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T Ashizawa

Publications and source records attributed to T Ashizawa.

At least 73 records · Page 4Linked to original sources
PubMed1998

The GAA triplet-repeat expansion in Friedreich ataxia interferes with transcription and may be associated with an unusual DNA structure.

Friedreich ataxia (FRDA), an autosomal recessive, neurodegenerative disease is the most common inherited ataxia. The vast majority of patients are homozygous for an abnormal expansion of a polymorphic GAA triplet repeat in the first intron of the X25 gene, which encodes a mitochondrial protein, frataxin. Cellular degeneration in FRDA may be caused by mitochondrial dysfunction, possibly due to abnormal iron accumulation, as observed in yeast cells deficient for a frataxin homologue. Using RNase protection assays, we have shown that patients homozygous for the expansion have a marked deficiency of mature X25 mRNA. The mechanism(s) by which the intronic GAA triplet expansion results in this reduction of X25 mRNA is presently unknown. No evidence was found for abnormal splicing of the expanded intron 1. Using cloned repeat sequences from FRDA patients, we show that the GAA repeat per se interferes with in vitro transcription in a length-dependent manner, with both prokaryotic and eukaryotic enzymes. This interference was most pronounced in the physiological orientation of transcription, when synthesis of the GAA-rich transcript was attempted. These results are consistent with the observed negative correlation between triplet-repeat length and the age at onset of disease. Using in vitro chemical probing strategies, we also show that the GAA triplet repeat adopts an unusual DNA structure, demonstrated by hyperreactivity to osmium tetroxide, hydroxylamine, and diethyl pyrocarbonate. These results raise the possibility that the GAA triplet-repeat expansion may result in an unusual yet stable DNA structure that interferes with transcription, ultimately leading to a cellular deficiency of frataxin.

Antisense Elements (Genetics)↗
PubMed1998

Antitumor activity of KW-2170, a novel pyrazoloacridone derivative.

5-(3-Aminopropyl)amino-7,10-dihydroxy-2-(2-hydroxethyl)-aminoethyl -6H-pyrazolo[4,5,1-de]acridin-6-one dihydroxy-chloride (KW-2170), a novel derivative of pyrazoloacridone, was selected and evaluated for its antitumor activity and toxicity in mice. KW-2170 exhibited antitumor activity superior to adriamycin (ADM) against Sarcoma 180, breast carcinoma MM102 and fibrosarcoma Meth A inoculated s.c. in mice. Its therapeutic index (LD10/ED50) was higher than that of ADM on two murine carcinoma models, MM102 and Meth A. KW-2170 showed significant antitumor activity against 17 human tumor xenografts of a total of 24 tumors tested and the total tumor response rate by treatment with KW-2170 was significantly higher than that by ADM (70.8 versus 58.3%). In particular, human lung carcinoma was highly sensitive to KW-2170, and a marked tumor regression was observed on Lu-65 and Lu-99 human lung carcinoma xenograft models. Ovary and pancreas carcinomas were also sensitive to the drug. Additionally, its therapeutic index was also high on these human carcinoma models in comparison with that of ADM. The best antitumor efficacy of KW-2170 was observed by a weekly treatment schedule followed by a single treatment schedule and a successive administration schedule also tended to be toxic to the hosts. KW-2170 exhibited very low cross-resistance against four lines of multidrug resistant tumors expressing high levels of P-glycoprotein, and the drug showed significant antitumor activity against ADM-resistant human ovary carcinoma A2780/ADM and against nasopharynx carcinoma KB-A1 xenografts which were not sensitive to ADM. These results indicate that KW-2170 has a very potent antitumor activity and is feasible as a new antitumor drug against ADM-refractory solid tumors in clinics.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Molecular and clinical studies in SCA-7 define a broad clinical spectrum and the infantile phenotype.

OBJECTIVE: To screen for the SCA-7 mutation in autosomal dominant cerebellar ataxia (ADCA) families and study genotype/phenotype correlations. BACKGROUND: The association of cerebellar ataxia and progressive pigmentary macular dystrophy clinically defines a distinct form of ADCA classified as SCA-7. SCA-7 is caused by expansion of a highly unstable CAG repeat that lies in the coding region of a novel gene on chromosome 3p12-13. METHODS: We screened 51 ADCA kindreds, in which SCA-1, SCA-2, SCA-3, and SCA6 mutations had been excluded, for the SCA-7 mutation using primers that specifically amplify the SCA-7 CAG repeat. RESULTS: The SCA-7 mutation was identified in 10 independent families. Normal alleles ranged from 7 to 16 repeats; expanded alleles ranged from 41 to 306 repeats. One allele with 36 repeats was found in an asymptomatic individual carrying an at-risk haplotype. SCA-7 presents a wide spectrum of clinical features including visual loss, dementia, hypoacusia, severe hypotonia, and auditory hallucinations. Juvenile SCA-7 occurs on maternal and paternal transmission of the mutation, whereas the infantile form occurs only on paternal transmission. An infant of African American descent carried the largest SCA-7 expansion (306 CAG repeats) and had severe hypotonia, congestive heart failure, patent ductus arteriosus, cerebral and cerebellar atrophy, and visual loss. CONCLUSION: These data show a wide spectrum of phenotypic abnormalities in SCA-7 and define an infantile phenotype caused by the largest CAG repeat expansion described to date.

