Consensus proposals for classification of the family Hepeviridae
Smith, D.B. ; Simmonds, P. ; Jameel, S. ; Emerson, S.U. ; Harrison, T.J. ; Meng, X.J. ; Okamoto, H. ; Poel, W.H.M. van der; Purdy, M.A. - \ 2014
Journal of General Virology 95 (2014). - ISSN 0022-1317 - p. 2223 - 2232.
hepatitis-e virus - genetic-variability - phylogenetic analysis - molecular-cloning - wild rats - c virus - hev - genotype - china - recombination
The family Hepeviridae consists of positive-stranded RNA viruses that infect a wide range of mammalian species, as well as chickens and trout. A subset of these viruses infects humans and can cause a self-limiting acute hepatitis that may become chronic in immunosuppressed individuals. Current published descriptions of the taxonomical divisions within the family Hepeviridae are contradictory in relation to the assignment of species and genotypes. Through analysis of existing sequence information, we propose a taxonomic scheme in which the family is divided into the genera Orthohepevirus (all mammalian and avian hepatitis E virus (HEV) isolates) and Piscihepevirus (cutthroat trout virus). Species within the genus Orthohepevirus are designated Orthohepevirus A (isolates from human, pig, wild boar, deer, mongoose, rabbit and camel), Orthohepevirus B (isolates from chicken), Orthohepevirus C (isolates from rat, greater bandicoot, Asian musk shrew, ferret and mink) and Orthohepevirus D (isolates from bat). Proposals are also made for the designation of genotypes within the human and rat HEVs. This hierarchical system is congruent with hepevirus phylogeny, and the three classification levels (genus, species and genotype) are consistent with, and reflect discontinuities in the ranges of pairwise distances between amino acid sequences. Adoption of this system would include the avoidance of host names in taxonomic identifiers and provide a logical framework for the assignment of novel variants.
Mechanisms involved in apoptosis of carp leukocytes upon in vitro and in vivo immunostimulation
Kepka, M. ; Verburg-van Kemenade, B.M.L. ; Homa, J. ; Chadzinska, M.K. - \ 2014
Fish and Shellfish Immunology 39 (2014)2. - ISSN 1050-4648 - p. 386 - 395.
cytochrome-c release - molecular-cloning - cyprinus-carpio - nitric-oxide - transgenic zebrafish - signal-transduction - ceramide generation - respiratory burst - human neutrophils - oxidative stress
During inflammation leukocyte activity must be carefully regulated, as high concentrations and/or prolonged action of pro-inflammatory mediators e.g. reactive oxygen species (ROS) can be detrimental not only for pathogens but also for host tissues. Programmed cell death – apoptosis is a most effective regulatory mechanism for down regulation of leukocyte activity, but little is known about this process in fish. We aimed to reveal the mechanisms of initiation and regulation of apoptosis in carp neutrophilic granulocytes and macrophages. During zymosan-induced peritonitis in carp, activated inflammatory neutrophilic granulocytes and monocytes/macrophages died by apoptosis. This correlated with a strong production of ROS, but pretreatment of the fish with NADPH oxidase inhibitor only slightly decreased late apoptosis. Interestingly in vitro incubation with zymosan or phorbol ester, but not lipopolisaccharide and poli I:C induced apoptosis of head kidney neutrophilic granulocytes. This coincided with loss of mitochondrial membrane potential. Moreover, in zymosan-stimulated neutrophilic granulocytes NADPH oxidase inhibitor not only reduced the production of ROS but also apoptosis. A similar effect was not observed in cells stimulated with phorbol ester, where DPI reduced ROS production, but not apoptosis. In PMA-stimulated neutrophilic granulocytes both the respiratory burst and apoptosis were reduced by protein kinase inhibitor. Furthermore, a short neutrophil stimulation either with PMA or with zymosan did induce caspase-independent apoptosis. These results show that in carp, apoptosis is an important regulatory process during in vitro and in vivo immunostimulation. In neutrophils, protein kinase, but not NADPH oxidase, is involved in PMA-induced apoptosis while apoptosis induced by zymosan is ROS-dependent.
Evaluation of immune and apoptosis related gene responses using an RNAi approach in vaccinated Penaeus monodon during oral WSSV infection
Kulkarni, A.D. ; Caipang, C.M.A. ; Kiron, V. ; Rombout, J.H.W.M. ; Fernandes, J.M.O. ; Brinchmann, M. - \ 2014
Marine Genomics 18 (2014)part A. - ISSN 1874-7787 - p. 55 - 65.
