Biomedical subjects

Y Takeshima

Publications and source records attributed to Y Takeshima.

At least 73 records · Page 4Linked to original sources

PubMed1996

p16/CDKN2 gene and p53 gene alterations in Japanese non-smoking female lung adenocarcinoma.

Primary lung adenocarcinomas in non-smoking females are increasing in the USA and Japan. Environmental factors such as passive smoking, asbestos, domestic radon, and hormonal effects have been implicated, but the etiology is still uncertain. We therefore analyzed point mutations of p16 gene, a newly characterized tumor suppressor gene, and compared the results with alterations of p53 gene in 28 primary lung adenocarcinomas in non-smoking Japanese females. There were no cases with somatic point mutation of p16 gene, except for one case with two germline mutations (silent mutations). In contrast, six out of 16 informative cases showed loss of heterozygosity of p53 gene using a TP53 microsatellite marker and 19 out of 28 cases showed expression of oncoprotein using DO-7 immunohistochemistry. These findings suggest that p16 gene alteration is a rare event in primary lung adenocarcinomas in Japanese non-smoking females, compared with alterations of the p53 gene.

Adenocarcinoma↗

PubMed1996

Alteration of the p53 gene of lung carcinomas with sarcomatous transformation (spindle cell carcinoma): analysis of four cases.

Lung carcinoma with sarcomatous transformation (LCST) is highly aggressive and characterized by local invasion and/or distant metastasis, which leads to a shorter survival than ordinary lung carcinomas. Therefore, to elucidate whether the malignant potential of the spindle cell element in LCST is associated with the alteration of the p53 gene, four cases were examined by analyses of overexpression of the p53 oncoprotein, mutation of the p53 gene and loss-of-heterozygosity (LOH) at chromosome 17p. In two cases overexpression of the p53 oncoprotein of the spindle cell component showed a higher degree of staining than that of the carcinoma component; LOH was identified in both carcinoma and sarcomatous components in one case, while in contrast, another case showed LOH in the sarcomatous component only. Mutations were clearly detected in two cases; one showed a CTT to CGT transversion in codon 194 of exon 6 in both components, whereas the other showed a CTG to CAG transversion in codon 265 of exon 8 in the sarcomatous component only. On the basis of these observations, it suggested that the sarcomatous component shows a higher frequency of p53 gene abnormalities in comparison to the carcinoma component. These results also suggested that the acquisition of malignant potential in the sarcomatous component, or the morphological alteration of carcinoma cells, is correlated with abnormalities associated with the p53 gene.

Adenocarcinoma↗

PubMed1996

Primary carcinoid tumor of the kidney with special reference to its histogenesis.

A case of primary carcinoid tumor of the kidney occurring in a 66-year-old woman is reported. The tumor was 10 x 10 x 9 cm in size, solid, yellowish-white in color, and associated with massive hemorrhagic necrosis. Histologically, it was composed of trabecular and anastomosing ribbon-like nests. The tumor cells showed argyrophilicity with the Grimelius stain and cytoplasmic positivity for neuroendocrine markers, (including chromogranin A, neuron-specific enolase, synaptophysin and Leu-7), prostatic acid phosphatase, keratin and vimentin. Numerous cytoplasmic neurosecretory granules were demonstrated ultrastructurally. These histologic and immunohistochemical findings are consistent with the theory that this tumor has a common phenotype with that of carcinoid tumor arising from the hindgut or cloaca.

Acid Phosphatase↗

PubMed1996

A case of Becker muscular dystrophy resulting from the skipping of four contiguous exons (71-74) of the dystrophin gene during mRNA maturation.

The mutations in one-third of both Duchenne and Becker muscular dystrophy patients remain unknown because they do not involve gross rearrangements of the dystrophin gene. Here we report the first example of multiple exon skipping during the splicing of dystrophin mRNA precursor encoded by an apparently normal dystrophin gene. A 9-year-old Japanese boy exhibiting excessive fatigue and high serum creatine kinase activity was examined for dystrophinopathy. An immunohistochemical study of muscle tissue biopsy disclosed faint and discontinuous staining of the N-terminal and rod domains of dystrophin but no staining at all of the C-terminal domain of dystrophin. The dystrophin transcript from muscle tissue was analyzed by the reverse transcriptase polymerase chain reaction. An amplified product encompassing exons 67-79 of dystrophin cDNA was found to be smaller than that of the wild-type product. Sequence analysis of this fragment showed that the 3' end of exon 70 was directly connected to the 5' end of exon 75 and, thus, that exons 71-74 were completely absent. As a result, a truncated dystrophin protein lacking 110 amino acids from the C-terminal domain should result from translation of this truncated mRNA, and the patient was diagnosed as having Becker muscular dystrophy at the molecular level. Genomic DNA was analyzed to identify the cause of the disappearance of these exons. Every exon-encompassing region could be amplified from genomic DNA, indicating that the dystrophin gene is intact. Furthermore, sequencing of these amplified products did not disclose any particular nucleotide change that could be responsible for the multiple exon skipping observed. Considering that exons 71-74 are spliced out alternatively in some tissue-specific isoforms, to suppose that the alternative splicing machinery is present in the muscle tissue of the index case and that it is activated by an undetermined mechanism is reasonable. These results illustrate a novel genetic anomaly that results in dystrophinopathy.

