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C Hashimoto

Publications and source records attributed to C Hashimoto.

At least 37 records · Page 2Linked to original sources
PubMed1998

Role of Nudel protease activation in triggering dorsoventral polarization of the Drosophila embryo.

The establishment of embryonic dorsoventral polarity in Drosophila depends on a signaling mechanism in which the signal for ventral development is locally produced. This mechanism requires the activity of the nudel gene in ovarian follicle cells, which provide dorsoventral positional information for the embryo. The nudel gene product, a large mosaic protein with a central serine protease domain, has been proposed to function in locally triggering a protease cascade that produces the ventral signal. Here we provide evidence that the serine protease activity of the Nudel protein is essential for embryonic dorsoventral polarity and that the active Nudel protease is generated by autoproteolytic cleavage of a zymogen form. Activation of the Nudel protease is independent of the other known proteases involved in dorsoventral polarity establishment and appears to occur symmetrically on the surface of the embryo. Our findings suggest that Nudel protease activation initiates the protease cascade that produces the ventral signal, but that spatial regulation occurring downstream of Nudel protease activation localizes the cascade to the ventral side of the embryo.

Animals↗
PubMed1998

NAD+ biosynthesis from tryptophan in the presence of nicotinic acid or vice versa by rat hepatocytes--effect of clofibrate-feeding.

NAD+ biosynthesis from tryptophan in the presence of nicotinic acid or vice versa by rat hepatocytes was investigated. In the control hepatocytes, NAD+ synthesis from tryptophan was not affected by nicotinic acid from 0.026 to 0.26 mM. NAD+ synthesis from nicotinic acid was slightly inhibited with varying concentrations of tryptophan from 0.1 to 1.0 mM. In the clofibrate-treated hepatocytes, NAD+ synthesis from tryptophan was greatly increased (234% of the control), while that from nicotinic acid was decreased (71.2% of the control). Both, NAD+ synthesis from tryptophan and that from nicotinic acid were decreased by the coexisting nicotinic acid or tryptophan. Total amount of NAD+ synthesized from tryptophan and nicotinic acid at their physiological concentrations was significantly higher than that in the control hepatocytes as a result of a large increase of NAD+ synthesized from tryptophan. When the metabolic flux of 0.1 or 0.5mM tryptophan was investigated, the glutarate pathway was suppressed in the clofibrate-treated hepatocytes, the quinolinic acid-NAD+ flux being elevated. Similarly to clofibrate, DEHP and CPP revealed an increase in NAD+ synthesis from tryptophan. Mutual relationship of NAD+ biosyntheses from tryptophan and nicotinic acid in rat hepatocytes is discussed and the relevance with peroxisomal proliferation is suggested.

Animals↗
PubMed1997

Molecular cloning and sequence analysis of the cDNA for the mouse counterpart to adult hamster liver purified growth inhibitory factor.

A cDNA clone encoding the mouse counterpart to adult hamster liver purified growth inhibitory factor (PGIF) was isolated from a mouse liver cDNA library by using antibodies raised against PGIF and sequenced. It contained a single open reading frame with a coding capacity for a 323 amino acid protein. Sequence analysis showed that it shared high homology with rat- and human liver arginases: the cDNA clone was 92% identical for rat arginase at the nucleotide level and was 93% identical to it at the deduced amino acid level. These results suggest that PGIF derived from adult hamster liver was identical or closely related to an isoform of hamster liver arginases.

Amino Acid Sequence↗
PubMed1997

Characterization of a 34 kDa immunoreactive peptide with the anti protein kinase C antibody which can recognize part of the C4 region.

Among the immunoreactive peptides from hamster tissues with the antibody (Ab-1) which can recognize part of the C4 conserved region of protein kinase C (PKC), a peptide with 34 kDa (34 kDa species) is constitutively detected in the soluble and the nuclear but not in the particulate fractions. The partially purified 34 kDa species demonstrates no phospholipid-dependent or independent PKC activity and no phorbol 12,13 dibutyrate (PDBu) binding, and is not phosphorylated under the assay condition of PKC activity. The species inhibits the enzymic activity of the partially purified PKC from hamster brain in an uncompetitive manner with Ki of 200 +/- 23 nM.

