Ludovit Skultety - Academia.edu (original) (raw)
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Papers by Ludovit Skultety
Journal of Proteome Research, 2010
Analytical Chemistry, 2008
Rapid and reliable detection, identification, and typing of bacterial species are necessary in re... more Rapid and reliable detection, identification, and typing of bacterial species are necessary in response to natural or terrorist-caused outbreaks of infectious diseases and play crucial roles in diagnosis and efficient treatment. We report here two proteomic approaches with a high potential in the detection and identification of Coxiella burnetii, the causative agent of Q fever. The first of them starts with the acetonitrile (ACN) and trichloroacetic acid extractions of inactivated C. burnetii cells followed by the detection of extracted molecules and ions derived from the inactivated cells by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. In the second approach, identification of the proteins extracted by ACN is accomplished after enzymatic digestion by electrospray tandem mass spectrometry coupled to a nanoscale ultraperformance liquid chromatography (LC-MS/MS). In order to observe morphological differences on the surface structures upon extraction, the inactivated and treated cells of the bacterium were examined by electron microscopy. The LC-MS/MS approach has allowed identification of 20 proteins in the ACN extracts of C. burnetii strain RSA 493 that were observed in more than 3 out of 10 experiments.
Acta virologica
The lipid A proximal carbohydrate region in a lipopolysaccharide (LPS II) isolated from Coxiella ... more The lipid A proximal carbohydrate region in a lipopolysaccharide (LPS II) isolated from Coxiella burnetii strain Nine Mile in avirulent phase II has been reinvestigated. So called "Kdo-like substance", reported to be present in LPS II (Schramek and Mayer, 1982) has been unambiguously identified as the 3-deoxy-D-manno-2-octulosonic acid (Kdo).
Proteomics, 2014
The Gram-negative pathogen Coxiella burnetii is an intracellular bacterium that replicates within... more The Gram-negative pathogen Coxiella burnetii is an intracellular bacterium that replicates within the phagolysosomal vacuoles of eukaryotic cells. This pathogen can infect a wide range of hosts, and is the causative agent of Q fever in humans. Surface-exposed and cell envelope associated proteins are thought to be important for both pathogenesis and protective immunity. Herein, we propose a complementary strategy consisting of (i) in silico prediction and (ii) inventory of the proteomic composition using three enrichment approaches coupled with protein identification. The efficiency of classical Triton X-114 phase partitioning was compared with two novel procedures; isolation of alkaline proteins by liquid-phase IEF, and cell surface enzymatic shaving using biofunctional magnetic beads. Of the 2026 protein sequences analyzed using seven distinct bioinformatic algorithms, 157 were predicted to be outer membrane proteins (OMP) and/or lipoproteins (LP). Using the three enrichment proto...
Acta virologica, 1994
The immunogenicity and protective efficacy of the phase I and phase II Coxiella burnetii whole ce... more The immunogenicity and protective efficacy of the phase I and phase II Coxiella burnetii whole cells (Cb I and Cb II) and their outer membrane components (OMC), i.e. phase I trichloroacetic acid extract (TCAE), phase I 29 K protein (PRO), phase I and II lipopolysaccharides (LPS I, LPS II), polysaccharides (PS I, PS II), and lipid A (LA I, LA II), were compared. The highest immune response was observed in BALB/c mice by Cb I in both humoral immunity and lymphocyte transformation assays, and in the protective effect as well. The immune response was also significant by Cb II, but their protective capacity was low. The OMC reacted variously. Only TCAE and PRO gave a high value of humoral immunity evaluated by the serological methods. All OMC reacted in the haemolytic plaque assay giving different responses. Lymphoproliferation of splenocytes was positive with all OMC using both Cb I and Cb II antigens with the exception of PS I and PS II in the case of Cb II antigen. The induction of pr...
