Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

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Learning-induced gene expression in the heads of two Nasonia species that differ in long-term memory formation
Hoedjes, K.M. ; Smid, H.M. ; Schijlen, E.G.W.M. ; Vet, L.E.M. ; Vugt, J.J.F.A. van - \ 2015
BMC Genomics 16 (2015). - ISSN 1471-2164
natural variation - antisense transcription - protein-synthesis - foraging success - parasitic wasps - drosophila - vitripennis - pathway - consolidation - opportunities
Background Cellular processes underlying memory formation are evolutionary conserved, but natural variation in memory dynamics between animal species or populations is common. The genetic basis of this fascinating phenomenon is poorly understood. Closely related species of Nasonia parasitic wasps differ in long-term memory (LTM) formation: N. vitripennis will form transcription-dependent LTM after a single conditioning trial, whereas the closely-related species N. giraulti will not. Genes that were differentially expressed (DE) after conditioning in N. vitripennis, but not in N. giraulti, were identified as candidate genes that may regulate LTM formation. Results RNA was collected from heads of both species before and immediately, 4 or 24 hours after conditioning, with 3 replicates per time point. It was sequenced strand-specifically, which allows distinguishing sense from antisense transcripts and improves the quality of expression analyses. We determined conditioning-induced DE compared to naïve controls for both species. These expression patterns were then analysed with GO enrichment analyses for each species and time point, which demonstrated an enrichment of signalling-related genes immediately after conditioning in N. vitripennis only. Analyses of known LTM genes and genes with an opposing expression pattern between the two species revealed additional candidate genes for the difference in LTM formation. These include genes from various signalling cascades, including several members of the Ras and PI3 kinase signalling pathways, and glutamate receptors. Interestingly, several other known LTM genes were exclusively differentially expressed in N. giraulti, which may indicate an LTM-inhibitory mechanism. Among the DE transcripts were also antisense transcripts. Furthermore, antisense transcripts aligning to a number of known memory genes were detected, which may have a role in regulating these genes. Conclusion This study is the first to describe and compare expression patterns of both protein-coding and antisense transcripts, at different time points after conditioning, of two closely related animal species that differ in LTM formation. Several candidate genes that may regulate differences in LTM have been identified. This transcriptome analysis is a valuable resource for future in-depth studies to elucidate the role of candidate genes and antisense transcription in natural variation in LTM formation.
Autophagy in adipose tissue and the beta cell: implications for obesity and diabetes
Stienstra, R. ; Haim, Y. ; Riahi, Y. ; Netea, M.G. ; Rudich, A. ; Leibowitz, G. - \ 2014
Diabetologia 57 (2014)8. - ISSN 0012-186X - p. 1505 - 1516.
endoplasmic-reticulum stress - unfolded protein response - pancreatic b-cell - insulin-secretion - proinflammatory response - protective role - kappa-b - pathway - metabolism - glucose
Autophagy is a lysosomal degradation pathway recycling intracellular long-lived proteins and damaged organelles, thereby maintaining cellular homeostasis. In addition to inflammatory processes, autophagy has been implicated in the regulation of adipose tissue and beta cell functions. In obesity and type 2 diabetes autophagic activity is modulated in a tissue-dependent manner. In this review we discuss the regulation of autophagy in adipose tissue and beta cells, exemplifying tissue-specific dysregulation of autophagy and its implications for the pathophysiology of obesity and type 2 diabetes. We will highlight common themes and outstanding gaps in our understanding, which need to be addressed before autophagy could be envisioned as a therapeutic target for the treatment of obesity and diabetes.
