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Biomedical subjects

M Kusumoto

Publications and source records attributed to M Kusumoto.

At least 37 records · Page 2Linked to original sources
PubMed2000

Reactivation of insertionally inactivated Shiga toxin 2 genes of Escherichia coli O157:H7 caused by nonreplicative transposition of the insertion sequence.

IS1203v is an insertion sequence which has been found in inactivated Shiga toxin 2 genes of Escherichia coli O157:H7. We analyzed the transpositional mechanism of IS1203v in order to investigate whether the Shiga toxin 2 genes inactivated by IS1203v could revert to the wild type. When the transposase activity of IS1203v was enhanced by artificial frameshifting, IS1203v was obviously excised from the Shiga toxin 2 gene in a circular form. The IS1203v circle consisted of the entire IS1203v, but an extra 3-bp sequence (ATC) intervened between the 5' and 3' ends of IS1203v. The extra 3-bp sequence was identical to a direct repeat which was probably generated upon insertion. Moreover, we detected the Shiga toxin 2 gene with a precise excision of IS1203v. In the wild-type situation, the transposition products of IS1203v could be observed by PCR amplification. These results show that IS1203v can transpose in a nonreplicative manner and that the Shiga toxin gene inactivated by this insertion sequence can revert to the wild type.

Bacterial Toxins↗
PubMed1999

Adenoviral GM-CSF Gene Transduction into Breast Cancer Cells Induced Long-Lasting Antitumor Immunity in Mice.

BACKGROUND: Immunogene therapy is regarded as a novel treatment that overcomes the limitation of preexisting adjuvant therapies and demonstrates the potential for the total elimination of cancer cells by affecting concealed tumor cells. The aim of this study was to examine the enhancement of antitumor immunity of irradiated granulocyte-macrophage colony-stimulating factor (GM-CSF) gene-transduced mouse breast cancer cells. METHODS: To study prophylactic vaccine effects, Balb/c mice were vaccinated subcutaneously with saline or irradiated mouse breast cancer cells (BalbMC, 1 x10&sup6; /mouse) infected with or without recombinant adenovirus harboring the GM-CSF gene (Adv/GM-CSF) on day-7. Mice were challenged with parental cells (1x10&sup5;/mouse) on day 0 in the flank opposite the vaccination site. RESULTS: BalbMC cells secreted GM-CSF after infection with Adv/GM-CSF. Vaccination with irradiated GM-CSF-secreting BalbMC completely protected syngeneic mouse challenged with live parental cells. On the other hand, vaccination with irradiated BalbMC protected 60% of mice from tumor development after challenge with parental cells. None of the tumor-free mice initially vaccinated with irradiated GM-CSF-producing BalbMC cells developed tumor upon repeated challenge with parental cells during the entire observation period. CONCLUSION: Our study demonstrated the feasibility of this immunotherapeutic approach as a novel adjuvant therapy after surgery for breast cancer.

Journal Article↗
PubMed1999

Crystal structure of an IRF-DNA complex reveals novel DNA recognition and cooperative binding to a tandem repeat of core sequences.

There has been growing interest in the role of the IRF (interferon regulatory factor) family of transcription factors in the regulation of immune responses, cytokine signaling, and oncogenesis. These members are characterized by their well-conserved DNA binding domains at the N-terminal regions. Here we report the 2.2 A resolution crystal structure of the DNA binding domain of one such family member, IRF-2, bound to DNA. The structure reveals its recognition sequence, AANNGAAA (here, recognized bases are underlined and in bold, and N indicates any base), and its cooperative binding to a tandem repeat of the GAAA core sequence induced by DNA structure distortions. These facts explain well the diverse binding properties of the IRF family members, which bind to both single and tandemly repeated sequences. Furthermore, we also identified the 'helix-hairpin-strand motif' at the C terminus of the recognition helix as a metal binding site that is commonly found in certain classes of DNA-interactive proteins. Our results provide new insights into the structure and function of this family of transcription factors.

Amino Acid Sequence↗
PubMed1999

Quantitative determination of apoptotic death in cultured human pancreatic cancer cells by propidium iodide and digitonin.

