Improvement of electrospray stability in negative ion mode for nano-PGC-LC-MS glycoanalysis via post-column make-up flow

Analysis of glycans via a porous graphitized carbon liquid chromatography (PGC-LC) coupled with electrospray ionization (tandem) mass spectrometry (ESI-MS(/MS)) is a powerful analytical method in the field of glycomics. Isobaric glycan structures can be identified reliably with the help of PGC-LC separation and subsequent identification by ESI-MS(/MS) in negative ion mode. In an effort to adapt PGC-LC-ESI-MS(/MS) to the nano-scale operation, spray instability along the nano-PGC-LC gradient was repeatedly observed on an LTQ Orbitrap Elite mass spectrometer equipped with a standard nano-electrospray ionization source. A stable electrospray was achieved with the implementation of a post-column make-up flow (PCMF). Thereby, acetonitrile was used to supplement the eluate from the nano-PGC-LC column. The improved spray stability enhanced detection and resolution of glycans during the analysis. This was in particular the case for smaller O-glycans which elute early in the high aqueous content regime of the nano-PGC-LC elution gradient. This study introduces PCMF as an easy-to-use instrumental adaptation to significantly improve spray stability in negative ion mode nano-PGC-LC-ESI-MS(/MS)-based analysis of glycans.     

Effects of insecticide application on parasitism rates of pollen beetle larvae (<Emphasis Type="Italic">Brassicogethes aeneus</Emphasis> (Fabricius)) by tersilochine parasitoids

Larval parasitoids can substantially reduce the population density of the pollen beetle [Brassicogethes aeneus (Fabricius), syn. Meligethes aeneus (Fabricius)]. The most abundant tersilochine parasitoids of pollen beetle are Tersilochus heterocerus, Phradis interstitialis and P. morionellus. The main activity of these parasitoids was observed in the period shortly before flowering to full flowering of oilseed rape. Insecticide applications during this period may have negative effects on parasitoids. In the present study, the effects of the insecticides Biscaya (a.i. thiacloprid), Mavrik (a.i. tau-fluvalinate) and Karate Zeon (a.i. lambda-cyhalothrin) applied during the bud or flowering stage of winter oilseed rape on parasitization of pollen beetle larvae by T. heterocerus were studied in 12 field trials at different locations in Germany in 2013–2015. The effects on parasitism by Phradis spp. were assessed in 2015. Parasitism of pollen beetle larvae by T. heterocerus was found in all field trials in all experimental years, but in most trials not before full flowering. Maximum percentage of parasitized larvae at different locations ranged between 3.4 and 16.8% in 2013, 8.3 and 22.4% in 2014 and from 11.1 to 29.1% in 2015. Levels of parasitism were not significantly different between the untreated control and insecticide treatments within each location. In contrast to T. heterocerus, Phradis spp. was not detected at all locations and not before flowering declining. In field trials at Lucklum and Puch, the maximum level of parasitism by Phradis spp. was 9.4 and 18.3%, respectively. No significant effect of insecticide application on parasitism by Phradis spp. was observed between the treatments. The results of this study showed that the insecticides used in the field trials did not affect parasitization of pollen beetle larvae by T. heterocerus and Phradis spp., regardless whether applied at the bud stage, at the beginning of flowering or full flowering.     

Impact of insecticides on oilseed rape bud infestation with eggs and larvae of pollen beetle (<Emphasis Type="Italic">Brassicogethes aeneus</Emphasis> (Fabricius))

