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.     

Systemic arterial supply to the left basal segment without the pulmonary artery: four consecutive cases.

Systemic arterial supply from the descending thoracic aorta to the basal segment of the left lower lobe without a pulmonary artery supply is a rare congenital anomaly within the spectrum of pulmonary sequestration cases. We encountered four consecutive cases, which were treated successfully by three basalectomies and one lower lobectomy to preserve lung function.     

Treatment strategy for synchronous metastases of colorectal cancer: is hepatic resection after an observation interval appropriate?

Background In cases of synchronous colorectal hepatic metastases, the primary colorectal cancer strongly influences on the metastases. Our treatment policy has been to conduct hepatic resection for the metastases at an interval of 3 months after colorectal resection. We examined the appropriateness of interval hepatic resection for synchronous hepatic metastasis. Materials and methods The subjects were 164 patients who underwent resection of hepatic metastasis of colorectal cancer (synchronous, 70 patients; metachronous, 94 patients). Background factors for hepatic metastasis and postoperative results were compared for synchronous and metachronous cases. Results The cumulative survival rate for 164 patients at 3, 5, and 10 years postoperatively was 71.9%, 51.8%, and 36.6%, and the post-resection recurrence rate in remnant livers was 26.8%. Interval resection for synchronous hepatic metastases was conducted in 49 cases after a mean interval of 131 days. No difference was seen in postoperative outcome between synchronous and metachronous cases. Conclusion The outcome was similarly favorable in cases of synchronous hepatic metastasis and in cases of metachronous metastasis. Delaying resection allows accurate understanding of the number and location of hepatic metastases, and is beneficial in determining candidates for surgery and in selecting surgical procedure.     

EFFECTS OF BKCa CHANNELS ON THE REDUCTION OF CYTOSOLIC Ca2+ IN cGMP-INDUCED RELAXATION OF GUINEAPIG TRACHEA

1. In order to examine the mechanisms of cGMP-induced relaxation in airway smooth muscle, the effects of atrial natriuretic peptide (ANP) and 8-brom cGMP on muscle tone were studied by measuring isometric tension, while the effects on cytosolic Ca²⁺ concentrations were studied by measuring the spectra of fura-2 loaded in guinea-pig tracheal strips. 2. Atrial natriuretic peptide and 8-brom cGMP caused a concentration-dependent inhibition of spontaneous tone in the guinea-pig trachea. The relaxant effects of these agents on spontaneous tone were markedly suppressed in the presence of iberiotoxin (IbTX), a selective inhibitor of large-conductance Ca²⁺-activated K⁺ (BKca) channels. Iberiotoxin (30 nmol/L) markedly affected the maximal effect induced by ANP and 8-brom cGMP and augmented EC₇₀ values for ANP and EC₅₀values for 8-brom cGMP approximately 27- and 17-fold, respectively. The inhibitory effects of IbTX on relaxation induced by these agents were diminished in the presence of 1 μmol/L nifedipine, an antagonist of voltage-operated Ca²⁺channels (VOCC). 3. The inhibitory action of ANP and 8-brom cGMP on spontaneous tone was not affected by the presence of 10 μmol/L glibenclamide, an inhibitor of ATP-sensitive K⁺ channels, and 100 nmol/L apamin, an inhibitor of small-conductance Ca²+-activated K⁺ channels. When these agents were applied to tissues precontracted by high (40mmol/L) K⁺, the relaxant effects of these agents markedly diminished. 4. The extracellular Ca²⁺-dependent contraction was inhibited in the presence of 0.3 μmoI/L ANP or 0.1 mmol/L 8-brom cGMP. Concentration—response curves to extracellular Ca²⁺ (0.03—2.4 mmol/L) were markedly diminished by exposure to these agents. The maximal effect induced by extracellular Ca²⁺ was affected by these agents. 5. Atrial natriuretic peptide caused an inhibition of spontaneous tone accompanied by a reduction in the intracellular Ca²⁺ concentration. In the presence of IbTX, the elimination of both muscle tone and cytosolic Ca²⁺ by ANP was suppressed. 6. We conclude that ANP and 8-brom cGMP activate BKca channels and that the inhibition of Ca²⁺ influx through VOCC, mediated by BKca channel activation, may be involved in cGMP-dependent bronchodilation.     

