removal rates (RRs). Silica slurries containing potassium permanganate gave RRs as high as 2000 nm/h at pH 4. Addition of copper sulfate to this slurry further enhanced the RRs to ˜3500 nm/h at pH 6

Cloning and characterization of the metE gene encoding S-adenosylmethionine synthetase from Bacillus subtilis.

PubMed Central

Yocum, R R; Perkins, J B; Howitt, C L; Pero, J

1996-01-01

The metE gene, encoding S-adenosylmethionine synthetase (EC 2.5.1.6) from Bacillus subtilis, was cloned in two steps by normal and inverse PCR. The DNA sequence of the metE gene contains an open reading frame which encodes a 400-amino-acid sequence that is homologous to other known S-adenosylmethionine synthetases. The cloned gene complements the metE1 mutation and integrates at or near the chromosomal site of metE1. Expression of S-adenosylmethionine synthetase is reduced by only a factor of about 2 by exogenous methioinine. Overproduction of S-adenosylmethionine synthetase from a strong constitutive promoter leads to methionine auxotrophy in B. subtilis, suggesting that S-adenosylmethionine is a corepressor of methionine biosynthesis in B. subtilis, as others have already shown for Escherichia coli. PMID:8755891

Cloning and characterization of the metE gene encoding S-adenosylmethionine synthetase from Bacillus subtilis.

PubMed

Yocum, R R; Perkins, J B; Howitt, C L; Pero, J

1996-08-01

The metE gene, encoding S-adenosylmethionine synthetase (EC 2.5.1.6) from Bacillus subtilis, was cloned in two steps by normal and inverse PCR. The DNA sequence of the metE gene contains an open reading frame which encodes a 400-amino-acid sequence that is homologous to other known S-adenosylmethionine synthetases. The cloned gene complements the metE1 mutation and integrates at or near the chromosomal site of metE1. Expression of S-adenosylmethionine synthetase is reduced by only a factor of about 2 by exogenous methioinine. Overproduction of S-adenosylmethionine synthetase from a strong constitutive promoter leads to methionine auxotrophy in B. subtilis, suggesting that S-adenosylmethionine is a corepressor of methionine biosynthesis in B. subtilis, as others have already shown for Escherichia coli.

Extensive enrichment of N-glycolylneuraminic acid in extracellular sialoglycoproteins abundantly synthesized and secreted by human cancer cells.

PubMed

Inoue, Sadako; Sato, Chihiro; Kitajima, Ken

2010-06-01

N-Glycolylneuraminic acid (Neu5Gc) is the second most populous sialic acid (Sia). The only known biosynthetic pathway of Neu5Gc is the hydroxylation of cytidine-5'-monophosphate-N-acetylneuraminic acid (CMP-Neu5Ac), catalyzed by CMP-Neu5Ac hydroxylase (CMAH). Neu5Gc is abundantly found in mammals except for human, in which CMAH is inactivated due to mutation in the CMAH gene. Evidence has accumulated to show occurrence of Neu5Gc-containing glycoconjugates in sera of cancer patients, human cancerous tissues and cultured human cell lines. Recently, occurrence of natural antibodies against Neu5Gc was shown in healthy humans and is a serious problem for clinical xenotransplantation and stem cell therapies. Studying human occurrence of Neu5Gc is of importance and interest in a broad area of medical sciences. In this study, using a fluorometric high performance liquid chromatography method, we performed quantitative analyses of Sias both inside and in the external environment of the cell and found that (i) incorporation of Neu5Gc was most prominent in soluble glycoproteins found both in the extracellular space and inside the cell as the major Sia compounds. (ii) Of the total Neu5Gc in the Sia compounds that the cells synthesized, 90% was found in the secreted sialoglycoproteins, whereas for Neu5Ac, 70% was found in the secreted sialoglycoproteins. (iii) The Neu5Gc ratio was higher in the secreted sialoglycoproteins (as high as 40% of total Sias) than in intracellular sialoglycoproteins. (iv) The majority of the secreted sialoglycoproteins was anchored on the culture dishes and solubilized by brief trypsin treatment. Based on these findings, a new idea on the mechanism of accumulation of Neu5Gc in cancer cells was proposed.

Genomic and molecular analysis of phage CMP1 from Clavibacter michiganensis subspecies michiganensis

PubMed Central

Wittmann, Johannes; Gartemann, Karl-Heinz; Eichenlaub, Rudolf

2011-01-01

Bacteriophage CMP1 is a member of the Siphoviridae family that infects specifically the plant-pathogen Clavibacter michiganensis subsp. michiganensis. The linear double- stranded DNA is terminally redundant and not circularly permuted. The complete nucleotide sequence of the bacteriophage CMP1 genome consists of 58,652 bp including the terminal redundant ends of 791 bp. The G+C content of the phage (57%) is significantly lower than that of its host (72.66%). 74 potential open reading frames were identified and annotated by different bioinformatic tools. Two large clusters which encode the early and the late functions could be identified which are divergently transcribed. There are only a few hypothetical gene products with conserved domains and significant similarity to sequences from the databases. Functional analyses confirmed the activity of four gene products, an endonuclease, an exonuclease, a single-stranded DNA binding protein and a thymidylate synthase. Partial genomic sequences of CN77, a phage of Clavibacter michiganensis subsp. nebraskensis, revealed a similar genome structure and significant similarities on the level of deduced amino acid sequences. An endolysin with peptidase activity has been identified for both phages, which may be good tools for disease control of tomato plants against Clavibacter infections. PMID:21687530

Genomic and molecular analysis of phage CMP1 from Clavibacter michiganensis subspecies michiganensis.

PubMed

Wittmann, Johannes; Gartemann, Karl-Heinz; Eichenlaub, Rudolf; Dreiseikelmann, Brigitte

2011-01-01

Bacteriophage CMP1 is a member of the Siphoviridae family that infects specifically the plant-pathogen Clavibacter michiganensis subsp. michiganensis. The linear double- stranded DNA is terminally redundant and not circularly permuted. The complete nucleotide sequence of the bacteriophage CMP1 genome consists of 58,652 bp including the terminal redundant ends of 791 bp. The G+C content of the phage (57%) is significantly lower than that of its host (72.66%). 74 potential open reading frames were identified and annotated by different bioinformatic tools. Two large clusters which encode the early and the late functions could be identified which are divergently transcribed. There are only a few hypothetical gene products with conserved domains and significant similarity to sequences from the databases. Functional analyses confirmed the activity of four gene products, an endonuclease, an exonuclease, a single-stranded DNA binding protein and a thymidylate synthase. Partial genomic sequences of CN77, a phage of Clavibacter michiganensis subsp. nebraskensis, revealed a similar genome structure and significant similarities on the level of deduced amino acid sequences. An endolysin with peptidase activity has been identified for both phages, which may be good tools for disease control of tomato plants against Clavibacter infections.

Investigation on interaction of Achatinin, a 9-O-acetyl sialic acid-binding lectin, with lipopolysaccharide in the innate immunity of Achatina fulica snails.

PubMed

Biswas, C; Sinha, D; Mandal, C

2000-01-01

Achatinin, a 9-O-acetyl sialic acid (9-O-AcSA) binding lectin, has been demonstrated to be synthesized in amoebocytes of Achatina fulica snails. This lectin was affinity-purified from Achatina amoebocytes lysate (AAL); it appeared as a single band on native polyacrylamide gel electrophoresis (PAGE) and showed 16 identical subunits of M.W. 15 kDa on sodium dodecyl sulphate (SDS)-PAGE. It was found to be homologous with an earlier reported lectin, Achatinin-H, derived from hemolymph of A. fulica snails (Sen, G., Mandal, C., 1995. The specificity of the binding site of Achatinin-H, a sialic-acid binding lectin from Achantia fulica. Carbohydr. Res., 268, 115-125). Homology between both lectins was confirmed by their similar electrophoretic mobilities, carbohydrate specificity and cross reactivity on immunodiffusion. Achatinin showed in vitro calcium dependent binding to two 9-O-acetylated sialoglyoconjugates (9-O-AcSG) on lipopolysaccharide (LPS) (Escherichia coli 055: B5) of M.W. 40 kDa and 27.5 kDa, which was abolished following de-O-acetylation. Based on the previously defined narrow sugar specificity of Achatinin towards 9-O-AcSAalpha2-->6GalNAc [Sen, G., Mandal, C., 1995. The specificity of the binding site of Achatinin-H, a sialic-acid binding lectin from Achatina fulica. Carbohydr. Res., 268, 115-125], we conclude that LPS contains this lectinogenic epitope at the terminal sugar moiety. The Achatinin-mediated hemagglutination inhibition of rabbit erythrocytes by LPS further confirmed it. The lectin exhibited bacteriostatic effect on Gram-negative bacteria E. coli, DH5alpha and C600. AAL was earlier reported to undergo coagulation in presence of pg level of LPS (Biswas, C., Mandal, C., 1999. The role of amoebocytes in the endotoxin-mediated coagulation in the innate immunity of Achatina fulica snail, Scand. J. Immunol. 49, 131-138). We now demonstrate that Achatinin participates in LPS-mediated coagulation of AAL as indicated by enhanced release of Achatinin from

Recoding aminoacyl-tRNA synthetases for synthetic biology by rational protein-RNA engineering.

PubMed

Hadd, Andrew; Perona, John J

2014-12-19

We have taken a rational approach to redesigning the amino acid binding and aminoacyl-tRNA pairing specificities of bacterial glutaminyl-tRNA synthetase. The four-stage engineering incorporates generalizable design principles and improves the pairing efficiency of noncognate glutamate with tRNA(Gln) by over 10(5)-fold compared to the wild-type enzyme. Better optimized designs of the protein-RNA complex include substantial reengineering of the globular core region of the tRNA, demonstrating a role for specific tRNA nucleotides in specifying the identity of the genetically encoded amino acid. Principles emerging from this engineering effort open new prospects for combining rational and genetic selection approaches to design novel aminoacyl-tRNA synthetases that ligate noncanonical amino acids onto tRNAs. This will facilitate reconstruction of the cellular translation apparatus for applications in synthetic biology.

Multivalent Interactions of Human Primary Amine Oxidase with the V and C22 Domains of Sialic Acid-Binding Immunoglobulin-Like Lectin-9 Regulate Its Binding and Amine Oxidase Activity

PubMed Central

Fair-Mäkelä, Ruth; Salo-Ahen, Outi M. H.; Guédez, Gabriela; Bligt-Lindén, Eva; Grönholm, Janne; Jalkanen, Sirpa; Salminen, Tiina A.

2016-01-01

Sialic acid-binding immunoglobulin-like lectin-9 (Siglec-9) on leukocyte surface is a counter-receptor for endothelial cell surface adhesin, human primary amine oxidase (hAOC3), a target protein for anti-inflammatory agents. This interaction can be used to detect inflammation and cancer in vivo, since the labeled peptides derived from the second C2 domain (C22) of Siglec-9 specifically bind to the inflammation-inducible hAOC3. As limited knowledge on the interaction between Siglec-9 and hAOC3 has hampered both hAOC3-targeted drug design and in vivo imaging applications, we have now produced and purified the extracellular region of Siglec-9 (Siglec-9-EC) consisting of the V, C21 and C22 domains, modeled its 3D structure and characterized the hAOC3–Siglec-9 interactions using biophysical methods and activity/inhibition assays. Our results assign individual, previously unknown roles for the V and C22 domains. The V domain is responsible for the unusually tight Siglec-9–hAOC3 interactions whereas the intact C22 domain of Siglec-9 is required for modulating the enzymatic activity of hAOC3, crucial for the hAOC3-mediated leukocyte trafficking. By characterizing the Siglec-9-EC mutants, we could conclude that R120 in the V domain likely interacts with the terminal sialic acids of hAOC3 attached glycans whereas residues R284 and R290 in C22 are involved in the interactions with the active site channel of hAOC3. Furthermore, the C22 domain binding enhances the enzymatic activity of hAOC3 although the sialic acid-binding capacity of the V domain of Siglec-9 is abolished by the R120S mutation. To conclude, our results prove that the V and C22 domains of Siglec-9-EC interact with hAOC3 in a multifaceted and unique way, forming both glycan-mediated and direct protein-protein interactions, respectively. The reported results on the mechanism of the Siglec-9–hAOC3 interaction are valuable for the development of hAOC3-targeted therapeutics and diagnostic tools. PMID:27893774

Assessment of maternal serum sialic acid levels in preterm versus term labor: a prospective-controlled clinical study.

PubMed

Ugur, Mete Gurol; Kurtul, Naciye; Balat, Ozcan; Ekici, Melek; Kul, Seval

2012-11-01

To compare total serum sialic acid (SA) levels between singleton pregnant women diagnosed with preterm labor between 24th and 36th weeks of pregnancy, singleton pregnant women at term, and their gestational age-matched controls. Thirty pregnants diagnosed with preterm labor (group I), 30 gestational age-matched control pregnants (group II), 30 pregnants with labor at term (group III), and 30 gestational age-matched control pregnants (group IV) were enrolled. Detailed history, demographic data (age, gravidity, parity, abortion), ultrasound parameters, cervical dilatation and effacement, fetal tococardiography, routine laboratory tests, and total SA levels were assessed. There was no statistically significant difference between the parameters other than SA. SA levels of the preterm labor group (group I) were significantly higher than the other three groups. We may suggest that pathways including SA or molecules containing SA in subclinical infection without the clinical manifestations of apparent infection may be involved in the pathogenesis of preterm birth. Future longitudinal studies are needed to investigate prediction performance and to better understand the role of SA in molecular mechanisms leading to preterm labor.

Crystal structure of an indole-3-acetic acid amido synthetase from grapevine involved in auxin homeostasis.

PubMed

Peat, Thomas S; Böttcher, Christine; Newman, Janet; Lucent, Del; Cowieson, Nathan; Davies, Christopher

2012-11-01

Auxins are important for plant growth and development, including the control of fruit ripening. Conjugation to amino acids by indole-3-acetic acid (IAA)-amido synthetases is an important part of auxin homeostasis. The structure of the auxin-conjugating Gretchen Hagen3-1 (GH3-1) enzyme from grapevine (Vitis vinifera), in complex with an inhibitor (adenosine-5'-[2-(1H-indol-3-yl)ethyl]phosphate), is presented. Comparison with a previously published benzoate-conjugating enzyme from Arabidopsis thaliana indicates that grapevine GH3-1 has a highly similar domain structure and also undergoes a large conformational change during catalysis. Mutational analyses and structural comparisons with other proteins have identified residues likely to be involved in acyl group, amino acid, and ATP substrate binding. Vv GH3-1 is a monomer in solution and requires magnesium ions solely for the adenlyation reaction. Modeling of IAA and two synthetic auxins, benzothiazole-2-oxyacetic acid (BTOA) and 1-naphthaleneacetic acid (NAA), into the active site indicates that NAA and BTOA are likely to be poor substrates for this enzyme, confirming previous enzyme kinetic studies. This suggests a reason for the increased effectiveness of NAA and BTOA as auxins in planta and provides a tool for designing new and effective auxins.

Structural control of caspase-generated glutamyl-tRNA synthetase by appended noncatalytic WHEP domains.

PubMed

Halawani, Dalia; Gogonea, Valentin; DiDonato, Joseph A; Pipich, Vitaliy; Yao, Peng; China, Arnab; Topbas, Celalettin; Vasu, Kommireddy; Arif, Abul; Hazen, Stanley L; Fox, Paul L

2018-06-08

Aminoacyl-tRNA synthetases are ubiquitous, evolutionarily conserved enzymes catalyzing the conjugation of amino acids onto cognate tRNAs. During eukaryotic evolution, tRNA synthetases have been the targets of persistent structural modifications. These modifications can be additive, as in the evolutionary acquisition of noncatalytic domains, or subtractive, as in the generation of truncated variants through regulated mechanisms such as proteolytic processing, alternative splicing, or coding region polyadenylation. A unique variant is the human glutamyl-prolyl-tRNA synthetase (EPRS) consisting of two fused synthetases joined by a linker containing three copies of the WHEP domain (termed by its presence in tryptophanyl-, histidyl-, and glutamyl-prolyl-tRNA synthetases). Here, we identify site-selective proteolysis as a mechanism that severs the linkage between the EPRS synthetases in vitro and in vivo Caspase action targeted Asp-929 in the third WHEP domain, thereby separating the two synthetases. Using a neoepitope antibody directed against the newly exposed C terminus, we demonstrate EPRS cleavage at Asp-929 in vitro and in vivo Biochemical and biophysical characterizations of the N-terminally generated EPRS proteoform containing the glutamyl-tRNA synthetase and most of the linker, including two WHEP domains, combined with structural analysis by small-angle neutron scattering, revealed a role for the WHEP domains in modulating conformations of the catalytic core and GSH- S -transferase-C-terminal-like (GST-C) domain. WHEP-driven conformational rearrangement altered GST-C domain interactions and conferred distinct oligomeric states in solution. Collectively, our results reveal long-range conformational changes imposed by the WHEP domains and illustrate how noncatalytic domains can modulate the global structure of tRNA synthetases in complex eukaryotic systems. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Moderate folic acid supplementation and MTHFD1-synthetase deficiency in mice, a model for the R653Q variant, result in embryonic defects and abnormal placental development.

PubMed

Christensen, Karen E; Hou, Wenyang; Bahous, Renata H; Deng, Liyuan; Malysheva, Olga V; Arning, Erland; Bottiglieri, Teodoro; Caudill, Marie A; Jerome-Majewska, Loydie A; Rozen, Rima

2016-11-01

Moderately high folic acid intake in pregnant women has led to concerns about deleterious effects on the mother and fetus. Common polymorphisms in folate genes, such as methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase (MTHFD1) R653Q, may modulate the effects of elevated folic acid intake. We investigated the effects of moderate folic acid supplementation on reproductive outcomes and assessed the potential interaction of the supplemented diet with MTHFD1-synthetase (Mthfd1S) deficiency in mice, which is a model for the R653Q variant. Female Mthfd1S +/+ and Mthfd1S +/- mice were fed a folic acid-supplemented diet (FASD) (5-fold higher than recommended) or control diets before mating and during pregnancy. Embryos and placentas were assessed for developmental defects at embryonic day 10.5 (E10.5). Maternal folate and choline metabolites and gene expression in folate-related pathways were examined. The combination of FASD and maternal MTHFD1-synthetase deficiency led to a greater incidence of defects in E10.5 embryos (diet × maternal genotype, P = 0.0016; diet × embryonic genotype, P = 0.054). The methylenetetrahydrofolate reductase (MTHFR) protein and methylation potential [ratio of S-adenosylmethionine (major methyl donor):S-adenosylhomocysteine) were reduced in maternal liver. Although 5-methyltetrahydrofolate (methylTHF) was higher in maternal circulation, the methylation potential was lower in embryos. The presence of developmental delays and defects in Mthfd1S +/- embryos was associated with placental defects (P = 0.003). The labyrinth layer failed to form properly in the majority of abnormal placentas, which compromised the integration of the maternal and fetal circulation and presumably the transfer of methylTHF and other nutrients. Moderately higher folate intake and MTHFD1-synthetase deficiency in pregnant mice result in a lower methylation potential in maternal liver and embryos and a greater

Effects of polyamine biosynthesis inhibitors on S-adenosylmethionine synthetase and S-adenosylmethionine decarboxylase activities in carrot cell cultures

Treesearch

S.C. Minocha; R. Minocha; A. Komamine

1991-01-01

Changes in the activites of S-adcnosylmethionine (SAM) synthetase (methionine adenosyltransferase, EC 2.5.1.6.) and SAM decarboxylase (EC 4.1.1.50) were studied in carrot (Daucus carota) cell cultures in response to 2,4-dichlorophenoxyacetic acid (2,4-D) and several inhibitors of polyamine biosynthesis. Activity of SAM synthetase increased...

Malonyl-CoA Synthetase, Encoded by ACYL ACTIVATING ENZYME13, Is Essential for Growth and Development of Arabidopsis[C][W][OA

PubMed Central

Chen, Hui; Kim, Hyun Uk; Weng, Hua; Browse, John

2011-01-01

Malonyl-CoA is the precursor for fatty acid synthesis and elongation. It is also one of the building blocks for the biosynthesis of some phytoalexins, flavonoids, and many malonylated compounds. In plants as well as in animals, malonyl-CoA is almost exclusively derived from acetyl-CoA by acetyl-CoA carboxylase (EC 6.4.1.2). However, previous studies have suggested that malonyl-CoA may also be made directly from malonic acid by malonyl-CoA synthetase (EC 6.2.1.14). Here, we report the cloning of a eukaryotic malonyl-CoA synthetase gene, Acyl Activating Enzyme13 (AAE13; At3g16170), from Arabidopsis thaliana. Recombinant AAE13 protein showed high activity against malonic acid (Km = 529.4 ± 98.5 μM; Vm = 24.0 ± 2.7 μmol/mg/min) but little or no activity against other dicarboxylic or fatty acids tested. Exogenous malonic acid was toxic to Arabidopsis seedlings and caused accumulation of malonic and succinic acids in the seedlings. aae13 null mutants also grew poorly and accumulated malonic and succinic acids. These defects were complemented by an AAE13 transgene or by a bacterial malonyl-CoA synthetase gene under control of the AAE13 promoter. Our results demonstrate that the malonyl-CoA synthetase encoded by AAE13 is essential for healthy growth and development, probably because it is required for the detoxification of malonate. PMID:21642549

Disruption of plastid acyl:acyl carrier protein synthetases increases medium chain fatty acid accumulation in seeds of transgenic Arabidopsis.

PubMed

Tjellström, Henrik; Strawsine, Merissa; Silva, Jillian; Cahoon, Edgar B; Ohlrogge, John B

2013-04-02

Engineering transgenic plants that accumulate high levels of medium-chain fatty acids (MCFA) has been least successful for shorter chain lengths (e.g., C8). We demonstrate that one limitation is the activity of acyl-ACP synthetase (AAE) that re-activates fatty acids released by acyl-ACP thioesterases. Seed expression of Cuphea pulcherrima FATB acyl-ACP thioesterase in a double mutant lacking AAE15/16 increased 8:0 accumulation almost 2-fold compared to expression in wild type. These results also provide an in planta demonstration that AAE enzymes participate not only in activation of exogenously added MCFA but also in activation of MCFA synthesized in plastids. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Fate of 1-(1',4'-cyclohexadienyl)-2-methylaminopropane (CMP) in soil: route-specific by-product in the clandestine manufacture of methamphetamine.

PubMed

Pal, Raktim; Megharaj, Mallavarapu; Kirkbride, K Paul; Naidu, Ravi

2012-02-01

We investigated the fate of 1-(1',4'-cyclohexadienyl)-2-methylaminopropane (CMP) in soil. CMP is the major route-specific byproduct in the clandestine manufacture of methamphetamine (MAP) by the use of excess alkali metal (e.g., lithium) in liquid ammonia, which is commonly referred to as the "Nazi method". This is one of the most common methods used in many countries for the illicit production of MAP. Knowledge on the fate of CMP in the terrestrial environment is essential to combat potential threats arising from illegal dumping of clandestine laboratory wastes. We report on the sorption-desorption, degradation, and metabolism patterns of CMP in three South Australian soils investigated in laboratory scale. CMP sorption in the test soils followed a Freundlich isotherm in the concentration range of 5 to 100μgmL(-1). Degradation studies showed that CMP was fairly unstable in both non-sterile and sterile soils, with half-life values typically less than one week. The role of biotic and abiotic soil processes in the degradation of CMP also varied significantly between the different soils, and with the length of the incubation period. Interestingly, but not surprisingly, the results showed that the CMP was not actually degraded to any simpler compounds but transformed to more persistent MAP. Thus, the main concern with Nazi method is the potential hazard from MAP rather than CMP if wastes are disposed of into the environment. Copyright © 2011 Elsevier B.V. All rights reserved.

Dexamethasone regulates glutamine synthetase expression in rat skeletal muscles

NASA Technical Reports Server (NTRS)

Max, Stephen R.; Konagaya, Masaaki; Konagaya, Yoko; Thomas, John W.; Banner, Carl; Vitkovic, Ljubisa

1986-01-01

The regulation of glutamine synthetase by glucocorticoids in rat skeletal muscles was studied. Administration of dexamethasone strikingly enhanced glutamine synthetase activity in plantaris and soleus muscles. The dexamethasone-mediated induction of glutamine synthetase activity was blocked to a significant extent by orally administered RU38486, a glucocorticoid antagonist, indicating the involvement of intracellular glucocorticoid receptors in the induction. Northern blot analysis revealed that dexamethasone-mediated enhancement of glutamine synthetase activity involves dramatically increased levels of glutamine synthetase mRNA. The induction of glutamine synthetase was selective in that glutaminase activity of soleus and plantaris muscles was not increased by dexamethasone. Furthermore, dexamethasone treatment resulted in only a small increase in glutamine synthetase activity in the heart. Accordingly, there was only a slight change in glutamine synthetase mRNA level in this tissue. Thus, glucocorticoids regulate glutamine synthetase gene expression in rat muscles at the transcriptional level via interaction with intracellular glutamine production by muscle and to mechanisms underlying glucocorticoid-induced muscle atrophy.

Boronic acid-tethered amphiphilic hyaluronic acid derivative-based nanoassemblies for tumor targeting and penetration.

PubMed

Jeong, Jae Young; Hong, Eun-Hye; Lee, Song Yi; Lee, Jae-Young; Song, Jae-Hyoung; Ko, Seung-Hak; Shim, Jae-Seong; Choe, Sunghwa; Kim, Dae-Duk; Ko, Hyun-Jeong; Cho, Hyun-Jong

2017-04-15

(3-Aminomethylphenyl)boronic acid (AMPB)-installed hyaluronic acid-ceramide (HACE)-based nanoparticles (NPs), including manassantin B (MB), were fabricated for tumor-targeted delivery. The amine group of AMPB was conjugated to the carboxylic acid group of hyaluronic acid (HA) via amide bond formation, and synthesis was confirmed by spectroscopic methods. HACE-AMPB/MB NPs with a 239-nm mean diameter, narrow size distribution, negative zeta potential, and >90% drug encapsulation efficiency were fabricated. Exposed AMPB in the outer surface of HACE-AMPB NPs (in the aqueous environment) may react with sialic acid of cancer cells. The improved cellular accumulation efficiency, in vitro antitumor efficacy, and tumor penetration efficiency of HACE-AMPB/MB NPs, compared with HACE/MB NPs, in MDA-MB-231 cells (CD44 receptor-positive human breast adenocarcinoma cells) may be based on the CD44 receptor-mediated endocytosis and phenylboronic acid-sialic acid interaction. Enhanced in vivo tumor targetability, infiltration efficiency, and antitumor efficacies of HACE-AMPB NPs, compared with HACE NPs, were observed in a MDA-MB-231 tumor-xenografted mouse model. In addition to passive tumor targeting (based on an enhanced permeability and retention effect) and active tumor targeting (interaction between HA and CD44 receptor), the phenylboronic acid-sialic acid interaction can play important roles in augmented tumor targeting and penetration of HACE-AMPB NPs. STATEMENT OF SIGNIFICANCE: (3-Aminomethylphenyl)boronic acid (AMPB)-tethered hyaluronic acid-ceramide (HACE)-based nanoparticles (NPs), including manassantin B (MB), were fabricated and their tumor targeting and penetration efficiencies were assessed in MDA-MB-231 (CD44 receptor-positive human adenocarcinoma) tumor models. MB, which exhibited antitumor efficacies via the inhibition of angiogenesis and hypoxia inducible factor (HIF)-1, was entrapped in HACE-AMPB NPs in this study. Phenylboronic acid located in the outer surface

United States Coast Guard Configuration Management Plan (CMP) for the Ports and Waterways Safety System (PAWSS) Project

DOT National Transportation Integrated Search

1997-01-01

Prepared ca. 1997. The Configuration Management Plan (CMP) provides configuration management instructions and guidance for the Vessel Traffic Service (VTS) system of the Ports and Waterways Safety System (PAWSS) project. The CMP describes in detail t...

Polydiacetylene liposomes with phenylboronic acid tags: a fluorescence turn-on sensor for sialic acid detection and cell-surface glycan imaging.

PubMed

Wang, Dong-En; Yan, Jiahang; Jiang, Jingjing; Liu, Xiang; Tian, Chang; Xu, Juan; Yuan, Mao-Sen; Han, Xiang; Wang, Jinyi

2018-03-01

Sialic acid (SA) located at the terminal end of glycans on cell membranes has been shown to play an important yet distinctive role in various biological and pathological processes. Effective methods for the facile, sensitive and in situ analysis of SA on living cell surfaces are of great significance in terms of clinical diagnostics and therapeutics. Here, a new polydiacetylene (PDA) liposome-based sensor system bearing phenylboronic acid (PBA) and 1,8-naphthalimide derived fluorophore moieties was developed as a fluorescence turn-on sensor for the detection of free SA in aqueous solution and the in situ imaging of SA-terminated glycans on living cell surfaces. In the sensor system, three diacetylene monomers, PCDA-pBA, PCDA-Nap and PCDA-EA, were designed and synthesized to construct the composite PDA liposome sensor. The monomer PCDA-pBA modified with PBA molecules was employed as a receptor for SA recognition, while the monomer PCDA-Nap containing a 1,8-naphthalimide derivative fluorophore was used for fluorescence signaling. When the composite PDA liposomes were formed, the energy transfer between the fluorophore and the conjugated backbone could directly quench the fluorescence of the fluorophore. In the presence of additional SA or SA abundant cells, the strong binding of SA with PBA moieties disturbed the pendent side chain conformation, resulting in the fluorescence restoration of the fluorophore. The proposed methods realized the fluorescence turn-on detection of free SA in aqueous solution and the in situ imaging of SA on living MCF-7 cell surfaces. This work provides a new potential tool for simple and selective analysis of SA on living cell membranes.

Crystal structures and biochemical analyses suggest a unique mechanism and role for human glycyl-tRNA synthetase in Ap4A homeostasis.

PubMed

Guo, Rey-Ting; Chong, Yeeting E; Guo, Min; Yang, Xiang-Lei

2009-10-16

Aminoacyl-tRNA synthetases catalyze the attachment of amino acids to their cognate tRNAs for protein synthesis. However, the aminoacylation reaction can be diverted to produce diadenosine tetraphosphate (Ap4A), a universal pleiotropic signaling molecule needed for cell regulation pathways. The only known mechanism for Ap4A production by a tRNA synthetase is through the aminoacylation reaction intermediate aminoacyl-AMP, thus making Ap4A synthesis amino acid-dependent. Here, we demonstrate a new mechanism for Ap4A synthesis. Crystal structures and biochemical analyses show that human glycyl-tRNA synthetase (GlyRS) produces Ap4A by direct condensation of two ATPs, independent of glycine concentration. Interestingly, whereas the first ATP-binding pocket is conserved for all class II tRNA synthetases, the second ATP pocket is formed by an insertion domain that is unique to GlyRS, suggesting that GlyRS is the only tRNA synthetase catalyzing direct Ap4A synthesis. A special role for GlyRS in Ap4A homeostasis is proposed.

Crystal Structures and Biochemical Analyses Suggest a Unique Mechanism and Role for Human Glycyl-tRNA Synthetase in Ap4A Homeostasis*

PubMed Central

Guo, Rey-Ting; Chong, Yeeting E.; Guo, Min; Yang, Xiang-Lei

2009-01-01

Aminoacyl-tRNA synthetases catalyze the attachment of amino acids to their cognate tRNAs for protein synthesis. However, the aminoacylation reaction can be diverted to produce diadenosine tetraphosphate (Ap4A), a universal pleiotropic signaling molecule needed for cell regulation pathways. The only known mechanism for Ap4A production by a tRNA synthetase is through the aminoacylation reaction intermediate aminoacyl-AMP, thus making Ap4A synthesis amino acid-dependent. Here, we demonstrate a new mechanism for Ap4A synthesis. Crystal structures and biochemical analyses show that human glycyl-tRNA synthetase (GlyRS) produces Ap4A by direct condensation of two ATPs, independent of glycine concentration. Interestingly, whereas the first ATP-binding pocket is conserved for all class II tRNA synthetases, the second ATP pocket is formed by an insertion domain that is unique to GlyRS, suggesting that GlyRS is the only tRNA synthetase catalyzing direct Ap4A synthesis. A special role for GlyRS in Ap4A homeostasis is proposed. PMID:19710017

Co-expression of sialic acid receptors compatible with avian and human influenza virus binding in emus (Dromaius novaehollandiae).

