The N-terminal pro region mediates retention of unprocessed type-I PME in the Golgi apparatus

The pectin matrix of the cell wall, a complex and dynamic network, impacts on cell growth, cell shape and signaling processes. A hallmark of pectin structure is the methylesterification status of its major component, homogalacturonan (HGA), which affects the biophysical properties and enzymatic turnover of pectin. The pectin methylesterases (PMEs), responsible for de-esterification, encompass a protein family of more than 60 isoforms in the Arabidopsis genome. The pivotal role of PME in the regulation of pectin properties also requires tight control at the post-translational level. Type-I PMEs are characterized by an N-terminal pro region, which exhibits homology with pectin methylesterase inhibitors (PMEIs). Here, we demonstrate that the proteolytic removal of the N-terminal pro region depends on conserved basic tetrad motifs, occurs in the early secretory pathway, and is required for the subsequent export of the PME core domain to the cell wall. In addition, we demonstrate the involvement of AtS1P, a subtilisin-like protease, in Arabidopsis PME processing. Our results indicate that the pro region operates as an effective retention mechanism, keeping unprocessed PME in the Golgi apparatus. Consequently, pro-protein processing could constitute a post-translational mechanism regulating PME activity.     

Genetic elements associated with antimicrobial resistance in enteropathogenic <Emphasis Type="Italic">Escherichia coli</Emphasis> (EPEC) from Brazil

Background  

We recently observed an association of resistance with a certain enteropathogenic Escherichia coli (EPEC) serotypes and identified a conjugative plasmid, similar to plasmid pED208, that was conserved among archival O111:H2/NM and O119:H2 strains of diverse geographical origin. In this study, we sought to determine the prevalence and distribution of this plasmid among a collection of EPEC isolates from Brazil, as well as to study the susceptibilities of these isolates to antimicrobial agents.     

Influence of metabolic syndrome on biomarkers of oxidative stress and inflammation in obese adults

Objective: Both obesity and the metabolic syndrome (MetS) have been independently linked with increased oxidative and inflammatory stress. This study tested the hypothesis that obesity with MetS is associated with greater oxidative and inflammatory burden compared with obesity alone. Research Methods and Procedures: Forty‐eight normal‐weight and 40 obese (20 without MetS; 20 with MetS) adults were studied. MetS was defined according to National Cholesterol Education Program Adult Treatment Panel III criteria. Plasma concentrations of oxidized low‐density lipoprotein, C‐reactive protein, tumor necrosis factor‐α, interleukin (IL)‐6, and IL‐18 were determined by enzyme immunoassay. Results: Plasma biomarkers of oxidative stress and inflammation were lowest in normal‐weight controls. Of note, obese MetS adults demonstrated significantly higher plasma concentrations of oxidized low‐density lipoprotein (62.3 ± 3.2 vs. 54.0 ± 4.0 U/L; p < 0.05), C‐reactive protein (3.0 ± 0.6 vs. 1.5 ± 0.3 mg/L; p < 0.01), tumor necrosis factor‐α (2.1 ± 0.1 vs. 1.6 ± 0.1 pg/mL; p < 0.05), IL‐6 (2.8 ± 0.4 vs. 1.4 ± 0.2 pg/mL; p < 0.01), and IL‐18 (253 ± 16 vs. 199 ± 16 pg/mL; p < 0.01), compared with obese adults without MetS. Discussion: These results suggest that MetS heightens oxidative stress and inflammatory burden in obese adults. Increased oxidative and inflammatory stress may contribute to the greater risk of coronary heart disease and cerebrovascular disease in obese adults with MetS.     

myo-Inositol phosphate isomers generated by the action of a phytate-degrading enzyme from Klebsiella terrigena on phytate

