Publications (25)94.08 Total impact
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Article: Heterosis-associated proteome analyses of maize (Zea mays L.) seminal roots by quantitative label-free LC-MS.
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ABSTRACT: Heterosis is the superior performance of heterozygous F1-hybrid plants compared to their homozygous genetically distinct parents. Seminal roots are embryonic roots that play an important role during early maize (Zea mays L.) seedling development. In the present study the most abundant soluble proteins of 2-4 cm seminal roots of the reciprocal maize F1-hybrids B73xMo17 and Mo17xB73 and their parental inbred lines B73 and Mo17 were quantified by label-free LC-MS/MS. In total, 1,918 proteins were detected by this shot-gun approach. Among those, 970 were represented by at least two peptides and were further analyzed. Eighty-five proteins displayed non-additive accumulation in at least one hybrid. The functional category protein metabolism was the most abundant class of non-additive proteins represented by 27 proteins. Within this category 16 of 17 non-additively accumulated ribosomal proteins showed high or above high parent expression in seminal roots. These results imply that an increased protein synthesis rate in hybrids might be related to the early manifestation of hybrid vigor in seminal roots. SIGNIFICANCE: In the present study a shot-gun proteomics approach allowed for the identification detection of 1,917 proteins and analysis of 970 seminal root proteins of maize that were represented by at least 2 peptides. The comparison of proteome complexity of reciprocal hybrids and their parental inbred lines indicate an increased protein synthesis rate in hybrids that may contribute to the early manifestation of heterosis in seminal roots.Journal of proteomics 04/2013; · 5.07 Impact Factor -
Article: Specification of cortical parenchyma and stele of maize primary roots by asymmetric levels of auxin, cytokinin, and cytokinin-regulated proteins.
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ABSTRACT: In transverse orientation, maize (Zea mays) roots are composed of a central stele that is embedded in multiple layers of cortical parenchyma. The stele functions in the transport of water, nutrients, and photosynthates, while the cortical parenchyma fulfills metabolic functions that are not very well characterized. To better understand the molecular functions of these root tissues, protein- and phytohormone-profiling experiments were conducted. Two-dimensional gel electrophoresis combined with electrospray ionization tandem mass spectrometry identified 59 proteins that were preferentially accumulated in the cortical parenchyma and 11 stele-specific proteins. Hormone profiling revealed preferential accumulation of indole acetic acid and its conjugate indole acetic acid-aspartate in the stele and predominant localization of the cytokinin cis-zeatin, its precursor cis-zeatin riboside, and its conjugate cis-zeatin O-glucoside in the cortical parenchyma. A root-specific beta-glucosidase that functions in the hydrolysis of cis-zeatin O-glucoside was preferentially accumulated in the cortical parenchyma. Similarly, four enzymes involved in ammonium assimilation that are regulated by cytokinin were preferentially accumulated in the cortical parenchyma. The antagonistic distribution of auxin and cytokinin in the stele and cortical parenchyma, together with the cortical parenchyma-specific accumulation of cytokinin-regulated proteins, suggest a molecular framework that specifies the function of these root tissues that also play a role in the formation of lateral roots from pericycle and endodermis cells.Plant physiology 11/2009; 152(1):4-18. · 6.53 Impact Factor -
Article: Acclimatory responses of the Daphnia pulex proteome to environmental changes. II. Chronic exposure to different temperatures (10 and 20 degrees C) mainly affects protein metabolism.
