Biomedical subjects

T Tsuzuki

Publications and source records attributed to T Tsuzuki.

At least 73 records · Page 4Linked to original sources

PubMed1997

Significance of the conserved amino acid sequence for human MTH1 protein with antimutator activity.

8-Oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP) is produced during normal cellular metabolism, and incorporation into DNA causes transversion mutation. Organisms possess an enzyme, 8-oxo-dGTPase, which catalyzes the hydrolysis of 8-oxo-dGTP to the corresponding nucleoside monophosphate, thereby preventing the occurrence of mutation. There are highly conserved amino acid sequences in prokaryotic and eukaryotic proteins containing this and related enzyme activities. To elucidate the significance of the conserved sequence, amino acid substitutions were introduced by site- directed mutagenesis of the cloned cDNA for human 8-oxo-dGTPase, and the activity and stability of mutant forms of the enzyme were examined. When lysine-38 was replaced by other amino acids, all of the mutants isolated carried the 8-oxo-dGTPase-negative phenotype. 8-Oxo-dGTPase-positive revertants, isolated from one of the negative mutants, carried the codon for lysine. Using the same procedure, the analysis was extended to other residues within the conserved sequence. At the glutamic acid-43, arginine-51 and glutamic acid-52 sites, all the positive revertants isolated carried codons for amino acids identical to those of the wild type protein. We propose that Lys-38, Glu-43, Arg-51 and Glu-52 residues in the conserved region are essential to exert 8-oxo-dGTPase activity.

Amino Acid Sequence↗

PubMed1997

Organization and expression of the mouse MTH1 gene for preventing transversion mutation.

An enzyme, 8-oxo-7,8-dihydrodeoxyguanosine triphosphatase (8-oxo-dGTPase), is present in various organisms and plays an important role in the control of spontaneous mutagenesis. The enzyme hydrolyzes 8-oxo-dGTP, an oxidized form of dGTP, to 8-oxo-dGMP, thereby preventing the occurrence of A:T to C:G transversion, caused by misincorporation. We isolated the mouse genomic sequence encoding the enzyme and elucidated its structure. The gene, named MTH1 for mutT homologue 1, is composed of at least five exons and spans approximately 9 kilobase pairs. A genomic region containing the pseudogene was also isolated. The promoter region for the gene is GC-rich, contains many AP-1 and AP-2 recognition sequences, and lacks a typical TATA box. Primer extension and S1 mapping analyses revealed the existence of multiple transcription initiation sites, among which a major site was defined as +1. The putative promoter region was placed upstream of the chloramphenicol acetyltransferase reporter gene, and control of expression of the gene was examined by introducing the construct into mouse NIH 3T3 cells. Deletion analysis indicated that a sequence from -321 to +9 carries the basic promoter activity while an adjacent region, spanning from +352 to +525 stimulates the frequency of transcription.

3T3 Cells↗

PubMed1997

Comparison of ras activation in prostate carcinoma in Japanese and American men.

BACKGROUND: Comparative studies of point mutations in K-, N-, and H-ras oncogenes were performed on prostate carcinoma from Japanese and American patients to clarify the racial difference. METHODS: We probed for mutations in 70 Japanese and 31 American specimens using polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis and immunohistochemistry for ras p21. RESULTS: Within the 70 Japanese specimens, eight mutations in codon 12 of K-ras (five GGT-->GTT transversions and three CGT-->GAT transitions) and one mutation in codon 12 of the N-ras gene (a GGT-->GTT transversion) were confirmed, whereas the American samples yielded only one definable mutation, a GGT-->GAT transition, in codon 12 of K-ras. CONCLUSIONS: The frequency of ras gene mutations in clinical carcinoma in Japanese men was higher than that in American men. It is suggested that there may be fundamental differences in the etiology of prostate carcinoma in Japan and the United States, perhaps based on genetics and/or environmental factors.

Adenocarcinoma↗

PubMed1997

Genomic alterations of human gliomas detected by restriction landmark genomic scanning.

