Masanori Narahara
Publications
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1.81Impact points
β-Citryl-L-glutamate acts as an iron carrier to activate aconitase activity.
Biological & pharmaceutical bulletin. 01/2011; 34(9):1455-64.
The compound β-citryl-L-glutamate (β-CG) was initially isolated from developing brains, though its functional roles remain unclear. In in vitro experiments, the [Fe(II)(β-CG)] complex activated aconitase in the presence of reducing reagents, whereas no Fe complex with citrate, glutamate, or deferoxa... [more] The compound β-citryl-L-glutamate (β-CG) was initially isolated from developing brains, though its functional roles remain unclear. In in vitro experiments, the [Fe(II)(β-CG)] complex activated aconitase in the presence of reducing reagents, whereas no Fe complex with citrate, glutamate, or deferoxamine displayed such an effect. β-CG and [Fe(II)(β-CG)] both bound to the fourth labile Fe atom (Fe(a)) in the [4Fe-4S] cluster of aconitase. Furthermore, [Fe(II)(β-CG)] reactivated aconitase damaged by ammonium peroxodisulfate (APS), while β-CG and citrate had no effect. These findings suggest that [Fe(II)(β-CG)] can transfer Fe to aconitase disassembled by APS. In intact mitochondria, both β-CG and [Fe(II)(β-CG)] bound to Fe(a) of aconitase, whereas only [Fe(II)(β-CG)] reactivated the enzyme disassembled by APS. In cultured neuronal cells, β-CG significantly enhanced cell viability by accelerating mitochondrial activity in primary cultures of neurons from newborn mouse cerebrum tissues. Thus, the β-CG plays a role as an Fe-carrier for mitochondrial aconitase, and then activates it. Taken together, these findings suggest that β-CG is an endogenous low molecular weight Fe chaperone for aconitase.
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1.81Impact points
Superoxide scavenging and xanthine oxidase inhibiting activities of copper-β-citryl-L-glutamate complex.
Biological & pharmaceutical bulletin. 01/2010; 33(12):1938-43.
β-Citryl-L-glutamate (β-CG) is a unique compound initially isolated from developing brains, which also appears in high concentrations during the period characterized by growth and differentiation of neurons in developing animals, and then decreases with maturation. However, its functional roles rema... [more] β-Citryl-L-glutamate (β-CG) is a unique compound initially isolated from developing brains, which also appears in high concentrations during the period characterized by growth and differentiation of neurons in developing animals, and then decreases with maturation. However, its functional roles remain unclear. The stability constant obtained in our previous pH titration studies showed that β-CG forms relatively strong complexes with copper. Reactive oxygen species (ROS) and nitric oxide (NO) have been suggested to act as mediators of the cell death that occurs in neurons during development of the nervous system. However, regulation of ROS and NO formation by Cu in the developing brain remains poorly understood. The activity of superoxide dismutase (SOD), a key superoxide scavenging enzyme, is low in the developing brain. Furthermore, xanthine oxidase (XO) has been implicated in diverse pathological situations due to its capability of generating both ROS and NO. Therefore, we examined the effects of β-CG and its Cu-complex on SOD and XO activities. We found that the [Cu(II)(β-CG)] complex had SOD activity and a strong competitive inhibition of XO, while reduced glutathione caused concentration-dependent decreases of the XO inhibitory activities in the [Cu(II)(β-CG)] complex.
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1.81Impact points
beta-Citryl-L-glutamate is an endogenous iron chelator that occurs naturally in the developing brain.
Biological & pharmaceutical bulletin. 01/2010; 33(5):729-37.
The compound beta-citryl-L-glutamate (beta-CG) was initially isolated from developing brains, while it has also been found in high concentrations in testes and eyes. However, its functional roles are unclear. To evaluate its coordination with metal ions, we performed pH titration experiments. The st... [more] The compound beta-citryl-L-glutamate (beta-CG) was initially isolated from developing brains, while it has also been found in high concentrations in testes and eyes. However, its functional roles are unclear. To evaluate its coordination with metal ions, we performed pH titration experiments. The stability constant, logbeta(pqr) for M(p)(beta-CG)(q)H(r) was calculated from pH titration data, which showed that beta-CG forms relatively strong complexes with Fe(III), Cu(II), Fe(II) and Zn(II). beta-CG was also found able to solubilize Fe more effectively from Fe(OH)(2) than from Fe(OH)(3). Therefore, we examined the effects of beta-CG on Fe-dependent reactive oxygen species (ROS)-generating systems, as well as the potential ROS-scavenging activities of beta-CG and metal ion-(beta-CG) complexes. beta-CG inhibited the Fe-dependent degradation of deoxyribose and Fe-dependent damage to DNA or plasmid DNA in a dose-dependent manner, whereas it had no effect on Cu-mediated DNA damage. In addition, thermodynamic data showed that beta-CG in a physiological pH solution is an Fe(II) chelator rather than an Fe(III) chelator. Taken together, these findings suggest that beta-CG is an endogenous low molecular weight Fe chelator.
