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O Chisaka

Publications and source records attributed to O Chisaka.

At least 19 recordsLinked to original sources
PubMed2001

Hoxa3 regulates integration of glossopharyngeal nerve precursor cells.

In vertebrates, certain Hox genes are known to control cellular identities along the anterior-posterior (A-P) axis in the developing hindbrain. In mouse Hoxa3 mutants, truncation of the glossopharyngeal (IXth) nerve or the fusion of the IXth and vagus (Xth) nerves was reported, although its underlying mechanism is largely unknown. To elucidate the mechanism of the IXth nerve defects, we reexamined the phenotype of Hoxa3 mutant embryos. In Hoxa3 mutants, we observed an abnormal caudal stream of the migrating Hoxa3-expressing neural crest cells at the prospective IXth nerve-forming area. Dorsomedial migration of the placode-derived neuronal precursor cells of the IXth nerve was also affected. Motor neurons at rhombomere 6 (r6), where those of the IXth nerve were positioned, often projected axons to the Xth nerve. In summary, the Hoxa3 gene has crucial roles in ensuring the correct axon projection pattern of all three components of the IXth nerve, i.e., motor neurons and sensory neurons of the proximal and distal ganglia.

Animals↗
PubMed2001

Role of cadherins in maintaining the compartment boundary between the cortex and striatum during development.

In ventricular cells of the mouse telencephalon, differential expression of cadherin cell adhesion molecules defines neighbouring regions; R-cadherin delineates the future cerebral cortex, while cadherin-6 delineates the lateral ganglionic eminence. By using cell labelling analyses in the whole embryo culture system, we demonstrated that the interface between R-cadherin and cadherin-6 expression is a boundary for cell lineage restriction at embryonic day 10.5. Interestingly, when a group of cells with exogenous cadherin-6 were generated to straddle the cortico-straital boundary by electroporation at embryonic day 11.0, ectopic cadherin-6-expressing cortical cells were sorted into the striatal compartment, and the reverse was the trend for ectopic R-cadherin-expressing striatal cells. Although cadherin-6 gene knockout mice engineered in this study showed no obvious phenotype in telencephalic compartmentalisation, the preferential sorting of ectopic cadherin-6-expressing cells was abolished in this mutant background. Thus, the differential expression pattern of cadherins in the embryonic telencephalon is responsible for maintaining the cortico-striatal compartment boundary.

Animals↗
PubMed2001

Targeted disruption of cadherin-11 leads to a reduction in bone density in calvaria and long bone metaphyses.

The migration and adhesion of osteoblasts requires several classical cadherins. Cadherin-11, one of the classical cadherins, was expressed in mouse osteoblasts in skull bone and femur, revealed by immunohistochemistry. To elucidate the function of cadherin-11 in osteoblastogenesis, cadherin-11 null mutant mice were investigated. Although apparently normal at birth, Alizarin red staining of null mutant mice showed a reduced calcified area at the frontal suture that caused a round-shaped calvaria with increasing animal age to 3 months. Consequently, there was a reduction in bone density at the femoral metaphyses and the diploë of calvaria in null mutant mice. In the in vitro culture of newborn calvarial cells, the calcified area of mutant cells was smaller than those derived from wild-type littermates. These results show that absence of cadherin-11 leads to reduced bone density in some parts of skeletons including calvaria and long bone metaphyses, and thus suggest that cadherin-11 plays roles in the regulation of osteoblast differentiation and in the mineralization of the osteoid matrix.

Animals↗
PubMed2000

Loss of cadherin-11 adhesion receptor enhances plastic changes in hippocampal synapses and modifies behavioral responses.