Alterations of genomic DNA in eight primary astrocytic tumors and two glioma cell lines were examined using a recently developed two-dimensional gel electrophoresis method called restriction landmark genomic scanning (RLGS). RLGS allows us to detect amplifications, deletions, and methylation in genomic DNA in one procedure without requiring any polymorphic markers. Approximately 2000 spots (landmark sites) in tumor specimens were compared with those in normal brain tissue. The 10 spots with intensified signal were reproducibly detected in at least 50% of primary tumors, implying amplification of corresponding DNA sequences. Conversely, 12 spots with reduced signal were observed in more than 50% of all tumors, suggesting inactivation by allelic loss, homozygous deletion, or CpG island methylation. These results suggest that common genetic alterations are closely correlated with the genesis or progression of human gliomas.

Brain Neoplasms↗

PubMed1997

Broncho-bronchiolitis obliterans as a complication of bone marrow transplantation: a clinicopathological study of eight autopsy cases. Nagoya BMT Group.

We identified eight patients with bronchiolitis obliterans (BO) in the autopsies of 81 bone marrow transplant (BMT) recipients. Rapidly progressive dyspnoea and cough were the main presenting symptoms in all eight patients, associated with overinflation and/or infiltrative opacity seen on chest X-ray and obstructive disorder revealed by pulmonary function tests. Early lesions were characterized by epithelial loss and an inflammatory infiltrate containing foamy histiocytes with mild luminal narrowing. Partial or total occlusion of the bronchiolar lumina by fibrous connective tissue was the feature of late lesions. Both changes were coexistent in all cases. In one case, small bronchi with cartilage were also affected by the obstructive process, showing bronchitis obliterans. All eight patients showed non-obstructive broncho-bronchiolitis characterized by denuding of respiratory epithelium, mural oedema and an inflammatory infiltrate in addition to BO, and these changes were also seen in 18 patients without BO. The submucosal glands of large bronchi and the trachea showed mucous retention and a mild inflammatory infiltrate in four of the eight patients. Coexistent infectious processes were seen in all cases, cytomegalovirus and Aspergillus being the most frequent organisms. BO probably develops as an immunopathological event related to graft-versus-host disease (GVHD) during the impaired immune status phase of the post-BMT period, possibly initiated by infection. Bronchial gland involvement in chronic GVHD is one of the factors responsible for this abnormal immune status.

Adolescent↗

PubMed1997

Analyses of human gliomas by restriction landmark genomic scanning.

The 16 primary gliomas were examined for changes in genomic DNA using a recently developed 2-dimensional gel electrophoresis method called restriction landmark genomic scanning (RLGS). This approach allows detection of DNA amplification, deletion, methylation and potentially other genetic rearrangements represented as decreases and increases in spot/fragment intensity on an autoradiogram. Approximately 2,000 landmark sites in tumor DNA were compared with those of DNA isolated from normal brain tissues. Seven spots showing intensified signal were consistently detected in at least 50% of tumors, implying activation of corresponding DNA sequences, and 8 additional spots having reduced signal were observed, again in more than 50% of all tumors, suggesting inactivation by the loss of 1 allele or homozygous deletion. Decreased signal may also infer relative CpG island methylation state. Of those spots consistently identified in tumors, 2 amplified and 4 reduced spots were found to be characteristic of low- and high-grade tumors, while the remaining 5 amplified and 4 reduced spots were associated with high-grade gliomas only, suggesting a link of specific mutations to degree of malignancy. A separate subset of glioblastomas evaluated, however, showed no alterations in these 'hot spots' which were detected in even low grade astrocytomas. The results demonstrate the genetic heterogeneity of glioblastoma and implicate the progression of neoplasia via differing genetic pathways.

Astrocytoma↗

PubMed1997

Genomic alterations in human glioma cell lines detected by restriction landmark genomic scanning.

Restriction landmark genomic scanning (RLGS) is a 2-dimensional gel analysis capable of detecting amplifications, deletions and rearrangements in genomic DNA. Using RLGS, we examined genomic DNA from each of 6 human-derived malignant glioma cell lines and from normal brain tissue samples. RLGS allows us to screen genomic DNAs as approximately 2,000 landmark sites in one procedure without any polymorphic markers. The resulting 2,000 spots in tumor samples were compared with those in normal brain. Six spots common to 5 of the 6 cell lines showed intensified signal, suggesting amplification of a tumor-specific DNA fragment. In addition, 25 spots common to 5 of the 6 lines showed a decrease in signal intensity, conversely indicating allelic loss of homozygous deletion. These results imply the existence of consistent genetic alterations in human glioma.

