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
The clinical course of malignant melanomas is frequently unpredictable, although a number of prognostically useful variables can be identified. There is a need for additional markers of prognostic value. In a series of 60 malignant cutaneous melanomas, we analysed the immunohistochemical expression of c-myc proto-oncogene, heat shock protein 70 (HSP70) and HLA-DR molecules in order to investigate their prognostic significance. C-myc, HSP70 and HLA-DR were expressed in 43.3%, 56.6% and 38.3% of all melanoma cases, respectively. Advanced Clark levels (Clark III–V) were significantly associated with c-myc expression rate (P<0.05), HSP70 detection (P<0.01) and HLA-DR positivity (P<0.01). Increased Breslow thickness (>1.5 mm) was related to HLA-DR expression (P<0.05). High mitotic rate was closely associated with c-myc positivity (P<0.05), while HSP70 and HLA-DR expression separately correlated to clinical stage of the disease (P<0.05). The evaluation of these variables may be of immunological and prognostic significance. They were found to be associated with melanocyte subpopulations of the vertical growth phase which are arguably characterized by an increased invasive potential. 相似文献
Ethanol-induced fatty liver in rats was attenuated by repeated running exercise, and the protective effect of exercise was associated with the synergistic expression of heat shock proteins (HSP72). Rats were placed in four groups of six. The two ethanol-fed groups of rats received a liquid diet (Lieber-DeCarli formulation) in which 36% of the calories were derived from ethanol. One group remained sedentary (S/E), whereas the other was trained to run on a rodent treadmill at a speed of 27 m/min, 1 hr/day, 5 days/week, for 7 weeks (R/E). Two other groups–one exercised as previously mentioned (R/C) and one sedentary (S/C)–received control-liquid diets in which the ethanol was isocalorically substituted with a dextran/maltose mixture. The degree of fatty infiltration in liver sections stained with hematoxylin and eosin was graded on a 0–4 scale and the data analyzed by ANOVA on ranks. Ethanol significantly induced fatty infiltration in the S/E group, whereas fatty infiltration in the livers of the R/E group was not different from the S/C group. Electrophoresis and Western blotting of liver homogenates demonstrated that HSP72 was not expressed in either the S/C or S/E groups and was only slightly expressed in the R/C group. The combination of exercise and ethanol, however, resulted in an elevated expression of HSP72 in the R/E group. The content of HSP73 was unaffected by any treatment. 相似文献
Seven mixed-breed dogs were challenged with either promastigotes or amastigotes of Leishmania donovani infantum strains recently isolated from naturally infected dogs. Different routes and numbers of parasites were utilized and each dog was monitored for at least 1 year post-infection. Anti-parasite specific antibody levels were measured by enzyme-linked immunosorbence, immunofluorescence, crossed-immune electrophoresis and Western blotting on crude antigen. Western blotting on two pure parasite proteins, dp72 and gp70-2, was also done. Mitogenic and antigen-specific stimulation of peripheral blood lymphocytes was monitored; and the haematological, clinical and parasitological parameters measured. Dogs challenged with amastigotes exhibited a more pronounced humoral response to leishmanial antigens. Only in one case was strong antigen-specific proliferation detected. Clinical signs of disease, including hypergammaglobulinaemia, enlarged lymph nodes and the presence of parasites, were also more apparent in the dogs challenged with amastigotes. None of the seven dogs died. Serum antibodies to leishmanial antigens were apparent between 1.5 to 3 months following challenge and correlated with the appearance of enlarged lymph nodes, hypergammaglobulinaemia and the presence of parasites in tissue biopsies. Serum antibodies remained chronically high in these dogs throughout the period of the study. Only one dog (1/3) challenged intravenously with promastigotes and the dog challenged intradermally with amastigotes produced transient antibody responses to leishmanial antigen. 相似文献
After synthesis and folding, peptides and proteins undergo changes in charge and conformation through nonenzymatic deamidation of asparaginyl and glutaminyl residues. Each amide has a specific deamidation rate that is genetically determined by the sequence of residues immediately adjacent in the peptide chain and by secondary, tertiary, and quaternary structure. By means of experimentally verified computations, we have determined the deamidation rates of 49 Drosophila peptides and proteins. These rates demonstrate that deamidation provides molecular clocks that are suitable for the regulation of Drosophila aging, development, and other biochemical processes. We have also determined the rates of deamidation for 17,886 other proteins from a wide variety of organisms. The distribution function of these deamidation rates demonstrates the suitability of amide residues as biomolecular clocks. 相似文献