Possible involvement of LKB1-AMPK signaling in non-homologous end joining.

LKB1/STK11 is a tumor suppressor gene responsible for Peutz-Jeghers syndrome, an inherited cancer disorder associated with genome instability. The LKB1 protein functions in the regulation of cell proliferation, polarization and differentiation. Here, we suggest a role of LKB1 in non-homologous end joining (NHEJ), a major DNA double-strand break (DSB) repair pathway. LKB1 localized to DNA ends upon the generation of micro-irradiation and I-SceI endonuclease-induced DSBs. LKB1 inactivation either by RNA interference or by kinase-dead mutation compromised NHEJ-mediated DNA repair by suppressing the accumulation of BRM, a catalytic subunit of the SWI/SNF complex, at DSB sites, which promotes the recruitment of an essential NHEJ factor, KU70. AMPK2, a major substrate of LKB1 and a histone H2B kinase, was recruited to DSBs in an LKB1-dependent manner. AMPK2 depletion and a mutation of H2B that disrupted the AMPK2 phoshorylation site impaired KU70 and BRM recruitment to DSB sites. LKB1 depletion induced the formation of chromosome breaks and radials. These results suggest that LKB1-AMPK signaling controls NHEJ and contributes to genome stability.

Histone acetylation by CBP and p300 at double-strand break sites facilitates SWI/SNF chromatin remodeling and the recruitment of non-homologous end joining factors.

Non-homologous end joining (NHEJ) is a major repair pathway for DNA double-strand breaks (DSBs) generated by ionizing radiation (IR) and anti-cancer drugs. Therefore, inhibiting the activity of proteins involved in this pathway is a promising way of sensitizing cancer cells to both radiotherapy and chemotherapy. In this study, we developed an assay for evaluating NHEJ activity against DSBs in chromosomal DNA in human cells to identify the chromatin modification/remodeling proteins involved in NHEJ. We showed that ablating the activity of the homologous histone acetyltransferases, CBP and p300, using inhibitors or small interfering RNAs-suppressed NHEJ. Ablation of CBP or p300 impaired IR-induced DSB repair and sensitized lung cancer cells to IR and the anti-cancer drug, etoposide, which induces DSBs that are repaired by NHEJ. The CBP/p300 proteins were recruited to sites of DSBs and their ablation suppressed acetylation of lysine 18 within histone H3, and lysines 5, 8, 12, and 16 within histone H4, at the DSB sites. This then suppressed the recruitment of KU70 and KU80, both key proteins for NHEJ, to the DSB sites. Ablation of CBP/p300 also impaired the recruitment of BRM, a catalytic subunit of the SWI/SNF complex involved in chromatin remodeling at DSB sites. These results indicate that CBP and p300 function as histone H3 and H4 acetyltransferases at DSB sites in NHEJ and facilitate chromatin relaxation. Therefore, inhibition CBP and p300 activity may sensitize cancer cells to radiotherapy and chemotherapy.

Unbalanced translocation, a major chromosome alteration causing loss of heterozygosity in human lung cancer.

Loss of heterozygosity (LOH) is a major genetic event causing inactivation of tumor suppressor genes in human carcinogenesis. To elucidate chromosomal mechanisms causing LOH, 201 LOHs in 10 cases of human lung cancer, which were detected by a genome-wide single nucleotide polymorphism array analysis, were investigated for responsible chromosome alterations by integrating information on breakpoints for DNA copy number changes obtained by array-comparative genome hybridization and on numerical and structural chromosomal alterations obtained by spectral karyotyping. The majority (80%) of LOHs were partial chromosome LOHs caused by structural chromosomal alterations, while the remaining (20%) were whole chromosome LOHs caused by whole chromosome deletions. Unbalanced translocation was defined as the most frequent alteration, and it accounted for 30% of all LOHs. Three other structural alterations-interstitial deletion (19%), mitotic recombination (9%) and gene conversion (6%)-also contributed to the occurrence of LOH, while terminal deletion contributed to only a small subset (1%). Since unbalanced translocation is a common chromosomal alteration in lung cancer cells, the results in the present study strongly indicate that a considerable fraction of LOHs detected in lung cancer cells are caused by unbalanced translocation.

Chemical and nutritional properties of hypoallergenic wheat flour.

The chemical and nutritional properties were investigated of hypoallergenic wheat flour (HWF) prepared by the cellulase-actinase treatment. HWF was composed mainly of oligopeptides and free amino acids, and its average molecular weight was lower than 1,000. Feeding tests on rats showed that, with respect to the PER, GOT and GPT activities and other nutritional indices, the HWF diet was almost equivalent to the control diet which had been prepared from normal wheat flour (NWF). No abnormality was apparent in the main organs after the HWF diet had been fed for 3 weeks. The small intestinal absorption of the HWF diet was found normal by measuring the free amino acid concentration in the intestinal tract and in the portal vein plasma. These data suggest that the absorption of amino acids from the HWF diet was comparable with or more efficient than that from a simulated free amino acid diet.

