Plant protein phosphorylation monitored by capillary liquid chromatography--element mass spectrometry

Many essential cellular functions such as growth rate, motility, and metabolic activity are linked to reversible protein phosphorylation, since they are controlled by signaling cascades based mainly on phosphorylation/dephosphorylation events. Quantification of global or site-specific protein phosphorylation is not straightforward with standard proteomic techniques. The coupling of capillary liquid chromatography (microLC) with ICP-MS (inductively coupled plasma-mass spectrometry) is a method which allows a quantitative screening of protein extracts for their phosphorus and sulfur content, and thus provides access to the protein phosphorylation degree. In extension of a recent pilot study, we analyzed protein extracts from the model organisms Arabidopsis thaliana and Chlamydomonas reinhardtii as representatives for multicellular and unicellular green photosynthetically active organisms. The results indicate that the average protein phosphorylation level of the algae C. reinhardtii is higher than that of A. thaliana. Both the average phosphorylation levels were found to be between the extreme values determined so far for prokaryotes (C. glutamicum, lowest levels) and eukaryotes (Mus musculus, highest levels). Tissue samples of A. thaliana representing different stages of plant development showed varying levels of protein phosphorylation indicating a different adjustment of the kinase/phosphatase system. We also utilized the microLC-ICP-MS technology to estimate the efficiency of a novel phosphoprotein enrichment method based on aluminum hydroxide, since the enrichment of phosphorylated species is often an essential step for their molecular characterization.     

mRNA degradation in bacteria

Messenger RNAs in prokaryotes exhibit short half-lives when compared with eukaryotic mRNAs. Considerable progress has been made during recent years in our understanding of mRNA degradation in bacteria. Two major aspects determine the life span of a messenger in the bacterial cell. On the side of the substrate, the structural features of mRNA have a profound influence on the stability of the molecule. On the other hand, there is the degradative machinery. Progress in the biochemical characterization of proteins involved in mRNA degradation has made clear that RNA degradation is a highly organized cellular process in which several protein components, and not only nucleases, are involved. In Escherichia coli, these proteins are organized in a high molecular mass complex, the degradosome. The key enzyme for initial events in mRNA degradation and for the assembly of the degradosome is endoribonuclease E. We discuss the identified components of the degradosome and its mode of action. Since research in mRNA degradation suffers from dominance of E. coli-related observations we also look to other organisms to ask whether they could possibly follow the E. coli standard model.     

Assembly of binding loops on aromatic templates as VCAM‐1 mimetics

The design and synthesis of cyclic mimetics of VCAM‐1 protein that reproduce the integrin‐binding domain are presented. The unprotected peptide precursor 37 – 43 , Thr‐Gln‐Ile‐Asp‐Ser‐Pro‐Leu, was grafted onto functional templates of type naphthalene, biphenyl and benzyl through the chemoselective formation of C‐ and N‐terminal oximes resulting in a mixture of four isomeric forms due to syn–anti isomerism of the oxime bonds. Some isomers could be monitored by HPLC and identified by NMR. The molecule containing a naphthalene‐derived template was found to inhibit the VCAM‐1/VLA‐4 interaction more efficiently than previously reported for sulfur‐bridged cyclic peptides containing similar sequences. The finding confirms the importance of incorporating conformational constraints between the terminal ends of the peptide loop 37 – 43 in the design of synthetic inhibitors of the VCAM‐1/integrin interaction. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Ablation of connexin43 in smooth muscle cells of the mouse intestine: functional insights into physiology and morphology

Connexin43 (Cx43) gap-junction channels are highly abundant in intestinal smooth muscle but their functional impact has not been studied so far. Here, we have aimed to elucidate the functional role of Cx43 in the tunica muscularis of the mouse intestine in vivo. Transgenic mice with conditional deletion of Cx43 in smooth muscle cells (SMC) were generated. Histological investigations by immunofluorescence analyses and organ-bath recordings to assess the contractility of intestinal tissue strips were carried out. Measurements of gastrointestinal transit and of the visceromotor response by utilizing a standardized colorectal distension model to quantify alterations of visceral sensory function were also performed in SMC-specific Cx43 null mice and control littermates. Histologically, we found thickening of the tunica muscularis and a 13-fold increase of neutrophil infiltration of the gastrointestinal wall of SMC-specific Cx43 null mice. These animals also exhibited a decrease of 29% in gastrointestinal transit time. In contrast, the visceromotor response to a standardized colorectal distension was elevated, as was the contractility in SMC-specific Cx43 null mice, compared with controls. Thus, SMC-specific ablation of Cx43 in mice leads to morphological and functional alterations of the intestinal tunica muscularis, to gastrointestinal motor dysfunction and to altered visceral sensory function. This study was supported by a grant from the German Research Association (Wi 270/25-1,2) to K.W. and in part by the IFORES program of the University Hospital, Essen, Germany.     

