Evaluation of nine genotypes of oilseed rape (Brassica napus L.) for larval infestation and performance of rape stem weevil (Ceutorhynchus napi Gyll.).

The rape stem weevil, Ceutorhynchus napi Gyll., is a serious pest of winter oilseed rape (Brassica napus L.) crops in Europe causing severe yield loss. In currently used oilseed rape cultivars no resistance to C. napi has been identified. Resynthesized lines of B. napus have potential to broaden the genetic variability and may improve resistance to insect pests. In this study, the susceptibility to C. napi of three cultivars, one breeding line and five resynthesized lines of oilseed rape was compared in a semi-field plot experiment under multi-choice conditions. Plant acceptance for oviposition was estimated by counting the number of C. napi larvae in stems. The larval instar index and the dry body mass were assessed as indicators of larval performance. The extent of larval feeding within stems was determined by the stem injury coefficient. Morphological stem traits and stem contents of glucosinolates were assessed as potential mediators of resistance. The resynthesized line S30 had significantly fewer larvae than the cultivars Express617 and Visby and the resynthesized lines L122 and L16. The low level of larval infestation in S30 was associated with a low larval instar and stem injury index. Low numbers of larvae were not correlated with the length or diameter of stems, and the level of stem glucosinolates. As indicated by the low larval infestation and slow larval development the resistance of S30 to C. napi is based on both antixenotic and antibiotic properties of the genotypes. The resynthesized line S30 should therefore be introduced into B. napus breeding programs to enhance resistance against C. napi.

Study of posttranslational modifications in lenticular alphaA-Crystallin of mice using proteomic analysis techniques.

In the present work the complexity in the 2D-gel protein pattern of murin lenticular alphaA-Crystallin was analyzed. An in depth study of the different protein isoforms was done combining different proteomic tools. Lens proteins of four different ages, from embryo to 100-week-old mice, were separated by large 2D-PAGE, revealing an increase in the number and intensity of the spots of alphaA-Crystallin during the process of aging. For further analyses the oldest mice were chosen. Comparison and evaluation of two different staining methods proved Imidazole-Zinc to be a good alternative to the generally used Coomassie stain. The characterization of the different alphaA-Crystallin protein species was done using nanoLC-ESI-MS/MS (liquid chromatography electrospray ionisation tandem mass spectrometry). Data interpretation was done by database searching, manual validation and a new MS/MS-interpretation tool for posttranslational modifications--the PTM-Explorer. Using this way, eight different phosphorylation sites were identified and localized; the identification of four of them was not published so far. Furthermore, quantitative N-terminal acetylation of alphaA-Crystallin and variable C-terminal truncation was observed, also not published in this extent yet. The results of the mass spectrometric analysis were validated by immunoblotting experiments using two different alphaA-Crystallin specific antibodies. In addition, a fluorescent phospho-specific stain was used to detect the protein spots including phosphorylation groups. Re-separation 2D-PAGE was done to round off the present study and explain the appearance of some of the protein spots in the gel as artifacts of the 2D-PAGE separation.

Tryptic transpeptidation products observed in proteome analysis by liquid chromatography-tandem mass spectrometry.

Commonly, prior to mass spectrometry based analysis of proteins or protein mixtures, the proteins are subjected to specific enzymatic proteolysis. For this purpose trypsin is most frequently used. However, the process of proteolysis is not unflawed. For example, some side activities of trypsin are known and have already been described in the literature (e.g., chymotryptic activity). Here, we describe the occurrence of transpeptidated peptides during standard proteome analysis using two-dimensional polyacrylamide gel electrophoresis followed by mass spectrometric protein identification. Different types of transpeptidated peptides have been detected. The most frequently observed transpeptidation reaction is N-terminal addition of arginine or lysine to peptides. Furthermore, addition of two amino acids to the N-terminus of a peptide has also been detected. Another transpeptidation that we observed, is combination of two peptides, which were originally located in different regions of the analyzed protein. Currently, the full amount of peptides generated by transpeptidation is not clear. However, it should be recognized that protein information is presently lost as these effects are not detectable with available database search software.

A peptide preconcentration approach for nano-high-performance liquid chromatography to diminish memory effects.