Rise-Time of FRET-Acceptor Fluorescence Tracks Protein Folding
Lindhoud, S. ; Westphal, A.H. ; Mierlo, C.P.M. van; Visser, A.J.W.G. ; Borst, J.W. - \ 2014
International Journal of Molecular Sciences 15 (2014)12. - ISSN 1661-6596 - p. 23836 - 23850.
resonance energy-transfer - beta parallel protein - single-molecule fluorescence - azotobacter-vinelandii - spectroscopic ruler - refractive-index - tryptophan residue - molten globule - wild-type - pathway
Uniform labeling of proteins with fluorescent donor and acceptor dyes with an equimolar ratio is paramount for accurate determination of Förster resonance energy transfer (FRET) efficiencies. In practice, however, the labeled protein population contains donor-labeled molecules that have no corresponding acceptor. These FRET-inactive donors contaminate the donor fluorescence signal, which leads to underestimation of FRET efficiencies in conventional fluorescence intensity and lifetime-based FRET experiments. Such contamination is avoided if FRET efficiencies are extracted from the rise time of acceptor fluorescence upon donor excitation. The reciprocal value of the rise time of acceptor fluorescence is equal to the decay rate of the FRET-active donor fluorescence. Here, we have determined rise times of sensitized acceptor fluorescence to study the folding of double-labeled apoflavodoxin molecules and show that this approach tracks the characteristics of apoflavodoxin's complex folding pathway.
Molecular dynamics simulation of energy migration between tryptophan residues in apoflavodoxin
Nunthaboot, N. ; Tanaka, F. ; Kokpol, S. ; Visser, N.V. ; Amerongen, H. van; Visser, A.J.W.G. - \ 2014
RSC Advances : An international journal to further the chemical sciences 4 (2014). - ISSN 2046-2069 - p. 31443 - 31451.
time-resolved fluorescence - azotobacter-vinelandii - anisotropy decay - force-field - flavodoxin - proteins - water - pathway - system - state
Molecular dynamics (MD) simulations over a 30 ns trajectory have been carried out on apoflavodoxin from Azotobacter vinelandii to compare with the published, experimental time-resolved fluorescence anisotropy results of F¨orster Resonance Energy Transfer (FRET) between the three tryptophan residues. MD analysis of atomic coordinates yielding both the time course of geometric parameters and the time-correlated second-order Legendre polynomial functions reflects immobilization of tryptophans in the protein matrix. However, one tryptophan residue (Trp167) undergoes flip-flop motion on the nanosecond timescale. The simulated time-resolved fluorescence anisotropy of tryptophan residues in apoflavodoxin implying a model of two unidirectional FRET pathways is in very good agreement with the experimental time-resolved fluorescence anisotropy, although the less efficient FRET pathway cannot be resolved and is hidden in the contribution of a slow protein motion.
Striga hermonthica MAX2 restores branching but not the Very Low Fluence Response in the Arabidopsis thaliana max2 mutant
Liu, Q. ; Zhang, Y. ; Matusovaa, R. ; Charnikhova, T. ; Amini, M. ; Jamil, M. ; Fernandez-Aparicio, M. ; Huang, K. ; Timko, M.P. ; Westwood, J.H. ; Ruyter-Spira, C.P. ; Krol, A.R. van der; Bouwmeester, H.J. - \ 2014
New Phytologist 202 (2014)2. - ISSN 0028-646X - p. 531 - 541.
arabidopsis seed-germination - box protein max2 - plant hormone - strigolactone - inhibition - photomorphogenesis - stimulants - karrikins - molecule - pathway
Seed germination of Striga spp. (witchweeds), one of the world’s most destructive parasitic weeds, cannot be induced by light but is specifically induced by strigolactones. It is not known whether Striga uses the same components for strigolactone signaling as host plants, whether it has endogenous strigolactone biosynthesis and whether there is post-germination strigolactone signaling in Striga. Strigolactones could not be detected in in vitro grown Striga, while for host-grown Striga, the strigolactone profile is dominated by a subset of the strigolactones present in the host. Branching of in vitro grown Striga is affected by strigolactone biosynthesis inhibitors. ShMAX2, the Striga ortholog of Arabidopsis MORE AXILLARY BRANCHING 2 (AtMAX2) – which mediates strigolactone signaling – complements several of the Arabidopsis max2-1 phenotypes, including the root and shoot phenotype, the High Irradiance Response and the response to strigolactones. Seed germination of max2-1 complemented with ShMAX2 showed no complementation of the Very Low Fluence Response phenotype of max2-1. Results provide indirect evidence for ShMAX2 functions in Striga. A putative role of ShMAX2 in strigolactone-dependent seed germination of Striga is discussed.