Child↗

PubMed1995

C to T transition at the first nucleotide of codon 63 of the beta-globin gene corresponding to hemoglobin M-Saskatoon in an Indonesian boy.

Hemoglobin (Hb) M-Saskatoon, a beta variant of methemoglobin, is characterized by mild hemolysis. It is caused by the substitution of a histidine by a tyrosine at the 63rd amino acid residue of the beta-globin chain. Amplification and sequence analysis of genomic beta-globin DNA from an Indonesian boy diagnosed as having the more severe disease thalassemia demonstrated the presence of a C to T transition at nucleotide 473 in one of the two beta-globin genes resulting in a histidine to tyrosine substitution at 63rd residue. This amino acid change matched with that reported in Hb M-Saskatoon. This nucleotide change abolished a recognition site for the restriction endonuclease NlaIII. NlaIII digestion of the corresponding beta-globin DNA amplified from the patient's parents indicated that the mutation was inherited through from his mother. This result shows that the world-wide distribution of Hb M-Saskatoon extends to Indonesia, where it was not previously identified. Possible causes of the unusually severe symptoms observed in the case are discussed.

Base Sequence↗

PubMed1995

Role of point mutation of the K-ras gene in tumorigenesis of B6C3F1 mouse lung lesions induced by urethane.

In order to elucidate the role of point mutation of the K-ras gene in the tumorigenetic process of lung tumors, an experimental model of lung lesions in mice induced by the administration of urethane was used. A total of 135 B6C3F1 male mice, 6 weeks old, were given urethane in the drinking water at 0, 6, 60, 600 or 1200 ppm, and were then killed after varying periods of time. The lung lesions were histologically characterized as hyperplasia, adenoma and adenocarcinoma. Point mutations in codons 12 and 61 of the K-ras gene were detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and confirmed by using dideoxy sequencing analysis. K-ras gene mutation was identified in 9 (23.7%) of 38 lesions classified as hyperplasia, 31 (46.3%) of 67 adenomas, and 3 (50%) of 6 adenocarcinomas. The most frequent mutation was an AT-to-TA transversion at the second base of codon 61 and this pattern accounted for 65% of the three mutant forms observed. These results suggest that the point mutation of K-ras gene is involved in all stages of mouse lung tumorigenesis, i.e., activation of this gene can also influence the later stages of lung lesions.

Adenocarcinoma↗

PubMed1995

Modulation of in vitro splicing of the upstream intron by modifying an intra-exon sequence which is deleted from the dystrophin gene in dystrophin Kobe.

Molecular analysis of dystrophin Kobe showed that exon 19 of the dystrophin gene bearing 52-bp deletion was skipped during splicing, although the known consensus sequences at the 5' and 3' splice sites of exon 19 were maintained (Matsuo, M., T. Masumura, H. Nishio, T. Nakajima, Y. Kitoh, T. Takumi, J. Koga, and H. Nakamura. 1991. J. Clin. Invest. 87:2127-2131). These data suggest that the deleted sequence of exon 19 may function as a cis-acting element for exact splicing for the upstream and downstream introns. To investigate this potential role of exon 19, an in vitro splicing system using artificial dystrophin mRNA precursors (pre-mRNAs) was established. Pre-mRNA containing exon 18, truncated intron 18, and exon 19 was spliced precisely in vitro, whereas splicing of intron 18 was almost completely abolished when the wild-type exon 19 was replaced by the dystrophin Kobe exon 19. Splicing of intron 18 was not fully reactivated when dystrophin Kobe exon 19 was restored to nearly normal length by inserting other sequences into the deleted site. These results suggest that the presence of the exon 19 sequence which is lost in dystrophin Kobe is more critical for splicing of intron 18 than the length of the exon 19 sequence. Characteristically, the efficiency of splicing of this intron seemed to correlate with the presence of polypurine tracks within the downstream exon 19. Moreover, an antisense 31-mer 2'-O-methyl ribonucleotide complementary to the 5' half of the deleted sequence in dystrophin Kobe exon 19 inhibited splicing of wild-type pre-mRNA in a dose- and time-dependent manner. This first in vitro evidence that dystrophin pre-mRNA splicing can be modulated by an antisense oligonucleotide raises the possibility of a new therapeutic approach for Duchenne muscular dystrophy.

