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Biomedical subjects

H Fujisawa

Publications and source records attributed to H Fujisawa.

At least 55 records · Page 3Linked to original sources
PubMed2000

Immunohistochemical study of the distribution of Ca(2+)/calmodulin-dependent protein kinase phosphatase in the rat central nervous system.

Distribution of Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaM-K Pase) which dephosphorylate multifunctional Ca(2+)/calmodulin-dependent protein kinases (CaM-kinases) in the rat brain and spinal cord were examined immunohistochemically by using an antibody against this enzyme. CaM-K Pase was localized only in the cytoplasm as has been investigated in PC 12 cells, and was never observed in the nucleus. Immunostainability varied from cell group to cell group. Mitral cells in the olfactory bulb, pyramidal neurons in the fifth layer of the cerebral cortex, hippocampal and striatal interneurons, dorsal and ventral pallidal, entopeduncular, and the reticular part of the substantia nigra neurons were intensely immunolabeled. Motoneurons in all the cranial nerve nuclei and the anterior horn of the spinal cord also revealed intense immunolabeling. On the contrary, pyramidal neurons in the Ammon's horn of the hippocampal formation, granule cells in the olfactory bulb, dentate gyrus and cerebellar cortex, Purkinje cells, neurons in the medial habenular nucleus and the inferior olivary nucleus have not shown immunoreactivity. Axons in the white matter or nerve root of the cranial nerve nuclei were immunolabeled. Glial cells in the white matter also showed immunostaining. Because the substrate of CaM-K Pase is multifunctional CaM-kinase II, I and IV, localization of each CaM-kinase was compared with that of CaM-K Pase. The distribution of CaM-K Pase and these CaM-kinases was found to overlap in various regions in the brain and spinal cord. It was concluded, therefore, that CaM-K Pase could regulate the activity of these CaM-kinases by dephosphorylation, when they existed together in neurons.

Animals↗
PubMed2000

Determination of cell adhesion sites of neuropilin-1.

Neuropilin-1 is a type 1 membrane protein with three distinct functions. First, it can mediate cell adhesion via a heterophilic molecular interaction. Second, in neuronal cells, neuropilin-1 binds the class 3 semaphorins, which are neuronal chemorepellents, and plays a role in the directional guidance of axons. Neuropilin-1 is expected to form complexes with the plexinA subfamily members and mediate the semaphorin-elicited inhibitory signals into neurons. Third, in endothelial cells, neuropilin-1 binds a potent endothelial cell mitogen, vascular endothelial growth factor (VEGF)(165), and regulates vessel formation. Though the binding sites in neuropilin-1 for the class 3 semaphorins and VEGF(165) have been analyzed, the sites involved in cell adhesion activity of the molecule have not been identified. In this study, we produced a variety of mutant neuropilin-1s and tested their cell adhesion activity. We showed that the b1 and b2 domains within the extracellular segment of neuropilin-1 were required for the cell adhesion activity, and peptides with an 18-amino acid stretch in the b1 and b2 domains were sufficient to induce the cell adhesion activity. In addition, we demonstrated that the cell adhesion ligands for neuropilin-1 were proteins and distributed in embryonic mesenchymal cells but distinct from the class 3 semaphorins, VEGF, or plexins.

3T3 Cells↗
PubMed2000

Expression and putative role of neuropilin-1 in the early scaffold of axon tracts in embryonic Xenopus brain.