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ABSTRACT: Temperature affects essentially every aspect of the biology of poikilothermic animals including the energy and mass budgets, activity, growth, and reproduction. While thermal effects in ecologically important groups such as daphnids have been intensively studied at the ecosystem level and at least partly at the organismic level, much less is known about the molecular mechanisms underlying the acclimation to different temperatures. By using 2D gel electrophoresis and mass spectrometry, the present study identified the major elements of the temperature-induced subset of the proteome from differently acclimated Daphnia pulex. Specific sets of proteins were found to be differentially expressed in 10 degrees C or 20 degrees C acclimated D. pulex. Most cold-repressed proteins comprised secretory enzymes which are involved in protein digestion (trypsins, chymotrypsins, astacin, carboxypeptidases). The cold-induced sets of proteins included several vitellogenin and actin isoforms (cytoplasmic and muscle-specific), and an AAA+ ATPase. Carbohydrate-modifying enzymes were constitutively expressed or down-regulated in the cold. Specific sets of cold-repressed and cold-induced proteins in D. pulex can be related to changes in the cellular demand for amino acids or to the compensatory control of physiological processes. The increase of proteolytic enzyme concentration and the decrease of vitellogenin, actin and total protein concentration between 10 degrees C and 20 degrees C acclimated animals reflect the increased amino-acids demand and the reduced protein reserves in the animal's body. Conversely, the increase of actin concentration in cold-acclimated animals may contribute to a compensatory mechanism which ensures the relative constancy of muscular performance. The sheer number of peptidase genes (serine-peptidase-like: > 200, astacin-like: 36, carboxypeptidase-like: 30) in the D. pulex genome suggests large-scaled gene family expansions that might reflect specific adaptations to the lifestyle of a planktonic filter feeder in a highly variable aquatic environment.BMC Physiology 05/2009; 9:8. -
Article: Acclimatory responses of the Daphnia pulex proteome to environmental changes. I. Chronic exposure to hypoxia affects the oxygen transport system and carbohydrate metabolism.
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ABSTRACT: Freshwater planktonic crustaceans of the genus Daphnia show a remarkable plasticity to cope with environmental changes in oxygen concentration and temperature. One of the key proteins of adaptive gene control in Daphnia pulex under hypoxia is hemoglobin (Hb), which increases in hemolymph concentration by an order of magnitude and shows an enhanced oxygen affinity due to changes in subunit composition. To explore the full spectrum of adaptive protein expression in response to low-oxygen conditions, two-dimensional gel electrophoresis and mass spectrometry were used to analyze the proteome composition of animals acclimated to normoxia (oxygen partial pressure [Po2]: 20 kPa) and hypoxia (Po2: 3 kPa), respectively. The comparative proteome analysis showed an up-regulation of more than 50 protein spots under hypoxia. Identification of a major share of these spots revealed acclimatory changes for Hb, glycolytic enzymes (enolase), and enzymes involved in the degradation of storage and structural carbohydrates (e.g. cellubiohydrolase). Proteolytic enzymes remained constitutively expressed on a high level. Acclimatory adjustments of the D. pulex proteome to hypoxia included a strong induction of Hb and carbohydrate-degrading enzymes. The scenario of adaptive protein expression under environmental hypoxia can be interpreted as a process to improve oxygen transport and carbohydrate provision for the maintenance of ATP production, even during short episodes of tissue hypoxia requiring support from anaerobic metabolism.BMC Physiology 05/2009; 9:7. -
Article: Tissue specific control of the maize (Zea mays L.) embryo, cortical parenchyma, and stele proteomes by RUM1 which regulates seminal and lateral root initiation.
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ABSTRACT: The different root types of maize (Zea mays L.) originate from distinct tissues during development. The maize mutant rum1 (rootless with undetectable meristems 1) does not initiate seminal roots and lateral roots in the primary root. While seminal roots are laid down during embryogenesis, endodermis cells of the parenchyma, and pericycle cells of the stele contribute to the postembryonic initiation of lateral roots. In this study, tissue specific protein profiles of immature embryo, cortical parenchyma which includes endodermis, cortex and epidermis cell layers, and stele tissues were compared between wild-type and rum1 via 2-DE. Electrospray ionization tandem mass spectrometry (ESI-MS/MS) identified 86 proteins encoded by 69 genes that were differentially accumulated between wild-type and rum1 (Fc>or=2; FDR<10%) demonstrating that RUM1 affects the proteome composition of cortical parenchyma, stele and embryo tissues. While several protein isoforms, protein families or members of biochemical pathways regulated by RUM1 were differentially accumulated in at least two tissues, other proteins displayed tissue specific expression differences. Multiple members of the globulin gene family displayed, for example, embryo specific expression differences, while different glycolysis related enzymes were differentially expressed in all three analyzed tissues. Proteins related to signal transduction and cell fate were overrepresented in cortical parenchyma versus embryo and embryo versus stele tissues, respectively, and might imply tissue specific functions of these protein classes.Journal of Proteome Research 04/2009; 8(5):2285-97. · 5.11 Impact Factor -
Article: Inducible knockout mutagenesis reveals compensatory mechanisms elicited by constitutive BK channel deficiency in overactive murine bladder.
