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Biomedical subjects

Akira Fujimori

Publications and source records attributed to Akira Fujimori.

10 recordsLinked to original sources
PubMed2003

A sensitive transcriptome analysis method that can detect unknown transcripts.

We have developed an AFLP-based gene expression profiling method called 'high coverage expression profiling' (HiCEP) analysis. By making improvements to the selective PCR technique we have reduced the rate of false positive peaks to approximately 4% and consequently the number of peaks, including overlapping peaks, has been markedly decreased. As a result we can determine the relationship between peaks and original transcripts unequivocally. This will make it practical to prepare a database of all peaks, allowing gene assignment without having to isolate individual peaks. This precise selection also enables us to easily clone peaks of interest and predict the corresponding gene for each peak in some species. The procedure is highly reproducible and sensitive enough to detect even a 1.2-fold difference in gene expression. Most importantly, the low false positive rate enables us to analyze gene expression with wide coverage by means of four instead of six nucleotide recognition site restriction enzymes for fingerprinting mRNAs. Therefore, the method detects 70-80% of all transcripts, including non-coding transcripts, unknown and known genes. Moreover, the method requires no sequence information and so is applicable even to eukaryotes for which there is no genome information available.

Animals↗
PubMed2003

Growth retardation and skin abnormalities of the Recql4-deficient mouse.

Mutations in the Recql4 gene are very likely responsible for a subset of Rothmund-Thomson syndrome (RTS) cases, but until now there has been no animal model to confirm this. Knockout mice in which the Recql4 gene is disrupted at exons 5-8 exhibit embryonic lethality at embryonic day 3.5-6.5. We generated a helicase activity-inhibited mouse by deleting exon 13 of Recql4, which is one of the coding exons of the consensus RecQ-helicase domain. This domain is the primary site of mutations that have been identified in RTS patients. The exon 13-deleted Recql4-deficient mice are viable, but exhibit severe growth retardation and abnormalities in several tissues, and embryonic fibroblasts show a defect in cell proliferation. Abnormalities in the Recql4-deficient mice are similar to those in RTS patients, suggesting that defects in the Recql4 gene may indeed be responsible for RTS. We speculate that the loss of Recql4 helicase activity results in the prematurely aged appearance observed in some RecQ helicase diseases.

Adenosine Triphosphatases↗
PubMed2003

Mouse dexamethasone-induced RAS protein 1 gene is expressed in a circadian rhythmic manner in the suprachiasmatic nucleus.

We identified the Dexamethasone-induced RAS protein 1 (Dexras1) gene as a cycling gene in the suprachiasmatic nucleus (SCN). Investigation of the whole brain using in situ hybridization demonstrated the localization of the expression of the gene in the SCN, thalamus, piriform cortex and hippocampus. However, rhythmic expression of the gene was observed only in the SCN. The rhythmic change in gene expression during 1 day was approximately five-fold, and the maximum expression was observed during subjective night. Real-time PCR using the SCN, paraventricular nucleus and cortex confirmed these results. Next, we analyzed the expression of the Dexras1 gene in the SCN of cryptochrome (Cry) 1 and 2 double knockout mice. We found that the rhythmic expression disappeared. The results indicate that Dexras1 rhythmicity and levels are dependent upon CRYs. This is the first time that the G protein, which may be involved in the input pathway, has been isolated as a cycling gene in the SCN.

Animals↗
PubMed2003

Progression of renal failure with anaemia and multiple effects of angiotensin-converting enzyme inhibitor in rats with renal mass reduction.

