Phosphorylated alpha-synuclein is ubiquitinated in alpha-synucleinopathy lesions

alpha-Synuclein is one of the major components of intracellular fibrillary aggregates in the brains of a subset of neurodegenerative disorders, including Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, and Hallervorden-Spatz disease, which are referred to as alpha-synucleinopathies. We have shown previously (Fujiwara, H., Hasegawa, M., Dohmae, N., Kawashima, A., Masliah, E., Goldberg, M. S., Shen, J., Takio, K., and Iwatsubo, T. (2002) Nat. Cell Biol. 4, 160-164) that alpha-synuclein deposited in synucleinopathy brains is extensively phosphorylated at Ser-129 and migrates at 15 kDa. Here we examined the biochemical characteristics of the additional, higher molecular mass species of phosphorylated alpha-synuclein-positive polypeptides that also are recovered in the Sarkosyl-insoluble fraction of synucleinopathy and migrate at about 22 and 29 kDa. These 22 and 29 kDa bands were positive for three different anti-ubiquitin antibodies and comigrated perfectly with in vitro ubiquitinated alpha-synuclein that may correspond to mono- and diubiquitinated alpha-synuclein, respectively. Furthermore, cyanogen bromide cleavage of the 22 and 29 kDa polypeptides shifted the mobility to 19 and 26 kDa, respectively, and they retained immunoreactivity for both ubiquitin and alpha-synuclein. Finally, protein sequence analysis showed that the 19 kDa band contained two amino-terminal sequences of alpha-synuclein and ubiquitin. These results strongly suggest that phosphorylated alpha-synuclein is targeted to mono- and diubiquitination in synucleinopathy brains, which may have implications for mechanisms of these diseases.     

Mitochondrial reactive oxygen species regulate spatial profile of proinflammatory responses in lung venular capillaries

Cytokine-induced lung expression of the endothelial cell (EC) leukocyte receptor P-selectin initiates leukocyte rolling. To understand the early EC signaling that induces the expression, we conducted real-time digital imaging studies in lung venular capillaries. To compare receptor- vs nonreceptor-mediated effects, we infused capillaries with respectively, TNF-alpha and arachidonate. At concentrations adjusted to give equipotent increases in the cytosolic Ca(2+), both agents increased reactive oxygen species (ROS) production and EC P-selectin expression. Blocking the cytosolic Ca(2+) increases abolished ROS production; blocking ROS production abrogated P-selectin expression. TNF-alpha, but not arachidonate, released Ca(2+) from endoplasmic stores and increased mitochondrial Ca(2+). Furthermore, Ca(2+) depletion abrogated TNF-alpha responses partially, but arachidonate responses completely. These differences in Ca(2+) mobilization by TNF-alpha and arachidonate were reflected in spatial patterning in the capillary in that the TNF-alpha effects were localized at branch points, while the arachidonate effects were nonlocalized and extensive. Furthermore, mitochondrial blockers inhibited the TNF-alpha- but not the arachidonate-induced responses. These findings indicate that the different modes of Ca(2+) mobilization determined the spatial patterning of the proinflammatory response in lung capillaries. Responses to TNF-alpha revealed that EC mitochondria regulate the proinflammatory process by generating ROS that activate P-selectin expression.     

Permanent restoration of human skin treated with cultured epithelium grafting--wound healing by stem cell based tissue engineering--

The technique of epidermal cell culture developed by Green and colleagues made a breakthrough in the treatment of massive wounds in vivo with grown cells in vitro. In the past two decades, progress of culture methods and clinical practice have been made and now it is possible to treat extensive skin defect with large amounts of cultured epithelium. Since 1985, we have been successfully used cultured epidermis as autografts for the permanent coverage of full-thickness burn wounds or excised burn scars, giant nevi, tattoos and so on. Furthermore, cultured epidermis has been available as allografts to promote the healing of chronic skin ulcers or deep dermal burn. In this paper we describe our clinical experience of cultured epithelium grafting for the treatment of wounds and predict new trial of wound management and regeneration based on tissue engineering concept.     

Enzyme inhibitors to increase poly-3-hydroxybutyrate production by transgenic tobacco

Chemical regulation of secondary-metabolite synthesis was investigated through the improvement of poly-3-hydroxybutyrate (PHB) production in transgenic tobacco plants by the use of enzyme inhibitors. Two tobacco lines, BC3 and rCAB8, that produce PHB in both the cytosol and plastids were used. An acetyl-CoA carboxylase inhibitor, D-(+)-Quizalofop-ethyl, increased PHB accumulation in both lines 2-fold. The accumulation rate of plastidial PHB in the rCAB8 line was 2.5-fold higher than that of cytosolic PHB in the BC3 line. A specific inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase, mevastatin, also increased PHB accumulation but only in the BC3 line. These results indicated that chemical regulation of the native metabolic flows by the specific enzyme inhibitors increased secondary-metabolite production in the transgenic tobacco plants we used.     

