J. Kopecky

Crop Research Institute, Praha, Praha, Czech Republic

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Publications (243)676.99 Total impact

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    ABSTRACT: Th17 cells constitute a subset of CD4(+) T-lymphocytes playing a crucial role in protection against extracellular bacteria and fungi. They are also associated with tissue injury in autoimmune and inflammatory diseases. Here we report that serpin from the tick Ixodes ricinus, IRS-2, inhibits Th17 differentiation by impairment of IL-6/STAT-3 signalling pathway. Following activation, mature dendritic cells produce an array of cytokines including pleiotropic cytokine IL-6, which triggers IL-6 signalling pathway. The major transcription factor activated by IL-6 is STAT-3. We show that IRS-2 selectively inhibits production of IL-6 in dendritic cells stimulated with Borrelia spirochetes, which leads to attenuated STAT-3 phosphorylation and finally to impaired Th17 differentiation. Presented results extend the knowledge about the effect of tick salivary serpins on innate immunity cells and their function in driving adaptive immune response. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
    Infection and Immunity 02/2015; 83(5). DOI:10.1128/IAI.03065-14 · 4.16 Impact Factor
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    ABSTRACT: Dietary intake of omega-3 fatty acids is associated with considerable health benefits, including the prevention of metabolic disorders such as cardiovascular disease and type 2 diabetes. Furthermore, incorporation of the main omega-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), at the systemic level has been found to be more efficient when these fatty acids are supplied in the form of marine phospholipids compared to triglycerides. In this work, the uptake of omega-3 fatty acids and their incorporation in specific lipids were studied in adipose, skeletal muscle, and liver tissues of mice given high-fat diets with or without omega-3 supplements in the form of phospholipids or triglycerides using time-of-flight secondary ion mass spectrometry (TOF-SIMS). The results demonstrate significant uptake of EPA and DHA, and the incorporation of these fatty acids in specific lipid molecules, in all three tissue types in response to the dietary omega-3 supplements. Moreover, the results indicate reduced concentrations of arachidonic acid (AA) and depletion of lipids containing AA in tissue samples from mice given supplementary omega-3, as compared to the control mice. The effect on the lipid composition, in particular the DHA uptake and AA depletion, was found to be significantly stronger when the omega-3 supplement was supplied in the form of phospholipids, as compared to triglycerides. TOF-SIMS was found to be a useful technique for screening the lipid composition and simultaneously obtaining the spatial distributions of various lipid classes on tissue surfaces.
    Analytical and Bioanalytical Chemistry 02/2015; DOI:10.1007/s00216-015-8515-7 · 3.58 Impact Factor
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    ABSTRACT: Carpoglyphus lactis is a stored product mite infesting saccharide rich stored commodities including dried fruits, wine, beer, milk products, jams and honey. The association with microorganisms can improve the survival of mites on dried fruits. The microbial communities associated to C. lactis were studied in specimens originating from the packages of dried apricot, plums and figs and compared to the laboratory strain reared on house dust mite diet (HDMd). Clone libraries of bacterial 16S rRNA gene and fungal ITS region were constructed and analyzed by OTU approach. The 16S rRNA gene libraries differed among the compared diets. The sequences classified to the genera Leuconostoc, Elizabethkingia, Ewingella, Erwinia, Bacillus and Serratia were prevailing in mites sampled from the dried fruits. The ITS library showed smaller differences between the laboratory strain on HDMd and the isolates from dried fruits packages, with the exception of the mite strain from dried plums. The population growth was used as an indirect indicator of fitness and decreased in the order from yeast diet to HDMd, and dried fruits. The treatment and pretreatment of mites by antibiotics did not reveal the presence of antagonistic bacteria which might slow down the C. lactis population growth. The shifts of the microbial community in the gut of C. lactis were induced by the diet changes. The identified yeasts and bacteria are suggested as the main food source of stored product mites on dried fruits. The study describes the adaptation of C. lactis to feeding on dried fruits including the interaction with microorganisms. We also identified potentially pathogenic bacteria carried by the mites to dried fruits for human consumption. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    Journal of Applied Microbiology 12/2014; 118(2). DOI:10.1111/jam.12714 · 2.39 Impact Factor
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    ABSTRACT: The effect of Ixodes ricinus tick saliva on the production of various cytokines and chemokines by mouse splenocytes was tested by a cytokine array. We demonstrated a strong upregulation of three chemokines, monocyte chemoattractant protein-1 (MCP-1), thymus-derived chemotactic agent 3 (TCA-3) and macrophage inflammatory protein 2 (MIP-2). MCP-1 could be induced by tick saliva itself. While TCA-3 and MIP-2 are engaged in Th2 polarization of the host immune response associated with tick feeding, MCP-1 may act as a histamine release factor, increasing blood flow into the feeding lesion thus facilitating tick engorgement in the late, rapid feeding phase.This article is protected by copyright. All rights reserved.