A comparison of the University of Wisconsin solution and the modified Kawakami solution for initial flush and coronary perfusion in long-term canine heart preservation.

BACKGROUND: We compared two different solutions, the University of Wisconsin (UW) solution (intracellular-like) and the modified Kawakami (mK) solution (extracellular-like), for initial flush of coronary vascular beds before simple storage and following coronary perfusion. METHODS: After a right thoracotomy in the 4th intercostal space, the donor heart was isolated by ligating the azygos vein and venae cavae, and cross-clamping the aorta. Cardiac arrest was then obtained with a cold GIK solution. Following initial flush of coronary vascular beds, the donor heart was resected, stored utilizing a combination of simple immersion and coronary perfusion, and then transplanted. A total of 48 mongrel dogs was divided into three groups each using different solutions for the initial flush of coronary vascular beds and for coronary perfusion. In group I (n=10) the UW solution was used for both initial flush and coronary perfusion. In group II (n=7) the mK solution was used for both initial flush and coronary perfusion, and in group III (n=7) the UW solution was used for initial flush and the mK solution for coronary perfusion. Intracellular high-energy phosphate was surveyed by 31P-nuclear magnetic resonance spectroscopy. RESULTS: After 12-hour simple immersion and 1-hour coronary perfusion, phosphocreatine and adenosine triphosphate were significantly (p<0.05) higher in group III than in groups I and II. The high-energy phosphate levels of the graft tissue were better in groups I and III than in group II. Orthotopic transplantation was then performed using 10 preserved grafts in group I and seven preserved grafts of group III. After transplantation, left ventricular (LV) pressure of group I animals recovered to 82.3% and group III recovered to 95.8% of the control values. LV dp/dt of group I and III animals recovered to 76.5% and 96.7% of the control values, respectively. CONCLUSIONS: The UW solution, which is acceptable for both initial flush and simple storage, is not suitable for continuous coronary perfusion even for a short period due to its high viscosity. A combination of the UW solution both for initial flush and the following cold simple immersion and the mK solution for continuous coronary perfusion is appropriate for long-term preservation of the canine heart.

The effects of the hypothermic management of brain dead dogs on preserving graft viability in heart transplantation.

The effect of hypothermic management for brain dead dogs on preserving graft viability was evaluated through preservation and transplantation. After the occurrence of brain death, 43 dogs were divided into two groups; the normothermic group (37.2+/-0.3 degrees C) and the hypothermic group (31.8+/-0.3 degrees C) according to the esophageal temperature. After the 6-hour management of brain dead donors, the heart beat was arrested using a cardioplegic solution followed by coronary vascular bed washout. The donor heart was then harvested and preserved for 12 hours with simple immersion into the University of Wisconsin solution. Following preservation, orthotopic transplantation was performed in six grafts randomly selected from each group. During the 6-hour management of brain dead dogs; 1) heart rates, rate-pressure products, and the total amount of catecholamine were significantly (p<0.05) lower in the hypothermic group than in the normothermic group, and 2) lactate contents collected from the coronary sinus blood and O2-extraction rates of the heart tended to be lower in the hypothermic group than in the normothermic group. During 12 hours of preservation, intracellular pH and creatine phosphate contents were higher in the hypothermic group than in the normothermic group. Following orthotopic transplantation, the animals in the hypothermic group showed a significantly (p<0.05) higher recovery rate of left ventricular (LV) pressure and the maximum rate of the rise of LV pressure compared with normothermic group animals. We conclude that the hypothermic management of brain dead dogs may be effective in preserving graft viability and may provide a clinical application for heart transplantation with acceptable outcomes.

Blood pressure, insulin, and haematocrit values in Japanese subjects over 60 years of age.

Many studies have suggested an association between blood pressure (BP) and both insulinaemia and haematocrit values in Western populations. However, relatively few data regarding such associations for the Japanese population are available. We investigated the relationship between BP and various parameters, including insulin and haematocrit, in 269 healthy Japanese individuals (113 men and 156 women) over 60 years of age. The data were analysed by stepwise multiple regression analysis. In men, the most important determinant of systolic BP (SBP) and diastolic BP (DBP) was the plasma insulin concentration (beta = 0.32, F = 12.4, P < 0.001 and beta = 0.32, F = 13, P < 0.001, respectively), and haematocrit was found to be associated with DBP (beta = 0.21, F = 5.3, P < 0.05). In women, fasting insulins were unrelated to BP, but fasting plasma glucose and triglyceride concentrations and age were associated with SBP (beta = 0.29, F = 15.5, P < 0.001, beta = 0.27, F = 14.3, P < 0.001, and beta = 0.2, F = 7.8, P < 0.01, respectively), and haematocrit, fasting plasma glucose concentration and age were associated with DBP (beta = 0.29, F = 14, P < 0.001, beta = 0.2, F = 6.9, P < 0.01 and beta = 0.2, F = 6.3, P < 0.05, respectively). These results suggest that the insulin-BP associations in healthy Japanese subjects over 60 years of age differ according to sex, and that haematocrit is an important determinant of DBP in both sexes in our population.