Animals↗
PubMed1997

The Xenopus homeobox gene twin mediates Wnt induction of goosecoid in establishment of Spemann's organizer.

We describe the isolation of the Xenopus homeobox gene twin (Xtwn), which was identified in an expression cloning screen for molecules with dorsalizing activities. Injection of synthetic Xtwn mRNA restores a complete dorsal axis in embryos lacking dorsal structures and induces a complete secondary dorsal axis when ectopically expressed in normal embryos. The sequence homology, expression pattern and gain-of-function phenotype of Xtwn is most similar to the previously isolated Xenopus homeobox gene siamois (Xsia) suggesting that Xtwn and Xsia comprise a new subclass of homeobox genes important in dorsal axis specification. We find that Xtwn is able to activate the Spemann organizer-specific gene goosecoid (gsc) via direct binding to a region of the gsc promoter previously shown to mediate Wnt induction. Since Xtwn expression is strongly induced in ectodermal (animal cap) cells in response to overexpression of a dorsalizing Wnt molecule, we examined the possibility that Xtwn might be a direct target of a Wnt signal transduction cascade. First, we demonstrate that purified LEF1 protein can interact, in vitro, with consensus LEF1/TCF3-binding sites found within the Xtwn promoter. Second, these binding sites were shown to be required for Wnt-mediated induction of a Xtwn reporter gene containing these sites. As LEF1/TCF3 family transcription factors have previously been shown to directly mediate Wnt signaling, these results suggest that Xtwn induction by Wnt may be direct. Finally, in UV-hyperventralized embryos, expression of endogenous Xtwn is confined to the vegetal pole and a Xtwn reporter gene is hyperinduced vegetally in a LEF1/TCF3-binding-site-dependent manner. These results suggest that cortical rotation distributes Wnt-like dorsal determinants to the dorsal side of the embryo, including the dorsal marginal zone, and that these determinants may directly establish Spemann's organizer in this region.

Activins↗
PubMed1996

Distribution and subcellular localization of a growth inhibitory factor in hamster liver and its intracellular partner(s).

The subcellular, intralobular distributions and intracellular partner(s) of a factor which inhibits the proliferation of cell growth (Hashimoto C. et al. (1994) Biochim. Biophys. Acta 1221, 107-117) were determined in hamster livers, using a combination of immunological and biochemical techniques. The IgG fraction from an antiserum raised against the growth inhibitory factor with 37 kDa was shown to be highly specific for the antigen. The nuclear and cytosolic fractions demonstrated inhibitory effects on cell growth and Western blot analysis revealed that both fractions contained the immunoreactive 37 kDa protein with the anti-inhibitory factor IgG but microsomal and mitochondrial fractions did not. The nuclear and cytoplasmic localization of the inhibitory factor were further confirmed by immunochemical staining mediated through the immune IgG and an avidin-biotinylated horseradish peroxidase complex, the parenchymal liver cells were clearly stained, but endothelial and connective tissue cells were not. Although some staining was evident throughout the liver parenchyma, the hepatocytes with most intensively stained nuclei were located in the periportal region. In the liver from hamsters 6 days old or the regenerating hamster livers 3 days after partial hepatectomy, the staining intensity was low and the number of hepatocytes with the inhibitory factor positive nuclei was very few compared with the adult hamster livers. In primary cultures of the isolated hepatocytes from adult hamster the inhibitory factor disappeared from nuclei after incubation for 24-48 h. The extracts of hepatic nuclei from adult hamsters were immunoprecipitated with either the anti-growth inhibitory factor IgG or a monoclonal antibody to the RM protein. The growth inhibitory factor and the RB protein coprecipitated in each case, implying that the proteins were complexed with each other in the nuclei. The RB protein family is composed of two sets of species, an un- or underphosphorylated species and a hyperphosphorylated one. It was suggested that the factor bound preferentially to the un- or underphosphorylated member of the family.

