Characterization of the biological functions of a transcription factor, c-myc intron binding protein 1 (MIBP1).

The c-myc intron binding protein 1 (MIBP1) is a gigantic zinc finger protein comprising 2,437 amino acids and belonging to the MHC binding protein (MBP) family. MIBP1 is suggested to be a transcription factor involved in various biological functions. We show here that MIBP1 represses c-myc transcription from the major promoter, P2.

Inhibitory effect of a novel angiotensin II type 1 receptor antagonist RNH-6270 on growth of vascular smooth muscle cells from spontaneously hypertensive rats: different anti-proliferative effect to angiotensin-converting enzyme inhibitor.

The current study was undertaken to evaluate the anti-proliferative effect of a novel angiotensin II type 1 (AT1) receptor antagonist, RNH-6270, on exaggerated growth of vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR), in comparison with the effects of an angiotensin-converting enzyme (ACE) inhibitor. RNH-6270 and temocapril significantly inhibited basal DNA synthesis in VSMCs from SHRs in a dose-dependent manner, but not in cells from Wistar-Kyoto (WKY) rats. SHR-derived VSMC showed a hyperresponse of DNA synthesis to serum and angiotensin II compared with that of WKY rats-derived VSMC.

Delineation of the critical interval for the familial exudative vitreoretinopathy gene by linkage and haplotype analysis.

Familial exudative vitreoretinopathy (FEVR) is an ocular disorder characterized by deficient vascularization of the peripheral retina and causes visual loss attributable to various types of retinal detachment. The locus of the gene responsible for the autosomal dominant form of FEVR (EVR1) has been assigned to 11q13-23. However, a detailed evaluation of the critical region has not been made.

Polar alteration of short tandem repeats (STRs) in mammalian cells.

Instability of short tandem repeats (STRs) in DNA during replication is observed in all organisms examined, and is causatively involved in various human diseases. We explore the mechanisms involved in instability by examining length changes occurring during the replication of [(CA)(20)TA](n) and [(CAG)(20)TAG](n), in human cells. We show that the majority of alterations consist of an insertion or deletion of one repeat unit, and base substitutions or length changes involving many repeat units are rare.

Outside-to-inside signal through the membrane TNF-alpha induces E-selectin (CD62E) expression on activated human CD4+ T cells.

The membrane TNF-alpha is known to serve as a precursor of the soluble form of TNF-alpha. Although it has been reported the biological functions of the membrane TNF-alpha as a ligand, the outside-to-inside (reverse) signal transmitted through membrane TNF-alpha is poorly understood. Here we report a novel function mediated by outside-to-inside signal via membrane TNF-alpha into the cells expressing membrane TNF-alpha.

Precise estimation of allele frequencies of single-nucleotide polymorphisms by a quantitative SSCP analysis of pooled DNA.

We show that single-nucleotide polymorphisms (SNPs) of moderate to high heterozygosity (minor allele frequencies >10%) can be efficiently detected, and their allele frequencies accurately estimated, by pooling the DNA samples and applying a capillary-based SSCP analysis. In this method, alleles are separated into peaks, and their frequencies can be reliably and accurately quantified from their peak heights (SD <1.8%).

Microsatellite genotyping of post-PCR fluorescently labeled markers.

We show that a post-PCR multicolor fluorescence-labeling technique is applicable to multiplex microsatellite genotyping. Forty-three dinucleotide microsatellite markers, which are located on 11q13-23, a candidate region for dominant familial exudative vitreoretinopathy (FEVR), were used to evaluate the quality of the marker profile produced by this technique. Thirty-eight people from six families with this disease were subjects for genotyping.

Analysis of p53 tumour suppressor gene somatic mutations in rheumatoid arthritis synovium.

Objective: In order to study the role of the p53 tumour suppressor gene in the proliferation of rheumatoid arthritis (RA) synovium, we analysed the mutation of p53 in the synovial fibroblast-like type B synoviocyte from RA patients.

Methods: Synovial fibroblast-like type B synoviocytes were prepared from the synovial tissues from nine Japanese patients with RA. The p53 cDNA region from exons 4-11 was screened for mutations by the streamlined mutation detection method in which polymerase chain reaction (PCR) products are post-labelled and are analysed by automated capillary electrophoresis using single-strand conformation polymorphism conditions, followed by direct sequencing of the subclones of the PCR products.

ATM mutations in patients with ataxia telangiectasia screened by a hierarchical strategy.

ATM has been identified as a gene that is responsible for ataxia telangiectasia (AT), a pleiotropic disorder of autosomal recessive inheritance. While many mutations of this gene in AT patients of various ethnicities have been reported, data on Japanese patients are scarce. In this report, we present the results of a thorough survey of ATM mutations in 14 unrelated AT patients, with an emphasis on Japanese subjects.

A streamlined mutation detection system: multicolor post-PCR fluorescence labeling and single-strand conformational polymorphism analysis by capillary electrophoresis.

