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.

Nuclear pore formation depends on membrane curvature. The membrane deforming activity of Nup53 is required for nuclear pore complex (NPC) assembly during interphase.

Many microorganisms excrete typical cytoplasmic proteins into the culture supernatant. As none of the classical secretion systems appears to be involved, this type of secretion was referred to as "nonclassical protein secretion." Here, we demonstrate that in Staphylococcus aureus the major autolysin plays a crucial role in release of cytoplasmic proteins. Comparative secretome analysis revealed that in the wild type S. aureus strain, 22 typical cytoplasmic proteins were excreted into the culture supernatant, although in the atl mutant they were significantly decreased. The presence or absence of prophages had little influence on the secretome pattern. In the atl mutant, secondary peptidoglycan hydrolases were increased in the secretome; the corresponding genes were transcriptionally up-regulated suggesting a compensatory mechanism for the atl mutation. Using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a cytoplasmic indicator enzyme, we showed that all clinical isolates tested excreted this protein. In the wall teichoic acid-deficient tagO mutant with its increased autolysis activity, GAPDH was excreted in even higher amounts than in the WT, confirming the importance of autolysis in excretion of cytoplasmic proteins. To answer the question of how discriminatory the excretion of cytoplasmic proteins is, we performed a two-dimensional PAGE of cytoplasmic proteins isolated from WT. Surprisingly, the most abundant proteins in the cytoplasm were not found in the secretome of the WT, suggesting that there exists a selection mechanism in the excretion of cytoplasmic proteins. As the major autolysin binds at the septum site, we assume that the proteins are preferentially released at and during septum formation.

Emergence of carbapenem-non-susceptible extended-spectrum b-lactamase-producing Klebsiella pneumoniae isolates at the university hospital of Tü bingen, Germany The spread of Gram-negative bacteria with plasmid-borne extended-spectrum b-lactamases (ESBLs) has become a worldwide problem. This study analysed a total of 366 ESBL-producing Enterobacteriaceae strains isolated from non-selected patient specimens at the university hospital of Tü bingen in the period January 2003 to December 2007. Although the overall ESBL rate was comparatively low (1.6 %), the percentages of ESBL-producing Enterobacter spp. and Escherichia coli increased from 0.8 and 0.5 %, respectively, in 2003 to 4.6 and 3.8 % in 2007. In particular, the emergence was observed of one carbapenem-resistant ESBL-producing E. coli isolate and five carbapenem-non-susceptible ESBL-positive Klebsiella pneumoniae isolates, in two of which carbapenem resistance development was documented in vivo under a meropenemcontaining antibiotic regime. The possible underlying mechanism for this carbapenem resistance in three of the K. pneumoniae isolates was loss of the Klebsiella porin channel protein OmpK36 as shown by PCR analysis. The remaining two K. pneumoniae isolates exhibited increased expression of a tripartite AcrAB-TolC efflux pump as demonstrated by SDS-PAGE and mass spectrometry analysis of bacterial outer-membrane extracts, which, in addition to other unknown mechanisms, may contribute towards increasing the carbapenem MIC values further. Carbapenem-non-susceptible ESBL isolates may pose a new problem in the future due to possible outbreak situations and limited antibiotic treatment options. Therefore, a systematic exploration of intestinal colonization with ESBL isolates should be reconsidered, at least for haemato-oncological departments from where four of the five carbapenem-non-susceptible ESBL isolates originated. INTRODUCTIONIn contrast to the USA and the Western Pacific area, the prevalence of extended-spectrum b-lactamases (ESBLs) in Germany was comparably low in 2001 (8.2 % of Klebsiella pneumoniae and 0.8 % of Escherichia coli isolates; Sturenburg & Mack, 2003). However, the prevalence of ESBL-producing Gram-negative bacteria is increasing, both in hospitals and in the community (Sturenburg & Mack, 2003;Pitout et al., 2005).ESBLs confer resistance to amino-and ureidopenicillins, oximino-cephalosporins and monobactams. As co-resistance to non-b-lactam antimicrobials is common among ESBL isolates, the treatment options are limited. Among the E. coli and K. pneumoniae isolates predominantly found to produce ESBLs in Europe, carbapenem-non-susceptible isolates are becoming more widespread (

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 twodimensional 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.

Overall, our findings uncover a novel function of the anaphylatoxin C3a for platelet function and thrombus formation, highlighting a detrimental role of imbalanced complement activation in cardiovascular diseases.

Carrot (Daucus carota L.) cells respond to treatment with fungal elicitors by synthesizing wallbound p-hydroxybenzoic acid (p-HBA). The biosynthetic pathway to p-HBA is still hypothetical. Tracer experiments with L-phenylalanine indicate the involvement of the general phenylpropanoid pathway. 3,4 (Methylenedioxy) innamic acid, an inhibitor of hydrocycinnamate CoA ligase, inhibits the accumulation of anthocyanins in carrot, while it does not interfere with p-HBA synthesis. Thus p-HBA biosynthesis does not appear to involve CoA thioesters. In the present report the sequence of enzymic reactions leading to p-HBA was investigated in vitro using protein preparations from cells treated with a fungal elicitor from Pythium aphanidermatum (Edson) Fitzp. The side-chain degradation from p-coumaric acid to p-HBA is not analogous to the β-oxidation of fatty acids and involves p-hydroxybenzaldehyde as an intermediate. The final step from p-hydroxybenzaldehyde to p-HBA is catalyzed by an NAD-dependent p-hydroxybenzaldehyde dehydrogenase (EC 1.2.1.-). This reaction was characterized with regard to cofactor requirements, pH and temperature optima. The in-vitro formation of p-HBA from p-coumaric acid and the activity of the hydroxybenzaldehyde dehydrogenase are moderately elicitor-induced but to a much lesser extent than phenylalanine ammonialyase, which is the starting enzyme of the general phenylpropanoid pathway.

Mitogenic factors are known to stimulate the Na + /H + -exchanger (NHE), leading to cytosolic alkalinization and/or cell swelling. Conversely, a hallmark of apoptosis is cell shrinkage and CD95-induced apoptosis has been reported to be paralleled by cytosolic acidification. To assess whether the CD95-receptor regulates NHE activity in Jurkat T-lymphocytes, we performed conventional BCECF fluorescence measurements and SNARF flow cytometric analysis (FACS). The recoveries from acidifications following application of butyrate or a NH 3 pulse were both abolished by a specific NHE-inhibitor, HOE694, indicating that they fully depend on NHE activity. Thus they were taken as a measure of NHE activity. CD95-receptor stimulation caused a cytosolic acidification and blunted the recovery from acidification following application of butyrate or a NH 3 pulse. Moreover, the NHE-dependent alkalinization following osmotic cell shrinkage was almost abolished by CD95-receptor stimulation. As apparent from the effect of osmotic cell shrinkage, inhibition of the NHE by CD95-receptor stimulation was absent in Lck 56 -deficient J-CaM1.6 cells and restored by retransfection of J-CaM1.6-cells with Lck 56 . CD95-receptor stimulation led within 4 h to a decrease of cellular ATP which could contribute to NHE inhibition. Treatment of Jurkat cells with the NHE inhibitor HOE694 accelerated CD95-induced DNA fragmentation. In conclusion, CD95-receptor stimulation inhibits NHE activity through a mechanism that depends directly or indirectly on the activation of the Src-like kinase Lck 56 . This effect contributes to CD95-induced cytosolic acidification, DNA fragmentation and cell shrinkage.