Cellular stability of Rho-GTPases glucosylated by Clostridium difficile toxin B

Mono-glucosylation of Rho, Rac, and Cdc42 by Clostridium difficile toxin B (TcdB) induces changes of actin dynamics and apoptosis. When fibroblasts were treated with TcdB, an apparent decrease of the cellular Rac1 level was observed when applying anti-Rac1(Mab 102). This decrease was not based on degradation as inhibition of the proteasome by lactacystin did not stabilise cellular Rac1 levels. The application of anti-Rac1 (Mab 23A8) showed that the cellular Rac1 level slightly increased in TcdB-treated fibroblasts; thus, the apparent loss of cellular Rac1 was not due to degradation but due to impaired recognition of glucosylated Rac1 by anti-Rac1 (Mab 102). In contrast, recognition of RhoA by anti-RhoA (Mab 26C4) and Cdc42 by anti-Cdc42 (Mab 44) was not altered by glucosylation; a transient decrease of cellular RhoA and Cdc42 in TcdB-treated fibroblasts was indeed due to proteasomal degradation, as inhibition of the proteasome by lactacystin stabilised both cellular RhoA and Cdc42 levels. The finding that the apparent decrease of Rac1 reflects Rac1 glucosylation offers a valuable tool to determine Rac1 glucosylation.     

Comparative immunological characterization of various photosynthetic cytochromes c from pro- and eukaryotic algae

Photosynthetic c-type cytochromes isolated from various pro- and eukaryotic algae have been compared by an immunochemical method. Thereby the extent of cross-reactivity of several cytochromes with antisera to cytochrome c from Spirulina platensis, Bumilleriopsis filiformis, and Scenedesmus acutus was quantitatively determined by antigen-binding tests. When immunological relationship is taken as a measure of structural relationship, the following conclusions can be drawn: (1) c-type cytochromes from Anabaena variabilis, Nostoc muscorum, Calothrix membranacea, and Spirulina platensis show large differences in cross-reactivity. (2) The acidic Spirulina cytochrome c is fairly closely related to the two eukaryotic cytochromes assayed here.Abbreviations SAUG Sammlung von Algenkulturen am Pflanzenphysiologischen Institut der Universität Göttingen, FRG - PCC Pasteur Culture Collection     

Artemisinin biosynthesis in growing plants of Artemisia annua. A 13CO2 study

Artemisinin from Artemisia annua has become one of the most important drugs for malaria therapy. Its biosynthesis proceeds via amorpha-4,11-diene, but it is still unknown whether the isoprenoid precursors units are obtained by the mevalonate pathway or the more recently discovered non-mevalonate pathway. In order to address that question, a plant of A. annua was grown in an atmosphere containing 700 ppm of ¹³CO₂ for 100 min. Following a chase period of 10 days, artemisinin was isolated and analyzed by ¹³C NMR spectroscopy. The isotopologue pattern shows that artemisinin was predominantly biosynthesized from (E,E)-farnesyl diphosphate (FPP) whose central isoprenoid unit had been obtained via the non-mevalonate pathway. The isotopologue data confirm the previously proposed mechanisms for the cyclization of (E,E)-FPP to amorphadiene and its oxidative conversion to artemisinin. They also support deprotonation of a terminal allyl cation intermediate as the final step in the enzymatic conversion of FPP to amorphadiene and show that either of the two methyl groups can undergo deprotonation.     

JKTBP1 is involved in stabilization and IRES-dependent translation of NRF mRNAs by binding to 5' and 3' untranslated regions

Heterogeneous nuclear ribonucleoprotein D-like protein (JKTBP) 1 was implicated in cap-independent translation by binding to the internal ribosome entry site in the 5′ untranslated region (UTR) of NF-κB-repressing factor (NRF). Two different NRF mRNAs have been identified so far, both sharing the common 5′ internal ribosome entry site but having different length of 3′ UTRs. Here, we used a series of DNA and RNA luciferase reporter constructs comprising 5′, 3′ or both NRF UTRs to study the effect of JKTBP1 on translation of NRF mRNA variants. The results indicate that JKTBP1 regulates the level of NRF protein expression by binding to both NRF 5′ and 3′ UTRs. Using successive deletion and point mutations as well as RNA binding studies, we define two distinct JKTBP1 binding elements in NRF 5′ and 3′ UTRs. Furthermore, JKTBP1 requires two distinct RNA binding domains to interact with NRF UTRs and a short C-terminal region for its effect on NRF expression. Together, our study shows that JKTBP1 contributes to NRF protein expression via two disparate mechanisms: mRNA stabilization and cap-independent translation. By binding to 5′ UTR, JKTBP1 increases the internal translation initiation in both NRF mRNA variants, whereas its binding to 3′ UTR elevated primarily the stability of the major NRF mRNA. Thus, JKTBP1 is a key regulatory factor linking two pivotal control mechanisms of NRF gene expression: the cap-independent translation initiation and mRNA stabilization.     

Gait changes precede overt arthritis and strongly correlate with symptoms and histopathological events in pristane-induced arthritis

Introduction  

Pristane-induced arthritis (PIA) in the rat has been described as an animal model of inflammatory arthritis which exhibits features similar to rheumatoid arthritis in humans, such as a chronic, destructive, and symmetrical involvement of peripheral joints. However, so far little is known about the earliest inflammatory events and their influence on locomotor behaviour during the course of PIA. To investigate this issue a detailed analysis of the pathologic changes occurring during the prodromal and early stages of PIA was performed.     

Biosynthesis of calystegines: 15N NMR and kinetics of formation in root cultures of Calystegia sepium

Calystegines are nortropane alkaloids bearing between three and five hydroxyl groups in various positions. [15N]Tropinone was administered to root cultures of Calystegia sepium and the incorporation into calystegines was followed. Increase of label in calystegines was measured by one-dimensional 15N NMR and inverse-detected 2D NMR techniques. The results show that tropinone and pseudotropine are metabolites in the biosynthetic pathway of calystegines. The velocity of calystegine accumulation was followed kinetically by transfer of root cultures from 15N-enriched medium to 14N-medium and analysis by GC-MS. A constant calystegine formation with no interference by excretion or degradation was observed. A biosynthetic rate for individual calystegines at each time point was calculated, the maximum was 0.4 mg/day/g of biomass. This allowed the velocity of individual biosynthetic steps to be estimated.