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Methods: Hybrid cells containing a fragment of human chromosome 8 in scid cells (RD13B2) were used. DNA-PK activity was measured by an in vitro assay. Immunoprecipitation of the nuclear extract was performed with an anti-Ku80 antibody. Proteins co-precipitated with Ku80 were separated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and detected by Western blotting using anti-heat shock protein (HSP)72 and anti-heat shock cognate protein (HSC)73 antibodies. HSC73 was overexpressed with the pcDNA3.1 vector. Short hairpin (sh)RNA was used to downregulate HSC73 and HSP72.
Results: The activity of heat-inactivated DNA-PK recovered to about 50% of control during an additional incubation at 37?°C after heat treatment at 44?°C for 15?min in the presence of cycloheximide (which inhibits de novo protein synthesis). Maximal recovery was observed within 3?h of incubation at 37?°C after heat treatment. Constitutively expressed HSC73, which folds newly synthesized proteins, reached maximal levels 3?h after heat treatment using a co-immunoprecipitation assay with the Ku80 protein. Inhibiting HSC73, but not HSP72, expression with shRNA decreased the recovery of DNA-PK activity after heat treatment.
Conclusions: These results suggest that de novo protein synthesis is unnecessary for recovery of some heat-inactivated DNA-PK. Rather, it might be reactivated by the molecular chaperone activity of HSC73, but not HSP72. 相似文献
Background
On April 1, 2016, the Ministerial ordinance was enforced, and musculoskeletal examination of the extremities was made mandatory. From 2008, the University of us started musculoskeletal direct examination. To expand the examination, from 2016, we started to use the marksheet-type questionnaire. This study aimed to report the results of a musculoskeletal examination and investigate the association between musculoskeletal examination and age/gender and reports the reliability of the collected questionnaire data.Methods
Direct musculoskeletal examination was performed in K school by 7 orthopedic surgeons. A marksheet-type screening questionnaire was distributed to all the elementary and junior high school students in Tsukuba and Hitachiomiya cities. The rates of abnormal findings for scoliosis, standing flexion, full squatting with the heels on the floor, general joint laxity, and standing on one leg, torticollis, and flat feet were calculated. We compared the results of the questionnaire and direct examination and calculated sensitivity, specificity, and odds ratio.Results
A total of 1844 students in K school had direct examination, and 22,494 questionnaires were able to correct in Tsukuba and Hitachiomiya cities. The rates of abnormal findings in direct examination/questionnaire in scoliosis, standing flexion, full squat, general joint laxity, standing on one leg, torticollis and flat foot were 18.7% (344/1842)/5.1% (1094/21441), 20.2% (372/1841)/26.6% (5817/22078), 6.2% (114/1832)/6.9% (1516/22101), 7.5% (1648/22252), 4.9% (1100/22077), 2.2% (31/1844)/1.2% (272/21687), and 12.5% (231/1842)/8.7% (1785/20871), respectively. Sensitivities of the questionnaire for scoliosis, stand flexion, full squatting, torticollis, and flat feet were 16.8% (53/316), 67.9% (250/368), 48.2% (55/114), 18.9% (7/37), and 32.2% (65/202), respectively.Conclusion
We reported the result of musculoskeletal examination. Accuracy and reliability of this questionnaire were not satisfactory. To perform high quality musculoskeletal examinations, we will aim to increase the quality of screening methods. 相似文献![点击此处可从《Journal of neurology, neurosurgery, and psychiatry》网站下载免费的PDF全文](/ch/ext_images/free.gif)