Base Sequence↗

PubMed1994

High-level expression of human superoxide dismutase in the cyanobacterium Anacystis nidulans 6301.

A chemically synthesized gene encoding human CuZn superoxide dismutase (hSOD) was cloned into the shuttle vector pBAX18R and expressed in Anacystis nidulans 6301 (Synechococcus sp. strain PCC 6301) under the control of a ribulose-1,5-bisphosphate carboxylase/oxygenase gene (rbc) promoter derived from A. nidulans 6301. The sequences immediately upstream from the hSOD coding region and the distances between the ribosomal binding site and ATG initiation codon strongly affected the expression of the hSOD gene in A. nidulans cells. Optimal expression of hSOD was obtained with the expression vector pBAXSOD8-I, which contained a GGAGAG sequence. In defined conditions, irradiation with light increased hSOD enzyme activity in the transformants > 18-fold and the level of the hSOD protein reached a value of about 3% of the total soluble protein. The transformants that expressed hSOD acquired the ability to extenuate photooxidative damage induced by methyl viologen.

Amino Acid Sequence↗

PubMed1994

Twenty seven nucleotide deletion within exon 11 of the erythrocyte band 3 gene in Indonesian ovalocytosis.

We here report the molecular characterization of an Indonesian ovalocytosis. The analysis of genomic gene by polymerase chain reaction shows that the individual has two amplified products from a region encompassing exon 11 of the erythrocyte band 3 gene. The sequence of the larger product matched completely with that of normal individuals. In the sequence of the smaller product, 27 nucleotides within exon 11 disappeared. The deletion removes a total of nine amino acids in the boundary of cytoplasmic and membrane domains of band 3 protein, a membrane anion transporter protein. This is the first report to confirm the heterogeneous presence of an altered membrane band 3 protein in Indonesian ovalocytosis.

Anion Exchange Protein 1, Erythrocyte↗

PubMed1994

p53 mutations in lung cancers from Japanese mustard gas workers.

Mustard gas (MG) is a mutagenic and carcinogenic alkylating agent, and is a known risk factor for occupational lung cancer. Our hypothesis is that lung cancers from MG workers contain mutations (G:C to A:T transitions) as the result of MG-produced DNA promutagenic adducts in the p53 tumor suppressor gene. We analyzed 12 primary lung cancers from Japanese MG factory workers and 12 lung cancers from non-exposed individuals. Genomic DNA was isolated from archival paraffin-embedded tissues. Exons 5-8 were amplified by polymerase chain reaction using p53-specific primers, and sequenced by dideoxy termination methods. Six out of 12 lung cancers from MG workers contained a total of eight somatic point mutations: two cases had double G:C to A:T transitions; one had a G:C to T:A transversion; one case had an A:T to G:C transition; and two cases had single base deletions. Four of the six mutated purines occurred on the non-transcribed, DNA-coding strand. Out of 12 unexposed cases, there were six single base mutations in six cancers, and no double mutations. The p53 mutational frequency in the MG-exposed cases is similar to the non-exposed controls and the usual smoking-related lung cancers reported previously. However, the distinctive double mutations (G:C to A:T transition) observed in two cases are unusual and may be related to MG exposure.

Aged↗

PubMed1994

A novel expression vector for the cyanobacterium, Synechococcus PCC 6301.

A cyanobacterial expression vector was constructed using ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) promoter and terminator sequences derived from Synechococcus PCC 6301. recombinant plasmid, designated pARUB19, has an ampicillin-resistant (ApR) gene as a selectable marker and four unique restriction sites to allow the insertion of foreign genes. Using this vector, the luciferase gene from the firefly, Photinus pyralis, was introduced into Synechococcus PCC 6301 cells. The luciferase expression vector could be maintained stably in the host cells. Light production of luciferin/luciferase was detected in the transformants. Luciferase amounted to 1.2% of the total soluble protein. This plasmid may facilitate higher levels of foreign gene expression in Synechococcus PCC 6301.