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1.81Impact points
Triglyceride accumulation by peroxisome proliferators in rat hepatocytes.
Biological & pharmaceutical bulletin. 05/2007; 30(4):627-32.
Peroxisome proliferators (PxPs) induce peroxisomal beta-oxidation (Px-ox) in the liver of rodents and have a hypolipidemic function. To investigate hypolipidemic effect of PxPs, the relationship between TG fluctuation and Px-ox activity, as an indicator of the function of PxPs, was studied in primar... [more] Peroxisome proliferators (PxPs) induce peroxisomal beta-oxidation (Px-ox) in the liver of rodents and have a hypolipidemic function. To investigate hypolipidemic effect of PxPs, the relationship between TG fluctuation and Px-ox activity, as an indicator of the function of PxPs, was studied in primary cultured rat hepatocytes. Nafenopin (Nf) treatment of hepatocytes caused an increase in Px-ox activity in association with cellular TG accumulation in a time-dependent manner with a coefficient of r=0.918. This relationship between the activity and cellular TG were obtained using structurally diverse PxPs with a correlation coefficient of r=0.747. Treatment of the hypolipidemic drug, but non-PxP Pravastatin, decreased TG in the medium, but did not have the effects on cellular TG and Px-ox activity. The total amount of TG and diacylglycerol acyltransferase activity, the last enzyme in the TG de novo synthesis pathway, were not affected by Nf treatment. When hepatocytes were cultured with Brefeldin A, cellular TG was accumulated, the same as with Nf, however, Px-ox activity was not enhanced. Nf treatment markedly decreased the level of apolipoprotein B (apo B) in very low density lipoprotein (VLDL) fractions prepared from conditioned media and increased that of cellular apoB by Western blot analysis. Microsomal triglyceride transfer protein activity was not influenced by Nf. Together, with regards to TG lowering effect of PxPs, it is suggested that PxPs cause hepatocellular accumulation of TG without effects on TG biosynthesis and VLDL construction, and they might have inhibitory effect on VLDL secretion process.
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3.54Impact points
Suppression of Sox6 in P19 cells leads to failure of neuronal differentiation by retinoic acid and induces retinoic acid-dependent apoptosis.
FEBS letters. 12/2004; 577(1-2):60-6.
The Sox6 gene is a member of the Sox gene family, which encodes transcription factors, and previous studies have suggested that it plays an important role in the development of the central nervous system. Aggregation of embryonic carcinoma P19 cells with retinoic acid (RA) results in the development... [more] The Sox6 gene is a member of the Sox gene family, which encodes transcription factors, and previous studies have suggested that it plays an important role in the development of the central nervous system. Aggregation of embryonic carcinoma P19 cells with retinoic acid (RA) results in the development of neurons, glia, and fibroblast-like cells. Sox6 mRNA increases rapidly in P19 cells during RA induction and then decreases during differentiation into neuronal cells. To investigate whether Sox6 expression is essential for neuronal differentiation, we established Sox6-suppressed P19 (P19[anti-Sox6]) cells by transfection of antisense-Sox6 cDNA. Most of the P19[anti-Sox6] cells showed no neurites and were not stained by the anti-MAP 2 antibody, while the suppression of Sox6 expression nearly totally blocked neuronal differentiation in P19 cells. Further, Sox6 suppression caused RA-dependent apoptosis by P19[anti-Sox6] cells: RA-treated P19[anti-Sox6] cells showed chromatin condensation, DNA fragmentation, and an increase in caspase-3-like activity. Thus, Sox6 is considered essential for neuronal differentiation and may play an important role in the early stages of neuronal differentiation or apoptosis.