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ABSTRACT: The large-conductance, voltage-dependent and Ca(2+)-dependent K(+) (BK) channel links membrane depolarization and local increases in cytosolic free Ca(2+) to hyperpolarizing K(+) outward currents, thereby controlling smooth muscle contractility. Constitutive deletion of the BK channel in mice (BK(-/-)) leads to an overactive bladder associated with increased intravesical pressure and frequent micturition, which has been revealed to be a result of detrusor muscle hyperexcitability. Interestingly, time-dependent and smooth muscle-specific deletion of the BK channel (SM-BK(-/-)) caused a more severe phenotype than displayed by constitutive BK(-/-) mice, suggesting that compensatory pathways are active in the latter. In detrusor muscle of BK(-/-) but not SM-BK(-/-) mice, we found reduced L-type Ca(2+) current density and increased expression of cAMP kinase (protein kinase A; PKA), as compared with control mice. Increased expression of PKA in BK(-/-) mice was accompanied by enhanced beta-adrenoceptor/cAMP-mediated suppression of contractions by isoproterenol. This effect was attenuated by about 60-70% in SM-BK(-/-) mice. However, the Rp isomer of adenosine-3',5'-cyclic monophosphorothioate, a blocker of PKA, only partially inhibited enhanced cAMP signaling in BK(-/-) detrusor muscle, suggesting the existence of additional compensatory pathways. To this end, proteome analysis of BK(-/-) urinary bladder tissue was performed, and revealed additional compensatory regulated proteins. Thus, constitutive and inducible deletion of BK channel activity unmasks compensatory mechanisms that are relevant for urinary bladder relaxation.FEBS Journal 03/2009; 276(6):1680-97. · 3.79 Impact Factor -
Article: Cationic composition and acid-base state of the extracellular fluid, and specific buffer value of hemoglobin from the branchiopod crustacean Triops cancriformis.
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ABSTRACT: Recent insights into the allosteric control of oxygen binding in the extracellular hemoglobin (Hb) of the tadpole shrimp Triops cancriformis raised the question about the physico-chemical properties of the protein's native environment. This study determined the cationic composition and acid-base state of the animal's extracellular fluid. The physiological concentrations of potential cationic effectors (calcium, magnesium) were more than one order of magnitude below the level effective to increase Hb oxygen affinity. The extracellular fluid in the pericardial space had a typical bicarbonate concentration of 7.6 mM but a remarkably high CO(2) partial pressure of 1.36 kPa at pH 7.52 and 20 degrees C. The discrepancy between this high CO(2) partial pressure and the comparably low values for water-breathing decapods could not solely be explained by the hemolymph-sampling procedure but may additionally arise from differences in cardiovascular complexity and efficiency. T. cancriformis hemolymph had a non-bicarbonate buffer value of 2.1 meq L(-1) pH(-1). Hb covered 40-60% of the non-bicarbonate buffering power. The specific buffer value of Hb of 1.1 meq (mmol heme)(-1) pH(-1) suggested a minimum requirement of two titratable histidines per heme-binding domain, which is supported by available information from N-terminal sequencing and expressed sequence tags.Journal of Comparative Physiology B 01/2009; 179(3):369-81. · 1.97 Impact Factor -
Article: Alterations in Protein Expression of Arabidopsis thaliana Cell Cultures During Hyper- and Simulated Micro-Gravity
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ABSTRACT: Callus cell cultures of Arabidopsis thaliana exposed to hypergravity (8×g), 2D clinorotation and random positioning exhibit changes in gene expression (Martzivanou et al., Protoplasma 229:155–162, 2006). In a recent investigation we could show that after 2h of exposure also the protein complement shows treatment-related changes which are indicative for reactive oxygen species being involved in the perception of/response to changes in the gravitational field. In the present study we have extended these investigations for a period of up to 16h of exposure. We report on changes in abundance of 28 proteins which have been identified by nano HPLC-ESI-MS/MS, and which were altered in amount after 2h of treatment. According to changes between 2 and 16h we could distinguish four groups of proteins which either declined, increased from down-regulated to control levels, showed a transient decline or a transient increase. With regard to function, our data indicate stress relief or adaptation to a new gravitational steady state under prolonged exposure. The latter assumption is supported by the appearance of a new set of 19 proteins which is changed in abundance after 8h of hypergravity. A comparative analysis of the different treatments showed some similarities in response between 8×g centrifugation and 2D clinorotation, while random positioning showed the least responses.Microgravity - Science and Technology 12/2008; 21(1):191-196. · 0.59 Impact Factor -
Article: Long-term immunity against actual poxviral HLA ligands as identified by differential stable isotope labeling.
