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Biomedical subjects

T Miyakawa

Publications and source records attributed to T Miyakawa.

At least 19 recordsLinked to original sources
PubMed2006

Reproducibility of a method for analysis of morphological changes in perioral soft tissue in children using video cameras.

The purpose of the present study was to determine the appropriate reference facial expression and to examine the reproducibility of our system using two commercially available video cameras in consideration of functional diagnosis of perioral muscles. The following items were investigated: 1) influence of forward and backward movements and the experiment sequence from measurement to analysis, 2) usefulness of head fixation by a restraining device during video recording for evaluation of morphological changes in facial expression, 3) conditions for establishment of reference points for determination of movements of perioral soft tissue, and 4) reproducibility of perioral soft tissue movements as assessed by changes in landmarks. The results of this study showed that accurate static and dynamic evaluation of perioral soft tissue is possible by using our method of video recording with the subject's head restrained and our method of analysis. The results also showed that centric occlusion of the most appropriate reference facial expression as for measurement of perioral soft tissue movements and that protrusion of the lips and lateral extension of the angel of the mouth are facial expressions with high degrees of reproducibility.

Child, Preschool↗
PubMed2004

Squamous metaplasia of Paget's disease.

A patient had triple extramammary Paget's disease of both axillary and genital regions. Right inguinal lymphadenopathy was found 1 year after excision of all the skin lesions. Excisional biopsy of the lymph node demonstrated a mixture of Paget cells and atypical squamoid cells with horn pearls suggestive of keratinization. The squamoid cells were positive for cytokeratin 10, a marker of suprabasal epidermis, and also positive for laminin gamma2 which is often expressed in invasive squamous cell carcinoma. The coexistence of these different cells within the same tumour island suggested that the squamoid cells derived from metaplasia of Paget cells.

Aged↗
PubMed2003

M1-M5 muscarinic receptor knockout mice as novel tools to study the physiological roles of the muscarinic cholinergic system.

A large body of evidence indicates that muscarinic acetylcholine receptors (mAChRs) play critical roles in regulating the activity of many important functions of the central and peripheral nervous systems. However, identification of the physiological and pathophysiological roles of the individual mAChR subtypes (M(1)-M(5)) has proven a difficult task, primarily due to the lack of ligands endowed with a high degree of receptor subtype selectivity and the fact that most tissues and organs express multiple mAChRs. To circumvent these difficulties, we used gene targeting technology to generate mutant mouse lines containing inactivating mutations of the M(1)-M(5) mAChR genes. The different mAChR mutant mice and the corresponding wild-type control animals were subjected to a battery of physiological, pharmacological, behavioral, biochemical, and neurochemical tests. The M(1)-M(5) mAChR mutant mice were viable and reproduced normally. However, each mutant line displayed specific functional deficits, suggesting that each mAChR subtype mediates distinct physiological functions. These results should offer new perspectives for the rational development of novel muscarinic drugs.

Animals↗
PubMed2001

Forebrain-specific calcineurin knockout selectively impairs bidirectional synaptic plasticity and working/episodic-like memory.

Calcineurin is a calcium-dependent protein phosphatase that has been implicated in various aspects of synaptic plasticity. By using conditional gene-targeting techniques, we created mice in which calcineurin activity is disrupted specifically in the adult forebrain. At hippocampal Schaffer collateral-CA1 synapses, LTD was significantly diminished, and there was a significant shift in the LTD/LTP modification threshold in mutant mice. Strikingly, although performance was normal in hippocampus-dependent reference memory tasks, including contextual fear conditioning and the Morris water maze, the mutant mice were impaired in hippocampus-dependent working and episodic-like memory tasks, including the delayed matching-to-place task and the radial maze task. Our results define a critical role for calcineurin in bidirectional synaptic plasticity and suggest a novel mechanistic distinction between working/episodic-like memory and reference memory.

Animals↗
PubMed2001

Repression of rRNA synthesis due to a secretory defect requires the C-terminal silencing domain of Rap1p in Saccharomyces cerevisiae.