Metabolomics reveals organ-specific metabolic rearrangements during early tomato seedling development
Gomez-Roldan, M.V. ; Engel, B. ; Vos, R.C.H. de; Vereijken, P.F.G. ; Astola, L. ; Groenenboom, M.A.C. ; Geest, H.C. van de; Bovy, A.G. ; Molenaar, J. ; Eeuwijk, F.A. van; Hall, R.D. - \ 2014
Metabolomics 10 (2014)5. - ISSN 1573-3882 - p. 958 - 974.
transcriptome coexpression analysis - mass-spectrometry - fruit-development - integrated analysis - network analysis - systems biology - arabidopsis - pathway - expression - tool
Tomato seedlings (Solanum lycopersicum cv.MoneyMaker), grown under strictly controlled conditions, have been used to study alterations occurring in secondary metabolite biosynthetic pathways following developmental and environmental perturbations. Robustness and reproducibility of the system were confirmed using detailed statistical analyses of the metabolome. LCMS profiling was applied using whole germinated seeds as well as cotyledons, hypocotyls and roots from 3 to 9 days old seedlings to generate relative levels of 433 metabolites, of which 62 were annotated. Initial focus was given to the polyphenol pathway and several additional mass signals have been putatively annotated using high mass resolution fragmentation. Clear organ and developmental stage—specific differences were observed. Seeds accumulated saponin-like compounds; roots accumulated mainly alkaloids; cotyledons contained mainly glycosylated flavonols and; hypocotyls contained mainly anthocyanins. For each organ, the developmental changes in metabolite profiles were described by using linear mixed models. Across three independent experiments, 85 % of the metabolites showed similar developmental trends. This tomato seedling system has given us valuable additional insights into the complexity of seedling secondary metabolism. How metabolic profiles reflect an interplay between depletion of stored molecules and de novo synthesis and how the overall picture for this important crop plant contrasts to e.g. Arabidopsis are emphasised.
Biosynthetic Origin of the Antibiotic Cyclocarbamate Brabantamide A (SB-253514) in Plant-Associated Pseudomonas
Schmidt, Y. ; Voort, M. van der; Crüsemann, M. ; Piel, J. ; Josten, M. ; Sahl, H.G. ; Miess, H. ; Raaijmakers, J.M. ; Gross, H. - \ 2014
ChemBioChem 15 (2014)2. - ISSN 1439-4227 - p. 259 - 266.
fluorescens dsm 11579 - baeyer-villiger monooxygenases - phospholipase a(2) - natural-product - genome sequences - inhibitors - analogs - bacteria - pathway - rhizosphere
Within the framework of our genome-based program to discover new antibiotic lipopeptides from Pseudomonads, brabantamides A–C were isolated from plant-associated Pseudomonas sp. SH-C52. Brabantamides A–C displayed moderate to high in vitro activities against Gram-positive bacterial pathogens. Their shared structure is unique in that they contain a 5,5-bicyclic carbamate scaffold. Here, the biosynthesis of brabantamide A (SB-253514) was studied by a combination of bioinformatics, feeding experiments with isotopically labelled precursors and in vivo and in vitro functional analysis of enzymes encoded in the biosynthetic pathway. The studies resulted in the deduction of all biosynthetic building blocks of brabantamide A and revealed an unusual feature of this metabolite: its biosynthesis occurs via an initially formed linear di-lipopeptide that is subsequently rearranged by a novel FAD-dependent Baeyer–Villiger monooxygenase.