Improvement of embryonic dopaminergic neurone survival in culture and after grafting into the striatum of hemiparkinsonian rats by CEP-1347

Transplantation of embryonic nigral tissue ameliorates functional deficiencies in Parkinson's disease (PD). A main constraint of neural grafting is the poor survival of dopaminergic neurones grafted into patients. Studies in rats indicated that many grafted neurones die by apoptosis. CEP-1347 is a mixed-lineage-kinase (MLK) inhibitor with neuroprotective action in several in vitro and in vivo models of neuronal apoptosis. We studied the effect of CEP-1347 on the survival of embryonic rat dopaminergic neurones in culture, and after transplantation in hemiparkinsonian rats. CEP-1347 and the alternative MLK inhibitor CEP-11004 significantly increased the survival of dopaminergic neurones in primary cultures from rat ventral mesencephalon and in Mn2+-exposed PC12 cells, a surrogate model of dopaminergic lethal stress. Moreover, combined treatment of the grafting cell suspension and the host animal with CEP-1347 significantly improved the long-term survival of rat dopaminergic neurones transplanted into the striatum of hemiparkinsonian rats. Also, the protective effect of CEP-1347 resulted in an increase in total graft size and in enhanced fibre outgrowth. Thus, treatment with CEP-1347 improved dopaminergic cell survival under severe stress and might be useful to improve the positive outcome of transplantation therapy in PD and reduce the amount of human tissue required.     

Application of a full body inertial measurement system in alpine skiing: a comparison with an optical video based system

This study aims at determining the accuracy of a full body inertial measurement system in a real skiing environment in comparison with an optical video based system. Recent studies have shown the use of inertial measurement systems for the determination of kinematical parameters in alpine skiing. However, a quantitative validation of a full body inertial measurement system for the application in alpine skiing is so far not available. For the purpose of this study, a skier performed a test-run equipped with a full body inertial measurement system in combination with a DGPS. In addition, one turn of the test-run was analyzed by an optical video based system. With respect to the analyzed angles, a maximum mean difference of 4.9° was measured. No differences in the measured angles between the inertial measurement system and the combined usage with a DGPS were found. Concerning the determination of the skier's trajectory, an additional system (e.g., DGPS) must be used. As opposed to optical methods, the main advantages of the inertial measurement system are the determination of kinematical parameters without the limitation of restricted capture volume, and small time costs for the measurement preparation and data analysis.     

Functional Heterologous Production of Reductive Dehalogenases from Desulfitobacterium hafniense Strains

The anaerobic dehalogenation of organohalides is catalyzed by the reductive dehalogenase (RdhA) enzymes produced in phylogenetically diverse bacteria. These enzymes contain a cobamide cofactor at the active site and two iron-sulfur clusters. In this study, the tetrachloroethene (PCE) reductive dehalogenase (PceA) of the Gram-positive Desulfitobacterium hafniense strain Y51 was produced in a catalytically active form in the nondechlorinating, cobamide-producing bacterium Shimwellia blattae (ATCC 33430), a Gram-negative gammaproteobacterium. The formation of recombinant catalytically active PceA enzyme was significantly enhanced when its dedicated PceT chaperone was coproduced and when 5,6-dimethylbenzimidazole and hydroxocobalamin were added to the S. blattae cultures. The experiments were extended to D. hafniense DCB-2, a reductively dehalogenating bacterium harboring multiple rdhA genes. To elucidate the substrate spectrum of the rdhA3 gene product of this organism, the recombinant enzyme was tested for the conversion of different dichlorophenols (DCP) in crude extracts of an RdhA3-producing S. blattae strain. 3,5-DCP, 2,3-DCP, and 2,4-DCP, but not 2,6-DCP and 3,4-DCP, were reductively dechlorinated by the recombinant RdhA3. In addition, this enzyme dechlorinated PCE to trichloroethene at low rates.     

Suitability of Macrolampis firefly and Pyrearinus click beetle luciferases for bacterial light off toxicity biosensor

Bioluminescence is widely used in biosensors. For water toxicity analysis, the naturally bioluminescent bacteria Vibrio fischeri have been used extensively. We investigated the suitability of two new beetle luciferases for Escherichia coli light off biosensors: Macrolampis firefly and Pyrearinus termitilluminans click beetle luciferases. The bioluminescence detection assay using this system is very sensitive, being comparable or superior to V. fischeri. The luciferase of P. termitilluminans produces a strong and sustained bioluminescence that is useful for less sensitive and inexpensive assays that require integration of the emission, whereas Macrolampis luciferase displays a flash-like luminescence that is useful for fast and more sensitive assays. The effect of heavy metals and sanitizing agents was analyzed. Zinc, copper, 1-propanol, and iodide had inhibitory effects on bioluminescence and growth assays; however, in these cases the bioluminescence was not a very reliable indicator of cell growth and metabolic activity because these agents also inhibited the luciferase. On the other hand, mercury and silver strongly affected cell bioluminescence and growth but not the luciferase activity, indicating that bioluminescence was a reliable indicator of cell growth and metabolic activity in this case. Finally, bioluminescent E. coli immobilized in agarose matrix gave a more stable format for environmental assays.