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71.
A Aliverti T Jansen G Zanetti S Ronchi R G Herrmann B Curti 《European journal of biochemistry》1990,191(3):551-555
A cDNA clone for the preprotein of spinach ferredoxin:NADP+ reductase has been modified to allow the expression in Escherichia coli of the mature flavoprotein form the lacks the transit peptide. An expression vector, pFNR1, was constructed by subcloning the fragment into the plasmid pDS12/RBSII, SphI. In the crude extracts of transformed cells after induction, two active holoproteins of 35 kDa and 32 kDa, respectively, were found. The 32-kDa protein, purified by immunoaffinity chromatography, was found to lack the first 28 residues of the spinach protein sequence and to have a methionine as the N-terminal residue instead of Val29. A new expression plasmid, pFNR2, was obtained by in vitro mutagenesis of the codon GTG for Val29 to the synonymous GTT; in this case, only the 35-kDa protein was expressed by transformed cells. Both the 35-kDa and 32-kDa enzymes were purified and characterized. All the properties analyzed of the cloned 35-kDa enzyme were very similar to those of the spinach flavoprotein. The 32-kDa form showed the same catalytic efficiency of the spinach enzyme as a diaphorase but its interaction with oxidized ferredoxin was partially impaired. 相似文献
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Grassano A. Ronchi A.L. Lorda G. Balatti A.P. 《World journal of microbiology & biotechnology》1999,15(1):93-97
The growth rate of different strains of Bradyrhizobium and Rhizobium was studied in media containing amaranth seed meal instead of yeast extract. Results obtained in erlenmeyer flasks and stirred fermenters show that both Bradyrhizobium japonicum strains E109, E110, 5019, 587 and Rhizobium melilotistrains B36, B323, B399, Lq22, Lq42, Lq51 and U322, grow satisfactorily in amaranth seed meal medium. Cell count obtained for the strains tested was greater than 4 × 1010 viable cells.ml–1. Amaranth seed meal (4 g.l–1) is a suitable component for culture media that can be used instead of yeast extract. 相似文献
74.
Tedeschi G Cappelletti G Negri A Pagliato L Maggioni MG Maci R Ronchi S 《Proteomics》2005,5(9):2422-2432
Nitric oxide (NO) is a precursor of reactive nitrating species, peroxynitrite and nitrogen dioxide, which modify proteins to generate oxidized species such as 3-nitrotyrosine that has been used as a hallmark of peroxynitrite-mediated oxidative stress on proteins. In the last few years however, a growing body of evidence indicates that NO also regulates a myriad of physiologic responses by modifying tyrosine residues. Looking for the molecular event triggered by NO in nerve growth factor (NGF)-induced neuronal differentiation, we recently reported that in differentiating PC12 cells, the cytoskeleton becomes the main cellular fraction containing nitrotyrosinated proteins, and alpha-tubulin is the major target. In the present work, we focus on the investigation of the sites of tyrosine nitration in alpha-tubulin purified by two-dimensional gel electrophoresis following anti-alpha-tubulin immunoprecipitation of protein extract from NGF-treated PC12 cells. Using Western blotting and matrix-assisted laser desorption/ionization-time of flight analysis, we show for the first time, both in vivo and in vitro, that nitration can occur on alpha-tubulin at sites other than the C-terminus and we positively identify Tyr 161 and Tyr 357 as two specific amino acids endogenously nitrated. 相似文献
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M. Di Paola Lipucci D. Fossi F. Tognoni V. Nuti Ronchi 《Plant Cell, Tissue and Organ Culture》1987,10(1):3-10
Juvenile leaves of Cupressus arizonica Green (3–5 mm in length) from eight week old seedlings, were cultured on liquid medium supplemented with isopentenyladenine (2 mgl-1). Buds formed from the explants after three weeks of culture, but further growth occurred only after transfer to half-strength medium without plant growth regulators. Histological analysis at different times of culture, showed an early mitotic activity within transfusion tissue, followed by dedifferentiation of epidermal and mesophyll parenchyma cells at the basal zone of the leaves. The differentiation of vascular nodules always preceded bud formation. The difficulty of conifers to root and grow beyond plantlet stage is discussed. 相似文献
79.
Abstract Cyto-histological changes accompanying the formation of adventitious buds in excised cotyledons of Lactuca sativa were studied during the first 12 days after planting in vitro. Prospective proliferating cells can first be recognized, already on the first day after planting, by a marked increase in nuclear and nucleolar volumes, followed on the second day by a burst of cell divisions involving particularly mesophyll cells. Then lignified elements develop together with meristematic center, forming a callus-like tissue in the inner part of the cotyledons. At the third day of culture, the epidermal cells start to divide with a periclinal wall followed by an anticlinal division. In the following days of culture the epidermal cells, which divide mainly with periclinal walls, form layers of cells below the surface, gradually filling up the intercellular spaces. From the 8th day on, the buds protude above the surface and develops into shoots. These results are discussed in relation to DNA content of nuclei of Lactuca sativa cotyledons and to the time course of cell division and tracheary element formation. The very regular sequence of changes associated with the initiation and development of the bud makes the in vitro culture of Lactuca cotyledons an appropriate System for histochemical and biochemical studies. 相似文献
80.
Frederick C. Streich Jr. Virginia P. Ronchi J. Patrick Connick Arthur L. Haas 《The Journal of biological chemistry》2013,288(12):8209-8221
Ligation of polyubiquitin chains to proteins is a fundamental post-translational modification, often resulting in targeted degradation of conjugated proteins. Attachment of polyubiquitin chains requires the activities of an E1 activating enzyme, an E2 carrier protein, and an E3 ligase. The mechanism by which polyubiquitin chains are formed remains largely speculative, especially for RING-based ligases. The tripartite motif (TRIM) superfamily of ligases functions in many cellular processes including innate immunity, cellular localization, development and differentiation, signaling, and cancer progression. The present results show that TRIM ligases catalyze polyubiquitin chain formation in the absence of substrate, the rates of which can be used as a functional readout of enzyme function. Initial rate studies under biochemically defined conditions show that TRIM32 and TRIM25 are specific for the Ubc5 family of E2-conjugating proteins and, along with TRIM5α, exhibit cooperative kinetics with respect to Ubc5 concentration, with submicromolar [S]0.5 and Hill coefficients of 3–5, suggesting they possess multiple binding sites for their cognate E2-ubiquitin thioester. Mutation studies reveal a second, non-canonical binding site encompassing the C-terminal Ubc5α-helix. Polyubiquitin chain formation requires TRIM subunit oligomerization through the conserved coiled-coil domain, but can be partially replaced by fusing the catalytic domain to GST to promote dimerization. Other results suggest that TRIM32 assembles polyubiquitin chains as a Ubc5-linked thioester intermediate. These results represent the first detailed mechanistic study of TRIM ligase activity and provide a functional context for oligomerization observed in the superfamily. 相似文献