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Biomedical subjects

N Takada

Publications and source records attributed to N Takada.

At least 73 records · Page 4Linked to original sources
PubMed1999

Isolation and functional characterization of the 5'-upstream region of mouse P/Q-type Ca2+ channel alpha1A subunit gene.

The omega-agatoxin-IVA-sensitive P/Q-type Ca2+ channel is predominantly expressed in the nervous system. To dissect the molecular mechanisms underlying the neuron-specific expression of the P/Q-type channel, we have isolated and characterized the 5'-upstream region of the mouse alpha1A subunit gene. A transcription start site appeared to exist at -269 bp upstream from the start codon as found by 5' RACE analysis. The proximal promoter of the alpha1A subunit gene lacks a typical TATA box, but contains several transcription factor binding sequences, including two Sp1 sites. When linked to a placental alkaline phosphatase (PLAP) reporter gene to examine the promoter activity, the 6.3-kb (-6,273 to +269) 5'-upstream region, but not a smaller 3.0-kb construct (-3, 021 to +269), was able to drive the reporter gene in neuron-like PC12 cells. In contrast, neither of these constructs enhanced the PLAP expression in fibroblast NIH3T3 cells. The sequence between 6.3 and 3.0 kb of the 5'-upstream region did not show promoter activity in either of the cell lines, but enhanced TK promoter activity in PC12 cells, though not in NIH3T3 cells. These results suggest that neuron-specific elements of the alpha1A subunit gene are likely to be located in the distal upstream regions (-6,273 to -3,021) of the 5'-upstream sequence.

3T3 Cells↗
PubMed1999

Identification of a transactivation activity in the COOH-terminal region of p73 which is impaired in the naturally occurring mutants found in human neuroblastomas.

p73 is a recently cloned tumor suppressor gene that is highly homologous to p53, and the products of both possess similar functions in inhibiting cell growth and inducing apoptosis. Interestingly, the COOH-terminal region of p53 displays no significant homology with that of p73. Moreover, p73 has an additional segment at its COOH terminus. Recently, we have found two mutations of p73 with amino acid substitution (P405R and P425L) in primary neuroblastomas. Because the region (amino acid residues 382-491) contains a glutamine- and proline-rich domain, we hypothesized that it has a transactivation function, and the mutations found in tumors result in loss of function. To test it, we used the yeast GAL4 DNA-binding fusion system. Yeast transformants expressing a GAL4-p73(1-112) or a GAL4-p73alpha(380-513) fusion protein were grown in SD medium lacking histidine and tryptophan and exhibited a significant induction of beta-galactosidase activity. Transient transfection experiments revealed that both of fusion proteins could induce the chloramphenicol acetyltransferase activity in mammalian cells, indicating that the COOH-terminal as well as NH2-terminal regions of p73 had significantly high levels of transactivation activity. Furthermore, the former activity was severely impaired in two naturally occurring mutant forms found in neuroblastomas. These suggest that, unlike p53, p73 has two domains with transactivation function, one in the NH2-terminal region and the other in the COOH-terminal region. Loss of function mutation in the latter might be involved in tumorigenesis and/or tumor progression.

3' Untranslated Regions↗
PubMed1999

Genetic heterogeneity of the immunogenic viral capsid protein region of human parvovirus B19 isolates obtained from an outbreak in a pediatric ward.

Whereas human parvovirus B19 commonly infects children and causes erythema infectiosum, it causes more severe diseases when it infects adults. In order to examine whether different clinical outcomes of B19 infection can be ascribed to the viral genetic heterogeneity, we have determined the nucleotide sequence of highly immunogenic portions of the B19 genome obtained from six patients with various clinical manifestations in a single outbreak. Our observations demonstrated that although the B19 sequences showed a significant heterogeneity, it was not correlated with the clinical manifestation. It was thus suggested that the host immune response to B19 infection may be a major determinant of clinical presentations associated with acute B19 infection.