Several factors such as proteinuria and renal fibrosis may be important in the progression of many forms of chronic renal diseases. The purposes of the current study were to investigate the progressive renal failure of the rats with surgical renal mass reduction (RMR) and the effect of the angiotensin-converting enzyme (ACE) inhibitor, lisinopril, and to document correlation of several factors associated with progressive renal failure. Rats were subtotal (5/6) nephrectomized by resection of the renal poles and sham-operated. The functional, histological and haematological changes of the rats were studied for up to 10 weeks. After 2 weeks of RMR, oral administration of lisinopril (10 mg kg(-1) per day) was performed for 8 weeks. RMR resulted in progressive renal failure with proteinuria, monocyte/macrophage (ED1+) infiltration, anaemia as assessed by haemoglobin and haematocrit (Htc), renal hypertrophy as assessed by left kidney to body weight ratio (BKW/BW), and renal fibrosis as assessed by glomerular lesions and tubulointerstitial changes. Lisinopril exhibited renoprotection with antiproteinuric effect and inhibition of monocyte/macrophage (ED1+) infiltration. However, beneficial effect of lisinopril on anaemia was not observed. At 10 weeks after surgery, severity of proteinuria positively correlated with plasma creatinine (Pcr), BKW/BW, histological damage, and systolic blood pressure, and negatively correlated with haemoglobin. Severity of tubulointerstitial changes positively correlated with Pcr and blood urea nitrogen, and negatively correlated with haemoglobin and Htc. Moreover, monocyte/macrophage (ED1+) infiltration positively correlated with severity of proteinuria and tubulointerstitial changes. These findings strongly support that proteinuria, monocyte/macrophage infiltration and renal fibrosis appear to play principal roles in the progressive renal failure with anaemia and renoprotection of ACE inhibition may be mediated by multiple actions of ACE inhibitor. The present study confirms that rats with RMR is useful to explore target molecules for renoprotective drugs and evaluate renoprotective effect of new molecular entities.

Analysis of Variance↗
PubMed2002

Targeted disruption of Np95 gene renders murine embryonic stem cells hypersensitive to DNA damaging agents and DNA replication blocks.

NP95, which contains a ubiquitin-like domain, a cyclin A/E-Cdk2 phosphorylation site, a retinoblastoma (Rb) binding motif, and a ring finger domain, has been shown to be colocalized as foci with proliferating cell nuclear antigen in early and mid-S phase nuclei. We established Np95 nulligous embryonic stem cells by replacing the exons 2-7 of the Np95 gene with a neo cassette and by selecting out a spontaneously occurring homologous chromosome crossing over with a higher concentration of neomycin. Np95-null cells were more sensitive to x-rays, UV light, N-methyl-N"-nitro-N-nitrosoguanidine (MNNG), and hydroxyurea than embryonic stem wild type (Np95(+/+)) or heterozygously inactivated (Np95(+/-)) cells. Expression of transfected Np95 cDNA in Np95-null cells restored the resistance to x-rays, UV, MNNG, or hydroxyurea concurrently to a level similar to that of Np95(+/-) cells, although slightly below that of wild type (Np95(+/+)) cells. These findings suggest that NP95 plays a role in the repair of DNA damage incurred by these agents. The frequency of spontaneous sister chromatid exchange was significantly higher for Np95-null cells than for Np95(+/+) cells or Np95(+/-) cells (p < 0.001). We conclude that NP95 functions as a common component in the multiple response pathways against DNA damage and replication arrest and thereby contributes to genomic stability.

Animals↗
PubMed2002

Conserved domains in the chicken homologue of BRCA2.

Germline mutation of the BRCA2 gene causes a high risk of developing breast and ovarian cancer. Although the BRCA2 protein has been implicated in homologous recombination (HR) of DNA and in transcription, it is still unclear how mutation leads to tumorigenesis. We have identified a non-mammalian homologue of BRCA2 from chicken, which encodes a protein with 3397 amino acids (aa) and shows only 40% identity to human BRCA2. However, comparison of the mammalian and chicken sequences revealed remarkably high homology in several segments. These include a N-terminal region (approximately 100 aa), which was previously shown to possess intrinsic transcriptional activity, and a C-terminal region (aa residue approximately 2480-approximately 3180 in human BRCA2), which has not been clearly assigned any function. In contrast, although the eight BRC repeats of mammalian BRCA2 are believed to play an important role in HR by interacting with Rad51, the BRC3, BRC5, and BRC6 repeats exhibit virtually no similarity to their mammalian counterparts. Among 311 missense mutations listed as unclassified variants in the NIH Breast Cancer Information Core database, only 83 of these sites are identical in chicken BRCA2. Thus, chicken BRCA2 may provide a means to identify domains and residues associated with cancer predisposition.