Bacterial Proteins↗

PubMed1994

Identification of a novel first exon in the human dystrophin gene and of a new promoter located more than 500 kb upstream of the nearest known promoter.

The dystrophin gene, which is mutated in patients with Duchenne and Becker muscular dystrophies, is the largest known human gene. Five alternative promoters have been characterized until now. Here we show that a novel dystrophin isoform with a different first exon can be produced through transcription initiation at a previously unidentified alternative promoter. The case study presented is that of a patient with Duchenne muscular dystrophy who had a deletion extending from the 5' end of the dystrophin gene to exon 2, including all promoters previously mapped in the 5' part of the gene. Transcripts from lymphoblastoid cells were found to contain sequences corresponding to exon 3, indicating the presence of new promoter upstream of this exon. The nucleotide sequence of amplified cDNA corresponding to the 5' end of the new transcript indicated that the 5' end of exon 3 was extended by 9 codons, only the last (most 3') of which codes for methionine. The genomic nucleotide sequence upstream from the new exon, as determined using inverse polymerase chain reaction, revealed the presence of sequences similar to a TATA box, an octamer motif and an MEF-2 element. The identified promoter/exon did not map to intron 2, as might have been expected, but to a position more than 500 kb upstream of the most 5' of the previously identified promoters, thereby adding 500 kb to the dystrophin gene. The sequence of part of the new promoter region is very similar to that of certain medium reiteration frequency repetitive sequences. These findings may help us understand the molecular evolution of the dystrophin gene.

Amino Acid Sequence↗

PubMed1994

Amino-terminal deletion of 53% of dystrophin results in an intermediate Duchenne-Becker muscular dystrophy phenotype.

We report a Japanese boy with muscular dystrophy whose clinical symptoms were intermediate between those usually considered typical of Duchenne and Becker muscular dystrophies. The patient had a large inframe deletion extending from exons 3 to 41 of the dystrophin gene, which would be expected to cause the production of a dystrophin protein composing only 53% of the normal polypeptide chain. Such an inframe deletion would be expected to cause Becker muscular dystrophy. We did not obtain evidence for alternative splicing or for RNA editing. Immunocytochemical analysis of skeletal muscle showed that a dystrophin-related polypeptide was detectable with antibody directed against the carboxyl-terminal part of the polypeptide but not with antibodies directed against the amino-terminal part, although labeling by antibody against the carboxyl-terminal was faint and patchy. The severity of the disease in this case may be due to the lack of the amino-terminal, actin-binding domain of dystrophin.

Adolescent↗

PubMed1994

A novel point mutation (G-1 to T) in a 5' splice donor site of intron 13 of the dystrophin gene results in exon skipping and is responsible for Becker muscular dystrophy.

The mutations in one-third of Duchenne and Becker muscular dystrophy patients remain unknown, as they do not involve gross rearrangements of the dystrophin gene. We now report a defect in the splicing of precursor mRNA (pre-mRNA), resulting from a maternally inherited mutation of the dystrophin gene in a patient with Becker muscular dystrophy. This defect results from a G-to-T transversion at the terminal nucleotide of exon 13, within the 5' splice site of intron 13, and causes complete skipping of exon 13 during processing of dystrophin pre-mRNA. The predicted polypeptide encoded by the aberrant mRNA is a truncated dystrophin lacking 40 amino acids from the amino-proximal end of the rod domain. This is the first report of an intraexon point mutation that completely inactivates a 5' splice donor site in dystrophin pre-mRNA. Analysis of the genomic context of the G-1-to-T mutation at the 5' splice site supports the exon-definition model of pre-mRNA splicing and contributes to the understanding of splice-site selection.

Adult↗

PubMed1993

p53 protein accumulates frequently in early bronchial neoplasia.