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ABSTRACT: Viral peptides are presented by HLA class I on infected cells to activate CD8(+) T cells. Several immunogenic peptides have been identified indirectly by epitope prediction and screening of T cell responses to poxviral vectors, including modified vaccinia virus Ankara (MVA) currently being tested as recombinant or smallpox vaccines. However, for the development of optimal vaccination and immunomonitoring strategies, it is essential to characterize the actual viral HLA ligand repertoire of infected cells. We used an innovative approach to identify naturally processed MVA HLA ligands by differential HPLC-coupled mass spectrometry. We describe 12 viral peptides presented by HLA-A*0201 and 3 by HLA-B*0702. All HLA-A*0201 ligands participated in the memory response of MVA-immune donors, and several were immunogenic in Dryvax vaccinees. Eight epitopes were novel. Viral HLA ligand presentation and viral protein abundance did not correlate. All ligands were expressed early during the viral life cycle, and a pool of three of these mediated stronger protection against a lethal challenge in mice as compared with late epitopes. This highlights the reliability of the comparative mass spectrometry-based technique to identify relevant viral CD8(+) T cell epitopes for optimizing the monitoring of protective immune responses and the development of effective peptide-based vaccines.The Journal of Immunology 12/2008; 181(9):6371-83. · 5.79 Impact Factor -
Article: Analysis of nonadditive protein accumulation in young primary roots of a maize (Zea mays L.) F(1)-hybrid compared to its parental inbred lines.
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ABSTRACT: Heterosis describes the superior performance of heterozygous F(1)-hybrids compared to their homozygous parental inbred lines. Heterosis is already manifested during early maize (Zea mays L.) primary root development. In this study, the most abundant soluble proteins have been investigated before the phenotypic manifestation of heterosis in 3.5-day-old primary roots in the flint inbred line UH002, the dent inbred line UH301 and the corresponding hybrid UH301 x UH002. In CBB-stained 2-DE gels, 150 of 304 detected proteins (49%) were accumulated in a nonadditive fashion in the hybrid compared to the average of their parental inbred lines (Student's t-test: p < 0.05). Remarkably, expression of 51% (76/150) of the nonadditively accumulated proteins exceeded the high parent or was below the low parent. ESI-MS/MS identified 75 of the 76 proteins that belonged to these expression classes. The most abundant functional classes among the 75 proteins that were encoded by 60 different genes were metabolism (58%) and disease and defense (19%). Nonadditive protein accumulation in primary roots of maize hybrids might be associated with heterosis manifestation. Identification of these proteins could therefore contribute to the better understanding of the molecular basis of heterosis.Proteomics 08/2008; 8(18):3882-94. · 4.43 Impact Factor -
Article: Human parvovirus B19 NS1 protein modulates inflammatory signaling by activation of STAT3/PIAS3 in human endothelial cells.