Plant proteomics is a relatively new research field that focuses on large-scale functional analys... more Plant proteomics is a relatively new research field that focuses on large-scale functional analysis of plant proteins. This new research field has already demonstrated immense potential for getting significantly deeper insight into the functional interaction of plant proteins and their roles in plant growth and development. The knowledge of key proteins responsible for valuable crop traits in the context of
General physiology and biophysics, 2014
Diterpenoid triepoxide - Triptolide (TTL) - increased protein levels of the noradrenaline transpo... more Diterpenoid triepoxide - Triptolide (TTL) - increased protein levels of the noradrenaline transporter in three pheochromocytoma cell lines. This transporter is involved in the apoptosis induction through the inhibition of a transcription factor NF-kappa B. Nevertheless, calcium release from the endoplasmic reticulum can also induce inner mitochondrial pathway of apoptosis in variety of cells. Therefore, the aim of this work was to evaluate an involvement of calcium and, more specifically, the intracellular calcium transport systems in the apoptosis induction in pheochrocytoma cell line PC12. We observed significantly increased amount of reticular calcium in TTL-treated cells compared to control, untreated cells. Surprisingly, gene expression of the IP3 receptors was not changed after the TTL treatment, but ryanodine receptor of the type 2 (RyR2) was downregulated and sarco/endoplasmic reticulum calcium ATPase type 3 (SERCA 3) was upregulated in TTL- treated cells, compared to untreated controls. SERCA 3 blocking with the specific blocker thapsigargin prevented increase in apoptosis observed by the TTL treatment. Decrease in the ATP production by a replacement of glucose in the cultivation medium for its nonutilizable analog 2-deoxyglucose also prevented induction of the apoptosis in TTL-treated PC12 cells. Thus, these results suggest that upregulation of the SERCA 3 is ultimately involved in the TTL-induced apoptosis in PC12 cells.
Proteome Science, 2012
Coxiella burnetii is Gram-negative bacterium responsible for the zoonosis Q-fever. While it has a... more Coxiella burnetii is Gram-negative bacterium responsible for the zoonosis Q-fever. While it has an obligate intracellular growth habit, it is able to persist for extended periods outside of a host cell and can resist environmental conditions that would be lethal to most prokaryotes. It is these extracellular bacteria that are the infectious stage encountered by eukaryotic hosts. The intracellular form has evolved to grow and replicate within acidified parasitophorous vacuoles. The outer coat of C. burnetii comprises a complex lipopolysaccharide (LPS) component that includes the unique methylated-6-deoxyhexose, virenose. Although potentially important as a biomarker for C. burnetii, the pathway for its biosynthesis remains obscure. The 6-deoxyhexoses constitute a large family integral to the LPS of many eubacteria. It is believed that precursors of the methylated-deoxyhexoses traverse common early biosynthetic steps as nucleotide-monosaccharides. As a prelude to a full biosynthetic characterization, we present herein the results from bioinformatics-based, proteomics-supported predictions of the pathway for virenose synthesis. Alternative possibilities are considered which include both GDP-mannose and TDP-glucose as precursors. We propose that biosynthesis of the unique C. burnetii biomarker, virenose, involves an early pathway similar to that of other C-3'-methylated deoxysugars which then diverges depending upon the nucleotide-carrier involved. The alternatives yield either the D- or L-enantiomers of virenose. Both pathways require five enzymatic steps, beginning with either glucose-6-phosphate or mannose-6-phosphate. Our in silico results comprise a model for virenose biosynthesis that can be directly tested. Definition of this pathway should facilitate the development of therapeutic agents useful for treatment of Q fever, as well as allowing improvements in the methods for diagnosing this highly infectious disease.
Journal of Proteomics, 2011
Journal of Proteomics, 2011
Journal of Proteome Research, 2010
European Journal of Epidemiology, 1994
Carbohydrate Research, 1998
Analytical Chemistry, 2008
European Journal of Mass Spectrometry, 2007
Fragmentation mechanisms of phytoalexin analogs, including brassitin and brassinin and their gluc... more Fragmentation mechanisms of phytoalexin analogs, including brassitin and brassinin and their glucosylated analogs, have been studied by electrospray (ESI) ion trap (IT) multistage (MS(n), n = 1-4) mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight (MALDI ToF/ToF) and ESI-Q/ToF tandem mass spectrometry techniques. At the fragmentation of sodium adducts a hitherto not described process has been elucidated The proposed mechanism of this process includes cyclization of the brassitin and brassinin cationized adducts through a six-membered cycle of the molecules and the elimination of isocyanate or isothiocyanate from the thio- or dithiocarbamate moiety, giving rise to [M + Na - 43](+) or [M + Na - 59](+) adducts. The elimination of NH=C=O or NH=C=S molecules has been confirmed by the high resolution measurement of the elemental composition of the ions produced and quantum-chemical calculations of the six-membered transition state. Other fragmentation routes include cleavage of an alkane linker, while numerous characteristic hexopyranose pathways are taking place in the glucosylated compounds. The presented theoretical data on the ESI and MALDI behavior of the saccharidic, as well as of the indole aglycon parts, can facilitate structural elucidation of the analogous compounds.