Although the general principles of axon guidance in vitro are understood, little is known about how axons respond to the myriad of cues in vivo and navigate axon pathways within the complex milieu of the embryonic brain. Although neuropilin-1 is an axon guidance receptor for chemorepulsive ligands in the class 3 subfamily of semaphorins, its role in directing axon growth in vivo is unknown. In the present study, we have examined the expression and role of neuropilin-1 in the embryonic forebrain of Xenopus. Neuropilin-1 was selectively expressed by a subset of axons in the early scaffold of axon tracts. These axons arise from the presumptive telencephalic nucleus, cross the rostral midline by means of the postoptic commissure, and enter the major longitudinal tract of the prosencephalon, the tract of the postoptic commissure. At the level of the mesencephalon, these axons diverge and enter one of two axon tracts: either the ventral longitudinal tract or the ventral commissure. This same population of axons also expresses NOC-2, a novel glycoform of the neural cell adhesion molecule N-CAM. We have previously revealed the presence of a chemorepulsive activity underlying the pathway followed by these axons as they cross the ventral commissure. When neuropilin-1 was overexpressed after blastomere injections of synthetic RNA transcripts, NOC-2 axons entered the ventral commissure but failed to cross the midline. Instead, these axons were inhibited from growing ventrally within the commissural pathway. These results suggest that the level of neuropilin-1 in the NOC-2 subpopulation of axons is critical for determining whether these axons reach the ventral midline. Thus, neuropilin-1 may a specific role in directing the growth of NOC-2 axons across the ventral midline in the early embryonic mesencephalon.

Animals↗
PubMed2000

Structural analysis of the bacteriophage T3 head-to-tail connector.

The connector protein of bacteriophage T3, p8, has been overexpressed in Escherichia coli. Purification of the oligomers built by several copies of p8 reveals a mixed population of dodecamers and tridecamers. The percentages of these two types of oligomers differ in every culture growth, indicating that assembly of this protein depends upon the conditions of the expression system. Those cultures that generated a majority of dodecamers allowed, after purification of the connectors, the two-dimensional crystallization of the dodecamers in a tetragonal arrangement, while the tridecamers did not form crystals. The processing and averaging of several images of frozen-hydrated crystals and their internal phase comparison shows that the crystals are arranged in a P42(1)2 space group, with cell unit dimensions of 165 x 165 A. The three-dimensional reconstruction generated with images of crystals ranging from 0 degrees to 60 degrees tilt reveals a wide domain surrounded by 12 protrusions and a narrow domain that serves to interact with the tail of the bacteriophage. A channel runs along the connector wide enough to allow the translocation of a double-stranded DNA molecule into the prohead. The general structure of the T3 connector is very similar to those obtained for other nonrelated bacteriophages and strongly suggests that the shape of this important viral structure is intimately related to its function.

Capsid↗
PubMed2000

Sema3C and netrin-1 differentially affect axon growth in the hippocampal formation.

The interaction between outgrowing neurons and their targets is a central element in the development of the afferent and efferent connections of the hippocampal system. This requires that axonal growth cones recognize specific guidance cues in the appropriate target area. At present, little is known about the mechanisms that determine the lamina-specific termination of hippocampal afferents. In order to understand the role of different guidance factors, we analyzed the effects of Sema3C and Netrin-1 on explants from the entorhinal cortex, dentate gyrus, cornu ammonis regions CA1 and CA3 and medial septum in a collagen coculture assay. Our observations suggest that both semaphorins and netrin play important roles in the neuron-target interactions in the hippocampal system. Sema3C is involved in the control of the ingrowth of the septohippocampal projection. We also show that netrin-1 is involved in attracting commissural neurons from dentate gyrus/hilus and CA3 to their target area in the contralateral hippocampus.

Animals↗
PubMed2000

Repulsive axon guidance molecule Sema3A inhibits branching morphogenesis of fetal mouse lung.

