Structural basis of Ets1 activation by Runx1.

Runx1 is required for definitive hematopoiesis and is well known for its frequent chromosomal translocations and point mutations in leukemia. Runx1 regulates a variety of genes via Ets1 activation on an Ets1•Runx1 composite DNA sequence. The structural basis of such regulation remains unresolved. To address this problem, we determined the crystal structure of the ternary complex containing Runx1(1-242) and Ets1(296-441) bound to T-cell receptor alpha (TCRα) enhancer DNA. In the crystal, an Ets1-interacting domain of Runx1 is bound to the Ets1 DNA-binding domain and displaced an entire autoinhibitory module of Ets1, revealing a novel mechanism of Ets1 activation. The DNA-binding and transcriptional studies with a variety of structure-guided Runx1 mutants confirmed a critical role of direct Ets1•Runx1 interaction in Ets1 activation. More importantly, the discovered mechanism provides a plausible explanation for how the Ets1•Runx1 interaction effectively activates not only a wild-type Ets1, but also a highly inhibited phosphorylated form of Ets1.

First results on 236U levels in global fallout.

The global fallout (236)U level in soil was deduced from measurements of (236)U, (239+240)Pu and (137)Cs in surface soils which are solely influenced by global fallout. A total of 12 soil cores from the depths of 0-10, 0-20 and 0-30 cm were collected at a flat forest area in Japan. Concentrations of (239+240)Pu and (238)U were determined by alpha-particle spectrometry, while the (236)U/(238)U ratio was measured with accelerator mass spectrometry (AMS). Consistent (236)U/(239)Pu ratios between 0.212 and 0.253 were found. Using this ratio, the total global fallout of (236)U on the earth is estimated to be as much as ca. 900 kg. This knowledge will contribute to the promotion of research on U isotopes, including (236)U, for the fields of geo-resources, waste management and geochemistry.

Increase in the stability of serine acetyltransferase from Escherichia coli against cold inactivation and proteolysis by forming a bienzyme complex.

Cysteine synthetase from Escherichia coli is a bienzyme complex composed of serine acetyltransferase (SAT) and O-acetylserine sulfhydrylase-A (OASS). The effects of the complex formation on the stability of SAT against cold inactivation and proteolysis were investigated. SAT was reversibly inactivated on cooling to 0 degrees C. Ultracentrifugal analysis showed that SAT (a hexamer) was dissociated mostly into two trimers on cooling to 0 degrees C in the absence of OASS, while in the presence of OASS one trimer of the SAT subunits formed a complex with one dimer of OASS subunits. In the presence of OASS, not only the cold inactivation rate was reduced but also the reactivation rate was increased. Furthermore, SAT became stable against proteolytic attack by alpha-chymotrypsin and V8 protease by forming the complex with OASS. On the other hand, SAT was degraded by trypsin in the same manner both in the presence and in the absence of OASS. The different tendency in the stability against proteolysis with the different proteases was discussed with respect to the substrate specificity of the proteases and amino acid sequence of the C-terminal region of SAT that interacts with OASS.

Pharmacokinetics of granisetron in adults and children with malignant diseases.

Granisetron (GRN) is widely used for patients with various cancers who suffer from chemotherapy-induced vomiting and nausea. The, pharmacokinetics of GRN has not been fully evaluated in such patients, however, and its dosage regimen is still controversial. In this study, we determined GRN levels in serum and urine from lung cancer patients and children suffering from cancer after intravenous infusion. In lung cancer patients, the interindividual variations in t(1/2beta), area under the concentration-time curve (AUC), and Vd(beta) were relatively smaller than expected from previous reports on healthy subjects, while t(1/2beta) was prolonged more than 5-fold in healthy subjects. Urinary excretion of unchanged GRN in lung cancer patients was ca. 15% of dose, consistent with previous reports, and one individual demonstrated an even higher urinary excretion (ca. 45%). The pharmacokinetic parameters of GRN in child cancer patients varied markedly among individuals, and some child patients had smaller t(1/2beta) than adult patients. In these cases, GRN should be administered at shorter intervals. These results suggested that a pharmacokinetic study of GRN was necessary for planning a dosage regimen and managing chemotherapy-induced vomiting and nausea.

Characteristics of serine acetyltransferase from Escherichia coli deleting different lengths of amino acid residues from the C-terminus.

