Biomedical subjects

Hiroyuki Sasaki

Publications and source records attributed to Hiroyuki Sasaki.

At least 37 records · Page 2Linked to original sources

PubMed2005

Tsix silences Xist through modification of chromatin structure.

X inactivation is controlled by Xist and its antisense gene, Tsix, neither of which encodes a protein. Xist is essential for X inactivation to occur in cis, and its differential expression is a key event in the initiation of X inactivation. Xist and Tsix are imprinted in the extraembryonic tissues of mouse embryos so that they are expressed from the paternal and maternal X, respectively, resulting in the preferential inactivation of the paternal X. Targeted disruption of Tsix causes ectopic expression of Xist, suggesting that Tsix negatively regulates Xist in cis. However, the molecular mechanism of this antisense regulation remains unknown. Here, we demonstrate that Tsix transcriptionally silences Xist in both embryonic and extraembryonic tissues of mouse embryos. Moreover, we show that disruption of Tsix impairs establishment of repressive epigenetic modifications and chromatin structure at the Xist locus. We propose that Tsix silences Xist through modification of the chromatin structure.

Animals↗

PubMed2005

QT PRODACT: inter-facility variability in electrocardiographic and hemodynamic parameters in conscious dogs and monkeys.

In safety pharmacology studies, the effect of a test compound on the electrocardiogram is routinely examined by using conscious dogs. However, the results may be widely variable. The monkey, on the other hand, has scarcely been used for such studies; and as yet, there have not been reported studies on monkeys conducted at several facilities with a standard protocol. In the present study, we examined inter-facility variabilities in electrocardiographic and hemodynamic parameters as described below. We analyzed the data from 8 facilities (9 test groups) on dogs and 5 facilities (7 test groups) on monkeys. This data was obtained from the studies employing the following standard protocol: dl-Sotalol or a vehicle (0.5 w/v% methylcellulose solution) was given to animals; and the PR interval, QRS duration, QT interval, heart rate, and mean blood pressure were determined time-sequentially before and after administration of the vehicle or dl-sotalol in each test group. dl-Sotalol produced a prolongation of the maximum mean QTcF interval in conscious dogs and QTcB interval in conscious monkeys by more than 10% in every test group. No difference in the corrected QT interval among the test groups was observed in dogs, but a difference was observed in monkeys.

Animals↗

PubMed2005

The effects of trial repetition and individual characteristics on decision making under uncertainty.

The authors examined participants' preferences between certain and uncertain outcomes in a multistage gambling task and the effects of individual difference characteristics on those preferences. In Study 1, 144 participants made choices in single-stage gambles under gain and loss conditions and replicated the certainty effect in a previous study (D. Kahneman & A. Tversky, 1979). In Study 2, 94 participants engaged in a multistage gambling task using the same decision problems as those in Study 1. They also answered a questionnaire consisting of the Japanese version (M. Kamahara, K. Higuchi, & N. Shimizu, 2001) of the Locus of Control Scale (J. B. Rotter, 1966), the Reflection-Impulsivity Scale (K. Takigiku & A. Sakamoto, 2001), and a Japanese version (M. Terasaki, K. Shiomi, Y. Kishimoto, & K. Hiraoka, 1987) of the Sensation-Seeking Scale (M. Zuckerman, 1994). The results indicated that the certainty effect (Kahneman & Tversky) disappears in multistage gambling tasks and that differences in reflection-impulsivity and in gender influence the process of decision making under gain conditions. These results are discussed in terms of the decision strategies and cognitive biases involved in the multistage gambling task.

Adolescent↗

PubMed2004

Large-scale identification and mapping of nuclear matrix-attachment regions in the distal imprinted domain of mouse chromosome 7.

