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The bacterial chemotaxis protein CheY is activated in vivo by the covalent
phosphorylation of a single aspartate residue at position 57. However, this
phosphate linkage is unstable (t1/2 approximately 20 s at room
temperature), thereby precluding many biochemical analyses. Here we present
a synthetic scheme to prepare an analog of CheY-phosphate (Che Y-P) with
chemical stability of the phosphate linkage enhanced by several orders of
magnitude relative to the native protein. Starting with CheY D57C, a
site-specific mutant of CheY with a unique cysteine residue in place of the
aspartate at position 57, two sequential disulfide exchange reactions were
performed to form the final product 'CheY D57C-SPO3' with a thiophosphate
moiety covalently bonded to the protein in a disulfide linkage. Mass
spectral analysis showed that the desired analog was present at 70-80% of
the total protein. The disulfide linkage had a t1/2 of 8 days at 4 degrees
C. Biochemical characterization of CheY D57C-SPO3 included assessment of
conformational properties using tryptophan fluorescence, evaluation of
metal binding properties and measurement of binding interactions with the
chemotaxis proteins CheZ and FliM. Despite possessing a phosphoryl group at
a nearly identical location as native CheY-phosphate, the analog was unable
to emulate CheY-phosphate function, thereby supporting the idea that there
are very precise geometric requirements for successful CheY activation.
相似文献
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The Special Surveillance Breast Program (SSBP) is a long-term, comprehensive, multidisciplinary program for women determined to be at high risk for the development of breast cancer. Because the women who attend this program are otherwise healthy but concerned about their risk for breast cancer, current and relevant information is required to address their concerns regarding the possibility of developing breast cancer. The purpose of this article is to describe the risk factors that indicate eligibility for the program, the structure of the program, and the results of an assessment that identified the needs of this special population. 相似文献
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JR Passweg G Socié W Hinterberger A Bacigalupo JC Biggs BM Camitta RE Champlin RP Gale E Gluckman EC Gordon-Smith JM Hows JP Klein ML Nugent R Pasquini PA Rowlings B Speck A Tichelli MJ Zhang MM Horowitz MM Bortin 《Canadian Metallurgical Quarterly》1997,90(2):858-864
Bone marrow transplants for severe aplastic anemia were first performed in the 1970s. Transplant regimens, supportive care, and patient selection have changed substantially since then. Our objective was to determine the impact of these changes on transplant outcome. We studied 1,305 recipients of HLA-identical sibling transplants for aplastic anemia between 1976 and 1992, reported to the IBMTR by 179 centers. We compared survival of transplants performed in three intervals (1976 through 1980 [n = 186], 1981 through 1987 [n = 648], and 1988 through 1992 [n = 471]) using Cox proportional hazards regression. Five-year survival (+/-95% confidence interval) increased from 48% +/- 7% in the 1976-1980 cohort to 66% +/- 6% in the 1988-1992 cohort (P < .0001). Risks of graft-versus-host disease (GVHD) and interstitial pneumonia decreased over time, but the risk of graft failure did not. Higher long-term survival resulted primarily from decreased mortality in the first 3 months posttransplantation. Late mortality risks were low and changed little over the intervals studied. In multivariate analysis, changes in transplantation strategies accounted for most but not all of the improved outcome. Use of cyclosporine to prevent GVHD was the most important factor. Changes in patient selection did not seem to explain improved survival. Survival after HLA-identical sibling bone marrow transplantations for aplastic anemia has improved since 1976. Changes in GVHD prophylaxis account for much of this improvement. Other changes may also operate. 相似文献
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RE Bonshek 《Canadian Metallurgical Quarterly》1997,8(6):363-365
OBJECTIVE: To assess whether accident and emergency (A&E) nurses using the Ottawa Ankle Rules could detect all ankle fractures. DESIGN: Prospective observational study. SETTING: A&E department of a university teaching hospital. SUBJECTS: All patients who presented with ankle injuries who were initially assessed by a nurse taught the Ottawa Ankle Rules. OUTCOME MEASURES: (1) The numbers of patients referred by the nurse for ankle radiography; (2) of these, the number with ankle fractures; (3) of those not sent for radiography initially by the nurse, the number who subsequently had x rays (ordered by the doctor) and had a fracture; (4) of those having no x rays, the number who reattended later. RESULTS: 324 patients were eligible; 238 had x rays at the request of the nurse (73%); 48 of these (20%) were diagnosed as having a fracture. Of those 86 patients not sent for radiography by the nurse, 19 subsequently had x ray examinations at the request of a doctor and no fracture was detected. Of the 67 not sent for radiography, none returned within the subsequent eight weeks. CONCLUSIONS: Nurses can apply the Ottawa Ankle Rules safely without missing acute fractures; that is, of those who were not sent for radiography by nurses, none subsequently reattended the A&E department or the trauma service of the Bristol Royal Infirmary during the following two months. 相似文献
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Although our understanding of molecular events in septic shock is growing exponentially, bedside management has changed only incrementally over the last 20 years. In pediatric and adult patients alike, treatment continues to be largely supportive. Morbidity and mortality, though gradually improving, continue to be high. The major similarities, as well as the minor differences, between pediatric and adult septic shock are reviewed in this article, with an emphasis on current clinical practice and recent clinical investigations of novel therapies. 相似文献