Brain Neoplasms↗

PubMed1997

Nodular granulomatous phlebitis of the skin: a fourth type of tuberculid.

We present five cases of granulomatous phlebitis of the skin and compare them with a case of miliary tuberculosis with granulomatous phlebitis. All five patients were hypersensitive to purified protein derivative, but without active tuberculosis. Although anti-tuberculous drugs were effective, no tubercle bacilli were isolated from the skin. Clinically, subcutaneous nodules were felt along the course of the leg vein. Histologically, epithelioid cell granulomas with Langhans' giant cells were observed within the walls of the cutaneous veins. In a later stage, granulomatous panniculitis was often associated. Using the polymerase chain reaction method. Mycobacterium tuberculosis DNA was detected in four of the five cases of granulomatous phlebitis of the skin. Granulomatous phlebitis of the skin seems to represent a relatively early phase of delayed-type hypersensitivity reactions to Mycobacterium tuberculosis and may represent a distinct entity different from other types of tuberculid-a new tuberculid. Nevertheless, before making the diagnosis, the possibility of true tuberculosis must always be excluded. Nodular granulomatous phlebitis of the skin would be an appropriate name for the newly described condition.

Aged↗

PubMed1997

Nitrogen dioxide in indoor ice skating facilities: an international survey.

An international survey of nitrogen dioxide (NO2) levels inside indoor ice skating facilities was conducted. One-week average NO2 concentrations were measured inside and outside of 332 ice rinks located in nine countries. Each rink manager also completed a questionnaire describing the building, the resurfacing machines, and their use patterns. The (arithmetic) mean NO2 level for all rinks in the study was 228 ppb, with a range of 1-2,680 ppb, based on a sample collected at breathing height and adjacent to the ice surface. The mean of the second indoor sample (collected at a spectator's area) was 221 ppb, with a range of 1-3,175 ppb. The ratio of the indoor to outdoor NO2 concentrations was above 1 for 95% of the rinks sampled, indicating the presence of an indoor NO2 source (mean indoor:outdoor ratio = 20). Estimates of short-term NO2 concentrations indicated that as many as 40% of the sampled rinks would have exceeded the World Health Organization 1-hour guideline value of 213 ppb NO2 for indoor air. Statistically significant associations were observed between NO2 levels and the type of fuel used to power the resurfacer, the absence of a catalytic converter on a resurfacer, and the use of an ice edger. There were also indications that decreased use of mechanical ventilation, increased number of resurfacing operations per day, and smaller rink volumes were associated with increased NO2 levels. In rinks where the main resurfacer was powered by propane, the NO2 concentrations were higher than in those with gasoline-powered resurfacers, while the latter had NO2 concentrations higher than in those using diesel. Rinks where the main resurfacer was electric had the lowest indoor NO2 concentrations, similar to the levels measured outdoors.

Air Pollution, Indoor↗

PubMed1997

Alkylation-induced apoptosis of embryonic stem cells in which the gene for DNA-repair, methyltransferase, had been disrupted by gene targeting.

An enzyme O6-methylguanine-DNA methyltransferase (MGMT) catalyzes transfer of a methyl group from O6-methylguanine and O4-methylthymine of alkylated DNA to its own molecule, thereby repairing the pre-mutagenic lesions in a single step reaction. Making use of gene targeting, we developed mouse embryonic stem (ES) cell lines deficient in the methyltransferase. Quantitative immunoblot analysis and enzyme assay revealed that MGMT-/- cells, in which both alleles were disrupted, contained no methyltransferase protein while cells with one intact allele (MGMT+/-) contained about half the amount of protein carried by the parental MGMT+/+ cells. MGMT-/- cells have an extremely high degree of sensitivity to simple alkylating agents, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and N-methyl-N-nitrosourea (MNU), whereas MGMT+/- cells are slightly more sensitive to these agents, as compared with findings from normal cells. A high frequency of mutation was induced in MGMT-/- cells on exposure to a relatively low dose of MNNG. Electrophoretic analyses of the DNAs as well as fluorochrome staining of the cells revealed that MGMT-/- cells treated with MNNG undergo apoptotic death, which occurs after G2-M arrest in the second cycle of cell proliferation.

Alkylation↗

PubMed1997

High incidence of nitrosamine-induced tumorigenesis in mice lacking DNA repair methyltransferase.