Semaphorin III/collapsin-1 (Sema3A) guides a specific subset of neuronal growth cones as a repulsive molecule. In this study, we have investigated a possible role of non-neuronal Sema3A in lung morphogenesis. Expression of mRNAs of Sema3A and neuropilin-1 (NP-1), a Sema3A receptor, was detected in fetal and adult lungs. Sema3A-immunoreactive cells were found in airway and alveolar epithelial cells of the fetal and adult lungs. Immunoreactivity for NP-1 was seen in fetal and adult alveolar epithelial cells as well as endothelial cells. Immunoreactivity of collapsin response mediator protein CRMP (CRMP-2), an intracellular protein mediating Sema3A signaling, was localized in alveolar epithelial cells, nerve tissue and airway neuroendocrine cells. The expression of CRMP-2 increased during the fetal, neonate and adult periods, and this pattern paralleled that of NP-1. In a two-day culture of lung explants from fetal mouse lung (E11.5), with exogenous Sema3A at a dose comparable to that which induces growth cone collapse of dorsal root ganglia neurons, the number of terminal buds was reduced in a dose-dependent manner when compared with control or untreated lung explants. This decrease was not accompanied with any alteration of the bromodeoxyuridine-positive DNA-synthesizing fraction. A soluble NP-1 lacking the transmembrane and intracellular region, neutralized the inhibitory effect of Sema3A. The fetal lung explants from neuropilin-1 homozygous null mice grew normally in vitro regardless of Sema3A treatment. These results provide evidence that Sema3A inhibits branching morphogenesis in lung bud organ cultures via NP-1 as a receptor or a component of a possible multimeric Sema3A receptor complex.

Animals↗
PubMed2000

Loss of heterozygosity on chromosome 10 is more extensive in primary (de novo) than in secondary glioblastomas.

Glioblastomas develop de novo (primary glioblastomas) or through progression from low-grade or anaplastic astrocytoma (secondary glioblastomas). There is increasing evidence that these glioblastoma subtypes develop through different genetic pathways. Primary glioblastomas are characterized by EGFR and MDM2 amplification/overexpression, PTEN mutations, and p16 deletions, whereas secondary glioblastomas frequently contain p53 mutations. Loss of heterozygosity (LOH) on chromosome 10 (LOH#10) is the most frequent genetic alteration in glioblastomas; the involvement of tumor suppressor genes, other than PTEN, has been suggested. We carried out deletion mappings on chromosome 10, using PCR-based microsatellite analysis. LOH#10 was detected at similar frequencies in primary (8/17; 47%) and secondary glioblastomas (7/13; 54%). The majority (88%) of primary glioblastomas with LOH#10 showed LOH at all informative markers, suggesting loss of the entire chromosome 10. In contrast, secondary glioblastomas with LOH#10 showed partial or complete loss of chromosome 10q but no loss of 10p. These results are in accordance with the view that LOH on 10q is a major factor in the evolution of glioblastoma multiform as the common phenotypic end point of both genetic pathways, whereas LOH on 10p is largely restricted to the primary (de novo) glioblastoma.

Adult↗
PubMed2000

Pemphigoid nodularis with IgA autoantibodies against the intracellular domain of desmoglein 1.

Pemphigoid nodularis is a rare variant of bullous pemphigoid. We report a 49-year-old Japanese male with clinical and histopathological features of pemphigoid nodularis including circulating and in vivo-bound IgG antibasement membrane zone antibodies and IgA anti-intercellular antibodies. Although the precise molecular target of the IgG autoantibodies could not be determined, intriguingly, immunoblotting showed that the IgA in the patient's serum reacted with the intracellular domain of desmoglein 1, the target antigen in cases of pemphigus foliaceus. However, the IgA did not react with the extracellular domain of desmoglein 1 in sensitive enzyme-linked immunosorbent assay studies using a baculovirus system. These results suggest therefore that these IgA antibodies may possibly not be pathogenic. The mechanism for the production of different autoantibodies is unknown, but this case provides further illustration of the atypical skin immunoreactants often seen in this unusual subtype of bullous pemphigoid.

Anti-Inflammatory Agents↗
PubMed2000

EAT-20, a novel transmembrane protein with EGF motifs, is required for efficient feeding in Caenorhabditis elegans.

The pharynx of Caenorhabditis elegans is a neuromuscular organ responsible for feeding, concentrating food by its pumping movement. A class of mutants, the eat mutants, are defective in this behavior. We have identified a novel eat gene, eat-20, encoding a unique transmembrane protein with three EGF motifs. Staining with a specific polyclonal antibody reveals that EAT-20 is expressed predominantly in the pharyngeal muscles and a subset of neurons. Some hypodermal cells also express EAT-20. eat-20 mutant animals are starved, have smaller brood sizes, and have prolonged egg-laying periods. The starvation apparently results from pharyngeal pumping defects, including a reduced pumping rate and "slippery pumping," in which the contents of the pharynx sometimes move rostrally. However, electrical activity of eat-20 mutants appears normal by electropharyngeogram.