A novel class of fungal lipoxygenases
Heshof, R. ; Jylhä, S. ; Haarmann, T. ; Jørgensen, A.L.W. ; Dalsgaard, T.K. ; Graaff, L.H. de - \ 2014
Applied Microbiology and Biotechnology 98 (2014)3. - ISSN 0175-7598 - p. 1261 - 1270.
manganese lipoxygenase - cloning - hydroperoxide - stereocontrol - oxygenation - oxylipins - pathway
Lipoxygenases (LOXs) are well-studied enzymes in plants and mammals. However, fungal LOXs are less studied. In this study, we have compared fungal LOX protein sequences to all known characterized LOXs. For this, a script was written using Shell commands to extract sequences from the NCBI database and to align the sequences obtained using Multiple Sequence Comparison by Log-Expectation. We constructed a phylogenetic tree with the use of Quicktree to visualize the relation of fungal LOXs towards other LOXs. These sequences were analyzed with respect to the signal sequence, C-terminal amino acid, the stereochemistry of the formed oxylipin, and the metal ion cofactor usage. This study shows fungal LOXs are divided into two groups, the Ile- and the Val-groups. The Ile-group has a conserved WRYAK sequence that appears to be characteristic for fungal LOXs and has as a C-terminal amino acid Ile. The Val-group has a highly conserved WL-L/F-AK sequence that is also found in LOXs of plant and animal origin. We found that fungal LOXs with this conserved sequence have a Val at the C-terminus in contrast to other LOXs of fungal origin. Also, these LOXs have signal sequences implying these LOXs will be expressed extracellularly. Our results show that in this group, in addition to the Gaeumannomyces graminis and the Magnaporthe salvinii LOXs, the Aspergillus fumigatus LOX uses manganese as a cofactor
Auxin: special! Preface
McSteen, P. ; Scheres, B. ; Zhao, Y.D. - \ 2013
Journal of Experimental Botany 64 (2013)9. - ISSN 0022-0957 - p. 2539 - 2540.
arabidopsis - biosynthesis - pathway
Silencing an N-Acyltransferase-Like Involved in Lignin Biosynthesis in Nicotiana attenuata Dramatically Alters Herbivory-Induced Phenolamide Metabolism
Gaquerel, E. ; Kotkar, H. ; Onkokesung, N. ; Galis, I. ; Baldwin, I.T. - \ 2013
PLoS One 8 (2013)5. - ISSN 1932-6203
arabidopsis-thaliana - jasmonic acid - insect herbivores - mediated defenses - down-regulation - pathway - tobacco - accumulation - evolution - shikimate
In a transcriptomic screen of Manduca sexta-induced N-acyltransferases in leaves of Nicotiana attenuata, we identified an N-acyltransferase gene sharing a high similarity with the tobacco lignin-biosynthetic hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyl transferase (HCT) gene whose expression is controlled by MYB8, a transcription factor that regulates the production of phenylpropanoid polyamine conjugates (phenolamides, PAs). To evaluate the involvement of this HCT-like gene in lignin production as well as the resulting crosstalk with PA metabolism during insect herbivory, we transiently silenced (by VIGs) the expression of this gene and performed non-targeted (UHPLC-ESI/TOF-MS) metabolomics analyses. In agreement with a conserved function of N. attenuata HCT-like in lignin biogenesis, HCT-silenced plants developed weak, soft stems with greatly reduced lignin contents. Metabolic profiling demonstrated large shifts (up to 12% deregulation in total extracted ions in insect-attacked leaves) due to a large diversion of activated coumaric acid units into the production of developmentally and herbivory-induced coumaroyl-containing PAs (N',N''-dicoumaroylspermidine, N',N''-coumaroylputrescine, etc) and to minor increases in the most abundant free phenolics (chlorogenic and cryptochlorogenic acids), all without altering the production of well characterized herbivory-responsive caffeoyl- and feruloyl-based putrescine and spermidine PAs. These data are consistent with a strong metabolic tension, exacerbated during herbivory, over the allocation of coumaroyl-CoA units among lignin and unusual coumaroyl-containing PAs, and rule out a role for HCT-LIKE in tuning the herbivory-induced accumulation of other PAs. Additionally, these results are consistent with a role for lignification as an induced anti-herbivore defense.
