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Biomedical subjects

F Otsuka

Publications and source records attributed to F Otsuka.

At least 145 records · Page 8Linked to original sources
PubMed1994

Purification and characterization of a protein that binds to metal responsive elements of the human metallothionein IIA gene.

Metal responsive element (MRE) is a cis-acting DNA motif located in the upstream region of vertebrate metallothionein genes, which can confer metal responsiveness on downstream heterologous promoters. A protein that binds to the MRE sequence in a zinc-dependent manner (zinc regulatory factor; ZRF) was purified 16,000-fold from HeLa cell nuclear extracts by means of the avidin-biotin method, in which a complex formed between ZRF and a biotinylated probe containing MRE was trapped by streptavidin-agarose beads, and ZRF was recovered by salt extraction. By repeating the method three times, a homogeneous 116-kDa protein was obtained whose recovery was zinc-dependent and MRE sequence-specific. UV cross-linking analysis also revealed that a protein that specifically binds to MRE has the same molecular mass as the purified protein. Zinc-dependent and MRE sequence-specific footprints of ZRF were obtained on MREa and MREb in the upstream region of the human metallothionein IIA gene. The ZRF-MRE complex dissociates by the addition of chelating reagents, suggesting a direct role of zinc ions in the DNA binding of ZRF. Partial amino acid sequences of ZRF were found to be highly homologous to those of a mouse MRE-binding protein, mMTF-1.

Amino Acid Sequence↗
PubMed1994

Elevated chromosome aberration frequency after X-ray exposure of cultured fibroblasts derived from patients with porokeratosis.

Porokeratosis (PK) is a rare genetic skin disorder inherited as an autosomal dominant trait and regarded as a disease predisposing to cancer. To evaluate chromosomal radiosensitivity of PK cells, we examined chromosome aberration frequency after X-irradiation of cultured skin fibroblasts derived from PK patients and controls. Without X-ray exposure, frequencies of chromosome-type aberrations (exchanges or deletions) were not different between the patients and controls. Following X-ray irradiation, frequencies of deletions in the patient group were significantly increased, whereas those of exchanges were not elevated. No differences in chromatid-type aberration frequency were found between the patients and controls with or without exposure to X-ray. The observed radiosensitivity, though not as high as in ataxia telangiectasia (AT) cells, agrees well with the previously reported higher radiosensitivity of PK fibroblasts in survival analysis.

Analysis of Variance↗
PubMed1994

Identification of a novel cDNA clone encoding protein tyrosine kinase in murine skin.

Tyrosine phosphorylation is widely recognized as playing important roles in cell differentiation, proliferation, and carcinogenesis. We have used the polymerase chain reaction (PCR) method to identify protein tyrosine kinases that are expressed in the skin. Mixed oligonucleotide probes were used to amplify and screen a neonatal murine skin cDNA pool for clones encoding amino acid contiguities whose conservation is characteristic of the protein tyrosine kinase family. When the PCR products were sequenced, 13 distinct clones were found, of which one is novel to date and has provisionally been named tks (for tyrosine kinase identified from skin). Sequence homology comparison showed that the tks gene is homologous to the src and fes/fps families. Northern blotting using PCR products of tks as a probe revealed that the mRNA of tks is detected ubiquitously and weakly in other tissues such as brain, lung, liver, thymus and kidney. This fact suggests that the tks gene is expressed in widely distributed cell types.

Amino Acid Sequence↗
PubMed1994

Proton radiotherapy of skin carcinomas.

At the Proton Medical Research Center, University of Tsukuba, we performed a pilot study of proton-beam radiotherapy in 12 patients with the following types of carcinoma: Bowen's disease (4), oral verrucous carcinoma (5), and squamous cell carcinoma (3). They received total doses of 51-99.2 Gy in fractions of 2-12.5 Gy. All of the tumours responded well to the treatment. All four lesions of Bowen's disease, three of the five oral verrucous carcinomas, and the three squamous cell carcinomas completely regressed following irradiation. Two squamous cell carcinomas recurred during the follow-up period. One recurrent squamous cell carcinoma was successfully treated by a salvage surgical operation, and in the other case the patient refused further therapy. In two verrucous carcinomas there was 90% regression of tumour volume. No severe radiation-related complication occurred. As proton radiotherapy produces good local tumour control without significant morbidity to the surrounding normal tissues, it may prove to be a useful therapeutic modality for the treatment of skin carcinomas.

Aged↗
PubMed1994

Xeroderma pigmentosum variant: DNA ploidy analysis of various skin tumors and normal-appearing skin in a patient.