spot-syndrome-virus - double-stranded-rna - black tiger shrimp - toll-like receptors - litopenaeus-vannamei - molecular-cloning - innate immunity - fenneropenaeus-chinensis - expression analysis - vibrio-anguillarum
In the present study RNA interference was used to elucidate the connection between two endogenous genes [Penaeus monodon Rab7 (PmRab7) or P. monodon inhibitor of apoptosis (PmIAP)], and selected immune/apoptosis-related genes in orally ‘vaccinated’ shrimp after white spot syndrome virus (WSSV) infection. P. monodon were vaccinated by feeding them with formalin inactivated WSSV-coated feed. Thereafter, PmRab7 or PmIAP genes were silenced by injecting the shrimps with their respective dsRNA. The resulting groups of shrimps, Rab7 and IAP, were orally infected with WSSV and the expression of three immune-relevant genes in Rab7 group and five apoptosis-related genes in IAP group was evaluated. In the Rab7 group, PmToll, PmPPAE 2 and Pm penaeidin genes were down-regulated. The IAP-silenced shrimps were characterized by down-regulation of Pm caspase, PmERp57, Pm14-3-3 e, Pm ald, and up-regulation of PmSTAT. Thus, silencing of PmRab7/PmIAP has provided important clues on their relationship with selected immune/apoptosis genes in orally vaccinated P. monodon during WSSV infection.
Protein profiling in the gut of Penaeus monodon gavaged with oral WSSV-vaccines and live white spot syndrom virus
Kulkarni, A.D. ; Kiron, V. ; Rombout, J.H.W.M. ; Brinchmann, M. ; Fernandes, J.M.O. ; Sudheer, N.S. ; Singh, B.I.S. - \ 2014
Proteomics 14 (2014)13-14. - ISSN 1615-9853 - p. 1660 - 1673.
differentially expressed genes - myosin light-chain - fenneropenaeus-chinensis - litopenaeus-vannamei - proteomic analysis - molecular-cloning - arginine kinase - binding-protein - phosphoglycerate kinase - marsupenaeus-japonicus
White spot syndrome virus (WSSV) is a pathogen that causes considerable mortality of the farmed shrimp, Penaeus monodon. Candidate ‘vaccines’, WSSV envelope protein VP28 and formalin-inactivated WSSV, can provide short-lived protection against the virus. In this study, P. monodon was orally intubated with the aforementioned vaccine candidates, and protein expression in the gut of immunised shrimps was profiled. The alterations in protein profiles in shrimps infected orally with live-WSSV were also examined. Seventeen of the identified proteins in the vaccine and WSSV-intubated shrimps varied significantly compared to those in the control shrimps. These proteins, classified under exoskeletal, cytoskeletal, immune-related, intracellular organelle part, intracellular calcium-binding or energy metabolism, are thought to directly or indirectly affect shrimp's immunity. The changes in the expression levels of crustacyanin, serine proteases, myosin light chain, and ER protein 57 observed in orally vaccinated shrimp may probably be linked to immunoprotective responses. On the other hand, altered expression of proteins linked to exoskeleton, calcium regulation and energy metabolism in WSSV-intubated shrimps is likely to symbolise disturbances in calcium homeostasis and energy metabolism.
Zebrafish ISG15 Exerts a Strong Antiviral Activity against RNA and DNA Viruses and Regulates the Interferon Response
Langevin, C. ; Aa, L.M. van der; Houel, A. ; Torhy, C. ; Briolat, V. ; Lunazzi, A. ; Harmache, A. ; Bremont, M. ; Levraud, J.P. ; Boudinot, P. - \ 2013
Journal of Virology 87 (2013)18. - ISSN 0022-538X - p. 10025 - 10036.
ubiquitin-like protein - induced 15-kda protein - stimulated gene isg15 - ifn-gamma immunity - i interferon - rig-i - mycobacterial disease - expression analysis - molecular-cloning - infected-cells
ISG15, a 15-kDa interferon-induced protein that participates in antiviral defenses of mammals, is highly conserved among vertebrates. In fish, as in mammals, viral infection and interferon treatment induce isg15 expression. The two ubiquitin-like domains of ISG15 and the presence of a consensus LRLRGG sequence in the C-terminal region, which is required for the covalent conjugation to a substrate protein, are also conserved in fish. Our data demonstrate that overexpression of zebrafish ISG15 (zf-ISG15) in EPC cells is sufficient to inhibit viral infection by RNA viruses belonging to the genera Novirhabdovirus and Birnavirus and by DNA viruses of the genus Iridovirus. In coexpression experiments with IHNV proteins, we demonstrate specific ISGylation of phosphoprotein and nonvirion protein. Mutation of the glycine residues in the consensus LRLRGG motif abolishes zf-ISG15 conjugation to these proteins and the cellular protection against viral infection, thus connecting ISGylation and ISG15-dependent viral restriction. Additionally, zf-ISG15 overexpression triggers induction of the rig-I and viperin genes as well as, to a lesser extent, the IFN gene. Overall, our data demonstrate the antiviral effect of a fish ISG15 protein, revealing the conservation among vertebrates of an ISGylation mechanism likely directed against viruses. Furthermore, our findings indicate that zf-ISG15 affects the IFN system at several levels, and its study shall shed further light on the evolution of the complex regulation of the innate antiviral response in vertebrate cells.