Some properties of serine acetyltransferases (SATs) from Escherichia coli, deleting 10-25 amino acid residues from the C-terminus (SATdeltaC10-deltaC25) were investigated. The specific activity depended only slightly on the length of the C-terminal region deleted. Although the sensitivity of SATdeltaC10 to inhibition by L-cysteine was similar to that for the wild-type SAT, it became less with further increases in the length of the amino acid residues deleted. SATdeltaC10 was inactivated on cooling to 0 degrees C and dissociated into dimers or trimers in the same manner as the wild-type SAT, but Met-256-le mutant SAT as well as SATdeltaC14, SATdeltaC20, and SATdeltaC25 were stable. Since SATdeltaC10, SATdeltaC14, and SATdeltaC25 did not form a complex with O-acetylserine sulfhydrylase-A (OASS-A) in a way similar to SATdeltaC20, it was indicated that 10 amino acid residues or fewer from the C-terminus of the wild-type SAT are responsible for the complex formation with OASS-A. The C-terminal peptide of the 10 amino acid residues interacted competitively with OASS-A with respect to OAS although its affinity was much lower than that for the wild-type SAT.

Effects of bienzyme complex formation of cysteine synthetase from escherichia coli on some properties and kinetics.

Some properties and kinetics of the free and bound serine acetyltransferases (SATs) and O-acetylserine sulfhydrylase-As (OASS-As) from Escherichia coli were investigated. In some cases, SATdeltaC20, deleting 20 amino acid residues from the C-terminus of the wild-type SAT (Biosci. Biotechnol. Biochem., 63, 168-179 (1999)) was tested for comparison. The optimum pH and stability against some reagents for the free and bound wild-type SATs were similar except for the resistance to cold inactivation. The kinetics for the wild-type SAT and SATdeltaC20 followed a Ping-Pong Bi Bi mechanism with a mixed-type inhibition by L-cysteine. The kinetics and kinetic constants for the wild-type SAT were not changed by the complex formation with OASS-A. The optimum pH for OASS-A was shifted towards an alkaline pH by the complex formation. Thermal stability and stability against some reagents for the free and bound OASS-As were almost the same. On the other hand, the maximum velocity for OASS-A was lowered and dissociation constants for the substrates and products were increased by forming the complex with the wild-type SAT, although the kinetics for the free and bound enzymes followed the same Ping-Pong Bi Bi mechanism. From comparisons of computed courses of L-cysteine formation from L-serine using SAT (wild-type SAT and SATdeltaC20) and OASS-A with the experimental results and changes in the stability of the wild-type SAT by the complex formation, we discuss the role and significance of a complex formation for the cysteine synthetase.

[New operative technique to resect of peripheral bronchogenic carcinoma with chest wall invasion using Kent's retractor].

We report the new operative technique for resection of peripheral bronchogenic carcinoma with chest wall invasion using Kent's retractor and hook suspender. Standard muscle sparing thoracotomy cannot obtain the adequate operative field in the posterior chest wall under the scapula because of sparing the latissimus dorsi muscle. To obtain a sufficient operative field at the operation for en bloc chest wall resection, especially with the area of the posterior chest wall under the scapula, resection of the trapezius muscle, rhomboideus major muscle, and latissimus dorsi muscle has been added to muscle-sparing thoracotomy. Through our new technique using Kent's retractor and hook suspender that has been used in the abdominal surgery, assistant surgeon can be eliminated in favor of hanging up the scapula, which is able to be performed much easier to obtain a good operative field. To use the Kent's retractor and hook suspender, the scapula is lifted upward and cranially. With these procedures, the proximal portion of the first, second and third ribs in the posterior chest wall covering by the scapula is able to resect from vertebrocostal junction with muscle-sparing thoracotomy. We examined the apparatus for a case of the right upper lung carcinoma with invasion to the posterior chest wall. We performed right upper lobectomy with chest wall in the area of the second, third and forth ribs with suspending the scapula by Kent's retractor after muscle-sparing thoracotomy.

[Cases of sternal metastasis except from breast cancer].

Surgical resection is the option for sternal metastasis from breast cancer. But surgical treatment is the subject of controversy for sternal metastasis except from breast cancer. Four cases of secondary malignant tumors of the sternum except from breast cancer were reported. Primary lesions included thyroid cancer, gastric cancer, malignant fibrous histiocytoma of the chest wall and multiple myeloma. In 3 cases, operation were done and they survived 11 months, 13 months and 3 years respectively. In case of secondary malignant tumor of the sternum except from breast cancer, surgical treatment should be considered.