Amino Acid Sequence↗
PubMed2002

Cloning of hamster type XVII collagen cDNA, and pathogenesis of anti-type XVII collagen antibody and complement in hamster bullous pemphigoid.

Bullous pemphigoid is an inflammatory subepidermal blistering skin disease associated with an IgG autoimmune response to the type XVII collagen. The immunopathologic features of bullous pemphigoid can be reproduced in mice by the passive transfer of anti-type XVII collagen antibodies. In this model, it is thought that blister formation depends upon complement activation, neutrophil recruitment, and some proteolytic enzymes. In this study, we cloned hamster type XVII collagen cDNA, which contains a 4296 bp coding region and which is predicted to be a transmembrane protein with an extracellular collagenous domain, residing in type II orientation. Antipeptide antibodies (anti-1191 IgG) were obtained against a segment of hamster type XVII collagen homologous with the human type XVII collagen autoantibody-reactive site. The antipeptide antibodies were passively transferred to neonatal Syrian hamsters. The injected hamsters developed a microscopic subepidermal blister as seen previously in the mice. In order to test whether antigen-antibody complexes and complement initiate the subepidermal blister formation, we carried out experiments in vitro on condition that inflammatory cells were completely eliminated. Complement activation in sera was inhibited either by heating (at 56 degrees C for 30 min) or by preincubating with cobra venom factor. When the hamster skin was incubated with fresh anti-1191 antisera, separation of dermal-epidermal junction was observed. The anti-1191 IgG failed to induce C3 deposition and dermal-epidermal junction separation, however, if the anti-1191 IgG was added alone or complement activation in sera was inhibited. Under these conditions, IgG but not C3 was deposited on the basement membrane. These results strongly suggest that antigen-antibody complexes and complement initiate dermal-epidermal junction separation.

Amino Acid Sequence↗
PubMed2002

Effects of angiotensin II type 1 receptor antagonist, YM358, on cardiac hypertrophy and dysfunction after myocardial infarction in rats.

This study was undertaken to investigate the effects of an angiotensin II type 1 receptor antagonist, YM358 (2,7-diethyl-5-[[2'(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-5H-pyrazolo [1,5-b] [1,2,4]triazole potassium salt monohydrate), on cardiac hypertrophy and dysfunction in rats with heart failure after myocardial infarction (MI). One day after the coronary ligation, rats were randomized, and administered YM358 or vehicle for 2, 4 or 8 weeks. In MI rats, mean blood pressure, left ventricular (LV) systolic pressure, and the first derivative of LV pressure significantly decreased, and LV end-diastolic pressure (LVEDP) markedly increased after 2 to 8 week treatment of YM358. From 2 weeks after the ligation, ratios of cardiac weight and lung weight to body weight (BW) significantly increased, which indicated the progression of cardiac hypertrophy and lung congestion in MI rats. Two weeks after the ligation, YM358 did not improve LV function, although it decreased the elevated LVEDP and cardiac weights/BW ratios 8 weeks after the ligation. These results indicated that long-term treatment with YM358 improves the reduced cardiac function and reduces cardiac hypertrophy after MI, and may be useful for the treatment of congestive heart failure.

Angiotensin Receptor Antagonists↗
PubMed2002

Sequence analysis of 193.4 and 83.9 kbp of mouse and chicken genomic DNAs containing the entire Prkdc (DNA-PKcs) gene.

The catalytic subunit of DNA-dependent protein kinase plays critical roles in nonhomologous end joining in repair of DNA double-strand breaks and V(D)J recombination. In addition to the SCID phenotype, it has been suggested that the molecule contributes to the polymorphic variations in radiosensitivity and susceptibility to cancer in mouse strains. Here we show the nucleotide sequence of approximately 193-kbp and 84-kbp genomic regions encoding the entire Prkdc gene (also known as DNA-PKcs) in the mouse and chicken, respectively. A large retroposon was found in intron 51 in the mouse but not in the human or chicken. Comparative analyses of the genome strongly suggested that the region contains only two genes for Prkdc and Mcm4; however, several conserved sequences and cis elements were also predicted.

Animals↗