Animals↗
PubMed1996

The maternal nudel protein of Drosophila has two distinct roles important for embryogenesis.

The nudel gene is maternally required to define dorsoventral polarity of the Drosophila embryo. It encodes an unusual mosaic protein with a protease domain that may trigger the protease cascade required for ventral development. We describe phenotypic and molecular analyses of nudel mutations that provide further insight into nudel protein function. Surprisingly, nudel mutations primarily cause either dorsalized embryos in which dorsal cell fates are expanded over ventral and lateral cell fates or fragile eggs that fail to develop beyond early embryonic stages. The nudel protein is therefore required not only for embryonic dorsoventral polarity but also for structural integrity of the egg. Complementation and antagonistic interactions between nudel alleles suggest that the nudel protein is functionally modular and that protein-protein interactions are important for nudel protein function. Three nudel mutations that produce dorsalized embryos map to the protease domain of nudel, suggesting that this domain is specifically required for defining embryonic dorsoventral polarity. Finally, certain combinations of nudel alleles simultaneously produce completely dorsalized and normal embryos yet very few embryos of intermediate mutant phenotypes. The unusual biphasic distribution of phenotypes may indicate that nudel activity above a threshold is required to generate embryonic dorsoventral polarity.

Alleles↗
PubMed1995

Molecular mechanisms of Spemann's organizer formation: conserved growth factor synergy between Xenopus and mouse.

Mesoderm induction assays in Xenopus have implicated growth factors such as activin, Vg1, Xwnt-8, and noggin as important in directing the formation of dorsal mesoderm (Spemann's organizer). Because these growth factors are structurally very different, they presumably act through distinct cell surface receptors that initiate different intracellular signaling cascades. A consequence of all of these signaling pathways, however, seems to be the induction of goosecoid (gsc) gene expression. To understand how integration of these different signaling pathways results in formation of Spemann's organizer, we sought to identify growth factor-responsive elements within the gsc promoter. Through microinjection of reporter genes we have identified two cis-acting elements, a distal element (DE) and a proximal element (PE), that are required for activin/BVg1 and Wnt induction, respectively. We have shown that the DE mediates activin induction in the absence of protein synthesis and therefore constitutes the first activin response element identified to interpret transforming growth factor-beta (TGF-beta) superfamily member signaling directly. Using a reporter gene construct containing a multimerized DE, we find that an activin/BVg1-type signaling cascade is active throughout the vegetal hemisphere and marginal zone but not in the animal hemisphere. We demonstrate further that both the distal and proximal elements are essential for high-level transcription of the gsc gene, specifically in dorsal mesoderm, strongly suggesting that establishment of Spemann's organizer requires synergistic input from activin/BVg1-like and Wnt signaling pathways. Finally, mechanisms of establishing the organizer are likely to be conserved throughout vertebrate evolution.

Amino Acid Sequence↗
PubMed1995

Disruption of BMP signals in embryonic Xenopus ectoderm leads to direct neural induction.