Controlling pore assembly of staphylococcal gamma-haemolysin by low temperature and by disulphide bond formation in double-cysteine LukF mutants

Staphylococcal LukF and Hlg2 are water-soluble monomers of gamma-haemolysin that assemble into oligomeric pores on the erythrocyte membranes. Here, we have created double-cysteine LukF mutants, in which single disulphide bonds connect either the prestem domain and the cap domain (V12C-T136C, Cap-Stem), or two beta-strands within the prestem domain (T117C-T136C, Stem-Stem) to control pore assembly of gamma-haemolysin at intermediate stages. The disulphide-trapped mutants were inactive in erythrocyte lysis, but gained full haemolytic activity if the disulphide bonds were reduced. The disulphide bonds blocked neither the membrane binding ability nor the intermediate prepore oligomerization, but efficiently inhibited the transition from prepores to pores. The prepores of Cap-Stem were dissociated into monomers in 1% SDS. In contrast, the prepores of Stem-Stem were stable in SDS and had ring-shaped structures similar to those of wild-type LukF, as observed by transmission electron microscopy. The transition of both mutants from prepores to pores could even be achieved by reducing disulphide bonds at low temperature (2 degrees C), whereas prepore oligomerization was effectively inhibited by low temperature. Finally, real-time transition of Stem-Stem from prepores to pores on ghost cells, visualized using a Ca2+-sensitive fluorescent indicator (Rhod2), was shown by the sequential appearance of fluorescence spots, indicating pore-opening events. Taken together, these data indicate that the prepores are legitimate intermediates during gamma-haemolysin pore assembly, and that conformational changes around residues 117 and 136 of the prestem domain are essential for pore formation, but not for membrane binding or prepore oligomerization. We propose a mechanism for gamma-haemolysin pore assembly based on the demonstrated intermediates.     

Identification of the several new radicals formed in the reaction mixture of oxidized linoleic acid with ferrous ions using HPLC-ESR and HPLC-ESR-MS

ESR, HPLC-ESR and HPLC-ESR-MS analyses were performed for the reaction mixtures of oxidized linoleic acid with ferrous ions combined use of spin trapping technique. More than 14 peaks were detected on the HPLC-ESR elution profile. In addition to 7-carboxyheptyl and pentyl radicals, several new radicals such as 7-carboxy1dihydroxyheptyl, 1,5-dihydroxypentyl, 8-carboxy-1-hydroxyoctyl, 7-carboxy-1-hydroxyheptyl, 1-hydroxypentyl and 1-hydroxyhexyl were identified using HPLC-ESR and HPLC-ESR-MS.     

Non-invasive analysis of reactive oxygen species generated in NH4OH-induced gastric lesions of rats using a 300 MHz in vivo ESR technique

Free radicals are reportedly involved in mucosal injury, including NH_{>^{••- derived from neutrophils caused gastric lesion formation, while Opact">•-} or H2O2 derived from the xanthine oxidase system in endothelial cells was involved in neutrophil infiltration.}     

Characterization of factors involved in the production of 2(E)-nonenal during mashing

To characterize the factors involved in the production of volatile aldehydes during mashing, a model mashing experiment was done. After we inactivated the endogenous lipoxygenase (LOX) activity in the mash by mashing at 70 degrees C for 30 min, further incubation with recombinant barley LOX-1 stimulated the accumulation of 2(E)-nonenal; however, this effect was significantly reduced by boiling the mash sample. The result suggests that both LOX-1 and a heat-stable enzymatic factor are involved in the production of 2(E)-nonenal during mashing. Malt contained fatty acid hydroperoxide lyase-like activity (HPL-like activity) that transformed 9-hydroperoxy-10(E), 12(Z)-octadecadienoic and 13-hydroperoxy-9(Z), 11(E)-octadecadienoic acid into 2(E)-nonenal and hexanal, respectively. Proteinase K sensitivity tests showed that they are distinct factors. 9-HPL-like activity survived through the mashing at 70 degrees C for 30 min but was inactivated by boiling, suggesting it will be the heat-stable enzymatic factor found in the model mashing experiment.