Biomedical subjects

Ken-ichi Watanabe

Publications and source records attributed to Ken-ichi Watanabe.

30 records · Page 2Linked to original sources

PubMed2003

Induction of apoptosis in the vestibule of guinea pigs after middle ear inoculation with lipopolysaccharide (LPS).

OBJECTIVE: During the process of apoptosis, double-stranded DNA is broken into single-stranded DNA (ssDNA) by the action of caspases and caspase activated deoxyribonuclease (CAD). We immunohistochemically examined the apoptotic changes induced by endotoxin in the vestibule of guinea pigs. METHODS: Lipopolysaccharide (LPS) (5 mg/ml, 0.2 ml), a bacterial endotoxin, was transtympanically injected into the middle ear. 48 h after injection of LPS, animals were sacrificed by intracardiac perfusion of fixative. The temporal bones were then removed and immunohistochemically stained for ssDNA, CAD and caspase 3. RESULTS: ssDNA was detected after 48 h in the dark cell area of the LPS group, not in the sensory epithelium. CAD was observed both in the dark cell area and the sensory epithelium. Caspase 3 was also detected both in the dark cell area and the sensory epithelium. CONCLUSION: Our findings suggest that apoptosis is involved in the dysfunction of the vestibule under inflammatory conditions.

Animals↗

PubMed2003

Improved synthesis of pure [18F]fluoro-compounds for PET studies from bromo-compounds.

For preparing [18F]labeled compounds free from bromo-compounds for PET (positron emission tomography) studies, we propose the following two processes in which no separation is needed between the fluoro-compound and bromo-compound : (1). the bromo-compound is first converted into O-tosylate, which is then [18F]fluorinated; and (2). after [18F]fluorination of the bromo-compound, its excess is converted into an easily separable compound, e.g., phthalimide-compound. Direct tosylation with silver tosylate was effected in 65-85% yield for most commercial bromo-compounds, and with potassium phthalimide, the bromo-compounds in cold run were converted easily into phthalimide-compounds in 84-90% yield.

Chemistry, Organic↗

PubMed2003

Germ cell-specific expression of microphthalmia-associated transcription factor mRNA in mouse testis.

The gene coding for microphthalmia-associated transcription factor (Mitf) contains many promoters that could generate multiple Mitf isoforms with distinct amino-termini, such as ubiquitously expressed Mitf-A and Mitf-H. To gain further insight into Mitf isoform multiplicity and the regulation of the promoter usage of the Mitf gene, we have analyzed the function of the amino-terminal domains of Mitf isoforms and the expression of Mitf mRNA in mouse postnatal testis, which is characterized by spermatogenesis and by a cool temperature because of its unique location. Here we show that the amino-terminal domain of Mitf-A possesses a transactivation activity, as judged by yeast expression analysis. We also show the expression of Mitf-A and Mitf-D mRNAs in testis by PCR-based methods. Moreover, in situ hybridization analysis revealed that an Mitf mRNA, probably representing Mitf-A and/or Mitf-D, is expressed in germ cells, including spermatogonia, spermatocytes that undergo meiosis, and round spermatids with the haploid genome, but is undetectable in elongated spermatids with remodeled and condensed chromatin. Notably, Mitf mRNA is undetectable in somatic Leydig cells and peritubular cells. Therefore, multiple promoters may direct differential expression of the Mitf gene in the testis and contribute to functional diversity of Mitf isoforms.

Animals↗

PubMed2003

Individuals exhibiting conspicuous nevi (lentigo simplex) are resistant to allergic rhinitis/conjunctivitis (pollinosis), but those who do not show increased susceptibility to pollinosis.

We investigated 115 individuals in Tokyo from two groups of persons with conspicuous nevi on the head, neck, and arms, etc. (nevus-positive group) and persons who do not exhibit such nevi (nevi-negative group) interviewing them to determine whether they suffered from allergic rhinitis/conjunctivitis (pollinosis) and examining them for the presence and extent of nevi. There was a distinct correlation: only 5 persons among 85 individuals of the nevus-positive group suffered from pollinosis, and all 30 people in the nevus-negative group had pollinosis. Thus, individuals exhibiting conspicuous nevi are resistant to pollinosis, but those who do not show increased susceptibility to pollinosis.

Adolescent↗

PubMed2003

Traumatic perilymph fistula in infants: a case report.