Adult↗
PubMed1999

p73 at chromosome 1p36.3 is lost in advanced stage neuroblastoma but its mutation is infrequent.

p73, a novel p53 family member, is a recently identified candidate neuroblastoma (NBL) suppressor gene mapped at chromosome 1p36.33 and was found to inhibit growth and induce apoptosis in cell lines. To test the hypothesis that p73 is a NBL suppressor gene, we analysed the p73 gene in primary human NBLs. Loss of heterozygosity (LOH) for p73 was observed in 19% (28/151) of informative cases which included 92 mass-screening (MS) tumors. The high frequency of p73 LOH was significantly associated with sporadic NBLs (9% vs 34%, P<0.001), N-myc amplification (10% vs 71%, P<0.001), and advanced stage (14% vs 28%, P<0.05). Both p73alpha and p73beta transcripts were detectable in only 46 of 134 (34%) NBLs at low levels by RT-PCR methods, while they were easily detectable in most breast cancers and colorectal cancers under the same conditions. They found no correlation between p73 LOH and its expression levels (P>0.1). We found two mutations out of 140 NBLs, one somatic and one germline, which result in amino acid substitutions in the C-terminal region of p73 which may affect transactivation functions, though, in the same tumor samples, no mutation of the p53 gene was observed as reported previously. These results suggest that allelic loss of the p73 gene may be a later event in NBL tumorigenesis. However, p73 is infrequently mutated in primary NBLs and may hardly function as a tumor suppressor in a classic Knudson's manner.

Chromosome Mapping↗
PubMed1999

A novel tripartite structure comprising a mariner-like element and two additional retrotransposons found in the Bombyx mori genome.

A clone of a DNA-mediated mobile element (transposon) corresponding to a mariner-like element (MLE) was obtained by carrying out the polymerase chain reaction with genomic DNA of Bombyx mori using a Hyalophora cecropia MLE sequence as a primer. This clone had a size of about 4.2 kb and, after sequencing, was found to contain an RNA-mediated, shorter retrotransposon named L1Bm, which was in turn integrated with a much longer retrotransposon named BMC1. Thus, the mobile elements made a novel tripartite structure. The BMC1 and L1Bm moieties of the composite structure each contained a 63-bp conserved sequence which was subsequently found to be highly conserved in all BMC1 and L1Bm elements registered so far. We propose that the 63-bp stretch may be a recognition site for a retrotransposition mechanism conducted by a reverse transcriptase and an endonuclease complex. On the basis of this inference, we propose a model that predicts how different types of BMC1 and L1Bm elements are dispersed in the genome. In addition, a phylogenetic tree made from the current and extant BMC1 and L1Bm sequences indicated that these elements can be classified into Subfamilies I and II.

Animals↗
PubMed1999

Utility of endoscopic ultrasonography in assessing the indications for endoscopic surgery of submucosal esophageal tumors.

BACKGROUND: The submucosal tumor (SMT) of the esophagus presents good indications for minimal invasive surgery, such as endoscopic or thoracoscopic resection. However, it is essential that the layer of origin be ascertained. The SMT can be detected as hypoechoic lesions in the wall of the esophagus, and the intramural location can be estimated in terms of destruction or deviation of the ultrasonic layer structure of the esophagus. METHODS: To test the efficacy of this approach, a series of patients were examined by an endoscopic ultrasonography (EUS). RESULTS: In one patient, the lesion was diagnosed as located in the muscularis mucosa, and endoscopic resection was performed. In another four patients, the lesions appeared to be in the proper muscle layer, and enucleation was carried out under thoracoscopy or open surgery. Histologically, all of the lesions were leiomyomas, and the EUS diagnosis was correct in all of the patients. CONCLUSIONS: Thus, the intramural condition of SMTs can be estimated and indications for endoscopic or thoracoscopic resection assessed by this approach.

Biopsy↗
PubMed1999

Direct and rapid detection by PCR of Erysipelothrix sp. DNAs prepared from bacterial strains and animal tissues.

A PCR method for rapid screening of Erysipelothrix spp. in the slaughterhouse was carried out by using four species-specific sets of oligonucleotide primers after initial amplification with the primer set MO101-MO102, which amplifies the 16S rRNA sequences of all four Erysipelothrix species. The DNA sequences coding for the rRNA gene cluster, including 16S rRNA, 23S rRNA, and the noncoding region downstream of 5S rRNA, were determined in order to design primers for the species-specific PCR detection system. The homology among the 4.5-kb DNA sequences of the rRNA genes of Erysipelothrix rhusiopathiae serovar 2 (DNA Data Bank of Japan accession no. AB019247), E. tonsillarum serovar 7 (accession no. AB019248), E. rhusiopathiae serovar 13 (accession no. AB019249), and E. rhusiopathiae serovar 18 (accession no. AB019250) ranged from 96.0 to 98.4%. The PCR amplifications were specific and were able to distinguish the DNAs from each of the four Erysipelothrix species. The results of PCR tests performed directly with tissue specimens from diseased animals were compared with the results of cultivation tests, and the PCR tests were completed within 5 h. The test with this species-specific system based on PCR amplification with the DNA sequences coding for the rRNA gene cluster was an accurate, easy-to-read screening method for rapid diagnosis of Erysipelothrix sp. infection in the slaughterhouse.

