Pektin Methylesterasen (PMEs) und PME- Inhibitor (PMEI)-verwandte Proteine im Maispollen : Genexpression, subzelluläre Lokalisation und funktionelle Charakterisierung

Pektin Methylesterasen (PMEs) und Pektin Methylesterase Inhibitor verwandte Proteine (PMEI-RPs) im Maispollen: Genexpression, subzelluläre Lokalisation und funktionelle Charakterisierung Während der Pollenentwicklung und dem Pollenschlauchwachstum spielen dynamische Veränderungen der Pektinzusammensetzung in der Zellwand eine entscheidende Rolle. Besonders die durch Pektin Methylesterasen vermittelte Demethylierung der Pektinmatrix hat Auswirkungen auf die physikalischen Eigenschaften der Zellwand. Durch MALDI- bzw. ESI-TOF Untersuchungen von Maispollenproben und Datenbankrecherchen wurden unterschiedliche Pektin Methylesterasen identifiziert. Nach Erhalt der vollständigen cDNSs wurden diese in bakterielle und eukaryotische Expressionsvektoren kloniert. Von den vier identifizierten PMEs besitzen zwei Isoformen eine für Typ I charakteristische Prodomäne. Die anderen zwei Isoformen besitzen keine Prodomäne und gehören zu den Typ II PMEs. Mit Hilfe der rekombinanten Proteine sollen, mittels in vitro Untersuchungen, die biologische(n) Funktion(en) aufgeklärt werden. Mittels semiquantitativer RT-PCR wurden die Expressionsstärken und die Pflanzenorgane, in denen die entsprechenden Gene transkribiert werden, ermittelt. PMEI-RPs konnten nur in Antheren und sich entwickelnden Pollen nachgewiesen werden. Die PMEs konnten sowohl in Maisblättern, Pollen, als auch in weiteren Pflanzenorganen nachgewiesen werden. Die subzelluläre Lokalisation der PMEI-RPs wurde zusätzlich durch Fraktionierungen, durch immunocytochemischen Untersuchungen und durch transiente Expression von PMEI-RP::Reportergen-Chimären untersucht. Erstaunlicherweise führten die rekombinanten und nativen PMEI-RPs weder zu einer Inhibition der PME-Aktivität noch zu einer Inhibition mit Invertasen (Zellwand und Vakuolen-Isoformen). Zusätzlich wurden ß-1,3-Glucanasen, Polygalacturonasen und Inulasen durch in vitro Tests als mögliche Zielproteine ausgeschlossen. In Größenausschluss-Chromatographien unterschiedlicher Maispollen-Proben und rekombinanter PMEI-RPs konnten nur monomere PMEI-RPs bei ihrem erwarteten Mr ohne Hinweis auf einen Bindepartner nachgewiesen werden. Versuche mit Chemikalien, die Proteinkomplexe quervernetzen, führten ebenfalls zu keinen nachweisbaren PMEI-RP Zielproteinkomplexen. Es liegt nahe, dass einige Isoformen der kürzlich identifizierten komplexen PMEI-RP Familien entweder andere Zielproteine als Pektin Methylesterasen und Invertasen haben, oder alternativ, weitere noch unbekannte Funktionen aufweisen. Die Expression der PMEI-RP2 Isoform führte zu einer Reduktion des Pollenschlauchwachstums, was auf eine Interaktion von PMEI-RPs mit PMEs deuten könnte

Functional Dissection of the Apicomplexan Glideosome Molecular Architecture

SummaryThe glideosome of apicomplexan parasites is an actin- and myosin-based machine located at the pellicle, between the plasma membrane (PM) and inner membrane complex (IMC), that powers parasite motility, migration, and host cell invasion and egress. It is composed of myosin A, its light chain MLC1, and two gliding-associated proteins, GAP50 and GAP45. We identify GAP40, a polytopic protein of the IMC, as an additional glideosome component and show that GAP45 is anchored to the PM and IMC via its N- and C-terminal extremities, respectively. While the C-terminal region of GAP45 recruits MLC1-MyoA to the IMC, the N-terminal acylation and coiled-coil domain preserve pellicle integrity during invasion. GAP45 is essential for gliding, invasion, and egress. The orthologous Plasmodium falciparum GAP45 can fulfill this dual function, as shown by transgenera complementation, whereas the coccidian GAP45 homolog (designated here as) GAP70 specifically recruits the glideosome to the apical cap of the parasite