Mammalian imprinted genes, which are expressed from only one of the parental alleles, have a tendency to form clusters and are regulated by long-range mechanisms. Nuclear matrix-attachment regions (MARs), the anchor points of loop domains, are involved in coordination of gene expression and could play a role in regulation of imprinted domains. We have identified and mapped a total of 52 MARs in a 1-Mb imprinted domain on mouse distal chromosome 7 using our cosmid contigs and an in vitro MAR assay. We find two MAR clusters (comprising 20 and 19 MARs), one of which is mapped in the Th-Ins2 intergenic region, coincident with the boundary between the two imprinted subdomains. However, the imprinted/non-imprinted boundaries are not associated with a MAR. Based on the sequence information, we find that many of the MARs are rich in long interspersed nuclear elements. In addition, comparisons of the results obtained with several MAR-prediction software programs reveal good performance of ChrClass in terms of both sensitivity and specificity. This study presents the first large-scale mapping of MARs in an imprinted domain and provides a platform for understanding the roles of MARs in imprinting.

Animals↗

PubMed2004

Structural and functional analysis of a 0.5-Mb chicken region orthologous to the imprinted mammalian Ascl2/Mash2-Igf2-H19 region.

Previous studies revealed that Igf2 and Mpr/Igf2r are imprinted in eutherian mammals and marsupials but not in monotremes or birds. Igf2 lies in a large imprinted cluster in eutherians, and its imprinting is regulated by long-range mechanisms. As a step to understand how the imprinted cluster evolved, we have determined a 490-kb chicken sequence containing the orthologs of mammalian Ascl2/Mash2, Ins2 and Igf2. We found that most of the genes in this region are conserved between chickens and mammals, maintaining the same transcriptional polarities and exon-intron structures. However, H19, an imprinted noncoding transcript, was absent from the chicken sequence. Chicken ASCL2/CASH4 and INS, the orthologs of the imprinted mammalian genes, showed biallelic expression, further supporting the notion that imprinting evolved after the divergence of mammals and birds. The H19 imprinting center and many of the local regulatory elements identified in mammals were not found in chickens. Also, a large segment of tandem repeats and retroelements identified between the two imprinted subdomains in mice was not found in chickens. Our findings show that the imprinted genes were clustered before the emergence of imprinting and that the elements associated with imprinting probably evolved after the divergence of mammals and birds.

Amino Acid Sequence↗

PubMed2004

A 210-kb segment of tandem repeats and retroelements located between imprinted subdomains of mouse distal chromosome 7.

Mammalian genes subject to genomic imprinting often form clusters and are regulated by long-range mechanisms. The distal imprinted domain of mouse chromosome 7 is orthologous to the Beckwith-Wiedemann syndrome domain in human chromosome 11p15.5 and contains at least 13 imprinted genes. This domain consists of two subdomains, which are respectively regulated by an imprinting center. We here report the finished-quality sequence of a 0.6-Mb region encompassing the more centromeric subdomain. The sequence contains four imprinted genes (Ascl2/Mash2, Ins2, Igf2 and H19) and reveals previously unidentified CpG islands and tandem repeats, which may be features of imprinted genes. Most interestingly, a unique 210-kb segment consisting almost exclusively of tandem repeats and retroelements is identified. This segment, located between Th and Ins2, has features of heterochromatin-forming DNA and is highly methylated at CpG sites. The segment exhibits asynchronous replication on the parental chromosomes, a feature of the imprinted domains. We propose that this repeat segment could serve either as a boundary between the two subdomains or as a target for epigenetic chromatin modifications that regulate imprinting.

Animals↗

PubMed2004

Oligomeric Aip2p/Dld2p modifies the protein conformation of both properly folded and misfolded substrates in vitro.