    Parasite Immunology 12/2014; 37(2). DOI:10.1111/pim.12168 · 1.85 Impact Factor
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    ABSTRACT: Type I interferon (IFN), mainly produced by dendritic cells (DC), is critical in the host defence against tick-transmitted pathogens. Here we report that salivary cysteine protease inhibitor from the hard tick Ixodes scapularis, sialostatin L2, affects IFN-β mediated immune reactions in mouse dendritic cells. Following IFN receptor ligation, the Janus activated kinases/signal transducer and activator of transcription (JAK/STAT) pathway is activated. We show that sialostatin L2 attenuates phosphorylation of STATs in spleen dendritic cells upon addition of recombinant IFN-β. LPS stimulated dendritic cells release IFN-β which in turn leads to the induction of IFN-stimulated genes (ISG) through JAK/STAT pathway activation. The induction of two ISG, interferon regulatory factor 7 (IRF-7) and IP-10, was suppressed by sialostatin L2 in LPS-stimulated dendritic cells. Finally, the interference of sialostatin L2 with IFN action led to the enhanced replication of tick-borne encephalitis virus in DC. In summary, we present here that tick salivary cystatin negatively affects IFN-β responses which may consequently increase the pathogen load after transmission via tick saliva.
    Parasite Immunology 11/2014; 37(2). DOI:10.1111/pim.12162 · 1.85 Impact Factor
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    ABSTRACT: BACKGROUND:Bacteria are associated with the gut, fat bodies and reproductive organs of stored product mites (Acari: Astigmata). The mites are pests due to the production of allergens. Addition of antibiotics to diets can help to characterize the association between mites and bacteria. METHODOLOGY AND PRINCIPAL FINDINGS: Ampicillin, neomycin and streptomycin were added to the diets of mites and the effects on mite population growth (Acarus siro, Lepidoglyphus destructor and Tyrophagus putrescentiae) and associated bacterial community structure were assessed. Mites were treated by antibiotic supplementation (1 mgg-1 of diet) for 21 days and numbers of mites and bacterial communities were analyzed and compared to the untreated control. Bacterial quantities, determined by real-time PCR, significantly decreased in antibiotic treated specimens from 5 to 30 times in A. siro and T. putrescentiae, while no decline was observed in L. destructor. Streptomycin treatment eliminated Bartonella-like bacteria in the both A. siro and T. putrescentiae and Cardinium in T. putrescentiae. Solitalea-like bacteria proportion increased in the communities of neomycin and streptomycin treated A. siro specimens. Kocuria proportion increased in the bacterial communities of ampicillin and streptomycin treated A. siro and neomycin and streptomycin treated L. destructor. CONCLUSIONS/SIGNIFICANCE: The work demonstrated the changes of mite associated bacterial community under antibiotic pressure in pests of medical importance. Pre-treatment of mites by 1 mgg-1 antibiotic diets improved mite fitness as indicated accelerated population growth of A. siro pretreated streptomycin and neomycin and L. destructor pretreated by neomycin. All tested antibiotics supplemented to diets caused the decrease of mite growth rate in comparison to the control diet.