The metabolite chemotype of Nicotiana benthamiana transiently expressing artemisinin biosynthetic pathway genes is a function of CYP71AV1 type and relative gene dosage
Ting, H.M. ; Wang, B. ; Ryden, A.M. ; Woittiez, L.S. ; Verstappen, F.W.A. ; Ruyter-Spira, C.P. ; Beekwilder, M.J. ; Bouwmeester, H.J. ; Krol, A.R. van der; Herpen, T.W.J.M. van - \ 2013
New Phytologist 199 (2013)2. - ISSN 0028-646X - p. 352 - 366.
molecular-cloning - annua l - amorpha-4,11-diene synthase - dihydroartemisinic acid - mass-spectrometry - key role - arabidopsis - identification - metabolomics - precursors
Artemisia annua, which produces the anti-malaria compound artemisinin, occurs as high-artemisinin production (HAP) and low-artemisinin production (LAP) chemotypes. Understanding the basis of the difference between these chemotypes would assist breeding and optimising artemisinin biosynthesis. Here we present a systematic comparison of artemisinin biosynthesis genes that may be involved in determining the chemotype (CYP71AV1, DBR2 and ALDH1). These genes were isolated from the two chemotypes and characterized using transient expression in planta. The enzyme activity of DBR2 and ALDH1 from the two chemotypes did not differ, but structural differences in CYP71AV1 from LAP and HAP chemotypes (AMOLAP and AMOHAP, respectively) resulted in altered enzyme activity. AMOLAP displays a seven amino acids N-terminal extension compared with AMOHAP. The GFP fusion of both proteins show equal localization to the ER but AMOHAP may have reduced stability. Upon transient expression in Nicotiana benthamiana, AMOLAP displayed a higher enzyme activity than AMOHAP. However, expression in combination with the other pathway genes also resulted in a qualitatively different product profile ('chemotype'); that is, in a shift in the ratio between the unsaturated and saturated (dihydro) branch of the pathway.
The Irish potato famine pathogen Phytophthora infestans translocates the CRN8 kinase into host plant cells
Damme, M. van; Bozkurt, T.O. ; Cakir, C. ; Schornack, S. ; Sklenar, J. ; Jones, A.M.E. ; Kamoun, S. - \ 2012
PLoS Pathogens 8 (2012)8. - ISSN 1553-7366
toxoplasma-gondii - protein-kinase - iii effectors - tyrosine-phosphatase - oomycete effectors - molecular-cloning - virulence factor - rhoptry protein - innate immunity - rxlr effectors
Phytopathogenic oomycetes, such as Phytophthora infestans, secrete an arsenal of effector proteins that modulate plant innate immunity to enable infection. We describe CRN8, a host-translocated effector of P. infestans that has kinase activity in planta. CRN8 is a modular protein of the CRN effector family. The C-terminus of CRN8 localizes to the host nucleus and triggers cell death when the protein is expressed in planta. Cell death induction by CRN8 is dependent on its localization to the plant nucleus, which requires a functional nuclear localization signal (NLS). The C-terminal sequence of CRN8 has similarity to a serine/threonine RD kinase domain. We demonstrated that CRN8 is a functional RD kinase and that its autophosphorylation is dependent on an intact catalytic site. Co-immunoprecipitation experiments revealed that CRN8 forms a dimer or multimer. Heterologous expression of CRN8 in planta resulted in enhanced virulence by P. infestans. In contrast, in planta expression of the dominant-negative CRN8 R469A; D470A resulted in reduced P. infestans infection, further implicating CRN8 in virulence. Overall, our results indicate that similar to animal parasites, plant pathogens also translocate biochemically active kinase effectors inside host cells.
Expansion of the gamma-gliadin gene family in Aegilops and Triticum
Goryunova, S.V. ; Salentijn, E.M.J. ; Chikida, N.N. ; Kochieva, E.Z. ; Meer, I.M. van der; Gilissen, L.J.W.J. ; Smulders, M.J.M. - \ 2012
BMC Evolutionary Biology 12 (2012). - ISSN 1471-2148 - 12 p.