Pharmacokinetics of etoposide after intrathoracic instillation to lung cancer patients with pleural effusion.

OBJECTIVE: To examine etoposide (VP16) levels in serum and pleural effusion after intravenous infusion or intrathoracic instillation to lung cancer patients. METHODS: Four patients were administered VP16 by intrathoracic instillation and three patients were administered it intravenously. Serum, urine, and pleural effusion were collected and VP16 levels in the biological fluids were determined by HPLC. Pharmacokinetic parameters were calculated. RESULTS: VP16 distributed rapidly into pleural effusion after intravenous infusion. In two of three patients, VP16 levels in pleural effusion were maintained at constant levels more than 24 hours in spite of the decline in serum VP16 levels. After intrathoracic instillation, VP16 in pleural effusion reached high levels and eliminated slowly. Serum levels of VP16 were relatively low compared with those in pleural effusion. CONCLUSION: It was demonstrated that intrathoracic instillation of VP16 might be useful for managing malignant pleural effusion and reducing systemic side-effects by cutting down the dose.

Purification and characterization of serine acetyltransferase from Escherichia coli partially truncated at the C-terminal region.

Incubation of serine acetyltransferase (SAT) from Escherichia coli at 25 degrees C in the absence of protease inhibitors yielded a truncated SAT. The truncated SAT was much less sensitive to feedback inhibition than the wild-type SAT. Analyses of the N- and C-terminal amino acid sequences found that the truncated SAT designated as SAT delta C20 was a resultant form of the wild-type SAT cleaved between Ser 253 and Met 254, deleting 20 amino acid residues from the C-terminus. Based on these findings, we constructed a plasmid containing an altered cysE gene encoding the truncated SAT. SAT delta C20 was produced using the cells of E. coli JM70 transformed with the plasmid and purified to be homogeneous on an SDS-polyacrylamide gel. Properties of the purified SAT delta C20 were investigated in comparison with those of the wild-type SAT and Met-256-Ile mutant SAT, which was isolated by Denk and Böck but not purified (J. Gen. Microbiol., 133, 515-525 (1987)). SAT delta C20 was composed of four identical subunits like the wild-type SAT and Met-256-Ile mutant SAT. Specific activity, optimum pH for reaction, thermal stability, and stability to reagents for SAT delta C20 were similar those for the wild-type SAT and Met-256-Ile mutant SAT. However, SAT delta C20 did not form a complex with O-acetylserine sulfhydrylase-A (OASS-A), a counterpart of the cysteine synthetase and did not reduce OASS activity in contrast to the wild-type SAT and Met-256-Ile mutant SAT.

Effect of 3-methylcholanthrene administration on expression of cytochrome P-450 isoforms induced by phenobarbital in rat hepatocytes.

The effects of an inducer on expression of cytochrome P-450 (P-450) isoforms induced antecedently by another inducer are unknown. Thus, we examined the amount of phenobarbital (PB)-inducible P-450 isoforms (P-450 2B1/2B2) in hepatocytes from rats injected first with PB and then with 3-methylcholanthrene (MC) (PB+MC-treated animals) by quantitative immunohistochemistry. In addition, expression of P-450 2B2 mRNA was examined by in situ hybridization. In PB-treated animals, P-450 2B1/2B2 content increased in perivenular and midzonal hepatocytes. In PB+MC-treated animals, however, the PB-induced increase in 2B1/2B2 content was suppressed in perivenular hepatocytes but promoted in midzonal hepatocytes. The hybridization signal for P-450 2B2 mRNA appeared almost exclusively in perivenular hepatocytes after 24 hr of PB injection and disappeared after 48 hr of injection. In PB+MC-treated animals, however, strong hybridization signal was observed in midzonal and perivenular hepatocytes after 48 hr of PB injection. The promotion of the increase in P-450 2B1/2B2 content in midzonal hepatocytes in PB+MC-treated animals probably corresponds to the strong hybridization signal, whereas there appeared to be a divergence between the intensity of the signal and the content in perivenular hepatocytes. The results indicate that MC administration drastically influences the pattern of expression of P-450 isoforms induced by PB in perivenular and midzonal hepatocytes.

