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
Sensitivity of normotensive Wistar rats and NISAG rats (with hereditary arterial hypertension) to heat stress is compared
at the organism and cell levels. High temperature sensitivity of NISAG rats correlates with a low content of the main heat
shock protein HSP70. This relationship can serve as a biochemical marker of predisposition to arterial hypertension.
Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 124, No. 8, pp. 171–173, August, 1997 相似文献
The expression of two heat shock proteins, HSP72 and p57, in addition to ubiquitin, has been studied immunocytochemically in nine amyotrophic lateral sclerosis (ALS) cases and 10 age-matched controls. HSP72 and p57 antibodies did not identify the characteristic ubiquitin-immunoreactive inclusions present in anterior horn cells in ALS spinal cord. Antibodies to HSP72, but not to p57 or ubiquitin, strongly labelled structures corresponding to polyglucosan bodies in spinal grey matter. Such immunoreactive profiles were more abundant in ALS cases, although they were also present in control material. They were sometimes identified by haematoxylin and eosin and periodic acid Schiff reaction, but were not labeled by phosphotungstic acid haematoxylin or by antibodies to glial fibrillary acidic protein. Although ubiquitin, HSP72 and p57 are stress-induced proteins, they are expressed differently and might therefore have different significance in neuronal degeneration. 相似文献
Objectives Cutaneous burns are dynamic injuries with a central zone of necrosis surrounded by a zone of ischemia. Conversion of this ischemic zone to full necrosis over the days following injury is due in part to highly reactive oxygen radicals. Curcumin is a component of the Oriental spice turmeric that has been shown to have antioxidant and antiapoptotic properties. The authors hypothesized that treatment of burns with curcumin would reduce the conversion of the ischemic zone to full necrosis. Methods This was a randomized controlled experiment. Twenty Sprague‐Dawley rats were used. Two burns were created on each animal's dorsum using a brass comb with four rectangular prongs preheated in boiling water and applied for 30 seconds, resulting in four rectangular 10 × 20–mm full‐thickness burns separated by three 5 × 20–mm unburned interspaces (zone of ischemia). Animals were randomized to curcumin or vehicle by oral gavage 30 minutes before injury and at 24, 48, and 72 hours after injury. Wounds were observed at one, two, and three days after injury for visual evidence of necrosis in the unburned interspaces. Full‐thickness biopsy specimens from the interspaces were evaluated with hematoxylin and eosin staining seven days after injury for evidence of necrosis. The percentage of interspaces that progressed to necrosis was compared with chi‐square tests. Results Forty comb burns with 120 unburned interspaces were created, evenly distributed between curcumin and vehicle alone. The percentage of interspaces that progressed to full‐thickness necrosis at one, two, three, and seven days after injury in the curcumin and vehicle groups were 30% versus 63% (p = 0.003), 30% versus 70% (p < 0.001), 63% versus 95% (p = 0.02), and 63% versus 95% (p = 0.02), respectively. Conclusions Pretreatment of rats with oral curcumin followed by once‐daily oral treatment for three days reduced the percentage of unburned skin interspaces that progressed to full necrosis. 相似文献