p53 mutations are common in human lung cancer and frequently generate levels of p53 protein that are detectable by immunohistochemistry. For this reason, p53 protein accumulation is a candidate biomarker, but little is known about its timing or frequency in multistage bronchial carcinogenesis. We studied human lung tissues containing preinvasive squamous neoplasms from 34 donors with and without lung cancer. Nuclear p53 protein was present in 0% of normal mucosas, 6.7% of squamous metaplasias, 29.5% of mild dysplasias, 26.9% of moderate dysplasias, 59.7% of severe dysplasias, 58.5% of carcinomas in situ, 67.5% of microinvasive carcinomas, and 79.5% of invasive tumors. These data indicate that (a) p53 protein accumulates in about 30% of the earliest recognized neoplastic lesions (i.e., mild dysplasia), (b) there is an increasing frequency of p53 protein accumulation starting with mild dysplasia, and (c) p53 protein accumulates infrequently in normal or metaplastic mucosa. In a subset of six patients whose most advanced lesion was carcinoma in situ without evidence of invasive cancer, p53 protein was detected in 0% of normal mucosas, 8.3% of squamous metaplasias, 37.5% of mild dysplasias, 12.5% of moderate dysplasias, 93.8% of severe dysplasias, and 55% of carcinoma in situ lesions. These data show clearly that p53 alterations can occur before invasion and suggest that the frequency is similar to that observed in the full series. Since two-thirds or more of lung cancers have p53 alterations, the timing and frequency of p53 protein accumulation make the p53 tumor suppressor gene an attractive marker for early diagnosis and evaluation of chemoprevention agents.

Biomarkers, Tumor↗

PubMed1992

Ovarian sex cord tumor with annular tubules--a case report and review of the literature in Japanese.

A case of ovarian sex cord tumor with annular tubules (SCTAT) in a 41-year-old female is reported. The patient's chief complaint was menstrual irregularity, but the serum values of estradiol and testosterone were not elevated. On gross examination, the left ovary measured 8 x 7 x 6 cm in size and its cut surface was yellowish gray or yellow and solid with no cystic area. Histologically, the tumor was composed of simple and complex annular tubules, lined by columnar cells containing abundant lipid droplets. Eosinophilic materials in the center of the annular tubules were continuous with the basement membrane in the periphery, and showed immunoreactivity for type IV collagen. Also, hollow tubules, consisting of columnar cells with a truncated luminal surface and elongated trabeculae, composed of columnar cells with clear cytoplasm, were noted. These structures were reminiscent of Sertoli cell tumor. Based on these histological observations, it appears that SCTAT is a sex cord/stromal tumor made up of cells with differentiation in the direction of Sertoli cells rather than granulosa cells.

Adult↗

PubMed1992

[An autopsied case of progressive systemic sclerosis with anti Wa antibody who showed a rapid progression].

An anti Wa antibody was reported as a new t-RNA related protein antibody in 1986. This autoantibody is now considered specific for the diagnosis of progressive systemic sclerosis (PSS). Up to date only 5 cases with anti Wa antibody have been identified. We report here an autopsied case of PSS with this antibody. A 53 years old female was admitted to our hospital because of dry cough and dyspnea in Sep 1987. There were fine crackles and chest X ray revealed interstitial pneumonia. The progressive respiratory failure was treated by steroid pulse therapy effectively. Sclerotic skin changes of hand began to appear in Sep 1988 and rapidly progressed to arms, chest and forehead by Dec 1988. A skin biopsy confirmed PSS changes. An anti Wa antibody was detected by double immunodiffusion and the protein antigen was associated with t-RNA when immunoprecipitation was conducted. She died of heart failure in July 1989. An autopsy revealed the diffuse fibrotic change of the heart and the lung. Cases with anti Wa antibody were shortly reviewed from the literature.

Autoantibodies↗

PubMed1991

Quantitative risk assessment of carcinogenicity of urethane (ethyl carbamate) on the basis of long-term oral administration to B6C3F1 mice.

A carcinogenicity study of urethane was performed for quantitative assessment of its risk in humans. Three hundred 6-week-old male B6C3F1 mice were divided into 6 groups, each consisting of 50 mice, and urethane was given ad libitum in drinking water at levels of 0 (control), 0.6, 3, 6, 60 and 600 ppm for 70 weeks. The tumors with a clear dose-response relationship were lung tumor (alveolar/bronchiolar adenoma or carcinoma) and liver tumor (hemangioma or angiosarcoma). The incidences of these two types of tumor were applied to estimation of the virtually safe dose (VSD) at the level of 10(-6) by using four mathematical models (Logit, Probit, Weibull and Multistage models). The VSD based on the incidences of lung tumor by using the Logit model was estimated to be 1.8 x 10(-4) mg/kg body weight/day. On the other hand the VSD based on those of liver tumor by using the Weibull model was 7.2 x 10(-5) mg/kg body weight/day. Thus, the VSDs based on the incidences of the two different types of tumor using the most compatible mathematical model in each case, as judged from the P-values, were similar.

Adenoma↗