PubMed

Gujjar, Naveen; Chothe, Shubhada K; Gawai, Shashikant; Nissly, Ruth; Bhushan, Gitanjali; Kanagaraj, Vijayarani; Jayarao, Bhushan M; Kathaperumal, Kumanan; Subbiah, Madhuri; Kuchipudi, Suresh V

2017-01-01

Influenza A viruses (IAVs) continue to threaten animal and human health with constant emergence of novel variants. While aquatic birds are a major reservoir of most IAVs, the role of other terrestrial birds in the evolution of IAVs is becoming increasingly evident. Since 2006, several reports of IAV isolations from emus have surfaced and avian influenza infection of emus can lead to the selection of mammalian like PB2-E627K and PB2-D701N mutants. However, the potential of emus to be co-infected with avian and mammalian IAVs is not yet understood. As a first step, we investigated sialic acid (SA) receptor distribution across major organs and body systems of emu and found a widespread co-expression of both SAα2,3Gal and SAα2,6Gal receptors in various tissues that are compatible with avian and human IAV binding. Our results suggest that emus could allow genetic recombination and hence play an important role in the evolution of IAVs. Copyright © 2016 Elsevier Inc. All rights reserved.

An acyl-CoA synthetase in Mycobacterium tuberculosis involved in triacylglycerol accumulation during dormancy.

PubMed

Daniel, Jaiyanth; Sirakova, Tatiana; Kolattukudy, Pappachan

2014-01-01

Latent infection with dormant Mycobacterium tuberculosis is one of the major reasons behind the emergence of drug-resistant strains of the pathogen worldwide. In its dormant state, the pathogen accumulates lipid droplets containing triacylglycerol synthesized from fatty acids derived from host lipids. In this study, we show that Rv1206 (FACL6), which is annotated as an acyl-CoA synthetase and resembles eukaryotic fatty acid transport proteins, is able to stimulate fatty acid uptake in E. coli cells. We show that purified FACL6 displays acyl-coenzyme A synthetase activity with a preference towards oleic acid, which is one of the predominant fatty acids in host lipids. Our results indicate that the expression of FACL6 protein in Mycobacterium tuberculosis is significantly increased during in vitro dormancy. The facl6-deficient Mycobacterium tuberculosis mutant displayed a diminished ability to synthesize acyl-coenzyme A in cell-free extracts. Furthermore, during in vitro dormancy, the mutant synthesized lower levels of intracellular triacylglycerol from exogenous fatty acids. Complementation partially restored the lost function. Our results suggest that FACL6 modulates triacylglycerol accumulation as the pathogen enters dormancy by activating fatty acids.

Mutations during the Adaptation of H9N2 Avian Influenza Virus to the Respiratory Epithelium of Pigs Enhance Sialic Acid Binding Activity and Virulence in Mice.

PubMed

Yang, W; Punyadarsaniya, D; Lambertz, R L O; Lee, D C C; Liang, C H; Höper, D; Leist, S R; Hernández-Cáceres, A; Stech, J; Beer, M; Wu, C Y; Wong, C H; Schughart, K; Meng, F; Herrler, G

2017-04-15

The natural reservoir for influenza viruses is waterfowl, and from there they succeeded in crossing the barrier to different mammalian species. We analyzed the adaptation of avian influenza viruses to a mammalian host by passaging an H9N2 strain three times in differentiated swine airway epithelial cells. Using precision-cut slices from the porcine lung to passage the parental virus, isolates from each of the three passages (P1 to P3) were characterized by assessing growth curves and ciliostatic effects. The only difference noted was an increased growth kinetics of the P3 virus. Sequence analysis revealed four mutations: one each in the PB2 and NS1 proteins and two in the HA protein. The HA mutations, A190V and T212I, were characterized by generating recombinant viruses containing either one or both amino acid exchanges. Whereas the parental virus recognized α2,3-linked sialic acids preferentially, the HA190 mutant bound to a broad spectrum of glycans with α2,6/8/9-linked sialic acids. The HA212 mutant alone differed only slightly from the parental virus; however, the combination of both mutations (HA190+HA212) increased the binding affinity to those glycans recognized by the HA190 mutant. Remarkably, only the HA double mutant showed a significantly increased pathogenicity in mice. In contrast, none of those mutations affected the ciliary activity of the epithelial cells which is characteristic for virulent swine influenza viruses. Taken together, our results indicate that shifts in the HA receptor affinity are just an early adaptation step of avian H9N2 strains; further mutational changes may be required to become virulent for pigs. IMPORTANCE Swine play an important role in the interspecies transmission of influenza viruses. Avian influenza A viruses (IAV) of the H9N2 subtype have successfully infected hosts from different species but have not established a stable lineage. We have analyzed the adaptation of IAV-H9N2 virus to target cells of a new host by

Cloning and expression in Escherichia coli of isopenicillin N synthetase genes from Streptomyces lipmanii and Aspergillus nidulans.

PubMed Central

Weigel, B J; Burgett, S G; Chen, V J; Skatrud, P L; Frolik, C A; Queener, S W; Ingolia, T D

1988-01-01

beta-Lactam antibiotics such as penicillins and cephalosporins are synthesized by a wide variety of microbes, including procaryotes and eucaryotes. Isopenicillin N synthetase catalyzes a key reaction in the biosynthetic pathway of penicillins and cephalosporins. The genes encoding this protein have previously been cloned from the filamentous fungi Cephalosporium acremonium and Penicillium chrysogenum and characterized. We have extended our analysis to the isopenicillin N synthetase genes from the fungus Aspergillus nidulans and the gram-positive procaryote Streptomyces lipmanii. The isopenicillin N synthetase genes from these organisms have been cloned and sequenced, and the proteins encoded by the open reading frames were expressed in Escherichia coli. Active isopenicillin N synthetase enzyme was recovered from extracts of E. coli cells prepared from cells containing each of the genes in expression vectors. The four isopenicillin N synthetase genes studied are closely related. Pairwise comparison of the DNA sequences showed between 62.5 and 75.7% identity; comparison of the predicted amino acid sequences showed between 53.9 and 80.6% identity. The close homology of the procaryotic and eucaryotic isopenicillin N synthetase genes suggests horizontal transfer of the genes during evolution. Images PMID:3045077

Aminoacyl-tRNA synthetases database Y2K

PubMed Central

Szymanski, Maciej; Barciszewski, Jan

2000-01-01

The aminoacyl-tRNA synthetases (AARS) are a diverse group of enzymes that ensure the fidelity of transfer of genetic information from DNA into protein. They catalyse the attachment of amino acids to transfer RNAs and thereby establish the rules of the genetic code by virtue of matching the nucleotide triplet of the anticodon with its cognate amino acid. Currently, 818 AARS primary structures have been reported from archaebacteria, eubacteria, mitochondria, chloroplasts and eukaryotic cells. The database is a compilation of the amino acid sequences of all AARSs, known to date, which are available as separate entries or alignments of related proteins via the WWW at http://rose.man.poznan.pl/aars/index.html PMID:10592262

Aminoacyl-tRNA synthetases database Y2K.

PubMed

Szymanski, M; Barciszewski, J

2000-01-01

The aminoacyl-tRNA synthetases (AARS) are a diverse group of enzymes that ensure the fidelity of transfer of genetic information from DNA into protein. They catalyse the attachment of amino acids to transfer RNAs and thereby establish the rules of the genetic code by virtue of matching the nucleotide triplet of the anticodon with its cognate amino acid. Currently, 818 AARS primary structures have been reported from archaebacteria, eubacteria, mitochondria, chloro-plasts and eukaryotic cells. The database is a compilation of the amino acid sequences of all AARSs, known to date, which are available as separate entries or alignments of related proteins via the WWW at http://rose.man.poznan.pl/aars/index.html

Targeting of CD22-positive B-cell lymphoma cells by synthetic divalent sialic acid analogues.

PubMed

Schweizer, Astrid; Wöhner, Miriam; Prescher, Horst; Brossmer, Reinhard; Nitschke, Lars

2012-10-01

CD22 is an inhibitory co-receptor of the B-cell receptor (BCR) on B cells. Since CD22 is ubiquitously expressed in the B-cell lineage and CD22 endocytosis can be triggered efficiently, antibodies and antibody-based immunotoxins against CD22 are used to target B cells both in B-cell lymphomas and leukemias, as well as in autoimmune diseases. CD22 recognizes α2,6-linked sialic acids as endogenous ligands. We have developed new synthetic sialosides as ligands for human CD22. These sialosides bind CD22 on human B cells with high affinity and can efficiently enhance IgM-triggered Ca(2+) signaling. We coupled these sialosides to Pseudomonas exotoxin A to generate a novel CD22 ligand-based immunotoxin. This sialoside-exotoxin-A construct can specifically kill CD22-positive B-cell lymphoma cells. It binds specifically to CD22-positive B-cell lymphoma cells and is dominant over endogenous cis-ligands on the B-cell surface. The sialoside-exotoxin-A construct is efficiently internalized by endocytosis into B-cell lymphoma cell lines. Thus we show the development of a new therapeutic compound for targeting CD22 on human B cells, both for B-cell lymphoma, as well as for B-cell-mediated autoimmune diseases. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Connected Mathematics Project (CMP). What Works Clearinghouse Intervention Report

ERIC Educational Resources Information Center

What Works Clearinghouse, 2010

2010-01-01

The "Connected Mathematics Project" ("CMP") is a mathematics curriculum designed for students in grades 6-8. Each grade level of the curriculum is a full-year program and covers numbers, algebra, geometry/measurement, probability, and statistics. The curriculum uses an investigative approach, and students utilize interactive…

Structural insights into the polyphyletic origins of glycyl tRNA synthetases

DOE PAGES

Valencia-Sánchez, Marco Igor; Rodríguez-Hernández, Annia; Ferreira, Ruben; ...

2016-05-23

Glycyl tRNA synthetase (GlyRS) provides a unique case among class II aminoacyl tRNA synthetases, with two clearly widespread types of enzymes: a dimeric (α 2) species present in some bacteria, archaea, and eukaryotes; and a heterotetrameric form (α 2β 2) present in most bacteria. Although the differences between both types of GlyRS at the anticodon binding domain level are evident, the extent and implications of the variations in the catalytic domain have not been described, and it is unclear whether the mechanism of amino acid recognition is also dissimilar. Here, we show that the α-subunit of the α 2β 2more » GlyRS from the bacterium Aquifex aeolicus is able to perform the first step of the aminoacylation reaction, which involves the activation of the amino acid with ATP. The crystal structure of the α-subunit in the complex with an analog of glycyl adenylate at 2.8 Å resolution presents a conformational arrangement that properly positions the cognate amino acid. This work shows that glycine is recognized by a subset of different residues in the two types of GlyRS. Furthermore, a structural and sequence analysis of class II catalytic domains shows that bacterial GlyRS is closely related to alanyl tRNA synthetase, which led us to define a new subclassification of these ancient enzymes and to propose an evolutionary path of α 2β 2 GlyRS, convergent with α 2 GlyRS and divergent from AlaRS, thus providing a possible explanation for the puzzling existence of two proteins sharing the same fold and function but not a common ancestor.« less

Versatility of acyl-acyl carrier protein synthetases

DOE PAGES

Beld, Joris; Finzel, Kara; Burkart, Michael D.

2014-10-09

The acyl carrier protein (ACP) requires posttranslational modification with a 4'-phosphopantetheine arm for activity, and this thiol-terminated modification carries cargo between enzymes in ACP-dependent metabolic pathways. In this paper, we show that acyl-ACP synthetases (AasSs) from different organisms are able to load even, odd, and unnatural fatty acids onto E. coli ACP in vitro. Vibrio harveyi AasS not only shows promiscuity for the acid substrate, but also is active upon various alternate carrier proteins. AasS activity also extends to functional activation in living organisms. We show that exogenously supplied carboxylic acids are loaded onto ACP and extended by the E.more » coli fatty acid synthase, including unnatural fatty acid analogs. These analogs are further integrated into cellular lipids. Finally, in vitro characterization of four different adenylate-forming enzymes allowed us to disambiguate CoA-ligases and AasSs, and further in vivo studies show the potential for functional application in other organisms.« less

Protein-inorganic hybrid nanoflowers as ultrasensitive electrochemical cytosensing interfaces for evaluation of cell surface sialic acid.

PubMed

Cao, Hongmei; Yang, Da-Peng; Ye, Daixin; Zhang, Xianxia; Fang, Xueen; Zhang, Song; Liu, Baohong; Kong, Jilie

2015-06-15

The identification of biocompatible nanomaterials with high conductivities as sensing interfaces is important in developing novel electrochemical cytosensors. We prepared a novel protein-inorganic nanomaterial-bovine serum albumin (BSA) incorporated Ag nanoflowers with three-dimensional porous architectures, using a simple biomimetic method. The BSA-incorporated Ag nanoflowers were modified on a glassy carbon electrode (GCE) surface and conjugated with a targeting lectin molecule, i.e., Sambucus nigra agglutinin (SNA), for sensing DLD-1 human colon cancer cells. The BSA-incorporated Ag nanoflowers were a suitable platform, and showed improved cell-immobilization capacity, and good biocompatibility, with retention of activity of the immobilized cells. These properties are attributed to the large surface area of the porous structure and the natural BSA layer acting as a biocompatible support. The attachment of DLD-1 cells to the GCE increased the electron-transfer resistance, with a good correlation with the logarithm of the concentration from 1.35×10(2) to 1.35×10(7) cells mL(-1), with a low detection limit of 40 cells mL(-1). Based on the affinity between SNA and sialic acid (SA), the UV-vis absorption spectrum of the one-step reaction between SA and acidic ninhydrin indicated that the average number of SA molecules on a single living DLD-1 cell surface was approximately 2.16×10(12). This proposed cytosensing strategy had good reproducibility, acceptable precision, and high specificity for SA-over-expressed cells, indicating that it has potential applications for the early monitoring of tumor cells and convenient evaluation of SA on living cells. Copyright © 2015 Elsevier B.V. All rights reserved.

Identification of Bacillus subtilis men mutants which lack O-succinylbenzoyl-coenzyme A synthetase and dihydroxynaphthoate synthase.

PubMed Central

Meganathan, R; Bentley, R; Taber, H

1981-01-01

Menaquinone (vitamin K2)-deficient mutants of Bacillus subtilis, whose growth requirement is satisfied by 1,4-dihydroxy-2-naphthoic acid but not by o-succinylbenzoic acid (OSB), have been analyzed for enzymatic defects. Complementation analysis of cell-free extracts of the mutants revealed that there are two groups, as already indicated by genetic analysis. The missing enzyme in each group was identified by complementation of the cell-free extracts with o-succinylbenzoyl-coenzyme A (CoA) synthetase and dihydroxynaphthoate synthase extracted from Mycobacterium phlei. Mutants found to lack dihydroxynaphthoate synthase, and which therefore complement with dihydroxynaphthoate synthase of M. phlei, were designated as menB; those lacking o-succinylbenzoyl-CoA synthetase, and therefore complementing with o-succinylbenzoyl-CoA synthetase, were designated as menE. The menB mutants RB413 (men-325) and RB415 (men-329), when incubated with [2,3-14C2]OSB, produced only the spirodilactone form of OSB in a reaction that was CoA and adenosine 5'-triphosphate dependent. PMID:6780515

Structure of the prolyl-tRNA synthetase from the eukaryotic pathogen Giardia lamblia

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

Larson, Eric T.; Kim, Jessica E.; Napuli, Alberto J.

2012-09-01

The structure of Giardia prolyl-tRNA synthetase cocrystallized with proline and ATP shows evidence for half-of-the-sites activity, leading to a corresponding mixture of reaction substrates and product (prolyl-AMP) in the two active sites of the dimer. The genome of the human intestinal parasite Giardia lamblia contains only a single aminoacyl-tRNA synthetase gene for each amino acid. The Giardia prolyl-tRNA synthetase gene product was originally misidentified as a dual-specificity Pro/Cys enzyme, in part owing to its unexpectedly high off-target activation of cysteine, but is now believed to be a normal representative of the class of archaeal/eukaryotic prolyl-tRNA synthetases. The 2.2 Å resolutionmore » crystal structure of the G. lamblia enzyme presented here is thus the first structure determination of a prolyl-tRNA synthetase from a eukaryote. The relative occupancies of substrate (proline) and product (prolyl-AMP) in the active site are consistent with half-of-the-sites reactivity, as is the observed biphasic thermal denaturation curve for the protein in the presence of proline and MgATP. However, no corresponding induced asymmetry is evident in the structure of the protein. No thermal stabilization is observed in the presence of cysteine and ATP. The implied low affinity for the off-target activation product cysteinyl-AMP suggests that translational fidelity in Giardia is aided by the rapid release of misactivated cysteine.« less

Mineral nitrogen sources differently affect root glutamine synthetase isoforms and amino acid balance among organs in maize.

PubMed

Prinsi, Bhakti; Espen, Luca

2015-04-03

Glutamine synthetase (GS) catalyzes the first step of nitrogen assimilation in plant cell. The main GS are classified as cytosolic GS1 and plastidial GS2, of which the functionality is variable according to the nitrogen sources, organs and developmental stages. In maize (Zea mays L.) one gene for GS2 and five genes for GS1 subunits are known, but their roles in root metabolism are not yet well defined. In this work, proteomic and biochemical approaches have been used to study root GS enzymes and nitrogen assimilation in maize plants re-supplied with nitrate, ammonium or both. The plant metabolic status highlighted the relevance of root system in maize nitrogen assimilation during both nitrate and ammonium nutrition. The analysis of root proteomes allowed a study to be made of the accumulation and phosphorylation of six GS proteins. Three forms of GS2 were identified, among which only the phosphorylated one showed an accumulation trend consistent with plastidial GS activity. Nitrogen availabilities enabled increments in root total GS synthetase activity, associated with different GS1 isoforms according to the nitrogen sources. Nitrate nutrition induced the specific accumulation of GS1-5 while ammonium led to up-accumulation of both GS1-1 and GS1-5, highlighting co-participation. Moreover, the changes in thermal sensitivity of root GS transferase activity suggested differential rearrangements of the native enzyme. The amino acid accumulation and composition in roots, xylem sap and leaves deeply changed in response to mineral sources. Glutamine showed the prevalent changes in all nitrogen nutritions. Besides, the ammonium nutrition was associated with an accumulation of asparagine and reducing sugars and a drop in glutamic acid level, significantly alleviated by the co-provision with nitrate. This work provides new information about the multifaceted regulation of the GS enzyme in maize roots, indicating the involvement of specific isoenzymes/isoforms, post

Genetic engineering of CHO cells for viral resistance to minute virus of mice.

PubMed

Mascarenhas, Joaquina X; Korokhov, Nikolay; Burger, Lisa; Kassim, Ademola; Tuter, Jason; Miller, Daniel; Borgschulte, Trissa; George, Henry J; Chang, Audrey; Pintel, David J; Onions, David; Kayser, Kevin J

2017-03-01

Contamination by the parvovirus minute virus of mice (MVM) remains a challenge in Chinese hamster ovary (CHO) biopharmaceutical production processes. Although infrequent, infection of a bioreactor can be catastrophic for a manufacturer, can impact patient drug supply and safety, and can have regulatory implications. We evaluated engineering a CHO parental cell line (CHOZN ® GS -/- ) to create a new host cell line that is resistant to MVM infection by modifying the major receptors used by the virus to enter cells. Attachment to a cell surface receptor is a key first step in the infection cycle for many viruses. While the exact functional receptor for MVM binding to CHO cell surface is unknown, sialic acid on the cell surface has been implicated. In this work, we used the zinc finger nuclease gene editing technology to validate the role of sialic acid on the cell surface in the binding and internalization of the MVM virus. Our approach was to systematically mutate genes involved in cell surface sialylation and then challenge each cell line for their ability to resist viral entry and propagation. To test the importance of sialylation, the following genes were knocked out: the CMP-sialic acid transporter, solute carrier family 35A1 (Slc35a1), the core 1-β-1,3-galactosyltransferase-1 specific chaperone (Cosmc), and mannosyl (α-1,3-)-glycoprotein β-1,2-N-acetylglucosaminyltransferase (Mgat1) as well as members of the sialyltransferase family. Slc35a1 is responsible for transporting sialic acid into the Golgi. Knocking out function of this gene in a cell results in asialylated glycan structures, thus eliminating the ability of MVM to bind to and enter the cell. The complete absence of sialic acid on the Slc35a1 knockout cell line led to complete resistance to MVM infection. The Cosmc and Mgat1 knockouts also show significant inhibition of infection likely due to their effect on decreasing cell surface sialic acid. Previously in vitro glycan analysis has been used to

Structural Insights into the Polyphyletic Origins of Glycyl tRNA Synthetases*♦

PubMed Central

Valencia-Sánchez, Marco Igor; Rodríguez-Hernández, Annia; Ferreira, Ruben; Santamaría-Suárez, Hugo Aníbal; Arciniega, Marcelino; Dock-Bregeon, Anne-Catherine; Moras, Dino; Beinsteiner, Brice; Brieba, Luis G.; Grøtli, Morten

2016-01-01

Glycyl tRNA synthetase (GlyRS) provides a unique case among class II aminoacyl tRNA synthetases, with two clearly widespread types of enzymes: a dimeric (α2) species present in some bacteria, archaea, and eukaryotes; and a heterotetrameric form (α2β2) present in most bacteria. Although the differences between both types of GlyRS at the anticodon binding domain level are evident, the extent and implications of the variations in the catalytic domain have not been described, and it is unclear whether the mechanism of amino acid recognition is also dissimilar. Here, we show that the α-subunit of the α2β2 GlyRS from the bacterium Aquifex aeolicus is able to perform the first step of the aminoacylation reaction, which involves the activation of the amino acid with ATP. The crystal structure of the α-subunit in the complex with an analog of glycyl adenylate at 2.8 Å resolution presents a conformational arrangement that properly positions the cognate amino acid. This work shows that glycine is recognized by a subset of different residues in the two types of GlyRS. A structural and sequence analysis of class II catalytic domains shows that bacterial GlyRS is closely related to alanyl tRNA synthetase, which led us to define a new subclassification of these ancient enzymes and to propose an evolutionary path of α2β2 GlyRS, convergent with α2 GlyRS and divergent from AlaRS, thus providing a possible explanation for the puzzling existence of two proteins sharing the same fold and function but not a common ancestor. PMID:27226617

Downregulation of acetyl-CoA synthetase 2 is a metabolic hallmark of tumor progression and aggressiveness in colorectal carcinoma.

PubMed

Bae, Jeong Mo; Kim, Jung Ho; Oh, Hyeon Jeong; Park, Hye Eun; Lee, Tae Hun; Cho, Nam-Yun; Kang, Gyeong Hoon

2017-02-01

Acetyl-CoA synthetase-2 is an emerging key enzyme for cancer metabolism, which supplies acetyl-CoA for tumor cells by capturing acetate as a carbon source under stressed conditions. However, implications of acetyl-CoA synthetase-2 in colorectal carcinoma may differ from other malignancies, because normal colonocytes use short-chain fatty acids as an energy source, which are supplied by fermentation of the intestinal flora. Here we analyzed acetyl-CoA synthetase-2 mRNA expression by reverse-transcription quantitative PCR in paired normal mucosa and tumor tissues of 12 colorectal carcinomas, and subsequently evaluated acetyl-CoA synthetase-2 protein expression by immunohistochemistry in 157 premalignant colorectal lesions, including 60 conventional adenomas and 97 serrated polyps, 1,106 surgically resected primary colorectal carcinomas, and 23 metastatic colorectal carcinomas in the liver. In reverse-transcription quantitative PCR analysis, acetyl-CoA synthetase-2 mRNA expression was significantly decreased in tumor tissues compared with corresponding normal mucosa tissues. In acetyl-CoA synthetase-2 immunohistochemistry analysis, all 157 colorectal polyps showed moderate-to-strong expression of acetyl-CoA synthetase-2. However, cytoplasmic acetyl-CoA synthetase-2 expression was downregulated (acetyl-CoA synthetase-2 low expression) in 771 (69.7%) of 1,106 colorectal carcinomas and 21 (91.3%) of 23 metastatic lesions. The colorectal carcinomas with acetyl-CoA synthetase-2-low expression were significantly associated with advanced TNM stage, poor differentiation, and frequent tumor budding. Regarding the molecular aspect, acetyl-CoA synthetase-2-low expression exhibited a tendency of frequent KRT7 expression and decreased KRT20 and CDX2 expression. In survival analysis, acetyl-CoA synthetase-2-low expression was an independent prognostic factor for poor 5-year progression-free survival (hazard ratio, 1.39; 95% confidence interval, 1.08-1.79; P=0.01). In conclusion

Computational Insights into the High-Fidelity Catalysis of Aminoacyl-tRNA Synthetases

NASA Astrophysics Data System (ADS)

Aboelnga, Mohamed M.

cysteine and valine, respectively. In chapter 8, an assessment QM/MM study using a variety of DFT functionals to represent the chemically active layer in aminoacylation mechanism of the unnatural amino acid ss-Hydroxynorvaline as catalyzed by Threonyl-tRNA synthetase has been carried out. Overall, it was found that substrate-assisted mechanisms are a common pathway for these enzymes. One important application of such information is to establish the criteria required for any candidate to inhibit the catalytic functions of aaRS, which was applied in chapter 9 to screen potential competitive inhibitors able to efficiently block the bacterial Threonyl-tRNA synthetases. The investigations reported herein should provide atomistic details into the fundamental catalytic mechanisms of the ubiquitous and ancient aaRS enzymes. Consequently, they will also help enable a much-needed deeper understanding of the underlying chemical principles of catalysis in general.

Homology of aspartyl- and lysyl-tRNA synthetases.

PubMed Central

Gampel, A; Tzagoloff, A

1989-01-01

The yeast nuclear gene MSD1 coding for mitochondrial aspartyl-tRNA synthetase has been cloned and sequenced. The identity of the gene is confirmed by the following evidence. (i) The primary structure of the protein derived from the gene sequence is similar to that of the yeast cytoplasmic aspartyl-tRNA synthetase. (ii) In situ disruption of MSD1 in a respiratory-competent haploid strain of yeast induces a pleiotropic phenotype consistent with a lesion in mitochondrial protein synthesis. (iii) Mitochondria from a mutant with a disrupted chromosomal copy of MSD1 are unable to acylate mitochondrial aspartyl-tRNA. The primary structures of the cytoplasmic and mitochondrial aspartyl-tRNA synthetases are similar to the yeast cytoplasmic lysyl-tRNA synthetase, suggesting that the two types of synthetases may have a common evolutionary origin. Searches of the current protein banks also have revealed a high degree of sequence similarity of the lysyl-tRNA synthetase to the product of the Escherichia coli herC gene and to the partial sequence of a protein encoded by an unidentified reading frame located adjacent to the E. coli frdA gene. Based on the sequence similarities and the map positions of the herC and frdA loci, we propose herC to be the structural gene of the constitutively expressed lysyl-tRNA synthetase of E. coli and the unidentified reading frame to be the structural gene of the heat-inducible lysyl-tRNA synthetase. Images PMID:2668951

Genetically encoded fluorescent coumarin amino acids

DOEpatents

Wang, Jiangyun; Xie, Jianming; Schultz, Peter G.

2010-10-05

The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetases that can incorporate the coumarin unnatural amino acid L-(7-hydroxycoumarin-4-yl) ethylglycine into proteins produced in eubacterial host cells such as E. coli. The invention provides, for example but not limited to, novel orthogonal synthetases, methods for identifying and making the novel synthetases, methods for producing proteins containing the unnatural amino acid L-(7-hydroxycoumarin-4-yl)ethylglycine and related translation systems.

Genetically encoded fluorescent coumarin amino acids

DOEpatents

Wang, Jiangyun [San Diego, CA; Xie, Jianming [San Diego, CA; Schultz, Peter G [La Jolla, CA

2012-06-05

The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetases that can incorporate the coumarin unnatural amino acid L-(7-hydroxycoumarin-4-yl)ethylglycine into proteins produced in eubacterial host cells such as E. coli. The invention provides, for example but not limited to, novel orthogonal synthetases, methods for identifying and making the novel synthetases, methods for producing proteins containing the unnatural amino acid L-(7-hydroxycoumarin-4-yl)ethylglycine and related translation systems.

Highly sensitive detection of influenza virus by boron-doped diamond electrode terminated with sialic acid-mimic peptide.

PubMed

Matsubara, Teruhiko; Ujie, Michiko; Yamamoto, Takashi; Akahori, Miku; Einaga, Yasuaki; Sato, Toshinori

2016-08-09

The progression of influenza varies according to age and the presence of an underlying disease; appropriate treatment is therefore required to prevent severe disease. Anti-influenza therapy, such as with neuraminidase inhibitors, is effective, but diagnosis at an early phase of infection before viral propagation is critical. Here, we show that several dozen plaque-forming units (pfu) of influenza virus (IFV) can be detected using a boron-doped diamond (BDD) electrode terminated with a sialic acid-mimic peptide. The peptide was used instead of the sialyloligosaccharide receptor, which is the common receptor of influenza A and B viruses required during the early phase of infection, to capture IFV particles. The peptide, which was previously identified by phage-display technology, was immobilized by click chemistry on the BDD electrode, which has excellent electrochemical characteristics such as low background current and weak adsorption of biomolecules. Electrochemical impedance spectroscopy revealed that H1N1 and H3N2 IFVs were detectable in the range of 20-500 pfu by using the peptide-terminated BDD electrode. Our results demonstrate that the BDD device integrated with the receptor-mimic peptide has high sensitivity for detection of a low number of virus particles in the early phase of infection.

Adenylosuccinate synthetase: recent developments.

PubMed

Honzatko, R B; Stayton, M M; Fromm, H J

1999-01-01

By exerting strategic control on purine nucleotide biosynthesis, and by engaging GTP-dependent transphosphorylation of IMP to activate loss of an oxygen atom during catalysis, adenylosuccinate synthetase remains as enzyme that justifiably fascinates students of enzyme catalysis. This review describes how the balanced application of X-ray crystallography and enzyme kinetics has advanced the comprehension of the catalytic and regulatory properties of adenylosuccinate synthetase. Detailed analysis has demonstrated the formation of 6-phosphoryl-IMP, an intermediate originally postulated over 40 years ago on the basis of oxygen-18 exchange experiments showing that position-6 oxygen of IMP becomes incorporated into phosphate. Inferences about the participation of amino acid side-chains that stabilize 6-P-IMP during catalysis have also been confirmed by site-directed mutagenesis and examination of such mutations on various kinetic parameters. Moreover, the action of certain regulatory ligands have also been viewed at atomic level resolution. For example, magnesium ion and GDP can induce conformational changes linked to the stabilization of one of two known conformations of the so-called 40s loop. Another significant finding is that two magnesium ions play fundamental roles: one binding with high affinity to the substrate GTP, and a second binding with lower affinity to the co-substrate aspartate. These structural and kinetic studies have also formed the basis for clarifying the action of various inhibitors and potentially important pharmacologic agents with this key regulatory enzyme. Finally, this review explores the current status of investigations on gene structure and gene expression in a number of organisms.

Overexpression of Human Fatty Acid Transport Protein 2/Very Long Chain Acyl-CoA Synthetase 1 (FATP2/Acsvl1) Reveals Distinct Patterns of Trafficking of Exogenous Fatty Acids

PubMed Central

Melton, Elaina M.; Cerny, Ronald L.; DiRusso, Concetta C.; Black, Paul N.

2014-01-01

In mammals, the fatty acid transport proteins (FATP1 through FATP6) are members of a highly conserved family of proteins, which function in fatty acid transport proceeding through vectorial acylation and in the activation of very long chain fatty acids, branched chain fatty acids and secondary bile acids. FATP1, 2 and 4, for example directly function in fatty acid transport and very long chain fatty acids activation while FATP5 does not function in fatty acid transport but activates secondary bile acids. In the present work, we have used stable isotopically labeled fatty acids differing in carbon length and saturation in cells expressing FATP2 to gain further insights into how this protein functions in fatty acid transport and intracellular fatty acid trafficking. Our previous studies showed the expression of FATP2 modestly increased C16:0-CoA and C20:4-CoA and significantly increased C18:3-CoA and C22:6-CoA after 4hr. The increases in C16:0-CoA and C18:3-CoA suggest FATP2 must necessarily partner with a long chain acyl CoA synthetase (Acsl) to generate C16:0-CoA and C18:3-CoA through vectorial acylation. The very long chain acyl CoA synthetase activity of FATP2 is consistent in the generation of C20:4-CoA and C22:6-CoA coincident with transport from their respective exogenous fatty acids. The trafficking of exogenous fatty acids into phosphatidic acid (PA) and into the major classes of phospholipids (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidyserine (PS)) resulted in distinctive profiles, which changed with the expression of FATP2. The trafficking of exogenous C16:0 and C22:6 into PA was significant where there was 6.9- and 5.3-fold increased incorporation, respectively, over the control; C18:3 and C20:4 also trended to increase in the PA pool while there were no changes for C18:1 and C18:2. The trafficking of C18:3 into PC and PI trended higher and approached significance. In the case of C20:4, expression of

Overexpression of human fatty acid transport protein 2/very long chain acyl-CoA synthetase 1 (FATP2/Acsvl1) reveals distinct patterns of trafficking of exogenous fatty acids.