Traumatic perilymph fistula is reported to be rare in infants because of the small size of the infant external meatus. We treated an infant with a traumatic perilymph fistula in the right ear. A metallic wire had penetrated the tympanic membrane. Horizontal-rotatory nystagmus was also observed. Computed tomographic images revealed dislocation of the ossicles. The perilymph fistula was closed under general anesthesia. The incus-stapes joint was separated and the footplate of the stapes was dislocated. Leakage of the perilymph fluid was apparent from the oval window and this fistula was closed with connective tissue. The perforation of the tympanic membrane was closed with temporal fascia. After surgery, the spontaneous nystagmus disappeared. The patient is under observation as an outpatient and is growing normally.

Child, Preschool↗

PubMed2002

p73-dependent expression of DAN during cisplatin-induced cell death and osteoblast differentiation.

We previously reported that DAN, a founding member of the DAN family of secreted proteins, acts as an inhibitor of cell cycle progression and is closely involved in retinoic acid-induced neuroblastoma differentiation. In this study, we found that DAN as well as p73, the recently identified p53 family member, was up-regulated during osteoblast differentiation. Additionally, the expression of DAN was increased in response to cisplatin-induced cell death of neuroblastoma SH-SY5Y cells. Consistent with the previous reports, p73 was accumulated after the treatment with cisplatin. Intriguingly, we found a putative p53/p73-binding site in the 5'-upstream region of the human DAN gene. A luciferase reporter assay and an in vitro DNA-binding experiment revealed that this canonical p53/p73-binding site was a functional responsive element and was specific for p73. Our results suggest that there exists a functional association between DAN and p73 during osteoblast differentiation as well as cisplatin-induced cell death.

Animals↗

PubMed2002

Microphthalmia-associated transcription factor interacts with LEF-1, a mediator of Wnt signaling.

Wnt signals regulate differentiation of neural crest cells through the beta-catenin associated with a nuclear mediator of the lymphoid-enhancing factor 1 (LEF-1)/T-cell factors (TCFs) family. Here we show the interaction between the basic helix-loop-helix and leucine-zipper region of microphthalmia-associated transcription factor (MITF) and LEF-1. MITF is essential for melanocyte differentiation and its heterozygous mutations cause auditory-pigmentary syndromes. Functional cooperation of MITF with LEF-1 results in synergistic transactivation of the dopachrome tautomerase (DCT) gene promoter, an early melanoblast marker. This activation depends on the separate cis-acting elements, which are also responsible for the induction of the DCT promoter by lithium chloride that mimics Wnt signaling. beta-catenin is required for efficient transactivation, but dispensable for the interaction between MITF and LEF-1. The interaction with MITF is unique to LEF-1 and not detectable with TCF-1. LEF-1 also cooperates with the MITF-related proteins, such as TFE3, to transactivate the DCT promoter. This study therefore suggests that the MITF/TFE3 family is a new class of nuclear modulators for LEF-1, which may ensure efficient propagation of Wnt signals in many types of cells.

Amino Acid Sequence↗

PubMed2002

Identification of the p33(ING1)-regulated genes that include cyclin B1 and proto-oncogene DEK by using cDNA microarray in a mouse mammary epithelial cell line NMuMG.

The candidate tumor suppressor p33(ING1) plays an important role in inducinggrowth arrest at G(0)-G(1) phase of the cell cycle and/or promoting apoptosis in cancerous cells. p33(ING1) is reported to act as a transcriptional cofactor by associating with tumor suppressor p53, HAT, or histone deacetyltransferase, suggesting that p33(ING1) is involved in chromatin-mediated transcriptional regulation. However, the molecular mechanism of p33(ING1)-mediated transcriptional regulation is poorly understood. Here we analyzed expression profiles in mouse mammary epithelial cells (NMuMG) by using a cDNA microarray consisting of 2304 mouse cDNAs after inducing transformation with antisense inhibitor of growth 1 (ING1) in retrovirus vector. The subsequent confirmation of the altered expression levels of the selected genes by semiquantitative reverse transcription-PCR demonstrated that overexpression of the antisense ING1 stimulated expression of 14 genes, which included cyclin B1, 12-O-tetradecanoylphorbol-13-acetate-inducible sequence 11, proto-oncogene DEK, and osteopontin, whereas we have detected transcriptional repression of 5 genes, including TPT1. In addition, adenovirus-mediated overexpression of ING1 in NMuMG cells resulted in down-regulation of cyclin B1, 12-O-tetradecanoylphorbol-13-acetate-inducible sequence 11, DEK, and osteopontin, whereas the levels of TPT1 expression were increased. The further analysis using p53(-/-) SAOS2 cells showed that the p33(ING1)-induced cyclin B1 down-regulation was p53 dependent. Thus, our cDNA microarray analysis suggested that p33(ING1) targets the multiple genes, including proto-oncogene DEK and cyclin B1, at least some of which are regulated in a p53-dependent manner, in the cells undergoing cell growth or apoptosis.