Laparoscopic minilaparotomy pancreaticoduodenectomy with lymphadenectomy using an abdominal wall-lift method.

Because of technical difficulties attributable to the lack of appropriate techniques, the high cost of laparoscopic instruments, and the need for numerous disposable stapling devices, laparoscopic pancreaticoduodenectomy has been performed in only two patients. To solve the above problems, we devised a method of laparoscopic minilaparotomy using an abdominal wall-lift method and have successfully used it to perform pancreaticoduodenectomy with lymphadenectomy in a patient with distal choledochal cancer.

[Hypothermic management of brain dead dogs].

For preventing graft failure, the effects of hypothermic management of brain dead dogs was investigated. Forty-three brain dead dogs were divided into two groups according to the degree of esophageal temperature; a normothermic group (37.2 +/- 0.3 degree C, mean +/- SEM, n = 22), and a hypothermic group (31.8 +/- 0.3 degree C, n = 21) which was obtained by introducing ice slush in the peritoneal cavity. During the management of brain dead dogs, 1) heart rate, pressure product, and a total amount of catecholamine were significantly lower (p < 0.05) in the hypothermic group than in the normothermic group, 2) mean blood pressure, the maximum rate of the rise of left ventricle (LVdp/dt) and cardiac output were not different between both groups, 3) lactate content in the coronary sinus, and O2-extraction rate of the heart tended to be lower in the hypothermic group than in the normothermic group. After transplantation, the recovery of cardiac function was better in the hypothermic group than in the normothermic group. Hypothermic management of brain dead dogs may safely decrease cardiac stress, and keep cardiac aerobic circumstances.

Long-term heart preservation by the combined method of simple immersion and coronary perfusion.

BACKGROUND: The combined method of cold storage and coronary perfusion for prolonged preservation of donor hearts was evaluated through preservation and transplantation with the use of 10 pair of adult mongrel dogs. METHODS: In situ initial flush for cooling and coronary vascular washout was performed with a University of Wisconsin solution. The heart was then removed and immersed into a cold (4degrees C) University of Wisconsin solution. After 12-hour cold storage, 1-hour preservation was added by coronary perfusion using a 4degree C oxygenated University of Wisconsin solution. High-energy phosphate (phosphocreatine, beta-adenosine triphosphate) and phosphate were measured by 31P-nuclear magnetic resonance spectroscopy at 0-hour cold storage, 12-hour cold storage, and immediately after coronary perfusion. RESULTS: Phosphocreatine and beta-adenosine triphosphate decreased to 12.1 +/- 24.2% (mean +/ standard deviation), 37.4% +/- 25.0%, respectively, after 12-hour cold storage, and significantly increased to 94.0% +/- 48.7% (p < 0.001), 48.8% (p < 0.05), respectively after 1-hour coronary perfusion. Disorder of nuclear arrangement and edema of muscular cells, which were observed after cold storage, were histologically restored after 1-hour coronary perfusion. After transplantation of preserved grafts, left ventricular pressure and left ventricular rate of pressure rise were evaluated in the graft with 12-hour cold storage only (group A) and in the graft with 12-hour cold storage and 1-hour coronary perfusion (group B). Left ventricular pressure 2 hours after transplantation in group B recovered to 76.1% significantly (p < 0.01), compared with 51.9% in group A. Significantly higher values of left ventricular rate of pressure rise 2 hours after transplantation was observed in group B (83.0% +/ 12.7%) compared with group A (68.2% +/- 12.5%). CONCLUSIONS: These results indicate that the combined method of cold storage and coronary perfusion may be effective for myocardial protection during long-term preservation.

Laparoscopic retrograde cholecystectomy (from fundus downward) facilitated by lifting the liver bed up to the diaphragm for inflammatory gallbladder.