    PLoS ONE 11/2014; 9(11):e112919. DOI:10.1371/journal.pone.0112919 · 3.53 Impact Factor
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    ABSTRACT: Obesity-associated low-grade inflammation of white adipose tissue (WAT) contributes to development of insulin resistance and other disorders. Accumulation of immune cells, especially macrophages, and macrophage polarization from M2 to M1 state, affect intrinsic WAT signaling, namely anti-inflammatory and proinflammatory cytokines, fatty acids (FA), and lipid mediators derived from both n-6 and n-3 long-chain PUFA such as (i) arachidonic acid (AA)-derived eicosanoids and endocannabinoids, and (ii) specialized pro-resolving lipid mediators including resolvins derived from both eicosapentaenoic (EPA) and docosahexaenoic acid (DHA), lipoxins (AA metabolites), protectins and maresins (DHA metabolites). In this respect, potential differences in modulating adipocyte metabolism by various lipid mediators formed by inflammatory M1 macrophages typical of obese state, and non-inflammatory M2 macrophages typical of lean state remain to be established. Studies in mice suggest that (i) transient accumulation of M2 macrophages could be essential for the control of tissue FA levels during activation of lipolysis, (ii) a currently unidentified M2 macrophage-borne signaling molecule(s) could inhibit lipolysis and re-esterification of lipolyzed FA back to triacylglycerols (TAG/FA cycle), and (iii) the egress of M2 macrophages from rebuilt WAT and removal of the negative feedback regulation could allow for a full unmasking of metabolic activities of adipocytes. Thus, M2 macrophages could support remodeling of WAT to a tissue containing metabolically flexible adipocytes endowed with a high capacity of both TAG/FA cycling and oxidative phosphorylation. This situation could be exemplified by a combined intervention using mild calorie restriction and dietary supplementation with EPA/DHA, which enhances the formation of "healthy" adipocytes.
    Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids 10/2014; 1851(4). DOI:10.1016/j.bbalip.2014.09.023 · 4.50 Impact Factor
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    ABSTRACT: Long-chain n-3 polyunsaturated fatty acids (LC n-3 PUFA) in the diet protect against insulin resistance and obesity. Fibroblast growth factor-21 (Fgf21) is a hormonal factor released mainly by the liver that has powerful anti-diabetic effects. Here, we tested whether the beneficial metabolic effects of LC n-3 PUFA involve the induction of Fgf21. C57BL/6 J mice were exposed to an obesogenic, corn-oil-based, high-fat diet (cHF), or a diet in which corn oil was replaced with a fish-derived LC n-3 PUFA concentrate (cHF + F) using two experimental settings: short-term (3 weeks) and long-term treatment (8 weeks). CHF + F reduced body weight gain, insulinemia, and triglyceridemia compared to cHF. cHF increased plasma Fgf21 levels and hepatic Fgf21 gene expression compared with controls, but these effects were less pronounced or absent in cHF + F-fed mice. In contrast, hepatic expression of peroxisome proliferator-activated receptor (PPAR)-α target genes were more strongly induced by cHF + F than cHF, especially in the short-term treatment setting. The expression of genes encoding Fgf21, its receptors, and Fgf21 targets was unaltered by short-term LC n-3 PUFA treatment, with the exception of Ucp1 (uncoupling protein 1) and adiponectin genes, which were specifically up-regulated in white fat. In the long-term treatment setting, the expression of Fgf21 target genes and receptors was not differentially affected by LC n-3 PUFA. Collectively, our findings indicate that increased Fgf21 levels do not appear to be a major mechanism through which LC n-3 PUFA ameliorates high-fat-diet-associated metabolic disorders.
    Lipids 09/2014; 49(11). DOI:10.1007/s11745-014-3948-x · 2.35 Impact Factor
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    ABSTRACT: Sodium channels (SCs) in mites and insects are target sites for pesticides, including pyrethroids. Point mutations in the SC gene have been reported to change the structural conformation of the protein and its sensitivity to pesticides. To find mutations in the SC gene of the mite Varroa destructor (VmNa), the authors analysed the VmNa gene sequences available in GenBank and prepared specific primers for the amplification of two fragments containing the regions coding for (i) the domain II S4-S6 region (bp 2805-3337) and (ii) the domain III S4-3' terminus region (bp 4737-6500), as determined according to the VmNa cDNA sequence AY259834. Sensitive and resistant mite populations did not differ in the amino acid sequences of the III S4-3' terminus VmNa region. However, differences were found in the IIS4-IIS6 fragment. In the resistant population, the mutation C(3004) → G resulted in the substitution L(1002) → V (codon ctg → gtg) at the position equivalent to that of the housefly L925 in the domain II S5 helix. Additionally, the mutation F(1052) → L (codon ttc → ctc) at the position equivalent to that of the housefly F975 in the domain II P-loop connecting segments S5 and S6 was detected in both the resistant and sensitive populations. All individuals that survived the tau-fluvalinate treatment in the bioassay harboured the L(1002) → V mutation combined with the F(1052), while dead individuals from both the sensitive and resistant populations harboured mostly the L(1002) residue and either of the two residues at position 1052. © 2013 Society of Chemical Industry.