A Trichome-Specific Linoleate Lipoxygenase Expressed During Pyrethrin Biosynthesis in Pyrethrum
Ramirez, A.M. ; Yang, T. ; Bouwmeester, H.J. ; Jongsma, M.A. - \ 2013
Lipids 48 (2013)10. - ISSN 0024-4201 - p. 1005 - 1015.
chrysanthemum-cinerariaefolium - tanacetum-cinerariifolium - jasmonic acid - gene - arabidopsis - seedlings - defense - pathway - stress - growth
The lipid precursor alcohols of pyrethrins—jasmolone, pyrethrolone and cinerolone—have been proposed as sharing parts of the oxylipin pathway with jasmonic acid. This implies that one of the first committed steps of pyrethrin biosynthesis is catalyzed by a lipoxygenase, catalyzing the hydroperoxidation of linolenic acid at position 13. Previously, we showed that the expression and activity of chrysanthemyl diphosphate synthase (TcCDS), the enzyme catalyzing the first committed step in the biosynthesis of the acid moiety of pyrethrins, is trichome-specific and developmentally regulated in flowers. In the present study we characterized the expression pattern of 25 lipoxygenase EST contigs, and subsequently carried out the molecular cloning of two pyrethrum lipoxygenases, TcLOX1 and TcLOX2, that have a similar pattern to TcCDS. Only recombinant TcLOX1 catalyzed the peroxidation of the linolenic acid substrate. Just as TcCDS, TcLOX1, are exclusively expressed in trichomes. Phylogenetic analysis showed that the enzyme shared the highest homology with chloroplast-localized 13-type-lipoxygenases that are involved in maintaining basal levels of jasmonate.
Botrytis cinerea mutants deficient in D-galacturonic acid catabolism have a perturbed virulence on Nicotiana benthamiana and Arabidopsis, but not on tomato
Zhang, L. ; Kan, J.A.L. van - \ 2013
Molecular Plant Pathology 14 (2013)1. - ISSN 1464-6722 - p. 19 - 29.
cell-wall polysaccharides - plant defensin gene - salicylic-acid - filamentous fungi - thaliana - pathogen - resistance - pathway - camalexin - biosynthesis
d-Galacturonic acid is the most abundant monosaccharide component of pectic polysaccharides that comprise a significant part of most plant cell walls. Therefore, it is potentially an important nutritional factor for Botrytis cinerea when it grows in and t
Deep RNA Sequencing of the Skeletal Muscle Transcriptome in Swimming Fish
Palstra, A.P. ; Beltran, S. ; Burgerhout, E. ; Brittijn, S.A. ; Magnoni, L.J. ; Henkel, C.V. ; Jansen, A. ; Thillart, G.E.E.J.M. ; Spaink, H.P. ; Planas, J.V. - \ 2013
PLoS One 8 (2013)1. - ISSN 1932-6203
trout oncorhynchus-mykiss - rainbow-trout - growth-hormone - luteinizing-hormone - profiling analysis - androgen receptor - in-vitro - expression - differentiation - pathway
Deep RNA sequencing (RNA-seq) was performed to provide an in-depth view of the transcriptome of red and white skeletal muscle of exercised and non-exercised rainbow trout (Oncorhynchus mykiss) with the specific objective to identify expressed genes and quantify the transcriptomic effects of swimming-induced exercise. Pubertal autumn-spawning seawater-raised female rainbow trout were rested (n = 10) or swum (n = 10) for 1176 km at 0.75 body-lengths per second in a 6,000-L swim-flume under reproductive conditions for 40 days. Red and white muscle RNA of exercised and non-exercised fish (4 lanes) was sequenced and resulted in 15–17 million reads per lane that, after de novo assembly, yielded 149,159 red and 118,572 white muscle contigs. Most contigs were annotated using an iterative homology search strategy against salmonid ESTs, the zebrafish Danio rerio genome and general Metazoan genes. When selecting for large contigs (>500 nucleotides), a number of novel rainbow trout gene sequences were identified in this study: 1,085 and 1,228 novel gene sequences for red and white muscle, respectively, which included a number of important molecules for skeletal muscle function. Transcriptomic analysis revealed that sustained swimming increased transcriptional activity in skeletal muscle and specifically an up-regulation of genes involved in muscle growth and developmental processes in white muscle. The unique collection of transcripts will contribute to our understanding of red and white muscle physiology, specifically during the long-term reproductive migration of salmonids.