Abattoirs↗
PubMed1999

Primary hypomagnesemia caused by isolated magnesium malabsorption: atypical case in adult.

Isolated magnesium malabsorption is a rare disorder, which bas been described in no more than 30 patients worldwide. Patients with this disorder typically present with convulsion and diarrhea in early infancy. Hypomagnesemia and hypocalcemia were found in a 35-year-old man with muscle cramps, who bad been diagnosed as primary hypoparathyroidism. Oral magnesium therapy corrected the low serum calcium, magnesium and parathyroid hormone levels. We report an atypical case of isolated magnesium malabsorption in an adult.

Administration, Oral↗
PubMed1999

A possible barrier function of the articular surface.

Since MacConaill first reported the existence of a thin additional layer of the articular cartilage and named it the lamina splendens, there have been various opinions as to the role of this layer in the lubrication of the articular surface. We studied the superficial portion of the articular cartilage in the 20 day-old and 30 day-old rats using light and transmission electron microscopy. Furthermore, we studied the articular cartilage of the rat whose "cover layer" had been removed mechanically. Also, intraarticular latex beads injection, intraarticular dye injection using lithium carmine and supravital staining experiments were performed. On day 20, dye injected intraarticularly was clearly observed by light microscopy in chondrocytes situated in the deeper layers. The dye injected in the 30 day-old rats, however, was not seen in the chondrocytes but was found only in the superficial layer. Dye was found in the chondrocytes when supravital staining was performed in the articular cartilage of 30 day-old rats after mechanical removal of the cover layer. By transmission electron microscopy, a superficial layer consisted of fine filamentous structures was observed on the articular surface of the 30 day-old rats. The cover layer was destroyed by intraarticular injected latex beads in 30 day-old rats. These findings strongly support the idea that the cover layer acts as a barrier against substances which invade from the surface of the articular cartilage. The development period of the cover layer coincides with the initiation of weight bearing, and joint cartilage debris and pressure changes might further promote maturation.

Animals↗
PubMed1998

Molecular cloning and functional characterization of a novel receptor-activated TRP Ca2+ channel from mouse brain.

Characterization of mammalian homologues of Drosophila TRP proteins, which induce light-activated Ca2+ conductance in photoreceptors, has been an important clue to understand molecular mechanisms underlying receptor-activated Ca2+ influx in vertebrate cells. We have here isolated cDNA that encodes a novel TRP homologue, TRP5, predominantly expressed in the brain. Recombinant expression of the TRP5 cDNA in human embryonic kidney cells dramatically potentiated extracellular Ca2+-dependent rises of intracellular Ca2+ concentration ([Ca2+]i) evoked by ATP. These [Ca2+]i transients were inhibited by SK&F96365, a blocker of receptor-activated Ca2+ entry, and by La3+. Expression of the TRP5 cDNA, however, did not significantly affect [Ca2+]i transients induced by thapsigargin, an inhibitor of endoplasmic reticulum Ca2+-ATPases. ATP stimulation of TRP5-transfected cells pretreated with thapsigargin to deplete internal Ca2+ stores caused intact extracellular Ca2+-dependent [Ca2+]i transients, whereas ATP suppressed [Ca2+]i in thapsigargin-pretreated control cells. Furthermore, in ATP-stimulated, TRP5-expressing cells, there was no significant correlation between Ca2+ release from the internal Ca2+ store and influx of extracellular Ca2+. Whole-cell mode of patch-clamp recording from TRP5-expressing cells demonstrated that ATP application induced a large inward current in the presence of extracellular Ca2+. Omission of Ca2+ from intrapipette solution abolished the current in TRP5-expressing cells, whereas 10 nM intrapipette Ca2+ was sufficient to support TRP5 activity triggered by ATP receptor stimulation. Permeability ratios estimated from the zero-current potentials of this current were PCa:PNa:PCs = 14.3:1. 5:1. Our findings suggest that TRP5 directs the formation of a Ca2+-selective ion channel activated by receptor stimulation through a pathway that involves Ca2+ but not depletion of Ca2+ store in mammalian cells.