Cadherins organize symmetrical junctions between the pre- and postsynaptic membranes in central synapses. One of them, cadherin-11 (cad11), is expressed in the limbic system of the brain, most strongly in the hippocampus. Immunohistochemical studies of the hippocampus showed that cad11 proteins were densely distributed in its synaptic neuropil zones; in cultured hippocampal neurons, their distribution often overlapped with that of synaptophysin, and also occasionally with that of GluR1 at spines. To assess the role of cad11 in synaptic formation and/or function, we analyzed brains of cad11-deficient mice. In these mice, long-term potentiation (LTP) in the CA1 region of the hippocampus was, unexpectedly, enhanced; and the level of LTP saturation was increased. In behavioral tests, the mutant mice showed reduced fear- or anxiety-related responses. These results suggest that the cad11-mediated junctions may modulate synaptic efficacy, confining its dynamic changes to a limited range, or these junctions are required for normal development of synaptic organization in the hippocampus.

Animals↗
PubMed1999

Adhesive subdivisions intrinsic to the epithelial somites.

Developing somites express two subtypes of classic cadherin adhesion receptors, N-cadherin and cadherin-11 (cad11). To investigate the role of these adhesion molecules in somite morphogenesis, we analyzed the somites of mice whose N-cadherin and cad11 genes were disrupted. The epithelial somites of N-cadherin null mutant mice were fragmented as reported, whereas those of cad11(-/-) mice showed no structural anomaly. In mice double homozygous for N-cadherin and cad11 mutation, however, somites were further fragmented into smaller clusters than in the N-cadherin-deficient mice, suggesting that these two cadherins cooperate in the maintenance of epithelial somites. Despite the disorganization of epithelial structures, dorsoventral polarity markers were expressed in their correct patterns in all of these mutant somites. Uncx4.1, whose expression is localized only in the caudal region of each somite, was also expressed in a normal pattern in the mutant somites. However, the staining for Uncx4.1 revealed that, in the N-cadherin mutants, each somite tended to be cleaved at the border between the Uncx4. 1-positive and -negative regions and that the cleaved subunits maintained the clustered state, often exhibiting epithelioid morphology. This separation of the rostral and caudal regions was observed as soon as the epithelial somites had been formed. In the N-cadherin/cad11 double-homozygous mutants, this tendency was also observed, although each half of the somite further disintegrated into randomly arranged cell clusters. These results suggest that cells of the rostral and caudal regions of each epithelial somite have an activity to aggregate independently or separate from one another and that one role of N-cadherin and cad11 is to connect the two halves into a single unit.

Animals↗
PubMed1999

Expression profiling by iAFLP: A PCR-based method for genome-wide gene expression profiling.

The availability of comprehensive sets of genes has prompted the researchers to carry out systematic collection of gene expression data. RT-PCR has the highest specificity and sensitivity for transcript detection among all available methods. Low throughput, especially when quantitative data are desired, has precluded RT-PCR from genome-wide application. Here we report a PCR-based expression profiling method, introduced amplified fragment length polymorphism (iAFLP), that has the same specificity and sensitivity as RT-PCR and a throughput level comparable to that of DNA-microarray hybridization. In this method, restricted ends of total cDNAs from six sources were ligated with adaptors having various length of short insertions to a common sequence (polymorphic adaptors). Amplification of a pool of these differentially adapted cDNAs with a gene-specific primer and an adaptor primer allows us to quantitate the abundance of any transcript in six mRNA sources. Using three different primer colors this technique allows quantitation of expression of 864 genes across six different sources per day with a single autosequencer, which is comparable to the throughput of microarray analysis in terms of number of genes x number of sources.

Animals↗
PubMed1999

Lumbosacral plexus in Hoxa9 knockout mice with special reference to their nerve variations identified according to whether they were interphenotypic or intergenotypic differences.

