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
Objective: To investigate whether cockroach allergen extract can stimulate Protease-activated receptor 2 (PAR-2) expressed in mouse lung fibroblast.Materials: We established an immortalized lung fibroblast cell line, DM5, from PAR-2 deficient mice. By stable transfection with either an empty vector (DM5/EV) or an expression vector encoding mouse PAR-2 cDNA (DM5/Par2), a pair of lung fibroblast cell lines with or without functional PAR-2 expression were prepared.Treatment: The cells were exposed to cockroach allergen extract {up to 800 protein nitrogen unit (PNU)/ml}, trypsin (up to 100 nM), SLIGRL agonist peptide (up to 500 M), and trans-cinnamoyl-LIGRO agonist peptide (up to 400 M).Methods: The cells were loaded with Fluo-3 calcium indicator and mobilization of intracellular calcium with the stimuli was monitored by a fluorometric plate reader. Extracellular signal-regulated kinase (ERK) phosphorylation was examined by Western blot analysis using an anti-phospho ERK antibody.Results: The cockroach extract induced intracellular calcium transients in a concentration dependent manner in DM5/Par2 but not in DM5/EV. The activity was abolished when the cockroach extract was heat denatured or pre-incubated with PMSF (phenylmethanesulfonyl fluoride) prior to the assay. Concomitantly, ERK phosphorylation was seen in DM5/Par2 with the cockroach extract but not with a heat-denatured extract. The responses were sensitive to an inositol-1,4,5 triphosphate antagonist (2-APB) indicating that calcium was mobilized from intracellular store.Conclusions: Cockroach allergen extract can activate PAR-2 and thereby stimulate mouse lung fibroblasts likely through protease(s). The present study proposes a potential mechanism for cockroach antigens, similar to house dust mite antigens, in the etiology of respiratory diseases.Received 29 February 2004; returned for revision 12 April 2004; accepted by M. Katori 22 April 2004 相似文献
Clinical and Experimental Nephrology - The progression of chronic kidney disease (CKD) depends on the extent of fibrosis in the kidneys; however, a renal biopsy is necessary to evaluate the... 相似文献
Human SART-1 ( hSART-1 ) gene encodes a 125 kD protein with a leucine-zipper motif expressed in the nucleus of all proliferating cells, and a 43 kD protein expressed in the cytosol of most epithelial cancers. In this study, two rodent genes ( rSART-1 and mSART-1 ) homologous to hSART-1 were cloned from cDNA libraries of murine brain and a rat tumor cell line, respectively. mSART-1 and rSART-1 were highly homologous to hSART-1 with 86% and 84% identity at the nucleotide level, and 95% and 91% at the protein level, respectively. The leucine zipper domain and two basic amino acid portions that bind DNA, as well as peptide sequences recognized by human cyto-toxic T lymphocytes (CTLs), were all conserved in these rodent genes. Nuclear protein homologous to the 125 kD hSART-1800 protein, but not to the 43 kD cytosol SART-1259 protein, was detectable with specific antibody in the nuclear fractions of rodent tumor cell lines, and normal rodent fetal liver and testis. These rodent genes should be a novel tool for studies on the biological roles of the SART-1 gene, and also in the construction of animal models of specific immuno-therapy using SART-1 gene products. 相似文献
A 63-year-old woman complicated with liver cirrhosis and pancytopenia was admitted for aortic and mitral valve replacement. As laboratory findings at time of admission showed pancytopenia with Hb of 7.3 g/dl, WBC of 2,200/mm3, and platelet of 6.2 x 10(4)/mm3, splenectomy was first conducted and the blood cells and platelet increased in number. At 27 days after splenectomy, double vale replacement was performed without blood transfusion and her postoperative course was unevenfull. It is considered that preoperative splenectomy is useful in management of patients complicated with hypersplenism and pancytopenia. 相似文献
Mucosal cancer in the gastrointestinal tract generally has low risk of lymph node metastasis. Endoscopic mucosal resection (EMR) and endoscopic submucosal dissection (ESD) are techniques of local excision of neoplasia confined to the mucosal layer. Specimens from EMR/ESD contribute to several diagnoses, and histologic results affect treatment decisions. A combined laparoscopic and endoscopic approach to neoplasia with a nonexposure technique allows full-thickness resection of the stomach wall without exposing the gastric lumen to the peritoneal cavity, preventing cancer cell dissemination to the peritoneal cavity. This article reviews EMR/ESD and describes a new full-thickness resection method using the nonexposure technique (CLEAN-NET). 相似文献
PURPOSE: The purpose of this work was to provide a scientific basis for specific immunotherapy of colon cancer. EXPERIMENTAL DESIGN: This study focused on identification of colon tumor-associated antigens and HLA-A2-restricted and tumor-reactive cytotoxic T lymphocytes (CTLs) generated from tumor-infiltrating lymphocytes of a colon cancer patient. A gene expression cloning method was used to identify genes coding for tumor antigens. Fifty-six peptides with HLA-A2-binding motifs encoded by these proteins were examined for their ability to induce HLA-A2-restricted and tumor-reactive CTLs. RESULTS: We identified the following three genes coding for proliferation-related proteins: thymidylate synthase (TYMS), which is involved in chemoresistance (5-fluorouracil); 5'-aminoimidazole-4-carboxamide-1-beta-d-ribonucleotide transfolmylase/inosinicase (AICRT/I); and phosphoglycerate kinase 1 (PKG1), which was secreted by tumor cells and involved in the angiogenic process. TYMS was preferentially expressed in tumor cells, whereas AICRT/I and PKG1 were equally expressed in both cancer cells and normal tissues at the mRNA level. Among 56 peptides with HLA-A2-binding motifs encoded by these proteins, 8 peptides were recognized by the CTLs, and 5 of 8 peptides were also recognized by the CTL precursors without ex vivo activation in the peripheral blood of colon cancer patients. Furthermore, four of them (one each from TYMS and PKG1 and two from AICRT/1) possessed the ability to induce HLA-A2-restricted and peptide-specific CTLs cytotoxic to colon tumor cells in peripheral blood mononuclear cells of colon cancer patients. CONCLUSIONS: TYMS and PGK1, as well as their epitope peptides, might be appropriate target molecules for specific immunotherapy of HLA-A2(+) colon cancer patients because of the positive role of TYMS and PGK1 in chemoresistance (5-fluorouracil) and angiogenesis of tumor cells, respectively. 相似文献