Functional and biophysical analyses of the class XIV Toxoplasma gondii Myosin D

Summary: The obligate intracellular parasite Toxoplasma gondii uses gliding motility to migrate across the biological barriers of the host and to invade cells. This unique form of locomotion requires an intact actin cytoskeleton and involves at least one motor protein (TgMyoA) that belongs to the class XIV of the myosin superfamily. TgMyoA is anchored in the inner membrane complex and is essential for the gliding motion, host cell invasion and egress of T. gondii tachyzoites. TgMyoD is the smallest T. gondii myosin and is structurally very closely related to TgMyoA. We show here that TgMyoD exhibits similar transient kinetic properties as the fast single-headed TgMyoA. To determine if TgMyoD also contributes to parasite gliding motility, the TgMyoD gene was disrupted by double homologous recombination. In contrast to TgMyoA, TgMyoD gene is dispensable for tachyzoite propagation and motility. Parasites lacking TgMyoD glide normally and their virulence is not compromised in mice. The fact that TgMyoD is predominantly expressed in bradyzoites explains the absence of a phenotype observed with myodko in tachyzoites and does not exclude a role of this motor in gliding that would be restricted to the cyst forming but nevertheless motile stage of the parasit

Toxoplasma gondii myosins B/C: one gene, two tails, two localizations, and a role in parasite division

In apicomplexan parasites, actin-disrupting drugs and the inhibitor of myosin heavy chain ATPase, 2,3-butanedione monoxime, have been shown to interfere with host cell invasion by inhibiting parasite gliding motility. We report here that the actomyosin system of Toxoplasma gondii also contributes to the process of cell division by ensuring accurate budding of daughter cells. T. gondii myosins B and C are encoded by alternatively spliced mRNAs and differ only in their COOH-terminal tails. MyoB and MyoC showed distinct subcellular localizations and dissimilar solubilities, which were conferred by their tails. MyoC is the first marker selectively concentrated at the anterior and posterior polar rings of the inner membrane complex, structures that play a key role in cell shape integrity during daughter cell biogenesis. When transiently expressed, MyoB, MyoC, as well as the common motor domain lacking the tail did not distribute evenly between daughter cells, suggesting some impairment in proper segregation. Stable overexpression of MyoB caused a significant defect in parasite cell division, leading to the formation of extensive residual bodies, a substantial delay in replication, and loss of acute virulence in mice. Altogether, these observations suggest that MyoB/C products play a role in proper daughter cell budding and separation

Correlation effects and resonances in molecular photoionization

Vita.The photoionization cross sections of CS$sb2$ leading to the $Xsp2Pisb{g}$, $Asp2Pisb{u}$, and $Bsp2Sigmasb{u}sp+$, and $Csp2Sigmasb{g}sp+$ states of CS$sb2sp+$ have been computed in the multichannel frozen core Hartree-Fock approximation. The results were obtained using the iterative Schwinger variational method using a partial wave expansion up to l = 120. We have found shape resonances in the $kpisb{g}$ and $kpisb{u}$ continuum channels. In particular, the $kpisb{g}$ resonance is seen to affect dramatically both total cross sections and photoelectron asymmetry parameters in all other channels when interchannel coupling is included. The form of the resonant wave function indicates that this resonance is caused by low lying virtual d orbitals on the sulfur atoms. We have found the position of the corresponding poles of the single channel T matrix in the complex plane. We have determined that the position of the pole is relatively insensitive to the interatomic bond lengths. This is indicative of the atomic nature of the resonant state. Vibrationally resolved calculation for the various vibrational transitions show that both the $ksppi$ shape resonances located well above threshold and the $ksigma$ shape resonances below threshold are insensitive to bond length. We have also found near constant vibrational branching ratios in all channels and polarization components. These results indicate that caution must be applied when using bond length sensitivity as an exclusive means to determine the nature of the shape resonance. We have implemented a multichannel complete-active-space configuration-interaction (MCCASCI) approximation to study electron-correlation effects in molecular photoionization. This approach includes both target relaxation and correlation due to coupling between different asymptotic scattering channels. The method employs a numerical CASCI using a single center expansion to evaluate all integrals. We present results of a detailed MCCASCI-Schwinger study of the photoionization of molecular nitrogen in the photon energy region of 19-26 eV, including up to nine coupled electronic channels. The results are in good agreement with the available experimental data. In particular, we obtain for the first time the experimental structure in the (3$sigmasb{g})sp{-1}$ photoelectron asymmetry parameter near 22eV by including the $Csp2Sigmasb{u}sp+$ ion state of N$sb2sp+$