The lumbosacral plexus in specific Hox gene mutant mice originating from C57bl/60, i.e., Hoxa9 +/+, +/- and -/- genotypes (10 specimens of each), were dissected minutely and detailed examinations were made of the eight nerves in the plexus and the lowest intercostal nerve. We identified three types of nerve variation in mice with Hox mutations: interphenotypic variations, intergenotypic variations and common variations regardless of the genotype axial phenotype. The interphenotypic variations involved a caudal shift of the nerve origin in mice with the Hoxa9 -/- axial phenotype. We divided these variations into three patterns according to whether or not nerve configurations in mice with the wild and/or Hoxa9 -/- axial phenotypes were consistent, as follows: 1) the iliohypogastric and pudendal nerve morphologies of either of the phenotypes were consistent; 2) the femoral, obturator and sciatic nerve morphologies of the wild axial phenotype were consistent, but those of the -/- phenotype showed several variations and 3) both phenotypes showed several variations of the ilioinguinal and genitofemoral nerves. The intergenotypic variations, were limited to two examples: the common trunk formation of the ilioinguinal and iliohypogastric nerves in mice with the +/- and -/- genotypes, regardless of their axial phenotypes, and the territory of the ilioinguinal nerve touched the lateral margin of the thigh in the wild genotype, but extended beyond the margin onto the posterior aspect in the -/- genotype. The common variations regardless of either axial phenotype or genotype, included the lateral cutaneous nerve of the thigh which showed a limited, common variation in both of the nerve origin, ramification pattern and territory among either the three genotypes or two axial phenotypes. We consider the interphenotypic variations to be consistent with previous experimental findings showing a discrepancy between the nerve origins and their cues for nerve pathfinding. However, this developmental sequence did not seem to apply to the intergenotypic or common variations.

Animals↗
PubMed1998

Analysis of the murine Hoxa-9 cDNA: an alternatively spliced transcript encodes a truncated protein lacking the homeodomain.

Hoxa-9 is one of the homeo box (Hox) genes exhibiting similarity to the Drosophila Abdominal B gene. So far, only partial nucleotide sequences have been reported for mouse Hoxa-9 cDNA (Rubin et al., (1987) Mol. Cell. Biol. 7, 3836-3841). Here, we have determined the nucleotide sequence of the 5'-region of mouse Hoxa-9 cDNA and its genomic structure. Mouse Hoxa-9 cDNA contains a complete ORF encoding a protein of 271aa exhibiting 96.7% identity to its human counterpart. Interestingly, an alternatively spliced transcript (Hoxa-9T) was identified by RT-PCR. Sequence analysis revealed that 173bp within the Hoxa-9 ORF was missing from the Hoxa-9T cDNA. This additional splicing would potentially result in a frameshift, leading to the production of a truncated protein lacking the homeobox. Northern blot analysis revealed that the probe containing the homeodomain hybridized to two major transcripts (2.5 and 1.9kb) in the trunk region of 12.5 dpc embryos, and adult kidney and large intestine. On the other hand, the probe containing the additional intron detected only 2.5kb transcript in the same tissues, indicating that 1.9kb transcript corresponds to Hoxa-9T mRNA. We have also determined the transcriptional start site of Hoxa-9T.

Alternative Splicing↗
PubMed1997

Cadherin-6 expression transiently delineates specific rhombomeres, other neural tube subdivisions, and neural crest subpopulations in mouse embryos.

Mammalian cadherin-6 (K-cadherin, cad6) was originally identified by means of the polymerase chain reaction, but its biological functions have not yet been determined. We analyzed the expression pattern of the mouse homologue of this cadherin during development and found that it was transiently expressed in restricted rhombomeres and in other subdivisions of the neural plate and tube. In the midbrain and anterior hindbrain of E8.0-8.5 embryos, cad6 was expressed only in neural crest-generating regions. In contrast, in the posterior hindbrain and contiguous spinal cord of these embryos, cad6 occurred throughout the neural plate, forming a sharp anterior limit at the future rhombomere 4 and 5 boundary. Subsequently, this neural plate expression became confined to rhombomere 6, although most of the neural crest-generating areas remained positive throughout the body. Neural crest cells expressing cad6 migrated out of the neural tube, and subsequently accumulated mainly along peripheral nerves. We then studied the effect of Hoxa-1 mutation on the expression of cad6, as their expressions spatiotemporally overlapped with each other in the early posterior hindbrain. In E8.0-8.5 Hoxa-1 mutants, cad6 expression was suppressed in the region of rhombomeres 4 to 6, although that in the other regions was not essentially affected. At later stages, however, cad6-positive crest cells appeared and migrated out of rhombomeres 4 to 6, indicating that the suppression of cad6 expression was transient and restricted to early stages. Importantly, this effect of the Hoxa-1 mutation concurred with the timing of the expression of this gene. We also studied Hoxa-3 mutants, but found no effect of this mutation on the cad6 expression pattern. These findings suggest that cad6 may contribute to the formation of the segmental structure of the early brain through its ability to confer specific adhesiveness on cells and that Hoxa-1 may be required for early cad6 expression in the posterior hindbrain.