BACKGROUND: The patient with xeroderma pigmentosum provides an appropriate human model for the evaluation of tumor development and progression. METHODS: We describe a patient with variant-type xeroderma pigmentosum who developed actinic keratoses, a squamous cell carcinoma, and its metastasis into a lymph node. DNA ploidy was measured and analyzed in cells of these tumors and the patient's sun-exposed as well as sun-shielded skin. RESULTS: The sun-shielded skin showed a normal diploid DNA distribution pattern, while the sun-exposed skin had an increased number of hyperdiploid cells. The actinic keratosis showed further increased hyperdiploid cells. The squamous cell carcinoma showed a large number of hyperdiploid cells and formed an aneuploid cell fraction. Histographically, the aneuploid cell fraction became more apparent and was revealed to be a major fraction in the metastatic squamous cell carcinoma. CONCLUSIONS: The changes in the DNA ploidy pattern well reflect the clinical and histologic characteristics of the respective skin conditions and likely provide the cellular basis for the sequential or stepwise carcinogenic process in the patient's xeroderma pigmentosum skin. Further studies are necessary to determine whether the present results explain the similar carcinogenic process in sun-damaged skin of the nonpredisposed general population.

Aneuploidy↗
PubMed1994

Nuclear proteins binding to the human metallothionein-IIA gene upstream sequences.

Metallothionein genes are known to be transcriptionally regulated by a variety of factors such as heavy metals, glucocorticoids and cytokines, and have multiple regulatory elements in their 5'-flanking region. To study the interactions between these sequences and regulatory factors, HeLa cell nuclear proteins were analyzed by band-shift assay using a 95-base pair (bp) DNA probe containing a part of the human MT-IIA gene upstream sequences. Consequently, two Zn-dependent DNA-binding proteins were detected. One of these showed properties almost identical with those of zinc regulatory factor (ZRF), which had been detected using an oligonucleotide probe containing the metal responsive element (MRE); namely, this protein is activated only by Zn, and requires not only MRE but also its flanking sequences for optimal DNA-binding. The other protein appears to be Sp1, based on its recognition sequences specificity. In addition, by Southwestern blotting analysis of nuclear extracts using the 95-bp probe or MRE oligonucleotide probe, we detected a Zn-dependent DNA-binding protein with a molecular mass of 116 kDa, which is likely to be ZRF. Analysis of HeLa cell nuclear proteins fractionated by glycerol gradient centrifugation showed that ZRF is distinct from another MRE-binding protein, MREBP.

DNA-Binding Proteins↗
PubMed1993

Structure determination of glycosphingolipids of cultured human keratinocytes.

From cultured human keratinocytes, seven glycolipid fractions were isolated by DEAE and silica-gel column chromatographies, and further by HPLC on a silica-gel column. By means of 1H-NMR spectroscopy, fast atom bombardment mass spectrometry and GLC-mass spectrometry, one fraction was determined to contain acylglucosylceramides, which consist of amide linked omega-hydroxy fatty acids (C30:0, C30:1, C32:1 and C34:1), fatty acids linked to the omega-hydroxy fatty acids through ester linkages (C14:1, C16:1, C18:1 and C18:2), a long-chain base (d18-sphingenine), and beta-glucose. Five of the other fractions contained glucosylceramides, and the seventh fraction contained a mixture of glucosylceramides and galactosylceramides. Glucosylceramides containing long-chain omega-hydroxy fatty acids, which are assumed to be immediate precursors of the acylglucosylceramides, were hardly detected in these glycolipid fractions. Six glucosylceramide fractions were separated due to differences in their fatty acids and sphingosines. On comparison with the results reported in our previous paper, the acylglucosylceramide content of the cultured human keratinocytes was about half that of human epidermis. Under the culture conditions used, the human keratinocytes did not differentiate into granular or horny cells. Taken together, the results suggest that the synthesis of acylglucosylceramides is not activated much in the cultured keratinocytes, but would be more activated in differentiated cells.

Adult↗
PubMed1993

Epithelioid cell melanomas have greater DNA ploidy abnormalities than spindle cell melanomas: cytological evidence for a higher malignant potential of the former.

The cellular DNA ploidy of 30 cases of malignant melanoma was measured by 4',6-diamidino-2-phenylindole-DNA (DAPI-DNA) microfluorometry. DNA histograms and DNA index values were compared among melanomas of different cell morphology. Epithelioid cell melanomas often showed greater DNA aneuploidy than spindle cell melanomas in terms of histographic pattern. DNA index values of the former (mean +/- standard error, 1.84 +/- 0.31) were significantly higher than those of the latter (1.55 +/- 0.24; P < 0.05). The DNA index values of mixed-type melanomas were intermediate. These results indicate that the epithelioid cell melanomas have greater DNA ploidy abnormalities which are usually correlated with a greater malignant potential in pigmentary neoplasms. Thus our results confirm clinical evidence that melanoma patients with the epithelioid type of cells have a poorer prognosis than those with the spindle type of cells.

Adult↗
PubMed1993

Bound lipids liberated by alkaline hydrolysis after exhaustive extraction of pulverized clavus.