Oligomeric actin-interacting protein 2 (Aip2p) [Nat. Struct. Biol. 2 (1995) 28]/D-lactate dehydrogenase protein 2 (Dld2p) [Yeast 15 (1999) 1377, Biochem. Biophys. Res. Commun. 295 (2002) 910] exhibits the unique grapple-like structure with an ATP-dependent opening [Biochem. Biophys. Res. Commun. 320 (2004) 1271], which is required for the F-actin conformation modifying activity in vitro and in vivo [Biochem. Biophys. Res. Commun. 319 (2004) 78]. To further investigate the molecular nature of oligomeric Aip2p/Dld2p, the substrate specificity of its binding and protein conformation modifying activity was examined. In the presence of 1mM ATP or AMP-PNP, oligomeric Aip2p/Dld2p bound to all substrates so far examined, and modified the conformation of actin, DNase I, the mature form of invertase, prepro-alpha-factor, pro-alpha-factor, and mitochondrial superoxide dismutase, as determined by the trypsin susceptibility assay. Of note, the activity could modify even the conformation of pathogenic highly aggregated polypeptides, such as recombinant prion protein in beta-sheet form, alpha-synuclein, and amyloid beta (1-42) in the presence of ATP. The in vivo protein conformation modifying activity, however, depends on the growth stage; the most significant substrate modification activity was observed in yeast cells at the log phase, suggesting the presence of a cofactor/s in yeast cells, where F-actin is supposed to be a major target in vivo. These data further support our previous notion that the oligomeric Aip2p/Dld2p may belong to an unusual class of molecular chaperones [Biochem. Biophys. Res. Commun. 320 (2004) 1271], which can target both properly folded and misfolded proteins in an ATP-dependent manner in vitro.

Actins↗

PubMed2004

Compartmentalization established by claudin-11-based tight junctions in stria vascularis is required for hearing through generation of endocochlear potential.

Claudins are cell adhesion molecules working at tight junctions (TJs) that are directly involved in compartmentalization in multicellular organisms. The cochlea includes a rather peculiar compartment filled with endolymph. This compartment is characterized by high K+ concentration (approximately 150 mM) and a positive endocochlear potential (approximately 90 mV; EP), both indispensable conditions for cochlear hair cells to transduce acoustic stimuli to electrical signals. These conditions are thought to be generated by the stria vascularis, which is adjacent to the endolymph compartment. The stria vascularis itself constitutes an isolated compartment delineated by two epithelial barriers, marginal and basal cell layers. Because TJs of basal cells are primarily composed of claudin-11, claudin-11-deficient (Cld11-/-) mice were generated with an expectation that the compartmentalization in stria vascularis in these mice would be affected. Auditory brainstem response measurements revealed that Cld11-/- mice suffered from deafness; although no obvious gross morphological malformations were detected in Cld11-/- cochlea, freeze-fracture replica electron microscopy showed that TJs disappeared from basal cells of the stria vascularis. In good agreement with this, tracer experiments showed that the basal cell barrier was destroyed without affecting the marginal cell barrier. Importantly, in the endolymph compartment of Cld11-/- cochlea, the K+ concentration was maintained around the normal level (approximately 150 mM), whereas the EP was suppressed down to approximately 30 mV. These findings indicated that the establishment of the stria vascularis compartment, especially the basal cell barrier, is indispensable for hearing ability through the generation/maintenance of EP but not of a high K+ concentration in the endolymph.

Alleles↗

PubMed2004

Membrane-embedded C-terminal segment of rat mitochondrial TOM40 constitutes protein-conducting pore with enriched beta-structure.

TOM40 is the central component of the preprotein translocase of the mitochondrial outer membrane (TOM complex). We purified recombinant rat TOM40 (rTOM40), which was refolded in Brij35 after solubilization from inclusion bodies by guanidine HCl. rTOM40 (i) consisted of a 63% beta-sheet structure and (ii) bound a matrix-targeted preprotein with high affinity and partially translocated it into the rTOM40 pore. This partial translocation was inhibited by stabilization of the mature domain of the precursor. (iii) rTOM40 bound preprotein initially through ionic interactions, followed by salt-resistant non-ionic interactions, and (iv) exhibited presequence-sensitive, cation-specific channel activity in reconstituted liposomes. Based on the domain structure of rTOM40 deduced by protease treatment, we purified the elastase-resistant and membrane-embedded C-terminal segment (rTOM40(DeltaN165)) as a recombinant protein with 62% beta-structure that exhibited properties comparable with those of full-size rTOM40. We concluded that the membrane-embedded C-terminal half of rTOM40 constitutes the preprotein recognition domain with an enriched beta-structure, which forms the preprotein conducting pore containing a salt-sensitive cis-binding site and a salt-resistant trans-binding site.