For the first time a dual pathway for dephosphorylation of myo-inositol hexakisphosphate by a histidine acid phytase was established. The phytate-degrading enzyme of Klebsiella terrigena degrades myo-inositol hexakisphosphate by stepwise dephosphorylation, preferably via D-Ins(1,2,4,5,6)P5, D-Ins(1,2,5,6)P4, D-Ins(1,2,6)P3, D-Ins(1,2)P2 and alternatively via D-Ins(1,2,4,5,6)P5, Ins(2,4,5,6)P4, D-Ins(2,4,5)P3, D-Ins(2,4)P2 to finally Ins(2)P. It was estimated that more than 98% of phytate hydrolysis occurs via D-Ins(1,2,4,5,6)P5. Therefore, the phytate-degrading enzyme from K. terrigena has to be considered a 3-phytase (EC 3.1.3.8). A second dual pathway of minor importance could be proposed that is in accordance with the results obtained by analysis of the dephosphorylation products formed by the action of the phytate-degrading enzyme of K. terrigena on myo-inositol hexakisphosphate. It proceeds preferably via D-Ins(1,2,3,5,6)P5, D-Ins(1,2,3,6)P4, Ins(1,2,3)P3, D-Ins(2,3)P2 and alternatively via D-Ins(1,2,3,5,6)P5, D-Ins(2,3,5,6)P4, D-Ins(2,3,5)P3, D-Ins(2,3)P2 to finally Ins(2)P. D-Ins(2,3,5,6)P4, D-Ins(2,3,5)P3, and D-Ins(2,4)P2 were reported for the first time as intermediates of enzymatic phytate dephosphorylation. A role of the phytate-degrading enzyme from K. terrigena in phytate breakdown could not be ruled out. Because of its cytoplasmatic localization and the suggestions for substrate recognition, D-Ins(1,3,4,5,6)P5 might be the natural substrate of this enzyme and, therefore, may play a role in microbial pathogenesis or cellular myo-inositol phosphate metabolism.     

Coating of poly(p-xylylene) by PLA-PEO-PLA triblock copolymers with excellent polymer-polymer adhesion for stent applications

Poly(p-xylylene) (PPX) was deposited by chemical vapor deposition (CVD) on stainless steel substrates. These PPX films were coated by solution casting of poly(lactide)-poly(ethylene oxide)-poly(lactide) triblock copolymers (PLA-PEO-PLA) loaded with 14C-labeled paclitaxel. Adhesion of PLA-PEO-PLA on PPX substrate coatings was measured using the blister test method. Excellent adhesion of the block copolymers on PPX substrates was found. Stress behavior and film integrity of PLA-PEO-PLA was compared to pure PLA on unexpanded and expanded stent bodies and was found to be superior for the block copolymers. The release of paclitaxel from the biodegradable coatings was studied under physiological conditions using the scintillation counter method. Burst release of paclitaxel was observed from PLA-PEO-PLA layers regardless of composition, but an increase in paclitaxel loading was observed with increasing content of PEO.     

Discovery of novel steroidal histamine H3 receptor antagonists/inverse agonists

Emerging from an HTS campaign, novel steroid-based histamine H₃ receptor antagonists were identified and characterized. Structural moieties of the hit compounds were combined to improve binding affinities which resulted in compound 4 as lead molecule. During the lead optimization due to the versatile modifications of diamino steroid derivatives, several in vitro potent compounds with subnanomolar binding affinities to histamine H₃ receptors were found. The unfavorable binding to rat muscarinic receptors was successfully reduced by tuning the basicity. Compound 20 showed significant in vivo activity in the rat dipsogenia model and could serve as a pharmacological tool in the future.     

Identifying the Real Minority Carrier Lifetime in Nonideal Semiconductors: A Case Study of Kesterite Materials

Time‐resolved photoluminescence (TRPL) is a powerful characterization technique to study carrier dynamics and quantify absorber quality in semiconductors. The minority carrier lifetime, which is critically important for high‐performance solar cells, is often derived from TRPL analysis. However, here it is shown that various nonideal absorber properties can dominate the TRPL signal making reliable extraction of the minority carrier lifetime not possible. Through high‐resolution intensity‐, temperature‐, voltage‐dependent, and spectrally resolved TRPL measurements on absorbers and devices it is shown that photoluminescence (PL) decay times for kesterite materials are dominated by minority carrier detrapping. Therefore, PL decay times do not correspond to the minority carrier lifetime for these materials. The lifetimes measured here are on the order of hundreds of picoseconds in contrast to the nanosecond lifetimes suggested by the decay curves. These results are supported with additional measurements, device simulation, and comparison with recombination limited PL decays measured on Cu(In,Ga)Se₂. The kesterite material system is used as a case study to demonstrate the general analysis of TRPL data in the limit of various measurement conditions and nonideal absorber properties. The data indicate that the current bottleneck for kesterite solar cells is the minority carrier lifetime.     