One of the main insect pests in oilseed rape is the pollen beetle (Brassicogethes aeneus (Fabricius), syn. Meligethes aeneus). To maximize efficiency of control of this pest, insecticides are required that ideally, not just prevent yield losses by bud feeding of overwintered pollen beetles, but simultaneously minimize the reproduction of the pest, thereby reducing the size of the following generation infesting next year’s oilseed rape. The neonicotinoid active substance thiacloprid is known to reduce bud infestation with eggs and larvae. However, the mechanisms by which this occurs as well as the effects of other active substances are not known. In this study, the effects of the neonicotinoid insecticide Biscaya (a.i. thiacloprid) and the pyrethroids Mavrik (tau-fluvalinate) and Karate Zeon (lambda-cyhalothrin) applied at the bud stage of winter oilseed rape with recommended field rates on infestation of buds with eggs and larvae of pollen beetles were tested in field trials in Germany in 2013–2015. In additional greenhouse experiments, it was investigated whether effects on bud infestation were caused by lethal effects on pollen beetle or by insecticidal residues on plants causing sublethal effects. In the field trials, application of Biscaya and Mavrik significantly reduced the percentage of buds containing eggs and larvae in contrast to Karate Zeon. In 2014 and 2015, 14 days after application, bud infestation on the main raceme was reduced by 86 and 82%, respectively, in Biscaya-treated plots and by 51 and 71%, respectively, in Mavrik-treated plots compared to the untreated plots. In the greenhouse experiments, the lowest percentage of bud infestation with eggs and larvae was recorded on Biscaya-treated plants whereas on Mavrik-treated plants, there was no significant difference compared with the control. The results of the field trials show that Biscaya and Mavrik reduced oilseed rape bud infestation with eggs and larvae of pollen beetles primarily by lethal effects on overwintered pollen beetles or by repellency. However, Biscaya had additional effects on egg laying, which was supported by the greenhouse experiments.     

The 23-kDa light-stress-regulated heat-shock protein of Chenopodium rubrum L. is located in the mitochondria

The 23-kDa nuclear-encoded heat-shock protein (HSP) of Chenopodium rubrum L. is regulated by light at the posttranslational level. Higher light intensities are more effective in inducing the accumulation of the mature protein under heat-shock conditions. Based on this and other properties the protein was considered to belong to the group of small chloroplastic HSPs. However, we have now obtained the following evidence that this 23-kDa HSP is localized in the mitochondria: (i) Immunogold-labelled protein was almost exclusively restricted to the mitochondria in electron microscope thin sections. (ii) Using purified, isolated mitochondria from potato tubers the in-vitro-synthesized translation product of 31 kDa was readily transported into mitochondria where it was processed to the 23-kDa product. (iii) The protein could be detected by Western blotting in a preparation of washed mitochondria of Chenopodium, while under the same conditions no signal could be obtained in a preparation of isolated chloroplasts. (iv) Finally, sequence comparison with the published sequences of mitochondrial proteins by Lenne et␣al. (1995, Biochem J 311:805–813) and LaFayette et␣al. (1996, Plant Mol Biol 30:159–169) showed clearly that the 23-kDa protein is considerably more similar to these two proteins than to the group of plastid small HSPs. From these data we infer that mitochondria are involved in the response of the plants to high light stress under heat-shock conditions. Received: 11 July 1996 / Accepted: 24 August 1996     

Continuous production of erythrulose using transketolase in a membrane reactor

Transketolase can be used for synthesis of chiral intermediates and carbohydrates. However the enzyme is strongly deactivated by the educts. This deactivation depends on the reactor employed. An enzyme membrane reactor allows the continuous production of L-erythrulose with high conversion and stable operational points. A productivity (space-time yield) of 45g L d was reached.     

Analysis of the chloroplast protein complexes by blue-native polyacrylamide gel electrophoresis (BN-PAGE)

Blue-native polyacrylamide gel electrophoresis (BN-PAGE) is a powerful procedure for the separation and characterization of the protein complexes from mitochondria. Membrane proteins are solubilized in the presence of aminocaproic acid and n-dodecylmaltoside and Coomassie-dyes are utilized before electrophoresis to introduce a charge shift on proteins. Here, we report a modification of the procedure for the analysis of chloroplast protein complexes. The two photosystems, the light-harvesting complexes, the ATP synthase, the cytochrome b ₆ f complex and the ribulose-bisphosphate carboxylase/oxygenase are well resolved. Analysis of the protein complexes on a second gel dimension under denaturing conditions allows separation of more than 50 different proteins which are part of chloroplast multi-subunit enzymes. The resolution capacity of the blue-native gels is very high if compared to 'native green gel systems' published previously. N-terminal amino acid sequences of single subunits can be directly determined by cyclic Edman degradation as demonstrated for eight proteins. Analysis of chloroplast protein complexes by blue-native gel electrophoresis will allow the generation of 'protein maps' from different species, tissues and developmental stages or from mutant organelles. Further applications of blue-native gel electrophoresis are discussed.     

