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21.
Johanna Stern Amaranta Kahn Yael Vazana Melina Shamshoum Sarah Mora?s Raphael Lamed Edward A. Bayer 《PloS one》2015,10(5)
Degradation of cellulose is of major interest in the quest for alternative sources of renewable energy, for its positive effects on environment and ecology, and for use in advanced biotechnological applications. Due to its microcrystalline organization, celluose is extremely difficult to degrade, although numerous microbes have evolved that produce the appropriate enzymes. The most efficient known natural cellulolytic system is produced by anaerobic bacteria, such as C. thermocellum, that possess a multi-enzymatic complex termed the cellulosome. Our laboratory has devised and developed the designer cellulosome concept, which consists of chimaeric scaffoldins for controlled incorporation of recombinant polysaccharide-degrading enzymes. Recently, we reported the creation of a combinatorial library of four cellulosomal modules comprising a basic chimaeric scaffoldin, i.e., a CBM and 3 divergent cohesin modules. Here, we employed selected members of this library to determine whether the position of defined cellulolytic enzymes is important for optimized degradation of a microcrystalline cellulosic substrate. For this purpose, 10 chimaeric scaffoldins were used for incorporation of three recombinant Thermobifida fusca enzymes: the processive endoglucanase Cel9A, endoglucanase Cel5A and exoglucanase Cel48A. In addition, we examined whether the characteristic properties of the T. fusca enzymes as designer cellulosome components are unique to this bacterium by replacing them with parallel enzymes from Clostridium thermocellum. The results support the contention that for a given set of cellulosomal enzymes, their relative position within a scaffoldin can be critical for optimal degradation of microcrystaline cellulosic substrates. 相似文献
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Michael Andrew Pest Courtney Alice Pest Melina Rodrigues Bellini Qingping Feng Frank Beier 《PloS one》2015,10(11)
Background
Osteoarthritis (OA) is a degenerative joint disease with poorly understood etiology and pathobiology. Mitogen activated protein kinases (MAPKs) including ERK and p38 play important roles in the mediation of downstream pathways involved in cartilage degenerative processes. Dual specificity phosphatase 1 (DUSP1) dephosphorylates the threonine/serine and tyrosine sites on ERK and p38, causing deactivation of downstream signalling. In this study we examined the role of DUSP1 in spontaneous OA development at 21 months of age using a genetically modified mouse model deficient in Dusp1 (DUSP1 knockout mouse).Results
Utilizing histochemical stains of paraffin embedded knee joint sections in DUSP1 knockout and wild type female and male mice, we showed similar structural progression of cartilage degeneration associated with OA at 21 months of age. A semi-quantitative cartilage degeneration scoring system also demonstrated similar scores in the various aspects of the knee joint articular cartilage in DUSP1 knockout and control mice. Examination of overall articular cartilage thickness in the knee joint demonstrated similar results between DUSP1 knockout and wild type mice. Immunostaining for cartilage neoepitopes DIPEN, TEGE and C1,2C was similar in the cartilage lesion sites and chondrocyte pericellular matrix of both experimental groups. Likewise, immunostaining for phosphoERK and MMP13 showed similar intensity and localization between groups. SOX9 immunostaining demonstrated a decreased number of positive cells in DUSP1 knockout mice, with correspondingly decreased staining intensity. Analysis of animal walking patterns (gait) did not show a discernable difference between groups.Conclusion
Loss of DUSP1 does not cause changes in cartilage degeneration and gait in a mouse model of spontaneous OA at 21 months of age. Altered staining was observed in SOX9 immunostaining which may prove promising for future studies examining the role of DUSPs in cartilage and OA, as well as models of post-traumatic OA. 相似文献24.
Geobacillus, a bacterial genus, is represented by over 25 species of Gram-positive isolates from various man-made and natural thermophilic
areas around the world. An isolate of this genus (M-7) has been acquired from a thermal area near Yellowstone National Park,
MT and partially characterized. The cells of this organism are globose (ca. 0.5 μ diameter), and they are covered in a matrix
capsule which gives rise to elongate multicelled bacilliform structures (ranging from 3 to 12 μm) as seen by light and atomic
force microscopy, respectively. The organism produces unique petal-shaped colonies (undulating margins) on nutrient agar,
and it has an optimum pH of 7.0 and an optimum temperature range of 55–65°C. The partial 16S rRNA sequence of this organism
has 97% similarity with Geobacillus stearothermophilus, one of its closest relatives genetically. However, uniquely among all members of this genus, Geobacillus sp. (M-7) produces volatile organic substances (VOCs) that possess potent antibiotic activities. Some of the more notable
components of the VOCs are benzaldehyde, acetic acid, butanal, 3-methyl-butanoic acid, 2-methyl-butanoic acid, propanoic acid,
2-methyl-, and benzeneacetaldehyde. An exposure of test organisms such as Aspergillus fumigatus, Botrytis cinerea, Verticillium dahliae, and Geotrichum candidum produced total inhibition of growth on a 48-h exposure to Geobacillus sp.(M-7) cells (ca.107) and killing at a 72-h exposure at higher bacterial cell concentrations. A synthetic mixture of those available volatile
compounds, at the ratios occurring in Geobacillus sp. (M-7), mimicked the bioactivity of this organism. 相似文献
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Self-organization of MTOCs replaces centrosome function during acentrosomal spindle assembly in live mouse oocytes 总被引:8,自引:0,他引:8