Animals↗

PubMed2002

Physical interaction of p73 with c-Myc and MM1, a c-Myc-binding protein, and modulation of the p73 function.

p73 shares high sequence homology with the tumor suppressor p53. Like p53, ectopic overexpression of p73 induces cell cycle arrest and/or apoptosis, and these biological activities are linked to its sequence-specific transactivation function. The COOH-terminal region of p73 is unique and has a function to modulate DNA-binding ability and transactivation activity. To identify and characterize cellular proteins that interact with the COOH-terminal region of p73 alpha and regulate its activity, we employed a yeast-based two-hybrid screen with a human fetal brain cDNA library. We found MM1, a nuclear c-Myc-binding protein, was associated with p73 alpha in both yeast two-hybrid and in vitro pull-down assays. In mammalian cells, MM1 co-immunoprecipitated with p73 alpha, whereas p73 beta and tumor suppressor p53 did not interact with MM1. Overexpression of MM1 in p53-deficient osteosarcoma SAOS-2 cells enhanced the p73 alpha-dependent transcription from the p53/p73-responsive Bax and PG13 promoters, whereas p73 beta- and p53-mediated transcriptional activation was unaffected in the presence of MM1. MM1 also stimulated the p73 alpha-mediated growth suppression in SAOS-2 cells. More importantly, we found that c-Myc was physically associated with p73 alpha and significantly impaired the transcriptional activity of p73 alpha on Bax and p21(waf1) promoters. Expression of MM1 strongly reduced the c-Myc-mediated inhibitory activity on p73 alpha. These results suggest that MM1 may act as a molecular partner for p73 to prevent the c-Myc-mediated inhibitory effect on its activity.

Base Sequence↗

PubMed2002

Inhibitors of fibril formation and cytotoxicity of beta-amyloid peptide composed of KLVFF recognition element and flexible hydrophilic disrupting element.

Beta-Amyloid peptide (Abeta) is the main protein components of neuritic plaques and its neurotoxicity would be exposed by formation of aggregate. The aggregation inhibitors composed of an Abeta recognition element (KLVFF) and a flexible hydrophilic disrupting element (aminoethoxy ethoxy acetate and aspartate) are designed and chemically synthesized. The inhibitory effects are examined by a pigment binding assay using Congo red or thioflavin T. The present compounds suppress the formation of aggregate, and the compound DDX3 is an especially effective inhibitor. In addition, the synthesized compounds efficiently suppress the cytotoxicity of Abeta against IMR-32 neuroblastoma cells in vitro.

Amyloid beta-Peptides↗

PubMed2002

Immunoreactivity for myeloperoxidase (MPO) in the vestibule after the injection of bacterial lipopolysaccharide into the middle ear.

OBJECTIVE: In this study, the effect of endotoxin on the vestibule of the ear of guinea pigs was immunohistochemically examined. METHODS: Bacterial lipopolysaccharide was injected into the middle ear transtympanically. After 48 h, the animals were sacrificed by intracardiac perfusion of fixative; then, the temporal bones were removed and processed for immunohistochemical staining with the anti-myeloperoxidase antibody. RESULTS: Myeloperoxidase could be detected after 48 h in the sensory epithelium and the dark cell area. CONCLUSION: It is reported that radical oxygen species, which are cytotoxic, are detected under inflammatory conditions. Our results suggest that myeloperoxidase and reactive oxygen species are involved in vestibular dysfunction under inflammation.

Animals↗

PubMed2002

Local injection of OK-432 in the treatment of ranula: a case report.

We treated a 57-year-old woman for ranula. After aspirating the cyst's contents, we administered 0.1 KE/ml of OK-432 via local injection. One month later, the cyst had still persisted, so we repeated the procedure. After 2 weeks, the ranula began to shrink markedly, and at 4 weeks it had almost disappeared. No recurrence of the ranula was observed during the subsequent 1 year of follow-up. Following each injection, the patient developed transient fever and local swelling but no serious complications. Our experience suggests that OK-432 injection is an effective treatment for ranula. However, because this treatment causes the cyst to collapse rather than disappear completely, patients should be regularly monitored over the long term.

Antineoplastic Agents↗