Kippei Ohgaki

Architectural role of mitochondrial transcription factor A in maintenance of human mitochondrial DNA.

ABSTRACT Mitochondrial transcription factor A (TFAM), a transcription factor for mitochondrial DNA (mtDNA) that also possesses the property of nonspecific DNA binding, is essential for maintenance of mtDNA. To clarify the role of TFAM, we repressed the expression of endogenous TFAM in HeLa cells by RNA interference. The amount of TFAM decreased maximally to about 15% of the normal level at day 3 after RNA interference and then recovered gradually. The amount of mtDNA changed closely in parallel with the daily change in TFAM while in organello transcription of mtDNA at day 3 was maintained at about 50% of the normal level. TFAM lacking its C-terminal 25 amino acids (TFAM-ΔC) marginally activated transcription in vitro. When TFAM-ΔC was expressed at levels comparable to those of endogenous TFAM in HeLa cells, mtDNA increased twofold, suggesting that TFAM-ΔC is as competent in maintaining mtDNA as endogenous TFAM under these conditions. The in organello transcription of TFAM-ΔC-expressing cells was no more than that in the control. Thus, the mtDNA amount is finely correlated with the amount of TFAM but not with the transcription level. We discuss an architectural role for TFAM in the maintenance of mtDNA in addition to its role in transcription activation.

Alcohol drinking, cigarette smoking, and the development of squamous cell carcinoma of the esophagus: molecular mechanisms of carcinogenesis

Esophageal cancer is the eighth most common incident cancer in the world and ranks sixth among all cancers in mortality. Esophageal cancers are classified into two histological types; esophageal squamous cell carcinoma (ESCC), and adenocarcinoma, and the incidences of these types show a striking variety of geographic distribution, possibly reflecting differences in exposure to specific environmental factors. Both alcohol consumption and cigarette smoking are major risk factors for the development of ESCC. Acetaldehyde is the most toxic ethanol metabolite in alcohol-associated carcinogenesis, while ethanol itself stimulates carcinogenesis by inhibiting DNA methylation and by interacting with retinoid metabolism. Cigarette smoke contains more than 60 carcinogens and there are strong links between some of these carcinogens and various smoking-induced cancers; these mechanisms are well established. Synergistic effects of cigarette smoking and alcohol consumption are also observed in carcinogenesis of the upper aerodigestive tract. Of note, intensive molecular biological studies have revealed the molecular mechanisms involved in the development of ESCC, including genetic and epigenetic alterations. However, a wide range of molecular changes is associated with ESCC, possibly because the esophagus is exposed to many kinds of carcinogens including alcohol and cigarette smoke, and it remains unclear which alterations are the most critical for esophageal carcinogenesis. This brief review summarizes the general mechanisms of alcohol- and smoking-induced carcinogenesis and then discusses the mechanisms of the development of ESCC, with special attention to alcohol consumption and cigarette smoking.

ERAL1 is associated with mitochondrial ribosome and elimination of ERAL1 leads to mitochondrial dysfunction and growth retardation.

ERAL1, a homologue of Era protein in Escherichia coli, is a member of conserved GTP-binding proteins with RNA-binding activity. Depletion of prokaryotic Era inhibits cell division without affecting chromosome segregation. Previously, we isolated ERAL1 protein as one of proteins which were associated with mitochondrial transcription factor A by using immunoprecipitation. In this study, we analysed the localization and function of ERAL1 in mammalian cells. ERAL1 was localized in mitochondrial matrix and associated with mitoribosomal proteins including the 12S rRNA. siRNA knockdown of ERAL1 decreased mitochondrial translation, caused redistribution of ribosomal small subunits and reduced 12S rRNA. The knockdown of ERAL1 in human HeLa cells elevated mitochondrial superoxide production and slightly decreased mitochondrial membrane potential. The knockdown inhibited the growth of HeLa cells with an accumulation of apoptotic cells. These results suggest that ERAL1 is localized in a small subunit of the mitochondrial ribosome, plays an important role in the small ribosomal constitution, and is also involved in cell viability.