PubMed

Melton, Elaina M; Cerny, Ronald L; DiRusso, Concetta C; Black, Paul N

2013-11-01

In mammals, the fatty acid transport proteins (FATP1 through FATP6) are members of a highly conserved family of proteins, which function in fatty acid transport proceeding through vectorial acylation and in the activation of very long chain fatty acids, branched chain fatty acids and secondary bile acids. FATP1, 2 and 4, for example directly function in fatty acid transport and very long chain fatty acids activation while FATP5 does not function in fatty acid transport but activates secondary bile acids. In the present work, we have used stable isotopically labeled fatty acids differing in carbon length and saturation in cells expressing FATP2 to gain further insights into how this protein functions in fatty acid transport and intracellular fatty acid trafficking. Our previous studies showed the expression of FATP2 modestly increased C16:0-CoA and C20:4-CoA and significantly increased C18:3-CoA and C22:6-CoA after 4h. The increases in C16:0-CoA and C18:3-CoA suggest FATP2 must necessarily partner with a long chain acyl CoA synthetase (Acsl) to generate C16:0-CoA and C18:3-CoA through vectorial acylation. The very long chain acyl CoA synthetase activity of FATP2 is consistent in the generation of C20:4-CoA and C22:6-CoA coincident with transport from their respective exogenous fatty acids. The trafficking of exogenous fatty acids into phosphatidic acid (PA) and into the major classes of phospholipids (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidyserine (PS)) resulted in distinctive profiles, which changed with the expression of FATP2. The trafficking of exogenous C16:0 and C22:6 into PA was significant where there was 6.9- and 5.3-fold increased incorporation, respectively, over the control; C18:3 and C20:4 also trended to increase in the PA pool while there were no changes for C18:1 and C18:2. The trafficking of C18:3 into PC and PI trended higher and approached significance. In the case of C20:4, expression of

Effect of replacing the aspartic acid/glutamic acid residues of bullfrog sialic acid binding lectin with asparagine/glutamine and arginine on the inhibition of cell proliferation in murine leukemia P388 cells.

PubMed

Ogawa, Yuko; Iwama, Masanori; Ohgi, Kazuko; Tsuji, Tsutomu; Irie, Masachika; Itagaki, Tadashi; Kobayashi, Hiroko; Inokuchi, Norio

2002-06-01

The sialic acid binding lectin from bullfrog oocytes (cSBL) is known to have anti-tumor activity. In a previous report, to elucidate the relationship between the net charge and anti-tumor activity of cSBL, we examined the effect of chemical modifications of cSBL with a water-soluble carbodiimide in the presence of various nucleophiles. The results suggested that the anti-tumor activity and internalization into tumor cells increased with an increase in the net charge of cSBL. However, in the chemically modified cSBL, a modification site was observed on average in two of the carboxyl groups of cSBL. To confirm these previous results and to determine which modified carboxyl group contributes to the increase in anti-tumor activity, we prepared mutants with substitutions of Asn/Gln and Arg at three acidic amino acid residues of cSBL and studied their anti-tumor activity and internalization efficiency. The results showed the enhancing effect of charge on anti-tumor activity and internalization, and suggested that the replacement of D24 and E88 of cSBL with arginine is more effective than that of E97. The double mutant D24RE88R showed comparable anti-tumor activity to the ethylenediamine-modified cSBL reported previously. The mutant was well-characterized as a pure cSBL derivative suitable for studying the mechanism of the anti-tumor action of cSBL.

Function of membranous lysyl-tRNA synthetase and its implication for tumorigenesis.

PubMed

Young, Ho Jeon; Lee, Jung Weon; Kim, Sunghoon

2016-12-01

Aminoacyl-tRNA synthetases (ARSs) are essential enzymes that conjugate specific amino acids to their cognate tRNAs for protein synthesis. Besides their catalytic activity, recent studies have uncovered many additional functions of these enzymes through their interactions with diverse cellular factors. Among human ARSs, cytosolic lysyl-tRNA synthetase (KRS) is often highly expressed in cancer cells and tissues, and facilitates cancer cell migration and invasion through the interaction with the 67kDa laminin receptor on the plasma membrane. Specific modulation of this interaction by small molecule inhibitors has revealed a new way to control metastasis. Here, we summarize the pro-metastatic functions of KRS and their patho-physiological implications. Copyright © 2016 Elsevier B.V. All rights reserved.

Connected Mathematics Project (CMP). What Works Clearinghouse Intervention Report. Updated

ERIC Educational Resources Information Center

What Works Clearinghouse, 2017

2017-01-01

"Connected Mathematics Project" (CMP) is a math curriculum for students in grades 6-8. It uses interactive problems and everyday situations to explore mathematical ideas, with a goal of fostering a problem-centered, inquiry-based learning environment. At each grade level, the curriculum covers numbers, algebra, geometry/measurement,…

Mis-Regulation of 3-Deoxy-d-Arabino-Heptulosonate 7-Phosphate Synthetase Does Not Account for Growth Inhibition by Phenylalanine in Agmenellum quadruplicatum

PubMed Central

Jensen, Roy A.; Stenmark-Cox, S.; Ingram, Lonnie O.

1974-01-01

The growth of the blue-green bacterium, Agmenellum quadruplicatum, is inhibited in the presence of l-phenylalanine. This species has a single, constitutively synthesized 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthetase. l-Phenylalanine inhibits DAHP synthetase non-competitively with respect to both substrate reactants. Other aromatic amino acids do not inhibit the activity of DAHP synthetase. A common expectation for branch-point enzymes such as DAHP synthetase is a balanced pattern of feedback control by all of the ultimate end products. It seemed likely that growth inhibition might equate with defective regulation within the branched aromatic pathway. Accordingly, the possibility was examined that mis-regulation of DAHP synthetase by l-phenylalanine in wild-type cells causes starvation for precursors of the other aromatic end products. However, the molecular basis for growth inhibition cannot be attributed to l-phenylalanine inhibition of DAHP synthetase for the following reasons: (i) DAHP synthetase enzymes from l-phenylalanine-resistant mutants are more, rather than less, sensitive to feedback inhibition by l-phenylalanine. (ii) Shikimate not only fails to antagonize inhibition, but is itself inhibitory. (iii) Neither the sensitivity nor the completeness of l-phenylalanine inhibition of the wild-type enzyme in vitro appears sufficient to account for the potent inhibition of growth in vivo by l-phenylalanine. The dominating effect of l-phenylalanine in the control of DAHP synthetase appears to reflect a mechanism that prevents rather than causes growth inhibition by l-phenylalanine. The alteration of the control of DAHP synthetase in mutants selected for resistance to growth inhibition by l-phenylalanine did indicate that the cause for this metabolite vulnerability can be localized within the aromatic amino acid pathway. Apparently, an aromatic intermediate (between shikimate and the end products) accumulates in the presence of l

Unnatural reactive amino acid genetic code additions

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

Deiters, Alexander; Cropp, T. Ashton; Chin, Jason W.

This invention provides compositions and methods for producing translational components that expand the number of genetically encoded amino acids in eukaryotic cells. The components include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, orthogonal pairs of tRNAs/synthetases and unnatural amino acids. Proteins and methods of producing proteins with unnatural amino acids in eukaryotic cells are also provided.

Unnatural reactive amino acid genetic code additions

DOEpatents

Deiters, Alexander; Cropp, Ashton T; Chin, Jason W; Anderson, Christopher J; Schultz, Peter G

2013-05-21

This invention provides compositions and methods for producing translational components that expand the number of genetically encoded amino acids in eukaryotic cells. The components include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, pairs of tRNAs/synthetases and unnatural amino acids. Proteins and methods of producing proteins with unnatural amino acids in eukaryotic cells are also provided.

Unnatural reactive amino acid genetic code additions

DOEpatents

Deiters, Alexander [La Jolla, CA; Cropp, T Ashton [San Diego, CA; Chin, Jason W [Cambridge, GB; Anderson, J Christopher [San Francisco, CA; Schultz, Peter G [La Jolla, CA

2011-02-15

This invention provides compositions and methods for producing translational components that expand the number of genetically encoded amino acids in eukaryotic cells. The components include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, orthogonal pairs of tRNAs/synthetases and unnatural amino acids. Proteins and methods of producing proteins with unnatural amino acids in eukaryotic cells are also provided.

Unnatural reactive amino acid genetic code additions

DOEpatents

Deiters, Alexander; Cropp, T. Ashton; Chin, Jason W.; Anderson, J. Christopher; Schultz, Peter G.

2014-08-26

This invention provides compositions and methods for producing translational components that expand the number of genetically encoded amino acids in eukaryotic cells. The components include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, orthogonal pairs of tRNAs/synthetases and unnatural amino acids. Proteins and methods of producing proteins with unnatural amino acids in eukaryotic cells are also provided.

Influence of endogenous pyrogen on the cerebral prostaglandin-synthetase system.

PubMed

Ziel, R; Krupp, P

1976-11-15

The biotransformation of arachidonic acid to prostaglandins in vitro is specifically augmented by endogenous pyrogen to a degree depending on the concentration applied, providing that the microsomal fraction of the cerebral cortex is used as prostaglandin-synthetase system. This effect is inhibited by non-steroidal anti-inflammatory agents. These findings are compatible with the hypothesis that prostaglandins might act as mediators of the febrile reaction induced by endogenous pyrogen.

Glutathione synthetase deficiency: a family report.

PubMed Central

Pejaver, R K; Watson, A H

1994-01-01

Glutathione synthetase deficiency is a rare inborn error of metabolism. Low levels of and at times unstable molecules of glutathione synthetase leads to glutathione deficiency affecting various systems of the body. The inheritance is thought to be of autosomal recessive variety. We diagnosed the condition in a neonate and proceeded to investigate the family. The results are discussed below. PMID:8158601

Pharmacological studies on the TXA2 synthetase inhibitor (E)-3-[p-(1H-imidazol-1-ylmethyl)phenyl]-2-propenoic acid (OKY-046).

PubMed

Hiraku, S; Taniguchi, K; Wakitani, K; Omawari, N; Kira, H; Miyamoto, T; Okegawa, T; Kawasaki, A; Ujiie, A

1986-07-01

The effects of (E)-3-[p-(1H-imidazol-1-ylmethyl)phenyl]-2-propenoic acid (OKY-046) on thromboxane A2 (TXA2) synthetase in vitro and on experimental animal models of sudden death and cerebral infarction were studied. IC50 values of OKY-046 for the TXA2 synthetase of human, rabbit, dog and guinea pig washed platelets were 0.004, 0.004, 0.26 and 2.4 microM, respectively. OKY-046 at concentrations up to 1 mM, however, did not inhibit prostacyclin (PGI2) synthetase from bovine aorta microsomes or cyclooxygenase and PGE2 isomerase from sheep seminal vesicle microsomes. Similarly, platelet 12-lipoxygenase was not affected by OKY-046. Evidence for a re-direction of arachidonate metabolism from thromboxane synthesis toward PGI2 synthesis was obtained using rat peritoneal cells. Namely, OKY-046 increased PGI2 production accompanied by an inhibition of TXA2 production at a concentration of more than 1 microM. OKY-046 at a dose of 0.1 mg/kg (i.v.) in dogs inhibited the aortic and mesenteric arterial contraction of rabbit induced by the addition of arachidonate to extracorporated blood of the dogs. OKY-046 at a dose of 0.3 mg/kg (i.v.) prevented the arachidonate-induced sudden death and also decreased the incidence of cerebral infarction induced by injection of arachidonate into the internal carotid artery in rabbits. Aspirin also decreased the incidence of cerebral infarction at a dose of 30 mg/kg (i.v.). These results suggest that OKY-046 may be valuable for the treatment of cerebrovascular and cardiovascular diseases associated with vasoconstriction and thrombosis due to TXA2.

Aminoacyl-tRNA synthetases as drug targets in eukaryotic parasites☆

PubMed Central

Pham, James S.; Dawson, Karen L.; Jackson, Katherine E.; Lim, Erin E.; Pasaje, Charisse Flerida A.; Turner, Kelsey E.C.; Ralph, Stuart A.

2013-01-01

Aminoacyl-tRNA synthetases are central enzymes in protein translation, providing the charged tRNAs needed for appropriate construction of peptide chains. These enzymes have long been pursued as drug targets in bacteria and fungi, but the past decade has seen considerable research on aminoacyl-tRNA synthetases in eukaryotic parasites. Existing inhibitors of bacterial tRNA synthetases have been adapted for parasite use, novel inhibitors have been developed against parasite enzymes, and tRNA synthetases have been identified as the targets for compounds in use or development as antiparasitic drugs. Crystal structures have now been solved for many parasite tRNA synthetases, and opportunities for selective inhibition are becoming apparent. For different biological reasons, tRNA synthetases appear to be promising drug targets against parasites as diverse as Plasmodium (causative agent of malaria), Brugia (causative agent of lymphatic filariasis), and Trypanosoma (causative agents of Chagas disease and human African trypanosomiasis). Here we review recent developments in drug discovery and target characterisation for parasite aminoacyl-tRNA synthetases. PMID:24596663

Salivary Lipid Peroxidation and Total Sialic Acid Levels in Smokers and Smokeless Tobacco Users as Maraş Powder

PubMed Central

Kurtul, Naciye; Gökpınar, Engin

2012-01-01

Maraş powder (MP), a different type of smokeless tobacco (ST) and prepared from a tobacco of species Nicotiana rustica Linn, is widely used in Turkey. We aimed to investigate the effects of MP on salivary total sialic acid (TSA) and malondialdehyde (MDA) levels and to compare these parameters in smokers and MP users (MPUs). The salivary TSA and MDA concentrations were significantly higher in the smokers and MPU than those of control subjects and also in MPU than that of smokers. We have also observed that as the number of cigarettes consumed and MP amount increases, TSA and MDA levels increase too. In smokers, MDA values were significantly correlated with the number of cigarettes smoked and the duration of smoking. In MPU, both MDA and TSA levels were significantly correlated with the duration of MP use and the amount of daily consumed MP. We have concluded increased salivary TSA and MDA levels associated in MPU and smokers. Results can help to evaluate harmful effects of these habits. It is important to point out that bigger change in the measured parameters has been observed for MP use. This observation may be an important indication of harmful effects of ST use as MP. PMID:22577253

Inhibition of recombinant Pneumocystis carinii dihydropteroate synthetase by sulfa drugs.

PubMed Central

Hong, Y L; Hossler, P A; Calhoun, D H; Meshnick, S R

1995-01-01

Forty-four sulfa drugs were screened against crude preparations of recombinant Pneumocystis carinii dihydropteroate synthetase. The apparent Michaelis-Menten constants (Km) for p-aminobenzoic acid and 7,8-dihydro-6-hydroxymethylpterin pyrophosphate were 0.34 +/- 0.02 and 2.50 +/- 0.71 microM, respectively. Several sulfa drugs, including sulfathiazole, sulfachlorpyridazine, sulfamethoxypyridazine, and sulfathiourea, inhibited dihydropteroate synthetase approximately as well as sulfamethoxazole, as determined by the concentrations which cause 50% inhibition and/or by Ki. For all sulfones and sulfonamides tested, unsubstituted p-amino groups were necessary for activity, and sulfonamides containing an N1-heterocyclic substituent were found to be the most effective inhibitors. Folate biosynthesis in isolated intact P. carinii was approximately equally sensitive to inhibition by sulfamethoxazole, sulfachlorpyridazine, sulfamethoxypyridazine, sulfisoxazole, and sulfathiazole. Two of these drugs, sulfamethoxypyridazine and sulfisoxazole, are known to be less toxic than sulfamethoxazole and should be further evaluated for the treatment of P. carinii pneumonia. PMID:7486915

Medium-chain fatty acid synthesis in lactating-rabbit mammary gland. Intracellular concentration and specificity of medium-chain acyl thioester hydrolase.

PubMed Central

Knudsen, J

1979-01-01

The concentration of medium-chain acyl thioester hydrolase and of fatty acid synthetase was determined by rocket immunoelectrophoresis in nine different particle-free supernatant fractions from lactating-rabbit mammary gland. The molar ratio of the hydrolase to fatty acid synthetase was 1.99 +/- 0.66 (mean +/- S.D.). A rate-limiting concentration of malonyl-CoA was required to ensure the predominant synthesis of medium-chain fatty acids when 2 mol of the hydrolase was added per mol of fatty acid synthetase. The interaction of the hydrolase with fatty acid synthetase was concentration-dependent, though an optimum concentration of hydrolase to synthetase could not be obtained. The lactating-rabbit mammary gland hydrolase altered the pattern of fatty acids synthesized by fatty acid synthetases prepared from cow, goat, sheep and rabbit lactating mammary glands, rabbit liver and cow adipose tissue. PMID:574008

Immunocytochemical localization of glutamic acid decarboxylase (GAD) and glutamine synthetase (GS) in the area postrema of the cat. Light and electron microscopy

NASA Technical Reports Server (NTRS)

D'Amelio, Fernando E.; Mehler, William R.; Gibbs, Michael A.; Eng, Lawrence F.; Wu, Jang-Yen

1987-01-01

Morphological evidence is presented of the existence of the putative neurotransmitter gamma-aminobutyric acid (GABA) in axon terminals and of glutamine synthetase (GS) in ependymoglial cells and astroglial components of the area postrema (AP) of the cat. Purified antiserum directed against the GABA biosynthetic enzyme glutamic acid decarboxylase (GAD) and GS antiserum were used. The results showed that punctate structures of variable size corresponding to axon terminals exhibited GAD-immunoreactivity and were distributed in varying densities. The greatest accumulation occurred in the caudal and middle segment of the AP and particularly in the area subpostrema, where the aggregation of terminals was extremely dense. The presence of both GAD-immunoreactive profiles and GS-immunostained ependymoglial cells and astrocytes in the AP provide further evidence of the functional correlation between the two enzymes.

Propylisopropylacetic acid (PIA), a constitutional isomer of valproic acid, uncompetitively inhibits arachidonic acid acylation by rat acyl-CoA synthetase 4: a potential drug for bipolar disorder

PubMed Central

Modi, Hiren R.; Basselin, Mireille; Taha, Ameer Y.; Li, Lei O.; Coleman, Rosalind A.; Bialer, Meir; Rapoport, Stanley I.

2013-01-01

Background Mood stabilizers used for treating bipolar disorder (BD) selectively downregulate arachidonic acid (AA) turnover (deacylation-reacylation) in brain phospholipids, when given chronically to rats. In vitro studies suggest that one of these, valproic acid (VPA), which is teratogenic, reduces AA turnover by inhibiting the brain acyl-CoA synthetase (Acsl)-4 mediated acylation of AA to AA-CoA. We tested whether non-teratogenic VPA analogues might also inhibit Acsl-4 catalyzed acylation, and thus have potential anti-BD action. Methods Rat Acsl4-flag protein was expressed in E. coli, and the ability of three VPA analogues, propylisopropylacetic acid (PIA), propylisopropylacetamide (PID) and N-methyl-2,2,3,3-tetramethylcyclopropanecarboxamide (MTMCD), and of sodium butyrate, to inhibit conversion of AA to AA-CoA by Acsl4 was quantified using Michaelis-Menten kinetics. Results Acsl4-mediated conversion of AA to AA-CoA in vitro was inhibited uncompetitively by PIA, with a Ki of 11.4 mM compared to a published Ki of 25 mM for VPA, while PID, MTMCD and sodium butyrate had no inhibitory effect. Conclusions PIA's ability to inhibit conversion of AA to AA-CoA by Acsl4 in vitro suggests that, like VPA, PIA may reduce AA turnover in brain phospholipids in unanesthetized rats, and if so, may be effective as a non-teratogenic mood stabilizer in BD patients. PMID:23354024

Performance of colloidal silica and ceria based slurries on CMP of Si-face 6H-SiC substrates

NASA Astrophysics Data System (ADS)

Chen, Guomei; Ni, Zifeng; Xu, Laijun; Li, Qingzhong; Zhao, Yongwu

2015-12-01

Colloidal silica and ceria based slurries, both using KMnO4 as an oxidizer, for chemical mechanical polishing (CMP) of Si-face (0 0 0 1) 6H-SiC substrate, were investigated to obtain higher material removal rate (MRR) and ultra-smooth surface. The results indicate that there was a significant difference in the CMP performance of 6H-SiC between silica and ceria based slurries. For the ceria based slurries, a higher MRR was obtained, especially in strong acid KMnO4 environment, and the maximum MRR (1089 nm/h) and a smoother surface with an average roughness Ra of 0.11 nm was achieved using slurries containing 2 wt% colloidal ceria, 0.05 M KMnO4 at pH 2. In contrast, due to the attraction between negative charged silica particles and positive charged SiC surface below pH 5, the maximum MRR of silica based slurry was only 185 nm/h with surface roughness Ra of 0.254 nm using slurries containing 6 wt% colloidal silica, 0.05 M KMnO4 at pH 6. The polishing mechanism was discussed based on the zeta potential measurements of the abrasives and the X-ray photoelectron spectroscopy (XPS) analysis of the polished SiC surfaces.

A Designed Angiopoietin-1 Variant, Dimeric CMP-Ang1 Activates Tie2 and Stimulates Angiogenesis and Vascular Stabilization in N-glycan Dependent Manner

PubMed Central

Oh, Nuri; Kim, Kangsan; Jin Kim, Soo; Park, Intae; Lee, Jung-Eun; Suk Seo, Young; Joo An, Hyun; Min Kim, Ho; Young Koh, Gou

2015-01-01

Angiopoietin-1 (Ang1), a potential growth factor for therapeutic angiogenesis and vascular stabilization, is known to specifically cluster and activate Tie2 in high oligomeric forms, which is a unique and essential process in this ligand-receptor interaction. However, highly oligomeric native Ang1 and Ang1 variants are difficult to produce, purify, and store in a stable and active form. To overcome these limitations, we developed a simple and active dimeric CMP-Ang1 by replacing the N-terminal of native Ang1 with the coiled-coil domain of cartilage matrix protein (CMP) bearing mutations in its cysteine residues. This dimeric CMP-Ang1 effectively increased the migration, survival, and tube formation of endothelial cells via Tie2 activation. Furthermore, dimeric CMP-Ang1 induced angiogenesis and suppressed vascular leakage in vivo. Despite its dimeric structure, the potencies of such Tie2-activation-induced effects were comparable to those of a previously engineered protein, COMP-Ang1. We also revealed that these effects of dimeric CMP-Ang1 were affected by specified N-glycosylation in its fibrinogen-like domain. Taken together, our results indicate that dimeric CMP-Ang1 is capable of activating Tie2 and stimulating angiogenesis in N-glycan dependent manner. PMID:26478188

Abrasive Particle Trajectories and Material Removal Non-Uniformity during CMP and Filtration Characteristics of CMP Slurries - A Simulation and Experimental Study

NASA Astrophysics Data System (ADS)

Rastegar, Vahid

Nanoscale finishing and planarization are integral process steps in multilevel metallization designs for integrated circuit (IC) manufacturing since it is necessary to ensure local and global surface planarization at each metal layer before depositing the next layer. Chemical mechanical planarization (CMP) has been widely recognized as the most promising technology to eliminate topographic variation and has allowed the construction of multilevel interconnection structures with a more regularly stacked sequence, resulting in better device performance [1]. Understanding fundamental of the CMP mechanisms can offer guidance to the control and optimization of the polishing processes. CMP kinematics based on slurry distribution and particle trajectories have a significant impact on MRR profiles. In this work a mathematical model to describe particle trajectories during chemical mechanical polishing was developed and extended to account for the effect of larger particles, particle location changes due to slurry dispensing and in-situ conditioning. Material removal rate (MRR) and within wafer non-uniformity (WIWNU) were determined based on the calculated particle trajectory densities. Rotary dynamics and reciprocating motion were optimized to obtain best MRR uniformity. Edge-fast MRR profile was discussed based on mechanical aspect of CMP. Using the model, we also investigated the effect of variable rotational speeds of wafer and pad, and of large particles on WIWNU and scratch growth. It was shown that the presence of even a small portion of large particles can deteriorate the WIWNU significantly and also lead to more scratches. Furthermore, it was shown that the in-situ conditioning improves the uniformity of the polished wafers. Furthermore, a combined experimental and computational study of fibrous filters for removal of larger abrasive particles from aqueous dispersions, essential to minimize defects during chemical mechanical polishing, was performed. Dilute aqueous

Effect of photocatalytic oxidation technology on GaN CMP

NASA Astrophysics Data System (ADS)

Wang, Jie; Wang, Tongqing; Pan, Guoshun; Lu, Xinchun

2016-01-01

GaN is so hard and so chemically inert that it is difficult to obtain a high material removal rate (MRR) in the chemical mechanical polishing (CMP) process. This paper discusses the application of photocatalytic oxidation technology in GaN planarization. Three N-type semiconductor particles (TiO2, SnO2, and Fe2O3) are used as catalysts and added to the H2O2-SiO2-based slurry. By optical excitation, highly reactive photoinduced holes are produced on the surface of the particles, which can oxidize OH- and H2O absorbed on the surface of the catalysts; therefore, more OH* will be generated. As a result, GaN MRRs in an H2O2-SiO2-based polishing system combined with catalysts are improved significantly, especially when using TiO2, the MRR of which is 122 nm/h. The X-ray photoelectron spectroscopy (XPS) analysis shows the variation trend of chemical composition on the GaN surface after polishing, revealing the planarization process. Besides, the effect of pH on photocatalytic oxidation combined with TiO2 is analyzed deeply. Furthermore, the physical model of GaN CMP combined with photocatalytic oxidation technology is proposed to describe the removal mechanism of GaN.

Structural modeling of tissue-specific mitochondrial alanyl-tRNA synthetase (AARS2) defects predicts differential effects on aminoacylation

PubMed Central

Euro, Liliya; Konovalova, Svetlana; Asin-Cayuela, Jorge; Tulinius, Már; Griffin, Helen; Horvath, Rita; Taylor, Robert W.; Chinnery, Patrick F.; Schara, Ulrike; Thorburn, David R.; Suomalainen, Anu; Chihade, Joseph; Tyynismaa, Henna

2015-01-01

The accuracy of mitochondrial protein synthesis is dependent on the coordinated action of nuclear-encoded mitochondrial aminoacyl-tRNA synthetases (mtARSs) and the mitochondrial DNA-encoded tRNAs. The recent advances in whole-exome sequencing have revealed the importance of the mtARS proteins for mitochondrial pathophysiology since nearly every nuclear gene for mtARS (out of 19) is now recognized as a disease gene for mitochondrial disease. Typically, defects in each mtARS have been identified in one tissue-specific disease, most commonly affecting the brain, or in one syndrome. However, mutations in the AARS2 gene for mitochondrial alanyl-tRNA synthetase (mtAlaRS) have been reported both in patients with infantile-onset cardiomyopathy and in patients with childhood to adulthood-onset leukoencephalopathy. We present here an investigation of the effects of the described mutations on the structure of the synthetase, in an effort to understand the tissue-specific outcomes of the different mutations. The mtAlaRS differs from the other mtARSs because in addition to the aminoacylation domain, it has a conserved editing domain for deacylating tRNAs that have been mischarged with incorrect amino acids. We show that the cardiomyopathy phenotype results from a single allele, causing an amino acid change R592W in the editing domain of AARS2, whereas the leukodystrophy mutations are located in other domains of the synthetase. Nevertheless, our structural analysis predicts that all mutations reduce the aminoacylation activity of the synthetase, because all mtAlaRS domains contribute to tRNA binding for aminoacylation. According to our model, the cardiomyopathy mutations severely compromise aminoacylation whereas partial activity is retained by the mutation combinations found in the leukodystrophy patients. These predictions provide a hypothesis for the molecular basis of the distinct tissue-specific phenotypic outcomes. PMID:25705216

Identification of N-acetylneuraminic acid and its 9-O-acetylated derivative on the cell surface of Cryptococcus neoformans: influence on fungal phagocytosis.

PubMed Central

Rodrigues, M L; Rozental, S; Couceiro, J N; Angluster, J; Alviano, C S; Travassos, L R

1997-01-01

Sialic acids from sialoglycoconjugates present at the cell surface of Cryptococcus neoformans yeast forms were analyzed by high-performance thin-layer chromatography, binding of influenza A and C virus strains, enzymatic treatment, and flow cytofluorimetry with fluorescein isothiocyanate-labeled lectins. C. neoformans yeast forms grown in a chemically defined medium contain N-acetylneuraminic acid and its 9-O-acetylated derivative. A density of 3 x 10(6) residues of sialic acid per cell was found in C. neoformans. Sialic acids in cryptococcal cells are glycosidically linked to galactopyranosyl units as inferred from the increased reactivity of neuraminidase-treated yeasts with peanut agglutinin. N-Acetylneuraminic acids are alpha-2,6 and alpha-2,3 linked, as indicated by using virus strains M1/5 and M1/5 HS8, respectively, as agglutination probes. The alpha-2,6 linkage markedly predominated. These findings were essentially confirmed by the interaction of cryptococcal cells with the lectins Sambucus nigra agglutinin and Maackia amurensis agglutinin. We also investigated whether the sialyl residues present in C. neoformans are involved in the fungal interaction with a cationic solid-phase substrate and with mouse resident macrophages. Adhesion of yeast cells to poly-L-lysine was mediated, in part, by sialic acid residues, since the number of adherent cells was markedly reduced after treatment with bacterial neuraminidase. The enzymatic removal of sialic acids also made C. neoformans yeast cells more susceptible to endocytosis by macrophages. The results show that sialic acids are components of the cryptococcal cell surface that contribute to its negative charge and protect yeast forms against phagocytosis. PMID:9393779

Overexpression of human fatty acid transport protein 2/very long chain acyl-CoA synthetase 1 (FATP2/Acsvl1) reveals distinct patterns of trafficking of exogenous fatty acids

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

Melton, Elaina M.; Center for Cardiovascular Sciences, Albany Medical College, Albany, NY; Cerny, Ronald L.

Highlights: •Roles of FATP2 in fatty acid transport/activation contribute to lipid homeostasis. •Use of 13C- and D-labeled fatty acids provide novel insights into FATP2 function. •FATP2-dependent trafficking of FA into phospholipids results in distinctive profiles. •FATP2 functions in the transport and activation pathways for exogenous fatty acids. -- Abstract: In mammals, the fatty acid transport proteins (FATP1 through FATP6) are members of a highly conserved family of proteins, which function in fatty acid transport proceeding through vectorial acylation and in the activation of very long chain fatty acids, branched chain fatty acids and secondary bile acids. FATP1, 2 and 4,more » for example directly function in fatty acid transport and very long chain fatty acids activation while FATP5 does not function in fatty acid transport but activates secondary bile acids. In the present work, we have used stable isotopically labeled fatty acids differing in carbon length and saturation in cells expressing FATP2 to gain further insights into how this protein functions in fatty acid transport and intracellular fatty acid trafficking. Our previous studies showed the expression of FATP2 modestly increased C16:0-CoA and C20:4-CoA and significantly increased C18:3-CoA and C22:6-CoA after 4 h. The increases in C16:0-CoA and C18:3-CoA suggest FATP2 must necessarily partner with a long chain acyl CoA synthetase (Acsl) to generate C16:0-CoA and C18:3-CoA through vectorial acylation. The very long chain acyl CoA synthetase activity of FATP2 is consistent in the generation of C20:4-CoA and C22:6-CoA coincident with transport from their respective exogenous fatty acids. The trafficking of exogenous fatty acids into phosphatidic acid (PA) and into the major classes of phospholipids (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidyserine (PS)) resulted in distinctive profiles, which changed with the expression of FATP2. The

Regulation of the intersubunit ammonia tunnel in Mycobacterium tuberculosis glutamine-dependent NAD[superscript +] synthetase

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

Chuenchor, Watchalee; Doukov, Tzanko I.; Resto, Melissa

Glutamine-dependent NAD{sup +} synthetase is an essential enzyme and a validated drug target in Mycobacterium tuberculosis (mtuNadE). It catalyses the ATP-dependent formation of NAD{sup +} from NaAD{sup +} (nicotinic acid-adenine dinucleotide) at the synthetase active site and glutamine hydrolysis at the glutaminase active site. An ammonia tunnel 40 {angstrom} (1 {angstrom} = 0.1 nm) long allows transfer of ammonia from one active site to the other. The enzyme displays stringent kinetic synergism; however, its regulatory mechanism is unclear. In the present paper, we report the structures of the inactive glutaminase C176A variant in an apo form and in three synthetase-ligandmore » complexes with substrates (NaAD{sup +}/ATP), substrate analogue {l_brace}NaAD{sup +}/AMP-CPP (adenosine 5'-[{alpha},{beta}-methylene]triphosphate){r_brace} and intermediate analogues (NaAD{sup +}/AMP/PPi), as well as the structure of wild-type mtuNadE in a product complex (NAD{sup +}/AMP/PPi/glutamate). This series of structures provides snapshots of the ammonia tunnel during the catalytic cycle supported also by kinetics and mutagenesis studies. Three major constriction sites are observed in the tunnel: (i) at the entrance near the glutaminase active site; (ii) in the middle of the tunnel; and (iii) at the end near the synthetase active site. Variation in the number and radius of the tunnel constrictions is apparent in the crystal structures and is related to ligand binding at the synthetase domain. These results provide new insight into the regulation of ammonia transport in the intermolecular tunnel of mtuNadE.« less

Chemical-modification studies of a unique sialic acid-binding lectin from the snail Achatina fulica. Involvement of tryptophan and histidine residues in biological activity.