Bone morphogenetic proteins (BMPs), which have been implicated in the patterning of mesoderm, are members of the transforming growth factor-beta (TGF-beta) superfamily. We have investigated the roles of Xenopus BMP-7 (XBMP-7) and BMP-4 (XBMP-4), and activin (another TGF-beta-related molecule) in early development by generating dominant-negative versions of these growth factors. Mutations were generated by altering the cleavage sites that are required for maturation of the active dimeric forms of XBMP-7, XBMP-4, and activin. These mutant constructs, designated Cm-XBMP-7, Cm-XBMP-4, and Cm-activin, result in polypeptides that allow for dimerization of the subunits, but are incapable of maturation. Expression of Cm-XBMP-7 and Cm-XBMP-4, but not Cm-activin, in the ventral marginal zone of the Xenopus embryo results in the development of a secondary axis, similar to that seen by ectopic expression of the truncated BMP receptor. These results suggest that the cleavage mutants interfere with BMP signaling during mesodermal patterning. We also found that expression of Cm-XBMP-7 or Cm-XBMP-4 in animal cap ectoderm directly induces neuroectoderm. The neural induction was specific for Cm-XBMP-7 and Cm-XBMP-4 because ectopic expression of Cm-activin or Vg-1 did not mimic the same phenotype. Molecular study of neural patterning by Cm-XBMP-7 and Cm-XBMP-4 revealed that only anterior neuroectodermal markers are expressed in response to these Cm-XBMPs. These results suggest that the BMPs are involved in the specification of ectoderm in Xenopus development, and that neural induction requires the removal of BMP signals in the ectoderm. We propose that neural induction occurs by a default mechanism, whereby the inhibition of BMP signaling is required for the conversion of ectoderm to neuroectoderm in the developing Xenopus embryo.

Activins↗
PubMed1995

An unusual mosaic protein with a protease domain, encoded by the nudel gene, is involved in defining embryonic dorsoventral polarity in Drosophila.

Dorsoventral polarity of the Drosophila embryo is induced by a ventral extracellular signal, which is produced by a locally activated protease cascade within the extraembryonic perivitelline compartment. Local activation of the protease cascade depends on a positional cue that is laid down during oogenesis outside the oocyte. Here we present evidence that the nudel gene encodes an essential component of this cue. The nudel gene, which is expressed in follicle cells covering the oocyte, encodes an unusual mosaic protein resembling an extracellular matrix protein with a central serine protease domain. Our findings suggest that embryonic dorsoventral polarity is defined by a positional cue that requires the nudel protein to anchor and to trigger the protease cascade producing the polarity-inducing signal.

Amino Acid Sequence↗
PubMed1995

The Drosophila toll gene functions zygotically and is necessary for proper motoneuron and muscle development.

Toll is a maternally required Drosophila gene that encodes a transmembrane protein with an important function in embryonic dorsal-ventral patterning. The Toll protein is widely expressed zygotically, but its roles in late embryo-genesis have not been described in detail. We have examined the expression of Toll protein in the late embryonic central nervous system and somatic musculature. Toll is expressed in a dynamic pattern in teh musculature, initially in several muscle fibers in each hemisegment, with a later narrowing of expression to a single muscle fiber pair. Zygotic Toll mutants were used to investigate the development consequences of loss of Toll expression. We found that loss of one or both copies of the Toll gene leads to widespread defects in motoneuron number and muscle patterning. Loss of motoneurons prevents certain muscle fibers from receiving their wild-type innervation. Denervation in the mutants results in collateral sprouting from nearby nerve branches and leads to the appearance of ectopically placed motor endings. The limited expressivity observed suggests that Toll is only one of several genes required for proper motoneuron and muscle specification.

Animals↗
PubMed1995

Ventralization of the Drosophila embryo by deletion of extracellular leucine-rich repeats in the Toll protein.

Dorsoventral polarity of the Drosophila embryo is established by a signal transduction pathway in which the maternal transmembrane protein Toll appears to function as the receptor for a ventrally localized extracellular ligand. Certain dominant Toll alleles encode proteins that behave as partially ligand-independent receptors, causing embryos containing these proteins to become ventralized. In extracts of embryos derived from mothers carrying these dominant alleles, we detected a polypeptide of approximately 35 kDa in addition to full-length Toll polypeptides with antibodies to Toll. Our biochemical analyses suggest that the smaller polypeptide is a truncated form of Toll lacking extracellular domain sequences. To assay the biological activity of such a shortened form of Toll, we synthesized RNA encoding a mutant polypeptide lacking the leucine-rich repeats that comprise most of Toll's extracellular domain and injected this RNA into embryos. The truncated Toll protein elicited the most ventral cell fate independently of the wild-type Toll protein and its ligand. These results support the view that Toll is a receptor whose extracellular domain regulates the intrinsic signaling activity of its cytoplasmic domain.