Animals↗

PubMed2004

ICF syndrome in a girl with DNA hypomethylation but without detectable DNMT3B mutation.

A 3-year-old girl with phenotypic and cytogenetic manifestations of the ICF syndrome and DNA hypomethylation but without DNMT3B gene mutation is described. At age 3 months, she had an apneic spell that left her with spastic paraplegia and severe mental retardation. At age 8 months, she suffered meningococcal meningitis and sepsis. When seen by us at age 3 years with virilization, she had a cleft plate, macroglossia, and an atrial septal defect. An adenoma was surgically removed from the right adrenal cortex. Her serum immunoglobulin levels were normal except IgA at the low normal border. Her lymphocytes showed paracentromeric stretching of chromosomes 1 and 16 in 7% of metaphases, and multiradial figures involving these chromosomes in 1% of cells. Hypomethylation of classical satellite 2 DNA was observed with BstBI digestion, but in a lesser degree than those in the individuals with proven DNMT3B mutations. No mutation was found in the coding and promoter regions of the gene. Several alternative interpretations were considered to explain the low frequencies of chromosomal instabilities and the lower degree of DNA hypomethylation, and undetected DNA3B mutations. A mutation may be present in the gene but undetected, present in other DNA methyltransferases (DNMT) genes or in a DNMT-associated protein gene.

Abnormalities, Multiple↗

PubMed2004

Radixin deficiency causes deafness associated with progressive degeneration of cochlear stereocilia.

Ezrin/radixin/moesin (ERM) proteins cross-link actin filaments to plasma membranes to integrate the function of cortical layers, especially microvilli. We found that in cochlear and vestibular sensory hair cells of adult wild-type mice, radixin was specifically enriched in stereocilia, specially developed giant microvilli, and that radixin-deficient (Rdx(-)(/)(-)) adult mice exhibited deafness but no obvious vestibular dysfunction. Before the age of hearing onset ( approximately 2 wk), in the cochlea and vestibule of Rdx(-)(/)(-) mice, stereocilia developed normally in which ezrin was concentrated. As these Rdx(-)(/)(-) mice grew, ezrin-based cochlear stereocilia progressively degenerated, causing deafness, whereas ezrin-based vestibular stereocilia were maintained normally in adult Rdx(-)(/)(-) mice. Thus, we concluded that radixin is indispensable for the hearing ability in mice through the maintenance of cochlear stereocilia, once developed. In Rdx(-)(/)(-) mice, ezrin appeared to compensate for radixin deficiency in terms of the development of cochlear stereocilia and the development/maintenance of vestibular stereocilia. These findings indicated the existence of complicate functional redundancy in situ among ERM proteins.

Animals↗

PubMed2004

Oligomeric Aip2p/Dld2p forms a novel grapple-like structure and has an ATP-dependent F-actin conformation modifying activity in vitro.

In order to investigate the molecular mechanism of the F-actin conformation modifying activity [Biochem. Biophys. Res. Commun. 319 (2004) 78] of actin-interacting protein 2 (Aip2p) [Nat. Struct. Biol. 2 (1995) 28]/D-lactate dehydrogenase protein 2 (Dld2p) [Yeast 15 (1999) 1377; Biochem. Biophys. Res. Commun. 295 (2002) 910], the ultrastructure and the regulatory mechanism of the activity were further examined. Interestingly, a novel oligomeric grapple-like structure of 10-12 subunits with an ATP-dependent opening was observed. ATP regulates the opening and closing of the "gate" that forms the opening within oligomeric Aip2p/Dld2p, where binding to the substrate occurs while in the open form. In the presence of ATP (open state of oligomeric Aip2p/Dld2p), oligomeric Aip2p/Dld2p bound the F-actin fiber within the opening, whereas in the absence of ATP (closed state of oligomeric Aip2p/Dld2p), no binding was observed. Simultaneously, the oligomeric Aip2p/Dld2p increased the trypsin susceptibility of F-actin in an ATP-dependent manner. Use of the non-hydrolyzable ATP analogue AMP-PNP yielded similar results to those observed with ATP, suggesting that ATP binding rather than ATP hydrolysis is required for the protein conformation modifying reaction of oligomeric Aip2p/Dld2p. Endogenous Aip2p/Dld2p purified from Saccharomyces cerevisiae also exhibited such protein conformation modifying activity, but monomeric Aip2p/Dld2p with a C-terminal coiled-coil region-truncation failed to exhibit the activity. These data suggest that the oligomerization of Aip2p/Dld2p, which exhibits the unique grapple-like structure with an ATP-dependent opening, is required for the F-actin conformation modifying activity.