PubMed Central

Basu, S; Mandal, C; Allen, A K

1988-01-01

A unique sialic acid-binding lectin, achatininH (ATNH) was purified in single step from the haemolymph of the snail Achatina fulica by affinity chromatography on sheep submaxillary-gland mucin coupled to Sepharose 4B. The homogeneity was checked by alkaline gel electrophoresis, immunodiffusion and immunoelectrophoresis. Amino acid analysis showed that the lectin has a fairly high content of acidic amino acid residues (22% of the total). About 1.3% of the residues are half-cystine. The glycoprotein contains 21% carbohydrate. The unusually high content of xylose (6%) and fucose (2.7%) in this snail lectin is quite interesting. The protein was subjected to various chemical modifications in order to detect the amino acid residues and carbohydrate residues present in its binding sites. Modification of tyrosine and arginine residues did not affect the binding activity of ATNH; however, modification of tryptophan and histidine residues led to a complete loss of its biological activity. A marked decrease in the fluorescence emission was found as the tryptophan residues of ATNH were modified. The c.d. data showed the presence of an identical type of conformation in the native and modified agglutinin. The modification of lysine and carboxy residues partially diminished the biological activity. The activity was completely lost after a beta-elimination reaction, indicating that the sugars are O-glycosidically linked to the glycoprotein's protein moiety. This result confirms that the carbohydrate moiety also plays an important role in the agglutination property of this lectin. Images Fig. 3. PMID:3140796

Group B Streptococcus Engages an Inhibitory Siglec through Sialic Acid Mimicry to Blunt Innate Immune and Inflammatory Responses In Vivo

PubMed Central

Chang, Yung-Chi; Olson, Joshua; Beasley, Federico C.; Tung, Christine; Zhang, Jiquan; Crocker, Paul R.; Varki, Ajit; Nizet, Victor

2014-01-01

Group B Streptococcus (GBS) is a common agent of bacterial sepsis and meningitis in newborns. The GBS surface capsule contains sialic acids (Sia) that engage Sia-binding immunoglobulin-like lectins (Siglecs) on leukocytes. Here we use mice lacking Siglec-E, an inhibitory Siglec of myelomonocytic cells, to study the significance of GBS Siglec engagement during in vivo infection. We found GBS bound to Siglec-E in a Sia-specific fashion to blunt NF-κB and MAPK activation. As a consequence, Siglec-E-deficient macrophages had enhanced pro-inflammatory cytokine secretion, phagocytosis and bactericidal activity against the pathogen. Following pulmonary or low-dose intravenous GBS challenge, Siglec-E KO mice produced more pro-inflammatory cytokines and exhibited reduced GBS invasion of the central nervous system. In contrast, upon high dose lethal challenges, cytokine storm in Siglec-E KO mice was associated with accelerated mortality. We conclude that GBS Sia mimicry influences host innate immune and inflammatory responses in vivo through engagement of an inhibitory Siglec, with the ultimate outcome of the host response varying depending upon the site, stage and magnitude of infection. PMID:24391502

Three-Dimensional Vibration Isolator for Suppressing High-Frequency Responses for Sage III Contamination Monitoring Package (CMP)

NASA Technical Reports Server (NTRS)

Li, Y.; Cutright, S.; Dyke, R.; Templeton, J.; Gasbarre, J.; Novak, F.

2015-01-01

The Stratospheric Aerosol and Gas Experiment (SAGE) III - International Space Station (ISS) instrument will be used to study ozone, providing global, long-term measurements of key components of the Earth's atmosphere for the continued health of Earth and its inhabitants. SAGE III is launched into orbit in an inverted configuration on SpaceX;s Falcon 9 launch vehicle. As one of its four supporting elements, a Contamination Monitoring Package (CMP) mounted to the top panel of the Interface Adapter Module (IAM) box experiences high-frequency response due to structural coupling between the two structures during the SpaceX launch. These vibrations, which were initially observed in the IAM Engineering Development Unit (EDU) test and later verified through finite element analysis (FEA) for the SpaceX launch loads, may damage the internal electronic cards and the Thermoelectric Quartz Crystal Microbalance (TQCM) sensors mounted on the CMP. Three-dimensional (3D) vibration isolators were required to be inserted between the CMP and IAM interface in order to attenuate the high frequency vibrations without resulting in any major changes to the existing system. Wire rope isolators were proposed as the isolation system between the CMP and IAM due to the low impact to design. Most 3D isolation systems are designed for compression and roll, therefore little dynamic data was available for using wire rope isolators in an inverted or tension configuration. From the isolator FEA and test results, it is shown that by using the 3D wire rope isolators, the CMP high-frequency responses have been suppressed by several orders of magnitude over a wide excitation frequency range. Consequently, the TQCM sensor responses are well below their qualification environments. It is indicated that these high-frequency responses due to the typical instrument structural coupling can be significantly suppressed by a vibration passive control using the 3D vibration isolator. Thermal and contamination

Arabidopsis thaliana GH3.15 acyl acid amido synthetase has a highly specific substrate preference for the auxin precursor indole-3-butyric acid.

PubMed

Sherp, Ashley M; Westfall, Corey S; Alvarez, Sophie; Jez, Joseph M

2018-03-23

Various phytohormones control plant growth and development and mediate biotic and abiotic stress responses. Gretchen Hagen 3 (GH3) acyl acid amido synthetases are plant enzymes that typically conjugate amino acids to indole-3-acetic acid (IAA) or jasmonic acid (JA) to inactivate or activate these phytohormones, respectively; however, the physiological and biological roles of many of these enzymes remain unclear. Using a biochemical approach, we found that the Arabidopsis thaliana GH3.15 (AtGH3.15) preferentially uses indole-3-butyric acid (IBA) and glutamine as substrates. The X-ray crystal structure of the AtGH3.15·AMP complex, modeling of IBA in the active site, and biochemical analysis of site-directed mutants provide insight on active site features that lead to AtGH3.15's preference for IBA. Assay-based in planta analysis of AtGH3.15-overexpressing lines indicated that their root elongation and lateral root density were resistant to IBA treatment but not to treatment with either IAA or JA. These findings suggest that AtGH3.15 may play a role in auxin homeostasis by modulating the levels of IBA for peroxisomal conversion to IAA. Analysis of AtGH3.15 promoter-driven yellow fluorescent protein reporter lines revealed that AtGH3.15 is expressed at significant levels in seedlings, roots, and parts of the siliques. We conclude that AtGH3.15 is unique in the GH3 protein family for its role in modifying IBA in auxin homeostasis and that it is the first GH3 protein shown to primarily modify a plant growth regulator other than IAA and JA. © 2018 Sherp et al.

42 CFR 417.422 - Eligibility to enroll in an HMO or CMP.

Code of Federal Regulations, 2010 CFR

2010-10-01

... SERVICES (CONTINUED) MEDICARE PROGRAM HEALTH MAINTENANCE ORGANIZATIONS, COMPETITIVE MEDICAL PLANS, AND HEALTH CARE PREPAYMENT PLANS Enrollment, Entitlement, and Disenrollment under Medicare Contract § 417.422... entered into a contract under subpart L of this part; (d) During an enrollment period of the HMO or CMP...

Characterization of cDNAs and genomic DNAs for human threonyl- and cysteinyl-tRNA synthetases

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

Cruzen, M.E.

1993-01-01

Techniques of molecular biology were used to clone, sequence and map two human aminoacyl-tRNA synthetase (aaRS) cDNAs: threonyl-tRNA synthetase (ThrRS) a class II enzyme and cysteinyl-tRNA synthetase (CysRS) a class I enzyme. The predicted protein sequence of human ThrRS is highly homologous to that of lower eukaryotic and prokaryotic ThRSs, particularly in the regions containing the three structural motifs common to all class II synthetases. Signature regions 1 and 2, which characterize the class IIa subgroup (SerRS, ThrRS and HisRS) are highly conserved from bacteria to human. Structural predictions for human ThrRS based on the known structure of the closelymore » related SerRS from E.coli implicate strongly conserved residues in the signature sequences to be important in substrate binding. The amino terminal 100 residues of the deduced amino acid sequence of ThrRS shares structural similarity to SerRS consistent with forming an antiparallel helix implicated in tRNA binding. The 5' untranslated sequence of the human ThrRS gene shares short stretches of common sequence with the gene for hamster HisRS including a binding site for the promoter specific transcription factor sp-1. The deduced amino acid sequence of human CysRS has a high degree of sequence identify to E. coli CysRS. Human CysRS possesses the classic characteristics of a class I synthetase and is most closely related to the MetRS subgroup. The amino terminal half of human CysRS can be modeled as a nucleotide binding fold and shares significant sequence and structural similarity to the other enzymes in this subgroup. The CysRS structural gene (CARS) was mapped to human chromosome 11p15.5 by fluorescent in situ hybridization. CARS is the first aaRS gene to be mapped to chromosome 11. The steady state of both CysRS and ThrRs mRNA were quantitated in several human tissues. Message levels for these enzymes appear to be subjected to differential regulation in different cell types.« less

Expression, purification, crystallization and preliminary X-ray diffraction analysis of the VP8* sialic acid-binding domain of porcine rotavirus strain OSU

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

Zhang, Yang-De, E-mail: zhangyd1960@yahoo.com.cn; Li, Hao; Liu, Hui

2007-02-01

Porcine rotavirus strain OSU VP8* domain has been expressed, purified and crystallized. X-ray diffraction data from different crystal forms of the VP8* domain have been collected to 2.65 and 2.2 Å resolution, respectively. The rotavirus outer capsid spike protein VP4 is utilized in the process of rotavirus attachment to and membrane penetration of host cells. VP4 is cleaved by trypsin into two domains: VP8* and VP5*. The VP8* domain is implicated in initial interaction with sialic acid-containing cell-surface carbohydrates and triggers subsequent virus invasion. The VP8* domain from porcine OSU rotavirus was cloned and expressed in Escherichia coli. Different crystalmore » forms (orthorhombic P2{sub 1}2{sub 1}2{sub 1} and tetragonal P4{sub 1}2{sub 1}2) were harvested from two distinct crystallization conditions. Diffraction data have been collected to 2.65 and 2.2 Å resolution and the VP8*{sub 65–224} structure was determined by molecular replacement.« less

Genetically programmed expression of proteins containing the unnatural amino acid phenylselenocysteine

DOEpatents

Wang, Jiangyun; Schultz, Peter G.

2010-09-07

The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetases that can incorporate the unnatural amino acid phenylselenocysteine into proteins produced in eubacterial host cells such as E. coli. The invention provides, for example but not limited to, novel orthogonal aminoacyl-tRNA synthetases, polynucleotides encoding the novel synthetase molecules, methods for identifying and making the novel synthetases, methods for producing proteins containing the unnatural amino acid phenylselenocysteine and translation systems. The invention further provides methods for producing modified proteins (e.g., lipidated proteins) through targeted modification of the phenylselenocysteine residue in a protein.

Genetically programmed expression of proteins containing the unnatural amino acid phenylselenocysteine

DOEpatents

Wang, Jiangyun; Schultz, Peter G.

2012-07-10

The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetases that can incorporate the unnatural amino acid phenylselenocysteine into proteins produced in eubacterial host cells such as E. coli. The invention provides, for example but not limited to, novel orthogonal aminoacyl-tRNA synthetases, polynucleotides encoding the novel synthetase molecules, methods for identifying and making the novel synthetases, methods for producing proteins containing the unnatural amino acid phenylselenocysteine and translation systems. The invention further provides methods for producing modified proteins (e.g., lipidated proteins) through targeted modification of the phenylselenocysteine residue in a protein.

Pseudomonas syringae Phytotoxins: Mode of Action, Regulation, and Biosynthesis by Peptide and Polyketide Synthetases

PubMed Central

Bender, Carol L.; Alarcón-Chaidez, Francisco; Gross, Dennis C.

1999-01-01

Coronatine, syringomycin, syringopeptin, tabtoxin, and phaseolotoxin are the most intensively studied phytotoxins of Pseudomonas syringae, and each contributes significantly to bacterial virulence in plants. Coronatine functions partly as a mimic of methyl jasmonate, a hormone synthesized by plants undergoing biological stress. Syringomycin and syringopeptin form pores in plasma membranes, a process that leads to electrolyte leakage. Tabtoxin and phaseolotoxin are strongly antimicrobial and function by inhibiting glutamine synthetase and ornithine carbamoyltransferase, respectively. Genetic analysis has revealed the mechanisms responsible for toxin biosynthesis. Coronatine biosynthesis requires the cooperation of polyketide and peptide synthetases for the assembly of the coronafacic and coronamic acid moieties, respectively. Tabtoxin is derived from the lysine biosynthetic pathway, whereas syringomycin, syringopeptin, and phaseolotoxin biosynthesis requires peptide synthetases. Activation of phytotoxin synthesis is controlled by diverse environmental factors including plant signal molecules and temperature. Genes involved in the regulation of phytotoxin synthesis have been located within the coronatine and syringomycin gene clusters; however, additional regulatory genes are required for the synthesis of these and other phytotoxins. Global regulatory genes such as gacS modulate phytotoxin production in certain pathovars, indicating the complexity of the regulatory circuits controlling phytotoxin synthesis. The coronatine and syringomycin gene clusters have been intensively characterized and show potential for constructing modified polyketides and peptides. Genetic reprogramming of peptide and polyketide synthetases has been successful, and portions of the coronatine and syringomycin gene clusters could be valuable resources in developing new antimicrobial agents. PMID:10357851

Cyclopiazonic acid biosynthesis in Aspergillus sp.: characterization of a reductase-like R* domain in cyclopiazonate synthetase that forms and releases cyclo-acetoacetyl-L-tryptophan.

PubMed

Liu, Xinyu; Walsh, Christopher T

2009-09-15

The fungal neurotoxin alpha-cyclopiazonic acid (CPA), a nanomolar inhibitor of Ca2+-ATPase, has a pentacyclic indole tetramic acid scaffold that arises from one molecule of tryptophan, acetyl-CoA, malonyl-CoA, and dimethylallyl pyrophosphate by consecutive action of three enzymes, CpaS, CpaD, and CpaO. CpaS is a hybrid, two module polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) that makes and releases cyclo-acetoacetyl-L-tryptophan (cAATrp), the tetramic acid that serves as substrate for subsequent prenylation and oxidative cyclization to the five ring CPA scaffold. The NRPS module in CpaS has a predicted four-domain organization of condensation, adenylation, thiolation, and reductase* (C-A-T-R*), where R* lacks the critical Ser-Tyr-Lys catalytic triad of the short chain dehydrogenase/reductase (SDR) superfamily. By heterologous overproduction in Escherichia coli of the 56 kDa Aspergillus flavus CpaS TR* didomain and the single T and R* domains, we demonstrate that CpaS catalyzes a Dieckmann-type cyclization on the N-acetoacetyl-Trp intermediate bound in thioester linkage to the phosphopantetheinyl arm of the T domain to form and release cAATrp. This occurs without any participation of NAD(P)H, so R* does not function as a canonical SDR family member. Use of the T and R* domains in in trans assays enabled multiple turnovers and evaluation of specific mutants. Mutation of the D3803 residue in the R* domain, conserved in other fungal tetramate synthetases, abolished activity both in in trans and in cis (TR*) activity assays. It is likely that cyclization of beta-ketoacylaminoacyl-S-pantetheinyl intermediates to released tetramates represents a default cyclization/release route for redox-incompetent R* domains embedded in NRPS assembly lines.

Structural characterization of the Mycobacterium tuberculosis biotin biosynthesis enzymes 7,8-diaminopelargonic acid synthase and dethiobiotin synthetase .

PubMed

Dey, Sanghamitra; Lane, James M; Lee, Richard E; Rubin, Eric J; Sacchettini, James C

2010-08-10

Mycobacterium tuberculosis (Mtb) depends on biotin synthesis for survival during infection. In the absence of biotin, disruption of the biotin biosynthesis pathway results in cell death rather than growth arrest, an unusual phenotype for an Mtb auxotroph. Humans lack the enzymes for biotin production, making the proteins of this essential Mtb pathway promising drug targets. To this end, we have determined the crystal structures of the second and third enzymes of the Mtb biotin biosynthetic pathway, 7,8-diaminopelargonic acid synthase (DAPAS) and dethiobiotin synthetase (DTBS), at respective resolutions of 2.2 and 1.85 A. Superimposition of the DAPAS structures bound either to the SAM analogue sinefungin or to 7-keto-8-aminopelargonic acid (KAPA) allowed us to map the putative binding site for the substrates and to propose a mechanism by which the enzyme accommodates their disparate structures. Comparison of the DTBS structures bound to the substrate 7,8-diaminopelargonic acid (DAPA) or to ADP and the product dethiobiotin (DTB) permitted derivation of an enzyme mechanism. There are significant differences between the Mtb enzymes and those of other organisms; the Bacillus subtilis DAPAS, presented here at a high resolution of 2.2 A, has active site variations and the Escherichia coli and Helicobacter pylori DTBS have alterations in their overall folds. We have begun to exploit the unique characteristics of the Mtb structures to design specific inhibitors against the biotin biosynthesis pathway in Mtb.

Plasmodium falciparum mitochondria import tRNAs along with an active phenylalanyl-tRNA synthetase.

PubMed

Sharma, Arvind; Sharma, Amit

2015-02-01

The Plasmodium falciparum protein translation enzymes aminoacyl-tRNA synthetases (aaRSs) are an emergent family of drug targets. The aaRS ensemble catalyses transfer of amino acids to cognate tRNAs, thus providing charged tRNAs for ribosomal consumption. P. falciparum proteome expression relies on a total of 36 aaRSs for the three translationally independent compartments of cytoplasm, apicoplast and mitochondria. In the present study, we show that, of this set of 36, a single genomic copy of mitochondrial phenylalanyl-tRNA synthetase (mFRS) is targeted to the parasite mitochondria, and that the mFRS gene is exclusive to malaria parasites within the apicomplexan phyla. Our protein cellular localization studies based on immunofluorescence data show that, along with mFRS, P. falciparum harbours two more phenylalanyl-tRNA synthetase (FRS) assemblies that are localized to its apicoplast and cytoplasm. The 'extra' mFRS is found in mitochondria of all asexual blood stage parasites and is competent in aminoacylation. We show further that the parasite mitochondria import tRNAs from the cytoplasmic tRNA pool. Hence drug targeting of FRSs presents a unique opportunity to potentially stall protein production in all three parasite translational compartments.

Characterization of the N-Acetyl-5-neuraminic Acid-binding Site of the Extracytoplasmic Solute Receptor (SiaP) of Nontypeable Haemophilus influenzae Strain 2019

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

Johnston, Jason W.; Coussens, Nathan P.; Allen, Simon

Nontypeable Haemophilus influenzae is an opportunistic human pathogen causing otitis media in children and chronic bronchitis and pneumonia in patients with chronic obstructive pulmonary disease. The outer membrane of nontypeable H. influenzae is dominated by lipooligosaccharides (LOS), many of which incorporate sialic acid as a terminal nonreducing sugar. Sialic acid has been demonstrated to be an important factor in the survival of the bacteria within the host environment. H. influenzae is incapable of synthesizing sialic acid and is dependent on scavenging free sialic acid from the host environment. To achieve this, H. influenzae utilizes a tripartite ATP-independent periplasmic transporter. Inmore » this study, we characterize the binding site of the extracytoplasmic solute receptor (SiaP) from nontypeable H. influenzae strain 2019. A crystal structure of N-acetyl-5-neuraminic acid (Neu5Ac)-bound SiaP was determined to 1.4 {angstrom} resolution. Thermodynamic characterization of Neu5Ac binding shows this interaction is enthalpically driven with a substantial unfavorable contribution from entropy. This is expected because the binding of SiaP to Neu5Ac is mediated by numerous hydrogen bonds and has several buried water molecules. Point mutations targeting specific amino acids were introduced in the putative binding site. Complementation with the mutated siaP constructs resulted either in full, partial, or no complementation, depending on the role of specific residues. Mass spectrometry analysis of the O-deacylated LOS of the R127K point mutation confirmed the observation of reduced incorporation of Neu5Ac into the LOS. The decreased ability of H. influenzae to import sialic acid had negative effects on resistance to complement-mediated killing and viability of biofilms in vitro, confirming the importance of sialic acid transport to the bacterium.« less

Arabidopsis CER8 encodes LONG-CHAIN ACYL-COA SYNTHETASE 1 (LACS1) that has overlapping functions with LACS2 in plant wax and cutin synthesis.

PubMed

Lü, Shiyou; Song, Tao; Kosma, Dylan K; Parsons, Eugene P; Rowland, Owen; Jenks, Matthew A

2009-08-01

Plant cuticle is an extracellular lipid-based matrix of cutin and waxes, which covers aerial organs and protects them from many forms of environmental stress. We report here the characterization of CER8/LACS1, one of nine Arabidopsis long-chain acyl-CoA synthetases thought to activate acyl chains. Mutations in LACS1 reduced the amount of wax in all chemical classes on the stem and leaf, except in the very long-chain fatty acid (VLCFA) class wherein acids longer than 24 carbons (C(24)) were elevated more than 155%. The C(16) cutin monomers on lacs1 were reduced by 37% and 22%, whereas the C(18) monomers were increased by 28% and 20% on stem and leaf, respectively. Amounts of wax and cutin on a lacs1-1 lacs2-3 double mutant were much lower than on either parent, and lacs1-1 lacs2-3 had much higher cuticular permeability than either parent. These additive effects indicate that LACS1 and LACS2 have overlapping functions in both wax and cutin synthesis. We demonstrated that LACS1 has synthetase activity for VLCFAs C(20)-C(30), with highest activity for C(30) acids. LACS1 thus appears to function as a very long-chain acyl-CoA synthetase in wax metabolism. Since C(16) but not C(18) cutin monomers are reduced in lacs1, and C(16) acids are the next most preferred acid (behind C(30)) by LACS1 in our assays, LACS1 also appears to be important for the incorporation of C(16) monomers into cutin polyester. As such, LACS1 defines a functionally novel acyl-CoA synthetase that preferentially modifies both VLCFAs for wax synthesis and long-chain (C(16)) fatty acids for cutin synthesis.

Antipeptide antibodies that can distinguish specific subunit polypeptides of glutamine synthetase from bean (Phaseolus vulgaris L.)

NASA Technical Reports Server (NTRS)

Cai, X.; Henry, R. L.; Takemoto, L. J.; Guikema, J. A.; Wong, P. P.; Spooner, B. S. (Principal Investigator)

1992-01-01

The amino acid sequences of the beta and gamma subunit polypeptides of glutamine synthetase from bean (Phaseolus vulgaris L.) root nodules are very similar. However, there are small regions within the sequences that are significantly different between the two polypeptides. The sequences between amino acids 2 and 9 and between 264 and 274 are examples. Three peptides (gamma 2-9, gamma 264-274, and beta 264-274) corresponding to these sequences were synthesized. Antibodies against these peptides were raised in rabbits and purified with corresponding peptide-Sepharose affinity chromatography. Western blot analysis of polyacrylamide gel electrophoresis of bean nodule proteins demonstrated that the anti-beta 264-274 antibodies reacted specifically with the beta polypeptide and the anti-gamma 264-274 and anti-gamma 2-9 antibodies reacted specifically with the gamma polypeptide of the native and denatured glutamine synthetase. These results showed the feasibility of using synthetic peptides in developing antibodies that are capable of distinguishing proteins with similar primary structures.

Glucocorticoid receptor-mediated induction of glutamine synthetase in skeletal muscle cells in vitro

NASA Technical Reports Server (NTRS)

Max, Stephen R.; Thomas, John W.; Banner, Carl; Vitkovic, Ljubisa; Konagaya, Masaaki

1987-01-01

The regulation by glucocorticoids of glutamine synthetase in L6 muscle cells in culture is studied. Glutamine synthetase activity was strikingly enhanced by dexamethasone. The dexamethasone-mediated induction of glutamine synthetase activity was blocked by RU38486, a glucocorticoid antagonist, indicating the involvement of intracellular glucocorticoid receptors in the induction process. RU38486 alone was without effect. Northern blot analysis revealed that dexamethasone-mediated enhancement of glutamine synthetase activity involves increased levels of glutamine synthetase mRNA. Glucocorticoids regulate the expression of glutamine synthetase mRNA in cultured muscle cells via interaction with intracellular receptors. Such regulation may be relevant to control of glutamine production by muscle.

How does Pseudomonas fluorescens avoid suicide from its antibiotic pseudomonic acid?: Evidence for two evolutionarily distinct isoleucyl-tRNA synthetases conferring self-defense.

PubMed

Yanagisawa, Tatsuo; Kawakami, Makoto

2003-07-11

Two isoleucyl-tRNA synthetases (IleRSs) encoded by two distinct genes (ileS1 and ileS2) were identified in pseudomonic acid (mupirocin)-producing Pseudomonas fluorescens. The most striking difference between the two IleRSs (IleRS-R1 and IleRS-R2) is the difference in their abilities to resist pseudomonic acid. Purified IleRS-R2 showed no sensitivity to pseudomonic acid even at a concentration of 5 mm, 105 times higher than the Ki value of IleRS-R1. The amino acid sequence of IleRS-R2 exhibits eukaryotic features that are originally found in eukaryotic proteins. Escherichia coli cells transformed with the ileS2 gene exerted pseudomonic acid resistance more than did those transformed with ileS1. Cells transformed with both genes became almost as resistant as P. fluorescens. These results suggest that the presence of IleRS-R2 could be the major reason why P. fluorescens is intrinsically resistant to the antibiotic. Here we suggest that the evolutionary scenario of the eukaryotic ileS2 gene can be explained by gene acquisition and that the pseudomonic acid producer may have maintained the ileS2 gene to protect itself from pseudomonic acid.

Invariant amino acids in the Mur peptide synthetases of bacterial peptidoglycan synthesis and their modification by site-directed mutagenesis in the UDP-MurNAc:L-alanine ligase from Escherichia coli.

PubMed

Bouhss, A; Mengin-Lecreulx, D; Blanot, D; van Heijenoort, J; Parquet, C

1997-09-30

The comparison of the amino acid sequences of 20 cytoplasmic peptidoglycan synthetases (MurC, MurD, MurE, MurF, and Mpl) from various bacterial organisms has allowed us to detect common invariants: seven amino acids and the ATP-binding consensus sequence GXXGKT/S all at the same position in the alignment. The Mur synthetases thus appeared as a well-defined class of closely functionally related proteins. The conservation of a constant backbone length between certain invariants suggested common structural motifs. Among the other enzymes catalyzing a peptide bond formation driven by ATP hydrolysis to ADP and Pi, only folylpoly-gamma-l-glutamate synthetases presented the same common conserved amino acid residues, except for the most N-terminal invariant D50. Site-directed mutageneses were carried out to replace the K130, E174, H199, N293, N296, R327, and D351 residues by alanine in the MurC protein from Escherichia coli taken as model. For this purpose, plasmid pAM1005 was used as template, MurC being highly overproduced in this genetic setting. Analysis of the Vmax values of the mutated proteins suggested that residues K130, E174, and D351 are essential for the catalytic process whereas residues H199, N293, N296, and R327 were not. Mutations K130A, H199A, N293A, N296A, and R327A led to important variations of the Km values for one or more substrates, thereby indicating that these residues are involved in the structure of the active site and suggesting that the binding order of the substrates could be ATP, UDP-MurNAc, and alanine. The various mutated murC plasmids were tested for their effects on the growth, cell morphology, and peptidoglycan cell content of a murC thermosensitive strain at 42 degrees C. The observed effects (complementation, altered morphology, and reduced peptidoglycan content) paralleled more or less the decreased values of the MurC activity of each mutant.

Orthogonal translation components for the in vivo incorporation of unnatural amino acids

DOEpatents

Schultz, Peter G.; Alfonta, Lital; Chittuluru, Johnathan R.; Deiters, Alexander; Groff, Dan; Summerer, Daniel; Tsao, Meng -Lin; Wang, Jiangyun; Wu, Ning; Xie, Jianming; Zeng, Huaqiang; Seyedsayamdost, Mohammad; Turner, James

2015-08-11

The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetase that can incorporate unnatural amino acid into proteins produced in eubacterial host cells such as E. coli, or in a eukaryotic host such as a yeast cell. The invention provides, for example but not limited to, novel orthogonal synthetases, methods for identifying and making the novel synthetases, methods for producing proteins containing unnatural amino acids, and translation systems.

Orthogonal translation components for the in vivo incorporation of unnatural amino acids

DOEpatents

Schultz, Peter G.; Xie, Jianming; Zeng, Huaqiang

2012-07-10

The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetases that can incorporate unnatural amino acids into proteins produced in eubacterial host cells such as E. coli, or in a eukaryotic host such as a yeast cell. The invention provides, for example but not limited to, novel orthogonal synthetases, methods for identifying and making the novel synthetases, methods for producing proteins containing unnatural amino acids, and translation systems.

Molecular design of boronic acid-functionalized squarylium cyanine dyes for multiple discriminant analysis of sialic acid in biological samples: selectivity toward monosaccharides controlled by different alkyl side chain lengths.

PubMed

Ouchi, Kazuki; Colyer, Christa L; Sebaiy, Mahmoud; Zhou, Jin; Maeda, Takeshi; Nakazumi, Hiroyuki; Shibukawa, Masami; Saito, Shingo

2015-02-03

We designed a new series of boronic acid-functionalized squarylium cyanine dyes (SQ-BA) with different lengths of alkyl chain residues, suitable for multiple discriminant analysis (MDA) of sialic acid (Neu5Ac) in biological samples. The SQ-BA dyes form aggregates based on hydrophobic interactions, which result in quenched fluorescence in aqueous solutions. When the boronic acid binds with saccharides, the fluorescence intensity increases as a result of dissociation to the emissive monomeric complex. We inferred that different dye aggregate structures (H-aggregates and J-aggregates) were induced depending on the alkyl chain length, so that monosaccharides would be recognized in different ways (especially, multipoint interaction with J-aggregates). A distinctive emission enhancement of SQ-BA dyes with shorter-alkyl-chains in the presence of Neu5Ac was observed (2.4-fold fluorescence enhancement; with formation constant 10(1.7) M(-1)), with no such enhancement for SQ-BA dyes with longer-alkyl-chain. In addition, various enhancement factors for other monosaccharides were observed depending on the alkyl chain length. Detailed thermodynamic and NMR studies of the SQ-BA complexes revealed the unique recognition mechanism: the dye aggregate with a shorter-alkyl-chain causes the slipped parallel structure and forms a stable 2:1 complex with Neu5Ac, as distinct from longer-alkyl-chain dyes, which form a 1:1 monomeric complex. MDA using the four SQ-BA dyes was performed for human urine samples, resulting in the successful discrimination between normal and abnormal Neu5Ac levels characteristic of disease. Thus, we successfully controlled various responses to similar monosaccharides with a novel approach that chemically modified not the boronic acid moiety itself but the length of the alkyl chain residue attached to the dye in order to generate specificity.

The role of functionally defective rare germline variants of sialic acid acetylesterase in autoimmune Addison's disease

PubMed Central

Gan, Earn H; MacArthur, Katie; Mitchell, Anna L; Pearce, Simon H S

2012-01-01

Background Autoimmune Addison's disease (AAD) is a rare condition with a complex genetic basis. A panel of rare and functionally defective genetic variants in the sialic acid acetylesterase (SIAE) gene has recently been implicated in several common autoimmune conditions. We performed a case–control study to determine whether these rare variants are associated with a rarer condition, AAD. Method We analysed nine SIAE gene variants (W48X, M89V, C196F, C226G, R230W, T312M, Y349C, F404S and R479C) in a United Kingdom cohort of 378 AAD subjects and 387 healthy controls. All samples were genotyped using Sequenom iPlex chemistry to characterise primer extension products. Results A heterozygous rare allele at codon 312 (312*M) was found in one AAD patient (0.13%) but was not detected in the healthy controls. The commoner, functionally recessive variant at codon 89 (89*V) was found to be homozygous in two AAD patients but was only found in the heterozygous state in controls. Taking into account all nine alleles examined, 4/378 (1.06%) AAD patients and 1/387 (0.25%) healthy controls carried the defective SIAE alleles, with a calculated odds ratio of 4.13 (95% CI 0.44–97.45, two-tailed P value 0.212, NS). Conclusion We demonstrated the presence of 89*V homozygotes and the 312*M rare allele in the AAD cohort, but overall, our analysis does not support a role for rare variants in SIAE in the pathogenesis of AAD. However, the relatively small collection of AAD patients limits the power to exclude a small effect. PMID:23011869

Site specific incorporation of heavy atom-containing unnatural amino acids into proteins for structure determination

DOEpatents

Xie, Jianming [San Diego, CA; Wang, Lei [San Diego, CA; Wu, Ning [Boston, MA; Schultz, Peter G [La Jolla, CA

2008-07-15

Translation systems and other compositions including orthogonal aminoacyl tRNA-synthetases that preferentially charge an orthogonal tRNA with an iodinated or brominated amino acid are provided. Nucleic acids encoding such synthetases are also described, as are methods and kits for producing proteins including heavy atom-containing amino acids, e.g., brominated or iodinated amino acids. Methods of determining the structure of a protein, e.g., a protein into which a heavy atom has been site-specifically incorporated through use of an orthogonal tRNA/aminoacyl tRNA-synthetase pair, are also described.