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ABSTRACT: The pathogenic mechanism by which parvovirus B19 may induce inflammatory cardiomyopathy (iCMP) is complex but is known to involve inflammatory processes, possibly including activation of JAK/STAT signaling. The nonstructural B19 protein NS1 acts as a transactivator triggering signaling cascades that eventually lead to activation of interleukin 6 (IL-6). We examined the impact of NS1 on modulation of STAT signaling in human endothelial cells (HMEC-1). The NS1 sequences were identified from B19 DNA isolated from the myocardia of patients with fatal iCMP. B19 infection as well as NS1 overexpression in HMEC-1 cells produced a significant upregulation in the phosphorylation of both tyrosine(705) and serine(727) STAT3 (P < 0.05). The increased STAT3 phosphorylation was accompanied by dimerization, nuclear translocation, and DNA binding of pSTAT3. In contrast, NS1 expression did not result in increased STAT1 activation. Notably, the expression levels of the negative regulators of STAT activation, SOCS1 and SOCS3, were not altered by NS1. However, the level of PIAS3 was upregulated in NS1-expressing HMEC-1 cells. Analysis of the transcriptional activation of target genes revealed that NS1-induced STAT3 signaling was associated with upregulation of genes involved in immune response (e.g., the IFNAR1 and IL-2 genes) and downregulation of genes associated with viral defense (e.g., the OAS1 and TYK2 genes). Our results demonstrate that B19 NS1 modulates the STAT/PIAS pathway. The NS1-induced upregulation of STAT3/PIAS3 in the absence of STAT1 phosphorylation and the lack of SOCS1/SOCS3 activation may contribute to the mechanisms by which B19 evades the immune response and establishes persistent infection in human endothelial cells. Thus, NS1 may play a critical role in the mechanism of viral pathogenesis in B19-associated iCMP.Journal of Virology 06/2008; 82(16):7942-52. · 5.40 Impact Factor -
Article: An exocyst complex functions in plant cell growth in Arabidopsis and tobacco.
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ABSTRACT: The exocyst, an octameric tethering complex and effector of Rho and Rab GTPases, facilitates polarized secretion in yeast and animals. Recent evidence implicates three plant homologs of exocyst subunits (SEC3, SEC8, and EXO70A1) in plant cell morphogenesis. Here, we provide genetic, cell biological, and biochemical evidence that these and other predicted subunits function together in vivo in Arabidopsis thaliana. Double mutants in exocyst subunits (sec5 exo70A1 and sec8 exo70A1) show a synergistic defect in etiolated hypocotyl elongation. Mutants in exocyst subunits SEC5, SEC6, SEC8, and SEC15a show defective pollen germination and pollen tube growth phenotypes. Using antibodies directed against SEC6, SEC8, and EXO70A1, we demonstrate colocalization of these proteins at the apex of growing tobacco pollen tubes. The SEC3, SEC5, SEC6, SEC8, SEC10, SEC15a, and EXO70 subunits copurify in a high molecular mass fraction of 900 kD after chromatographic fractionation of an Arabidopsis cell suspension extract. Blue native electrophoresis confirmed the presence of SEC3, SEC6, SEC8, and EXO70 in high molecular mass complexes. Finally, use of the yeast two-hybrid system revealed interaction of Arabidopsis SEC3a with EXO70A1, SEC10 with SEC15b, and SEC6 with SEC8. We conclude that the exocyst functions as a complex in plant cells, where it plays important roles in morphogenesis.The Plant Cell 06/2008; 20(5):1330-45. · 8.99 Impact Factor -
Article: Identification and functional analysis of cyclooxygenase-1 as a molecular target of boswellic acids.
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ABSTRACT: Boswellic acids (BAs) are assumed as the anti-inflammatory principles of Boswellia species. Initially, it was found that BAs inhibit leukotriene biosynthesis and 5-lipoxygenase (EC number 1.13.11.34), whereas suppression of prostaglandin formation and inhibition of cyclooxygenases (COX, EC number 1.14.99.1) has been excluded. Recently, we demonstrated that BAs also interfere with platelet-type 12-lipoxygenase. Here, we show that BAs, preferably 3-O-acetyl-11-keto-beta-BA (AKBA), concentration-dependently inhibit COX-1 product formation in intact human platelets (IC(50)=6 microM) as well as the activity of isolated COX-1 enzyme in cell-free assays (IC(50)=32 microM). The inhibitory effect of AKBA is reversible, and increased levels of arachidonic acid (AA) as substrate for COX-1 impair the efficacy. COX-1 in platelet lysates or isolated COX-1 selectively bound to an affinity matrix composed of immobilized BAs linked via glutaric acid to sepharose and this binding was reversed by ibuprofen or AA. Automated molecular docking of BAs into X-ray structures of COX-1 yielded positive Chemscore values for BAs, indicating favorable binding to the active site of the enzyme. In contrast, COX-2 was less efficiently inhibited by BAs as compared to COX-1, and pull-down experiments as well as docking studies exclude strong affinities of BAs towards COX-2. In conclusion, BAs, in particular AKBA, directly interfere with COX-1 and may mediate their anti-inflammatory actions not only by suppression of lipoxygenases, but also by inhibiting cyclooxygenases, preferentially COX-1.Biochemical Pharmacology 02/2008; 75(2):503-13. · 4.70 Impact Factor -
Article: Transcriptomic and proteomic analyses of pericycle cells of the maize primary root.