Alleles↗
PubMed1995

[Specific quantification of gamma-seminoprotein-alpha 1 antichymotrypsin complex in serum by monoclonal antibody-based enzyme immunoassay].

Gamma-seminoprotein (gamma-Sm) is one of the serine proteinases in seminal plasma, and is well known as a tumor marker of prostate cancer. In the blood, a major portion of gamma-Sm combines with alpha 1 antichymotrypsin (ACT), a serine proteinase inhibitor. We developed a sensitive enzyme immunoassay (EIA) for the gamma-Sm-ACT complex using two different monoclonal antibodies (MoAbs). One MoAb, prepared against gamma-Sm, is used for the capture, and the other, prepared against ACT, is conjugated with horseradish peroxidase and used for detection. The detectable range of this assay in clinical applications was from 0.2 to 50 units/ml. The intra-assay coefficients of variation (CV%) obtained from ten repeated assays of three sera were 3.6 to 5.5%. The mean gamma-Sm-ACT complex concentration in the sera of normal individuals was determined to be 0.86 +/- 0.11 units/ml for males (n = 50) and 0.11 +/- 0.08 units/ml for females (n = 54). There was no significant increase in the level of the complex with increasing age in males. The ratio of the gamma-Sm-ACT complex to free gamma-Sm tended to be significantly higher in patients with prostate cancer than in those with benign prostatic hyperplasia.

Antibodies, Monoclonal↗
PubMed1994

Isolation and characterization of a growth inhibitory factor from hamster liver.

Among lysates from various organs and tissues of adult hamsters only lysates from liver demonstrated an inhibitory effect on the cell growth of SV40-transformed hamster fibroblasts in culture. Lysates from the liver of fetal hamsters and those from 7-day-old hamsters did not demonstrate any inhibitory effect on the cell growth. Lysates from the remnant liver 3 days after partial hepatectomy did not show any inhibitory effect on the cell growth but lysates from the remnant liver 14 days after the operation came to show an appreciable inhibitory effect on the cell growth. An inhibitor of the cell growth was purified from adult hamster liver by ammonium sulfate precipitation, DEAE-, hydroxyl apatite-, phenyl Sepharose- and Sephadex G75 column chromatography. The cell growth inhibitor thus prepared was shown to be pure by an ion-exchange chromatography, SDS-PAGE and analytical isoelectric focusing. The inhibitor was found to have a molecular mass of 37 kDa and an isoelectric point of approx. 7.5 and to cause reversible arrest of the transformed fibroblasts predominantly in the G0/G1 phase of the cell cycle at the concentration of approx. 0.9 microgram per ml.

Animals↗
PubMed1994

Transgenic expression of IL-10 in pancreatic islet A cells accelerates autoimmune insulitis and diabetes in non-obese diabetic mice.

To study the paracrine effect of IL-10 on autoimmune insulitis and diabetes, we produced IL-10 transgenic non-obese diabetic (NOD) mice (NOD-IL-10) in which murine IL-10 was expressed in pancreatic islet A cells under the control of a rat glucagon promoter without directly manipulating pancreatic islet B cells. Among 11 founder mice, four of four males and three of seven females developed diabetes by 10 weeks of age. Histological analysis of six NOD-IL-10 revealed severe insulitis and prominent ductal proliferation. NOD-IL-10 also showed spotty lymphocytic infiltration in the lung and liver in four of six founder mice. The onset of diabetes in NOD-IL-10 was remarkably earlier than that of 14 weeks of age at the earliest in female non-transgenic NOD mice. When the NOD-IL-10 mouse was backcrossed to C57BL/6 mice, none of the resulting F1, B-N2 or B-N3 generation toward C57BL/6 mice showed diabetes even at 39 weeks of age, in spite of the presence of peri-insulitis and prominent ductal proliferation, while two of four mice of the N-N2 generation toward NOD mice showed early-onset diabetes. Thus, transgenic paracrine expression of IL-10 in situ in the NOD genetic background enhances autoimmune insulitis and diabetes in their onset and severity, ignoring gender difference. Because expression of IL-10 was detected by polymerase chain reaction in pancreatic islets of non-transgenic NOD mice after 5 weeks of age, IL-10 secreted in situ is regarded to enhance cell-mediated autoimmune diabetes, in spite of established in vitro anti-Th1 activity of IL-10.