restriction fragment analysis - repeated nucleotide-sequences - t-cell epitopes - phylogenetic analysis - common wheat - bread wheat - molecular-cloning - rna interference - storage proteins - chloroplast dna
Background - The gamma-gliadins are considered to be the oldest of the gliadin family of storage proteins in Aegilops/Triticum. However, the expansion of this multigene family has not been studied in an evolutionary perspective. Results - We have cloned 59 gamma-gliadin genes from Aegilops and Triticum species (Aegilops caudata L., Aegilops comosa Sm. in Sibth. & Sm., Aegilops mutica Boiss., Aegilops speltoides Tausch, Aegilops tauschii Coss., Aegilops umbellulata Zhuk., Aegilops uniaristata Vis., and Triticum monococcum L.) representing eight different genomes: Am, B/S, C, D, M, N, T and U. Overall, 15% of the sequences contained internal stop codons resulting in pseudogenes, but this percentage was variable among genomes, up to over 50% in Ae. umbellulata. The most common length of the deduced protein, including the signal peptide, was 302 amino acids, but the length varied from 215 to 362 amino acids, both obtained from Ae. speltoides. Most genes encoded proteins with eight cysteines. However, all Aegilops species had genes that encoded a gamma-gliadin protein of 302 amino acids with an additional cysteine. These conserved nine-cysteine gamma-gliadins may perform a specific function, possibly as chain terminators in gluten network formation in protein bodies during endosperm development. A phylogenetic analysis of gamma-gliadins derived from Aegilops and Triticum species and the related genera Lophopyrum, Crithopsis, and Dasypyrum showed six groups of genes. Most Aegilops species contained gamma-gliadin genes from several of these groups, which also included sequences from the genera Lophopyrum, Crithopsis, and Dasypyrum. Hordein and secalin sequences formed separate groups. Conclusions - We present a model for the evolution of the gamma-gliadins from which we deduce that the most recent common ancestor (MRCA) of Aegilops/Triticum-Dasypyrum-Lophopyrum-Crithopsis already had four groups of gamma-gliadin sequences, presumably the result of two rounds of duplication of the locus.
A century of poultry genetics
Tixier-Boichard, M. ; Leenstra, F.R. ; Flock, D. ; Hocking, A.D. ; Weigend, S. - \ 2012
Worlds Poultry Science Journal 68 (2012)2. - ISSN 0043-9339 - p. 307 - 321.
major histocompatibility complex - chicken genome - nucleolar organizer - molecular-cloning - linked factors - linkage map - egg-white - rfp-y - diversity - selection
The 20th Century saw an astonishing advance in our understanding of genetics and the scientific basis of the genetic improvement of farm animals. The application of genetic principles to chickens in the 1950s and 1960s led to a rapid change in the productivity and efficiency of laying hens and broiler chickens, turkeys and ducks. Subsequently, the application of increasingly powerful computers and sophisticated mathematical algorithms has increased the range of traits that could be successfully incorporated into breeding programs. Random sample tests of the performance of laying hens enjoyed a period of popularity and more recently the few remaining tests included husbandry systems in addition to strain evaluation. Characterisation of avian blood groups has led to the identification of the B21 haplotype that confers resistance to Marek's disease and to selection for this locus in commercial lines. The decade following the millennium saw the publication of the genome sequence of the chicken and the identification of millions of single nucleotide polymorphisms that, coupled with technological advances, made the application of whole genome selection practical in poultry. In parallel, the molecular basis for some Mendelian traits described a century ago is now being deciphered. Similar technologies have been applied to study genetic diversity in chickens and have provided insights into the evolution and domestication of chicken breeds. Finally, in this review, the recent development of the European Poultry Genetics Symposia coordinated by Working Group 3 ‘Genetics and Breeding’ that was based on combining the British Poultry Breeders Round Table and AVIAGEN from West and Eastern Europe, is discussed
Mechanism and control of Genipa americana seed germination
Queiroz, S.E.E. ; Silva, E.A.A. da; Davide, A.C. ; Jose, A.C. ; Silva, A.T. ; Fraiz, A.C.R. ; Faria, J.M.R. ; Hilhorst, H.W.M. - \ 2012
Physiologia Plantarum 144 (2012)3. - ISSN 0031-9317 - p. 263 - 276.
endo-beta-mannanase - tomato lycopersicon-esculentum - endosperm cap - abscisic-acid - cell-wall - micropylar endosperm - radicle emergence - molecular-cloning - embryo - gene
Genipa americana (Rubiaceae) is important for restoration of riparian forest in the Brazilian Cerrado. The objective was to characterize the mechanism and control of germination of G. americana to support uniform seedling production. Morphology and morphometrics of seeds, embryo and endosperm were assessed by light and scanning electron microscopy during germination. Imbibition and germination curves were generated and over the same time interval endosperm digestion and resistance were measured by puncture force analysis and activity assay of endo-ß-mannanase in water and in abscisic acid. The gene encoding for endo-ß-mannanase was partially cloned and its expression monitored by qRT-PCR. Embryos displayed growth prior to radicle protrusion. A two-phase increase in endo-ß-mannanase activity coincided with the two stages of weakening of the micropylar endosperm. The second stage also coincided with growth of the embryo prior to radicle protrusion. Enzyme activity was initiated in the micropylar endosperm but spread to the lateral endosperm. ABA
Origin and evolution of TRIM proteins: new insights from the complete TRIM repertoire of zebrafish and pufferfish
Boudinot, P. ; Aa, L.M. van der; Jouneau, L. ; Pasquier, L. du; Pontarotti, P. ; Benmansour, A. ; Levraud, J.P. - \ 2011
PLoS ONE 6 (2011)7. - ISSN 1932-6203 - 28 p.