Amino Acid Sequence↗
PubMed1993

Effect of X protein on transactivation of hepatitis B virus promoters and on viral replication.

The X gene product of hepatitis B virus (HBV) transactivates a wide variety of promoters, including four promoters on the HBV genome (Rossner, 1992, J. Med. Virol. 36, 101-117). We compared their transactivation efficiencies and investigated whether the spatial organization of the promoters with respect to other cis-acting elements might influence their activities. Eight reporter plasmid constructs containing the bacterial chloramphenicol acetyltransferase (CAT) gene were designed such that four had the isolated HBV promoters linked to the CAT gene. In the other four, the CAT gene was inserted downstream to each of the four promoters retained in context in the HBV genome. Cells of the human hepatoblastoma line HepG2 were transfected with each one of these reporters together with an effector plasmid, pRSVX, which allowed expression of X protein. All of these promoters could be stimulated by X protein by approximately 2- to 3.5-fold irrespective of their spatial context in the HBV genome. Mutational analysis of in-frame ATG codons in the X gene provides evidence that transactivator product(s) are produced by internal initiation of translation. Transfection of HepG2 cells with HBV genomes bearing a stop mutation in the X gene at codon 118 resulted in poor production of all viral components. Their syntheses were restored upon transfection of the wild-type X gene.

Base Sequence↗
PubMed1993

Loss of Hox-A1 (Hox-1.6) function results in the reorganization of the murine hindbrain.

Targeted disruption of the murine hox-A1 gene results in severe defects in the formation of the hindbrain and associated cranial ganglia and nerves. Carbocyanine dye injections were used to trace afferent and efferent projections to and from the hindbrain in hox-A1-/hox-A1- mutant mice. Defects were observed in the position of efferent neurons in the hindbrain and in their projection patterns. In situ hybridization was used to analyze the transcription pattern of genes expressed within specific rhombomeres. Krox-20, int-2 (fgf-3), and hox-B1 all display aberrant patterns of expression in hox-A1- mutant embryos. The observed morphological and molecular defects suggest that there are changes in the formation of the hindbrain extending from rhombomere 3 through rhombomere 8 including the absence of rhombomere 5. Also, motor neurons identified by their axon projection patterns which would normally be present in the missing rhombomere appear to be respecified to or migrate into adjacent rhombomeres, suggesting a role for hox-A1 in the specification of cell identity and/or cell migration in the hindbrain.

Animals↗
PubMed1992

Developmental defects of the ear, cranial nerves and hindbrain resulting from targeted disruption of the mouse homeobox gene Hox-1.6.

Gene targeting in mouse embryo-derived stem cells has been used to generate mice with a disruption in the homeobox gene Hox-1.6. Mice heterozygous at the Hox-1.6 locus appear normal, whereas Hox-1.6-/Hox-1.6- mice die at or shortly after birth. These homozygotes exhibit profound defects in the formation of the external, middle and inner ears as well as in specific hindbrain nuclei, and in cranial nerves and ganglia. The affected tissues lie within a narrow region along the anteroposterior axis of the mouse but are of diverse embryonic origin. The set of defects associated with the disruption of Hox-1.6 is distinct from and nonoverlapping with that of the closely linked Hox-1.5 gene. But both mutations cause loss, rather than homeotic transformation, of tissues and structures.

Animals↗
PubMed1992

Hepatitis B virus-DNA transfected myeloma cell-specific cytotoxic T cells in chronic hepatitis B patients.