    Pest Management Science 06/2014; 70(6). DOI:10.1002/ps.3679 · 2.74 Impact Factor
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    Jan Kopecký, Marta Nesvorná, Jan Hubert
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    ABSTRACT: Bacteria of the genus Bartonella are carried by haematophagous mites, ticks, fleas and flies, and attack the erythrocytes of mammals. Here we describe a Bartonella-like clade, a distinct group related to Bartonellaceae, in stored-product mites (Acari: Astigmata) and a predatory mite Cheyletus eruditus (Acari: Prostigmata) based on the analysis of cloned 16S rRNA gene sequences. By using the clade-specific primers, closely related Bartonella-like 16S rRNA sequences were amplified from both laboratory colonies and field strains of three synanthropic mite species (Acarus siro, Lepidoglyphus destructor and Tyrophagus putrescentiae) and a predatory mite. Altogether, sequences of Bartonella-like bacteria were found in 11 strains, but were not detected in Dermatophagoides farinae and D. pteronyssinus and two strains of L. destructor. All obtained sequences formed a separate cluster branching as a sister group to Bartonellaceae and related to other separate clusters comprising uncultured bacterial clones from human skin and hemipteran insects (Nysius plebeius and Nysius sp.). The classification of sequences into operational taxonomic units (OTUs) showed a difference between A. siro and T. putrescentiae suggesting that the Bartonella-like bacteria are different in these two mite species. However, species specific sequences in separate OTUs were observed also for C. eruditus. Possible symbiotic interactions between Bartonella-like bacteria and their mite hosts are discussed.
    Experimental and Applied Acarology 04/2014; 64(1). DOI:10.1007/s10493-014-9811-1 · 1.82 Impact Factor
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    P Flachs, M Rossmeisl, J Kopecky
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    ABSTRACT: Type 2 diabetes (T2D) as well as cardiovascular disease (CVD) represent major complications of obesity and associated metabolic disorders (metabolic syndrome). This review focuses on the effects of long-chain n-3 polyunsaturated fatty acids (omega-3) on insulin sensitivity and glucose homeostasis, which are improved by omega-3 in many animal models of metabolic syndrome, but remain frequently unaffected in humans. Here we focus on: (i) mechanistic aspects of omega-3 action, reflecting also our experiments in dietary obese mice; and (ii) recent studies analysing omega-3's effects in various categories of human subjects. Most animal experiments document beneficial effects of omega-3 on insulin sensitivity and glucose metabolism even under conditions of established obesity and insulin resistance. Besides positive results obtained in both cross-sectional and prospective cohort studies on healthy human populations, also some intervention studies in prediabetic subjects document amelioration of impaired glucose homeostasis by omega-3. However, the use of omega-3 to reduce a risk of new-onset diabetes in prediabetic subjects still remains to be further characterized. The results of a majority of clinical trials performed in T2D patients suggest that omega-3 have none or marginal effects on metabolic control, while effectively reducing hypertriglyceridemia in these patients. Despite most of the recent randomized clinical trials do not support the role of omega-3 in secondary prevention of CVD, this issue remains still controversial. Combined interventions using omega-3 and antidiabetic or hypolipidemic drugs should be further explored and considered for treatment of patients with T2D and other diseases.