Correlation effects and resonances in molecular photoionization

Vita.The photoionization cross sections of CS$sb2$ leading to the $Xsp2Pisb{g}$, $Asp2Pisb{u}$, and $Bsp2Sigmasb{u}sp+$, and $Csp2Sigmasb{g}sp+$ states of CS$sb2sp+$ have been computed in the multichannel frozen core Hartree-Fock approximation. The results were obtained using the iterative Schwinger variational method using a partial wave expansion up to l = 120. We have found shape resonances in the $kpisb{g}$ and $kpisb{u}$ continuum channels. In particular, the $kpisb{g}$ resonance is seen to affect dramatically both total cross sections and photoelectron asymmetry parameters in all other channels when interchannel coupling is included. The form of the resonant wave function indicates that this resonance is caused by low lying virtual d orbitals on the sulfur atoms. We have found the position of the corresponding poles of the single channel T matrix in the complex plane. We have determined that the position of the pole is relatively insensitive to the interatomic bond lengths. This is indicative of the atomic nature of the resonant state. Vibrationally resolved calculation for the various vibrational transitions show that both the $ksppi$ shape resonances located well above threshold and the $ksigma$ shape resonances below threshold are insensitive to bond length. We have also found near constant vibrational branching ratios in all channels and polarization components. These results indicate that caution must be applied when using bond length sensitivity as an exclusive means to determine the nature of the shape resonance. We have implemented a multichannel complete-active-space configuration-interaction (MCCASCI) approximation to study electron-correlation effects in molecular photoionization. This approach includes both target relaxation and correlation due to coupling between different asymptotic scattering channels. The method employs a numerical CASCI using a single center expansion to evaluate all integrals. We present results of a detailed MCCASCI-Schwinger study of the photoionization of molecular nitrogen in the photon energy region of 19-26 eV, including up to nine coupled electronic channels. The results are in good agreement with the available experimental data. In particular, we obtain for the first time the experimental structure in the (3$sigmasb{g})sp{-1}$ photoelectron asymmetry parameter near 22eV by including the $Csp2Sigmasb{u}sp+$ ion state of N$sb2sp+$

Revealing the interface nature of ZDDP tribofilm by X-ray photoelectron spectroscopy and atom probe tomography

Purpose To decrease wear and friction, zinc dialkyldithiophosphate (ZDDP) has been used in engine oil for several decades, but the mechanism of the tribofilm formation is still unclear. The purpose of this study is to characterize the chemical details of the tribofilm by using high-resolution approaching. Design/methodology/approach An ISO VG 100 mineral oil mixed with ZDDP was used in sliding tests on cylindrical roller bearings. Tribofilm formation was observed after 2 h of the sliding test. X-ray photoelectron spectroscopy (XPS) and atom probe tomography (APT) were used for chemical analysis of the tribofilm. Findings The results show that the ZDDP tribofilm consists of the common ZDDP elements along with iron oxides. A considerable amount of zinc and a small amount of sulfur were observed. In particular, an oxide interlayer with sulfur enrichment was revealed by APT between the tribofilm and the steel substrate. The depth profile of the chemical composition was obtained, and a tribofilm of approximately 40 nm thickness was identified by XPS. Originality/value A sulfur enrichment at the interface is observed by APT, which is beneath an oxygen enrichment. The clear evidence of the S interlayer confirms the hard and soft acids and bases principle