Amino Acid Sequence↗
PubMed2000

Loss of heterozygosity on chromosome 19 in secondary glioblastomas.

Glioblastomas develop rapidly de novo (primary glioblastomas) or slowly through progression from low-grade or anaplastic astrocytoma (secondary glioblastomas). Recent studies have shown that these glioblastoma subtypes develop through different genetic pathways. Primary glioblastomas are characterized by EGFR amplification/overexpression, PTEN mutation, homozygous p16 deletion, and loss of heterozygosity (LOH) on entire chromosome 10, whereas secondary glioblastomas frequently contain p53 mutations and show LOH on chromosome 10q. In this study, we analyzed LOH on chromosomes 19q, 1p, and 13q, using polymorphic microsatellite markers in 17 primary glioblastomas and in 13 secondary glioblastomas that progressed from low-grade astrocytomas. LOH on chromosome 19q was frequently found in secondary glioblastomas (7 of 13, 54%) but rarely detected in primary glioblastomas (1 of 17, 6%, p = 0.0094). The common deletion was 19q13.3 (between D19S219 and D19S902). These results suggest that tumor suppressor gene(s) located on chromosome 19q are frequently involved in the progression from low-grade astrocytoma to secondary glioblastoma, but do not play a major role in the evolution of primary glioblastomas. LOH on chromosome 1p was detected in 12% of primary and 15% of secondary glioblastomas. LOH on 13q was detected in 12% of primary and in 38% of secondary glioblastomas and typically included the RB locus. Except for 1 case, LOH 13q and 19q were mutually exclusive.

Astrocytoma↗
PubMed2000

Studies on the phosphorylation of protein kinase B by Ca(2+)/calmodulin-dependent protein kinases.

Protein kinase B (PKB) was recently reported to be activated on the phosphorylation of Thr(308) by Ca(2+)/calmodulin-dependent protein kinase kinase alpha (CaM-kinase kinase alpha), suggesting that PKB was regulated through not only the phosphoinositide 3-kinase pathway but also the Ca(2+)/calmodulin protein kinase pathway. The activation of PKB by CaM-kinase kinase alpha was as high as 300-fold after incubation for 30 min under the phosphorylation conditions, and still increased thereafter, suggesting that the maximal activation of PKB on phosphorylation of the Thr(308) residue is several hundred fold. On the other hand, the V(max) value of CaM-kinase kinase alpha for the phosphorylation of PKB was more than two orders of magnitude lower than that for CaM-kinase IV, although the K(m) values for PKB and CaM-kinase IV were not significantly different, raising the question of whether or not PKB is a physiological substrate of CaM-kinase kinase alpha. Besides CaM-kinase kinase alpha, CaM-kinase II also remarkably activated PKB. However, the specific activities of CaM-kinase kinase alpha and CaM-kinase II as to the activation of PKB were more than three orders of magnitude lower than that of 3-phosphoinositide-dependent protein kinase 1 (PDK1).

Animals↗
PubMed2000

Inference about misclassification probabilities from repeated binary responses.

Repeated binary responses provide efficient information for two purposes: (1) estimating two misclassification (false-positive and false-negative error) probabilities and (2) testing the hypothesis that either is zero in a reliability study. We focus on the assessment of reliability of a diagnostic test when there is no gold standard. This paper uses a latent class model and illustrates some of its properties. In addition, application to data containing variation among individuals is considered. We apply this model to the serological data on the MNSs blood group of atomic bomb survivors and their children. The results provide valuable information for examining measurement reliability.

Adult↗
PubMed2000

Alteration of the retinotectal projection map by the graft of mesencephalic floor plate or sonic hedgehog.