Journal of Proteome Research, 2010
Analytical Chemistry, 2008
Rapid and reliable detection, identification, and typing of bacterial species are necessary in re... more Rapid and reliable detection, identification, and typing of bacterial species are necessary in response to natural or terrorist-caused outbreaks of infectious diseases and play crucial roles in diagnosis and efficient treatment. We report here two proteomic approaches with a high potential in the detection and identification of Coxiella burnetii, the causative agent of Q fever. The first of them starts with the acetonitrile (ACN) and trichloroacetic acid extractions of inactivated C. burnetii cells followed by the detection of extracted molecules and ions derived from the inactivated cells by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. In the second approach, identification of the proteins extracted by ACN is accomplished after enzymatic digestion by electrospray tandem mass spectrometry coupled to a nanoscale ultraperformance liquid chromatography (LC-MS/MS). In order to observe morphological differences on the surface structures upon extraction, the inactivated and treated cells of the bacterium were examined by electron microscopy. The LC-MS/MS approach has allowed identification of 20 proteins in the ACN extracts of C. burnetii strain RSA 493 that were observed in more than 3 out of 10 experiments.
Acta virologica
The lipid A proximal carbohydrate region in a lipopolysaccharide (LPS II) isolated from Coxiella ... more The lipid A proximal carbohydrate region in a lipopolysaccharide (LPS II) isolated from Coxiella burnetii strain Nine Mile in avirulent phase II has been reinvestigated. So called "Kdo-like substance", reported to be present in LPS II (Schramek and Mayer, 1982) has been unambiguously identified as the 3-deoxy-D-manno-2-octulosonic acid (Kdo).
Proteomics, 2014
The Gram-negative pathogen Coxiella burnetii is an intracellular bacterium that replicates within... more The Gram-negative pathogen Coxiella burnetii is an intracellular bacterium that replicates within the phagolysosomal vacuoles of eukaryotic cells. This pathogen can infect a wide range of hosts, and is the causative agent of Q fever in humans. Surface-exposed and cell envelope associated proteins are thought to be important for both pathogenesis and protective immunity. Herein, we propose a complementary strategy consisting of (i) in silico prediction and (ii) inventory of the proteomic composition using three enrichment approaches coupled with protein identification. The efficiency of classical Triton X-114 phase partitioning was compared with two novel procedures; isolation of alkaline proteins by liquid-phase IEF, and cell surface enzymatic shaving using biofunctional magnetic beads. Of the 2026 protein sequences analyzed using seven distinct bioinformatic algorithms, 157 were predicted to be outer membrane proteins (OMP) and/or lipoproteins (LP). Using the three enrichment proto...
Acta virologica, 1994
The immunogenicity and protective efficacy of the phase I and phase II Coxiella burnetii whole ce... more The immunogenicity and protective efficacy of the phase I and phase II Coxiella burnetii whole cells (Cb I and Cb II) and their outer membrane components (OMC), i.e. phase I trichloroacetic acid extract (TCAE), phase I 29 K protein (PRO), phase I and II lipopolysaccharides (LPS I, LPS II), polysaccharides (PS I, PS II), and lipid A (LA I, LA II), were compared. The highest immune response was observed in BALB/c mice by Cb I in both humoral immunity and lymphocyte transformation assays, and in the protective effect as well. The immune response was also significant by Cb II, but their protective capacity was low. The OMC reacted variously. Only TCAE and PRO gave a high value of humoral immunity evaluated by the serological methods. All OMC reacted in the haemolytic plaque assay giving different responses. Lymphoproliferation of splenocytes was positive with all OMC using both Cb I and Cb II antigens with the exception of PS I and PS II in the case of Cb II antigen. The induction of pr...