Animals↗
PubMed1993

Discordant and heterogeneous expression of GPI-anchored membrane proteins on leukemic cells in a patient with paroxysmal nocturnal hemoglobinuria.

We performed a flow cytometric analysis using monoclonal antibodies to decay accelerating factor (DAF) and CD59/membrane attack complex inhibitory factor (CD59/MACIF) in order to investigate the leukemic cells and erythrocytes from a patient with paroxysmal nocturnal hemoglobinuria (PNH) who developed acute myelocytic leukemia. In May 1990, the leukemic cells comprised 70% of the mononuclear cells in the bone marrow and 76% of those in the peripheral blood. They consisted of a mixture of positive and negative populations, including single DAF-positive cells. In August 1990, almost 100% of the peripheral mononuclear cells were leukemic blasts, and these consisted of a single population with reduced DAF expression. Single-color flow cytometric analysis showed that the leukemic cells lacked CD59/MACIF, while control leukemic cells (n = 3) expressed both DAF and CD59/MACIF. Leukemic blasts from this patient and six control patients expressed lymphocyte function-associated antigen 3 and FcIII receptors (CD 16) both before and after treatment with phosphatidylinositol-specific phospholipase C. The patient's erythrocytes lacking DAF and CD59/MACIF expression corresponded to the proportion of complement-sensitive cells at the onset of acute leukemia. These DAF- and CD59/MACIF-deficient erythrocytes disappeared almost completely with progression of the leukemia. In conclusion, it appears that the expression of glycosylphosphatidylinositol-linked membrane proteins by leukemic cells was heterogeneous and discordant in our patient, and that the leukemic cells were derived from the PNH clone because of their deficiency of CD59/MACIF. It is also suggested that DAF could compete more effectively than CD59/MACIF for a limited number of anchor molecules available on the proliferating leukemic cells.

Antigens, CD↗
PubMed1993

Diagnosis of paroxysmal nocturnal haemoglobinuria by phenotypic analysis of erythrocytes using two-colour flow cytometry with monoclonal antibodies to DAF and CD59/MACIF.

We investigated the relationship between the complement lysis sensitivity test and two-colour flow cytometric analysis using monoclonal antibodies to decay accelerating factor (DAF) and CD59/membrane attack complex inhibitory factor (MACIF) in patients with paroxysmal nocturnal haemoglobinuria (PNH) and other haematological diseases. Flow cytometry showed that all 59 PNH patients had two or three erythrocyte populations, while all 74 patients with other haematological diseases and all 31 healthy volunteers had a single erythrocyte population. We compared the percentage of PNH III erythrocytes in the lysis test with the percentage of negative cells shown by flow cytometry in 52 PNH patients, and found a significant correlation (r = 0.960, P < 0.001). However, in 13 patients the erythrocyte phenotypes did not correspond in both tests. This was generally related to difficulty of detecting PNH II erythrocytes in the lysis test. In the PNH patients the ranges of mean fluorescence intensity for the negative, intermediate and positive erythrocyte populations were respectively 1.1-2.5, 2.2-29, and 61-600 for CD59/MACIF positivity and 1.9-7.2, 3.6-22. and 31-350 for DAF positivity. In contrast, the mean intensities in healthy volunteers ranged from 190 to 720 for CD59/MACIF and from 150 to 350 for DAF. These findings suggest that PNH can be diagnosed and phenotypic analysis of PNH erythrocytes can be performed by respectively assessing the fluorescence profiles and mean fluorescence intensities of both proteins using flow cytometry. Flow cytometry may provide a superior diagnostic method to the traditional tests for PNH.

Adult↗