uPA deficiency exacerbates muscular dystrophy in MDX mice

Duchenne muscular dystrophy (DMD) is a fatal and incurable muscle degenerative disorder. We identify a function of the protease urokinase plasminogen activator (uPA) in mdx mice, a mouse model of DMD. The expression of uPA is induced in mdx dystrophic muscle, and the genetic loss of uPA in mdx mice exacerbated muscle dystrophy and reduced muscular function. Bone marrow (BM) transplantation experiments revealed a critical function for BM-derived uPA in mdx muscle repair via three mechanisms: (1) by promoting the infiltration of BM-derived inflammatory cells; (2) by preventing the excessive deposition of fibrin; and (3) by promoting myoblast migration. Interestingly, genetic loss of the uPA receptor in mdx mice did not exacerbate muscular dystrophy in mdx mice, suggesting that uPA exerts its effects independently of its receptor. These findings underscore the importance of uPA in muscular dystrophy.     

Analysis of planarian Adh3 supports an intron-rich architecture and tissue-specific expression for the urbilaterian ancestral form

The alcohol dehydrogenase class 3 enzyme (ADH3) is the presumed ancestral form of the medium-chain dehydrogenase-reductase ADH family. This enzyme has been involved in formaldehyde and nitric oxide metabolism of a variety of deuterostomes and ecdysozoan protostomes. We have now characterized the structure and expression of the Adh3 gene in the lophotrochozoan Schmidtea mediterranea, a freshwater planarian. The planarian gene expands over 8.7 kb and is organized into 7 exons. The 1340 bp long Adh3cDNA contains a 1137 bp open reading frame corresponding to a deduced protein of 379 amino acids. The protein sequence is consistent with that expected for a typical class III enzyme. Twenty out of the twenty-two amino acid positions associated with enzymatic roles are strictly preserved, which suggests that the enzymatic capabilities have been conserved. In situ hybridization experiments show that Adh3 is expressed along the intestine of S. mediterranea specimens. This is consistent with the pattern observed in invertebrates and in contrast with the widespread expression of vertebrate Adh3. The comparative study across bilateria, which now includes a lophotrochozoan representative, further supports the idea that the urbilaterian Adh3 ancestor showed an intron-rich architecture and tissue-specific expression, and strengthens the view that widespread expression of Adh3 was a vertebrate innovation.     

Prion protein does not redox-silence Cu2+, but is a sacrificial quencher of hydroxyl radicals

Oxidative stress is believed to play a central role in the pathogenesis of prion diseases, a group of fatal neurodegenerative disorders associated with a conformational change in the prion protein (PrP(C)). The precise physiological function of PrP(C) remains uncertain; however, Cu(2+) binds to PrP(C) in vivo, suggesting a role for PrP(C) in copper homeostasis. Here we examine the oxidative processes associated with PrP(C) and Cu(2+). (1)H NMR was used to monitor chemical modifications of PrP fragments. Incubation of PrP fragments with ascorbate and CuCl(2) showed specific metal-catalyzed oxidation of histidine residues, His(96/111), and the methionine residues, Met(109/112). The octarepeat region protects His(96/111) and Met(109/112) from oxidation, suggesting that PrP(90-231) might be more prone to chemical modification. We show that Cu(2+/+) redox cycling is not 'silenced' by Cu(2+) binding to PrP, as indicated by H(2)O(2) production for full-length PrP. Surprisingly, although detection of Cu(+) indicates that the octarepeat region of PrP is capable of reducing Cu(2+) even in the absence of ascorbate, H(2)O(2) is not generated unless ascorbate is present. Full-length PrP and fragments cause a dramatic reduction in detectable hydroxyl radicals in an ascorbate/Cu(2+)/O(2) system; however, levels of H(2)O(2) production are unaffected. This suggests that PrP does not affect levels of hydroxyl radical production via Fentons cycling, but the radicals cause highly localized chemical modification of PrP(C).     

Localization of aluminium in tea (Camellia sinensis) leaves using low energy X-ray fluorescence spectro-microscopy

Information on localization of Al in tea leaf tissues is required in order to better understand Al tolerance mechanism in this Al-accumulating plant species. Here, we have used low-energy X-ray fluorescence spectro-microscopy (LEXRF) to study localization of Al and other low Z-elements, namely C, O, Mg, Si and P, in fully developed leaves of the tea plant [Camellia sinensis (L.) O. Kuntze]. Plants were grown from seeds for 3?months in a hydroponic solution, and then exposed to 200?μM AlCl₃ for 2?weeks. Epidermal-mesophyll and xylem phloem regions of 20?μm thick cryo-fixed freeze-dried tea-leaf cross-sections were raster scanned with 1.7 and 2.2?keV excitation energies to reach the Al–K and P–K absorption edges. Al was mainly localized in the cell walls of the leaf epidermal cells, while almost no Al signal was obtained from the leaf symplast. The results suggest that the retention of Al in epidermal leaf apoplast represent the main tolerance mechanism to Al in tea plants. In addition LEXRF proved to be a powerful tool for localization of Al in plant tissues, which can help in our understanding of the processes of Al uptake, transport and tolerance in plants.     

