Purpose: Mouse double-stranded DNA-dependent protein kinase (DNA-PK) activity is heat sensitive. Recovery of heat-inactivated DNA repair activity is a problem after combination therapy with radiation and heat. We investigated the mechanism of recovery of heat-inactivated DNA-PK activity.
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
Summary— The regulation and role of the intracellular Ca2+ pools were studied in rat peritoneal mast cells. Cytosolic free calcium concentration ([Ca2+]i) was monitored in fura-2 loaded mast cells. In the presence of Ca2+ and K+, compound 48/80 induced a biphasic increase in [Ca2+]i composed of a fast transient phase and an apparent sustained phase. The sustained phase was partially inhibited by the addition of Mn2+. DTPA, a cell-impermeant chelator of Mn2+, reversed this inhibition, suggesting that a quenching of fura-2 fluorescence occurs in the extracellular medium. In the absence of extracellular Ca2+, the transient phase, but not the sustained one, could be preserved, provided that mast cells were depolarized. The transient phase was completely abolished by thapsigargin, a microsomal Ca2+-ATPase inhibitor. Maximum histamine release induced by either compound 48/80 or antigen was obtained in the absence of added Ca2+ only when mast cells were depolarized. These histamine releases were inhibited by low doses (< 30 nM) of thapsigargin. Thapsigargin at higher doses induced histamine release which was unaffected by changing the plasma membrane potential, but was completely dependent on extracellular Ca2+, showing that a Ca2+ influx is required for thapsigargin-induced exocytosis. Together, these results suggest that the mobilization of Ca2+ from thapsigargin sensitive-intracellular pools induced by compound 48/80 or antigen is sufficient to trigger histamine release. The modulation of these pools by the plasma membrane potential suggest their localization is close to the plasma membrane. 相似文献
Summary The efficiency of cold storage red blood cells (CSRBC) or whole blood at −80 °C used in 27 Rh(D) negative patients during
surgical operation was reported. The Rh(D) negative patients received the transfusion of CSRBC or whole blood stored at −80
°C for 180 to 360 days. The changes in the indexes, such as blood TB, DB, K+, Na+, BUN, Cr, urine protein (URPO), UOB, Hb, HCT, serum total protein, relative to hemolytic reaction and blood volume before
and after transfusion were observed. The results showed that after transfusion of CSRBC or whole blood 27 cases were negative
for urine protein and UOB, and the levels of BUN and Cr were normal (P>0.05). Blood TB, DB, Hb, and HCT were increased, while pH, blood K+ and blood Na+ was normal with the difference being not significant before and after operation (P>0.05). Plasma protein was decreased, but there was no significant difference before and after operation (P>0.05). It was suggested that CSRBC or whole blood at −80 °C could be safely infused to the Rh(D) negative patients without
side effects during the surgical operation.
YU Zhongqing, male, born in 1957, Technician in Charge 相似文献