major histocompatibility complex - mhc class-i - leukocyte receptor complex - adaptive immune-system - e3 ubiquitin-ligase - teleost fish - retroviral restriction - positive selection - molecular-cloning - monkey trim5-alpha
Tripartite motif proteins (TRIM) constitute a large family of proteins containing a RING-Bbox-Coiled Coil motif followed by different C-terminal domains. Involved in ubiquitination, TRIM proteins participate in many cellular processes including antiviral immunity. The TRIM family is ancient and has been greatly diversified in vertebrates and especially in fish. We analyzed the complete sets of trim genes of the large zebrafish genome and of the compact pufferfish genome. Both contain three large multigene subsets - adding the hsl5/trim35-like genes (hltr) to the ftr and the btr that we previously described - all containing a B30.2 domain that evolved under positive selection. These subsets are conserved among teleosts. By contrast, most human trim genes of the other classes have only one or two orthologues in fish. Loss or gain of C-terminal exons generated proteins with different domain organizations; either by the deletion of the ancestral domain or, remarkably, by the acquisition of a new C-terminal domain. Our survey of fish trim genes in fish identifies subsets with different evolutionary dynamics. trims encoding RBCC-B30.2 proteins show the same evolutionary trends in fish and tetrapods: they evolve fast, often under positive selection, and they duplicate to create multigenic families. We could identify new combinations of domains, which epitomize how new trim classes appear by domain insertion or exon shuffling. Notably, we found that a cyclophilin-A domain replaces the B30.2 domain of a zebrafish fintrim gene, as reported in the macaque and owl monkey antiretroviral TRIM5a. Finally, trim genes encoding RBCC-B30.2 proteins are preferentially located in the vicinity of MHC or MHC gene paralogues, which suggests that such trim genes may have been part of the ancestral MHC.
Nicotiana benthamiana as a Production Platform for Artemisinin Precursors
Herpen, T.W.J.M. van; Cankar, K. ; Nogueira, M. ; Bosch, H.J. ; Bouwmeester, H.J. ; Beekwilder, M.J. - \ 2010
PLoS ONE 5 (2010)12. - ISSN 1932-6203 - 11 p.
antimalarial-drug artemisinin - expression system - molecular-cloning - plants - biosynthesis - annua - reductase - tobacco - yield - acid
Background Production of pharmaceuticals in plants provides an alternative for chemical synthesis, fermentation or natural sources. Nicotiana benthamiana is deployed at commercial scale for production of therapeutic proteins. Here the potential of this plant is explored for rapid production of precursors of artemisinin, a sesquiterpenoid compound that is used for malaria treatment. Methodology/Principal Findings Biosynthetic genes leading to artemisinic acid, a precursor of artemisinin, were combined and expressed in N. benthamiana by agro-infiltration. The first committed precursor of artemisinin, amorpha-4,11-diene, was produced upon infiltration of a construct containing amorpha-4,11-diene synthase, accompanied by 3-hydroxy-3-methylglutaryl-CoA reductase and farnesyl diphosphate synthase. Amorpha-4,11-diene was detected both in extracts and in the headspace of the N. benthamiana leaves. When the amorphadiene oxidase CYP71AV1 was co-infiltrated with the amorphadiene-synthesizing construct, the amorpha-4,11-diene levels strongly decreased, suggesting it was oxidized. Surprisingly, no anticipated oxidation products, such as artemisinic acid, were detected upon GC-MS analysis. However, analysis of leaf extracts with a non-targeted metabolomics approach, using LC-QTOF-MS, revealed the presence of another compound, which was identified as artemisinic acid-12-ß-diglucoside. This compound accumulated to 39.5 mg.kg-1 fwt. Apparently the product of the heterologous pathway that was introduced, artemisinic acid, is further metabolized efficiently by glycosyl transferases that are endogenous to N. benthamiana. Conclusion/Significance This work shows that agroinfiltration of N. bentamiana can be used as a model to study the production of sesquiterpenoid pharmaceutical compounds. The interaction between the ectopically introduced pathway and the endogenous metabolism of the plant is discussed.
Aspergillus nidulans-galactosidase of glycoside hydrolase family 36 catalyses the formation of -galacto-oligosaccharides by transglycosylation
Nakai, H. ; Baumann, M.J. ; Petersen, B.O. ; Westphal, Y. ; Hachem, M.A. ; Dilokpimol, A. ; Duus, J.O. ; Schols, H.A. ; Svensson, B. - \ 2010
FEBS Journal 277 (2010)17. - ISSN 1742-464X - p. 3538 - 3551.