Chromosome segregation in mammalian oocytes is driven by a microtubule spindle lacking centrosomes. Here, we analyze centrosome-independent spindle assembly by quantitative high-resolution confocal imaging in live maturing mouse oocytes. We show that spindle assembly proceeds by the self-organization of over 80 microtubule organizing centers (MTOCs) that form de novo from a cytoplasmic microtubule network in prophase and that functionally replace centrosomes. Initially distributed throughout the ooplasm, MTOCs congress at the center of the oocyte, where they contribute to a massive, Ran-dependent increase of the number of microtubules after nuclear envelope breakdown and to the individualization of clustered chromosomes. Through progressive MTOC clustering and activation of kinesin-5, the multipolar MTOC aggregate self-organizes into a bipolar intermediate, which then elongates and thereby establishes chromosome biorientation. Finally, a stable barrel-shaped acentrosomal metaphase spindle with oscillating chromosomes and astral-like microtubules forms that surprisingly exhibits key properties of a centrosomal spindle. 相似文献
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Hogg ME Varu VN Vavra AK Popowich DA Banerjee MN Martinez J Jiang Q Saavedra JE Keefer LK Kibbe MR 《Free radical biology & medicine》2011,50(9):1065-1074
Nitric oxide (NO)-based therapies decrease neointimal hyperplasia; however, studies have been performed only in male animal models. Thus, we sought to evaluate the effect of NO on vascular smooth muscle cells (VSMC) in vitro and neointimal hyperplasia in vivo based on sex and hormone status. In hormone-replete medium, male VSMC proliferated at greater rates than female VSMC. In hormone-depleted medium, female VSMC proliferated at greater rates than male VSMC. However, in both hormone environments, NO inhibited proliferation and migration to a greater extent in male compared to female VSMC. These findings correlated with greater G0/G1 cell cycle arrest and changes in cell cycle protein expression in male compared to female VSMC after exposure to NO. Next, the rat carotid artery injury model was used to assess the effect of NO on neointimal hyperplasia in vivo. Consistent with the in vitro data, NO was significantly more effective at inhibiting neointimal hyperplasia in hormonally intact males compared to females using weight-based dosing. An increased weight-based dose of NO in females was able to achieve efficacy equal to that in males. Surprisingly, NO was less effective at inhibiting neointimal hyperplasia in castrated animals of both sexes. In conclusion, these data suggest that NO inhibits neointimal hyperplasia more effectively in males compared to females and in hormonally intact compared to castrated rats, indicating that the effects of NO in the vasculature may be sex- and hormone-dependent. 相似文献
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Sulfurtransferases/rhodaneses (Str) comprise a group of enzymes widely distributed in all phyla which catalyze in vitro the
transfer of a sulfur atom from suitable sulfur donors to nucleophilic sulfur acceptors. The best characterized Str is bovine
rhodanese (EC 2.8.1.1) which catalyses in vitro the transfer of a sulfane sulfur atom from thiosulfate to cyanide, leading
to the formation of sulfite and thiocyanate. Plants as well as other organisms contain many proteins carrying a typical rhodanese
pattern or domain forming multi-protein families (MPF). Despite the presence of Str activities in many living organisms, the
physiological role of the members of this MPF has not been established unambiguously. While in mammals these proteins are
involved in the elimination of toxic cyanogenic compounds, their ubiquity suggests additional physiological functions. In
plants, Str are localized in the cytoplasm, mitochondria, plastids, and nucleus. Str probably also transfer reduced sulfur
onto substrates as large as peptides or proteins. Several studies in different organisms demonstrate a protein–protein interaction
with members of the thioredoxin MPF indicating a role of Str in maintenance of the cellular redox homeostasis. The increased
expression of several members of the Str MPF in various stress conditions could be a response to oxidative stress. In summary,
data indicate that Str are involved in various essential metabolic reactions. 相似文献
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Rohloff P Miranda K Rodrigues JC Fang J Galizzi M Plattner H Hentschel J Moreno SN 《PloS one》2011,6(4):e18390
Acidocalcisomes are acidic calcium stores found in diverse organisms, being conserved from bacteria to humans. They possess an acidic matrix that contains several cations bound to phosphates, which are mainly present in the form of short and long polyphosphate chains. Their matrix is acidified through the action of proton pumps such as a vacuolar proton ATPase and a vacuolar proton pyrophosphatase. Calcium uptake occurs through a Ca(2+)/H(+) countertransporting ATPase located in the membrane of the organelle. Acidocalcisomes have been identified in a variety of microorganisms, including Apicomplexan parasites such as Plasmodium and Eimeria species, and in Toxoplasma gondii. We report the purification and characterization of an acidocalcisome fraction from T. gondii tachyzoites after subcellular fractionation and further discontinuous iodixanol gradient purification. Proton and calcium transport activities in the fraction were characterized by fluorescence microscopy and spectrophotometric methods using acridine orange and arsenazo III, respectively. This work will facilitate the understanding of the function of acidocalcisomes in Apicomplexan parasites, as we can now isolate highly purified fractions that could be used for proteomic analysis to find proteins that may clarify the biogenesis of these organelles. 相似文献