Adolescent↗
PubMed1998

UCH9, a new antitumor antibiotic produced by Streptomyces: I. Producing organism, fermentation, isolation and biological activities.

We developed a microbial prescreen using Bacillus stearothermophilus NUB3620 and bacteriophage TP-68 to detect potential antitumor compounds acting on DNA or topoisomerases. During the course of screening microbial cultures for their antibacteriophage activities, we found that Streptomyces sp. isolated from a soil sample collected in Iwakuni city, Yamaguchi prefecture, Japan, produced a new antitumor antibiotic, UCH9. UCH9 was isolated from culture broth by a combination of EtOAc extraction and column chromatography. UCH9 has a new structure related to the antitumor antibiotic chromomycins. It exhibited antimicrobial activity against Gram-positive organisms. UCH9 also showed cytotoxic activity against HeLa S3 cells with an IC50 value of 13 nM and exhibited antitumor activity in vivo against mouse leukemia P388.

Animals↗
PubMed1998

[Nurses' knowledge of alcohol-related problems and their treatment: a comparison of nurses in general hospital and alcoholism treatment units].

Nurses have the most contact with patients in the clinical settings, and they play an important role in the guidance and education of patients. Nurses' basic knowledge of alcohol-related problems greatly influences early discovery of and early intervention against alcohol-related problems in general units (GU). In a study undertaken mainly to understand the status of nurses' knowledge in GU, a survey was performed to compare the knowledge and attitude of nurses working in GU and those with working in units specializing in the treatment of alcoholism (AU). For reference, we used the results of separate surveys performed previously on the general population. The results showed that, compared with nurses in AU, nurses in GU (1) were more tolerant of drinking, but (2) were more stigmatic concerning alcoholism, and (3) had little knowledge concerning alcohol-related problems. Nurses in GU tended to have less knowledge about alcohol-related problems than the general population. Moreover, the knowledge of nurses in AU could not be considered sufficient. This study suggested the need for basic education concerning alcohol-related problems and their treatment aimed at nurses.

Adult↗
PubMed1998

Molecular basis of neuromuscular diseases.

For many neuromuscular disorders, the chromosomal location is known, the causal gene has been identified, and direct application of this knowledge may be made in a clinical setting. The benefits resulting from molecular-based methods include improved diagnostic accuracy and genetic counseling for patients and other at risk family members. This chapter discusses in detail four of the most frequently encountered neuromuscular disorders. These diseases include spinal muscular atrophy, Charcot-Marie-Tooth neuropathy, Duchenne/Becker type muscular dystrophy, and myotonic dystrophy.

Charcot-Marie-Tooth Disease↗
PubMed1997

Autosomal dominant cerebellar ataxia (SCA6) associated with small polyglutamine expansions in the alpha 1A-voltage-dependent calcium channel.

A polymorphic CAG repeat was identified in the human alpha 1A voltage-dependent calcium channel subunit. To test the hypothesis that expansion of this CAG repeat could be the cause of an inherited progressive ataxia, we genotyped a large number of unrelated controls and ataxia patients. Eight unrelated patients with late onset ataxia had alleles with larger repeat numbers (21-27) compared to the number of repeats (4-16) in 475 non-ataxia individuals. Analysis of the repeat length in families of the affected individuals revealed that the expansion segregated with the phenotype in every patient. We identified six isoforms of the human alpha 1A calcium channel subunit. The CAG repeat is within the open reading frame and is predicted to encode glutamine in three of the isoforms. We conclude that a small polyglutamine expansion in the human alpha 1A calcium channel is most likely the cause of a newly classified autosomal dominant spinocerebellar ataxia, SCA6.

Alleles↗
PubMed1997

UCE6, a new antitumor antibiotic with topoisomerase I-mediated DNA cleavage activity produced by actinomycetes: producing organism, fermentation, isolation and biological activity.

A novel antitumor antibiotic, UCE6 (1,3,8,10,11-pentahydroxy-2-methyl-10-(2-oxo-4-hydroxypentyl)na phthacene-5, 12-dione) with topoisomerase I-mediated DNA cleavage activity, was isolated from the culture broth of actinomycetes strain UOE6. Addition of silicone oil antifoam agent, KS69 (2%), to the fermentation enhanced the production of UCE6 by approximately 3 fold. A total of 1.15 g of UCE6 was recovered as reddish orange crystals from a 100 liter fermentation supplemented with 2% KS69. UCE6 exhibited growth inhibitory activity against HeLa S3, HCT116 and Lu-65 cells comparable to that of camptothecin.

Actinomyces↗
PubMed1996

Molecular detection of new mutations, resolution of ambiguous results and complex genetic counseling issues in Huntington disease.