Amino Acid Sequence↗
PubMed1998

Differential distribution of TRP Ca2+ channel isoforms in mouse brain.

Mammalian homologues of the Drosophila TRP proteins, which are essential for light-activated, phosphatidyl-inositide (PI)-dependent Ca2+ conductance in Drosophila photoreceptors, were molecularly identified, to investigate receptor-activated Ca2+ influx in the mammalian nervous system. Two cloned mouse TRP homologues, TRP3 and TRP4, structurally related to the voltage-dependent Na+ channel, were expressed predominantly in the brain, where a sharp contrast in the distribution of the RNA transcripts for TRP isoforms was demonstrated by in situ hybridization analysis. TRP3 mRNA was concentrated in cerebellar Purkinje cells and sparsely localized in the cerebellar granule layer, pontine nuclei, and thalamus, whereas TRP4 mRNA was abundantly expressed in hippocampal CA1 pyramidal neurons, dentate gyrus granule cells, and cerebral cortical neurons, and in the septal nuclei and the mitral layer of olfactory bulb. The distinct spatial patterns of TRP isoforms implicate that neurons are highly heterogeneous in receptor-activated Ca2+ influx responsible for the second phase of PI-mediated rise in intracellular Ca2+.

Amino Acid Sequence↗
PubMed1998

Expression and localization of ornithine decarboxylase in reversible papillomatosis induced by uracil in rat bladder.

Direct mechanical irritation by uracil calculi formed following feeding of 3% uracil in the diet to male rats produces severe papillary hyperplasia (papillomatosis, which is reversible) of bladder epithelium. To evaluate the mechanism of the appearance of uracil-induced papillomatosis, we examined the changes of the enzyme activity and the localization of ornithine decarboxylase (ODC), as well as polyamine biosynthesis, and epithelial proliferation, that accompany the sequential bladder epithelial changes following administration and withdrawal of uracil. Moreover, expression of ODC mRNA was investigated using northern blotting and localization of ODC mRNA was demonstrated using in situ hybridization. ODC activity during uracil administration was maintained at a high level compared to that in normal epithelium, but sharply decreased after cessation of uracil treatment. The accumulation of ODC protein was observed in the proliferating bladder epithelium by immunohistochemical examination and western blotting analysis, and even after cessation of treatment, the protein binding to anti-ODC antibody remained mildly elevated. Sequential changes of proliferating cell nuclear antigen (PCNA)-positive cells in the epithelium during the development and disappearance of papillomatosis correlated with ODC activity. ODC mRNA was expressed strongly in the proliferating epithelium in rats treated with uracil and weakly in normal epithelium, in accordance with the location of ODC protein. Consequently, our data demonstrate that cell proliferation in the development of papillomatosis is closely associated with polyamine metabolism, and moreover suggest that ODC activity is up-regulated at a post-translational step.

Animals↗
PubMed1998

Lyme disease Borrelia species in northeastern China resemble those isolated from far eastern Russia and Japan.

Fifty-nine Borrelia burgdorferi sensu lato culture isolates collected from northeastern China were characterized by 5S-23S rRNA intergenic spacer restriction fragment length polymorphism (RFLP) analysis and reactivity with monoclonal antibodies (MAbs). Among 59 culture isolates, 30 (50.8%) were Borrelia garinii and 17 (28.8%) were Borrelia afzelii, 2 were mixtures composed of B. garinii with RFLP pattern B and B. garinii with pattern C, and 9 were mixtures composed of B. garinii and B. afzelii. One isolate, ChY13p, produced a unique pattern and was identified as B. garinii based on analyses of 16S rRNA gene sequence, flagellin PCR-RFLP typing, and MAb reactivities. No Borrelia burgdorferi sensu stricto or Borrelia japonica isolates were detected. The results indicate that Lyme disease Borrelia species in northeastern China resemble those of Borrelia isolates from far eastern Russia and Japan.

Base Sequence↗
PubMed1998

[A case of psoriatic arthritis associated with ankylosing spondylitis treated with bilateral total hip arthroplasty].