In order to safely and reliably perform laparoscopic cholecystectomy in severe inflammatory cases (e.g., acute or chronic cholecystitis), we have designed a method of suturing the liver bed to the diaphragm, lifting it cephalad so as to maintain a good operative field. Initially, we dissect the gallbladder fundus, fully dissecting the neck of the gallbladder from the liver and finally dissecting the cystic duct (laparoscopic retrograde cholecystectomy facilitated by lifting the liver bed up to the diaphragm; Lap-RC). This method is different from laparoscopic standard cholecystectomy (Lap-SC), in which dissection of the cystic duct is done first. One hundred and twenty-nine consecutive laparoscopic cholecystectomies for various gallbladder diseases were carried out at Nerima General Hospital between August 1991 and June 1994. Fifteen cases of Lap-RC and six cases of Lap-SC in a severe inflammatory group were comparatively evaluated. Thirteen cases of Lap-RC and 92 cases of Lap-SC in a noninflammatory group were also comparatively evaluated. The rates of conversion to laparotomy were 0% in Lap-RC cases (0/15) and 33% in Lap-SC cases (3/9) in the severe inflammatory group. The incidences of major postoperative complications were 0% in Lap-RC cases (0/15) and 17% in Lap-SC cases (1/6) in the severe inflammatory group. In conclusion, Lap-RC showed satisfactory results in terms of both safety and reliability in patients with severe inflammatory disease.

Laparoscopic and minilaparotomy proximal gastrectomy and esophagogastrostomy: technique and case report.

A laparoscopic and minilaparotomy proximal gastrectomy with esophagogastrostomy (end-to-side anastomosis) was performed on a 56-year-old woman with a leiomyoma located just below the esophagogastric junction. Gastroscopic examination revealed a leiomyoma (diameter of 2.5 cm) just below the esophagogastric junction. We considered a laparoscopic proximal gastrectomy safer than a laparoscopic partial gastrectomy because of the risk of postoperative anastomotic stenosis in this case. Therefore, the patient underwent laparoscopic minilaparotomy proximal gastrectomy with esophagogastrostomy. On postoperative day 1, she was able to walk. On postoperative day 4, she started on a clear liquid diet and was discharged on postoperative day 14. During her postoperative recovery, the patient experienced little pain and did not request narcotic analgesia.

Inflammatory responses in open versus laparoscopic herniorrhaphy.

The aim of this study is to verify whether differences could be found or not as concerns of the postoperative surgical stress between the conventional open herniorrhaphy and laparoscopic herniorrhaphy. We therefore compared the immunological and inflammatory responses to open and laparoscopic procedures in this study. The sex, age, weight, and height characteristics of group 1 (open surgery group) matched those of group 2 (laparoscopic surgery group), and no statistically significant differences were found between them. Nor were there any statistically significant differences in operating time or the postoperative hospitalization. Blood samples were collected preoperatively [postoperative day (POD)0], POD 1 and 5, and the following parameters were assessed: total leukocyte (WBC) count, including neutrophil and lymphocyte subpopulations, HLA-DR+CD3+, IL-6, C-reactive protein (CRP), serum albumin, and body temperature. No differences were detected between two groups in leukocyte, neutrophil, or lymphocyte count, HLA-DR+CD3+, IL-6, albumin, or body temperature or any of the postoperative days. The only statistically significant difference was a higher CRP value in the laparoscopy group than the open group only on POD 1 (p < 0.05). It can be concluded that the laparoscopic approach appears to offer no advantages over the open approach to herniorrhaphy from the standpoint of immunological and inflammatory responses.

Laparoscopic minilaparotomy Billroth I gastrectomy with extraperigastric lymphadenectomy for early gastric cancer using an abdominal wall-lifting method.

Laparoscopic gastrectomy with extraperigastric lymphadenectomy for early gastric cancer has never been performed because of technical difficulties attributable to the lack of appropriate techniques, the high cost of laparoscopic instruments, and the need for numerous disposable stapling devices. In order to solve these problems, we have designed a method of laparoscopic minilaparotomy using an abdominal wall-lifting method, and a patient with early gastric cancer (depth of submucosa) underwent by this laparoscopic minilaparotomy distal gastrectomy with extraperigastric lymphadenectomy. During his postoperative recovery, the patient requested no narcotic analgesic, and was discharged on postoperative day 14.

Laparoscopic Billroth I gastrectomy for gastric ulcer: technique and case report.

A laparoscopic distal gastrectomy with gastroduodenostomy (Billroth I) was performed on a patient with intractable gastric ulcer. The patient was a 56-year-old man, complaining of severe epigastralgia, who had a 10-year history of peptic ulcer. Gastroscopy had revealed a UL-IV gastric ulcer on the lesser curvature at the angle of the stomach and a deformity of the bulbus. A diagnosis of intractable gastric ulcer was made, and the patient underwent laparoscopic distal gastrectomy and gastroduodenostomy. On postoperative day 1, he was able to walk. On postoperative day 4, he started on a clear liquid diet and was discharged on postoperative day 14.