molecular-cloning - bifidobacterium-adolescentis - n-acetylgalactosaminidase - escherichia-coli - pichia-pastoris - phanerochaete-chrysosporium - clostridium-perfringens - functional expression - lactobacillus-reuteri - thermotoga-maritima
The -galactosidase from Aspergillus nidulans (AglC) belongs to a phylogenetic cluster containing eukaryotic -galactosidases and -galacto-oligosaccharide synthases of glycoside hydrolase family 36 (GH36). The recombinant AglC, produced in high yield (0.65 g·L-1 culture) as His-tag fusion in Escherichia coli, catalysed efficient transglycosylation with -(1¿6) regioselectivity from 40 mm 4-nitrophenol -d-galactopyranoside, melibiose or raffinose, resulting in a 37–74% yield of 4-nitrophenol -d-Galp-(1¿6)-d-Galp, -d-Galp-(1¿6)--d-Galp-(1¿6)-d-Glcp and -d-Galp-(1¿6)--d-Galp-(1¿6)-d-Glcp-(1¿ß2)-d-Fruf (stachyose), respectively. Furthermore, among 10 monosaccharide acceptor candidates (400 mm) and the donor 4-nitrophenol -d-galactopyranoside (40 mm), -(1¿6) linked galactodisaccharides were also obtained with galactose, glucose and mannose in high yields of 39–58%. AglC did not transglycosylate monosaccharides without the 6-hydroxymethyl group, i.e. xylose, l-arabinose, l-fucose and l-rhamnose, or with axial 3-OH, i.e. gulose, allose, altrose and l-rhamnose. Structural modelling using Thermotoga maritima GH36 -galactosidase as the template and superimposition of melibiose from the complex with human GH27 -galactosidase supported that recognition at subsite +1 in AglC presumably requires a hydrogen bond between 3-OH and Trp358 and a hydrophobic environment around the C-6 hydroxymethyl group. In addition, successful transglycosylation of eight of 10 disaccharides (400 mm), except xylobiose and arabinobiose, indicated broad specificity for interaction with the +2 subsite. AglC thus transferred -galactosyl to 6-OH of the terminal residue in the -linked melibiose, maltose, trehalose, sucrose and turanose in 6–46% yield and the ß-linked lactose, lactulose and cellobiose in 28–38% yield. The product structures were identified using NMR and ESI-MS and five of the 13 identified products were novel, i.e. -d-Galp-(1¿6)-d-Manp; -d-Galp-(1¿6)-ß-d-Glcp-(1¿4)-d-Glcp; -d-Galp-(1¿6)-ß-d-Galp-(1¿4)-d-Fruf; -d-Galp-(1¿6)-d-Glcp-(1¿1)-d-Glcp; and -d-Galp-(1¿6)--d-Glcp-(1¿3)-d-Fruf.
Differential effects of human and plant N-acetylglucosaminyltransferase I (GnTI) in plants
Henquet, M.G.L. ; Heinhuis, B. ; Borst, J.W. ; Eigenhuijsen, J. ; Schreuder, M. ; Bosch, D. ; Krol, A.R. van der - \ 2010
Transgenic Research 19 (2010)4. - ISSN 0962-8819 - p. 535 - 547.
arabidopsis-thaliana - molecular-cloning - linked glycans - expression - golgi - transmembrane - mutant - beta-1,2-n-acetylglucosaminyltransferase-i - transformation - transferase
In plants and animals, the first step in complex type N-glycan formation on glycoproteins is catalyzed by N-acetylglucosaminyltransferase I (GnTI). We show that the cgl1-1 mutant of Arabidopsis, which lacks GnTI activity, is fully complemented by YFP-labeled plant AtGnTI, but only partially complemented by YFP-labeled human HuGnTI and that this is due to post-transcriptional events. In contrast to AtGnTI-YFP, only low levels of HuGnTI-YFP protein was detected in transgenic plants. In protoplast co-transfection experiments all GnTI-YFP fusion proteins co-localized with a Golgi marker protein, but only limited co-localization of AtGnTI and HuGnTI in the same plant protoplast. The partial alternative targeting of HuGnTI in plant protoplasts was alleviated by exchanging the membrane-anchor domain with that of AtGnTI, but in stably transformed cgl1-1 plants this chimeric GnTI still did not lead to full complementation of the cgl1-1 phenotype. Combined, the results indicate that activity of HuGnTI in plants is limited by a combination of reduced protein stability, alternative protein targeting and possibly to some extend to lower enzymatic performance of the catalytic domain in the plant biochemical environment
Nitric oxide hinders antibody clearance from the surface of Trypanoplasma borreli and increases susceptibility to complement-mediated lysis
Forlenza, M. ; Nakao, M. ; Wibowo, I. ; Joerink, M. ; Arts, J.A.J. ; Savelkoul, H.F.J. ; Wiegertjes, G.F. - \ 2009
Molecular Immunology 46 (2009)16. - ISSN 0161-5890 - p. 3188 - 3197.