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ABSTRACT: Each plant cell type expresses a unique transcriptome and proteome at different stages of differentiation dependent on its developmental fate. This study compared gene expression and protein accumulation in cell-cycle-competent primary root pericycle cells of maize (Zea mays) prior to their first division and lateral root initiation. These are the only root cells that maintain the competence to divide after they leave the meristematic zone. Pericycle cells of the inbred line B73 were isolated via laser capture microdissection. Microarray experiments identified 32 genes preferentially expressed in pericycle versus all other root cells that have left the apical meristem; selective subtractive hybridization identified seven genes preferentially expressed in pericycle versus central cylinder cells of the same root region. Transcription and protein synthesis represented the most abundant functional categories among these pericycle-specific genes. Moreover, 701 expressed sequence tags (ESTs) were generated from pericycle and central cylinder cells. Among those, transcripts related to protein synthesis and cell fate were significantly enriched in pericycle versus nonpericycle cells. In addition, 77 EST clusters not previously identified in maize ESTs or genomic databases were identified. Finally, among the most abundant soluble pericycle proteins separated via two-dimensional electrophoresis, 20 proteins were identified via electrospray ionization-tandem mass spectrometry, thus defining a reference dataset of the maize pericycle proteome. Among those, two proteins were preferentially expressed in the pericycle. In summary, these pericycle-specific gene expression experiments define the distinct molecular events during the specification of cell-cycle-competent pericycle cells prior to their first division and demonstrate that pericycle specification and lateral root initiation might be controlled by a different set of genes.Plant physiology 12/2007; 145(3):575-88. · 6.53 Impact Factor -
Article: TLR2/TLR4-independent neutrophil activation and recruitment upon endocytosis of nucleosomes reveals a new pathway of innate immunity in systemic lupus erythematosus.
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ABSTRACT: The nucleosome is a major autoantigen in systemic lupus erythematosus (SLE); it can be detected as a circulating complex in the serum, and nucleosomes have been suggested to play a key role in disease development. In the present study, we show for the first time that physiological concentrations of purified nucleosomes trigger innate immunity. The nucleosomes are endocytosed and induce the direct activation of human neutrophils (polymorphonuclear leukocytes (PMN)) as revealed by CD11b/CD66b up-regulation, IL-8 secretion, and increased phagocytic activity. IL-8 is a neutrophil chemoattractant detected in high concentrations in the sera of patients, and IL-8 secretion might thus result in enhanced inflammation, as observed in lupus patients, via an amplification loop. Nucleosomes act as free complexes requiring no immune complex formation and independently of the presence of unmethylated CpG DNA motifs. Both normal and lupus neutrophils are sensitive to nucleosome-induced activation, and activation is not due to endotoxin or high-mobility group box 1 contamination. In mice, i.p. injection of purified nucleosomes induces neutrophil activation and recruitment in a TLR2/TLR4-independent manner. Importantly, neutrophils have been suggested to link innate and adaptive immunity. Thus, nucleosomes trigger a previously unknown pathway of innate immunity, which may partially explain why peripheral tolerance is broken in SLE patients.The Journal of Immunology 01/2007; 177(11):7740-9. · 5.79 Impact Factor -
Article: Time-course of changes in amounts of specific proteins upon exposure to hyper-g, 2-D clinorotation, and 3-D random positioning of Arabidopsis cell cultures.