Enhanced protein secretion from insect cells by co-expression of the chaperone calreticulin and translation initiation factor eIF4E
Teng, C.Y. ; Chang, S.L. ; Oers, M.M. van; Wu, T.Y. - \ 2013
Molecular Biotechnology 54 (2013)1. - ISSN 1073-6085 - p. 68 - 78.
baculovirus expression system - ribosome entry site - endoplasmic-reticulum - quality-control - messenger-rna - calnexin - infection - pathway - er - glycoprotein
Host protein synthesis is shut down in the lytic baculovirus expression vector system (BEVS). This also affects host proteins involved in routing secretory proteins through the endoplasmic reticulum (ER)-Golgi system. It has been demonstrated that a secretory alkaline phosphatase–EGFP fusion protein (SEFP) can act as a traceable and sensitive secretory reporter protein in BEVS. In this study, a chaperone, calreticulin (CALR), and the translation initiation factor eIF4E were co-expressed with SEFP using a bicistronic baculovirus expression vector. We observed that the intracellular distribution of SEFP in cells co-expressing CALR was different from co-expressing eIF4E. The increased green fluorescence emitted by cells co-expressing CALR had a good correlation with the abundance of intracellular SEFP protein and an unconventional ER expansion. Cells co-expressing eIF4E, on the other hand, showed an increase in extracellular SEAP activity compared to the control. Utilization of these baculovirus expression constructs containing either eIF4E or CALR offers a significant advantage for producing secreted proteins for various biotechnological and therapeutic applications.
Disruption of the acetate kinase (ack) gene of Clostridium acetobutylicum results in delayed acetate production
Kuit, W. ; Minton, N.P. ; Lopez Contreras, A.M. ; Eggink, G. - \ 2012
Applied Microbiology and Biotechnology 94 (2012)3. - ISSN 0175-7598 - p. 729 - 741.
fermentative butanol production - enhanced butyric-acid - escherichia-coli - acetyl phosphate - solvent production - down-regulation - deleted mutant - atcc 824 - pathway - acetone
In microorganisms, the enzyme acetate kinase (AK) catalyses the formation of ATP from ADP by de-phosphorylation of acetyl phosphate into acetic acid. A mutant strain of Clostridium acetobutylicum lacking acetate kinase activity is expected to have reduced acetate and acetone production compared to the wild type. In this work, a C. acetobutylicum mutant strain with a selectively disrupted ack gene, encoding AK, was constructed and genetically and physiologically characterized. The ack (-) strain showed a reduction in acetate kinase activity of more than 97% compared to the wild type. The fermentation profiles of the ack (-) and wild-type strain were compared using two different fermentation media, CGM and CM1. The latter contains acetate and has a higher iron and magnesium content than CGM. In general, fermentations by the mutant strain showed a clear shift in the timing of peak acetate production relative to butyrate and had increased acid uptake after the onset of solvent formation. Specifically, in acetate containing CM1 medium, acetate production was reduced by more than 80% compared to the wild type under the same conditions, but both strains produced similar final amounts of solvents. Fermentations in CGM showed similar peak acetate and butyrate levels, but increased acetoin (60%), ethanol (63%) and butanol (16%) production and reduced lactate (-50%) formation by the mutant compared to the wild type. These findings are in agreement with the proposed regulatory function of butyryl phosphate as opposed to acetyl phosphate in the metabolic switch of solventogenic clostridia.