Huntington disease (HD) is an autosomal dominant neurodegenerative disorder caused by expansion of a variable length (CAG)n repeat in the 5' coding region of a novel gene on chromosome 4p16.3. We provide comprehensive molecular analysis of a sporadic case of HD in which a paternally derived normal length allele expanded to an affected length allele. Linkage analysis and paternity testing confirm the paternal origin of the expansion and demonstrate that unequal crossing over during meiosis is an unlikely mechanism for de novo expansion in HD. This case identifies a complex genetic counseling issue for the families of sporadic cases since calculations of recurrence risk are not possible at this time. In addition, we describe utilization of a combination of polymerase chain reaction (PCR) based assays for examination of both the CAG repeat and an adjacent variable length CCG repeat in the huntingtin gene. The combination of these assays can increase the accuracy of molecular diagnosis for HD and may clarify any ambiguous results obtained during molecular testing of HD families.

Female↗
PubMed1996

Effect of prior ethanol experience on dopamine overflow in accumbens of AA and ANA rats.

The purpose of this study was to investigate the effect of repeated ethanol administration on dopamine overflow in the nucleus accumbens of alcohol-preferring AA (Alko Alcohol) and alcohol-avoiding ANA (Alko Nonalcohol) rats. Dopamine is a possible mediator of the reinforcing effects of ethanol, but it has previously been shown that ethanol-naïve alcohol-preferring AA and alcohol-nonpreferring ANA rats do not differ in their dopaminergic reaction to an intraperitoneal ethanol injection (0.5-2.0 g/kg), as assessed by measuring extracellular dopamine in the nucleus accumbens with in vivo microdialysis. Here a group of AA rats drank 10% (v/v) ethanol voluntarily-continual access for 5-15 days, limited access for 3 weeks-while a yoked group of AA rats and a yoked group of ANA rats received the same amount intragastrically by intubation. The rats were implanted with guide cannulas on the fourth week of limited access. Dopamine overflow was monitored in the microdialysis perfusate after 1 g/kg i.p. ethanol. The AA and the ANA rats that received ethanol non-contingently showed the same dopaminergic response to this as naïve animals have before. The group that had ingested the ethanol voluntarily showed, however, a significantly smaller increase in dopamine after 1 g/kg ethanol i.p. This suggests that the active behavior associated with obtaining the contingent drug may have an important impact on the reactions of the dopamine system to the drug, producing different results than when the same drug is administered by other routes. The hypothesis that dopamine mediates ethanol reinforcement in AA rats is not supported by the results.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1996

Instability of the expanded (CTG)n repeats in the myotonin protein kinase gene in cultured lymphoblastoid cell lines from patients with myotonic dystrophy.

The mutation associated with myotonic dystrophy (DM) is the expansion of an unstable trinucleotide repeat, (CTG)n, in the 3'-untranslated region of the myotonin protein kinase gene. Although expanded repeats show both germline and somatic instability, the mechanisms of the instability are poorly understood. To establish a model system in which somatic instability of the DM repeat could be studied in more detail, we established lymphoblastoid cell lines (LBCL) from DM patients. Analysis of the DNA from DM LBCL using Southern blotting showed that the (CTG)n repeats were apparently stable up to 29 passages in culture. To study infrequent repeat size mutations that are undetectable due to the size heterogeneity, we established LBCL of single-cell origins by cloning using multiple steps of limiting dilution. After expansion to approximately 10(6) cells (equivalent to approximately 20 cell cycles), the DNAs of these cell lines were analyzed by the small pool PCR technique using primers flanking the (CTG)n repeat region. Two types of mutations of the expanded (CTG)n repeat alleles were detected: (1) frequent mutations that show small changes of the (CTG)n repeat size, resulting in alleles in a normal distribution around the progenitor allele, and (2) relatively rare mutations with large changes of the (CTG)n repeat size, with a bias toward contraction. The former may represent the mechanism responsible for the somatic heterogeneity of the (CTG)n repeat size observed in blood cells of DM patients. This in vitro experimental system will be useful for further studies on mechanisms involved in the regulation of the somatic stability of the (CTG)n repeats in DM.

Alleles↗
PubMed1996

Direct detection of expanded trinucleotide repeats using PCR and DNA hybridization techniques.

Recently, unstable trinucleotide repeats have been shown to be the etiologic factor in seven neuropsychiatric diseases, and they may play a similar role in other genetic disorders which exhibit genetic anticipation. We have tested one polymerase chain reaction (PCR)-based and two hybridization-based methods for direct detection of unstable DNA expansion in genomic DNA. This technique employs a single primer (asymmetric) PCR using total genomic DNA as a template to efficiently screen for the presence of large trinucleotide repeat expansions. High-stringency Southern blot hybridization with a PCR-generated trinucleotide repeat probe allowed detection of the DNA fragment containing the expansion. Analysis of myotonic dystrophy patients containing different degrees of (CTG)n expansion demonstrated the identification of the site of trinucleotide instability in some affected individuals without any prior information regarding genetic map location. The same probe was used for fluorescent in situ hybridization and several regions of (CTG)n/(CAG)n repeats in the human genome were detected, including the myotonic dystrophy locus on chromosome 19q. Although limited at present to large trinucleotide repeat expansions, these strategies can be applied to directly clone genes involved in disorders caused by large expansions of unstable DNA.

Blotting, Southern↗