The floor plate plays crucial roles in the specification and differentiation of neurons along the dorsal-ventral (DV) axis of the neural tube. The transplantation of the mesecephalic floor plate (mfp) into the dorsal mesencephalon in chick embryos alters the fate of the mesencephalon adjacent to the transplant from the tectum to the tegmentum, a ventral tissue of the mesencephalon. In this study, to test whether the mfp is involved in the specification of the DV polarity of the tectum and affects the projection patterns of retinal fibers to the tectum along the DV axis, we transplanted quail mfp into the dorsal mesencephalon of chick embryos, and analyzed projection patterns of dorsal and ventral retinal fibers to the tectum. In the embryos with the mfp graft, dorsal retinal fibers grew into the dorsal part of the tectum which is the original target for ventral but not dorsal retinal fibers and formed tight focuses there. In contrast, ventral retinal fibers did not terminate at any part of the tectum. Transplantation of Sonic hedgehog (Shh)-secreting quail fibroblasts into the dorsal mesencephalon also induced the ectopic tegmentum and altered the retinotectal projection along the DV axis, as the mfp graft did. These results suggest that some factors from the mesencephalic floor plate or the tegmentum, or Shh itself, play a crucial role in the establishment of the DV polarity of the tectum and the retinotectal projection map along the DV axis.

Animals↗
PubMed1999

Plexin-neuropilin-1 complexes form functional semaphorin-3A receptors.

Class 1 and 3 semaphorins repulse axons but bind to different cell surface proteins. We find that the two known semaphorin-binding proteins, plexin 1 (Plex 1) and neuropilin-1 (NP-1), form a stable complex. Plex 1 alone does not bind semaphorin-3A (Sema3A), but the NP-1/Plex 1 complex has a higher affinity for Sema3A than does NP-1 alone. While Sema3A binding to NP-1 does not alter nonneuronal cell morphology, Sema3A interaction with NP-1/Plex 1 complexes induces adherent cells to round up. Expression of a dominant-negative Plex 1 in sensory neurons blocks Sema3A-induced growth cone collapse. Sema3A treatment leads to the redistribution of growth cone NP-1 and plexin into clusters. Thus, physiologic Sema3A receptors consist of NP-1/plexin complexes.

Animals↗
PubMed1999

Cdc42 and Rac1 regulate the interaction of IQGAP1 with beta-catenin.

IQGAP1, a target of Cdc42 and Rac1 small GTPases, directly interacts with beta-catenin and negatively regulates E-cadherin-mediated cell-cell adhesion by dissociating alpha-catenin from the cadherin-catenin complex in vivo (Kuroda, S., Fukata, M., Nakagawa, M., Fujii, K., Nakamura, T., Ookubo, T., Izawa, I., Nagase, T., Nomura, N., Tani, H., Shoji, I., Matsuura, Y., Yonehara, S., and Kaibuchi, K. (1998) Science 281, 832-835). Here we investigated how Cdc42 and Rac1 regulate the IQGAP1 function. IQGAP1 interacted with the amino-terminal region (amino acids 1-183) of beta-catenin, which contains the alpha-catenin-binding domain. IQGAP1 dissociated alpha-catenin from the beta-catenin-alpha-catenin complex in a dose-dependent manner in vitro. Guanosine 5'-(3-O-thio)triphosphate (GTPgammaS).glutathione S-transferase (GST)-Cdc42 and GTPgammaS. GST-Rac1 inhibited the binding of IQGAP1 to beta-catenin in a dose-dependent manner in vitro, whereas neither GDP.GST-Cdc42, GDP. GST-Rac1, nor GTPgammaS.GST-RhoA did. The coexpression of dominant active Cdc42 with IQGAP1 suppressed the dissociation of alpha-catenin from the cadherin-catenin complex induced by the overexpression of IQGAP1 in L cells expressing E-cadherin (EL cells). Consistent with this, the overexpression of either dominant negative Cdc42 or Rac1 resulted in the reduction of E-cadherin-mediated cell adhesive activity in EL cells. These results indicate that Cdc42 and Rac1 negatively regulate the IQGAP1 function by inhibiting the interaction of IQGAP1 with beta-catenin, leading to stabilization of the cadherin-catenin complex.

Carrier Proteins↗