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3.54Impact points
Sox6 overexpression causes cellular aggregation and the neuronal differentiation of P19 embryonic carcinoma cells in the absence of retinoic acid.
FEBS letters. 03/2004; 560(1-3):192-8.
The Sox6 gene is a member of the Sox gene family that encodes transcription factors. Previous studies have suggested that Sox6 plays an important role in the development of the central nervous system. Aggregation of embryonic carcinoma P19 cells with retinoic acid (RA) results in the development of ... [more] The Sox6 gene is a member of the Sox gene family that encodes transcription factors. Previous studies have suggested that Sox6 plays an important role in the development of the central nervous system. Aggregation of embryonic carcinoma P19 cells with retinoic acid (RA) results in the development of neurons, glia and fibroblast-like cells. In this report, we have shown that Sox6 mRNA increased rapidly in P19 cells during RA induction and then decreased during the differentiation of P19 into neuronal cells. To explore the possible roles of Sox6 during this process, stably Sox6-overexpressing P19 cell lines (P19[Sox6]) were established. These P19[Sox6] had acquired both characteristics of the wild-type P19 induced by RA. First, P19[Sox6] cells showed a marked cellular aggregation in the absence of RA. Second, P19[Sox6] could differentiate into microtubule-associated protein 2 (MAP2)-expressing neuronal cells in the absence of RA. Sox6 expression could cause the activation of endogenous genes including the neuronal transcription factor Mash-1, the neuronal development-related gene Wnt-1, the neuron-specific cell adhesion molecule N-cadherin, and the neuron-specific protein MAP2, resulting in neurogenesis. Moreover, E-cadherin, a major cell adhesion molecule of wild-type P19, was strongly induced by Sox6, resulting in cellular aggregation without RA. Thus Sox6 may play a critical role in cellular aggregation and neuronal differentiation of P19 cells.
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1.81Impact points
cDNA cloning of the Sry-related gene Sox6 from rat with tissue-specific expression.
Biological & pharmaceutical bulletin. 07/2002; 25(6):705-9.
A cDNA encoding rat homologue of the previously characterized mouse Sox6 was isolated by a polymerase chain reaction (PCR) cloning strategy. Comparison of this eDNA with homologous mouse, human and rainbow trout cDNA exhibited an overall amino acid sequence identity of 99.6, 89.3 and 76.3% respectiv... [more] A cDNA encoding rat homologue of the previously characterized mouse Sox6 was isolated by a polymerase chain reaction (PCR) cloning strategy. Comparison of this eDNA with homologous mouse, human and rainbow trout cDNA exhibited an overall amino acid sequence identity of 99.6, 89.3 and 76.3% respectively. The leucine-zipper and HMG-box motif were almost completely conserved between these homologues. The expression of Sox6 was determined in rat by Northern hybridization and Real-time quantitative reverse transcription (RT)-PCR. rSox6 (rat Sox6) was specifically expressed in the neonatal brain and adult testis with Northern blotting. Real-time quantitative RT-PCR for the determination of Sox6 mRNA was examined. The rSox6 was expressed in the neonatal brain and adult testis as well as by Northern blotting and also expressed in the adult eyeball and slightly in the ovary.
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1.81Impact points
Immunocytochemical localization of beta-citryl-L-glutamate in primary neuronal cells and in the differentiation of P19 mouse embryonal carcinoma cells into neuronal cells.
Biological & pharmaceutical bulletin. 12/2000; 23(11):1287-92.
The immunocytochemical localization of beta-citryl-L-glutamate (beta-CG) in primary neuronal cells and in the differentiation of P19 cells was examined. 1: Cells with the morphological features of neurons in the primary culture were specifically stained with the anti-beta-CG antibody both in neurite... [more] The immunocytochemical localization of beta-citryl-L-glutamate (beta-CG) in primary neuronal cells and in the differentiation of P19 cells was examined. 1: Cells with the morphological features of neurons in the primary culture were specifically stained with the anti-beta-CG antibody both in neurites and in the cell body. 2: The neuronal cells differentiated from P19 cells were distinctly stained with the anti-beta-CG antibody both in neurites and in the cell body, while the non-neuronal cells were not. 3: The concentration of beta-CG was low in the P19 cells, but increased significantly with the differentiation of P19 cells into neurons. It was shown that beta-CG was localized exclusively in neurons. These findings suggest that beta-CG plays functional roles in the differentiation and growth of neuron.