We present here the efficacy of an in vitro cytotoxicity assay which can measure rapidly both apoptotic dead cells and cell growth rate, quantitatively. Using a multi-well plate reader, the fluorescence intensity of propidium iodide (PI) corresponding to dead cells and to total cells after digitonin treatment were measured in cultured human pancreatic cancer cells following exposure to etoposide. The percentage of dead cells measured by this assay was well correlated to that determined by Trypan blue staining. Furthermore, the cell growth rate determined simultaneously was also correlated to the cell number counted directly using a microscope. We demonstrate that this method, which was originally established for evaluating necrosis, could be applied to measure apoptotic cell death. Taken together, this simple assay is useful for testing the efficacy of anti-cancer agents and for investigating the molecular mechanisms of apoptosis in the cultured cells.

Antineoplastic Agents, Phytogenic↗
PubMed1999

Activation of telomerase and its diagnostic application in biopsy specimens from biliary tract neoplasms.

BACKGROUND: Telomerase activity has been reported to have potential as a useful diagnostic marker for cancer in various organs. The authors previously reported that telomerase activity in pancreatic juice differentiates pancreatic ductal carcinoma from adenoma and pancreatitis. In the current study, the usefulness of semiquantitatively determined telomerase activity in the diagnosis of malignant biliary tract neoplasms was investigated. METHODS: The samples examined included 61 surgically resected biliary tract tissues (11 gallbladder carcinomas, 5 bile duct carcinomas, 1 gallbladder adenoma, 30 cholecytitis cases, 7 cholesterol polyps, 1 normal gallbladder, and 6 normal common bile duct tissues), 42 bile samples from patients with biliary tract or pancreatic disease (19 cases of malignant biliary tract disease, 11 cases of benign biliary tract disease, 10 cases of malignant pancreatic disease, and 2 cases of benign pancreatic disease), and 14 bile duct biopsy specimens collected by percutaneous transhepatic choledochoscopy or endoscopic retrograde cholangiopancreatography (8 bile duct carcinoma specimens, 1 bile duct adenoma specimen, and 5 hepatolithiasis specimens). RESULTS: In biliary tract tissues, a telomerase ladder was detected in 73% of gallbladder carcinomas, 40% of bile duct carcinomas, and none of the other biliary tract tissues. One gallbladder adenoma showed a weak telomerase ladder. The telomerase ladder was detected in the bile sample from 1 patient (5.3%) with malignant biliary tract disease, none of the patients with benign biliary tract disease, 5 patients (50%) with malignant pancreatic disease, and none of the patients with benign pancreatic disease. In biopsy specimens, the telomerase ladder was detected in 75% of patients with bile duct carcinoma but not in any of the patients with hepatolithiasis. The median value of relative telomerase activity in the patients with bile duct carcinoma was significantly higher than that in the patients with hepatolithiasis. The diagnosis of bile duct carcinoma was confirmed preoperatively by histopathologic examination in only 25% of the biopsy specimens. CONCLUSIONS: The results of the current study indicate that telomerase is highly activated in biliary tract carcinomas and that the detection of a telomerase ladder in biopsy samples is an excellent tool for the diagnosis of bile duct carcinomas.

Amino Acid Sequence↗
PubMed1999

Identification of an insertion sequence, IS1203 variant, in a Shiga toxin 2 gene of Escherichia coli O157:H7.

An insertion sequence composed of 1310 bp was found in Shiga toxin 2 genes of some isolates of Escherichia coli O157:H7. This insertion sequence showed extremely high homology with IS1203 of E. coli O111:H(-). This IS1203 variant was inserted in the region encoding the amino-terminus of the B subunit with a duplication of 3 bp at the target site, resulting in inactivation of the Shiga toxin 2 gene.

Journal Article↗
PubMed1999

Susceptibility to hydrogen peroxide and catalase activity of root nodule bacteria.

The root nodule bacteria (free-living cells) tested had higher susceptibility to hydrogen peroxide (H2O2) than the other genera of aerobic or facultative anaerobic bacteria tested. The catalase activities tended to have a positive correlation with H2O2 resistance among all bacteria tested. Addition of a catalase inhibitor such as 3-amino-1, 2, 4-triazole increased the susceptibility to H2O2. These results suggest that the lower catalase activity brings about the higher susceptibility of root nodule bacteria to H2O2. Root nodule bacteria seemed to have two or three catalase isozymes during growth and their catalase activities were higher in log phase than in stationary phase, contrary to other genera of bacteria tested.

Amitrole↗
PubMed1999

Centrosome abnormalities in pancreatic ductal carcinoma.