Chloroplast DNA in Mature and Senescing Leaves: A Reappraisal

The fate of plastid DNA (ptDNA) during leaf development has become a matter of contention. Reports on little change in ptDNA copy number per cell contrast with claims of complete or nearly complete DNA loss already in mature leaves. We employed high-resolution fluorescence microscopy, transmission electron microscopy, semithin sectioning of leaf tissue, and real-time quantitative PCR to study structural and quantitative aspects of ptDNA during leaf development in four higher plant species (Arabidopsis thaliana, sugar beet [Beta vulgaris], tobacco [Nicotiana tabacum], and maize [Zea mays]) for which controversial findings have been reported. Our data demonstrate the retention of substantial amounts of ptDNA in mesophyll cells until leaf necrosis. In ageing and senescent leaves of Arabidopsis, tobacco, and maize, ptDNA amounts remain largely unchanged and nucleoids visible, in spite of marked structural changes during chloroplast-to-gerontoplast transition. This excludes the possibility that ptDNA degradation triggers senescence. In senescent sugar beet leaves, reduction of ptDNA per cell to ∼30% was observed reflecting primarily a decrease in plastid number per cell rather than a decline in DNA per organelle, as reported previously. Our findings are at variance with reports claiming loss of ptDNA at or after leaf maturation.In vascular plants, copy numbers of plastid genomes (plastomes) frequently range from <100 per cell in meristematic cells to several thousand per cell in fully developed diploid leaf parenchyma cells. Microscopy studies have shown that the multicopy organelle genomes are usually condensed in more or less distinct DNA regions (nucleoids) within the organelle matrix or stroma.During development, the ratio of nuclear to organelle genomes appears to be relatively stringently regulated (Herrmann and Possingham, 1980; Rauwolf et al., 2010). Disregarding greatly varying absolute values (summarized in Rauwolf et al., 2010; Liere and Börner, 2013), there is little dispute that the number of plastid genomes and nucleoids per organelle and cell increase during early leaf development in higher plants (Kowallik and Herrmann, 1972; Selldén and Leech, 1981; Baumgartner et al., 1989; Fujie et al., 1994; Li et al., 2006; Rauwolf et al., 2010). This increase is usually accompanied by an increase in both size and number of plastids per cell (Butterfass, 1979). By contrast, data about plastid DNA (ptDNA) amounts in chloroplasts and cells of mature, ageing, and senescent tissue differ and are highly controversial. Basically two patterns have been described: the maintenance of more or less constant amounts of ptDNA per cell and/or organelle (Li et al., 2006; Zoschke et al., 2007; Rauwolf et al., 2010; Udy et al., 2012) or a significant decrease in copy number brought about by either continued organelle and cell division without ptDNA replication (Lamppa and Bendich, 1979; Scott and Possingham, 1980; Tymms et al., 1983) or by ptDNA degradation (Baumgartner et al., 1989; Sodmergen et al., 1991). In a series of communications, Bendich and coworkers recently reported that ptDNA levels decline drastically before leaf maturation in several plant species. In Arabidopsis thaliana and maize (Zea mays), ptDNA levels were reported to decrease early and precipitously as leaves mature. It was concluded that, in fully expanded leaves, most chloroplasts contain no or only insignificant amounts of DNA long before the onset of leaf senescence (Oldenburg and Bendich, 2004; Rowan et al., 2004; Oldenburg et al., 2006; Shaver et al., 2006; Rowan et al., 2009). Retention of ptDNA was proposed to be dispensable after the photosynthetic machinery was established in that the plastome-encoded photosynthesis genes were no longer needed in adult leaves. Degradation or even entire loss of ptDNA was considered as an event during plastid and leaf development, common to all plants (Rowan et al., 2009). ptDNA degradation was also suggested to act as a signal inducing senescence (Sodmergen et al., 1991).A priori, there is no reason why different ptDNA patterns should not occur, and there is indeed evidence that organelle DNA can behave differently in different materials, both quantitatively and structurally (e.g., Selldén and Leech, 1981; Baumgartner et al., 1989). However, since contradictory data were reported for the same species that were grown under comparable, if not identical, conditions (Rowan et al., 2004, 2009; Li et al., 2006; Oldenburg et al., 2006; Shaver et al., 2006; Zoschke et al., 2007; Evans et al., 2010; Udy et al., 2012), it is apparent that some of them must reflect methodological insufficiencies of the experimental approaches employed.From