Actins↗

PubMed2004

JACOP, a novel plaque protein localizing at the apical junctional complex with sequence similarity to cingulin.

The apical junctional complex is composed of various cell adhesion molecules and cytoplasmic plaque proteins. Using a monoclonal antibody that recognizes a chicken 155-kDa cytoplasmic antigen (p155) localizing at the apical junctional complex, we have cloned a cDNA of its mouse homologue. The full-length cDNA of mouse p155 encoded a 148-kDa polypeptide containing a coiled-coil domain with sequence similarity to cingulin, a tight junction (TJ)-associated plaque protein. We designated this protein JACOP (junction-associated coiled-coil protein). Immunofluorescence staining showed that JACOP was concentrated in the junctional complex in various types of epithelial and endothelial cells. Furthermore, in the liver and kidney, JACOP was also distributed along non-junctional actin filaments. Upon immunoelectron microscopy, JACOP was found to be localized to the undercoat of TJs in the liver, but in some tissues, its distribution was not restricted to TJs but extended to the area of adherens junctions. Overexpression studies have revealed that JACOP was recruited to the junctional complex in epithelial cells and to cell-cell contacts and stress fibers in fibroblasts. These findings suggest that JACOP is involved in anchoring the apical junctional complex, especially TJs, to actin-based cytoskeletons.

Actins↗

PubMed2004

Establishment and characterization of cultured epithelial cells lacking expression of ZO-1.

In well polarized epithelial cells, closely related ZO-1 and ZO-2 are thought to function as scaffold proteins at tight junctions (TJs). In epithelial cells at the initial phase of polarization, these proteins are recruited to cadherin-based spotlike adherens junctions (AJs). As a first step to clarify the function of ZO-1, we successfully generated mouse epithelial cell clones lacking ZO-1 expression (ZO-1-/- cells) by homologous recombination. Unexpectedly, in confluent cultures, ZO-1-/- cells were highly polarized with well organized AJs/TJs, which were indistinguishable from those in ZO-1+/+ cells by electron microscopy. In good agreement, by immunofluorescence microscopy, most TJ proteins including claudins and occludin appeared to be normally concentrated at TJs of ZO-1-/- cells with the exception that a ZO-1 deficiency significantly up- or down-regulated the recruitment of ZO-2 and cingulin, another TJ scaffold protein, respectively, to TJs. When the polarization of ZO-1-/- cells was initiated by a Ca2+ switch, the initial AJ formation did not appear to be affected; however, the subsequent TJ formation (recruitment of claudins/occludin to junctions and barrier establishment) was markedly retarded. This retardation as well as the disappearance of cingulin were rescued completely by exogenous ZO-1 but not by ZO-2 expression. Quantitative evaluation of ZO-1/ZO-2 expression levels led to the conclusion that ZO-1 and ZO-2 would function redundantly to some extent in junction formation/epithelial polarization but that they are not functionally identical. Finally, we discussed advantageous aspects of the gene knock-out system with cultured epithelial cells in epithelial cell biology.

Adherens Junctions↗

PubMed2004

Essential role for de novo DNA methyltransferase Dnmt3a in paternal and maternal imprinting.