Site specific incorporation of keto amino acids into proteins

DOEpatents

Schultz, Peter G [La Jolla, CA; Wang, Lei [San Diego, CA

2011-03-22

Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

Site specific incorporation of keto amino acids into proteins

DOEpatents

Schultz, Peter G [La Jolla, CA; Wang, Lei [San Diego, CA

2008-10-07

Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

Site specific incorporation of keto amino acids into proteins

DOEpatents

Schultz, Peter G [La Jolla, CA; Wang, Lei [San Diego, CA

2011-12-06

Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

Site specific incorporation of keto amino acids into proteins

DOEpatents

Schultz, Peter G [La Jolla, CA; Wang, Lei [San Diego, CA

2012-02-14

Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

Selective and Specific Inhibition of the Plasmodium falciparum Lysyl-tRNA Synthetase by the Fungal Secondary Metabolite Cladosporin

PubMed Central

Hoepfner, Dominic; McNamara, Case W.; Lim, Chek Shik; Studer, Christian; Riedl, Ralph; Aust, Thomas; McCormack, Susan L.; Plouffe, David M.; Meister, Stephan; Schuierer, Sven; Plikat, Uwe; Hartmann, Nicole; Staedtler, Frank; Cotesta, Simona; Schmitt, Esther K.; Petersen, Frank; Supek, Frantisek; Glynne, Richard J.; Tallarico, John A.; Porter, Jeffrey A.; Fishman, Mark C.; Bodenreider, Christophe; Diagana, Thierry T.; Movva, N. Rao; Winzeler, Elizabeth A.

2012-01-01

Summary With renewed calls for malaria eradication, next-generation antimalarials need be active against drug-resistant parasites and efficacious against both liver- and blood-stage infections. We screened a natural product library to identify inhibitors of Plasmodium falciparum blood- and liver-stage proliferation. Cladosporin, a fungal secondary metabolite whose target and mechanism of action are not known for any species, was identified as having potent, nanomolar, antiparasitic activity against both blood and liver stages. Using postgenomic methods, including a yeast deletion strains collection, we show that cladosporin specifically inhibits protein synthesis by directly targeting P. falciparum cytosolic lysyl-tRNA synthetase. Further, cladosporin is >100-fold more potent against parasite lysyl-tRNA synthetase relative to the human enzyme, which is conferred by the identity of two amino acids within the enzyme active site. Our data indicate that lysyl-tRNA synthetase is an attractive, druggable, antimalarial target that can be selectively inhibited. PMID:22704625

Selective and specific inhibition of the plasmodium falciparum lysyl-tRNA synthetase by the fungal secondary metabolite cladosporin.

PubMed

Hoepfner, Dominic; McNamara, Case W; Lim, Chek Shik; Studer, Christian; Riedl, Ralph; Aust, Thomas; McCormack, Susan L; Plouffe, David M; Meister, Stephan; Schuierer, Sven; Plikat, Uwe; Hartmann, Nicole; Staedtler, Frank; Cotesta, Simona; Schmitt, Esther K; Petersen, Frank; Supek, Frantisek; Glynne, Richard J; Tallarico, John A; Porter, Jeffrey A; Fishman, Mark C; Bodenreider, Christophe; Diagana, Thierry T; Movva, N Rao; Winzeler, Elizabeth A

2012-06-14

With renewed calls for malaria eradication, next-generation antimalarials need be active against drug-resistant parasites and efficacious against both liver- and blood-stage infections. We screened a natural product library to identify inhibitors of Plasmodium falciparum blood- and liver-stage proliferation. Cladosporin, a fungal secondary metabolite whose target and mechanism of action are not known for any species, was identified as having potent, nanomolar, antiparasitic activity against both blood and liver stages. Using postgenomic methods, including a yeast deletion strains collection, we show that cladosporin specifically inhibits protein synthesis by directly targeting P. falciparum cytosolic lysyl-tRNA synthetase. Further, cladosporin is >100-fold more potent against parasite lysyl-tRNA synthetase relative to the human enzyme, which is conferred by the identity of two amino acids within the enzyme active site. Our data indicate that lysyl-tRNA synthetase is an attractive, druggable, antimalarial target that can be selectively inhibited. Copyright © 2012 Elsevier Inc. All rights reserved.

A role for long-chain acyl-CoA synthetase-4 (ACSL4) in diet-induced phospholipid remodeling and obesity-associated adipocyte dysfunction

USDA-ARS?s Scientific Manuscript database

OBJECTIVE: Regulation of fatty acid (FA) metabolism is central to adipocyte dysfunction during diet-induced obesity (DIO). Long-chain acyl-CoA synthetase-4 (ACSL4) has been hypothesized to modulate the metabolic fates of polyunsaturated FA (PUFA), including arachidonic acid (AA), but the in vivo act...

Design of S-Allylcysteine in Situ Production and Incorporation Based on a Novel Pyrrolysyl-tRNA Synthetase Variant.

PubMed

Exner, Matthias P; Kuenzl, Tilmann; To, Tuyet Mai T; Ouyang, Zhaofei; Schwagerus, Sergej; Hoesl, Michael G; Hackenberger, Christian P R; Lensen, Marga C; Panke, Sven; Budisa, Nediljko

2017-01-03

The noncanonical amino acid S-allyl cysteine (Sac) is one of the major compounds of garlic extract and exhibits a range of biological activities. It is also a small bioorthogonal alkene tag capable of undergoing controlled chemical modifications, such as photoinduced thiol-ene coupling or Pd-mediated deprotection. Its small size guarantees minimal interference with protein structure and function. Here, we report a simple protocol efficiently to couple in-situ semisynthetic biosynthesis of Sac and its incorporation into proteins in response to amber (UAG) stop codons. We exploited the exceptional malleability of pyrrolysyl-tRNA synthetase (PylRS) and evolved an S-allylcysteinyl-tRNA synthetase (SacRS) capable of specifically accepting the small, polar amino acid instead of its long and bulky aliphatic natural substrate. We succeeded in generating a novel and inexpensive strategy for the incorporation of a functionally versatile amino acid. This will help in the conversion of orthogonal translation from a standard technique in academic research to industrial biotechnology. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Actinobacterial Acyl Coenzyme A Synthetases Involved in Steroid Side-Chain Catabolism

PubMed Central

Casabon, Israël; Swain, Kendra; Crowe, Adam M.

2014-01-01

Bacterial steroid catabolism is an important component of the global carbon cycle and has applications in drug synthesis. Pathways for this catabolism involve multiple acyl coenzyme A (CoA) synthetases, which activate alkanoate substituents for β-oxidation. The functions of these synthetases are poorly understood. We enzymatically characterized four distinct acyl-CoA synthetases from the cholate catabolic pathway of Rhodococcus jostii RHA1 and the cholesterol catabolic pathway of Mycobacterium tuberculosis. Phylogenetic analysis of 70 acyl-CoA synthetases predicted to be involved in steroid metabolism revealed that the characterized synthetases each represent an orthologous class with a distinct function in steroid side-chain degradation. The synthetases were specific for the length of alkanoate substituent. FadD19 from M. tuberculosis H37Rv (FadD19Mtb) transformed 3-oxo-4-cholesten-26-oate (kcat/Km = 0.33 × 105 ± 0.03 × 105 M−1 s−1) and represents orthologs that activate the C8 side chain of cholesterol. Both CasGRHA1 and FadD17Mtb are steroid-24-oyl-CoA synthetases. CasG and its orthologs activate the C5 side chain of cholate, while FadD17 and its orthologs appear to activate the C5 side chain of one or more cholesterol metabolites. CasIRHA1 is a steroid-22-oyl-CoA synthetase, representing orthologs that activate metabolites with a C3 side chain, which accumulate during cholate catabolism. CasI had similar apparent specificities for substrates with intact or extensively degraded steroid nuclei, exemplified by 3-oxo-23,24-bisnorchol-4-en-22-oate and 1β(2′-propanoate)-3aα-H-4α(3″-propanoate)-7aβ-methylhexahydro-5-indanone (kcat/Km = 2.4 × 105 ± 0.1 × 105 M−1 s−1 and 3.2 × 105 ± 0.3 × 105 M−1 s−1, respectively). Acyl-CoA synthetase classes involved in cholate catabolism were found in both Actinobacteria and Proteobacteria. Overall, this study provides insight into the physiological roles of acyl-CoA synthetases in steroid catabolism and

42 CFR 417.544 - Physicians' services furnished directly by the HMO or CMP.

Code of Federal Regulations, 2010 CFR

2010-10-01

... attributable to salaries, wages, incentive payments, fringe benefits) must be distinguished from the cost of... 42 Public Health 3 2010-10-01 2010-10-01 false Physicians' services furnished directly by the HMO or CMP. 417.544 Section 417.544 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF...

Interim Action Proposed Plan for the Chemicals, Metals, and Pesticides (CMP) Pits Operable Unit

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

Bradley, J.

2002-06-18

The purpose of this Interim Action Proposed Plan (IAPP) is to describe the preferred interim remedial action for addressing the Chemicals, Metals, and Pesticides (CMP) Pits Operable Unit and to provide an opportunity for public input into the remedial action selection process.

Fluorescent molecularly imprinted polymers as plastic antibodies for selective labeling and imaging of hyaluronan and sialic acid on fixed and living cells.

PubMed

Panagiotopoulou, Maria; Kunath, Stephanie; Medina-Rangel, Paulina Ximena; Haupt, Karsten; Tse Sum Bui, Bernadette

2017-02-15

Altered glycosylation levels or distribution of sialic acids (SA) or hyaluronan in animal cells are indicators of pathological conditions like infection or malignancy. We applied fluorescently-labeled molecularly imprinted polymer (MIP) particles for bioimaging of fixed and living human keratinocytes, to localize hyaluronan and sialylation sites. MIPs were prepared with the templates D-glucuronic acid (GlcA), a substructure of hyaluronan, and N-acetylneuraminic acid (NANA), the most common member of SA. Both MIPs were found to be highly selective towards their target monosaccharides, as no cross-reactivity was observed with other sugars like N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, D-glucose and D-galactose, present on the cell surface. The dye rhodamine and two InP/ZnS quantum dots (QDs) emitting in the green and in the red regions were used as fluorescent probes. Rhodamine-MIPGlcA and rhodamine-MIPNANA were synthesized as monodispersed 400nm sized particles and were found to bind selectively their targets located in the extracellular region, as imaged by epifluorescence and confocal microscopy. In contrast, when MIP-GlcA and MIP-NANA particles with a smaller size (125nm) were used, the MIPs being synthesized as thin shells around green and red emitting QDs respectively, it was possible to stain the intracellular and pericellular regions as well. In addition, simultaneous dual-color imaging with the two different colored QDs-MIPs was demonstrated. Importantly, the MIPs were not cytotoxic and did not affect cell viability; neither was the cells morphology affected as demonstrated by live cell imaging. These synthetic receptors could offer a new and promising imaging tool to monitor disease progression. Copyright © 2016 Elsevier B.V. All rights reserved.

Site-specific incorporation of redox active amino acids into proteins

DOEpatents

Alfonta, Lital [San Diego, CA; Schultz, Peter G [La Jolla, CA; Zhang, Zhiwen [Austin, TX

2011-08-30

Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate redox active amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with redox active amino acids using these orthogonal pairs.

Site-specific incorporation of redox active amino acids into proteins

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

Alfonta, Lital; Schultz, Peter G.; Zhang, Zhiwen

Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate redox active amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with redox active amino acids using these orthogonal pairs.

Site-specific incorporation of redox active amino acids into proteins

DOEpatents

Alfonta, Lital [San Diego, CA; Schultz, Peter G [La Jolla, CA; Zhang, Zhiwen [San Diego, CA

2012-02-14

Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate redox active amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with redox active amino acids using these orthogonal pairs.

Site-specific incorporation of redox active amino acids into proteins

DOEpatents

Alfonta; Lital , Schultz; Peter G. , Zhang; Zhiwen

2010-10-12

Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate redox active amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with redox active amino acids using these orthogonal pairs.

Site-specific incorporation of redox active amino acids into proteins

DOEpatents

Alfonta, Lital [San Diego, CA; Schultz, Peter G [La Jolla, CA; Zhang, Zhiwen [San Diego, CA

2009-02-24

Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate redox active amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with redox active amino acids using these orthogonal pairs.

Comparative Study of Blood-Based Biomarkers, α2,3-Sialic Acid PSA and PHI, for High-Risk Prostate Cancer Detection.

PubMed

Ferrer-Batallé, Montserrat; Llop, Esther; Ramírez, Manel; Aleixandre, Rosa Núria; Saez, Marc; Comet, Josep; de Llorens, Rafael; Peracaula, Rosa

2017-04-17

Prostate Specific Antigen (PSA) is the most commonly used serum marker for prostate cancer (PCa), although it is not specific and sensitive enough to allow the differential diagnosis of the more aggressive tumors. For that, new diagnostic methods are being developed, such as PCA-3, PSA isoforms that have resulted in the 4K score or the Prostate Health Index (PHI), and PSA glycoforms. In the present study, we have compared the PHI with our recently developed PSA glycoform assay, based on the determination of the α2,3-sialic acid percentage of serum PSA (% α2,3-SA), in a cohort of 79 patients, which include 50 PCa of different grades and 29 benign prostate hyperplasia (BPH) patients. The % α2,3-SA could distinguish high-risk PCa patients from the rest of patients better than the PHI (area under the curve (AUC) of 0.971 vs. 0.840), although the PHI correlated better with the Gleason score than the % α2,3-SA. The combination of both markers increased the AUC up to 0.985 resulting in 100% sensitivity and 94.7% specificity to differentiate high-risk PCa from the other low and intermediate-risk PCa and BPH patients. These results suggest that both serum markers complement each other and offer an improved diagnostic tool to identify high-risk PCa, which is an important requirement for guiding treatment decisions.

Bacillus anthracis o-succinylbenzoyl-CoA synthetase: reaction kinetics and a novel inhibitor mimicking its reaction intermediate.

PubMed

Tian, Yang; Suk, Dae-Hwan; Cai, Feng; Crich, David; Mesecar, Andrew D

2008-11-25

o-Succinylbenzoyl-CoA (OSB-CoA) synthetase (EC 6.2.1.26) catalyzes the ATP-dependent condensation of o-succinylbenzoate (OSB) and CoA to form OSB-CoA, the fourth step of the menaquinone biosynthetic pathway in Bacillus anthracis. Gene knockout studies have highlighted this enzyme as a potential target for the discovery of new antibiotics. Here we report the first studies on the kinetic mechanism of B. anthracis OSB-CoA synthetase, classifying it as an ordered bi uni uni bi ping-pong mechanism. Through a series of pre-steady-state and steady-state kinetic studies in conjunction with direct binding studies, it is demonstrated that CoA, the last substrate to bind, strongly activates the first half-reaction after the first round of turnover. The activation of the first half-reaction is most likely achieved by CoA stabilizing conformations of the enzyme in the "F" form, which slowly isomerize back to the E form. Thus, the kinetic mechanism of OSB-CoA synthetase may be more accurately described as an ordered bi uni uni bi iso ping-pong mechanism. The substrate specificity of OSB-CoA synthetase was probed using a series of OSB analogues with alterations in the carboxylate groups. OSB-CoA shows a strong preference for OSB over all of the analogues tested as none were active except 4-[2-(trifluoromethyl)phenyl]-4-oxobutyric acid which exhibited a 100-fold decrease in k(cat)/K(m). On the basis of an understanding of OSB-CoA synthetase's kinetic mechanism and substrate specificity, a reaction intermediate analogue of OSB-AMP, 5'-O-{N-[2-(trifluoromethyl)phenyl]-4-oxobutyl}adenosine sulfonamide (TFMP-butyl-AMS), was designed and synthesized. This inhibitor was found to be an uncompetitive inhibitor to CoA and a mixed-type inhibitor to ATP and OSB with low micromolar inhibition constants. Collectively, these results should serve as an important forerunner to more detailed and extensive inhibitor design studies aimed at developing lead compounds against the OSB-CoA synthetase

Regulated capture by exosomes of mRNAs for cytoplasmic tRNA synthetases.

PubMed

Wang, Feng; Xu, Zhiwen; Zhou, Jie; Lo, Wing-Sze; Lau, Ching-Fun; Nangle, Leslie A; Yang, Xiang-Lei; Zhang, Mingjie; Schimmel, Paul

2013-10-11

Although tRNA synthetases are enzymes that catalyze the first step of translation in the cytoplasm, surprising functions unrelated to translation have been reported. These studies, and the demonstration of novel activities of splice variants, suggest a far broader reach of tRNA synthetases into cell biology than previously recognized. Here we show that mRNAs for most tRNA synthetases can be detected in exosomes. Also detected in exosomes was an mRNA encoding a unique splice variant that others had associated with prostate cancer. The exosomal mRNAs encoding the native synthetase and its cancer-associated splice variant could be translated in vitro and in mammalian cells into stable proteins. Other results showed that selection by exosomes of the splice variant mRNA could be regulated by an external stimulus. Thus, a broad and diverse regulated pool of tRNA synthetase-derived mRNAs is packaged for genetic exchange.

Mice lacking sialyltransferase ST3Gal-II develop late-onset obesity and insulin resistance

PubMed Central

Lopez, Pablo HH; Aja, Susan; Aoki, Kazuhiro; Seldin, Marcus M; Lei, Xia; Ronnett, Gabriele V; Wong, G William; Schnaar, Ronald L

2017-01-01

Sialyltransferases are a family of 20 gene products in mice and humans that transfer sialic acid from its activated precursor, CMP-sialic acid, to the terminus of glycoprotein and glycolipid acceptors. ST3Gal-II (coded by the St3gal2 gene) transfers sialic acid preferentially to the three positions of galactose on the Galβ1-3GalNAc terminus of gangliosides GM1 and GD1b to synthesize GD1a and GT1b, respectively. Mice with a targeted disruption of St3gal2 unexpectedly displayed late-onset obesity and insulin resistance. At 3 months of age, St3gal2-null mice were the same weight as their wild type (WT) counterparts, but by 13 months on standard chow they were visibly obese, 22% heavier and with 37% greater fat/lean ratio than WT mice. St3gal2-null mice became hyperglycemic and displayed impaired glucose tolerance by 9 months of age. They had sharply reduced insulin responsiveness despite equivalent pancreatic islet morphology. Analyses of insulin receptor (IR) tyrosine kinase substrate IRS-1 and downstream target Akt revealed decreased insulin-induced phosphorylation in adipose tissue but not liver or skeletal muscle of St3gal2-null mice. Thin-layer chromatography and mass spectrometry revealed altered ganglioside profiles in the adipose tissue of St3gal2-null mice compared to WT littermates. Metabolically, St3gal2-null mice display a reduced respiratory exchange ratio compared to WT mice, indicating a preference for lipid oxidation as an energy source. Despite their altered metabolism, St3gal2-null mice were hyperactive. We conclude that altered ganglioside expression in adipose tissue results in diminished IR sensitivity and late-onset obesity. PMID:27683310

Genetically programmed expression of proteins containing the unnatural amino acid phenylselenocysteine

DOEpatents

Wang, Jiangyun; Schultz, Peter G.

2013-03-12

The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetase that can incorporate the unnatural amino acid phenylselenocysteine into proteins produced in eubacterial host cells such as E. coli. The invention provides, for example but not limited to, novel orthogonal aminoacyl-tRNA synthetases, polynucleotides encoding the novel sythetases molecules, methods for identifying and making the novel synthetases, methods for producing containing the unnatural amino acid phenylselenocysteine and translation systems. The invention further provides methods for producing modified proteins (e.g., lapidated proteins) through targeted modification of the phenylselenocysteine residue in a protein.

ASN1-encoded asparagine synthetase in floral organs contributes to nitrogen filling in Arabidopsis seeds.

PubMed

Gaufichon, Laure; Marmagne, Anne; Belcram, Katia; Yoneyama, Tadakatsu; Sakakibara, Yukiko; Hase, Toshiharu; Grandjean, Olivier; Clément, Gilles; Citerne, Sylvie; Boutet-Mercey, Stéphanie; Masclaux-Daubresse, Céline; Chardon, Fabien; Soulay, Fabienne; Xu, Xiaole; Trassaert, Marion; Shakiebaei, Maryam; Najihi, Amina; Suzuki, Akira

2017-08-01

Despite a general view that asparagine synthetase generates asparagine as an amino acid for long-distance transport of nitrogen to sink organs, its role in nitrogen metabolic pathways in floral organs during seed nitrogen filling has remained undefined. We demonstrate that the onset of pollination in Arabidopsis induces selected genes for asparagine metabolism, namely ASN1 (At3g47340), GLN2 (At5g35630), GLU1 (At5g04140), AapAT2 (At5g19950), ASPGA1 (At5g08100) and ASPGB1 (At3g16150), particularly at the ovule stage (stage 0), accompanied by enhanced asparagine synthetase protein, asparagine and total amino acids. Immunolocalization confined asparagine synthetase to the vascular cells of the silique cell wall and septum, but also to the outer and inner seed integuments, demonstrating the post-phloem transport of asparagine in these cells to developing embryos. In the asn1 mutant, aberrant embryo cell divisions in upper suspensor cell layers from globular to heart stages assign a role for nitrogen in differentiating embryos within the ovary. Induction of asparagine metabolic genes by light/dark and nitrate supports fine shifts of nitrogen metabolic pathways. In transgenic Arabidopsis expressing promoter Ca MV 35S ::ASN1 fusion, marked metabolomics changes at stage 0, including a several-fold increase in free asparagine, are correlated to enhanced seed nitrogen. However, specific promoter Napin2S ::ASN1 expression during seed formation and a six-fold increase in asparagine toward the desiccation stage result in wild-type seed nitrogen, underlining that delayed accumulation of asparagine impairs the timing of its use by releasing amide and amino nitrogen. Transcript and metabolite profiles in floral organs match the carbon and nitrogen partitioning to generate energy via the tricarboxylic acid cycle, GABA shunt and phosphorylated serine synthetic pathway. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

42 CFR 417.440 - Entitlement to health care services from an HMO or CMP.

Code of Federal Regulations, 2010 CFR

2010-10-01

... HEALTH AND HUMAN SERVICES (CONTINUED) MEDICARE PROGRAM HEALTH MAINTENANCE ORGANIZATIONS, COMPETITIVE... Contract § 417.440 Entitlement to health care services from an HMO or CMP. (a) Basic rules. (1) Subject to... not converted to the risk portion of the contract, may enroll in a special supplemental plan, if...

The Electronic CardioMetabolic Program (eCMP) for Patients With Cardiometabolic Risk: A Randomized Controlled Trial

PubMed Central

Koliwad, Suneil; Poon, Tak; Xiao, Lan; Lv, Nan; Griggs, Robert; Ma, Jun

2016-01-01

Background Effective lifestyle interventions targeting high-risk adults that are both practical for use in ambulatory care settings and scalable at a population management level are needed. Objective Our aim was to examine the potential effectiveness, feasibility, and acceptability of delivering an evidence-based Electronic Cardio-Metabolic Program (eCMP) for improving health-related quality of life, improving health behaviors, and reducing cardiometabolic risk factors in ambulatory care high-risk adults. Methods We conducted a randomized, wait-list controlled trial with 74 adults aged ≥18 years recruited from a large multispecialty health care organization. Inclusion criteria were (1) BMI ≥35 kg/m2 and prediabetes, previous gestational diabetes and/or metabolic syndrome, or (2) BMI ≥30 kg/m2 and type 2 diabetes and/or cardiovascular disease. Participants had a mean age of 59.7 years (SD 11.2), BMI 37.1 kg/m2 (SD 5.4) and were 59.5% female, 82.4% white. Participants were randomized to participate in eCMP immediately (n=37) or 3 months later (n=37). eCMP is a 6-month program utilizing video conferencing, online tools, and pre-recorded didactic videos to deliver evidence-based curricula. Blinded outcome assessments were conducted at 3 and 6 months postbaseline. Data were collected and analyzed between 2014 and 2015. The primary outcome was health-related quality of life. Secondary outcomes included biometric cardiometabolic risk factors (eg, body weight), self-reported diet and physical activity, mental health status, retention, session attendance, and participant satisfaction. Results Change in quality of life was not significant in both immediate and delayed participants. Both groups significantly lost weight and reduced waist circumference at 6 months, with some cardiometabolic factors trending accordingly. Significant reduction in self-reported anxiety and perceived stress was seen in the immediate intervention group at 6 months. Retention rate was 93% at 3

The Electronic CardioMetabolic Program (eCMP) for Patients With Cardiometabolic Risk: A Randomized Controlled Trial.

PubMed

Azar, Kristen M J; Koliwad, Suneil; Poon, Tak; Xiao, Lan; Lv, Nan; Griggs, Robert; Ma, Jun

2016-05-27

Effective lifestyle interventions targeting high-risk adults that are both practical for use in ambulatory care settings and scalable at a population management level are needed. Our aim was to examine the potential effectiveness, feasibility, and acceptability of delivering an evidence-based Electronic Cardio-Metabolic Program (eCMP) for improving health-related quality of life, improving health behaviors, and reducing cardiometabolic risk factors in ambulatory care high-risk adults. We conducted a randomized, wait-list controlled trial with 74 adults aged ≥18 years recruited from a large multispecialty health care organization. Inclusion criteria were (1) BMI ≥35 kg/m(2) and prediabetes, previous gestational diabetes and/or metabolic syndrome, or (2) BMI ≥30 kg/m(2) and type 2 diabetes and/or cardiovascular disease. Participants had a mean age of 59.7 years (SD 11.2), BMI 37.1 kg/m(2) (SD 5.4) and were 59.5% female, 82.4% white. Participants were randomized to participate in eCMP immediately (n=37) or 3 months later (n=37). eCMP is a 6-month program utilizing video conferencing, online tools, and pre-recorded didactic videos to deliver evidence-based curricula. Blinded outcome assessments were conducted at 3 and 6 months postbaseline. Data were collected and analyzed between 2014 and 2015. The primary outcome was health-related quality of life. Secondary outcomes included biometric cardiometabolic risk factors (eg, body weight), self-reported diet and physical activity, mental health status, retention, session attendance, and participant satisfaction. Change in quality of life was not significant in both immediate and delayed participants. Both groups significantly lost weight and reduced waist circumference at 6 months, with some cardiometabolic factors trending accordingly. Significant reduction in self-reported anxiety and perceived stress was seen in the immediate intervention group at 6 months. Retention rate was 93% at 3 months and 86% at 6 months

The intrinsic antimicrobial activity of citric acid-coated manganese ferrite nanoparticles is enhanced after conjugation with the antifungal peptide Cm-p5

PubMed Central

Lopez-Abarrategui, Carlos; Figueroa-Espi, Viviana; Lugo-Alvarez, Maria B; Pereira, Caroline D; Garay, Hilda; Barbosa, João ARG; Falcão, Rosana; Jiménez-Hernández, Linnavel; Estévez-Hernández, Osvaldo; Reguera, Edilso; Franco, Octavio L; Dias, Simoni C; Otero-Gonzalez, Anselmo J

2016-01-01

Diseases caused by bacterial and fungal pathogens are among the major health problems in the world. Newer antimicrobial therapies based on novel molecules urgently need to be developed, and this includes the antimicrobial peptides. In spite of the potential of antimicrobial peptides, very few of them were able to be successfully developed into therapeutics. The major problems they present are molecule stability, toxicity in host cells, and production costs. A novel strategy to overcome these obstacles is conjugation to nanomaterial preparations. The antimicrobial activity of different types of nanoparticles has been previously demonstrated. Specifically, magnetic nanoparticles have been widely studied in biomedicine due to their physicochemical properties. The citric acid-modified manganese ferrite nanoparticles used in this study were characterized by high-resolution transmission electron microscopy, which confirmed the formation of nanocrystals of approximately 5 nm diameter. These nanoparticles were able to inhibit Candida albicans growth in vitro. The minimal inhibitory concentration was 250 µg/mL. However, the nanoparticles were not capable of inhibiting Gram-negative bacteria (Escherichia coli) or Gram-positive bacteria (Staphylococcus aureus). Finally, an antifungal peptide (Cm-p5) from the sea animal Cenchritis muricatus (Gastropoda: Littorinidae) was conjugated to the modified manganese ferrite nanoparticles. The antifungal activity of the conjugated nanoparticles was higher than their bulk counterparts, showing a minimal inhibitory concentration of 100 µg/mL. This conjugate proved to be nontoxic to a macrophage cell line at concentrations that showed antimicrobial activity. PMID:27563243

Genetic and Immunological Studies of Bacteriophage T4 Thymidylate Synthetase

PubMed Central

Krauss, S. W.; Stollar, B. D.; Friedkin, M.

1973-01-01

Thymidylate synthetase, which appears after infection of Escherichia coli with bacteriophage T4, has been partially purified. The phage enzyme is immunologically distinct from the host enzyme and has a molecular weight of 50,000 in comparison to 68,000 for the host enzyme. A system has been developed to characterize T4 td mutants previously known to have impaired expression of phage thymidylate synthetase. For this system, an E. coli host lacking thymidylate synthetase was isolated. Known genetic suppressors were transduced into this host. The resulting isogenic hosts were infected with phage T4 td mutants. The specific activities and amounts of cross-reacting material induced by several different types of phage mutants under conditions of suppression or non-suppression have been examined. The results show that the phage carries the structural gene specifying the thymidylate synthetase which appears after phage infection, and that the combination of plaque morphology, enzyme activity assays, and an assay for immunologically cross-reacting material provides a means for identifying true amber mutants of the phage gene. Images PMID:4575286

Acyl CoA synthetase 5 (ACSL5) ablation in mice increases energy expenditure and insulin sensitivity and delays fat absorption

USDA-ARS?s Scientific Manuscript database

Objective: The family of acyl-CoA synthetase enzymes (ACSL) activates fatty acids within cells to generate long chain fatty acyl CoA (FACoA). The differing metabolic fates of FACoAs such as incorporation into neutral lipids, phospholipids, and oxidation pathways are differentially regulated by the ...

Genetic Validation of Aminoacyl-tRNA Synthetases as Drug Targets in Trypanosoma brucei

PubMed Central

Kalidas, Savitha; Cestari, Igor; Monnerat, Severine; Li, Qiong; Regmi, Sandesh; Hasle, Nicholas; Labaied, Mehdi; Parsons, Marilyn; Stuart, Kenneth

2014-01-01

Human African trypanosomiasis (HAT) is an important public health threat in sub-Saharan Africa. Current drugs are unsatisfactory, and new drugs are being sought. Few validated enzyme targets are available to support drug discovery efforts, so our goal was to obtain essentiality data on genes with proven utility as drug targets. Aminoacyl-tRNA synthetases (aaRSs) are known drug targets for bacterial and fungal pathogens and are required for protein synthesis. Here we survey the essentiality of eight Trypanosoma brucei aaRSs by RNA interference (RNAi) gene expression knockdown, covering an enzyme from each major aaRS class: valyl-tRNA synthetase (ValRS) (class Ia), tryptophanyl-tRNA synthetase (TrpRS-1) (class Ib), arginyl-tRNA synthetase (ArgRS) (class Ic), glutamyl-tRNA synthetase (GluRS) (class 1c), threonyl-tRNA synthetase (ThrRS) (class IIa), asparaginyl-tRNA synthetase (AsnRS) (class IIb), and phenylalanyl-tRNA synthetase (α and β) (PheRS) (class IIc). Knockdown of mRNA encoding these enzymes in T. brucei mammalian stage parasites showed that all were essential for parasite growth and survival in vitro. The reduced expression resulted in growth, morphological, cell cycle, and DNA content abnormalities. ThrRS was characterized in greater detail, showing that the purified recombinant enzyme displayed ThrRS activity and that the protein localized to both the cytosol and mitochondrion. Borrelidin, a known inhibitor of ThrRS, was an inhibitor of T. brucei ThrRS and showed antitrypanosomal activity. The data show that aaRSs are essential for T. brucei survival and are likely to be excellent targets for drug discovery efforts. PMID:24562907

High fat diet-induced inflammation and oxidative stress are attenuated by N-acetylneuraminic acid in rats.