Hedgehog signaling via a calcitonin receptor-like receptor can induce arterial differentiation independently of VEGF signaling in zebrafish
Wilkinson, R.N. ; Koudijs, M.J. ; Patient, R.K. ; Ingham, P.W. ; Schulte-Merker, S. ; Eeden, F.J.M. van - \ 2012
Blood : journal of the American Society of Hematology 120 (2012)2. - ISSN 0006-4971 - p. 477 - 488.
endothelial-growth-factor - embryonic vascular development - hematopoietic stem-cells - aortic endothelium - sonic-hedgehog - pathway - notch - blood - gene - kinase
Multiple signaling pathways control the specification of endothelial cells (ECs) to become arteries or veins during vertebrate embryogenesis. Current models propose that a cascade of Hedgehog (Hh), vascular endothelial growth factor (VEGF), and Notch signaling acts instructively on ECs to control the choice between arterial or venous fate. Differences in the pheno-types induced by Hh, VEGF, or Notch inhibition suggest that not all of the effects of Hh on arteriovenous specification are mediated by VEGF. We establish that full derepression of the Hh pathway in ptc1;ptc2 mutants converts the posterior cardinal vein into a second arterial vessel that manifests intact arterial gene expression, intersegmental vessel sprouting, and HSC gene expression. Importantly, although VEGF was thought to be absolutely essential for arterial fates, we find that normal and ectopic arterial differentiation can occur without VEGF signaling in ptc1; ptc2 mutants. Furthermore, Hh is able to bypass VEGF to induce arterial differentiation in ECs via the calcitonin receptor-like receptor, thus revealing a surprising complexity in the interplay between Hh and VEGF signaling during arteriovenous specification. Finally, our experiments establish a dual function of Hh during induction of runx1(+) HSCs. (Blood. 2012;120(2):477-488)
Fluorescence of Alexa Fluor dye tracks protein folding
Lindhoud, S. ; Westphal, A.H. ; Borst, J.W. ; Visser, A.J.W.G. ; Mierlo, C.P.M. van - \ 2012
PLoS One 7 (2012)10. - ISSN 1932-6203 - 8 p.
azotobacter-vinelandii apoflavodoxin - resonance energy-transfer - beta parallel protein - molten-globule state - flavodoxin-ii - molecules - pathway - chains - intermediate - spectroscopy
Fluorescence spectroscopy is an important tool for the characterization of protein folding. Often, a protein is labeled with appropriate fluorescent donor and acceptor probes and folding-induced changes in Förster Resonance Energy Transfer (FRET) are monitored. However, conformational changes of the protein potentially affect fluorescence properties of both probes, thereby profoundly complicating interpretation of FRET data. In this study, we assess the effects protein folding has on fluorescence properties of Alexa Fluor 488 (A488), which is commonly used as FRET donor. Here, A488 is covalently attached to Cys69 of apoflavodoxin from Azotobacter vinelandii. Although coupling of A488 slightly destabilizes apoflavodoxin, the three-state folding of this protein, which involves a molten globule intermediate, is unaffected. Upon folding of apoflavodoxin, fluorescence emission intensity of A488 changes significantly. To illuminate the molecular sources of this alteration, we applied steady state and time-resolved fluorescence techniques. The results obtained show that tryptophans cause folding-induced changes in quenching of Alexa dye. Compared to unfolded protein, static quenching of A488 is increased in the molten globule. Upon populating the native state both static and dynamic quenching of A488 decrease considerably. We show that fluorescence quenching of Alexa Fluor dyes is a sensitive reporter of conformational changes during protein folding.
A petunia ABC protein controls strigolactone-dependent symbiotic signalling and branching
Kretzschmar, T. ; Kohlen, W. ; Sasse, J. ; Borghi, L. ; Schlegel, M. ; Bachelier, J.B. ; Reinhardt, D. ; Bours, R.M.E.H. ; Bouwmeester, H.J. ; Martinoia, E. - \ 2012
Nature 483 (2012)7389. - ISSN 0028-0836 - p. 341 - 344.