We report a case of a 40-year-old male with psoriatic arthritis associated with ankylosing spondylitis. At the age of 34, the patient suffered pain in his hips, knees, ankles, neck and low back 3 years after the onset of psoriasis vulgaris. The hip pain gradually became severe despite the medical treatment and physical therapy. On admission, the skin lesion of diffuse erythematous plaques with scales was observed on the trunk and extremities. Motion of the spine was markedly limited. We also noted limitation of motion in the bilateral hips and remarkable gait disturbance. In laboratory findings, rheumatoid factor and HLA-B 27 antigen were negative. Radiographs of the cervical spine showed typical bamboo spine as seen in ankylosing spondylitis. Obliteration of the sacroiliac joints and joint space narrowing in the hips with reactive sclerosis were revealed on the pelvic film. Bilateral cementless total hip arthroplasty with adductor tenotomy resulted in complete pain relief and a marked improvement in gait function 18 months after surgery. The appropriate reconstructive surgery was extremely helpful to increase daily activities of the patient in this case.

Adult↗
PubMed1998

Lyme disease spirochetes in ticks from northeastern China.

During May 1996, field surveys on Lyme disease spirochetes were conducted in Beijing, Shenyang, Fushun, and Inner Mongolia in northeastern China. The ticks collected consisted of 3 genera and 12 species. Of these, Ixodes persulcatus was dominant in sun-exposed vegetation in forests in Inner Mongolia; 57 Borrelia strains (55/123 unfed adults and 2/5 immature stages fed on a rodent) were obtained from this tick by BSK culture. Additionally, 2/2 Apodemus peninsulae were positive. Ixodes nipponensis, Ixodes pavlovskyi, Haemaphysalis douglasi, and Haemaphysalis megaspinosa, newly recorded in China, and other Haemaphysalis spp. were all negative for Borrelia. Based on a polymerase chain reaction restriction fragment length polymorphism analysis of the 45 strains successfully subcultured, these were classified as 29 Borrelia garinii and 16 Borrelia afelii. These strains seemed to be more closely related to Japanese strains in genetic features than to those from Europe. The result of sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggested more diversity in both genospecies, but Borrelia burgdorferi sensu stricto was not found.

Animals↗
PubMed1998

Mutational analysis of the p73 gene localized at chromosome 1p36.3 in colorectal carcinomas.

Genetic alteration of p53, which monitors DNA damage and operates cellular checkpoints, is a major factor in the development of human colorectal carcinoma (CRC). Recently, p73, a novel family member of p53, has been identified and found, like p53, to activate p21Waf1/Cip1 and to induce apoptosis. The p73 gene was mapped at chromosome 1p36.3 which is a region frequently deleted in CRCs and other cancers including neuroblastoma. To assess whether or not p73 is a tumor suppressor gene of CRC, we performed mutational analysis of p73 in 82 colorectal tumor tissues paired with constitutional DNA. Using a microsatellite marker for p73, the loss of heterozygosity (LOH) study was performed and allelic loss of p73 was found in 17% of the CRCs. RT-PCR single strand conformation polymorphism analysis showed no mutation except three polymorphisms in the p73 coding region. In addition, p73 was expressed at higher levels in the CRC tissues than in the normal mucosa or neuroblastoma tissues, though the transcripts were detectable only by the RT-PCR method. Our results suggest that, in CRCs, p73 may not play a role as a tumor suppressor, at least not in a classic Knudson manner.

Adolescent↗
PubMed1998

Characterization of an ascidian maternal T-box gene, As-mT.

The T-box genes encode a novel family of transcriptional factors that seem to play crucial roles in various processes of animal development, in particular, mesoderm formation of chordate embryos. The ascidian egg has been regarded as a typical mosaic egg, in which several types of embryonic cells are specified autonomously dependent on prelocalized maternal factors or determinants. In the present study, we investigated a possible role of a maternal T-box gene (As-mT) of the ascidian Halocynthia roretzi. A cDNA clone we obtained predicted As-mT protein of 891 amino acids with a distinct T-domain, which was divergent from those of other T-box genes. Expression of As-mT was exclusively maternal. Although the transcript became barely detectable by the gastrula stage, no zygotic expression was evident during embryogenesis. The maternal transcript was distributed rather evenly within eggs and early embryos without any special localization. Injection of synthetic As-mT mRNA into fertilized eggs induced retardation of embryogenesis. Although cleavage occurred normally, the initiation of gastrulation was delayed, and delay in the morphogenesis resulted in dumpy larvae. Expression of a muscle-specific actin gene, a notochord-specific Brachyury gene, and an epidermis-specific gene was not detected at the early gastrula stage, all of the three genes being expressed in normal embryos at that stage. However, the expression of these genes as well as a mesenchyme-specific gene and histochemical activity of endoderm-specific alkaline phosphatase were evident by the mid-tailbud stage.

Amino Acid Sequence↗