The enzyme O6-methylguanine-DNA methyltransferase repairs alkylation-induced DNA damage, O6-methylguanine and O4-methylthymine, the former being formed more frequently. Previously, by means of gene targeting, we generated mice in which alleles for methyltransferase were disrupted. We now use these mouse lines, which are totally deficient in methyltransferase activity, to examine protective effects of the enzyme against tumor formation. In gene-targeted female mice given an i.p. injection of 5 mg/kg of dimethylnitrosamine, a larger number of liver and lung tumors occurred, as compared with normal female mice treated in the same manner. In male mice given a lower dose of carcinogen, the difference between normal and gene-targeted mice was statistically insignificant although more tumors did form in the gene-targeted mice. Methyltransferase apparently afforded protection from nitrosamine-induced tumorigenesis.

Animals↗

PubMed1997

Changes in regional cortical temperature and cerebral blood flow after cortical spreading depression.

Regional cerebral blood flow (rCBF) measurement by laser Doppler flowmetry and cortical temperature measurement using thermoencephaloscopy (TES) were performed to investigate the relationship between the changes in rCBF and cortical temperature after induced cortical spreading depression (CSD) in rats. TES showed a gradually expanding thermoresponse like an extending circular wave after CSD induced by application of KCl. Similarly, a transient increase in rCBF spread from the site of stimulation with a velocity of propagation of 2.5 mm/min. Simultaneous monitoring of rCBF and cortical temperature showed that the transient increase in rCBF was associated with an initial decrease in cortical temperature, followed by an increase in cortical temperature. We suggest that the cortical temperature is regulated mainly by neurogenic control of the pial microvascular blood supply that is precisely adjusted to the metabolic needs of the cerebral cortex. Non-injured cortex with the fine vascular architecture must be preserved during neurosurgery to allow heat transfer from deep areas of the cortex.

Animals↗

PubMed1997

[A case of liposarcoma associated with chronic tuberculous empyema].

A case was 77-year-old male with left back pain who had received artificial pneumothorax therapy 40 years ago due to lung tuberculosis. He had a spindle shaped mass in his left chest wall which had connection to the chronic tuberculous cavity. Because malignant cell was not detected by biopsy, it was therapied as tuberculous abscess. Then the mass grew rapidly into 20 cm in diameter. Although surgical operation was carried out, it was impossible to remove it all. After the surgery, the pathological findings was liposarcoma. In spite of the radiotherapy, the patient died 4 months after the surgery. We should always keep in mind the early diagnosis of malignant tumor associated with chronic tuberculous empyema.

Aged↗

PubMed1997

[The effect of hypothermia on CSD propagation in rats].

In the cortical zone surrounding an ischemic or traumatic focus, CSD is a transient phenomenon involving interstitial ions, blood flow and metabolism and is believed to be completely reversible. However, it may extend to secondary brain injuries because CSD releases excitatory amino acids into the extracellular space. In order to prevent secondary brain injuries, it may be effective to block repeated CSD. This study was designed to determine whether hypothermia can block CSD propagation and whether this study is a potentially useful means for preventing secondary brain injuries. Male wistar rats weighing 270 g on average were used for the experiments. The animals were divided into two groups: hypothermic rats (33.5-34 degrees C, rectal temp.) and normothermic rats (37-37.5 degrees C). The changes in rCBF (regional Cerebral Blood Flow) were monitored in order to observe CSD. LDF (Laser Doppler Flowmetry) was used to measure rCBF. The two LDF probes were placed on the parietal cortex (4 mm apart). To elicit CSD, a needle stab injury was made on the cortex or a piece of paper soaked with 10% KCl was applied on the cortex. The velocity of CSD propagation was more prolonged in the hypothermic rats than in the normothermic rats (p < 0.01). There were smaller numbers of repeated CSD in the hypothermic rats than in the normothermic rats. Histological examination of the cerebral cortex revealed shrinkage neurons more distinctly in the normothermic rats than in hypothermic rats. From these results, we can speculate that hypothermic may block CSD propagation and that hypothermic therapy has the potential to prevent secondary brain injuries.

Animals↗

PubMed1997

[Three cases of primary lung cancer unexpectedly discovered during the operation of pneumothorax].