Determination of accuracy of quantitative immunohistochemical results with an antigen-immobilized filter model system.

To determine accuracy of quantitative immunohistochemical results, serially diluted liver cell lysates from methylcholanthrene (MC)-treated rats containing cytochrome P-450 (P-450) 1A were immobilized on nitrocellulose (NC) filters and stained by the indirect immunoperoxidase method under saturation conditions. The stained filters were processed for image analysis and the relationship between the resulting immunostaining intensity due to P-450 1A and the antigen amount immobilized on the filters was examined. The relationship examined in the filters fitted with an exponential curve. Thus, the intensity due to P-450 1A is not simply proportional to the antigen amount. Subsequently, immunostaining intensity due to P-450 1A was measured in sections from control and methylcholanthrene (MC)-treated rats by image analysis with an image processor, and the antigen content in cell lysates from control and MC-treated animals was measured immunochemically by quantitative single radial immunodiffusion. Although immunochemically measured P-450 1A content in the lysates increased markedly (26 times), average staining intensity in sections increased slightly (2-3 times) after MC injection. However, when the exponential curve obtained from the filter binding assay was used as a standard curve to convert staining intensity in sections to molar content of P-450 1A, the resulting content was compatible with the immunochemical content. The divergence between the increase in immunochemically measured P-450 1A content and that in immunostaining intensity in sections after MC injection is therefore attributed primarily to the curvilinear relationship between the intensity and the content.

[Thoracoscopic cutting needle biopsy].

We presented a new method of percutaneous cutting needle biopsy under the thoracoscope. Percutaneous cutting needles for lung tumor have been abandoned because the risk of hemorrhage, death, and pneumothorax is much greater with these instruments than the computed tomographic guided needle aspiration. On the other hand, thoracoscopic stapled lung wedge resection is easy to obtain a good sample for any lung disease, but should be needed two or three staples, and waste a time, in the case following lobectomy was needed for malignancy. This thoracoscopic cutting needle biopsy was easy to obtain a specimen for the frozened section diagnosis. Under the view of thoracoscope, percutaneous cutting needle biopsy is safety, saving the cost for staples and easy to control the bleeding from the lung. The indication of this thoracoscopic cutting needle biopsy is the case that could not obtain the precise diagnosis by the preoperative biopsy and imaging diagnosis might be suspicious a malignant tumor.

[Video-assisted right pneumonectomy for small cell carcinoma].

We presented a case of video-assisted right pneumonectomy for small cell carcinoma. A 54-year-old male was admitted because of an abnormal shadow on a chest rentogenogram occasionally. The bronchoscopic biopsy revealed squamous cell carcinoma at the right intermediate bronchus. His magnetic resonance imaging demonstrated a 5-cm right hilar mass involving with right pulmonary artery. The operation was carried out by video-assisted thoracotomy with small lateral thoracotomy. Pathological findings revealed oat cell carcinoma with a subcarinal node involvement postoperatively. His postoperative course was uneventful. He received adjuvant chemotherapy and 60 Gy radiation in the mediastinum. He is alive 12 months after surgery without any evidence of recurrence. The advantages of the video-assisted thoracotomy were reported less postoperative pain and discharged earlier than the standard thoracotomy. Video-assisted pneumonectomy had not disturbed him in the adjuvant therapy for oat cell carcinoma.

Preparation of liposomes that mimic the membrane of endoplasmic reticulum of rat hepatocytes.

To examine the interaction between biomembranes and membrane-bound proteins, large unilamellar liposomes have been required. In the present study, we prepared liposomes from a mixture of phospholipids having a phospholipid composition similar to that in the endoplasmic reticula (microsomes) of rat hepatocytes by eight different methods. The resulting liposomes were examined by a combination of the freeze-fracture-replica procedure with biochemical methods. The freeze-thawing method of Pick (1981) gave the best results; large unilamellar liposomes that mimic the membrane of endoplasmic reticulum were obtained. Liposomes made by this method are thus suitable for analysis of the interaction between the endoplasmic reticulum membrane and membrane-bound proteins.

[Video-assisted lobectomy for primary lung cancer].