a physiological point of view, the existence of DNA-deficient plastids in photosynthetically competent tissue seems unlikely. For instance, due to its susceptibility to photooxidative damage, the D1 protein (PsbA), a plastome-encoded core subunit of photosystem II, must be replaced continuously by a complex repair system to maintain photosynthesis (Prasil et al., 1992). This replacement requires de novo synthesis of the short-lived D1. There are no data available supporting an extreme mRNA stability, protein stability, or for another compensating biochemistry, preserving organelle functions for weeks or even months. The maximum mRNA half-life reported for psbA is in the range of 40 h (Kim et al., 1993).Resolving this controversy is of considerable scientific interest, both from a theoretical and an applied perspective. We therefore analyzed the fate of ptDNA in mature, ageing, and senescent leaves of four commonly studied higher plant species (Arabidopsis, sugar beet [Beta vulgaris], tobacco [Nicotiana tabacum], and maize; Figure 1) for which conflicting data have been reported. Four complementary methods were used for assessing the presence of ptDNA as well as its quantitative and morphological changes during leaf development: an improved 4′,6-diamidino-2-phenylindole (DAPI)–based fluorescence microscopy approach including deconvolution of fluorescence images, electron microscopy, semithin sectioning across leaf laminas, and real-time quantitative PCR (see Methods).Open in a separate windowFigure 1.Developmental Leaf Series of Sugar Beet, Tobacco, and Arabidopsis.(A) Sugar beet leaves, developmental stages II to VI (left to right; see text). Inset: leaf stages y1 and y3. Arrows indicate necrotic areas. Bar = 5 cm.(B) Tobacco leaves, developmental stages II and IV to VI. Inset: leaf stages y1 and y2. Bar = 5 cm; bar in inset = 1 cm.(C) Arabidopsis plants (left) from which leaves of developmental stages I to VI were taken. Bar = 4 cm.Figure 2, Supplemental Methods, and Supplemental Data Sets 1 to 4 present representative micrographs of developmental series of DAPI-stained chloroplasts in leaf spongy parenchyma cells of late ontogenetic stages from sugar beet, Arabidopsis, tobacco, and maize displaying clearly discernible nucleoid patterns. Figures 1A to 1C document some of the leaves from which samples were taken. Mesophyll cells of juvenile leaves investigated in our previous work (Li et al., 2006; Zoschke et al., 2007; Rauwolf et al., 2010) were included for comparison (Supplemental Data Sets 1 to 4, panels 1 to 37, 84 to 94, 112 to 117, and 123 to 128). The staining specificity of the fluorochrome was confirmed enzymatically. Treatment with DNase, but not DNase-free RNase or Proteinase K, either before or after staining with the fluorochrome, abolished the fluorescence but did not significantly affect chloroplast structure (compare with Rauwolf et al., 2010; see Methods).Open in a separate windowFigure 2.DAPI-DNA Fluorescence of Mature, Senescent, and Prenecrotic Leaf Mesophyll Cells or Cell Segments.Representative DAPI-stained squashed mesophyll cells of sugar beet ([A] to [C]), Arabidopsis ([D] to [F]), tobacco ([G] and [H]), and maize ([I] and [J]) leaflets or leaves (cell detail in [C], [E], [F], and [H]) of the developmental stages III/IV (I), IV ([A] and [D]), V ([B], [E], and [G]), and VI ([C], [F], [H], and [J]). Note that (E) represents a cell fragment of Supplemental Data Set 2, panel 102. Bar = 5 μm in (A), also for (B) to (J).     

Cyclo(l-Pro-d-Arg): a new antibacterial and antitumour diketopiperazine from Bacillus cereus associated with a rhabditid entomopathogenic

In continuation of our search for new antimicrobial secondary metabolites from Bacillus cereus associated with rhabditid entomopathogenic nematode, a new microbial diketopiperazine, cyclo(l-Pro-d-Arg), was isolated from the ethyl acetate extract of fermented modified nutrient broth. The chemical structures of the isolated compounds were identified based on their 1D, 2D NMR and high-resolution electrospray ionisation–mass spectroscopy data. Antibacterial activity of the compound was determined by minimum inhibitory concentration and disc diffusion method against medically important bacteria, and the compound was recorded to have significant antibacterial activity against test bacteria. The highest activity was recorded against Klebsiella pneumoniae (1 μg/mL). Cyclo(l-Pro-d-Arg) was recorded to have significant antitumor activity against HeLa cells (IC₅₀ value 50 μg/mL), and this compound was recorded to have no cytotoxicity against normal monkey kidney cells (VERO) up to 100 μg/mL). To the best of our knowledge, this is the first time that cyclo(l-Pro-d-Arg) has been isolated from a microbial natural source.