The occurrence of peptaibols and structurally related peptaibiotics in fungi and their mass spectrometric identification via diagnostic fragment ions.

Peptaibols and related peptide antibiotics (peptaibiotics) display diagnostically useful fragmentation patterns during mass spectrometry (FAB-MS, ESI-CID-MS/MS and CID-MSn]. The paper compiles fragmentation data of pseudo-molecular ions reported in the literature as a guide to the rational identification of recurrently isolated and new peptaibols and peptaibiotics. Taxonomic and ecological aspects of microorganisms producing peptaibols and peptaibiotics are discussed.     

EFFECTS OF SINGLE AND MULTIPLE EXPOSURES OF FERULIC ACID ON THE VEGETATIVE AND REPRODUCTIVE GROWTH OF PHASEOLUS VULGARIS BBL-290

Phaseolus vulgaris BBL-290 plants were grown in growth chambers in the Southeastern Plant Environment Laboratory and exposed to either single (at seedling, flower, or podfill) or multiple (biweekly or weekly) treatments of ferulic acid (FA). In the first experiment, plants were harvested one week after FA treatment (0, 1.0, 2.0 mM) and at final harvest (56 days old). FA delayed leaf expansion during the seedling and flowering stages. The total plant leaf area and the plant dry weight of plants treated with 1.0 and 2.0 mM FA as seedlings were reduced one week after treatment by 38–48%. The total plant leaf area and the plant dry weight of plants treated at flowering with 2.0 mM FA were reduced by 25% one week after treatment. Treatment with 2.0 mM FA at podfill caused the senescence and abscission of older leaves and reduced total plant leaf area, plant dry weight and mean pod dry weight by 54, 40, and 48%, respectively, one week after treatment. The plants treated at the seedling and flowering stages recovered by final harvest. In a subsequent experiment, FA (0, 0.50, 1.0, 1.5 mM) reduced total plant leaf area at the seedling and flowering stages but not at podfill. The youngest expanding leaves were most sensitive to FA at flowering. The leaf area of these leaves was reduced by 35 and 25%, one and two weeks after treatment, respectively. Their absolute growth rates were reduced from 31 to 56% one week after treatment at flowering. Their relative growth rates were reduced by 50% one week after treatment. Growth rates then recovered within two weeks after treatment. In the final experiment, biweekly exposures of FA (0.25, 0.50, 0.75, 1.0) reduced total plant leaf area but did not affect any other growth parameters. Weekly exposures of FA (0.25, 0.50, 0.75, 1.0) reduced total plant leaf area up to 34%, absolute growth rate up to 58%, leaf number up to 31% and pod number up to 58%. As the frequency of exposure to FA increased, the concentration necessary to affect bean plant growth and development decreased.     

Stofftransport im Ovar von Pyrrhocoris apterus L.