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1.81Impact points
Immunohistochemical and chemical changes of beta-citryl-L-glutamate in the differentiation of bovine lens epithelial cells into lens fiber cells.
Biological & pharmaceutical bulletin. 07/2000; 23(6):704-7.
Beta-citryl-L-glutamate (beta-CG) concentration was determined by HPLC during the differentiation of bovine lens epithelial cells into lens fiber cells in culture. beta-CG increased from 1 to 4 weeks of culture and then decreased slightly, while alpha-crystallin, a marker of lens cell differentiatio... [more] Beta-citryl-L-glutamate (beta-CG) concentration was determined by HPLC during the differentiation of bovine lens epithelial cells into lens fiber cells in culture. beta-CG increased from 1 to 4 weeks of culture and then decreased slightly, while alpha-crystallin, a marker of lens cell differentiation, increased rapidly 4 weeks after the culture and continued to increase gradually until week 11. In addition, the localization of beta-CG was immunohistochemically examined using anti-beta-CG antibody. Cells around lentoid bodies were stained with anti-beta-CG antibody, whereas cells in the bodies were stained strongly with anti-gamma-crystallin antibody. These findings suggest that beta-CG accumulated immediately before the differentiation of the bovine lens epithelial cells into lens fiber cells and may play a role in regulating the differentiation of lens cells.
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Expression of kininogen mRNAs and plasma kallikrein mRNA by cultured neurons, astrocytes and meningeal cells in the rat brain.
Immunopharmacology. 12/1999; 45(1-3):121-6.
Expression of kininogen mRNAs has been studied in cultures of three different types of cells in rat brain, including neurons and astrocytes from cerebral cortex and meningeal cells from the leptomeninges/choroid plexus. T-kininogen mRNA was expressed by meningeal cells, but not by neurons and astroc... [more] Expression of kininogen mRNAs has been studied in cultures of three different types of cells in rat brain, including neurons and astrocytes from cerebral cortex and meningeal cells from the leptomeninges/choroid plexus. T-kininogen mRNA was expressed by meningeal cells, but not by neurons and astrocytes, and the expression in meningeal cells was enhanced by culture with prostaglandin E2 (PGE2) or dibutyryl cAMP (Bt2cAMP). Low-molecular-weight kininogen mRNA was not detected in these cultures of cells, even after treatment with PGE2. Although expression of high-molecular-weight kininogen mRNA was very low in these cultures of cells, PGE2 or Bt2cAMP markedly stimulated its expression in cultures of meningeal cells and slightly in neurons, but not in astrocytes. We also found that expression of plasma kallikrein mRNA was strong in cultures of meningeal cells and slight in astrocytes, but absent in neurons. These results suggest that cells in the leptomeninges/choroid plexus are major sources of kininogens in rat brain which may function as precursor proteins for kinins and/or potent cysteine proteinase inhibitors during cerebral inflammation.
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2.56Impact points
Detection of bikunin mRNA in limited portions of rat brain.
Life sciences. 02/1999; 65(8):757-62.
Tissue distribution of bikunin mRNA, which encodes a Kunitz-type serine protease inhibitor of the inter-alpha-inhibitor family (IalphaI), was studied in rats and mice by the reverse-transcripsion polymerase chain reaction (RT-PCR). We found that the liver as well as other tissues, such as the kidney... [more] Tissue distribution of bikunin mRNA, which encodes a Kunitz-type serine protease inhibitor of the inter-alpha-inhibitor family (IalphaI), was studied in rats and mice by the reverse-transcripsion polymerase chain reaction (RT-PCR). We found that the liver as well as other tissues, such as the kidney, testis and adrenal gland, expressed bikunin mRNA. Although signals of bikunin mRNA were faint in the whole brain of rats and mice, distinct signals were found in limited portions of rat brain, such as the hippocampus, cerebral cortex and pituitary, but undetectable in cerebellum, medulla oblongata, hypothalamus, striatum, midbrain and choroid plexus. In three distinct types of cells, such as neurons, astrocytes and meningeal cells, in primary cultures isolated from the cerebral cortex and meninges of 1-day-old newborn rats, only neurons positively expressed bikunin mRNA. These results suggest that, in addition to peripheral tissues, neurons in the hippocampus and cerebral cortex produce bikunin, suggesting a potential role of bikunin/IalphaI family in these brain regions.