    Physiological research / Academia Scientiarum Bohemoslovaca 02/2014; 63 Suppl 1:S93-S118. · 1.49 Impact Factor
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    ABSTRACT: OBJECTIVE: Besides their role in lipid absorption, bile acids (BAs) can act as signalling molecules. Cholic acid was shown to counteract obesity and associated metabolic disorders in high-fat-diet (cHF)-fed mice while enhancing energy expenditure through induction of mitochondrial uncoupling protein 1 (UCP1) and activation of non-shivering thermogenesis in brown adipose tissue (BAT). In this study, the effects of another natural BA, chenodeoxycholic acid (CDCA), on dietary obesity, UCP1 in both interscapular BAT and in white adipose tissue (brite cells in WAT), were characterized in dietary-obese mice. RESEARCH DESIGN: To induce obesity and associated metabolic disorders, male 2-month-old C57BL/6J mice were fed cHF (35% lipid wt wt(-1), mainly corn oil) for 4 months. Mice were then fed either (i) for 8 weeks with cHF or with cHF with two different doses (0.5%, 1%; wt wt(-1)) of CDCA (8-week reversion); or (ii) for 3 weeks with cHF or with cHF with 1% CDCA, or pair-fed (PF) to match calorie intake of the CDCA mice fed ad libitum; mice on standard chow diet were also used (3-week reversion). RESULTS: In the 8-week reversion, the CDCA intervention resulted in a dose-dependent reduction of obesity, dyslipidaemia and glucose intolerance, which could be largely explained by a transient decrease in food intake. The 3-week reversion revealed mild CDCA-dependent and food intake-independent induction of UCP1-mediated thermogenesis in interscapular BAT, negligible increase of UCP1 in subcutaneous WAT and a shift from carbohydrate to lipid oxidation. CONCLUSIONS: CDCA could reverse obesity in cHF-fed mice, mainly in response to the reduction in food intake, an effect probably occuring but neglected in previous studies using cholic acid. Nevertheless, CDCA-dependent and food intake-independent induction of UCP1 in BAT (but not in WAT) could contribute to the reduction in adiposity and to the stabilization of the lean phenotype.
    International journal of obesity (2005) 12/2013; 38(8). DOI:10.1038/ijo.2013.230 · 5.39 Impact Factor
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    ABSTRACT: Very few soil quality indicators include disease suppressiveness criteria. We assessed whether 64 16S rRNA microarray probes whose signals correlated with tobacco black-root-rot suppressiveness in greenhouse analysis, could also discriminate suppressive from conducive soils under field conditions. Rhizobacterial communities of tobacco and wheat sampled in two years from four farmers’ fields of contrasted suppressiveness status were compared. The 64 previously-identified indicator probes correctly classified 72% of 29 field samples, with 9 probes for Azospirillum, Gluconacetobacter, Sphingomonadaceae, Planctomycetes, Mycoplasma, Lactobacillus crispatus and Thermodesulforhabdus providing the best prediction. The whole probe set (1033 probes) revealed strong effects of plant, field location and year on rhizobacterial community composition, and a smaller (7% variance) but significant effect of soil suppressiveness status. 17 additional probes correlating with suppressiveness status in the field (noticeably for Agrobacterium, Methylobacterium, Ochrobactrum) were selected, and combined with the 9 others they improved correct sample classification from 72% to 79% (100% tobacco and 63% wheat samples). Pseudomonas probes were not informative in the field, even those targeting biocontrol pseudomonads producing 2,4-diacetylphloroglucinol, nor was quantitative PCR for 2,4-diacetylphloroglucinol-synthesis gene phlD. This study shows that a subset of 16S rRNA probes targeting diverse rhizobacteria can be useful as suppressiveness indicators under field conditions.
    Environmental Microbiology Reports 11/2013; 6(4). DOI:10.1111/1758-2229.12131 · 3.26 Impact Factor
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    ABSTRACT: House dust mites produce antibacterial proteins suppressing bacterial growth. The 14.5-kDa bacteriolytic protein (UniProtKB Q8MWR6) has been known in Dermatophagoides pteronyssinus Trouessart. We have applied polymerase chain reaction and reverse transcription-PCR to detect a homologous gene sequence coding for a Q8MWR6-related protein in Dermatophagoides farinae (Hughes) using genomic DNA and total RNA, respectively. The resulting PCR product of expected size, 243 bp, was obtained from both Dermatophagoides spp., while no amplification was achieved from stored product mite samples. Sequence of the gene fragment from D. farinae showed 83% similarity to the previously described one in D. pteronyssinus. Successful amplification of the expected product from cDNA generated with oligo-dT primer implies that the NlpC/P60-like protein in Dermatophagoides mites is of eukaryotic or mite origin.