PubMed

Yida, Zhang; Imam, Mustapha Umar; Ismail, Maznah; Ismail, Norsharina; Ideris, Aini; Abdullah, Maizaton Atmadini

2015-10-24

Serum sialic acid levels are positively correlated with coronary artery disease and inflammation. Although sialic acid is a non-specific marker, it is considered sensitive likely due to its influence in sialylation of glycoprotein structures all over the body. We hypothesized that dietary supplementation with N-acetylneuraminic acid (Neu5Ac), a type of sialic acid, will have profound effects on high fat diet- (HFD-) induced inflammation and oxidative stress in view of the widespread incorporation of sialic acid into glycoprotein structures in the body. HFD-fed rats with or without simvastatin or Neu5Ac (50 and 400 mg/kg/day) were followed up for 12 weeks. Lipid profiles, and markers of inflammation (C-reactive protein, interleukin-6, and tumor necrosis factor alpha), insulin resistance (serum insulin and adiponectin, oral glucose tolerance test and homeostatic model of insulin resistance) and oxidative stress (total antioxidant status and thiobarbituric acid reactive species) in the serum and liver were determined, while mRNA levels of hepatic antioxidant and inflammation genes were also quantified. Serum levels of alanine transaminase, aspartate transaminase, alkaline phosphatase, urea, creatinine and uric acid were also assessed. HFD feeding caused hyperlipidemia and insulin resistance, and worsened liver and kidney functions. HFD feeding also potentiated inflammation and oxidative stress, partly through modulation of hepatic gene expression, while Neu5Ac especially at higher doses and simvastatin attenuated HFD-induced changes, although Neu5Ac showed better outcomes. Based on the present results, we surmised that Neu5Ac can prevent HFD-induced inflammation and oxidative stress, and may in fact be useful in the prevention of hyperlipidemia-associated inflammation and oxidative stress. However, the translational implications of these findings can only be determined after long-term effects are established. Hence, the use of Neu5Ac on obesity-related diseases

Genetics Home Reference: holocarboxylase synthetase deficiency

MedlinePlus

... holocarboxylase synthetase deficiency Orphanet: Multiple carboxylase deficiency Screening, Technology, and Research in Genetics Virginia Department of Health (PDF) Patient Support and Advocacy Resources (3 links) Children Living with Inherited Metabolic Diseases Organic Acidemia Association ...

Nucleotide synthetase ribozymes may have emerged first in the RNA world

PubMed Central

Ma, Wentao; Yu, Chunwu; Zhang, Wentao; Hu, Jiming

2007-01-01

Though the “RNA world” hypothesis has gained a central role in ideas concerning the origin of life, the scenario concerning its emergence remains uncertain. It has been speculated that the first scene may have been the emergence of a template-dependent RNA synthetase ribozyme, which catalyzed its own replication: thus, “RNA replicase.” However, the speculation remains uncertain, primarily because of the large sequence length requirement of such a replicase and the lack of a convincing mechanism to ensure its self-favoring features. Instead, we propose a nucleotide synthetase ribozyme as an alternative candidate, especially considering recent experimental evidence suggesting the possibility of effective nonenzymatic template-directed synthesis of RNA. A computer simulation was conducted to support our proposal. The conditions for the emergence of the nucleotide synthetase ribozyme are discussed, based on dynamic analysis on a computer. We suggest the template-dependent RNA synthetase ribozyme emerged later, perhaps after the emergence of protocells. PMID:17878321

[Improving the CMP appointment waiting time for children and adolescents].

PubMed

Cani, Pascale

2014-01-01

The increasing activity of mental health centres for children and adolescents and longer waiting times in obtaining a first appointment have led an area of child psychiatry to question the organisation of new consultation applications. Two CMP in the sector had a waiting period of over 40 days for half of the patients. Two improvement actions were implemented:the implementation of organisation and reception nurses and the development of a new applications management process. The evaluation after one year showed a decrease of half of the appointment waiting time without changing the non showed up rate.

Cylindrospermopsin and Saxitoxin Synthetase Genes in Cylindrospermopsis raciborskii Strains from Brazilian Freshwater

PubMed Central

Hoff-Risseti, Caroline; Dörr, Felipe Augusto; Schaker, Patricia Dayane Carvalho; Pinto, Ernani; Werner, Vera Regina; Fiore, Marli Fatima

2013-01-01

The Cylindrospermopsis raciborskii population from Brazilian freshwater is known to produce saxitoxin derivatives (STX), while cylindrospermopsin (CYN), which is commonly detected in isolates from Australia and Asia continents, has thus far not been detected in South American strains. However, during the investigation for the presence of cyrA, cyrB, cyrC and cyrJ CYN synthetase genes in the genomes of four laboratory-cultured C. raciborskii Brazilian strains, the almost complete cyrA gene sequences were obtained for all strains, while cyrB and cyrC gene fragments were observed in two strains. These nucleotide sequences were translated into amino acids, and the predicted protein functions and domains confirmed their identity as CYN synthetase genes. Attempts to PCR amplify cyrJ gene fragments from the four strains were unsuccessful. Phylogenetic analysis grouped the nucleotide sequences together with their homologues found in known CYN synthetase clusters of C. raciborskii strains with high bootstrap support. In addition, fragments of sxtA, sxtB and sxtI genes involved in STX production were also obtained. Extensive LC-MS analyses were unable to detect CYN in the cultured strains, whereas the production of STX and its analogues was confirmed in CENA302, CENA305 and T3. To our knowledge, this is the first study reporting the presence of cyr genes in South American strains of C. raciborskii and the presence of sxt and cyr genes in a single C. raciborskii strain. This discovery suggests a shift in the type of cyanotoxin production over time of South American strains of C. raciborskii and contributes to the reconstruction of the evolutionary history and diversification of cyanobacterial toxins. PMID:24015317

Treatment of renal colic by prostaglandin synthetase inhibitors and avafortan (analgesic antispasmodic).

PubMed

el-Sherif, A E; Foda, R; Norlen, L J; Yahia, H

1990-12-01

In a study of the pain-relieving effect of 3 drugs commonly used to treat acute renal colic in this hospital, intravenous indomethacin and intramuscular diclofenac (prostaglandin synthetase inhibitors) were compared with intravenous Avafortan (analgesic antispasmodic). As first-line analgesics, prostaglandin synthetase inhibitors, if given intravenously, offer an effective alternative to Avafortan. Of 145 patients studied, 32 required a second injection for complete relief of pain. Administering a second dose of prostaglandin synthetase inhibitors resulted in equally significant pain relief rate even though the route was intramuscular.

A core of three amino acids at the carboxyl-terminal region of glutamine synthetase defines its regulation in cyanobacteria.

PubMed

Saelices, Lorena; Robles-Rengel, Rocío; Florencio, Francisco J; Muro-Pastor, M Isabel

2015-05-01

Glutamine synthetase (GS) type I is a key enzyme in nitrogen metabolism, and its activity is finely controlled by cellular carbon/nitrogen balance. In cyanobacteria, a reversible process that involves protein-protein interaction with two proteins, the inactivating factors IF7 and IF17, regulates GS. Previously, we showed that three arginine residues of IFs are critical for binding and inhibition of GS. In this work, taking advantage of the specificity of GS/IFs interaction in the model cyanobacteria Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120, we have constructed a different chimeric GSs from these two cyanobacteria. Analysis of these proteins, together with a site-directed mutagenesis approach, indicates that a core of three residues (E419, N456 and R459) is essential for the inactivation process. The three residues belong to the last 56 amino acids of the C-terminus of Synechocystis GS. A protein-protein docking modeling of Synechocystis GS in complex with IF7 supports the role of the identified core for GS/IF interaction. © 2015 John Wiley & Sons Ltd.

Differential inhibition of adenylylated and deadenylylated forms of M. tuberculosis glutamine synthetase as a drug discovery platform

PubMed Central

Theron, A.; Roth, R. L.; Hoppe, H.; Parkinson, C.; van der Westhuyzen, C. W.; Stoychev, S.; Wiid, I.; Pietersen, R. D.; Baker, B.

2017-01-01

Glutamine synthetase is a ubiquitous central enzyme in nitrogen metabolism that is controlled by up to four regulatory mechanisms, including adenylylation of some or all of the twelve subunits by adenylyl transferase. It is considered a potential therapeutic target for the treatment of tuberculosis, being essential for the growth of Mycobacterium tuberculosis, and is found extracellularly only in the pathogenic Mycobacterium strains. Human glutamine synthetase is not regulated by the adenylylation mechanism, so the adenylylated form of bacterial glutamine synthetase is of particular interest. Previously published reports show that, when M. tuberculosis glutamine synthetase is expressed in Escherichia coli, the E. coli adenylyl transferase does not optimally adenylylate the M. tuberculosis glutamine synthetase. Here, we demonstrate the production of soluble adenylylated M. tuberulosis glutamine synthetase in E. coli by the co-expression of M. tuberculosis glutamine synthetase and M. tuberculosis adenylyl transferase. The differential inhibition of adenylylated M. tuberulosis glutamine synthetase and deadenylylated M. tuberulosis glutamine synthetase by ATP based scaffold inhibitors are reported. Compounds selected on the basis of their enzyme inhibition were also shown to inhibit M. tuberculosis in the BACTEC 460TB™ assay as well as the intracellular inhibition of M. tuberculosis in a mouse bone-marrow derived macrophage assay. PMID:28972974

A 4'-phosphopantetheinyl transferase mediates non-ribosomal peptide synthetase activation in Aspergillus fumigatus.

PubMed

Neville, Claire; Murphy, Alan; Kavanagh, Kevin; Doyle, Sean

2005-04-01

Aspergillus fumigatus is a significant human pathogen. Non-ribosomal peptide (NRP) synthesis is thought to be responsible for a significant proportion of toxin and siderophore production in the organism. Furthermore, it has been shown that 4'-phosphopantetheinylation is required for the activation of key enzymes involved in non-ribosomal peptide synthesis in other species. Here we report the cloning, recombinant expression and functional characterisation of a 4'-phosphopantetheinyl transferase from A. fumigatus and the identification of an atypical NRP synthetase (Afpes1), spanning 14.3 kb. Phylogenetic analysis has shown that the NRP synthetase exhibits greatest identity to NRP synthetases from Metarhizium anisolpiae (PesA) and Alternaria brassicae (AbrePsy1). Northern hybridisation and RT-PCR analysis have confirmed that both genes are expressed in A. fumigatus. A 120 kDa fragment of the A. fumigatus NRP synthetase, containing a putative thiolation domain, was cloned and expressed in the baculovirus expression system. Detection of a 4'-phosphopantetheinylated peptide (SFSAMK) from this protein, by MALDI-TOF mass spectrometric analysis after coincubation of the 4'-phosphopantetheinyl transferase with the recombinant NRP synthetase fragment and acetyl CoA, confirms that it is competent to play a role in NRP synthetase activation in A. fumigatus. The 4'-phosphopantetheinyl transferase also activates, by 4'-phosphopantetheinylation, recombinant alpha-aminoadipate reductase (Lys2p) from Candida albicans, a key enzyme involved in lysine biosynthesis.

Introduction of an Aliphatic Ketone into Recombinant Proteins in a Bacterial Strain that Overexpresses an Editing-Impaired Leucyl-tRNA Synthetase

PubMed Central

Tang, Yi; Wang, Pin; Van Deventer, James A.; Link, A. James; Tirrell, David A.

2011-01-01

A leucine analog containing a ketone has been incorporated into proteins in E. coli. Only E. coli strains overexpressing an editing-deficient leucyl-tRNA synthetase were capable of synthesizing proteins with the aliphatic ketone amino acid. Modification of ketone-containing proteins under mild conditions has been demonstrated. PMID:19670197

Atomistic scale nanoscratching behavior of monocrystalline Cu influenced by water film in CMP process

NASA Astrophysics Data System (ADS)

Shi, Junqin; Chen, Juan; Fang, Liang; Sun, Kun; Sun, Jiapeng; Han, Jing

2018-03-01

The effect of water film on the nanoscratching behavior of monocrystalline Cu was studied by molecular dynamics (MD) simulation. The results indicate that the friction force acting on abrasive particle increases due to the resistance of water film accumulating ahead of particle, but the water film with lubrication decreases friction force acting on Cu surface. The accumulation of water molecules around particle causes the anisotropy of ridge and the surface damage around the groove, and the water molecules remaining in the groove lead to the non-regular groove structure. The dislocation evolution displays the re-organization of the dislocation network in the nanoscratching process. The evaluation of removal efficiency shows the number of removed Cu atoms decreases with water film thickness. It is considered that an appropriate rather than a high removal efficiency should be adopted to evaluate the polishing process in real (chemical mechanical polishing) CMP. These results are helpful to reveal the polishing mechanism under the effect of water film from physical perspective, which benefits the development of ultra-precision manufacture and miniaturized components, as well as the innovation of CMP technology.

Two-dimensional cross correlation analysis of protein unfolding: Portrayal of the thermal denaturation of CMP kinases in the absence and presence of substrates

NASA Astrophysics Data System (ADS)

Schultz, Christian P.; Bârzu, Octavian; Mantsch, Henry H.

2000-03-01

The functional role of CMP kinases is to regenerate mono-phosphate nucleotides in cells by transferring phosphate residues from tri-phosphorylated nucleotides to monophosphorylated nucleotides. These enzymes possess two binding sites and maintain a highly conserved secondary structure. They are essential for cell survival. Herein we compare the infrared spectra of two similar, but not identical enzymes, the CMP kinases from Escherichia coli and Bacillus subtilis. A two-dimensional cross correlation analysis of the infrared spectra reveals differences in the denaturation behavior of the two proteins. Different secondary structure elements show different time-delayed or advanced unfolding events in the two enzymes. When bound to the active sites, the two nucleotide-substrates CMP and ATP exert a stabilizing effect on the structure of both proteins. The changes observed upon thermal denaturation are different for the two enzymes. Model 2D correlations are used to simulate the different denaturation of the two enzymes. Thermal denaturation and aggregation can be distinguished as two processes separated in time.

Increased Long Chain acyl-Coa Synthetase Activity and Fatty Acid Import Is Linked to Membrane Synthesis for Development of Picornavirus Replication Organelles

PubMed Central

Scott, Alison J.; Ford, Lauren A.; Pei, Zhengtong; Watkins, Paul A.; Ernst, Robert K.; Belov, George A.

2013-01-01

All positive strand (+RNA) viruses of eukaryotes replicate their genomes in association with membranes. The mechanisms of membrane remodeling in infected cells represent attractive targets for designing future therapeutics, but our understanding of this process is very limited. Elements of autophagy and/or the secretory pathway were proposed to be hijacked for building of picornavirus replication organelles. However, even closely related viruses differ significantly in their requirements for components of these pathways. We demonstrate here that infection with diverse picornaviruses rapidly activates import of long chain fatty acids. While in non-infected cells the imported fatty acids are channeled to lipid droplets, in infected cells the synthesis of neutral lipids is shut down and the fatty acids are utilized in highly up-regulated phosphatidylcholine synthesis. Thus the replication organelles are likely built from de novo synthesized membrane material, rather than from the remodeled pre-existing membranes. We show that activation of fatty acid import is linked to the up-regulation of cellular long chain acyl-CoA synthetase activity and identify the long chain acyl-CoA syntheatse3 (Acsl3) as a novel host factor required for polio replication. Poliovirus protein 2A is required to trigger the activation of import of fatty acids independent of its protease activity. Shift in fatty acid import preferences by infected cells results in synthesis of phosphatidylcholines different from those in uninfected cells, arguing that the viral replication organelles possess unique properties compared to the pre-existing membranes. Our data show how poliovirus can change the overall cellular membrane homeostasis by targeting one critical process. They explain earlier observations of increased phospholipid synthesis in infected cells and suggest a simple model of the structural development of the membranous scaffold of replication complexes of picorna-like viruses, that may be

Trans-oligomerization of duplicated aminoacyl-tRNA synthetases maintains genetic code fidelity under stress.

PubMed

Rubio, Miguel Ángel; Napolitano, Mauro; Ochoa de Alda, Jesús A G; Santamaría-Gómez, Javier; Patterson, Carl J; Foster, Andrew W; Bru-Martínez, Roque; Robinson, Nigel J; Luque, Ignacio

2015-11-16

Aminoacyl-tRNA synthetases (aaRSs) play a key role in deciphering the genetic message by producing charged tRNAs and are equipped with proofreading mechanisms to ensure correct pairing of tRNAs with their cognate amino acid. Duplicated aaRSs are very frequent in Nature, with 25,913 cases observed in 26,837 genomes. The oligomeric nature of many aaRSs raises the question of how the functioning and oligomerization of duplicated enzymes is organized. We characterized this issue in a model prokaryotic organism that expresses two different threonyl-tRNA synthetases, responsible for Thr-tRNA(Thr) synthesis: one accurate and constitutively expressed (T1) and another (T2) with impaired proofreading activity that also generates mischarged Ser-tRNA(Thr). Low zinc promotes dissociation of dimeric T1 into monomers deprived of aminoacylation activity and simultaneous induction of T2, which is active for aminoacylation under low zinc. T2 either forms homodimers or heterodimerizes with T1 subunits that provide essential proofreading activity in trans. These findings evidence that in organisms with duplicated genes, cells can orchestrate the assemblage of aaRSs oligomers that meet the necessities of the cell in each situation. We propose that controlled oligomerization of duplicated aaRSs is an adaptive mechanism that can potentially be expanded to the plethora of organisms with duplicated oligomeric aaRSs. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Bacillus anthracis o-succinylbenzoyl-CoA synthetase: reaction kinetics and a novel inhibitor mimicking its reaction intermediate †

PubMed Central

Tian, Yang; Suk, Dae-Hwan; Cai, Feng; Crich, David; Mesecar, Andrew D.

2009-01-01

O-succinylbenzoyl-CoA (OSB-CoA) synthetase (EC 6.2.1.26) catalyzes the ATP-dependent condensation of o-succinylbenzoate (OSB) and CoA to form OSB-CoA, the fourth step of the menaquinone biosynthetic pathway in Bacillus anthracis. Gene knockout studies have highlighted this enzyme as a potential target for the discovery of new antibiotics. Here we report the first studies on the kinetic mechanism of B. anthracis OSB-CoA synthetase, classifying it as an ordered Bi Uni Uni Bi ping-pong mechanism. Through a series of pre-steady-state and steady-state kinetic studies in conjunction with direct-binding studies, it is demonstrated that CoA, the last substrate to bind, strongly activates the first half-reaction after the first round of turnover. The activation of the first-half reaction is most likely achieved by CoA stabilizing conformations of the enzyme in the ‘F’ form, which slowly isomerize back to the E form. Thus, the kinetic mechanism of OSB-CoA synthetase may be more accurately described as an ordered Bi Uni Uni Bi Iso ping-pong mechanism. The substrate specificity of OSB-CoA synthetase was probed using a series of OSB analogs with alterations in the carboxylate groups. OSB-CoA shows a strong preference for OSB over all of the analogs tested as none were active except 4-(2-trifluoromethylphenyl)-4-oxobutyric acid which exhibited a 100-fold decrease in kcat/Km. Based on an understanding of OSB-CoA synthetase’s kinetic mechanism and substrate specificity, a reaction intermediate analog of OSB-AMP, 5’-O-(N-(2-trifluoromethylphenyl)-4-oxobutyl) adenosine sulfonamide (TFMP-butyl-AMS), was designed and synthesized. This inhibitor was found to be an uncompetitive inhibitor to CoA and a mixed-type inhibitor to ATP and OSB with low micromolar inhibition constants. Collectively, these results should serve as an important forerunner to more detailed and extensive inhibitor design studies aimed at developing lead compounds against the OSB-CoA synthetase class of

Antiviral activity of human oligoadenylate synthetases-like (OASL) is mediated by enhancing retinoic acid-inducible gene I (RIG-I) signaling

PubMed Central

Zhu, Jianzhong; Zhang, Yugen; Ghosh, Arundhati; Cuevas, Rolando A.; Forero, Adriana; Dhar, Jayeeta; Ibsen, Mikkel Søes; Schmid-Burgk, Jonathan Leo; Schmidt, Tobias; Ganapathiraju, Madhavi K.; Fujita, Takashi; Hartmann, Rune; Barik, Sailen; Hornung, Veit; Coyne, Carolyn B.; Sarkar, Saumendra N.

2014-01-01

SUMMARY Virus infection is sensed in the cytoplasm by retinoic acid-inducible gene I (RIG-I, also known as DDX58), which requires RNA and polyubiquitin binding to induce type I interferon (IFN), and activate cellular innate immunity. We show that the human IFN-inducible oligoadenylate synthetases-like (OASL) protein had antiviral activity and mediated RIG-I activation by mimicking polyubiquitin. Loss of OASL expression reduced RIG-I signaling and enhanced virus replication in human cells. Conversely, OASL expression suppressed replication of a number of viruses in a RIG-I-dependent manner and enhanced RIG-I-mediated IFN induction. OASL interacted and colocalized with RIG-I, and through its C-terminal ubiquitin-like domain specifically enhanced RIG-I signaling. Bone marrow derived macrophages from mice deficient for Oasl2 showed that among the two mouse orthologs of human OASL; Oasl2 is functionally similar to human OASL. Our findings show a mechanism by which human OASL contributes to host antiviral responses by enhancing RIG-I activation. PMID:24931123

Site-directed mutagenesis reveals transition-state stabilization as a general catalytic mechanism for aminoacyl-tRNA synthetases.

PubMed

Borgford, T J; Gray, T E; Brand, N J; Fersht, A R

1987-11-17

Some aminoacyl-tRNA synthetases of almost negligible homology do have a small region of similarity around four-residue sequence His-Ile(or Leu or Met)-Gly-His(or Asn), the HIGH sequence. The first histidine in this sequence in the tyrosyl-tRNA synthetase, His-45, has been shown to form part of a binding site for the gamma-phosphate of ATP in the transition state for the reaction as does Thr-40. Residue His-56 in the valyl-tRNA synthetase begins a HIGH sequence, and there is a threonine at position 52, one position closer to the histidine than in the tyrosyl-tRNA synthetase. The mutants Thr----Ala-52 and His----Asn-56 have been made and their complete free energy profiles for the formation of valyl adenylate determined. Difference energy diagrams have been constructed by comparison with the reaction of wild-type enzyme. The difference energy profiles are very similar to those for the mutants Thr----Ala-40 and His----Asn-45 of the tyrosyl-tRNA synthetase. Thr-52 and His-56 of the valyl-tRNA synthetase contribute little binding energy to valine, ATP, and Val-AMP. Instead, the wild-type enzyme binds the transition state and pyrophosphate some 6 kcal/mol more tightly than do the mutants. Preferential transition-state stabilization is thus an important component of catalysis by the valyl-tRNA synthetase. Further, by analogy to the tyrosyl-tRNA synthetase, the valyl-tRNA synthetase has a binding site for the gamma-phosphate of ATP in the transition state, and this is likely to be a general feature of aminoacyl-tRNA synthetases that have a HIGH region.

Twoplex 12/13 C6 aniline stable isotope and linkage-specific sialic acid labeling 2D-LC-MS workflow for quantitative N-glycomics.

PubMed

Albrecht, Simone; Mittermayr, Stefan; Smith, Josh; Martín, Silvia Millán; Doherty, Margaret; Bones, Jonathan

2017-01-01

Quantitative glycomics represents an actively expanding research field ranging from the discovery of disease-associated glycan alterations to the quantitative characterization of N-glycans on therapeutic proteins. Commonly used analytical platforms for comparative relative quantitation of complex glycan samples include MALDI-TOF-MS or chromatographic glycan profiling with subsequent data alignment and statistical evaluation. Limitations of such approaches include run-to-run technical variation and the potential introduction of subjectivity during data processing. Here, we introduce an offline 2D LC-MS E workflow for the fractionation and relative quantitation of twoplex isotopically labeled N-linked oligosaccharides using neutral 12 C 6 and 13 C 6 aniline (Δmass = 6 Da). Additional linkage-specific derivatization of sialic acids using 4-(4,6-dimethoxy-1,3,5-trizain-2-yl)-4-methylmorpholinium chloride offered simultaneous and advanced in-depth structural characterization. The potential of the method was demonstrated for the differential analysis of structurally defined N-glycans released from serum proteins of patients diagnosed with various stages of colorectal cancer. The described twoplex 12 C 6 / 13 C 6 aniline 2D LC-MS platform is ideally suited for differential glycomic analysis of structurally complex N-glycan pools due to combination and analysis of samples in a single LC-MS injection and the associated minimization in technical variation. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Characterization of Clostridium difficile Spores Lacking Either SpoVAC or Dipicolinic Acid Synthetase

PubMed Central

Donnelly, M. Lauren; Fimlaid, Kelly A.

2016-01-01

ABSTRACT The spore-forming obligate anaerobe Clostridium difficile is a leading cause of antibiotic-associated diarrhea around the world. In order for C. difficile to cause infection, its metabolically dormant spores must germinate in the gastrointestinal tract. During germination, spores degrade their protective cortex peptidoglycan layers, release dipicolinic acid (DPA), and hydrate their cores. In C. difficile, cortex hydrolysis is necessary for DPA release, whereas in Bacillus subtilis, DPA release is necessary for cortex hydrolysis. Given this difference, we tested whether DPA synthesis and/or release was required for C. difficile spore germination by constructing mutations in either spoVAC or dpaAB, which encode an ion channel predicted to transport DPA into the forespore and the enzyme complex predicted to synthesize DPA, respectively. C. difficile spoVAC and dpaAB mutant spores lacked DPA but could be stably purified and were more hydrated than wild-type spores; in contrast, B. subtilis spoVAC and dpaAB mutant spores were unstable. Although C. difficile spoVAC and dpaAB mutant spores exhibited wild-type germination responses, they were more readily killed by wet heat. Cortex hydrolysis was not affected by this treatment, indicating that wet heat inhibits a stage downstream of this event. Interestingly, C. difficile spoVAC mutant spores were significantly more sensitive to heat treatment than dpaAB mutant spores, indicating that SpoVAC plays additional roles in conferring heat resistance. Taken together, our results demonstrate that SpoVAC and DPA synthetase control C. difficile spore resistance and reveal differential requirements for these proteins among the Firmicutes. IMPORTANCE Clostridium difficile is a spore-forming obligate anaerobe that causes ∼500,000 infections per year in the United States. Although spore germination is essential for C. difficile to cause disease, the factors required for this process have been only partially characterized

A Repetitive DNA Element Regulates Expression of the Helicobacter pylori Sialic Acid Binding Adhesin by a Rheostat-like Mechanism

PubMed Central

Vallström, Anna; Olofsson, Annelie; Öhman, Carina; Rakhimova, Lena; Borén, Thomas; Engstrand, Lars; Brännström, Kristoffer; Arnqvist, Anna

2014-01-01

During persistent infection, optimal expression of bacterial factors is required to match the ever-changing host environment. The gastric pathogen Helicobacter pylori has a large set of simple sequence repeats (SSR), which constitute contingency loci. Through a slipped strand mispairing mechanism, the SSRs generate heterogeneous populations that facilitate adaptation. Here, we present a model that explains, in molecular terms, how an intergenically located T-tract, via slipped strand mispairing, operates with a rheostat-like function, to fine-tune activity of the promoter that drives expression of the sialic acid binding adhesin, SabA. Using T-tract variants, in an isogenic strain background, we show that the length of the T-tract generates multiphasic output from the sabA promoter. Consequently, this alters the H. pylori binding to sialyl-Lewis x receptors on gastric mucosa. Fragment length analysis of post-infection isolated clones shows that the T-tract length is a highly variable feature in H. pylori. This mirrors the host-pathogen interplay, where the bacterium generates a set of clones from which the best-fit phenotypes are selected in the host. In silico and functional in vitro analyzes revealed that the length of the T-tract affects the local DNA structure and thereby binding of the RNA polymerase, through shifting of the axial alignment between the core promoter and UP-like elements. We identified additional genes in H. pylori, with T- or A-tracts positioned similar to that of sabA, and show that variations in the tract length likewise acted as rheostats to modulate cognate promoter output. Thus, we propose that this generally applicable mechanism, mediated by promoter-proximal SSRs, provides an alternative mechanism for transcriptional regulation in bacteria, such as H. pylori, which possesses a limited repertoire of classical trans-acting regulatory factors. PMID:24991812

Deoxyribonucleic Acid Replication and Expression of Early and Late Bacteriophage Functions in Bacillus subtilis

PubMed Central

Pène, Jacques J.; Marmur, Julius

1967-01-01

The role of deoxyribonucleic acid (DNA) replication in the control of the synthesis of deoxycytidylate (dCMP) deaminase and lysozyme in Bacillus subtilis infected with bacteriophage 2C has been studied. These phage-induced enzymes are synthesized at different times during the latent period. It was shown by actinomycin inhibition that the formation of the late enzyme (lysozyme) required messenger ribonucleic acid (mRNA) synthesized de novo after the initiation of translation of mRNA which specifies the early function (dCMP deaminase). The inhibition of phage DNA synthesis by mitomycin C prevented the synthesis of lysozyme only when added before the onset of phage DNA replication, but it did not affect the synthesis or action of dCMP deaminase when added at any time during the latent period. Treatment of infected cells with mitomycin C after phage DNA synthesis had reached 8 to 10% of its maximal rate resulted in the production of normal amounts of lysozyme. These observations suggest that mRNA specifying early enzymes can be transcribed from parental (and probably also from progeny) DNA, whereas late functional messengers can be transcribed only after the formation of progeny DNA. PMID:4990039

Loss of Sialic Acid Binding Domain Redirects Protein σ1 to Enhance M Cell-Directed Vaccination

PubMed Central

Zlotkowska, Dagmara; Maddaloni, Massimo; Riccardi, Carol; Walters, Nancy; Holderness, Kathryn; Callis, Gayle; Rynda-Apple, Agnieszka; Pascual, David W.

2012-01-01

Ovalbumin (OVA) genetically fused to protein sigma 1 (pσ1) results in tolerance to both OVA and pσ1. Pσ1 binds in a multi-step fashion, involving both protein- and carbohydrate-based receptors. To assess the relative pσ1 components responsible for inducing tolerance and the importance of its sialic binding domain (SABD) for immunization, modified OVA-pσ1, termed OVA-pσ1(short), was deleted of its SABD, but with its M cell targeting moiety intact, and was found to be immunostimulatory and enhanced CD4+ and CD8+ T cell proliferation. When used to nasally immunize mice given with and without cholera toxin (CT) adjuvant, elevated SIgA and serum IgG responses were induced, and OVA-pσ1(s) was more efficient for immunization than native OVA+CT. The immune antibodies (Abs) were derived from elevated Ab-forming cells in the upper respiratory tissues and submaxillary glands and were supported by mixed Th cell responses. Thus, these studies show that pσ1(s) can be fused to vaccines to effectively elicit improved SIgA responses. PMID:22558374

Electrochemical characterization of bulk and thin film copper in ammonia- and nitric acid-based slurries for chemical mechanical planarization of interconnects

NASA Astrophysics Data System (ADS)

Sainio, Carlyn Anne

Copper will be replacing aluminum as the interconnect material in silicon integrated circuits. Chemical mechanical planarization (CMP) in combination with an inlaid metal interconnection scheme has been utilized to pattern copper interconnects. The thesis describes an attempt to understand the electrochemistry of copper in slurries used for CMP. Steady-state electrochemical potential measurements, linear polarization resistance determinations, and potentiodynamic and potentiostatic polarization scans have been used in order to characterize the mechanism by which copper is removed during CMP. Electrochemical measurements were implemented on a rotating disk assembly to simulate conditions during CMP. Experiments were performed on both bulk copper samples and blanket copper thin films sputter deposited onto silicon wafers. Electrochemical potential measurements have been used in conjunction with potential-pH diagrams to determine the possible copper species which are stable during CMP. Electrochemical results were correlated to CMP experiments to determine slurry compositions with optimum potential-pH ranges for copper planarization. The results indicate that such studies present an opportunity to isolate the electrochemical and chemical effects from the mechanical effects in the CMP of metals and to determine the dependencies of each of these effects on the other. CMP of copper was controlled by the removal of native or non-native surface films. High CMP rates were achieved by matching the rates of film formation and copper and film dissolution. During CMP, surface films are abraded, allowing increased dissolution of copper until the surface film reforms. When the surface was indented by abrasive particles, the corrosion rate of the exposed copper increased by two orders of magnitude. Etchants (i.e. ammonia or nitric acid) were necessary for high CMP rates (120-240 nm/min) and to minimize scratching. CMP rates of copper in 1 volume percent NHsb4OH and 0.7 volume

Mechanism-based inhibitors of MenE, an acyl-CoA synthetase involved in bacterial menaquinone biosynthesis†

PubMed Central

Lu, Xuequan; Zhang, Huaning; Tonge, Peter J.; Tan, Derek S.