Imprinted genes are epigenetically marked during gametogenesis so that they are exclusively expressed from either the paternal or the maternal allele in offspring. Imprinting prevents parthenogenesis in mammals and is often disrupted in congenital malformation syndromes, tumours and cloned animals. Although de novo DNA methyltransferases of the Dnmt3 family are implicated in maternal imprinting, the lethality of Dnmt3a and Dnmt3b knockout mice has precluded further studies. We here report the disruption of Dnmt3a and Dnmt3b in germ cells, with their preservation in somatic cells, by conditional knockout technology. Offspring from Dnmt3a conditional mutant females die in utero and lack methylation and allele-specific expression at all maternally imprinted loci examined. Dnmt3a conditional mutant males show impaired spermatogenesis and lack methylation at two of three paternally imprinted loci examined in spermatogonia. By contrast, Dnmt3b conditional mutants and their offspring show no apparent phenotype. The phenotype of Dnmt3a conditional mutants is indistinguishable from that of Dnmt3L knockout mice, except for the discrepancy in methylation at one locus. These results indicate that both Dnmt3a and Dnmt3L are required for methylation of most imprinted loci in germ cells, but also suggest the involvement of other factors.

Alleles↗

PubMed2004

Actin filaments play an essential role for transport of nascent HIV-1 proteins in host cells.

To investigate the role of actin filaments (F-actin) for human immunodeficiency virus type 1 (HIV-1) production in host cells, the effect of mycalolide B that is a novel actin-depolymerizing marine toxin was examined. Mycalolide B blocked the production of HIV-1 from primary infected T-lymphoblastoid and clonically infected monocytoid cells in a concentration-dependent manner. In the presence of 10 microM of mycalolide B, F-actins were disorganized and mostly disappeared in the host cells, and viral envelope- and capsid-proteins did not reach the plasma membrane, but were distributed in the cytoplasm forming aggregates. In electron micrographs, no HIV-1 virions were detected on the cell surface, but many lysosome-like vesicles containing electron dense granules were observed in the cytoplasm, implying that mycalolide B did not disturb the synthesis of viral proteins, but rather inhibited their transport processes of HIV-1 in the host cells.

Actin Cytoskeleton↗

PubMed2004

X-inactivation is stably maintained in mouse embryos deficient for histone methyl transferase G9a.

One of the two X chromosomes becomes inactivated during early development of female mammals. Recent studies demonstrate that the inactive X chromosome is rich in histone H3 methylated at Lys-9 and Lys-27, suggesting an important role for these modifications in X-inactivation. It has been shown that in the mouse Eed is required for maintenance of X-inactivation in the extraembryonic lineages. Interestingly, Eed associates with Ezh2 to form a complex possessing histone methyltransferase activity predominantly for H3 Lys-27. We previously showed that G9a is one of the histone methyltransferases specific for H3 Lys-9 and is essential for embryonic development. Here we examined X-inactivation in mouse embryos deficient for G9a. Expression of Xist, which is crucial for the initiation of X-inactivation, was properly regulated and the inactivated X chromosome was stably maintained even in the absence of G9a. These results demonstrate that G9a is not essential for X-inactivation.

Animals↗

PubMed2004

Electron tomography of fast frozen, stretched rigor fibers reveals elastic distortions in the myosin crossbridges.

As a first step toward freeze-trapping and 3-D modeling of the very rapid load-induced structural responses of active myosin heads, we explored the conformational range of longer lasting force-dependent changes in rigor crossbridges of insect flight muscle (IFM). Rigor IFM fibers were slam-frozen after ramp stretch (1000 ms) of 1-2% and freeze-substituted. Tomograms were calculated from tilt series of 30 nm longitudinal sections of Araldite-embedded fibers. Modified procedures of alignment and correspondence analysis grouped self-similar crossbridge forms into 16 class averages with 4.5 nm resolution, revealing actin protomers and myosin S2 segments of some crossbridges for the first time in muscle thin sections. Acto-S1 atomic models manually fitted to crossbridge density required a range of lever arm adjustments to match variably distorted rigor crossbridges. Some lever arms were unchanged compared with low tension rigor, while others were bent and displaced M-ward by up to 4.5 nm. The average displacement was 1.6 +/- 1.0 nm. "Map back" images that replaced each unaveraged 39 nm crossbridge motif by its class average showed an ordered mix of distorted and unaltered crossbridges distributed along the 116 nm repeat that reflects differences in rigor myosin head loading even before stretch.

Animals↗