Esophagectomy in patients 80 years of age and older with carcinoma of the thoracic esophagus

BackgroundThe purpose of this study was to clarify the indications for an esophagectomy in elderly patients (especially patients over 80 years of age) with esophageal cancer.MethodsA total of 668 patients with thoracic esophageal cancer who underwent an esophagectomy by the transthoracic approach were divided into three groups according to age, namely, groups I (>80 years, n = 16), II (70–79 years, n = 158), and III (≤69 years, n = 494). In group I, surgery was only done in patients with PS 0 or 1, as well as normal cardiac and pulmonary functions.ResultsThe incidence of preoperative pulmonary risk was 16% and 7% in groups II and III, respectively (P < 0.01). The morbidity rates of group II and III were 42% and 32%, respectively (P < 0.05). Pulmonary complications occurred in 18% and 10%, respectively, and cardiovascular complications occurred in 11% and 4%, respectively (P < 0.01). In group I, the morbidity and 30-day mortality rates were 25% and 0%, respectively, and pulmonary and cardiovascular complications occurred only in one patient each (6%). No significant differences were observed in cause-specific survival.ConclusionsIn the elderly, preoperative pulmonary risk is frequently present, and careful perioperative management is needed while paying special attention to pulmonary and cardiovascular complications. However, when the indications for surgery can be strictly determined, an esophagectomy is considered a viable treatment alternative with satisfactory prognosis even in patients 80 years of age and older without any increased morbidity or mortality.

The difference in p53 mutations between cancers of the upper and lower gastrointestinal tract.

Background:p53 gene mutations have been reported in over half of all human cancers and they appear to occur in the early stage of cancer, thus indicating the important role that such mutations may play in the carcinogenesis of the digestive tract. This study investigated the differences in p53 abnormalities between cancers of the upper and lower gastrointestinal tract. Materials and Methods: The DNA of 354 specimens of gastrointestinal cancer (esophagus 85, stomach 112, colon 157) was extracted and then p53 gene mutations were investigated by direct sequencing; the loss of heterozygosity was also synchronously analyzed in all cases. Results: (1) p53 gene mutation: p53 gene mutations were found in 41 samples (48.2%) in the esophagus, 18 samples (16.0%) in the stomach and 36 samples (22.9%) in the colon. p53 mutations were more frequently identified in well-differentiated cancers and a close correlation was recognized between p53 mutations and loss of heterozygosity. (2) Mutation spectrum: the ratio of transversion was 43.9% in esophagus, 33.3% in stomach and 25.0% in the colon tumors. Reciprocally, the ratio of transition was 31.7, 66.7, and 72.2%, respectively. Discussion: The frequency of p53 transversion mutations was extremely high in the upper digestive tract, whereas transition mutations were more frequently observed in the lower digestive tract. The investigation of the spectrum of mutations in p53 is therefore expected to lead to a better understanding of the agents responsible for inducing cancer.

The triangulating stapling technique for cervical esophagogastric anastomosis after esophagectomy.

PurposeTo evaluate the safety and feasibility of the triangulating stapling technique (TST) for cervical esophagogastric anastomosis after esophagectomy (CEGA).MethodsThe subjects were 123 patients who underwent transthoracic esophagectomy with three-field lymph node dissection and reconstruction with a 3.5-cm wide gastric tube, for thoracic esophageal cancer. We performed the TST for CEGA in 33 patients operated on after December, 2006 (TST group) and hand-sewn anastomosis in 90 patients operated on between 2002 and 2006 (HSA group).ResultsIn the TST group, CEGA was performed in an end-to-end fashion using three linear staplers. The first anastomosis was applied to the posterior walls of the remnant esophagus and gastric tube in an inverted fashion. The second and the third anastomoses were done in an everted fashion to make the anterior wall. The end-to-end HSA was performed with interrupted sutures using 4-0 absorbable material. Anastomotic leakage occurred in only 1 (3.0%) of the 33 TST patients, but in 13 (14.4%) of the 90 HSA patients (P = 0.07). The frequency of anastomotic stenosis was 9.1% and 25.6% in the TST and HSA groups, respectively (P < 0.05).ConclusionsCervical esophagogastric anastomosis using TST may reduce the frequency of anastomotic leakage and stenosis. This technique is a safe and reliable alternative for CEGA after esophagectomy.