To study the mechanisms of hepatitis B virus (HBV)-induced chronic hepatitis (B-CH), we took chronic hepatitis B patients' peripheral blood lymphocytes (PBL) and examined their cytotoxic activities against human myeloma cells (ARH77) transfected by HBV-DNA. Two different transfected cells, one expressing HBV envelope antigens (S6) and the other expressing HBV core antigens (C4), were prepared and used as targets in the in vitro cytotoxic test. We found that PBL of B-CH patients had specific cytotoxic activity against these target cells (S6, 22.0 +/- 4.8%; C4, 21.6 +/- 4.8%), whereas no remarkable cytotoxic activity was observed in non-B chronic hepatitis patients as well as asymptomatic chronic HBV carriers. These specific cytotoxic activities were inhibited with anti-CD3 antibody, hence these killer cells belonged to T cells (cytotoxic T cells; CTL). The requirement of HLA class 1 antigens to exert these CTL activities was demonstrated by the absence of CTL activity with PBL obtained from HLA-nonidentical B-CH patients and by the inhibition of their activities with anti-HLA class 1 antibody. Thus, our results indicate that, at least two different CTL, one recognizing envelop antigen and the other recognizing core antigen, exist in chronic hepatitis B patients.

Adolescent↗
PubMed1991

Regionally restricted developmental defects resulting from targeted disruption of the mouse homeobox gene hox-1.5.

Gene targeting in mouse embryo-derived stem cells has been used to disrupt the homeobox gene hox-1.5. Mice heterozygous at the hox-1.5 locus appear normal, whereas hox-1.5-/hox-1.5- mice die at or shortly after birth. These homozygotes are athymic, aparathyroid, have reduced thyroid and submaxillary tissue and exhibit a wide range of throat abnormalities. In addition, they often feature defects of the heart and arteries as well as craniofacial abnormalities. These deficiencies are remarkably similar to the pathology of the human congenital disorder DiGeorge's syndrome.

Abnormalities, Multiple↗
PubMed1990

Translation of hepatitis B virus DNA polymerase from the internal AUG codon, not from the upstream AUG codon for the core protein.

Hepatitis B virus DNA replicates via its own polymerase that also acts as reverse transcriptase (Summers and Mason, 1982). This enzyme is encoded by a 3.5 Kb mRNA transcript covering the whole genome. Since the same transcript also codes for the core protein, and since the core open reading frame (ORF) is located upstream of the pol ORF, it has been suggested that the polymerase is first produced as a core-pol fusion protein that subsequently undergoes cleavage. This is already known to be the case with retrovirus reverse transcriptase, for which a gag-pol fusion protein is made first and the latter protein is liberated by proteolytic cleavage. We investigated this problem using mutants that were modified at the translation initiation codon for the core and precore ORF. Our findings suggested that polymerase translation occurred from the internal AUG codon independently of core protein synthesis, and that obligatory production of the core-pol fusion protein is accordingly unlikely.

Base Sequence↗
PubMed1990

The essential region for assembly and particle formation in hepatitis B virus surface antigen produced in yeast cells.

The hepatitis B virus (HBV) genome carries a HBV surface antigen (HBsAg) gene that can encode a polypeptide of 226 amino acids (aa). This gene can be expressed in the yeast, Saccharomyces cerevisiae, and the products can be assembled into 22-nm particles indistinguishable from those recovered from a patient's serum. We constructed a set of deletion derivatives of the HBsAg gene, and examined the particle-forming ability of the resulting polypeptides by expressing the gene in yeast. Elimination of 9 aa from the N terminus had no effect, whereas the elimination of 21-80 aa decreased the ability to form particles, and the particles formed were correspondingly smaller. Elimination of 100 aa that delete the major hydrophobic domain of the molecule abolished the ability to form particles completely. Deletion of 53 aa from the C terminus showed little effect. However, deletions proceeding further toward the center of the molecule rendered the polypeptides unstable.

Amino Acid Sequence↗