carp cyprinus-carpio - blood-stream forms - common carp - molecular-cloning - bony fish - functional-characterization - trypanosoma-carassii - antigenic variation - immune-response - parasite
Trypanoplasma borreli is an extracellular blood parasite of carp belonging to the same Order (Kinetoplastida) as African trypanosomes. These mammalian parasites have developed different strategies to evade the host immune system including antigenic variation, immunosuppression and clearance of surface-bound antibodies. The latter mechanism allows trypanosomes to use their swimming movement to cause surface-bound antibodies to ‘sail’ and accumulate at the posterior end of the parasite, to be internalized via the flagellar pocket and be degraded. There is no evidence that T. borreli shows antigenic variation, but during the late phases of infection NO-mediated immunosuppression is observed. High levels of nitric oxide (NO) lead to extensive tissue nitration whereas the parasite itself is not affected. Therefore, the induction of NO has thus far been considered a parasite-driven response with immunosuppressive effects. In the present study, we show that the induction of NO, particularly during the early phase of T. borreli infections, should be re-considered an effective part of the host immune response. We show that T. borreli rapidly removes surface-bound IgM. In addition, moderate concentrations of NO, by hindering surface antibody clearance, maintain high the concentrations of membrane-bound IgM, thereby favoring antibody-dependent complement-mediated parasite lysis. We performed a comprehensive quantitative gene expression analysis of in total seven different complement factors involved in all three activation pathways, differentiating between 1 and 4 isoforms for each complement gene. Our gene expression analysis supports an important role for antibody-dependent complement-mediated lysis of T. borreli in vivo. To our knowledge, NO-dependent inhibition of antibody clearance from the surface of kinetoplastid parasites has not been investigated. Our data support a role for NO as an important player in host–parasite interactions, not only as immune suppressor (late response) but also as immune effector (early response) in infections with bloodstream parasites such as T. borreli
Receptor-Mediated and Lectin-Like Activities of Carp (Cyprinus carpio) TNF-¿
Forlenza, M. ; Magez, S. ; Scharsack, J.P. ; Westphal, A.H. ; Savelkoul, H.F.J. ; Wiegertjes, G.F. - \ 2009
The Journal of Immunology 183 (2009)8. - ISSN 0022-1767 - p. 5319 - 5332.
tumor-necrosis-factor - peripheral-blood lymphocytes - salmon salmo-salar - expression analysis - molecular-cloning - rainbow-trout - trypanoplasma-borreli - nitric-oxide - oncorhynchus-mykiss - trypanosoma-brucei
Functional characterization of TNF- in species other than mammalian vertebrates is limited, and TNF- has been studied in a limited number of fish species, primarily in vitro using recombinant proteins. Studies on TNF- from different fish species so far pointed to several inconsistencies, in particular with respect to some receptor-mediated activities of fish TNF-, such as the ability to directly activate phagocytes. In the present study a comprehensive analysis of in vitro as well as in vivo biological activities of two isoforms of carp TNF- was performed. Our results show that carp TNF- directly primes carp phagocytes and indirectly promotes typical receptor-mediated activities such as phagocyte activation by acting via endothelial cells. Additionally, for the first time in nonmammalian vertebrate species, the lectin-like activity of fish TNF- homologs was investigated. Our results show an evolutionary conservation of function of this receptor-independent activity of TNF- not only in cyprinid fish, but also in perciform and salmonid fish. The role of TNF- in vivo, during infections of carp with the blood parasite Trypanoplasma borreli, was examined using three fundamentally different but complementary approaches: (1) inhibition of TNF- expression, (2) overexpression of TNF-, and (3) inhibition of shedding of membrane-bound TNF-. Our results show that, also in fish, a tight regulation of TNF- expression is important, since depletion or excess of TNF- can make an important difference to survival of infection. Finally, we demonstrate a crucial protective role for membrane-bound TNF-, which has a yet unexploited function in fish. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact
Synthesis of Lewis X epitopes on plant N-glycans
Rouwendal, G.J.A. ; Florack, D.E.A. ; Hesselink, T. ; Cordewener, J.H.G. ; Helsper, J.P.F.G. ; Bosch, H.J. - \ 2009
Carbohydrate Research : an international journal 344 (2009)12. - ISSN 0008-6215 - p. 1487 - 1493.
dc-sign - fuc-tix - carbohydrate epitopes - beta-galactosidases - schistosoma-mansoni - molecular-cloning - transgenic plants - expression - antigen - alpha-1,3-fucosyl-transferase
Glycoproteins from tobacco line xFxG1, in which expression of a hybrid ß-(1¿4)-galactosyltransferase (GalT) and a hybrid a-(1¿3)-fucosyltransferase IXa (FUT9a) is combined, contained an abundance of hybrid N-glycans with Lewis X (LeX) epitopes. A comparison with N-glycan profiles from plants expressing only the hybrid ß-(1¿4)-galactosyltransferase suggested that the fucosylation of the LacNAc residues in line xFxG1 protected galactosylated N-glycans from endogenous plant ß-galactosidase activity
Breeding drought tolerant cowpea: constraints, accomplishments, and future prospects
Agbicodo, A.C.M.E. ; Fatokun, C.A. ; Muranaka, S. ; Visser, R.G.F. ; Linden, C.G. van der - \ 2009
Euphytica 167 (2009)3. - ISSN 0014-2336 - p. 353 - 370.