    Journal of Medical Entomology 07/2013; 50(4):679-933. DOI:10.1603/ME13027 · 1.82 Impact Factor
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    ABSTRACT: Objective:Resolution of low-grade inflammation of white adipose tissue (WAT) is one of the keys for amelioration of obesity-associated metabolic dysfunctions. We focused on the identification of adipokines, which could be involved at the early stages of resolution of WAT inflammation.Methods and ProcedureMale C57BL/6 J mice with obesity induced in response to a 22-week feeding corn oil-based high-fat (cHF) diet were divided into 4 groups and fed for 2 weeks control cHF diet or cHF-based diets supplemented with: (i) concentrate of n-3 long-chain polyunsaturated fatty acids (cHF+F); (ii) rosiglitazone (cHF+TZD); and (iii) both compounds (cHF+F+TZD).ResultsThe short-term combined intervention exerted additive effect in the amelioration of WAT inflammation in obese mice, namely in the epididymal fat, even in the absence of any changes in either adipocyte volume or fat mass. The combined intervention elicited hypolipidemic effect and induced adiponectin, while the responses to single interventions (cHF+F, cHF+TZD) were less pronounced. In addition, analysis in WAT lysates using protein arrays revealed that levels of a small set of adipose tissue-related proteins, namely macrophage inflammatory protein 1γ, endoglin, vascular cell adhesion molecule 1, and interleukin 1 receptor antagonist, changed in response to the anti-inflammatory interventions and were strongly reduced in the cHF+F+TZD mice. These results were verified using both the analysis of gene expression and ELISA in WAT lysates. In contrast with adiponectin, which showed changing plasma levels in response to dietary interventions, the levels of the above proteins were affected only in WAT.Conclusions:We identified several adipose tissue-related proteins, which are locally involved in resolution of low-grade inflammation and remodelling of WAT.International Journal of Obesity accepted article preview online, 12 June 2013; doi:10.1038/ijo.2013.108.
    International journal of obesity (2005) 06/2013; DOI:10.1038/ijo.2013.108 · 5.39 Impact Factor
  • J. Kopecky, R.A. Forrest
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    ABSTRACT: This paper presents the approach of a combined use of resonance integrals and average Maxwellian cross sections (MACS) at kT = 30 keV to test and validate the resolved resonance range or its reconstructed cross section curve. Based on these two integral measurements a sensitive and energy dependent test can be provided. These two integral quantities cover with their neutron spectra the energy region between En = 0.5 eV up to several hundred keV, respectively, with different weighting. Our principal motivation is to produce a validation tool, sensitive to the lower and upper parts of the resonance region through the difference in the applied 1/E and kT = 30 keV Maxwell–Boltzmann spectra of the resonance integral and MACS data.
    Fusion Engineering and Design 05/2013; 88(4):177–187. DOI:10.1016/j.fusengdes.2013.01.095 · 1.15 Impact Factor
  • Jan Hubert, Marta Nesvorná, Jan Kopecký
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    ABSTRACT: The effect of Tyrophagus putrescentiae on Fusarium poae transmission and fungal community composition was studied in nonsterile barley grain. The experiments included following treatments: control barley without mites; barley containing 10 or 50 mites without preincubation on F. poae (Tp10 and Tp50); barley containing 10 or 50 mites after preincubation on F. poae (FTp10 and FTp50). The number of mites, successful transfer of F. poae, and changes in the fungal communities were examined after 21 d of experiment. Increase of deoxynivalenol (DON) content in the barley was chosen as a criterion of successful F. poae transfer. The preincubation of T. putrescentiae on F. poae increased DON level approximately to 800 and 300 μg/kg of grain for FTp10 and FTp50, respectively. T. putrescentiae population growth in FTp10 was lower than in Tp10, while no difference was found between FTp50 and Tp50. Fungal communities were compared by amplification, cloning and sequencing of ITS fragments, and operational taxonomic units (OTU) analysis. The OTU analysis did not support the transfer of F. poae via mites. From the analyzed clones, only 13 cloned sequences clustered with F. poae in an OTU defined at distance level 0.07. The related clones originated from FTp10, Tp10, Tp50 and control treatments, but not from FTp50. However, the presence of F. poae in FTp50 was confirmed by PCR amplification with specific primers. The observation may be explained by different effect of mite population density, that is, in the high density, (FTp50 treatment) the fungus was overgrazed, while the lower population density (FTp10) supported F. poae transfer.