Functional interaction of reverse gyrase with single-strand binding protein of the archaeon Sulfolobus

Reverse gyrase is a unique hyperthermophile-specific DNA topoisomerase that induces positive supercoiling. It is a modular enzyme composed of a topoisomerase IA and a helicase domain, which cooperate in the ATP-dependent positive supercoiling reaction. Although its physiological function has not been determined, it can be hypothesized that, like the topoisomerase–helicase complexes found in every organism, reverse gyrase might participate in different DNA transactions mediated by multiprotein complexes. Here, we show that reverse gyrase activity is stimulated by the single-strand binding protein (SSB) from the archaeon Sulfolobus solfataricus. Using a combination of in vitro assays we analysed each step of the complex reverse gyrase reaction. SSB stimulates all the steps of the reaction: binding to DNA, DNA cleavage, strand passage and ligation. By co-immunoprecipitation of cell extracts we show that reverse gyrase and SSB assemble a complex in the presence of DNA, but do not make stable protein–protein interactions. In addition, SSB stimulates reverse gyrase positive supercoiling activity on DNA templates associated with the chromatin protein Sul7d. Furthermore, SSB enhances binding and cleavage of UV-irradiated substrates by reverse gyrase. The results shown here suggest that these functional interactions may have biological relevance and that the interplay of different DNA binding proteins might modulate reverse gyrase activity in DNA metabolic pathways.     

Effects of roscovitine on the nuclear and cytoskeletal components of calf oocytes and their subsequent development

Roscovitine, a potent inhibitor of M-phase promoting factor kinase activity, was used to maintain calf oocytes at the germinal vesicle stage for a 24h culture period. Cumulus-oocyte complexes were first prematured for 24h in the presence of different levels of roscovitine (12.5, 25, 50 and 100 microM). Roscovitine was shown to block germinal vesicle breakdown in calf oocytes in a concentration dependent manner. Significantly greater inhibitory effect was observed at 50 and 100 microM with 64.6% and 63.2% oocytes being blocked in the germinal vesicle stage when compared to the control (0.0%) and the 12.5 microM (2.9%) and 25 microM (18.8%) groups. However, this inhibitory effect of roscovitine was fully reversible since a substantial number of the oocytes resumed meiosis and reached the metaphase II stage after a further 24h of culture in a permissive medium. Cleavage rates and blastocyst yields were not significantly different for oocytes cultured under 50 microM roscovitine inhibition compared to oocytes not subjected to prematuration culture (rates of 76.7% cleavage and 8.7% blastocysts for control oocytes compared to 69.8% and 6.3%, respectively, for oocytes pretreated with 50 microM roscovitine). The morphology of the meiotic spindle was typical of metaphase II in 75.8% and 82.1% of the oocytes reaching the metaphase II stage after pretreatment with 50 microM roscovitine compared to control, respectively. A normal distribution of actin filaments was observed in 97.0% and 98.2% of oocytes exposed to 50 microM roscovitine compared to control, respectively. These results demonstrate the feasibility of maintaining calf oocytes in artificial meiotic arrest without compromising their subsequent developmental competence.     

A role for cyclic hydroxamates in aluminium resistance in maize?

Hydroxamate siderophores have been found to alleviate Al toxicity in bacteria. In Poaceae plants cyclic hydroxamates, like DIMBOA (2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one) and its derivatives have mostly been studied in relation to either defence against insects or allelopathy. In this study the influence of Al on concentrations of these benzoxazinoids (Bx) in root tips, whole roots and root xylem exudates of Zea mays L. varieties differing in Al resistance was analyzed by HPLC-MS. Aluminium resistant maize variety Sikuani maintained considerably higher Bx levels in root tips than the Al sensitive variety Bakero. In vitro binding of Al to DIMBOA was shown by fluorescence quenching. Addition of DIMBOA to Al-containing nutrient solution protected the sensitive maize against Al toxicity as shown by bioassays using callose and haematoxylin staining of root tips as stress indicators. This is the first study showing that Bx can detoxify Al in solution. Tissue analysis data provide first, circumstantial, support for a role of Bx in defence against Al toxicity also in planta.