2008-01-01

Menaquinone (vitamin K2) is an essential component of the electron transfer chain in many pathogens, including Mycobacterium tuberculosis and Staphylococcus aureus, and menaquinone biosynthesis is a potential target for antibiotic drug discovery. We report herein a series of mechanism-based inhibitors of MenE, an acyl-CoA synthetase that catalyzes adenylation and thioesterification of o-succinylbenzoic acid (OSB) during menaquinone biosynthesis. The most potent compound inhibits MenE with an IC50 value of 5.7 μM. PMID:18762421

Structural Biology of Non-Ribosomal Peptide Synthetases

PubMed Central

Miller, Bradley R.; Gulick, Andrew M.

2016-01-01

Summary The non-ribosomal peptide synthetases are modular enzymes that catalyze synthesis of important peptide products from a variety of standard and non-proteinogenic amino acid substrates. Within a single module are multiple catalytic domains that are responsible for incorporation of a single residue. After the amino acid is activated and covalently attached to an integrated carrier protein domain, the substrates and intermediates are delivered to neighboring catalytic domains for peptide bond formation or, in some modules, chemical modification. In the final module, the peptide is delivered to a terminal thioesterase domain that catalyzes release of the peptide product. This multi-domain modular architecture raises questions about the structural features that enable this assembly line synthesis in an efficient manner. The structures of the core component domains have been determined and demonstrate insights into the catalytic activity. More recently, multi-domain structures have been determined and are providing clues to the features of these enzyme systems that govern the functional interaction between multiple domains. This chapter describes the structures of NRPS proteins and the strategies that are being used to assist structural studies of these dynamic proteins, including careful consideration of domain boundaries for generation of truncated proteins and the use of mechanism-based inhibitors that trap interactions between the catalytic and carrier protein domains. PMID:26831698

Construction of hybrid peptide synthetases by module and domain fusions

PubMed Central

Mootz, Henning D.; Schwarzer, Dirk; Marahiel, Mohamed A.

2000-01-01

Nonribosomal peptide synthetases are modular enzymes that assemble peptides of diverse structures and important biological activities. Their modular organization provides a great potential for the rational design of novel compounds by recombination of the biosynthetic genes. Here we describe the extension of a dimodular system to trimodular ones based on whole-module fusion. The recombinant hybrid enzymes were purified to monitor product assembly in vitro. We started from the first two modules of tyrocidine synthetase, which catalyze the formation of the dipeptide dPhe-Pro, to construct such hybrid systems. Fusion of the second, proline-specific module with the ninth and tenth modules of the tyrocidine synthetases, specific for ornithine and leucine, respectively, resulted in dimodular hybrid enzymes exhibiting the combined substrate specificities. The thioesterase domain was fused to the terminal module. Upon incubation of these dimodular enzymes with the first tyrocidine module, TycA, incorporating dPhe, the predicted tripeptides dPhe-Pro-Orn and dPhe-Pro-Leu were obtained at rates of 0.15 min-1 and 2.1 min-1. The internal thioesterase domain was necessary and sufficient to release the products from the hybrid enzymes and thereby facilitate a catalytic turnover. Our approach of whole-module fusion is based on an improved definition of the fusion sites and overcomes the recently discovered editing function of the intrinsic condensation domains. The stepwise construction of hybrid peptide synthetases from catalytic subunits reinforces the inherent potential for the synthesis of novel, designed peptides. PMID:10811885

Construction of hybrid peptide synthetases by module and domain fusions.

PubMed

Mootz, H D; Schwarzer, D; Marahiel, M A

2000-05-23

Nonribosomal peptide synthetases are modular enzymes that assemble peptides of diverse structures and important biological activities. Their modular organization provides a great potential for the rational design of novel compounds by recombination of the biosynthetic genes. Here we describe the extension of a dimodular system to trimodular ones based on whole-module fusion. The recombinant hybrid enzymes were purified to monitor product assembly in vitro. We started from the first two modules of tyrocidine synthetase, which catalyze the formation of the dipeptide dPhe-Pro, to construct such hybrid systems. Fusion of the second, proline-specific module with the ninth and tenth modules of the tyrocidine synthetases, specific for ornithine and leucine, respectively, resulted in dimodular hybrid enzymes exhibiting the combined substrate specificities. The thioesterase domain was fused to the terminal module. Upon incubation of these dimodular enzymes with the first tyrocidine module, TycA, incorporating dPhe, the predicted tripeptides dPhe-Pro-Orn and dPhe-Pro-Leu were obtained at rates of 0.15 min(-1) and 2.1 min(-1). The internal thioesterase domain was necessary and sufficient to release the products from the hybrid enzymes and thereby facilitate a catalytic turnover. Our approach of whole-module fusion is based on an improved definition of the fusion sites and overcomes the recently discovered editing function of the intrinsic condensation domains. The stepwise construction of hybrid peptide synthetases from catalytic subunits reinforces the inherent potential for the synthesis of novel, designed peptides.

Inhibition of Long Chain Fatty Acyl-CoA Synthetase (ACSL) and Ischemia Reperfusion Injury

PubMed Central

Prior, Allan M.; Zhang, Man; Blakeman, Nina; Datta, Palika; Pham, Hung; Young, Lindon H.; Weis, Margaret T.; Hua, Duy H.

2014-01-01

Various triacsin C analogs, containing different alkenyl chains and carboxylic acid bioisoteres including 4-aminobenzoic acid, isothiazolidine dioxide, hydroxylamine, hydroxytriazene, and oxadiazolidine dione, were synthesized and their inhibitions of long chain fatty acyl-CoA synthetase (ACSL) were examined. Two methods, a cell-based assay of ACSL activity and an in situ [14C]-palmitate incorporation into extractable lipids were used to study the inhibition. Using an in vivo leukocyte recruitment inhibition protocol, the translocation of one or more cell adhesion molecules from the cytoplasm to the plasma membrane on either the endothelium or leukocyte or both was inhibited by inhibitors 1, 9, and triacsin C. The results suggest that inhibition of ACSL may attenuate the vascular inflammatory component associated with ischemia reperfusion injury and lead to a decrease of infarct expansion. PMID:24480468

Fatty acid-producing hosts

DOEpatents

Pfleger, Brian F; Lennen, Rebecca M

2013-12-31

Described are hosts for overproducing a fatty acid product such as a fatty acid. The hosts include an exogenous nucleic acid encoding a thioesterase and, optionally, an exogenous nucleic acid encoding an acetyl-CoA carboxylase, wherein an acyl-CoA synthetase in the hosts are functionally delected. The hosts prefereably include the nucleic acid encoding the thioesterase at an intermediate copy number. The hosts are preferably recominantly stable and growth-competent at 37.degree. C. Methods of producing a fatty acid product comprising culturing such hosts at 37.degree. C. are also described.

Genetics Home Reference: carbamoyl phosphate synthetase I deficiency

MedlinePlus

... synthetase I. This enzyme participates in the urea cycle, which is a sequence of biochemical reactions that occurs in liver cells. The urea cycle processes excess nitrogen, generated when protein is broken ...

Biosynthetic engineering of nonribosomal peptide synthetases.

PubMed

Kries, Hajo

2016-09-01

From the evolutionary melting pot of natural product synthetase genes, microorganisms elicit antibiotics, communication tools, and iron scavengers. Chemical biologists manipulate these genes to recreate similarly diverse and potent biological activities not on evolutionary time scales but within months. Enzyme engineering has progressed considerably in recent years and offers new screening, modelling, and design tools for natural product designers. Here, recent advances in enzyme engineering and their application to nonribosomal peptide synthetases are reviewed. Among the nonribosomal peptides that have been subjected to biosynthetic engineering are the antibiotics daptomycin, calcium-dependent antibiotic, and gramicidin S. With these peptides, incorporation of unnatural building blocks and modulation of bioactivities via various structural modifications have been successfully demonstrated. Natural product engineering on the biosynthetic level is not a reliable method yet. However, progress in the understanding and manipulation of biosynthetic pathways may enable the routine production of optimized peptide drugs in the near future. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Mutations in glycyl-tRNA synthetase impair mitochondrial metabolism in neurons.

PubMed

Boczonadi, Veronika; Meyer, Kathrin; Gonczarowska-Jorge, Humberto; Griffin, Helen; Roos, Andreas; Bartsakoulia, Marina; Bansagi, Boglarka; Ricci, Giulia; Palinkas, Fanni; Zahedi, René P; Bruni, Francesco; Kaspar, Brian; Lochmüller, Hanns; Boycott, Kym M; Müller, Juliane S; Horvath, Rita

2018-06-15

The nuclear-encoded glycyl-tRNA synthetase gene (GARS) is essential for protein translation in both cytoplasm and mitochondria. In contrast, different genes encode the mitochondrial and cytosolic forms of most other tRNA synthetases. Dominant GARS mutations were described in inherited neuropathies, while recessive mutations cause severe childhood-onset disorders affecting skeletal muscle and heart. The downstream events explaining tissue-specific phenotype-genotype relations remained unclear. We investigated the mitochondrial function of GARS in human cell lines and in the GarsC210R mouse model. Human-induced neuronal progenitor cells (iNPCs) carrying dominant and recessive GARS mutations showed alterations of mitochondrial proteins, which were more prominent in iNPCs with dominant, neuropathy-causing mutations. Although comparative proteomic analysis of iNPCs showed significant changes in mitochondrial respiratory chain complex subunits, assembly genes, Krebs cycle enzymes and transport proteins in both recessive and dominant mutations, proteins involved in fatty acid oxidation were only altered by recessive mutations causing mitochondrial cardiomyopathy. In contrast, significant alterations of the vesicle-associated membrane protein-associated protein B (VAPB) and its downstream pathways such as mitochondrial calcium uptake and autophagy were detected in dominant GARS mutations. The role of VAPB has been supported by similar results in the GarsC210R mice. Our data suggest that altered mitochondria-associated endoplasmic reticulum (ER) membranes (MAM) may be important disease mechanisms leading to neuropathy in this condition.

tRNA acceptor-stem and anticodon bases embed separate features of amino acid chemistry

PubMed Central

Carter, Charles W.; Wolfenden, Richard

2016-01-01

abstract The universal genetic code is a translation table by which nucleic acid sequences can be interpreted as polypeptides with a wide range of biological functions. That information is used by aminoacyl-tRNA synthetases to translate the code. Moreover, amino acid properties dictate protein folding. We recently reported that digital correlation techniques could identify patterns in tRNA identity elements that govern recognition by synthetases. Our analysis, and the functionality of truncated synthetases that cannot recognize the tRNA anticodon, support the conclusion that the tRNA acceptor stem houses an independent code for the same 20 amino acids that likely functioned earlier in the emergence of genetics. The acceptor-stem code, related to amino acid size, is distinct from a code in the anticodon that is related to amino acid polarity. Details of the acceptor-stem code suggest that it was useful in preserving key properties of stereochemically-encoded peptides that had developed the capacity to interact catalytically with RNA. The quantitative embedding of the chemical properties of amino acids into tRNA bases has implications for the origins of molecular biology. PMID:26595350

Valyl-tRNA synthetase modification-dependent restriction of bacteriophage T4.

PubMed Central

Olson, N J; Marchin, G L

1984-01-01

A strain of Escherichia coli, CP 790302, severely restricts the growth of wild-type bacteriophage T4. In broth culture, most infections of single cells are abortive, although a few infected cells exhibit reduced burst sizes. In contrast, bacteriophage T4 mutants impaired in the ability to modify valyl-tRNA synthetase develop normally on this strain. Biochemical evidence indicates that the phage-modified valyl-tRNA synthetase in CP 790302 is different from that previously described. Although the enzyme is able to support normal protein synthesis, a disproportionate amount of phage structural protein (serum blocking power) fails to mature into particles of the appropriate density. The results with host strain CP 790302 are consistent with either a gratuitous inhibition of phage assembly by faulty modification or abrogation of an unknown role that valyl-tRNA synthetase might normally play in viral assembly. PMID:6374167

3-Aminoquinoline/p-coumaric acid as a MALDI matrix for glycopeptides, carbohydrates, and phosphopeptides.

PubMed

Fukuyama, Yuko; Funakoshi, Natsumi; Takeyama, Kohei; Hioki, Yusaku; Nishikaze, Takashi; Kaneshiro, Kaoru; Kawabata, Shin-Ichirou; Iwamoto, Shinichi; Tanaka, Koichi

2014-02-18

Glycosylation and phosphorylation are important post-translational modifications in biological processes and biomarker research. The difficulty in analyzing these modifications is mainly their low abundance and dissociation of labile regions such as sialic acids or phosphate groups. One solution in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is to improve matrices for glycopeptides, carbohydrates, and phosphopeptides by increasing the sensitivity and suppressing dissociation of the labile regions. Recently, a liquid matrix 3-aminoquinoline (3-AQ)/α-cyano-4-hydroxycinnamic acid (CHCA) (3-AQ/CHCA), introduced by Kolli et al. in 1996, has been reported to increase sensitivity for carbohydrates or phosphopeptides, but it has not been systematically evaluated for glycopeptides. In addition, 3-AQ/CHCA enhances the dissociation of labile regions. In contrast, a liquid matrix 1,1,3,3-tetramethylguanidium (TMG, G) salt of p-coumaric acid (CA) (G3CA) was reported to suppress dissociation of sulfate groups or sialic acids of carbohydrates. Here we introduce a liquid matrix 3-AQ/CA for glycopeptides, carbohydrates, and phosphopeptides. All of the analytes were detected as [M + H](+) or [M - H](-) with higher or comparable sensitivity using 3-AQ/CA compared with 3-AQ/CHCA or 2,5-dihydroxybenzoic acid (2,5-DHB). The sensitivity was increased 1- to 1000-fold using 3-AQ/CA. The dissociation of labile regions such as sialic acids or phosphate groups and the fragmentation of neutral carbohydrates were suppressed more using 3-AQ/CA than using 3-AQ/CHCA or 2,5-DHB. 3-AQ/CA was thus determined to be an effective MALDI matrix for high sensitivity and the suppression of dissociation of labile regions in glycosylation and phosphorylation analyses.

Unnatural reactive amino acid genetic code additions

DOEpatents

Deiters, Alexander [La Jolla, CA; Cropp, T Ashton [Bethesda, MD; Chin, Jason W [Cambridge, GB; Anderson, J Christopher [San Francisco, CA; Schultz, Peter G [La Jolla, CA

2011-08-09

This invention provides compositions and methods for producing translational components that expand the number of genetically encoded amino acids in eukaryotic cells. The components include orthogonal tRNAs, orthogonal aminoacyl-tRNAsyn-thetases, pairs of tRNAs/synthetases and unnatural amino acids. Proteins and methods of producing proteins with unnatural amino acids in eukaryotic cells are also provided.

The action of Saraca asoca Roxb. de Wilde bark on the PGH2 synthetase enzyme complex of the sheep vesicular gland.

PubMed

Middelkoop, T B; Labadie, R P

1985-01-01

Extracts of S. asoca bark and pure compounds isolated from the bark were tested for properties that might inhibit the conversion of arachidonic acid by the PGH2 synthetase. They were assayed spectrophotometrically with adrenaline as cofactor. Methanol- and ethyl acetate extracts inhibited the conversion. The observed inhibition was confirmed in an oxygraphic assay. Two procyanidin dimers from the ethyl acetate extract showed enzyme catalyzed oxidation in our assay. The ether extract of the bark was also found to contain yet unknown substances which were capable of being oxidised by the PGH2 synthetase. The combined action of the components of the bark may explain the mode of action of the drug Asoka Aristha, the main ingredient of which is the bark of S. asoca. The drug is traditionally used in Sri Lanka to treat menorrhagia.

Acetate scavenging activity in Escherichia coli: interplay of acetyl-CoA synthetase and the PEP-glyoxylate cycle in chemostat cultures.

PubMed

Renilla, Sergio; Bernal, Vicente; Fuhrer, Tobias; Castaño-Cerezo, Sara; Pastor, José M; Iborra, José L; Sauer, Uwe; Cánovas, Manuel

2012-03-01

Impairment of acetate production in Escherichia coli is crucial for the performance of many biotechnological processes. Aerobic production of acetate (or acetate overflow) results from changes in the expression of central metabolism genes. Acetyl-CoA synthetase scavenges extracellular acetate in glucose-limited cultures. Once converted to acetyl-CoA, it can be catabolized by the tricarboxylic acid cycle or the glyoxylate pathway. In this work, we assessed the significance of these pathways on acetate overflow during glucose excess and limitation. Gene expression, enzyme activities, and metabolic fluxes were studied in E. coli knock-out mutants related to the glyoxylate pathway operon and its regulators. The relevance of post-translational regulation by AceK-mediated phosphorylation of isocitrate dehydrogenase for pathway functionality was underlined. In chemostat cultures performed at increasing dilution rates, acetate overflow occurs when growing over a threshold glucose uptake rate. This threshold was not affected in a glyoxylate-pathway-deficient strain (lacking isocitrate lyase, the first enzyme of the pathway), indicating that it is not relevant for acetate overflow. In carbon-limited chemostat cultures, gluconeogenesis (maeB, sfcA, and pck), the glyoxylate operon and, especially, acetyl-CoA synthetase are upregulated. A mutant in acs (encoding acetyl-CoA synthetase) produced acetate at all dilution rates. This work demonstrates that, in E. coli, acetate production occurs at all dilution rates and that overflow is the result of unbalanced synthesis and scavenging activities. The over-expression of acetyl-CoA synthetase by cAMP-CRP-dependent induction limits this phenomenon in cultures consuming glucose at low rate, ensuring the recycling of the acetyl-CoA and acetyl-phosphate pools, although establishing an energy-dissipating substrate cycle.

Identification, Expression and IAA-Amide Synthetase Activity Analysis of Gretchen Hagen 3 in Papaya Fruit (Carica papaya L.) during Postharvest Process

PubMed Central

Liu, Kaidong; Wang, Jinxiang; Li, Haili; Zhong, Jundi; Feng, Shaoxian; Pan, Yaoliang; Yuan, Changchun

2016-01-01

Auxin plays essential roles in plant development. Gretchen Hagen 3 (GH3) genes belong to a major auxin response gene family and GH3 proteins conjugate a range of acylsubstrates to alter the levels of hormones. Currently, the role of GH3 genes in postharvest physiological regulation of ripening and softening processes in papaya fruit is unclear. In this study, we identified seven CpGH3 genes in a papaya genome database. The CpGH3.1a, CpGH3.1b, CpGH3.5, CpGH3.6, and CpGH3.9 proteins were identified as indole-3-acetic acid (IAA)-specific amido synthetases. We analyzed the changes in IAA-amido synthetase activity using aspartate as a substrate for conjugation and found a large increase (over 5-fold) during the postharvest stages. Ascorbic acid (AsA) application can extend the shelf life of papaya fruit. Our data showed that AsA treatment regulates postharvest fruit maturation processes by promoting endogenous IAA levels. Our findings demonstrate the important role of GH3 genes in the regulation of auxin-associated postharvest physiology in papaya. PMID:27812360

Crystal structures of trypanosomal histidyl-tRNA synthetase illuminate differences between eukaryotic and prokaryotic homologs

PubMed Central

Merritt, Ethan A; Arakaki, Tracy L; Gillespie, J Robert; Larson, Eric T; Kelley, Angela; Mueller, Natascha; Napuli, Alberto J; Kim, Jessica; Zhang, Li; Verlinde, Christophe L M J; Fan, Erkang; Zucker, Frank; Buckner, Frederick S; Van Voorhis, Wesley C; Hol, Wim G J

2010-01-01

Crystal structures of histidyl-tRNA synthetase from the eukaryotic parasites Trypanosoma brucei and Trypanosoma cruzi provide a first structural view of a eukaryotic form of this enzyme, and reveal differences from bacterial homologs. Histidyl-tRNA synthetases in general contain an extra domain inserted between conserved motifs 2 and 3 of the Class II aminoacyl-tRNA synthetase catalytic core. The current structures show that the three dimensional topology of this domain is very different in bacterial and archaeal/eukaryotic forms of the enzyme. Comparison of apo and histidine-bound trypanosomal structures indicates substantial active site rearrangement upon histidine binding, but relatively little subsequent rearrangement after reaction of histidine with ATP to form the enzyme’s first reaction product, histidyladenylate. The specific residues involved in forming the binding pocket for the adenine moiety differ substantially both from the previously characterized binding site in bacterial structures and from the homologous residues in human histidyl-tRNA synthetases. The essentiality of the single histidyl-tRNA synthetase gene in T. brucei is shown by a severe depression of parasite growth rate that results from even partial suppression of expression by RNA interference. PMID:20132829

Increased PRPP synthetase activity in cultured rat hepatoma cells containing mutations in the hypoxanthine-guanine phosphoribosyltransferase gene.

PubMed

Graf, L H; McRoberts, J A; Harrison, T M; Martin, D W

1976-07-01

Nine independently derived clones of mutagenized rat hepatoma cells selected for resistance to 6-mercaptopurine (6-MP) or 6-thioguanine (6-ThioG) have been isolated. Each has severely reduced catalytic activity of hypoxanthine-guanine phosphoribosyltransferase (HPRT) and seven of them possess significantly increased activities of phosphoribosylpyrophosphate (PRPP) synthetase. The degrees of elevations of PRPP synthetase activities do not correlate with the degrees of deficiencies of HPRT activities. The cells from one of these clones, 1020/12, posses 40% of the normal HPRT catalytic activity and overproduce purines. We have extensively examined the cells from this clone. Immunotration studies of 1020/12 cells indicate that there is a mutation in the structural gene for HPRT. Although they possess increased specific catalytic activities of the enzyme. PRPP synthetase, the catalytic parameters, heat stability, and isoelectric pH of PRPP synthetase from 1020/12 cells are indistinguishable from those of the enzyme from wild-type cells. The cause of purine overproduction by 1020/12 cells appears to be the elevated PRPP synthetase activity, rather than a PRPP "sparing" effect stemming from reduced HPRT activity. Support for this idea is provided by the observation that the complete loss of HPRT activity in a clone derived from 1020/12 cells does not further enhance the levels of PRPP synthetase or purine overproduction. We propose that the elevated levels of PRPP synthetase activity in these HPRT deficient cells result from a mutational event in the structural gene for HPRT, and that this causes the disruption of a previously undescribed regulatory function of this gene on the expression of the PRPP synthetase gene.

Functional asymmetry in the lysyl-tRNA synthetase explored by molecular dynamics, free energy calculations and experiment

PubMed Central

Hughes, Samantha J; Tanner, Julian A; Hindley, Alison D; Miller, Andrew D; Gould, Ian R

2003-01-01

Background Charging of transfer-RNA with cognate amino acid is accomplished by the aminoacyl-tRNA synthetases, and proceeds through an aminoacyl adenylate intermediate. The lysyl-tRNA synthetase has evolved an active site that specifically binds lysine and ATP. Previous molecular dynamics simulations of the heat-inducible Escherichia coli lysyl-tRNA synthetase, LysU, have revealed differences in the binding of ATP and aspects of asymmetry between the nominally equivalent active sites of this dimeric enzyme. The possibility that this asymmetry results in different binding affinities for the ligands is addressed here by a parallel computational and biochemical study. Results Biochemical experiments employing isothermal calorimetry, steady-state fluorescence and circular dichroism are used to determine the order and stoichiometries of the lysine and nucleotide binding events, and the associated thermodynamic parameters. An ordered mechanism of substrate addition is found, with lysine having to bind prior to the nucleotide in a magnesium dependent process. Two lysines are found to bind per dimer, and trigger a large conformational change. Subsequent nucleotide binding causes little structural rearrangement and crucially only occurs at a single catalytic site, in accord with the simulations. Molecular dynamics based free energy calculations of the ATP binding process are used to determine the binding affinities of each site. Significant differences in ATP binding affinities are observed, with only one active site capable of realizing the experimental binding free energy. Half-of-the-sites models in which the nucleotide is only present at one active site achieve their full binding potential irrespective of the subunit choice. This strongly suggests the involvement of an anti-cooperative mechanism. Pathways for relaying information between the two active sites are proposed. Conclusions The asymmetry uncovered here appears to be a common feature of oligomeric aminoacyl

NMR analyses of the conformations of L-isoleucine and L-valine bound to Escherichia coli isoleucyl-tRNA synthetase

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

Kohda, D.; Kawai, G.; Yokoyama, S.

1987-10-06

The 400-MHz /sup 1/H NMR spectra of L-isoleucine and L-valine were measured in the presence of Escherichia coli isoleucyl-tRNA synthetase (IleRS). Because of chemical exchange of L-isoleucine or L-valine between the free state and the IleRS-bound state, a transferred nuclear Overhauser effect (TRNOE) was observed among proton resonances of L-isoleucine or L-valine. However, in the presence of isoleucyl adenylate tightly bound to the amino acid activation site of IleRS, no TRNOE for L-isoleucine or L-valine was observed. This indicates that the observed TRNOE is due to the interaction of L-isoleucine or L-valine with the amino acid activation site of IleRS.more » The conformations of these amino acids in the amino acid activation site of IleRS were determined by the analyses of time dependences of TRNOEs and TRNOE action spectra. The IleRS-bound L-isoleucine takes the gauche/sup +/ form about the C/sub ..cap alpha../-C/sub ..beta../ bond and the trans form about the C/sub ..beta../-C/sub ..gamma../sub 1// bond. The IleRS-bound L-valine takes the guache/sup -/ form about the C/sub ..cap alpha../-C/sub ..beta../ bond. Thus, the conformation of the IleRS-bound L-valine is the same as that of IleRS-bound L-isoleucine except for the delta-methyl group. The side chain of L-isoleucine or L-valine lies in an aliphatic hydrophobic pocket of the active site of IleRS. Such hydrophobic interaction with IleRS is more significant for L-isoleucine than for L-valine. The TRNOE analysis is useful for studying the amino acid discrimination mechanism of aminoacyl-tRNA synthetases.« less

42 CFR 417.558 - Emergency, urgently needed, and out-of-area services for which the HMO or CMP accepts...

Code of Federal Regulations, 2010 CFR

2010-10-01

... services for which the HMO or CMP accepts responsibility. 417.558 Section 417.558 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICARE PROGRAM HEALTH MAINTENANCE ORGANIZATIONS, COMPETITIVE MEDICAL PLANS, AND HEALTH CARE PREPAYMENT PLANS Medicare Payment: Cost...

Natural toxins that affect plant amino acid metabolism

USDA-ARS?s Scientific Manuscript database

A diverse range of natural compounds interfere with the synthesis and other aspects of amino acid metabolism. Some are amino acid analogues, but most are not. This review covers a number of specific natural phytotoxic compounds by molecular target site. Inhibition of glutamine synthetase is of part...

Characterization of a Bacillus subtilis surfactin synthetase knockout and antimicrobial activity analysis.

PubMed

Liu, Hongxia; Qu, Xiaoxu; Gao, Ling; Zhao, Shengming; Lu, Zhaoxin; Zhang, Chong; Bie, Xiaomei

2016-11-10

Gene knockout is an important approach to improve the production of antimicrobial compounds. B. subtilis PB2-LS10, derived from B. subtilis PB2-L by a surfactin synthetase (srf) genes knockout, exhibits stronger inhibitory action than its parental strain against all tested pathogenic bacteria and fungi. The antimicrobial extracts produced by B. subtilis PB2-L and B. subtilis PB2-LS10 respectively were characterized by the high-resolution LC-ESI-MS. To provide further insight into the distinct antimicrobial activities, we investigated the impact of the srf genes deletion on the growth and gene transcriptional profile of the strains. The mutant strain grew quickly and reached stationary phase 2h earlier than the wild-type. Prominent expression changes in the modified strain involved genes that were essential to metabolic pathways and processes. Genes related to amino acid transport, ATP-binding cassette (ABC) transporters and protein export were up-regulated in strain PB2-LS10. However, amino acid metabolism, carbohydrate metabolism and fatty acid metabolism were repressed. Because of its excellent antimicrobial activity, strain PB2-LS10 has potential for use in food preservation. Copyright © 2016 Elsevier B.V. All rights reserved.

A Survey of Glutamine Synthetase Activities in Tissues from Three Classes of Fish.

DTIC Science & Technology

1980-09-01

reveree side it necessay end identify by block enamaber) Glutamine synthetase, gamma-glutamyl transferase, osmoregulation , glutamate, glutamine...aspects of osmoregulation as well. The only known route of glutanmine synthesis n all species is activity of glutamine synthetase (EC 6.3.1.2) which...for osmoregulation . There is a relatively small difference n species which retain urea for osmoregulation . This may help to explain the relationship of

Mass Isotopomer Analysis of Metabolically Labeled Nucleotide Sugars and N- and O-Glycans for Tracing Nucleotide Sugar Metabolisms*

PubMed Central

Nakajima, Kazuki; Ito, Emi; Ohtsubo, Kazuaki; Shirato, Ken; Takamiya, Rina; Kitazume, Shinobu; Angata, Takashi; Taniguchi, Naoyuki

2013-01-01

Nucleotide sugars are the donor substrates of various glycosyltransferases, and an important building block in N- and O-glycan biosynthesis. Their intercellular concentrations are regulated by cellular metabolic states including diseases such as cancer and diabetes. To investigate the fate of UDP-GlcNAc, we developed a tracing method for UDP-GlcNAc synthesis and use, and GlcNAc utilization using 13C6-glucose and 13C2-glucosamine, respectively, followed by the analysis of mass isotopomers using LC-MS. Metabolic labeling of cultured cells with 13C6-glucose and the analysis of isotopomers of UDP-HexNAc (UDP-GlcNAc plus UDP-GalNAc) and CMP-NeuAc revealed the relative contributions of metabolic pathways leading to UDP-GlcNAc synthesis and use. In pancreatic insulinoma cells, the labeling efficiency of a 13C6-glucose motif in CMP-NeuAc was lower compared with that in hepatoma cells. Using 13C2-glucosamine, the diversity of the labeling efficiency was observed in each sugar residue of N- and O-glycans on the basis of isotopomer analysis. In the insulinoma cells, the low labeling efficiencies were found for sialic acids as well as tri- and tetra-sialo N-glycans, whereas asialo N-glycans were found to be abundant. Essentially no significant difference in secreted hyaluronic acids was found among hepatoma and insulinoma cell lines. This indicates that metabolic flows are responsible for the low sialylation in the insulinoma cells. Our strategy should be useful for systematically tracing each stage of cellular GlcNAc metabolism. PMID:23720760

Mitochondrial and cytoplasmic isoleucyl-, glutamyl- and arginyl-tRNA synthetases of yeast are encoded by separate genes.

PubMed

Tzagoloff, A; Shtanko, A

1995-06-01

Three complementation groups of a pet mutant collection have been found to be composed of respiratory-deficient deficient mutants with lesions in mitochondrial protein synthesis. Recombinant plasmids capable of restoring respiration were cloned by transformation of representatives of each complementation group with a yeast genomic library. The plasmids were used to characterize the complementing genes and to institute disruption of the chromosomal copies of each gene in respiratory-proficient yeast. The sequences of the cloned genes indicate that they code for isoleucyl-, arginyl- and glutamyl-tRNA synthetases. The properties of the mutants used to obtain the genes and of strains with the disrupted genes indicate that all three aminoacyl-tRNA synthetases function exclusively in mitochondrial proteins synthesis. The ISM1 gene for mitochondrial isoleucyl-tRNA synthetase has been localized to chromosome XVI next to UME5. The MSR1 gene for the arginyl-tRNA synthetase was previously located on yeast chromosome VIII. The third gene MSE1 for the mitochondrial glutamyl-tRNA synthetase has not been localized. The identification of three new genes coding for mitochondrial-specific aminoacyl-tRNA synthetases indicates that in Saccharomyces cerevisiae at least 11 members of this protein family are encoded by genes distinct from those coding for the homologous cytoplasmic enzymes.

Membrane anchoring of aminoacyl-tRNA synthetases by convergent acquisition of a novel protein domain.