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ABSTRACT: In previous studies it has been shown that callus cell cultures of Arabidopsis thaliana respond to changes in gravitational field strengths by altered gene expression. In this study an investigation was carried out into how different g conditions affect the proteome of such cells. For this purpose, callus cells were exposed to 8 g (centrifugation) and simulated microgravity (2-D clinorotation: fast rotating clinostat, yielding 0.0016 g at maximum; and 3-D random positioning) for up to 16 h. Extracts containing total soluble protein were subjected to 2-D SDS-PAGE. Image analysis of Sypro Ruby-stained gels showed that approximately 28 spots reproducibly and significantly (P <0.05) changed in amount after 2 h of hypergravity (18 up- and 10 down-regulated). These spots were analysed by electrospray ionization tandem mass spectrometry (ESI-MS/MS). In the case of 2-D clinorotation, 19 proteins changed in a manner similar to hypergravity, while random positioning affected only eight spots. Identified proteins were mainly stress related, and are involved in detoxification of reactive oxygen species, signalling, and calcium binding. Surprisingly, centrifugation and clinorotation showed homologies which were not detected for random positioning. The data indicate that simulation of weightlessness is different between clinorotation and random positioning.Journal of Experimental Botany 01/2007; 58(15-16):4357-63. · 5.36 Impact Factor -
Article: Native and subunit molecular mass and quarternary structure of the hemoglobin from the primitive branchiopod crustacean Triops cancriformis.
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ABSTRACT: Many branchiopod crustaceans are endowed with extracellular, high-molecular-weight hemoglobins whose exact structural characteristics have remained a matter of conjecture. By using a broad spectrum of techniques, we provide precise and coherent information on the hemoglobin of one of the phylogenetically 'oldest' extant branchiopods, the tadpole shrimp Triops cancriformis. The hemoglobin dissociated under reducing conditions into two subunits, designated TcHbA and TcHbB, with masses of 35,775+/-4 and 36,055+/-4 Da, respectively, determined by ESI-MS. Nonreducing conditions showed only two disulfide-bridged dimers, a homodimer of TcHbA, designated D1 (71,548+/-5 Da), and the heterodimer D2 (71,828+/-5 Da). Carbamidomethylation of free SH groups revealed the presence of three cysteines per subunit and indicated one intrasubunit and one intersubunit disulfide bridge. Ultracentrifugation and light-scattering experiments under nondenaturating conditions yielded mass estimates that suggested an uneven number of 17 subunits forming the native hemoglobin. This unrealistic number resulted from the presence of two size classes (16-mer and 18-mer), which were recognized by native PAGE and Ferguson plot analysis. ESI-MS revealed three hemoglobin isoforms with masses of 588.1 kDa, 662.0 kDa, and 665.0 kDa. The 16-mer and the smaller 18-mer species are supposed to be composed of TcHbA only, given the dominance of this subunit type in SDS/PAGE. Transmission electron microscopy of negatively stained specimens showed a population of compact molecules with geometrical extensions of 14, 16 and 9 nm. The proposed stoichiometric model of quarternary structure provides the missing link to achieve a mechanistic understanding of the structure-function relationships among the multimeric arthropodan hemoglobins.FEBS Journal 10/2006; 273(17):4055-71. · 3.79 Impact Factor -
Article: Comparative proteome analyses of maize (Zea mays L.) primary roots prior to lateral root initiation reveal differential protein expression in the lateral root initiation mutant rum1.
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ABSTRACT: The embryonically preformed primary root is the first root type of maize that emerges after germination. In this study the abundant soluble proteins of 2.5-day-old primary roots of wild-type and lateral root mutant rum1 seedlings were compared before the initiation of lateral roots. In CBB-stained 2-D gels, among 350 detected proteins 14 were identified as differentially accumulated (>twofold change; t-test: 95% significance) in wild-type versus rum1 primary roots. These proteins which were identified via ESI MS/MS are encoded by 12 different genes. Functionally, these proteins are involved in lignin biosynthesis, defense, and the citrate cycle. Nine of these genes were further analyzed at the RNA expression level. This study represents the first comparative proteomic analysis of maize primary roots prior to lateral root initiation and will contribute to a better understanding of the molecular basis of root development in cereals.PROTEOMICS 09/2006; 6(15):4300-8. · 4.51 Impact Factor -
Article: Plasmodia express two threonine-peptidase complexes during asexual development.