arbuscular-mycorrhizal fungi - medicago-truncatula - auxin transport - abscisic-acid - gene family - arabidopsis - pcr - germination - inhibition - pathway
Strigolactones were originally identified as stimulators of the germination of root-parasitic weeds1 that pose a serious threat to resource-limited agriculture2. They are mostly exuded from roots and function as signalling compounds in the initiation of arbuscular mycorrhizae3, which are plant–fungus symbionts with a global effect on carbon and phosphate cycling4. Recently, strigolactones were established to be phytohormones that regulate plant shoot architecture by inhibiting the outgrowth of axillary buds5, 6. Despite their importance, it is not known how strigolactones are transported. ATP-binding cassette (ABC) transporters, however, are known to have functions in phytohormone translocation7, 8, 9. Here we show that the Petunia hybrida ABC transporter PDR1 has a key role in regulating the development of arbuscular mycorrhizae and axillary branches, by functioning as a cellular strigolactone exporter. P. hybrida pdr1 mutants are defective in strigolactone exudation from their roots, resulting in reduced symbiotic interactions. Above ground, pdr1 mutants have an enhanced branching phenotype, which is indicative of impaired strigolactone allocation. Overexpression of Petunia axillaris PDR1 in Arabidopsis thaliana results in increased tolerance to high concentrations of a synthetic strigolactone, consistent with increased export of strigolactones from the roots. PDR1 is the first known component in strigolactone transport, providing new opportunities for investigating and manipulating strigolactone-dependent processes.
Activation of natural killer T Cells promotes M2 macrophage polarization in adipose tissue and improves systemic glucose tolerance via interleukin-4 (IL-4)/STAT6 protein signalling axis in obesity
Ji, Y. ; Sun, S. ; Xu, Aimin ; Bhargava, P. ; Yang, Liu ; Lam, K.S.L. ; Gao, Bin ; Lee, Chih-Hao ; Kersten, A.H. ; Qi, L. - \ 2012
Journal of Biological Chemistry 287 (2012)17. - ISSN 0021-9258 - p. 13561 - 13571.
invariant nkt cells - insulin-resistance - alternative activation - microbial infection - mice - inflammation - receptor - pathway - disease - fat
Natural killer T (NKT) cells are important therapeutic targets in various disease models and are under clinical trials for cancer patients. However, their function in obesity and type 2 diabetes remains unclear. Our data show that adipose tissues of both mice and humans contain a population of type 1 NKT cells, whose abundance decreases with increased adiposity and insulin resistance. Although loss-of-function of NKT cells had no effect on glucose tolerance in animals with prolonged high fat diet feeding, activation of NKT cells by lipid agonist a-galactosylceramide enhances alternative macrophage polarization in adipose tissue and improves glucose homeostasis in animals at different stages of obesity. Furthermore, the effect of NKT cells is largely mediated by the IL-4/STAT6 signaling axis in obese adipose tissue. Thus, our data identify a novel therapeutic target for the treatment of obesity-associated inflammation and type 2 diabetes.
Activation of antioxidant response element in mouse primary cortical cultures with sesquiterpene lactones isolated from Tanacetum parthenium
Fischedick, J.T. ; Standiford, M. ; Johnson, D.A. ; Vos, R.C.H. de; Todorovic, S. ; Banjanac, T. ; Verpoorte, R. ; Johnson, J.A. - \ 2012
Planta Medica 78 (2012)16. - ISSN 0032-0943 - p. 1725 - 1730.
biomimetic transformations - neurodegenerative disease - parthenolide - santamarine - anticancer - compositae - feverfew - pathway - target - cells
Tanacetum parthenium produces biologically active sesquiterpene lactones (SL). Nuclear factor E2-related factor 2 (Nrf2) is a transcription factor known to activate a series of genes termed the antioxidant response element (ARE). Activation of Nrf2/ARE may be useful for the treatment of neurodegenerative disease. In this study we isolated 11 SL from T. parthenium with centrifugal partition chromatography and semipreparative HPLC. Compounds were screened in vitro for their ability to activate the ARE on primary mouse cortical cultures as well as for their toxicity towards the cultures. All SL containing the a-methylene-¿-lactone moiety were able to activate the ARE and cause cellular toxicity. The structure-activity relationship among the SL isolated indicates that the guaianolides were more active and when lacking the endoperoxide functionality less toxic then the germacranolides.
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