PubMed

Olmedo-Verd, Elvira; Santamaría-Gómez, Javier; Ochoa de Alda, Jesús A G; Ribas de Pouplana, Lluis; Luque, Ignacio

2011-11-25

Four distinct aminoacyl-tRNA synthetases (aaRSs) found in some cyanobacterial species contain a novel protein domain that bears two putative transmembrane helices. This CAAD domain is present in glutamyl-, isoleucyl-, leucyl-, and valyl-tRNA synthetases, the latter of which has probably recruited the domain more than once during evolution. Deleting the CAAD domain from the valyl-tRNA synthetase of Anabaena sp. PCC 7120 did not significantly modify the catalytic properties of this enzyme, suggesting that it does not participate in its canonical tRNA-charging function. Multiple lines of evidence suggest that the function of the CAAD domain is structural, mediating the membrane anchorage of the enzyme, although membrane localization of aaRSs has not previously been described in any living organism. Synthetases containing the CAAD domain were localized in the intracytoplasmic thylakoid membranes of cyanobacteria and were largely absent from the plasma membrane. The CAAD domain was necessary and apparently sufficient for protein targeting to membranes. Moreover, localization of aaRSs in thylakoids was important under nitrogen limiting conditions. In Anabaena, a multicellular filamentous cyanobacterium often used as a model for prokaryotic cell differentiation, valyl-tRNA synthetase underwent subcellular relocation at the cell poles during heterocyst differentiation, a process also dependent on the CAAD domain.

Cloning and characterization of the human 5,10-methenyltetrahydrofolate synthetase-encoding cDNA.

PubMed

Dayan, A; Bertrand, R; Beauchemin, M; Chahla, D; Mamo, A; Filion, M; Skup, D; Massie, B; Jolivet, J

1995-11-20

Methenyltetrahydrofolate synthetase (MTHFS) catalyses the obligatory initial metabolic step in the intracellular conversion of 5-formyltetrahydrofolate to other reduced folates. We have isolated and sequenced a human MTHFS cDNA which is 872-bp long and codes for a 203-amino-acid protein of 23,229 Da. Escherichia coli BL21(DE3), transfected with pET11c plasmids containing an open reading frame encoding MTHFS, showed a 100-fold increase in MTHFS activity in bacterial extracts after IPTG induction. Northern blot studies of human tissues determined that the MTHFS mRNA was expressed preferentially in the liver and Southern blot analysis of human genomic DNA suggested the presence of a single-copy gene.

Influence of side chain conformation and configuration on glycosyl donor reactivity and selectivity as illustrated by sialic acid donors epimeric at the 7-position.

PubMed

Kancharla, Pavan K; Crich, David

2013-12-18

Two N-acetyl 4O,5N-oxazolidinone-protected sialyl thioglycosides epimeric at the 7-position have been synthesized and their reactivity and stereoselectivity in glycosylation reactions have been compared. It is demonstrated that the natural 7S-donor is both more reactive and more α-selective than the unnatural 7R-isomer. The difference in reactivity is attributed to the side chain conformation and specifically to the proximity of O7 to the anomeric center. In the natural 7S-isomer, O7 is closer to the anomeric center than in its unnatural 7R-epimer and, therefore, better able to support incipient positive charge at the locus of reaction. The difference in selectivity is also attributed to the side conformation, which in the unnatural 7R-series is placed perpendicularly above the α-face of the donor and so shields it to a greater extent than in the 7S-series. These observations are consistent with earlier conclusions on the influence of the side chain conformation on reactivity and selectivity derived from conformationally locked models in the glucose and galactose series and corroborate the suggestion that those effects are predominantly stereoelectronic rather than torsional. The possible relevance of side chain conformation as a factor in the influence of glycosylation stereoselectivity by remote protecting groups and as a control element in enzymic processes for glycosidic bond formation and hydrolysis are discussed. Methods for assignment of the anomeric configuration in the sialic acid glycosides are critically surveyed.

Regulation of glutamine synthetase activity in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 by the nitrogen source: effect of ammonium.

PubMed Central

Mérida, A; Candau, P; Florencio, F J

1991-01-01

Glutamine synthetase activity from Synechocystis sp. strain PCC 6803 is regulated as a function of the nitrogen source available in the medium. Addition of 0.25 mM NH4Cl to nitrate-grown cells promotes a clear short-term inactivation of glutamine synthetase, whose enzyme activity decreases to 5 to 10% of the initial value in 25 min. The intracellular levels of glutamine, determined under various conditions, taken together with the results obtained with azaserine (an inhibitor of transamidases), rule out the possibility that glutamine per se is responsible for glutamine synthetase inactivation. Nitrogen starvation attenuates the ammonium-mediated glutamine synthetase inactivation, indicating that glutamine synthetase regulation is modulated through the internal balance between carbon-nitrogen compounds and carbon compounds. The parallelism observed between the glutamine synthetase activity and the internal concentration of alpha-ketoglutarate suggests that this metabolite could play a role as a positive effector of glutamine synthetase activity in Synechocystis sp. Despite the similarities of this physiological system to that described for enterobacteria, the lack of in vivo 32P labeling of glutamine synthetase during the inactivation process excludes the existence of an adenylylation-deadenylylation system in this cyanobacterium. Images PMID:1676397

MS_RHII-RSD, a Dual-Function RNase HII-(p)ppGpp Synthetase from Mycobacterium smegmatis

PubMed Central

Murdeshwar, Maya S.

2012-01-01

In the noninfectious soil saprophyte Mycobacterium smegmatis, intracellular levels of the stress alarmones guanosine tetraphosphate and guanosine pentaphosphate, together termed (p)ppGpp, are regulated by the enzyme RelMsm. This enzyme consists of a single, bifunctional polypeptide chain that is capable of both synthesizing and hydrolyzing (p)ppGpp. The relMsm knockout strain of M. smegmatis (ΔrelMsm) is expected to show a (p)ppGpp null [(p)ppGpp0] phenotype. Contrary to this expectation, the strain is capable of synthesizing (p)ppGpp in vivo. In this study, we identify and functionally characterize the open reading frame (ORF), MSMEG_5849, that encodes a second functional (p)ppGpp synthetase in M. smegmatis. In addition to (p)ppGpp synthesis, the 567-amino-acid-long protein encoded by this gene is capable of hydrolyzing RNA·DNA hybrids and bears similarity to the conventional RNase HII enzymes. We have classified this protein as actRelMsm in accordance with the recent nomenclature proposed and have named it MS_RHII-RSD, indicating the two enzymatic activities present [RHII, RNase HII domain, originally identified as domain of unknown function 429 (DUF429), and RSD, RelA_SpoT nucleotidyl transferase domain, the SYNTH domain responsible for (p)ppGpp synthesis activity]. MS_RHII-RSD is expressed and is constitutively active in vivo and behaves like a monofunctional (p)ppGpp synthetase in vitro. The occurrence of the RNase HII and (p)ppGpp synthetase domains together on the same polypeptide chain is suggestive of an in vivo role for this novel protein as a link connecting the essential life processes of DNA replication, repair, and transcription to the highly conserved stress survival pathway, the stringent response. PMID:22636779

MS_RHII-RSD, a dual-function RNase HII-(p)ppGpp synthetase from Mycobacterium smegmatis.

PubMed

Murdeshwar, Maya S; Chatterji, Dipankar

2012-08-01

In the noninfectious soil saprophyte Mycobacterium smegmatis, intracellular levels of the stress alarmones guanosine tetraphosphate and guanosine pentaphosphate, together termed (p)ppGpp, are regulated by the enzyme Rel(Msm). This enzyme consists of a single, bifunctional polypeptide chain that is capable of both synthesizing and hydrolyzing (p)ppGpp. The rel(Msm) knockout strain of M. smegmatis (Δrel(Msm)) is expected to show a (p)ppGpp null [(p)ppGpp(0)] phenotype. Contrary to this expectation, the strain is capable of synthesizing (p)ppGpp in vivo. In this study, we identify and functionally characterize the open reading frame (ORF), MSMEG_5849, that encodes a second functional (p)ppGpp synthetase in M. smegmatis. In addition to (p)ppGpp synthesis, the 567-amino-acid-long protein encoded by this gene is capable of hydrolyzing RNA·DNA hybrids and bears similarity to the conventional RNase HII enzymes. We have classified this protein as actRel(Msm) in accordance with the recent nomenclature proposed and have named it MS_RHII-RSD, indicating the two enzymatic activities present [RHII, RNase HII domain, originally identified as domain of unknown function 429 (DUF429), and RSD, RelA_SpoT nucleotidyl transferase domain, the SYNTH domain responsible for (p)ppGpp synthesis activity]. MS_RHII-RSD is expressed and is constitutively active in vivo and behaves like a monofunctional (p)ppGpp synthetase in vitro. The occurrence of the RNase HII and (p)ppGpp synthetase domains together on the same polypeptide chain is suggestive of an in vivo role for this novel protein as a link connecting the essential life processes of DNA replication, repair, and transcription to the highly conserved stress survival pathway, the stringent response.

Root of the universal tree of life based on ancient aminoacyl-tRNA synthetase gene duplications.

PubMed

Brown, J R; Doolittle, W F

1995-03-28

Universal trees based on sequences of single gene homologs cannot be rooted. Iwabe et al. [Iwabe, N., Kuma, K.-I., Hasegawa, M., Osawa, S. & Miyata, T. (1989) Proc. Natl. Acad. Sci. USA 86, 9355-9359] circumvented this problem by using ancient gene duplications that predated the last common ancestor of all living things. Their separate, reciprocally rooted gene trees for elongation factors and ATPase subunits showed Bacteria (eubacteria) as branching first from the universal tree with Archaea (archaebacteria) and Eucarya (eukaryotes) as sister groups. Given its topical importance to evolutionary biology and concerns about the appropriateness of the ATPase data set, an evaluation of the universal tree root using other ancient gene duplications is essential. In this study, we derive a rooting for the universal tree using aminoacyl-tRNA synthetase genes, an extensive multigene family whose divergence likely preceded that of prokaryotes and eukaryotes. An approximately 1600-bp conserved region was sequenced from the isoleucyl-tRNA synthetases of several species representing deep evolutionary branches of eukaryotes (Nosema locustae), Bacteria (Aquifex pyrophilus and Thermotoga maritima) and Archaea (Pyrococcus furiosus and Sulfolobus acidocaldarius). In addition, a new valyl-tRNA synthetase was characterized from the protist Trichomonas vaginalis. Different phylogenetic methods were used to generate trees of isoleucyl-tRNA synthetases rooted by valyl- and leucyl-tRNA synthetases. All isoleucyl-tRNA synthetase trees showed Archaea and Eucarya as sister groups, providing strong confirmation for the universal tree rooting reported by Iwabe et al. As well, there was strong support for the monophyly (sensu Hennig) of Archaea. The valyl-tRNA synthetase gene from Tr. vaginalis clustered with other eukaryotic ValRS genes, which may have been transferred from the mitochondrial genome to the nuclear genome, suggesting that this amitochondrial trichomonad once harbored an

Combinatorial chemoenzymatic synthesis and high-throughput screening of sialosides.

PubMed

Chokhawala, Harshal A; Huang, Shengshu; Lau, Kam; Yu, Hai; Cheng, Jiansong; Thon, Vireak; Hurtado-Ziola, Nancy; Guerrero, Juan A; Varki, Ajit; Chen, Xi

2008-09-19

Although the vital roles of structures containing sialic acid in biomolecular recognition are well documented, limited information is available on how sialic acid structural modifications, sialyl linkages, and the underlying glycan structures affect the binding or the activity of sialic acid-recognizing proteins and related downstream biological processes. A novel combinatorial chemoenzymatic method has been developed for the highly efficient synthesis of biotinylated sialosides containing different sialic acid structures and different underlying glycans in 96-well plates from biotinylated sialyltransferase acceptors and sialic acid precursors. By transferring the reaction mixtures to NeutrAvidin-coated plates and assaying for the yields of enzymatic reactions using lectins recognizing sialyltransferase acceptors but not the sialylated products, the biotinylated sialoside products can be directly used, without purification, for high-throughput screening to quickly identify the ligand specificity of sialic acid-binding proteins. For a proof-of-principle experiment, 72 biotinylated alpha2,6-linked sialosides were synthesized in 96-well plates from 4 biotinylated sialyltransferase acceptors and 18 sialic acid precursors using a one-pot three-enzyme system. High-throughput screening assays performed in NeutrAvidin-coated microtiter plates show that whereas Sambucus nigra Lectin binds to alpha2,6-linked sialosides with high promiscuity, human Siglec-2 (CD22) is highly selective for a number of sialic acid structures and the underlying glycans in its sialoside ligands.

Crystallization and preliminary X-ray diffraction analysis of recombinant phosphoribosylpyrophosphate synthetase from the Thermophilic thermus thermophilus strain HB27

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

Abramchik, Yu. A.; Timofeev, V. I., E-mail: tostars@mail.ru; Muravieva, T. I.

2017-01-15

Phosphoribosylpyrophosphate synthetases (PRPP synthetases) are among the key enzymes essential for vital functions of organisms and are involved in the biosynthesis of purine and pyrimidine nucleotides, coenzymes, and the amino acids histidine and tryptophan. These enzymes are used in biotechnology for the combined chemoenzymatic synthesis of natural nucleotide analogs. Recombinant phosphoribosylpyrophosphate synthetase I from the thermophilic strain HB27 of the bacterium Thermus thermophilus (T. th HB27) has high thermal stability and shows maximum activity at 75°Ð¡, due to which this enzyme holds promise for biotechnological applications. In order to grow crystals and study them by X-ray crystallography, an enzyme sample,more » which was produced using a highly efficient producer strain, was purified by affinity and gel-filtration chromatography. The screening of crystallization conditions was performed by the vapor-diffusion technique. The crystals of the enzyme suitable for X-ray diffraction were grown by the counter-diffusion method through a gel layer. These crystals were used to collect the X-ray diffraction data set at the SPring-8 synchrotron radiation facility (Japan) to 3-Å resolution. The crystals belong to sp. gr. P2{sub 1} and have the following unitcell parameters: a = 107.7 Å, b = 112.6 Å, c = 110.2 Å, α = γ = 90°, β = 116.6°. The X-ray diffraction data set is suitable for determining the three-dimensional structure of the enzyme at 3.0-Å resolution.« less

Crystallization and preliminary X-ray diffraction analysis of recombinant phosphoribosylpyrophosphate synthetase from the Thermophilic thermus thermophilus strain HB27

NASA Astrophysics Data System (ADS)

Abramchik, Yu. A.; Timofeev, V. I.; Muravieva, T. I.; Sinitsyna, E. V.; Esipov, R. S.; Kuranova, I. P.

2017-01-01

Phosphoribosylpyrophosphate synthetases (PRPP synthetases) are among the key enzymes essential for vital functions of organisms and are involved in the biosynthesis of purine and pyrimidine nucleotides, coenzymes, and the amino acids histidine and tryptophan. These enzymes are used in biotechnology for the combined chemoenzymatic synthesis of natural nucleotide analogs. Recombinant phosphoribosylpyrophosphate synthetase I from the thermophilic strain HB27 of the bacterium Thermus thermophilus ( T. th HB27) has high thermal stability and shows maximum activity at 75°C, due to which this enzyme holds promise for biotechnological applications. In order to grow crystals and study them by X-ray crystallography, an enzyme sample, which was produced using a highly efficient producer strain, was purified by affinity and gel-filtration chromatography. The screening of crystallization conditions was performed by the vapor-diffusion technique. The crystals of the enzyme suitable for X-ray diffraction were grown by the counter-diffusion method through a gel layer. These crystals were used to collect the X-ray diffraction data set at the SPring-8 synchrotron radiation facility (Japan) to 3-Å resolution. The crystals belong to sp. gr. P21 and have the following unitcell parameters: a = 107.7 Å, b = 112.6 Å, c = 110.2 Å, α = γ = 90°, β = 116.6°. The X-ray diffraction data set is suitable for determining the three-dimensional structure of the enzyme at 3.0-Å resolution.

Quantification of N-acetyl- and N-glycolylneuraminic acids by a stable isotope dilution assay using high-performance liquid chromatography-tandem mass spectrometry.

PubMed

Allevi, Pietro; Femia, Eti Alessandra; Costa, Maria Letizia; Cazzola, Roberta; Anastasia, Mario

2008-11-28

The present report describes a method for the quantification of N-acetyl- and N-glycolylneuraminic acids without any derivatization, using their (13)C(3)-isotopologues as internal standards and a C(18) reversed-phase column modified by decylboronic acid which allows for the first time a complete chromatographic separation between the two analytes. The method is based on high-performance liquid chromatographic coupled with electrospray ion-trap mass spectrometry. The limit of quantification of the method is 0.1mg/L (2.0ng on column) for both analytes. The calibration curves are linear for both sialic acids over the range of 0.1-80mg/L (2.0-1600ng on column) with a correlation coefficient greater than 0.997. The proposed method was applied to the quantitative determination of sialic acids released from fetuin as a model of glycoproteins.

Identification of protein interfaces within the multi-aminoacyl-tRNA synthetase complex: the case of lysyl-tRNA synthetase and the scaffold protein p38.

PubMed

Rémion, Azaria; Khoder-Agha, Fawzi; Cornu, David; Argentini, Manuela; Redeker, Virginie; Mirande, Marc

2016-07-01

Human cytoplasmic lysyl-tRNA synthetase (LysRS) is associated within a multi-aminoacyl-tRNA synthetase complex (MSC). Within this complex, the p38 component is the scaffold protein that binds the catalytic domain of LysRS via its N-terminal region. In addition to its translational function when associated to the MSC, LysRS is also recruited in nontranslational roles after dissociation from the MSC. The balance between its MSC-associated and MSC-dissociated states is essential to regulate the functions of LysRS in cellular homeostasis. With the aim of understanding the rules that govern association of LysRS in the MSC, we analyzed the protein interfaces between LysRS and the full-length version of p38, the scaffold protein of the MSC. In a previous study, the cocrystal structure of LysRS with a N-terminal peptide of p38 was reported [Ofir-Birin Y et al. (2013) Mol Cell 49, 30-42]. In order to identify amino acid residues involved in interaction of the two proteins, the non-natural, photo-cross-linkable amino acid p-benzoyl-l-phenylalanine (Bpa) was incorporated at 27 discrete positions within the catalytic domain of LysRS. Among the 27 distinct LysRS mutants, only those with Bpa inserted in place of Lys356 or His364 were cross-linked with p38. Using mass spectrometry, we unambiguously identified the protein interface of the cross-linked complex and showed that Lys356 and His364 of LysRS interact with the peptide from Pro8 to Arg26 in native p38, in agreement with the published cocrystal structure. This interface, which in LysRS is located on the opposite side of the dimer to the site of interaction with its tRNA substrate, defines the core region of the MSC. The residues identified herein in human LysRS are not conserved in yeast LysRS, an enzyme that does not associate within the MSC, and contrast with the residues proposed to be essential for LysRS:p38 association in the earlier work.

A high-throughput screen against pantothenate synthetase (PanC) identifies 3-biphenyl-4-cyanopyrrole-2-carboxylic acids as a new class of inhibitor with activity against Mycobacterium tuberculosis.

PubMed

Kumar, Anuradha; Casey, Allen; Odingo, Joshua; Kesicki, Edward A; Abrahams, Garth; Vieth, Michal; Masquelin, Thierry; Mizrahi, Valerie; Hipskind, Philip A; Sherman, David R; Parish, Tanya

2013-01-01

The enzyme pantothenate synthetase, PanC, is an attractive drug target in Mycobacterium tuberculosis. It is essential for the in vitro growth of M. tuberculosis and for survival of the bacteria in the mouse model of infection. PanC is absent from mammals. We developed an enzyme-based assay to identify inhibitors of PanC, optimized it for high-throughput screening, and tested a large and diverse library of compounds for activity. Two compounds belonging to the same chemical class of 3-biphenyl-4- cyanopyrrole-2-carboxylic acids had activity against the purified recombinant protein, and also inhibited growth of live M. tuberculosis in manner consistent with PanC inhibition. Thus we have identified a new class of PanC inhibitors with whole cell activity that can be further developed.

Characterization of O-acetylation in sialoglycans by MALDI-MS using a combination of methylamidation and permethylation

NASA Astrophysics Data System (ADS)

Wu, Zhaoguan; Li, Henghui; Zhang, Qiwei; Liu, Xin; Zheng, Qi; Li, Jianjun

2017-04-01

O-Acetylation of sialic acid in protein N-glycans is an important modification and can occur at either 4-, 7-, 8- or 9-position in various combinations. This modification is usually labile under alkaline reaction conditions. Consequently, a permethylation-based analytical method, which has been widely used in glycomics studies, is not suitable for profiling O-acetylation of sialic acids due to the harsh reaction conditions. Alternatively, methylamidation can be used for N-glycan analysis without affecting the base-labile modification of sialic acid. In this report, we applied both permethylation and methylamidation approaches to the analysis of O-acetylation in sialic acids. It has been demonstrated that methylamidation not only stabilizes sialic acids during MALDI processing but also allow for characterization of their O-acetylation pattern. In addition, LC-MS/MS experiments were carried out to distinguish between the O-acetylated glycans with potential isomeric structures. The repeatability of methylamidation was examined to evaluate the applicability of the approach to profiling of O-acetylation in sialic acids. In conclusion, the combination of methylamidation and permethylation methodology is a powerful MALDI-TOF MS-based tool for profiling O-acetylation in sialic acids applicable to screening of N-glycans.

Evolutionary anomalies among the aminoacyl-tRNA synthetases

NASA Technical Reports Server (NTRS)

Doolittle, R. F.; Handy, J.; Bada, J. L. (Principal Investigator)

1998-01-01

Unexpected relationships among the various aminoacyl-tRNA synthetases continue to be uncovered. The question arises - is this mainly the result of promiscuous exchange, or is the confusion really a reflection of the differential loss of past duplications? Phylogenetic analysis may yet provide the answer.

Effects of GSH1 and GSH2 Gene Mutation on Glutathione Synthetases Activity of Saccharomyces cerevisiae.

PubMed

Xu, Wen; Jia, Haiyan; Zhang, Longmei; Wang, Haiyan; Tang, Hui; Zhang, Liping

2017-08-01

In this paper, three mutants from wild Saccharomyces cerevisiae HBU2.558, called U2.558, UN2.558, and UNA2.558, were screened by UV, sodium nitrite, Atmospheric and room temperature plasma, respectively. Glutathione production of the three mutants increased by 41.86, 72.09 and 56.76%, respectively. We detected the activity of glutathione synthetases and found that its activity was improved. Amino acid sequences of three mutant colonies were compared with HBU2.558. Four mutants: Leu51→Pro51 (L51P), Glu62→Val62 (E62V), Ala332→Glu332 (A332E) and Ser653→Gly653 (S653G) were found in the analysis of γ-glutamylcysteine ligase. L51 is located adjacently to the two active sites of GCL/E/Mg 2+ /ADP complex in the overall GCL structure. L51P mutant spread distortion on the β-sheet due to the fact that the φ was changed from -50.4° to -40.2°. A mutant Leu54→Pro54 (L54P) was found in the analysis of glutathione synthetase, and L54 was an amino acid located between an α-helix and a β-sheet. The results confirm that introduction of proline located at the middle of the β-sheet or at the N- or C-terminal between α-helix and β-sheet or, i.e., L51P and L54P, changed the φ, rigidity, hydrophobicity and conformational entropy, thus increased protein stability and improved the enzyme activity.

Synergetic effect of chelating agent and nonionic surfactant for benzotriazole removal on post Cu-CMP cleaning

NASA Astrophysics Data System (ADS)

Yanlei, Li; Yuling, Liu; Chenwei, Wang; Yue, Li

2016-08-01

The cleaning of copper interconnects after chemical mechanical planarization (CMP) process is a critical step in integrated circuits (ICs) fabrication. Benzotriazole (BTA), which is used as corrosion inhibitor in the copper CMP slurry, is the primary source for the formation of organic contaminants. The presence of BTA can degrade the electrical properties and reliability of ICs which needs to be removed by using an effective cleaning solution. In this paper, an alkaline cleaning solution was proposed. The alkaline cleaning solution studied in this work consists of a chelating agent and a nonionic surfactant. The removal of BTA was characterized by contact angle measurements and potentiodynamic polarization studies. The cleaning properties of the proposed cleaning solution on a 300 mm copper patterned wafer were also quantified, total defect counts after cleaning was studied, scanning electron microscopy (SEM) review was used to identify types of BTA to confirm the ability of cleaning solution for BTA removal. All the results reveal that the chelating agent can effectively remove the BTA residual, nonionic surfactant can further improve the performance. Project supported by the Natural Science Foundation of Hebei Province, China (No. F2015202267) and the Scientific Innovation Grant for Excellent Young Scientists of Hebei University of Technology (No. 2015007).

Compositions of orthogonal glutamyl-tRNA and aminoacyl-tRNA synthetase pairs and uses thereof

DOEpatents

Anderson, J Christopher [San Francisco, CA; Schultz, Peter G [La Jolla, CA; Santoro, Stephen [Cambridge, MA

2009-05-05

Compositions and methods of producing components of protein biosynthetic machinery that include glutamyl orthogonal tRNAs, glutamyl orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of glutamyl tRNAs/synthetases are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins using these orthogonal pairs.

Thermal modelling of stepwise anatexis in a thrust-thickened sialic crust

USGS Publications Warehouse

Zen, E.-A.

1988-01-01

One-dimensional modelling of the thermal history of a sialic crust thickened by multiple overstack thrusting of upper crustal material shows that anatexis is likely. both the uplift rate and the length of the incubation period between end of tectonism and start of uplift are important controls on the amount and temperature of the melt. Heat of fusion does not significantly affect the long-term thermal structure of the crust if the melt is not extracted because only a small fraction of conductive heat is converted to latent heat, though short-term thermal effects of latent heat can be locally important. Model results show that commonly <15% of mantle heat flux is converted to latent heat; even during peak melting in the most productive models, less than half of incremental mantle flux is converted. The results have obvious implications on the acceptability of proposed heat sources for crustal anatexis. Fusion could retard crustal temperature rise by nearly 100??C, but the system would recover except for situations of very rapid uplift. Understanding of the thermal evolution of a burial-uplift system requires knowledge not only of the timing of anatexis but of the pooling and movement of the magma, as well as the duration and nature of the incubation period; we are poorly equipped to measure these events. The model predicts that the characteristic time for anatexis is a thickened sialic crust is several tens of millions of years, comparable to the time lapse between orogenies; in making geological interpretations of magmatism, this time lag must be considered. -Author

Microbial biofilms for the removal of Cu²⁺ from CMP wastewater.

PubMed

Mosier, Aaron P; Behnke, Jason; Jin, Eileen T; Cady, Nathaniel C

2015-09-01

The modern semiconductor industry relies heavily on a process known as chemical mechanical planarization, which uses physical and chemical processes to remove excess material from the surface of silicon wafers during microchip fabrication. This process results in large volumes of wastewater containing dissolved metals including copper (Cu(2+)), which must then be filtered and treated before release into municipal waste systems. We have investigated the potential use of bacterial and fungal biomass as an alternative to the currently used ion-exchange resins for the adsorption of dissolved Cu(2+) from high-throughput industrial waste streams. A library of candidate microorganisms, including Lactobacillus casei and Pichia pastoris, was screened for ability to bind Cu(2+) from solution and to form static biofilm communities within packed-bed adsorption columns. The binding efficiency of these biomass-based adsorption columns was assessed under various flow conditions and compared to that of industrially used ion-exchange resins. We demonstrated the potential to regenerate the biomass within the adsorption columns through the use of a hydrochloric acid wash, and subsequently reuse the columns for additional copper binding. While the binding efficiency and capacity of the developed L. casei/P. pastoris biomass filters was inferior to ion-exchange resin, the potential for repeated reuse of these filters, coupled with the advantages of a more sustainable "green" adsorption process, make this technique an attractive candidate for use in industrial-scale CMP wastewater treatment. Copyright © 2015 Elsevier Ltd. All rights reserved.

Purification, gene cloning, and characterization of γ-butyrobetainyl CoA synthetase from Agrobacterium sp. 525a.

PubMed

Fujimitsu, Hiroshi; Matsumoto, Akira; Takubo, Sayaka; Fukui, Akiko; Okada, Kazuma; Mohamed Ahmed, Isam A; Arima, Jiro; Mori, Nobuhiro

2016-08-01

The report is the first of purification, overproduction, and characterization of a unique γ-butyrobetainyl CoA synthetase from soil-isolated Agrobacterium sp. 525a. The primary structure of the enzyme shares 70-95% identity with those of ATP-dependent microbial acyl-CoA synthetases of the Rhizobiaceae family. As distinctive characteristics of the enzyme of this study, ADP was released in the catalytic reaction process, whereas many acyl CoA synthetases are annotated as an AMP-forming enzyme. The apparent Km values for γ-butyrobetaine, CoA, and ATP were, respectively, 0.69, 0.02, and 0.24 mM.

Henoch-Schönlein purpura nephritis occurring postpartum in a patient with anti-PL-7 anti-synthetase syndrome.

PubMed

Nagai, Kojiro; Kishi, Jun; Morizumi, Shun; Minakuchi, Jun; Bando, Yoshimi; Nishioka, Yasuhiko; Doi, Toshio

2017-09-01

A 37-year-old pregnant woman developed purpura which was subsequently diagnosed as Henoch-Schönlein purpura (HSP). After childbirth, the patient developed proteinuria and hematuria. Further examination revealed that the HSP nephritis (HSPN) was associated with anti-threonyl-tRNA synthetase anti-synthetase syndrome. The onset of HSPN during pregnancy or after childbirth is rare. Moreover, to our knowledge, this is the first case to describe renal involvement in anti-synthetase syndrome.

Novel Reaction of Succinyl Coenzyme A (Succinyl-CoA) Synthetase: Activation of 3-Sulfinopropionate to 3-Sulfinopropionyl-CoA in Advenella mimigardefordensis Strain DPN7T during Degradation of 3,3′-Dithiodipropionic Acid ▿ †

PubMed Central

Schürmann, Marc; Wübbeler, Jan Hendrik; Grote, Jessica; Steinbüchel, Alexander

2011-01-01

The sucCD gene of Advenella mimigardefordensis strain DPN7T encodes a succinyl coenzyme A (succinyl-CoA) synthetase homologue (EC 6.2.1.4 or EC 6.2.1.5) that recognizes, in addition to succinate, the structural analogues 3-sulfinopropionate (3SP) and itaconate as substrates. Accumulation of 3SP during 3,3′-dithiodipropionic acid (DTDP) degradation was observed in Tn5::mob-induced mutants of A. mimigardefordensis strain DPN7T disrupted in sucCD and in the defined deletion mutant A. mimigardefordensis ΔsucCD. These mutants were impaired in growth with DTDP and 3SP as the sole carbon source. Hence, it was proposed that the succinyl-CoA synthetase homologue in A. mimigardefordensis strain DPN7T activates 3SP to the corresponding CoA-thioester (3SP-CoA). The putative genes coding for A. mimigardefordensis succinyl-CoA synthetase (SucCDAm) were cloned and heterologously expressed in Escherichia coli BL21(DE3)/pLysS. Purification and characterization of the enzyme confirmed its involvement during degradation of DTDP. 3SP, the cleavage product of DTDP, was converted into 3SP-CoA by the purified enzyme, as demonstrated by in vitro enzyme assays. The structure of 3SP-CoA was verified by using liquid chromatography-electrospray ionization-mass spectrometry. SucCDAm is Mg2+ or Mn2+ dependent and unspecific regarding ATP or GTP. In kinetic studies the enzyme showed highest enzyme activity and substrate affinity with succinate (Vmax = 9.85 ± 0.14 μmol min−1 mg−1, Km = 0.143 ± 0.001 mM). In comparison to succinate, activity with 3SP was only ca. 1.2% (Vmax = 0.12 ± 0.01 μmol min−1 mg−1) and the affinity was 6-fold lower (Km = 0.818 ± 0.046 mM). Based on the present results, we conclude that SucCDAm is physiologically associated with the citric acid cycle but is mandatory for the catabolic pathway of DTDP and its degradation intermediate 3SP. PMID:21515777

New Aminoacyl-tRNA Synthetase-like Protein in Insecta with an Essential Mitochondrial Function*♦

PubMed Central

Guitart, Tanit; Leon Bernardo, Teresa; Sagalés, Jessica; Stratmann, Thomas; Bernués, Jordi; Ribas de Pouplana, Lluís

2010-01-01

Aminoacyl-tRNA synthetases (ARS) are modular enzymes that aminoacylate transfer RNAs (tRNA) for their use by the ribosome during protein synthesis. ARS are essential and universal components of the genetic code that were almost completely established before the appearance of the last common ancestor of all living species. This long evolutionary history explains the growing number of functions being discovered for ARS, and for ARS homologues, beyond their canonical role in gene translation. Here we present a previously uncharacterized paralogue of seryl-tRNA synthetase named SLIMP (seryl-tRNA synthetase-like insect mitochondrial protein). SLIMP is the result of a duplication of a mitochondrial seryl-tRNA synthetase (SRS) gene that took place in early metazoans and was fixed in Insecta. Here we show that SLIMP is localized in the mitochondria, where it carries out an essential function that is unrelated to the aminoacylation of tRNA. The knockdown of SLIMP by RNA interference (RNAi) causes a decrease in respiration capacity and an increase in mitochondrial mass in the form of aberrant mitochondria. PMID:20870726

Engineering posttranslational proofreading to discriminate nonstandard amino acids