Zusammenfassung Synthese und Transport von DNS, RNS und Proteinen in einem telotrophen Ovar wurden autoradiographisch untersucht. Das Vorkommen von Endomitosen in Kernen sich differenzierender Nährzellen und des Follikelepithels können für Pyrrhocoris bestätigt werden, desgleichen der Transport von hochmolekularer RNS von den Nährzellkernen zu den Oocyten in der Phase euplasmatischen Wachstums. Ein Transport in jüngere Oocyten erfolgt nicht. Ein Beitrag des Follikelepithels zur RNS-Versorgung der Oocyten wie er für eine andere Wanze beschrieben wurde, ist bei Pyrrhocoris nicht feststellbar; gleichfalls im Gegensatz zu den Befunden an anderen Wanzen findet eine relativ schwache RNS-Synthese in allen Oocytenkernen statt. Protein wird mit unterschiedlicher Intensität in der gesamten Ovariole synthetisiert und von den Nährzellen in die Oocyten transportiert.Wie im polytrophen Ovar sind zwei RNS-Transporte unterscheidbar: Die langsamere Hauptfraktion hat anscheinend die gleiche, geringe Geschwindigkeit wie der Proteintransport; es wird daher angenommen, daß es sich um die passive Verfrachtung von Ribosomen im Strom des Euplasma handelt. Eine geringere Menge markierter RNS wird schneller durch das Cytoplasma transportiert und als messenger-RNS diskutiert.

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Summary Synthesis and transport of DNA, RNA, and proteins were studied in a telotrophic ovary using autoradiography. The occurrence of endomitosis in nuclei of differentiating nurse cells and follicular epithelium can be confirmed for Pyrrhocoris and also the transport of high molecular RNA from the nurse cell nuclei into the oocytes during their phase of euplasmatic growth. There is no transport into younger oocytes. In Pyrrhocoris no contribution of the follicular epithelium to the RNA supply of the oocytes is found as described for a different Heteroptere; contrary to the findings reported for other Heteroptera, a relatively low rate of RNA synthesis is detected in all oocyte nuclei. Protein is synthesized with different intensity in all parts of the ovariole and transported from the nurse cells into the oocytes.It is possible, like in the polytrophic ovary, to distinguish between two modes of RNA transport. The bulk of the RNA seems to moove at the same low speed as the proteins, presumably representing a passive movement of ribosomes in the flow of euplasma. A smaller fraction of labelled RNA is transported faster through the cytoplasm; it may consist of messenger RNA.

Die Untersuchungen wurden z.T. mit Mitteln durchgeführt, die die Deutsche Forschungsgemeinschaft Herrn Prof. Bier zur Verfügung stellte.     

Licht- und elektronenmikroskopische Untersuchungen zur Entwicklung der Hydropoten von Nelumbo nucifera

Die Hydropoten von Nelumbo nucifera werden in verschicdenen Entwicklungsstadien einer licht- und elektronenmikroskopischen Analyse unterzogen. Dabei erweisen sie sich als ein den Menyanthaceen-Hydropoten sehr nahcstehender Differenzierungstyp. Sowohl morphologisch-anatomisch als auch in ihrem Verhalten gegenüber den Vitalfarbstoffen Toluidinblau und Methylviolett zeigen sie eine große Ähnlichkeit mit den Hydropoten von Nymphoides peltata. Von den drüsenartigen, hydropotenähnlichen Epidermisdifferenzierungen auf der Blattunterseite der Nymphaeaceen unterscheiden sie sich grundlegend, da ihnen jede feinstrukturelle Andeutung einer Drüsenfunktion fehlt. Die Abglicderung der Gattung Nelumbo von den Nymphaeales erscheint u. a. dadurch gerechtfertigt Die Differenzierungsrichtung der an noch eingerollten submersen Blättern in Form einzelner Zellen auftretenden Hydropotenmeristemoide ist von dem Medium abhängig, in dem sich die Blätter weiterentwickeln. Im Wasser bleiben die jungen Hydropotenzellen in ihrem Streckungswachstum gegenüber den normalen Epidermiszellen stark zurück. Es kommt zu einer Auflockerung des Wandmatcrials in den Zellwandkuppen und zu einem Anstieg des Flavonol- und Gerbstoffgehaltes in den Hydropotenzellsäften. Ferner nimmt die Eigenschaft zu, kationische Farbstoffe elektroadsorptiv in der Zellwandkuppe anzureichern. Die Kutikula über den Hydropoten bleibt elektronenoptisch gut durchstrahlbar. An der Luft wachsen die jungen Hydropotenzellen zu normalen Epidermiszellen aus, wobei die am stärksten differenzierten Hydropotenzentren manchmal degenerieren