It is well known that emphysematous bulla is thought to be often associated with lung cancer. However, it is very rare that lung cancer predisposing to pneumothorax as initial manifestation. We performed surgical operations of four hundred and one cases of spontaneous pneumothorax, and discovered three cases of lung cancer during the operation. However, these three cases occupied the 30% of the patients with pneumothorax who were older than 65 years. The two of them were adenocarcinomas which were situated in the wall of bullae, but did not perforated the bullous wall. The other one was squamous cell carcinoma which was apart from the bullous lesion. This shows that we should always be careful of the associated lung cancer when we care elderly patients with pneumothorax.

Adenocarcinoma↗

PubMed1996

Alterations of retinoblastoma, p53, p16(CDKN2), and p15 genes in human astrocytomas.

BACKGROUND: Alterations of the suppressor genes, such as the retinoblastoma (RB), p53, p16(CDKN2), and p15 genes, have been reported in human gliomas. These genes have been suggested as the cell cycle regulatory genes at the G1-S checkpoint. METHODS: Alterations of the RB, p53, p16(CDKN2), and p15 genes in human astrocytomas were screened by single strand conformation polymorphism analysis of polymerase chain reaction products (PCR-SSCP analysis) and then confirmed by dideoxy sequencing. In addition, the expression of RB and p16 protein was examined by Western blot analysis. RESULTS: Aberrations of the RB gene were found in 3 of 23 surgical astrocytoma specimens (13%). Mutations were found at codon 754 in exon 22 (Val-->Gly), codon 519 in exon 17 (Thr-->Pro), and one base deletion at codon 903 resulting in stop codon at codon 905 in exon 26. These mutational locations were all near the regions associated with the functional domains of the RB gene. Aberrations of the p53 gene were found in 4 cases (17.4%). These mutations were found at codons 146 (Trp-->Gly) and 165 (Gln-->His) in exon 5, codon 73 (Val-->Glu) in exon 4, and codon 313 (Ser-->Asn) in exon 9. In addition, alterations of the p16(CDKN2) gene were found, with 5 cases (21.7%) having homozygous deletions, and 2 cases (8.7%) harboring point mutations. No p15 gene alteration was detected. The expression of p16 protein was undetectable in 10 cases (43.5%) by Western blot analysis, demonstrating an inverse correlation with the expression of RB protein. CONCLUSIONS: A few cases had overlapping alterations, and the incidence of one or more RB, p53, or p16(CDKN2) changes appeared to be relatively high in human astrocytomas. These results suggest that cell cycle regulatory gene alterations may play an important role in the development of gliomas.

Astrocytoma↗

PubMed1996

Targeted disruption of the Rad51 gene leads to lethality in embryonic mice.

The mouse Rad51 gene is a mammalian homologue of the Escherichia coli recA and yeast RAD51 genes, both of which are involved in homologous recombination and DNA repair. To elucidate the physiological role of RAD51 protein, the gene was targeted in embryonic stem (ES) cells. Mice heterozygous for the Rad51 null mutation were intercrossed and their offspring were genotyped. There were no homozygous (Rad51-/-) pups among 148 neonates examined but a few Rad51-/- embryos were identified when examined during the early stages of embryonic development. Doubly knocked-out ES cells were not detected under conditions of selective growth. These results are interpreted to mean that RAD51 protein plays an essential role in the proliferation of cell. The homozygous Rad51 null mutation can be categorized in cell-autonomous defects. Pre-implantational lethal mutations that disrupt basic molecular functions will thus interfere with cell viability.

Alleles↗

PubMed1996

Detection of ret homodimers in MEN 2A-associated pheochromocytomas.

Using transfection of NIH 3T3 cells, we have recently demonstrated that multiple endocrine neoplasia (MEN) 2A mutations activate the c-Ret protein by inducing its disulfide-linked homodimerization on the cell surface. To investigate whether the homodimers are present in original tumors, the expression of the c-Ret protein was analyzed in eight sporadic and two MEN 2A-associated pheochromocytomas, the latter two of which contained mutations in cysteine 618 or 634 of Ret. The c-Ret protein was expressed at variable levels in all pheochromocytomas examined. By labeling the c-Ret protein immunoprecipitated from tumor tissues with [gamma-32P]ATP in vitro, its homodimers were detected in pheochromocytomas from MEN 2A patients but not in a sporadic tumor. This result represents the first demonstration of Ret homodimers in original tumors.

Drosophila Proteins↗