A secretory defect causes specific transcriptional repression of both ribosomal protein and ribosomal RNA genes, suggesting the coupling of plasma membrane and ribosome syntheses. We previously reported that the rap1-17 allele, which produced C-terminally truncated Rap1p, derepressed transcription of ribosomal protein genes when the secretory pathway was blocked. In this paper, we demonstrate that the rap1-17 mutation also leads to significant attenuation of transcriptional repression of rRNA genes due to a secretory defect. In contrast, the rap1-2 temperature-sensitive allele containing a unique missense mutation in the middle of the coding sequence has only a weak effect on repression. These results suggest that the C-terminal silencing domain of Rap1p is required for transcriptional repression of rDNA in response to a secretory defect. We also demonstrated that transcriptional regulation of ribosomal protein genes in response to nitrogen limitation was not affected by the rap1-17 allele, suggesting that the mechanism of nitrogen response is distinct from that of the secretory response.

Alleles↗
PubMed2001

Differences between the tolerance characteristics of two anticonvulsant benzodiazepines in the amygdaloid-kindled rat.

The characteristics of the development of tolerance to the anticonvulsant effects of chronic treatment by dipotassium clorazepate and diazepam using amygdaloid-kindled rats were investigated. Dipotassium clorazepate (5 mg/kg) or diazepam (5 mg/kg) were intraperitoneally administered for 10 consecutive days. Tolerance to the anticonvulsant effect of dipotassium clorazepate developed in seizure stage on day 6, after-discharge duration on day 7 and seizure latency on day 4. In contrast, tolerance to the effects of diazepam developed more rapidly in seizure stage on day 4, after-discharge duration on day 4 and seizure latency on day 3. Thus tolerance to the anticonvulsive effect of dipotassium clorazepate developed relatively slower than that to diazepam. All rats had stage 5 convulsions 24 hr after cessation of the administration of dipotassium clorazepate and diazepam. Concomitant determinations of plasma concentrations of the main metabolite of dipotassium clorazepate and diazepam, desmethyldiazepam, showed no statistical difference during treatment, suggesting that the developed tolerance was not metabolic but functional.

Amygdala↗
PubMed2001

Hyperactivity and intact hippocampus-dependent learning in mice lacking the M1 muscarinic acetylcholine receptor.

Members of the muscarinic acetylcholine receptor family are thought to play key roles in the regulation of a large number of important functions of the CNS. However, the precise roles of the individual muscarinic receptor subtypes in modulating these processes are not well understood at present, primarily because of the lack of ligands with sufficient receptor subtype selectivity. To investigate the behavioral significance of the M(1) muscarinic receptor (M(1)R), which is abundantly expressed in the forebrain, we subjected M(1) receptor-deficient mice (M(1)R(-/-) mice) to a battery of behavioral tests. M(1)R(-/-) mice showed no significant impairments in neurological reflexes, motor coordination, pain sensitivity, and prepulse inhibition. Strikingly, however, M(1)R(-/-) mice consistently exhibited a pronounced increase in locomotor activity in various tests, including open field, elevated plus maze, and light/dark transition tests. Moreover, M(1)R(-/-) mice showed reduced immobilization in the Porsolt forced swim test and reduced levels of freezing after inescapable footshocks, suggesting that M(1)R(-/-) mice are hyperactive under stressful conditions as well. An increased number of social contacts was observed in a social interaction test. Surprisingly, M(1)R(-/-) mice displayed no significant cognitive impairments in the Morris water maze and in contextual fear conditioning. M(1)R(-/-) mice showed slight performance deficits in auditory-cued fear conditioning and in an eight-arm radial maze, most likely because of the hyperactivity phenotype displayed by the M(1)R(-/-) mice. Our results indicate that M(1) muscarinic receptors play an important role in the regulation of locomotor activity but appear to be less critical for cognitive processes, as generally assumed.

Animals↗
PubMed2001

Novel low molecular weight spirodiketopiperazine derivatives potently inhibit R5 HIV-1 infection through their antagonistic effects on CCR5.