Effective use of knowledge of human genome sequences in studies of hereditary diseases or cancer heavily depends on efficient methods for detection of mutations in individual samples. We describe here a simple and efficient mutation scanning system in which PCR products are post-labeled with two different fluorescent dyes in one tube, and analyzed by an automated capillary electrophoresis system using single-strand conformation polymorphism (SSCP) conditions (PLACE-SSCP). With the appropriate use of an internal control DNA, differences in electrophoretic mobilities between a reference and samples are precisely evaluated, then the presence of mutations is statistically judged.

SSCP analysis of long DNA fragments in low pH gel.

Sensitivity of single-strand conformation polymorphism analysis of PCR products (PCR-SSCP analysis) is known to be decreased when the DNA fragments are longer than 300 bp. We examined effects of buffer ions in an attempt to extend the length limit of the analysis. It has been noted that addition of glycerol to the gel containing Tris-borate buffer enhances the sensitivity, but the effects of glycerol have been left unexplained.

One-tube post-PCR fluorescent labeling of DNA fragments.

A method for fluorescent postlabeling of PCR products has been developed. The method uses Klenow fragment of DNA polymerase I that exchanges the 3'-terminal residue of PCR-amplified DNA fragment for fluorescent nucleotides. All reactions, including PCR, are performed in one tube simply by successive addition of reagents.

RNA-primed PCR.

We show that RNA can serve as a primer in PCR. Use of rTth DNA polymerase is essential because it has strong reverse transcriptase activity. RNA primers can be obtained by in vitro transcription and are less costly than DNA primers, which are chemically synthesized.

Detection of the PTC/retTPC oncogene in human thyroid cancers.

We have investigated the PTC/retTPC oncogene, an activated form of ret proto-oncogene with a specific rearrangement, in thyroid malignancies. Southern analysis was used to screen 36 thyroid papillary carcinomas (PC), 22 normal thyroid tissues from glands with PC elsewhere, three follicular carcinomas, eight follicular adenomas and 30 other non-malignant thyroids. Rearrangements were detected in four PCs (11%) using probes derived from the ret proto-oncogene.

Identification and analysis of the ret proto-oncogene promoter region in neuroblastoma cell lines and medullary thyroid carcinomas from MEN2A patients.

The human ret proto-oncogene (proto-ret), encoding a receptor tyrosine kinase, is highly expressed in neuroblastomas, medullary thyroid carcinomas (MTCs) and pheochromocytomas, which are all tumors of cells originating from the neural crest. In studies on the transcription mechanism of proto-ret, we identified the transcription start site and the promoter region by chloramphenicol acetyl transferase (CAT) assay. A sequence upstream from the transcription start site (-167 to +98 bp) showed definite promoter activity in both proto-ret mRNA-positive neuroblastoma NB39-nu cells and proto-ret mRNA-negative HeLa cells.

Expression of the ret proto-oncogene in human neuroblastoma cell lines and its increase during neuronal differentiation induced by retinoic acid.

Previously we observed specific expression of the ret proto-oncogene (proto-ret) in human neuroblastoma cell lines. A neuronal subline and non-neuronal sublines were isolated from the SK-N-SH cell line, which is composed of a heterogeneous cell population. Expression of proto-ret was detected in the neuronal subline, named SH-4305, but not in three non-neuronal sublines.

Tight linkage of the ret proto-oncogene with the multiple endocrine neoplasia type 2A locus.

The ret proto-oncogene has been mapped to 10q11.2 near the MEN2A locus by in situ hybridization. We carried out a linkage study of Japanese multiple endocrine neoplasia type 2A (MEN2A) families using a cosmid clone containing the ret proto-oncogene as probe.

Thalamic atrophy following cerebral infarction in the territory of the middle cerebral artery.

We investigated shrinkage of the ipsilateral thalamus following infarction in the territory of the middle cerebral artery in 33 patients who were admitted less than or equal to 2 days after the stroke and who were followed by computed tomography for greater than 1 year with no recurrences. The thalamic area was measured on the computed tomograms, and the ratio of the ipsilateral area to the contralateral area was calculated. All values were compared with values from the initial computed tomogram taken less than or equal to 2 days after the stroke.

Possible involvement of c-myc but not ras genes in hepatocellular carcinomas developing after spontaneous hepatitis in LEC rats.

LEC (Long-Evans with a cinnamon-like coat color) rats develop hepatocellular carcinomas (HCCs) spontaneously. We examined mutations of codons 12, 13, and 61 of the Ha-ras, Ki-ras, and N-ras genes in four HCCs by the polymerase chain reaction (PCR)-single-stranded DNA direct sequencing method. No ras gene mutations were observed, suggesting that ras activation is not involved in spontaneous hepatocarcinogenesis in LEC rats.

Specific expression of the ret proto-oncogene in human neuroblastoma cell lines.

The expression of the ret proto-oncogene (proto-ret), which possibly encodes two isoforms of a receptor-type tyrosine kinase, was examined in human tumor cell lines. Expression of the proto-ret mRNA was detected in all 11 neuroblastoma cell lines examined. The level of mRNA varied more than 100-fold in these neuroblastoma cell lines and was particularly high in three of them.