Granulocyte-Colony Stimulating Factor-Producing Gallbladder Cancer Without Recurrence More than 2 Years After Resection : Report of a Case

Patients with gallbladder carcinoma rarely have remarkable granulocytosis. Although surgical resection is the most effective treatment, patients with gallbladder carcinoma generally have a very poor prognosis. We report a case of a rapidly growing gallbladder tumor associated with remarkable granulocytosis in a patient who has survived without recurrence for more than 2 years since undergoing resection. The patient had remarkable leukocytosis of 51 500/mm3 with 89% granulocytes and an elevated granulocyte-colony stimulating factor (G-CSF) level of 800 pg/dl. We performed cholecystectomy with extended right lobectomy of the liver and lymph node dissection. A histological diagnosis of moderately differentiated gallbladder carcinoma with sarcomatous change invading the hepatic parenchyma was confirmed. An immunohistochemical examination using polyclonal antibody against G-CSF was performed to stain the tumor cells, which confirmed the diagnosis of a G-CSF-producing tumor.

DNA conformation‐dependent activities of human mitochondrial RNA polymerase

Mitochondrial RNA polymerase (POLRMT) is a core protein for mitochondrial DNA (mtDNA) transcription. In addition, POLRMT is assumed to be involved in replication, although its exact role is not yet clearly elucidated. We have found novel properties of human POLRMT using a reconstituted transcription system. Various lengths of RNA molecules were synthesized from templates even without a defined promoter sequence, when we used supercoiled circular double‐stranded DNA as a template. This promoter‐independent activity was as strong as the promoter‐dependent one. Promoter‐independent DNA conformation‐dependent transcription required TFB2M. On supercoiled templates, the promoter‐independent activity was strongly suppressed by a putatively physiological amount of TFAM, while promoter‐dependent transcription was inhibited to a lesser extent. These different inhibition patterns by TFAM may be important for prevention of random RNA synthesis in vivo. Promoter‐independent activity was also observed on relaxed circular single‐stranded DNA, where its activity no longer required TFB2M. RNA synthesis on single‐stranded DNA was weakly suppressed by a putatively physiological amount of TFAM but restored by the addition of mitochondrial single‐stranded DNA binding protein. We suggest that these properties of POLRMT could explain the characteristic features of mammalian mtDNA transcription and replication.

Surgical complications and the risk factors of totally laparoscopic distal gastrectomy

Purpose It has not yet been elucidated whether there are specific complications associated with totally laparoscopic distal gastrectomy (TLDG). We evaluated the complications and the risk factors associated with TLDG. Methods A retrospective analysis was performed on 138 consecutive patients who underwent TLDG. The clinical and operative data, which included the body mass index, respiratory function, hematological data, pathological data, and the experience of surgeon, were analyzed. Results Intraoperative and postoperative complications developed in 10 and 28 patients, respectively. A univariate analysis determined that the patient age, concurrent respiratory disease, and operation time were important risk factors. A multivariate analysis found no significant risk factors in this set, although the operation time was the most promising risk factor. Conclusions The present data suggest that TLDG can be performed with acceptable perioperative complication rates, although a longer operation time may cause a higher frequency of postoperative complications.

The impact of obesity on the use of a totally laparoscopic distal gastrectomy in patients with gastric cancer

Purpose Since a patients obesity can affect the mortality and morbidity of the surgery, less drastic surgeries may have a major benefit for obese individuals. This study evaluated the feasibility of performing a totally laparoscopic distal gastrectomy, with intracorporeal anastomosis, in obese patients suffering from gastric cancer. Materials and Methods This was a retrospective analysis of the 138 patients, who underwent a totally laparoscopic distal gastrectomy from April 2005 to March 2009, at the National Kyushu Cancer Center. The body mass index of 20 patients was ≥25, and in 118 patients, it was <25 kg/m2. Results The mean values of body mass index in the 2 groups were 27.3±2.2 and 21.4±2.3. Hypertension was significantly more frequent in the obese patients than in the non-obese patients. The intraoperative blood loss, duration of surgery, post-operative complication rate, post-operative hospital stay, and a number of retrieved lymph nodes were not significantly different between the two groups. Conclusions Intracorporeal anastomosis seemed to have a benefit for obese individuals. Totally laparoscopic gastrectomy is, therefore, considered to be a safe and an effective modality for obese patients.