Novel low molecular weight spirodiketopiperazine derivatives which potently inhibit R5 human immunodeficiency virus type 1 (HIV-1) infection through their antagonistic effects on CCR5 were identified. One such compound E913 (M(r) 484) specifically blocked the binding of macrophage inflammatory protein-1alpha (MIP-1alpha) to CCR5 (IC(50) 0.002 microm) and MIP-1alpha-elicited cellular Ca(2+) mobilization (IC(50) approximately 0.02 microm). E913 potently inhibited the replication of laboratory and primary R5 HIV-1 strains as well as various multidrug-resistant monocyte/macrophage tropic (R5) HIV-1 at IC(50) values of 0.03 to 0.06 microm. E913 was inactive against T cell tropic (X4) HIV-1; however, when combined with a CXCR4 antagonist AMD-3100, E913 potently and synergistically inhibited the replication of dualtropic HIV-1 and a 50:50 mixture of R5 and X4 HIV-1. Antagonism in anti-HIV-1 activity was not seen when E913 was combined with the reverse transcriptase inhibitor zidovudine or protease inhibitors. E913 proved to compete with the binding of antibodies to CCR5 which recognize the C-terminal half of the second extracellular loop (ECL2B) of CCR5. E913 and its analogs are acid-resistant and orally bioavailable in rodents. These data warrant that spirodiketopiperazine derivatives be further developed as potential therapeutics for HIV-1 infection.

Animals↗
PubMed2001

Ca(2+)-sensor region of IP(3) receptor controls intracellular Ca(2+) signaling.

Many important cell functions are controlled by Ca(2+) release from intracellular stores via the inositol 1,4,5-trisphosphate receptor (IP(3)R), which requires both IP(3) and Ca(2+) for its activity. Due to the Ca(2+) requirement, the IP(3)R and the cytoplasmic Ca(2+) concentration form a positive feedback loop, which has been assumed to confer regenerativity on the IP(3)-induced Ca(2+) release and to play an important role in the generation of spatiotemporal patterns of Ca(2+) signals such as Ca(2+) waves and oscillations. Here we show that glutamate 2100 of rat type 1 IP(3)R (IP(3)R1) is a key residue for the Ca(2+) requirement. Substitution of this residue by aspartate (E2100D) results in a 10-fold decrease in the Ca(2+) sensitivity without other effects on the properties of the IP(3)R1. Agonist-induced Ca(2+) responses are greatly diminished in cells expressing the E2100D mutant IP(3)R1, particularly the rate of rise of initial Ca(2+) spike is markedly reduced and the subsequent Ca(2+) oscillations are abolished. These results demonstrate that the Ca(2+) sensitivity of the IP(3)R is functionally indispensable for the determination of Ca(2+) signaling patterns.

Adenosine Triphosphate↗
PubMed2001

Mice lacking the M3 muscarinic acetylcholine receptor are hypophagic and lean.

Members of the muscarinic acetylcholine receptor family (M1-M5) have central roles in the regulation of many fundamental physiological functions. Identifying the specific receptor subtype(s) that mediate the diverse muscarinic actions of acetylcholine is of considerable therapeutic interest, but has proved difficult primarily because of a lack of subtype-selective ligands. Here we show that mice deficient in the M3 muscarinic receptor (M3R-/- mice) display a significant decrease in food intake, reduced body weight and peripheral fat deposits, and very low levels of serum leptin and insulin. Paradoxically, hypothalamic messenger RNA levels of melanin-concentrating hormone (MCH), which are normally upregulated in fasted animals leading to an increase in food intake, are significantly reduced in M3R-/- mice. Intra-cerebroventricular injection studies show that an agouti-related peptide analogue lacked orexigenic (appetite-stimulating) activity in M3R-/- mice. However, M3R-/- mice remained responsive to the orexigenic effects of MCH. Our data indicate that there may be a cholinergic pathway that involves M3-receptor-mediated facilitation of food intake at a site downstream of the hypothalamic leptin/melanocortin system and upstream of the MCH system.

Animals↗
PubMed2001

GSK-3 kinase Mck1 and calcineurin coordinately mediate Hsl1 down-regulation by Ca2+ in budding yeast.

