Reactivation of heat-inactivated Ku proteins by heat shock cognate protein HSC73

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.     

Chemotherapy in the treatment of advanced or recurrent olfactory neuroblastoma

Background: Olfactory neuroblastoma is a rare sino‐nasal tumor arising from the olfactory epithelium and is often characterized by local invasion or metastasis. The role of chemotherapy in the treatment of this tumor is unclear. The purpose of this study was to review our institution’s experience of chemotherapy for advanced or recurrent olfactory neuroblastoma. Methods: Twenty‐one patients with histologically proven olfactory neuroblastoma were treated at our institution between 1992 and 2002. Twelve of these patients received chemotherapy in the setting of unresectable or recurrent disease and were retrospectively reviewed for clinical characteristics, treatment outcome or survival. Results: Eight patients of the 12 patients received cisplatin‐based chemotherapy and the remaining four patients received chemotherapy consisting of docetaxel plus irinotecan (three patients) or cyclophosphamide, doxorubicin, and vincristine (1 patient). A partial response was achieved in five patients, with an overall response rate of 42%, although the chemotherapeutic regimens were heterogeneous. Two partial responses were obtained among the three patients who received docetaxel plus irinotecan. The response rate to chemotherapy was 83% in the younger age group (<40 years), as opposed to 0% in the older age group (≥40 years), and the difference between the two groups was statistically significant (P = 0.02). Conclusion: Our study indicated that olfactory neuroblastoma would be sensitive to chemotherapy, especially with young patients. Docetaxel plus irinotecan has the possibility of showing favorable response, and warrants further investigation.     

Monitoring of intracellular Ca2+ elevation in a single neural cell using a fluorescence microscope/video-camera system

For monitoring the changes in intracellular free Ca2+ concentration ([Ca2+]i), we developed a simple system combining a fluorescence microscope, an image intensifier, a video-camera, a cathode ray tube display and a photodiode, employing quin2 as a Ca2+ indicator. We recorded increases of the fluorescence intensity due to [Ca2+]i rises, when high K+ medium, neurotransmitter and Ca2+ ionophore were applied to the single cells of nervous system origin in culture. The present system is capable of simultaneous detection of the [Ca2+]i changes from multiple separate cells.     

Peripheral analgesic actions of opioid peptides and morphine analogues

Opiates and opioid peptides were administered in the order of 10(-9)-10(-6) mol peripherally, and their action on pain sensitivity was investigated by the modified formalin test which has two characteristic pain responses (the first and the second phase) in the mouse hindpaw. Opioid peptides (20-500 pmol) had dose-dependent analgesia against both first and second phases, and their action ranked dynorphin greater than [D-Ala2, Met5]-enkephalinamide greater than [Met5]-enkephalin. EKC and morphine (0.4-2.5 nmol) inhibited pain response of the first phase, but produced hyperalgesia in the second phase dose-dependently. Lidocaine hydrochloride had peripheral analgesic action, but was about 500-10000 times weaker than these substances. So, these peripheral analgesic actions have a different mechanism from that of local anesthetic action. N-methyl levallorphan which is thought to be a peripherally selective narcotic antagonist reversed these peripheral analgesic actions at the first and second phases and also prevented the hyperalgesic effects of EKC and morphine at the second phase. Naloxone reversed analgesia at only the first phase. These results suggest that an analgesic mechanism by opioids may exist at the peripheral site as well. Furthermore, it is estimated that a receptor exists which is antagonized by N-methyl levallorphan but not by naloxone and that there is a system of hyperalgesia by EKC and morphine in pain modulation.     

A surgical case of ventricular septal perforation after repairing left ventricular free wall rupture.

A 78-year-old woman with diagnosis of acute myocardial infarction (AMI) in the anteroseptal area fell into cardiogenic shock suddenly just before starting percutaneous coronary intervention (PCI). Echocardiography showed left ventricular free wall rupture, then an emergent operation was performed by sutureless patch repair using collagen fleece with fibrinogen-based impregnation. Eight days later from the initial operation, the onset of ventricular septal perforation (VSP) was recognized. Fifteen days after, the infarct exclusion technique with endocardial patch was performed. She has been doing well 4 months after the operation without residual shunt. To our best knowledge, this is the first surgical case report that free wall rupture of left ventricle and VSP which are serious complications after myocardial infarction happened in succession.