We had performed video-assisted lobectomy for six primary lung cancer cases. This report is presented our method of video-assisted lobectomy with a typical case. This case had a complain of positive sputum cytology at the regular examination. His chest rentogenogram was almost normal and the bronchoscopic appearance was revealed polypoid lesion at left B 3 orifice. His sputum cytology examined 3 years ago was positive. At that time we could not find the malignant site at the left B 3 orifice. So further examination was performed, but we did not find other metastatic site and mediastinal nodes involvement by chest computed tomography. Left upper lobectomy and lymph node dissection was performed by video-assisted thoracotomy with small lateral incision of about 8 centimeters on the fifth intercostal space and other two thoracotomy ports. This operation method was followed as same fashion as the standard lobectomy with double knotted pulmonary vein and artery without the usage of the automatic anastomotic device. His postoperative pathological study was revealed intramural bronchial cancer at B 3 orifice without node involvement. His postoperative course was uneventful. He had discharged at 18 postoperative day.

[Enhancement of rectal absorption of rifampicin by sodium para-aminosalicylate dihydrate in human subjects].

The suppositories of rifampicin (RFP) containing sodium para-aminosalicylate dihydrate (PAS-Na) were prepared in order to enhance the rectal absorption of RFP. By the addition of PAS-Na, the in vitro release of RFP from the suppositories was enhanced and the hardness of the suppositories decreased. The rectal absorption of RFP from the suppositories containing no PAS-Na (control suppositories) was significantly lower compared to oral administration of it (26%) in human subjects. When PAS-Na was added to the suppository (300 mg), both the area under the plasma concentration-time curve (AUC) and the maximum plasma concentration (Cmax) increased significantly compared to those of the control suppositories. The rectal absorption of PAS-Na itself from the suppositories seemed to be fast. PAS-Na might increase the absorption of RFP dissolved in the rectal fluid from the suppositories, but not affect the undissolved RFP.

[Problems in combined resection of adjacent organ in lung cancer: significance of preoperative and intraoperative ultrasonic examination].

Aggressive combined resections were carried out, on 24 lung cancer cases which showed invasion into surrounding organs. Those cases with wide infiltration of the ribs, and into surrounding intercostal tissues, and for those with invasion to diaphragm, the outcome of the operations was rather poor but, for those with invasion in pericardium and left atrium, fairly favorable results were obtained. These present results indicated that aggressive resections of adjacent organs is to be recommended for cases without N 2 infiltration. Experiences from these operations have taught us the importance and useful of diagnosis of the status of invasion of the tumor, during the operation by ultrasonogram (IUS), with direct contact of the probe to the thoracic wall or mediastinal organs, to discriminate the areas of chest wall and mediastinal organs that require resections. This method, in combination with esophagus ultrasonic endoscopy (EUS), enabled defining the infiltrated areas with accuracy far exceeding that obtained by tactile examination. Techniques of, and observations obtained by these examination methods are presented.

[A case of ruptured aneurysm of the descending thoracic aorta associated with active Behçet's disease].

A 63-year-old male receiving steroid for active Behçet's disease was admitted of massive hemoptysis. A pseudoaneurysm of about 5 cm in diameter caused by rupture of the aortic wall penetrating into the lung was detected on the descending thoracic aorta. Operation was performed using left atrium-femoral artery bypass with centrifugal pump. The technique of graft interposition was selected rather than patch closure to prevent postoperative anastomotic aneurysm. We consider that in the treatment of inflammatory aneurysm, use healthy aortic wall for suture and end-to-end anastomosis are very important. In spite of steroid supplementation, the inflammatory reaction get worse postoperatively and the dosage of steroid had to be increased to control inflammation. The patient is well 20 months after surgery.

[Effect of a newly developed platinum compound cis-1, 1-cyclobutane dicarboxylato-(2R)-2-methyl-1,4-butane diamine platinum (II), on human ovarian tumors transplanted into nude mice].

The therapeutic effect of cis-1, 1-cyclobutane dicarboxylato-(2R)-2-methyl-1,4-butane diamine platinum (II) (NK 121) on 3 human yolk sac tumors of the ovary (YST-1, YST-2 and YST-3), which were transplanted into nude mice, was compared with that of cisplatin (CDDP). While tumors YST-2 and YST-3 exhibited a broad comparable sensitivity to CDDP and NK 121, YST-1 was substantially more sensitive to CDDP than NK 121. A significant weight loss was noted in the non-tumor bearing nude mice treated with CDDP, as compared with the loss in both control and NK 121-treated groups. NK 121 showed a good antitumor activity without showing severe toxicities.