The centrosome plays an important role in microtubule nucleation and organization, ensuring the establishment of cell polarity and balanced chromosome segregation. Recent studies have suggested that the loss of cell polarity and/or chromosome missegregation (aneuploidy) in human malignant tumors could result from defects in centrosome function. Using immunofluorescence analysis with an antibody to gamma-tubulin (a well-characterized centrosomal component), we examined surgically resected human pancreatic tissues for centrosome abnormalities. The tissues included ductal carcinomas (n = 13), adenomas (n = 3), endocrine tumors (n = 3), chronic pancreatitis (n = 5), and normal pancreatic tissues (n = 12). We found that most (85%) carcinomas and some adenomas displayed abnormal centrosome profiles, characterized by an increase in size and number of centrosomes, and by their irregular distribution. In contrast, none of normal ductal and stromal tissues showed these abnormalities. These findings suggest that centrosome abnormalities may develop at a relatively early stage of pancreatic ductal carcinogenesis.

Adenocarcinoma↗
PubMed1999

Adenovirus-mediated p53 gene transduction inhibits telomerase activity independent of its effects on cell cycle arrest and apoptosis in human pancreatic cancer cells.

Evidence for a relationship between overexpression of wild-type p53 and telomerase activity remains controversial. We investigated whether p53 gene transduction could cause telomerase inhibition in pancreatic cancer cell lines, focusing on the relation of transduction to growth arrest, cell cycle arrest, and apoptotic cell death. The cells were infected with recombinant adenovirus expressing wild-type p53 or p21WAF1 at a multiplicity of infection of 100 or were continuously exposed to 10 microM VP-16, which is well known to induce apoptosis. Adenovirus-mediated p53 gene transduction caused G1 cell cycle arrest, apoptosis, and resultant growth inhibition in MIA PaCa-2 cells; the cell number 2 days after infection was 50% of preinfection value, and 13% of the cells were dead. Moreover, the transduction resulted in complete depression of telomerase activity through down-regulation of hTERT mRNA expression. In contrast, p21WAF1 gene transduction only arrested cell growth and cell cycle at G1 phase, and VP-16 treatment inhibited cell growth with G2-M arrest and apoptosis; after treatment, the cell number was 73% of pretreatment, and 12% of the cells were dead. Neither p21WAF1 gene transduction nor VP-16 treatment caused telomerase inhibition. Similar results were obtained in two other pancreatic cancer cell lines, SUIT-2 and AsPC-1. Thus, our results demonstrate that the p53 gene transduction directly inhibits telomerase activity, independent of its effects on cell growth arrest, cell cycle arrest, and apoptosis.

Adenoviridae↗
PubMed1998

9-Hydroxyellipticine inhibits telomerase activity in human pancreatic cancer cells.

There is increasing interest in identifying potent inhibitors of telomerase because the enzyme plays a crucial role in the development of cellular immortality and carcinogenesis. We hypothesized that 9-hydroxyellipticine (9-HE), an antitumor alkaloid, would inhibit telomerase activity because the drug has a unique mechanism of inhibiting phosphorylation of mutant p53 protein via inhibition of protein kinases, thereby restoring wild-type p53 function. This study was conducted to examine the effect of 9-HE on telomerase activity in human pancreatic cancer cells with differing p53 gene status. 9-HE treatment at relatively high concentrations resulted in rapid, complete inhibition of telomerase activity, irrespective of the p53 status. We conclude that 9-HE may exert a strong inhibitory effect on telomerase activity possibly through inhibition of protein kinases rather than through restoration of functional wild-type p53.

Antimetabolites, Antineoplastic↗
PubMed1998

Crystallographic Characterization of the DNA-Binding Domain of Interferon Regulatory Factor-2 Complexed with DNA.