Plant proteomics is a relatively new research field that focuses on large-scale functional analys... more Plant proteomics is a relatively new research field that focuses on large-scale functional analysis of plant proteins. This new research field has already demonstrated immense potential for getting significantly deeper insight into the functional interaction of plant proteins and their roles in plant growth and development. The knowledge of key proteins responsible for valuable crop traits in the context of
General physiology and biophysics, 2014
Diterpenoid triepoxide - Triptolide (TTL) - increased protein levels of the noradrenaline transpo... more Diterpenoid triepoxide - Triptolide (TTL) - increased protein levels of the noradrenaline transporter in three pheochromocytoma cell lines. This transporter is involved in the apoptosis induction through the inhibition of a transcription factor NF-kappa B. Nevertheless, calcium release from the endoplasmic reticulum can also induce inner mitochondrial pathway of apoptosis in variety of cells. Therefore, the aim of this work was to evaluate an involvement of calcium and, more specifically, the intracellular calcium transport systems in the apoptosis induction in pheochrocytoma cell line PC12. We observed significantly increased amount of reticular calcium in TTL-treated cells compared to control, untreated cells. Surprisingly, gene expression of the IP3 receptors was not changed after the TTL treatment, but ryanodine receptor of the type 2 (RyR2) was downregulated and sarco/endoplasmic reticulum calcium ATPase type 3 (SERCA 3) was upregulated in TTL- treated cells, compared to untreated controls. SERCA 3 blocking with the specific blocker thapsigargin prevented increase in apoptosis observed by the TTL treatment. Decrease in the ATP production by a replacement of glucose in the cultivation medium for its nonutilizable analog 2-deoxyglucose also prevented induction of the apoptosis in TTL-treated PC12 cells. Thus, these results suggest that upregulation of the SERCA 3 is ultimately involved in the TTL-induced apoptosis in PC12 cells.
Proteome Science, 2012
Coxiella burnetii is Gram-negative bacterium responsible for the zoonosis Q-fever. While it has a... more Coxiella burnetii is Gram-negative bacterium responsible for the zoonosis Q-fever. While it has an obligate intracellular growth habit, it is able to persist for extended periods outside of a host cell and can resist environmental conditions that would be lethal to most prokaryotes. It is these extracellular bacteria that are the infectious stage encountered by eukaryotic hosts. The intracellular form has evolved to grow and replicate within acidified parasitophorous vacuoles. The outer coat of C. burnetii comprises a complex lipopolysaccharide (LPS) component that includes the unique methylated-6-deoxyhexose, virenose. Although potentially important as a biomarker for C. burnetii, the pathway for its biosynthesis remains obscure. The 6-deoxyhexoses constitute a large family integral to the LPS of many eubacteria. It is believed that precursors of the methylated-deoxyhexoses traverse common early biosynthetic steps as nucleotide-monosaccharides. As a prelude to a full biosynthetic characterization, we present herein the results from bioinformatics-based, proteomics-supported predictions of the pathway for virenose synthesis. Alternative possibilities are considered which include both GDP-mannose and TDP-glucose as precursors. We propose that biosynthesis of the unique C. burnetii biomarker, virenose, involves an early pathway similar to that of other C-3'-methylated deoxysugars which then diverges depending upon the nucleotide-carrier involved. The alternatives yield either the D- or L-enantiomers of virenose. Both pathways require five enzymatic steps, beginning with either glucose-6-phosphate or mannose-6-phosphate. Our in silico results comprise a model for virenose biosynthesis that can be directly tested. Definition of this pathway should facilitate the development of therapeutic agents useful for treatment of Q fever, as well as allowing improvements in the methods for diagnosing this highly infectious disease.
Journal of Proteomics, 2011
Journal of Proteomics, 2011
Journal of Proteome Research, 2010
European Journal of Epidemiology, 1994
Carbohydrate Research, 1998
Analytical Chemistry, 2008
European Journal of Mass Spectrometry, 2007
Fragmentation mechanisms of phytoalexin analogs, including brassitin and brassinin and their gluc... more Fragmentation mechanisms of phytoalexin analogs, including brassitin and brassinin and their glucosylated analogs, have been studied by electrospray (ESI) ion trap (IT) multistage (MS(n), n = 1-4) mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight (MALDI ToF/ToF) and ESI-Q/ToF tandem mass spectrometry techniques. At the fragmentation of sodium adducts a hitherto not described process has been elucidated The proposed mechanism of this process includes cyclization of the brassitin and brassinin cationized adducts through a six-membered cycle of the molecules and the elimination of isocyanate or isothiocyanate from the thio- or dithiocarbamate moiety, giving rise to [M + Na - 43](+) or [M + Na - 59](+) adducts. The elimination of NH=C=O or NH=C=S molecules has been confirmed by the high resolution measurement of the elemental composition of the ions produced and quantum-chemical calculations of the six-membered transition state. Other fragmentation routes include cleavage of an alkane linker, while numerous characteristic hexopyranose pathways are taking place in the glucosylated compounds. The presented theoretical data on the ESI and MALDI behavior of the saccharidic, as well as of the indole aglycon parts, can facilitate structural elucidation of the analogous compounds.