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Bell Anthony J. Jr; Xin Hong; Taudte Susann; Shi Zhengshuang; Kallenbach Neville R. 《Protein engineering, design & selection : PEDS》2002,15(10):817-825
Using a cloned single domain of the high mobility group protein1 (HMGB1), we evaluated the effect of introducing metal bindingsite(s) on protein stability and function. An HMG domain isa conserved sequence of 80 amino acids rich in basic, aromaticand proline residues that is active in binding DNA in a sequence-or structure-specific manner. The design strategy focuses onanchoring selected regions of the protein, specifically loopsand turns in the molecule, using Hismetal ligands. Changesin secondary structure, thermostability and DNA binding propertiesof a series of such mutants were evaluated. The two most stablemutant constructs contain three surface histidine replacements(two metal binding sites) in the regions encompassing both turnsof the molecule. On ligation with the divalent nickel cation,the stability of these two triple histidine mutants (I38H/N51H/D55Hand G39H/N51H/D55H) increases by 1.3 and 1.6 kcal/mol, respectively,relative to the wild-type protein, although the creation ofbinding sites per se destabilizes the protein. The DNA-bindingproperties of the modified proteins are not impaired by theintroduction of the metal binding motifs. These results indicatethat it is feasible to stabilize protein tertiary structureusing appropriate placement of surface Hismetal bondswithout loss of function. 相似文献
223.
The proliferation and differentiation of oligodendrocyte progenitors are stringently controlled by an interacting network of growth and differentiation factors. Not much is known, however, about the intracellular signaling pathways activated in oligodendrocytes. In this study, we have examined the activation of mitogen-activated protein (MAP) kinase [also called extracellular signal-regulated protein kinases (ERKs)] in primary cultures of developing oligodendrocytes and in a primary oligodendrocyte cell line, CG4, in response to platelet-derived growth factor (PDGF) and basic fibroblast growth factor. MAP kinase activation was determined by an ingel protein kinase renaturation assay using myelin basic protein (MBP) as the substrate. The specificity of MAP kinase activation was further confirmed by an immune complex kinase assay using anti-MAP kinase antibodies. Stimulation of oligodendrocyte progenitors with the growth factors PDGF and basic fibroblast growth factor and a protein kinase C-activating tumor promoter, phorbol 12-myristate 13-acetate, resulted in a rapid activation of p42mapk (ERK2) and, to a lesser extent, p44mapk (ERK1). Immunoblot analysis with anti-phosphotyrosine antibodies revealed an increased Tyr phosphorylation of a 42-kDa phosphoprotein band cross-reacting with anti-MAP kinase antibodies. The phosphorylation of p42mapk in PDGF-treated oligodendrocyte progenitors was preceded by a robust autophosphorylation of the growth factor receptor. Immunoblot analysis with anti-pan-ERK antibodies indicated the presence of ERK-immunoreactive species other than p42mapk and p44mapk in oligodendrocytes. The presence of some of the same pan-ERK-immunoreactive species and certain renaturable MBP kinase activities was also demonstrable in myelin preparations from rat brain, suggesting that MAP kinases (and other MBP kinases) may function not only during oligodendrogenesis but also in myelinogenesis. 相似文献
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GF Combs C Garbisu BC Yee A Yee DE Carlson NR Smith AC Magyarosy T Leighton BB Buchanan 《Canadian Metallurgical Quarterly》1996,52(3):209-225
The bioavailability of selenium (Se) was determined in bacterial strains that reduce selenite to red elemental Se (SeO). A laboratory strain of Bacillus subtilis and a bacterial rod isolated from soil in the vicinity of the Kesterson Reservoir, San Joaquin Valley, CA, (Microbacterium arborescens) were cultured in the presence of 1 mM sodium selenite (Na2SeO3). After harvest, the washed, lyophilized B. Subtilis and M. arborescens samples contained 2.62 and 4.23% total Se, respectively, which was shown to consist, within error, entirely of SeO. These preparations were fed to chicks as supplements to a low-Se, vitamin E-free diet. Three experiments showed that the Se in both bacteria had bioavailabilities of approx 2% that of selenite. A fourth experiment revealed that gray SeO had a bioavailability of 2% of selenite, but that the bioavailability of red SeO depended on the way it was prepared (by reduction of selenite). When glutathione was the reductant, bioavailability resembled that of gray SeO and bacterial Se; when ascorbate was the reductant, bioavailability was twice that level (3-4%). These findings suggest that aerobic bacteria such as B. subtilis and M. arborescens may be useful for the bioremediation of Se-contaminated sites, i.e., by converting selenite to a form of Se with very low bioavailability. 相似文献
230.
A Asderakis T Augustine P Dyer C Short B Campbell NR Parrott RW Johnson 《Canadian Metallurgical Quarterly》1998,13(7):1799-1803
BACKGROUND: Dialysis can be life-saving for patients with end-stage renal failure. However, not only is it associated with significant morbidity and a greater mortality than transplantation, but it is also expensive. Therefore renal transplantation is generally regarded as the treatment of choice for patients in whom this form of renal replacement therapy is appropriate. Transplantation usually takes place after a variable period of dialytic therapy, but pre-emptive kidney transplantation (PKT) has established itself as an attractive alternative. MATERIALS AND METHODS: 1463 consecutive first kidney transplants performed between January 1980 and December 1995 in a single centre were analysed. The 161 patients (11%) transplanted without prior dialysis were compared with the 1302 patients who had been dialysed prior to being transplanted. The pre-emptive group did not differ from the dialysis group in respect of donor age, donor and recipient gender, HLA mismatch, or cold ischaemic time, although there were more live donor transplants within the pre-emptive group. RESULTS: Delayed graft function occurred more frequently in the dialysis group (25% vs 16%) but more patients experienced an acute rejection episode in the pre-emptive group (67 vs 55%). The actuarial graft survival in the pre-emptive group at 1, 5, and 10 years (84, 76 and 67%) was significantly higher than the respective values in the dialysis group (83, 69, and 56%). Within the live donor recipient cohort the survival advantage for the pre-emptive group was even more striking. CONCLUSION: Pre-emptive kidney transplantation not only avoids the risks, cost, and inconvenience of dialysis, but is also associated with better graft survival than transplantation after a period of dialysis, particularly within the live donor cohort. 相似文献