Interferon regulatory factors (IRFs) are transcription factors for interferon-related genes, which manifest both antiviral and tumor-suppressor activities and regulate cell growth in response to DNA damage. For the transcription initiation of the interferon-beta gene, IRFs form a macromolecular assembly bound to the promoter DNA, referred to as an enhancesome, together with several other transcription factors and DNA-binding proteins. The three-dimensional structure of IRF-DNA complex would provide insights into the structure and function of the enhancesome. In this study, we crystallized the DNA-binding domain of interferon regulatory factor-2 complexed with a DNA fragment. The crystals reproducibly grew by the vapor diffusion technique with 2-methyl-pentanediol from solutions containing small detergents, such as n-octyl-beta-d-glucoside. Cryocrystallographic experiments showed that crystals belong to space group P212121 with a = 90.66 Å, b = 101.01 Å, c = 171.58 Å and diffract up to 2.8 Å resolution. The absorption measurements of a solution in which the crystals were dissolved indicate that the DNA-binding domain binds to the DNA as a dimer. The calculated values of the solvent contents suggest that the protein-DNA complexes form a multimer in the crystal. These features may reflect the association of the complexes in the enhancesome. Copyright 1998 Academic Press.

Journal Article↗
PubMed1998

Telomerase activity detected in pancreatic juice 19 months before a tumor is detected in a patient with pancreatic cancer.

We report a patient with pancreatic cancer in whom telomerase activity had been detected in the pancreatic juice 19 months before he was diagnosed as having pancreatic cancer. A 61-yr-old alcoholic man complaining of epigastric and back pain was diagnosed as having groove pancreatitis based on the presence of inflammation in the pancreatic head and its extension to the duodenal mucosa with an associated elevated serum amylase level. All imaging modalities showed no sign of a tumor. However, high telomerase activity was detected in the pancreatic juice collected during endoscopic retrograde pancreatography. His symptoms subsided due to abstinence from alcohol. A tumor, however, was recognized on computed tomography 19 months later, at which time the patient immediately underwent a pylorus-preserving pancreaticoduodenectomy. The carcinoma was located mainly in the Santorini duct region. High telomerase activity in the pancreatic juice may precede clinical detection of pancreatic cancer and thus could be a useful early diagnostic marker for pancreatic cancer.

Biomarkers, Tumor↗
PubMed1998

Lack of pharmacokinetic interaction between mexiletine and omeprazole.

OBJECTIVE: To investigate the effect of omeprazole on the pharmacokinetics of mexiletine. METHODS: Nine healthy male Japanese volunteers participated in a crossover study. On day 1, the subjects received mexiletine 200 mg. On days 2-7, they received omeprazole 40 mg, and on day 8 they received mexiletine 200 mg and omeprazole 40 mg concomitantly. Serum concentrations of mexiletine were determined just before drug administration and at 1, 2, 3, 4, 6, 8, 12, and 24 hours on day 1 and day 8. RESULTS: No differences in mexiletine concentrations were observed between the two phases of the study. The mean AUCs after administration of mexiletine alone and in combination with omeprazole 40 mg/d were 6.26 and 6.20 ng.h/L, respectively. CONCLUSIONS: These findings suggest that omeprazole does not affect mexiletine metabolism.

Administration, Oral↗
PubMed1998

[MR images of mycobacterial infection].

With the progress of imaging technologies such as CT and MR imaging, we can obtain various informations from CT and MR images. Especially, thin-section high resolution CT (HRCT) provides very useful information for the diagnosis of mycobacterial infection and other diseases of pulmonary parenchyma. Advantages of MR images over HRCT are higher tissue contrast and multidirection capability. The T2-weighted images and Gd-DTPA enhanced T1-weighted images accurately reflect pathologic structure of the lesion. Therefore, MR images can add many useful findings to CT on the selected cases which are differentiation of tubercloma from lung cancer, chronic empyema from mesothelioma, aspergiloma from lung cancer and lymphadenopathy from lymph node metastasis. We describe the usefulness of MR images for diagnosing mycobacterial infection and its differentiation from other pulmonary diseases.

Diagnosis, Differential↗
PubMed1997

Correlation between measurements using a new halitosis monitor and organoleptic assessment.

Halitosis is known as unpleasant oral odor and is a health concern among the general public. Previously, we reported on a new portable monitor with a zinc-oxide, thin-film, semiconductor sensor which demonstrated simplicity of handling, high reproducibility and correspondence for organoleptic assessment. The results suggested its usefulness for the diagnosis of halitosis. Using the monitor, oral air samples of 94 subjects were measured in a field survey, and the values were compared with the organoleptic rates of corresponding samples assessed by two judges. A highly significant correlation (r = 0.824, P < 0.01) was demonstrated between the measures obtained by the two methods. The results suggest that the monitor is useful for not only a clinical study but also a field study of halitosis.

Adult↗