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ABSTRACT: Threonine-peptidases of the T1-family are multi-subunit complexes with broad substrate specificity. In eukaryotes, at least 14 genes encode subunits of the prototypic T1 threonine-peptidase, the proteasome. The proteasome determines the turnover of most proteins and thereby plays a fundamental role in diverse processes such as protein quality control, signal transduction, and cell cycle regulation. While eukaryotes and archaea possess a proteasome, bacteria generally express a second member of the T1-family, the proteasomal predecessor ClpQ/hslV that has a similar structure but is encoded by only one gene. The plasmodial genome is an exception because it encodes proteasomal subunits as well as a ClpQ/hslV-orthologe (Plasmodium falciparum-hslV; PfhslV). Structure, expression, and function of both types of peptidase-complex in P. falciparum are presently unknown. Our aim was to analyze both the coding sequences and derived proteins of both peptidase-complexes because highly specific and potent inhibitors can be designed against this class of enzymes. The proteasome was found expressed throughout the cell cycle, whereas PfhslV was detectable in schizonts and merozoites only. Treatment of P. falciparum with the threonine-peptidase inhibitor epoxomicin blocked two of three catalytically active proteasome subunits. This led to the accumulation of ubiquitinated proteins and, finally, to parasite death. In conclusion, we provide the first functional analysis of plasmodial threonine-peptidase-complexes and identify a lead compound for the development of a novel class of antimalarial drugs.Molecular and Biochemical Parasitology 08/2006; 148(1):79-85. · 2.55 Impact Factor -
Article: Molecular mass of macromolecules and subunits and the quaternary structure of hemoglobin from the microcrustacean Daphnia magna.
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ABSTRACT: The molecular masses of macromolecules and subunits of the extracellular hemoglobin from the fresh-water crustacean Daphnia magna were determined by analytical ultracentrifugation, multiangle laser light scattering and electrospray ionization mass spectrometry. The hemoglobins from hypoxia-incubated, hemoglobin-rich and normoxia-incubated, hemoglobin-poor Daphnia magna were analyzed separately. The sedimentation coefficient of the macromolecule was 17.4 +/- 0.1 S, and its molecular mass was 583 kDa (hemoglobin-rich animals) determined by AUC and 590.4 +/- 11.1 kDa (hemoglobin-rich animals) and 597.5 +/- 49 kDa (hemoglobin-poor animals), respectively, determined by multiangle laser light scattering. Measurements of the hemoglobin subunit mass of hemoglobin-rich animals by electrospray ionization mass spectrometry revealed a significant peak at 36.482 +/- 0.0015 kDa, i.e. 37.715 kDa including two heme groups. The hemoglobin subunits are modified by O-linked glycosylation in the pre-A segments of domains 1. No evidence for phosphorylation of hemoglobin subunits was found. The subunit migration behavior during SDS/PAGE was shown to be influenced by the buffer system used (Tris versus phosphate). The subunit mass heterogeneity found using Tris buffering can be explained by glycosylation of hemoglobin subunits. Based on molecular mass information, Daphnia magna hemoglobin is demonstrated to consist of 16 subunits. The quaternary structure of the Daphnia magna hemoglobin macromolecule was assessed by three-dimensional reconstructions via single-particle analysis based on negatively stained electron microscopic specimens. It turned out to be much more complex than hitherto proposed: it displays D4 symmetry with a diameter of approximately 12 nm and a height of about 8 nm.FEBS Journal 08/2006; 273(14):3393-410. · 3.79 Impact Factor
Top co-authors
Top Journals
- Biological Chemistry (2)
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Institutions
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2013
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Rheinische Friedrich-Wilhelms-Universität Bonn
- Institute of Crop Science and Resource Conservation (INRES)
Bonn, North Rhine-Westphalia, Germany
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2009
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Eberhard-Karls-Universität Tübingen
- Center for Plant Molecular Biology
Tübingen, Baden-Wuerttemberg, Germany
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2006
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Station Biologique de Roscoff
Roscoff, Brittany, France
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2004–2006
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Westfälische Wilhelms-Universität Münster
- Institut für Zoophysiologie
Münster, North Rhine-Westphalia, Germany
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