vigna-unguiculata l. - abscisic-acid biosynthesis - water-use efficiency - ascorbate peroxidase - transcription factor - genotypic differences - medicago-truncatula - freezing tolerance - enzymatic-activity - molecular-cloning
This review presents an overview of accomplishments on different aspects of cowpea breeding for drought tolerance. Furthermore it provides options to enhance the genetic potential of the crop by minimizing yield loss due to drought stress. Recent efforts have focused on the genetic dissection of drought tolerance through identification of markers defining quantitative trait loci (QTL) with effects on specific traits related to drought tolerance. Others have studied the relationship of the drought response and yield components, morphological traits and physiological parameters. To our knowledge, QTLs with effects on drought tolerance have not yet been identified in cowpea. The main reason is that very few researchers are working on drought tolerance in cowpea. Some other reasons might be related to the complex nature of the drought stress response, and partly to the difficulties associated with reliable and reproducible measurements of a single trait linked to specific molecular markers to be used for marker assisted breeding. Despite the fact that extensive research has been conducted on the screening aspects for drought tolerance in cowpea only very few¿like the `wooden box¿ technique¿have been successfully used to select parental genotypes exhibiting different mechanisms of drought tolerance. Field and pot testing of these genotypes demonstrated a close correspondence between drought tolerance at seedling and reproductive stages. Some researchers selected a variety of candidate genes and used differential screening methods to identify cDNAs from genes that may underlie different drought tolerance pathways in cowpea. Reverse genetic analysis still needs to be done to confirm the functions of these genes in cowpea. Understanding the genetics of drought tolerance and identification of DNA markers linked to QTLs, with a clear path towards localizing chromosomal regions or candidate genes involved in drought tolerance will help cowpea breeders to develop improved varieties that combine drought tolerance with other desired traits using marker assisted selection.
Transcriptome Analysis of Potato Tubers - Effects of Different Agricultural Practices
Dijk, J.P. van; Cankar, K. ; Scheffer, S.J. ; Beenen, H.G. ; Shepherd, L.V.T. ; Stewart, D. ; Davies, H.V. ; Wilkockson, S.J. ; Leifert, C. ; Gruden, K. ; Kok, E.J. - \ 2009
Journal of Agricultural and Food Chemistry 57 (2009)4. - ISSN 0021-8561 - p. 1612 - 1623.
gene-expression - immunocytochemical localization - proteinase-inhibitors - enzyme-inhibitors - molecular-cloning - microarray - family - safety - foods - glucosyltransferase
The use of profiling techniques such as transcriptomics, proteomics, and metabolomics has been proposed to improve the detection of side effects of plant breeding processes. This paper describes the construction of a food safety-oriented potato cDNA microarray (FSPM). Microarray analysis was performed on a well-defined set of tuber samples of two different potato varieties, grown under different, well-recorded environmental conditions. Data were analyzed to assess the potential of transcriptomics to detect differences in gene expression due to genetic differences or environmental conditions. The most pronounced differences were found between the varieties Sante and Lady Balfour, whereas differences due to growth conditions were less significant. Transcriptomics results were confirmed by quantitative PCR. Furthermore, the bandwidth of natural variation of gene expression was explored to facilitate biological and/or toxicological evaluation in future assessments.
Expression of the polymeric immunoglobulin receptor (pIgR) in mucosal tissues of common carp (Cyprinus carpio L.)
Rombout, J.H.W.M. ; Tuin, S.J.L. van der; Yang Guiwen, ; Schopman, N. ; Mroczek, A. ; Hermsen, G.J. ; Taverne-Thiele, J.J. - \ 2008
Fish and Shellfish Immunology 24 (2008)5. - ISSN 1050-4648 - p. 620 - 628.
transmembrane secretory component - poly-ig receptor - j-chain - molecular-cloning - messenger-rna - transport - sites - gene - exon
The mucosal immune system seems to be an important defence mechanism for fish but the binding of IgM in mucosal organs is poorly described in fish. In this study the gene encoding the polymeric Immunoglobulin Receptor (pIgR) in carp has been isolated and sequenced from a liver cDNA-library and aligned with other species. The pIgR of carp consists of 2 Ig domains, a transmembrane and an intracellular region, together 327 amino acids. In situ hybridisations with sense and anti-sense DIG-labelled pIgR RNA probes were performed on liver, gut and skin of common carp (Cyprinus carpio L.) and in these organs only anti-sense probes were found to hybridise. In liver the majority of hepatocytes was stained around the nucleus. In gut and skin, staining could be detected around the nucleus of the epithelial cells, but in gut also a subpopulation of lymphoid cells was stained in epithelium and lamina propria. The specific in situ hybridisation of the epithelia and hepatocytes coincides with the in situ binding of FITC-labelled carp IgM to the same cells. RT-PCR results indicate the expression of the pIgR gene in all lymphoid organs of carp, but not in muscle. Macrophages/neutrophils enriched by adherence or sorted B cells (MACS) did not show expression of the pIgR gene and are excluded as the pIgR expressing lymphoid cells in the intestine. The relevance of pIgR staining and gene expression in mucosal organs is discussed.