    Insect Science 04/2013; 21(1). DOI:10.1111/1744-7917.12036 · 1.51 Impact Factor
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    ABSTRACT: FAT/CD36 is a multifunctional glycoprotein that facilitates long-chain fatty acid (FA) uptake by cardiomyocytes and adipocytes and uptake of oxidized low density lipoproteins (oxLDL) by macrophages. CD36 also mediates FA-induced signaling to increase intracellular calcium in various cell types. The membrane impermeable sulfo-N-hydroxy-succinimidyl (NHS) ester of oleate (SSO) irreversibly binds CD36 and has been widely used to inhibit CD36-dependent FA uptake and signaling to calcium. The inhibition mechanism and whether SSO modification of CD36 involves the FA binding site remain unexplored. CHO cells expressing human CD36 were SSO-treated and the protein was pulled down, deglycosylated and resolved by electrophoresis. The CD36 band was extracted from the gel and digested before analysis by mass spectrometry. NHS derivatives react with primary or secondary amines on proteins to yield stable amide or imide bonds. Two oleoylated peptides, found only in SSO-treated samples, were identified with high contribution and confidence scores as carrying oleate modification of lysine 164 (K164). Lysine 164 lies within a predicted CD36 binding domain for FA and oxLDL. CHO cells expressing CD36 with mutated K164 had impaired CD36 function in FA uptake and FA-induced calcium release from the endoplasmic reticulum, supporting importance of K164 for both FA effects. Furthermore, consistent with importance of K164 for oxLDL binding, SSO inhibited oxLDL uptake by macrophages. In conclusion, SSO accesses K164 in the FA binding site on CD36 and initial modeling of this site is presented. The data suggest competition between FA and oxLDL for access to the CD36 binding pocket.
    Journal of Biological Chemistry 04/2013; 288(22). DOI:10.1074/jbc.M113.473298 · 4.60 Impact Factor

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Institutions

  • 2010–2014
    • Crop Research Institute
      Praha, Praha, Czech Republic
  • 2007–2014
    • University of South Bohemia in České Budějovice
      Budejovice, Jihočeský, Czech Republic
  • 1995–2014
    • Academy of Sciences of the Czech Republic
      • • Department of Adipose Tissue Biology
      • • Division of Autotrophic Microorganisms - Algatech
      • • Hydrobiologický ústav
      • • Biology Centre
      • • Laboratory of Biotransformation
      • • Fyziologický ústav
      Praha, Praha, Czech Republic
  • 2000–2008
    • The Police Academy of the Czech Republic in Prague
      Praha, Praha, Czech Republic
  • 2005
    • Institute for Clinical and Experimental Medicine (IKEM)
      • Diabetes Center
      Praha, Praha, Czech Republic
  • 1978–1998
    • Charles University in Prague
      • • Přírodovědecká fakulta
      • • Institute of Physiology (Pilsen)
      • • Institute of Medical Biochemistry (1. LF)
      Praha, Hlavni mesto Praha, Czech Republic
  • 1994
    • The Jackson Laboratory
      BHB, Maine, United States
  • 1992
    • Slovak Academy of Sciences
      Presburg, Bratislavský, Slovakia
  • 1981–1989
    • Stockholm University
      • Department of Biochemistry and Biophysics
      Tukholma, Stockholm, Sweden
  • 1982–1983
    • Utrecht University
      • Division of Physics of Man
      Utrecht, Utrecht, Netherlands