Aggressive resection for advanced pancreatic carcinoma

An aggressive pancreatectomy was performed on a 53 year old Japanese man with advanced cancer of the pancreas. The tumor originated from the body of the pancreas and invaded the stomach, duodenum, left kidney, transverse colon and common hepatic artery. An unexpected cancer was also found in the head of the pancreas during the operation. Therefore, total pancreatectomy, total gastrectomy, left adrenonephrectomy, resection of the left transverse colon and dissection of the regional lymph nodes were performed. Resection of the common hepatic artery was also performed, followed by an end-to-end anastomosis between the common hepatic artery and celiac trunk. The postoperative course was uneventful and the patient was doing well until nine months after the operation when multiple metastatic lesions were noted in the liver. He died 391 days after the operation from hepatic failure.     

In-vitro Negative Chronotropic and Inotropic Effects of a Novel Dihydropyridine Derivative,CD-832, in the Guinea-pig: Comparison with Calcium-channel Antagonists

The effects of CD-832 (4R-(-)-2-(nicotinoylamino)ethyl-3-nitroxypropyl-1,4-dihydro-2,6-dimethyl-4,3-nitrophenyl, 3,5-pyridine dicarboxylate), a novel dihydropyridine derivative, on guinea-pig isolated myocardial preparations have been compared with those of Ca²⁺-channel antagonists. All ten compounds induced concentration-dependent negative chronotropic effects on preparations of isolated right atria and negative inotropic effects on isolated right ventricular papillary muscles. The order of potency for the negative chronotropic effect was CD-832 > nicardipine = gallopamil > clentiazem > nifedipine = efonidipine > amlodipine = semotiadil > verapamil > diltiazem; that for the negative inotropic effect was nicardipine = gallopamil > nifedipine > verapamil > CD-832 > diltiazem > clentiazem > efonidipine = semotiadil > amlodipine. The ratio of the EC50 (the concentration of Ca²⁺ antagonist having 50% of the maximum effect) for the negative inotropic effect divided by the EC50 for the negative chronotropic effect, considered to be an index of selectivity for negative chronotropic effect, was higher for CD-832, amlodipine, efonidipine and semotiadil than for the other Ca²⁺ antagonists. The ratio for CD-832, nifedipine, nicardipine, efonidipine, amlodipine, verapamil, gallopamil, diltiazem, clentiazem and semotiadil was 11·4, 0·29, 0·87, 35·4, 37·1, 0·65, 0·87, 0·92, 7·11 and 30·0, respectively. These findings indicate that CD-832 and the newly developed Ca²⁺ antagonists including amlodipine, efonidipine, semotiadil and clentiazem were selective for a negative chronotropic effect rather than for a negative inotropic effect. This ‘chrono-selective’ effect of these drugs might be of benefit in the treatment of cardiovascular disorders.     

Immunological analysis of plasminogen activators from cultured human cancer cells

Summary Immunological similarities or differences between urokinase and plasminogen activators from 9 lines of cultured human caner cells with varying degrees of fibrinolytic activity were examined with antibodies against human urokinase.The antibodies completely inhibited the fibrinolytic activity of 4 lines of gastric cancer, 2 lines of lung cancer, 1 line of urinary bladder cancer and 1 line of renal cancer, indicating that the plasminogen activators from these cell lines were immunologically identical to urokinase. In 5 out of these cell lines, immunological identity was also confirmed by double diffusion analysis.The plasminogen activator from 1 line of lung cancer was found to be immunologically dissimilar to urokinase by a neutralization experiment and double diffusion analysis.These findings indicate that there are at least two immunologically distinguishable forms of plasminogen activators from human cancer cells.