In the present study, covalently bound lipids were found in clavus material and their lipid classes were determined by high-performance thin-layer chromatography (HPTLC). Clavus material, pulverized completely in a Mikro-Dismembrator II, was exhaustively extracted three times with chloroform/methanol (2:1, 1:1 and 1:2 v/v) at 80 degrees C for 1 h each time and this sequence of extractions was repeated to obtain the unbound lipid-free residue which was saponified and then extracted with chloroform. The extract proved to comprise several bands of lipids covalently bound through an ester-like linkage. These were identified as free fatty acids, cholesterol, ceramides and glucocerebrosides by HPTLC. However, omega-hydroxy fatty acids were not detected in the lipids. To analyse the fatty acids amide-linked to the bound ceramides, the latter were isolated by preparative HPTLC and subjected to mild acid hydrolysis. Since the bound ceramides constituted neither omega-hydroxy fatty acids nor alpha-hydroxy fatty acids, they were not identified as hydroxyl-acylsphingosines.

Chromatography, Thin Layer↗
PubMed1993

Unsuccessful attempt to detect genetic mutation in tuberous sclerosis utilizing the polymerase chain reaction.

Although tuberous sclerosis is supposed to be a phacomatosis inherited as an autosomal dominant trait, many cases develop without any affected parents or grandparents. In recent years, many vigorous investigations have been concentrated on finding the mutant gene, and possible candidate genes have been mapped on 9q34 and other chromosomes. In order to find a way of diagnosing asymptomatic carriers or patients, we tried to detect restriction fragment length polymorphisms (RFLPs) using the technique of polymerase chain reaction (PCR). We used a probe, MCOA12, which is located on 9q34 and has been known to show RFLPs in Caucasian tuberous sclerosis patients. However, we could not find a correlation between the phenotype and RFLP pattern in seven of eight families.

Base Sequence↗
PubMed1993

Porokeratosis large skin lesions are susceptible to skin cancer development: histological and cytological explanation for the susceptibility.

Porokeratosis (PK), an autosomal dominant inherited skin disorder, is known to develop malignant skin tumors on its skin lesions. Our recent literature survey has revealed that large PK skin lesions are frequently a precursor of malignant changes. In the study, large and small PK skin lesions were investigated in terms of histological features of the epidermis and of the cellular DNA content of epidermal cells. Large PK lesions frequently showed hypertrophic epidermis with many mitotic cells, while small lesions usually presented atrophic epidermis without such mitotic cells. Abnormal cells, like those containing hyperchromatic, large, and/or irregularly shaped nuclei, were present in the epidermis of both large and small lesions with a preponderance in the former over the latter. DNA polyploidy was seen more frequently in large PK lesions than in small ones. DNA index values were significantly higher in large lesions than in small ones. The histological features and DNA ploidy abnormalities probably reflect the higher proliferation and the greater potential for malignant changes of large PK skin lesions. Our study helps to explain the clinical evidence that large PK skin lesions are frequently a precursor of malignant skin tumors.

Adult↗
PubMed1993

Analysis of mammalian metallothionein isoforms by high-resolution SDS-gel electrophoresis.

Metallothioneins (MTs) are cysteine-rich heavy metal-binding proteins, whose possible functions are thought to be the protection against toxic metals as well as the regulation of essential metals. It is known that there are several MT isoforms, but the biological roles of the individual isoforms have not been elucidated. To facilitate the functional analysis of these isoforms, we improved an analytical method of MTs developed previously, which is based on a denaturing gel electrophoresis of chemically modified MTs. The established technique makes it possible not only to separate MT isoforms with a high resolution, but to estimate the levels of the individual isoforms by analyzing directly crude cell extracts. By this method, six MT isoforms were identified in the extracts of Cd-exposed human cells. It was also revealed that there is an apparent heterogeneity of the rat liver MT; five isoforms were identified in the liver extracts of Cd-injected rats. The present method will be useful in the functional analysis of the MT isoforms, as well as in a variety of aspects of the MT studies.

Animals↗
PubMed1993

Subungual pigmented nevus: evaluation of DNA ploidy in six cases.

The discrimination between subungual pigmented nevus and subungual melanoma in situ is still a clinical problem. We measured DNA ploidy in six cases of subungual melanotic lesions which exhibited the features of subungual pigmented nevus or lentigo simplex histologically. Five cases presented a diploid pattern with or without a slight increase of hyperdiploid cells. One case presented a polyploid pattern; it also exhibited histologically abnormal melanocytes with large nuclei and pigment-filled elongated dendrites. The DNA ploidy pattern and histologic features suggest that the lesion of this latter case contains abnormal melanocytes which probably have the potential to undergo a malignant transformation into a subungual melanoma. DNA ploidy analysis, therefore, is likely to provide information for evaluating the biologic behavior of subungual melanotic lesions.

Adult↗
PubMed1993

Difference in clinical features and HLA antigens between familial and non-familial vitiligo of non-segmental type.

Of 131 patients with non-segmental vitiligo studied, 29 (22%) had a family history of this disorder. The clinical features and HLA antigens were assessed, and a comparison made between patients with familial and those with non-familial, non-segmental vitiligo. Familial patients developed skin lesions significantly earlier than non-familial patients. There was a significant association between HLA-B46 and familial non-segmental vitiligo, whereas HLA-A31 and CW4 were found in non-familial patients. The differences in clinical features and HLA phenotypes suggest heterogeneity in the pathogenic process between familial and non-familial vitiligo patients.

Adolescent↗