An advanced method has been developed for the analysis of proteolytic digests of complex protein mixtures by reversed phase high-performance liquid chromatography coupled to mass spectrometry. The occurrence of memory effects was prevented by a parallel set of two precolumns employed for simultaneous separation and washing procedures. The system was tested extensively, and tryptic digests of three single proteins were analyzed. In addition, different solvent systems were evaluated for effective washing of the employed precolumns. Using the analytical strategy presented, a reliable identification of proteins in complex mixtures was obtained and not hampered by the occurrence of memory effects.

Identification of phosphorylation and acetylation sites in alphaA-crystallin of the eye lens ( mus musculus) after two-dimensional gel electrophoresis.

Posttranslational modifications are of great interest because of their relevance in biological systems as proteins are commonly activated or deactivated by phosphorylation, glycation and acetylation [1, 2]. During eye lens aging the number of the alphaA-crystallin isoproteins increases. This could be observed by the use of 2D-PAGE (two-dimensional gel electrophoresis). The number of alphaA-crystallin spots in the gel increased during eye lens aging. For further analysis the spots of 2D-PAGE were cut out and the identification of the proteins was done using nanoLC-ESI-MS/MS (liquid chromatography electrospray ionization tandem mass spectrometry). The created MS/MS-data were analyzed using the Sequest algorithm. Searches with different parameters were done to preferably get the complete sequence coverage and to identify posttranslational modifications of the alphaA-crystallin. The acetylated N-terminus of this protein could be detected. Furthermore, phosphorylation of serine 122 and 148 was identified in two different spots.

Enhanced stability of somatosensory evoked potentials attained in the median nerve by using temporal electrodes for intraoperative recording in patients in the semisitting position.

OBJECT: Findings published in case reports indicate that monitoring of median nerve somatosensory evoked potentials (MN-SSEPs) is unreliable in patients who undergo surgery while in the semisitting position due to the occurrence of changes in the potentials that are unrelated to neurological damage. The present study was designed to test the hypothesis that in these patients MN-SSEPs are more stable when recording electrodes are placed over the temporal region. METHODS: In 30 patients who underwent surgery in the semisitting position, MN-SSEPs were recorded intraoperatively by using electrodes placed over the temporal region as well as those placed at conventional recording sites. The authors analyzed MN-SSEP amplitudes and latencies at different recording sites and at distinct steps of the monitoring procedure. In 10 of the 30 patients a clinically significant attenuation (> 50%) of MN-SSEP amplitude was observed at conventional recording sites and this was obviously not related to neurological damage. In contrast, no significant changes were observed in MN-SSEPs recorded from electrodes located over the temporal region. CONCLUSIONS: In patients who undergo surgery in the semisitting position, the use of additional recording electrodes placed over the temporal region makes intraoperative MN-SSEP monitoring less prone to false-positive alarms and thus enhances the reliability of intraoperative MN-SSEP monitoring.

Observations on intraoperative somatosensory evoked potential (SEP) monitoring in the semi-sitting position.

BACKGROUND: Former case reports suggest that monitoring of median nerve somatosensory evoked potentials (M-SEP) is unreliable in patients operated in the semi-sitting position due to the occurrence of evoked potential changes unrelated to neurological damage. This study was designed to analyze these changes in greater detail and confirm that these changes are not caused by neurological damage. METHODS: M-SEP monitoring findings of 50 patients with surgery in the semi-sitting position were analyzed and compared with a group of 50 patients who underwent surgery in the supine position. M-SEP amplitudes and latencies at distinct steps of the monitoring procedure were used for further analysis. In 10 of the 50 semi-sitting patients, M-SEP were recorded additionally after surgery with the anesthetized patient in the supine position. RESULTS: Significant M-SEP changes occurred in the semi-sitting patients only. An amplitude loss of greater than 50% on at least one side was observed in 24 patients. The magnitude and the time course of the amplitude loss was considerably variable. A complete loss of the evoked potential was not observed in any case. In all 10 patients, M-SEP recovered completely when recorded in the supine position. CONCLUSIONS: In about half of the patients with M-SEP monitoring in the semi-sitting position, a significant amplitude loss occurs which is unrelated to neurological damage and presumably caused by subdural gas collections. There is no characteristic pattern of M-SEP changes which enables a differentiation of these 'artificial' alterations from true events. The only appropriate criterion to indicate an impending neurological damage in these patients seems to be a complete loss of the M-SEP potential.