The Ca2+-activated pathways of Saccharomyces cerevisiae induce a delay in the onset of mitosis through the activation of Swe1, a negative regulatory kinase that inhibits the Cdc28-Clb complex. Calcineurin and Mpk1 activate Swe1 at the transcriptional and post-translational level, respectively, and both pathways are essential for the cell cycle delay. Our genetic screening identified the MCK1 gene, which encodes a glycogen synthetase kinase-3 family protein kinase, as a component of the Ca2+ signaling pathway. Genetic analyses indicated that Mck1 functions downstream of the Mpk1 pathway and down-regulates Hsl1, an inhibitory kinase of Swe1. In medium with a high concentration of Ca2+, Hsl1 was delocalized from the bud neck and destabilized in a manner dependent on both calcineurin and Mck1. Calcineurin was required for the dephosphorylation of autophosphorylated Hsl1. The E3 ubiquitin ligase complex SCF(Cdc4), but not the anaphase-promoting complex (APC), was essential for Hsl1 destabilization. The Ca2+-activated pathway may play a role in the rapid inactivation of Hsl1 at the cell cycle stage(s) when APC activity is low.

Anaphase-Promoting Complex-Cyclosome↗
PubMed2001

Effects of high ELF magnetic fields on enzyme-catalyzed DNA and RNA synthesis in vitro and on a cell-free DNA mismatch repair.

Environmental electromagnetic fields have been implicated in human cancers. We examined whether high extremely low frequency (ELF) AC magnetic fields could affect DNA synthesis, transcription or repair, using in vitro model systems with defined sequences. The rate and fidelity of DNA polymerase catalyzed DNA synthesis, as well as of RNA polymerase catalyzed RNA synthesis, were not statistically significantly affected by 60 Hz 0.25-0.5 Tesla magnetic fields. The efficiency of mutS dependent mismatch repair with human cell extracts was also not affected by the magnetic field exposure. The results suggest that the core processes related to the transmission of genetic information are stable under high ELF magnetic fields.

Animals↗
PubMed2001

Exposure of Caenorhabditis elegans to extremely low frequency high magnetic fields induces stress responses.

Responses of the small heat shock protein gene, hsp-16, were examined in transgenic Caenorhabditis elegans exposed to electromagnetic fields. Expression of the hsp-16-lacZ gene was enhanced when transgenic animals were exposed to magnetic fields up to 0.5 T at 60 Hz. The hsp-16 promoter was more efficiently expressed at the embryonic than at the post-embryonic stage irrespective of exposure. Promoter activity was more sensitive to the stimulus in the intestine at the post-embryonic stage. Evidence is presented that the induction occurs at the transcriptional step of hsp-16.

Animals↗
PubMed2001

Neurogranin null mutant mice display performance deficits on spatial learning tasks with anxiety related components.

Neurogranin/RC3 is a protein that binds calmodulin and serves as a substrate for protein kinase C. Neuronally distributed in the hippocampus and forebrain, neurogranin is highly expressed in dendritic spines of hippocampal pyramidal cells, implicating this protein in long-term potentiation and in learning and memory processes. Null mutation of the neurogranin gene Ng generated viable knockout mice for analysis of the behavioral phenotype resulting from the absence of neurogranin protein. Ng -/- mice were normal on measures of general health, neurological reflexes, sensory abilities, and motor functions, as compared to wild type littermate controls. On the Morris water task, Ng -/- mice failed to reach acquisition criterion on the hidden platform test and did not show selective search on the probe trial. In the Barnes circular maze, another test for spatial navigation learning, Ng -/- mice showed impairments on some components of transfer, but normal performance on time spent around the target hole. Abnormal and idiosyncratic behaviors were detected, that appeared to represent an anxiogenic phenotype in Ng -/- mice, as measured in the light<-->dark exploration test and the open field center time parameter. These findings of apparent deficits in spatial learning and anxiety-like tendencies in Ng -/- support a role for neurogranin in the hippocampally-mediated interaction between stress and performance.

Animals↗
PubMed2001

Differential effect of Fyn tyrosine kinase deletion on offensive and defensive aggression.

Fyn tyrosine kinase is highly expressed in the limbic system and mice lacking Fyn tyrosine kinase showed increased fearfulness in a variety of tests for anxiety-related behaviors. To investigate the possible role of Fyn tyrosine kinase in aggression, we assessed the aggressive behaviors of the mice lacking the Fyn tyrosine kinase using the resident-intruder and restraint-induced target biting paradigms. The percentage of Fyn-deficient mice that attacked an inanimate target in a restraint tube was higher than that of the control mice. On the contrary, in the resident-intruder paradigm, the percentage of Fyn-deficient mice that attacked the intruder was lower and the Fyn-deficient mice showed a longer latency to attack an intruder. These results suggest a distinct role of Fyn tyrosine kinase in enhancing the offensive aggression and decreasing the defensive aggression. A possible influence of anxiety-phenotype of the Fyn-deficient mice on their abnormal aggressive behavior was discussed.

Aggression↗
PubMed2001

Fission yeast homologues of the B' subunit of protein phosphatase 2A: multiple roles in mitotic cell division and functional interaction with calcineurin.

BACKGROUND: Protein phosphatase 2A (PP2A) is a serine/threonine phosphatase distributed in eukaryotes from yeast to human, and plays pivotal roles in diverse cellular functions such as metabolism, cell cycle progression, gene expression and development. PP2A holoenzyme is a heterodimer of a catalytic subunit C and a regulatory subunit A, or a heterotrimer of C, A and a variable regulatory subunit consisting of three families; B, B', and PR72. Specific functions for each variable subunit are not well understood. RESULTS: Two fission yeast genes pbp1+ and pbp2+ homologous to the regulatory subunit B' were isolated. Physical in vivo interaction of the gene products with the catalytic subunit was demonstrated. A double disruption haploid mutant (Deltapbp1Deltapbp2) showed growth defect, cell shape and size abnormality, multiseptation and anucleated cell formation due to abnormality in septum positioning. These phenotypes were suppressed by human B' cDNA, indicating the striking conservation of the B' function from yeast to human. Over-expression of fission yeast B' led to growth defects, a loss of cell shape polarity, septal abnormality and anucleated cell formation. Deltapbp1Deltapbp2 and pbp1 null haploids were hypersensitive to calcineurin inhibitors, cyclosporin A and FK506, with which the mutants underwent arrest at post-anaphase and cell lysis. Double disruption of calcineurin and pbp1+, but not pbp2+, genes led to synthetic lethality. CONCLUSION: The fission yeast B' subunit of PP2A plays critical roles in cell shape control and septum formation, and shares essential functions with calcineurin for viability, possibly through their roles in cytokinesis and cell wall integrity.

Amino Acid Sequence↗
PubMed2001

PKC1, a protein kinase C homologue of Saccharomyces cerevisiae, participates in microtubule function through the yeast EB1 homologue, BIM1.

BACKGROUND: RSC is a chromatin-remodelling complex of Saccharomyces cerevisiae and essential for growth. Its catalytic subunit is encoded by the NPS1/STH1 gene. At the present time, little is known regarding the cellular function of RSC. RESULTS: To identify genes with functions related to NPS1, we screened high-copy suppressor genes for the temperature- and thiabendazole (TBZ)-sensitive mutant allele of NPS1, nps1-105. Amongst the suppressors we cloned PKC1/STT1 and BIM1 that encoded a homologue of mammalian protein kinase C and a conserved microtubule binding protein homologous to human EB1, respectively. Both the temperature sensitive mutation of PKC1, stt1, and the bim1 null mutation caused synthetic growth defects with nps1-105. A genetic analysis of the functional relationships between these genes revealed that PKC1 suppressed the defect of nps1-105 through the BIM1 function but not by the activation of the MPK1/MAPK pathway. The stt1 mutation alone showed TBZ sensitivity and delayed the G2-phase progression at semi-permissive temperatures. Both of these stt1 phenotypes were suppressed by the over-expression of BIM1. In addition, stt1 as well as nps1-105, mis-segregated a mini-chromosome at frequencies higher than the wild-type at a permissive temperature. The mis-segregation was enhanced in the nps1-105 stt1 double mutant. CONCLUSION: These results suggest that Pkc1p plays a role which is relevant to microtubule functions and that this role is mediated by a hitherto unknown PKC signalling pathway and by Bim1p

Blotting, Northern↗