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2.54Impact points
Presence of beta-citryl-L-glutamic acid in the lens: its possible role in the differentiation of lens epithelial cells into fiber cells.
Experimental eye research. 11/1995; 61(4):403-11.
The beta-CG concentration in the chicken brain was high during embryonic development and decreased rapidly to a lower level close to hatching, while the concentration in the eyeball which was also high during the embryonic life retained a fairly high level after hatching. The distribution of beta-CG... [more] The beta-CG concentration in the chicken brain was high during embryonic development and decreased rapidly to a lower level close to hatching, while the concentration in the eyeball which was also high during the embryonic life retained a fairly high level after hatching. The distribution of beta-CG in the bovine eye was determined. About 95% of total beta-CG content in the whole eye was localized in the lens. However, the distribution of beta-CG in the eye varied depending on species. beta-CG was exclusively localized in the lens in the eyes of fish and mammals, but distributed in both lens and retina in frogs. The molecule was localized in the retina rather than the lens in the chicken eye, although the concentrations was extremely low compared to those in the mammalian, amphibian and fish eyes. It was found that beta-CG is present ubiquitously in the lens or retina in various species. The distribution of beta-CG in the bovine lens was determined in the three cortex regions and nucleus. beta-CG was present at the highest concentration in the equatorial cortex, at a moderate concentration in the posterior and anterior cortex, and at the lowest concentration in the nucleus. Similar distribution patterns were also found in the rabbit and rat lens. When embryonic chick lens epithelial cells were cultured in the presence of fetal calf serum, the cells elongated, differentiated into fiber cells and formed lentoid bodies. The cells of lentoid bodies were stained strongly by the anti-beta-CG antibody, while cells around the structures were not. In addition, the beta-CG content in the lenses from the galactose cataractous rat decreased to about 20-30% of that in the normal lens. These findings suggest that beta-CG may play a role in the differentiation of epithelial cells into fiber cells.
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4.66Impact points
Purification and properties of beta-citryl-L-glutamate-hydrolysing enzyme from rat testis particulate.
Biochimica et biophysica acta. 08/1995; 1250(1):35-42.
beta-Citryl-L-glutamate-hydrolysing enzyme (beta-CGHE) was purified from rat testis particulate fraction 13,000-fold, at a yield of 7%. The enzyme was purified by ammonium sulfate fractionation, hydroxyapatite, chelating Sepharose, beta-CG-Sepharose affinity chromatography and Sephacryl S-300 gel fi... [more] beta-Citryl-L-glutamate-hydrolysing enzyme (beta-CGHE) was purified from rat testis particulate fraction 13,000-fold, at a yield of 7%. The enzyme was purified by ammonium sulfate fractionation, hydroxyapatite, chelating Sepharose, beta-CG-Sepharose affinity chromatography and Sephacryl S-300 gel filtration. The purified enzyme usually migrated as two periodic acid Schiff's-stained bands on native polyacrylamide gel-electrophoresis (PAGE) with molecular weights of 350 and 420 kDa. Both bands hydrolyzed beta-citryl-L-glutamate (beta-CG) to citrate and glutamate. The 420 kDa band was changed by digestion with N-glycosidase F, into a 350 kDa band on native PAGE. The purified enzyme was composed of 90, 100, 115 and 130 kDa subunits on SDS-PAGE under non-reduced conditions. The purified enzyme was pharmacologically similar to the beta-CGHE activity partially purified from rat testis. This enzyme required manganese ions for full activity and it was strongly inhibited by nucleotides such as ATP or GTP and phosphate ions. beta-CGHE was also potently inhibited by an excitatory amino acid agonist, L-quisqualate, but not by another agonists, N-methyl-D-aspartate and kinate. It had high substrate specificity for beta-CG. The antibodies against the purified enzyme reacted mainly to the 115 kDa band on the SDS-PAGE and precipitated the enzyme activity from the crude and purified enzyme solution.
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Expression of kininogen mRNAs and plasma kallikrein mRNA by cultured neurons, astrocytes and meningeal cells in the rat brain
Immunopharmacology.
Expression of kininogen mRNAs has been studied in cultures of three different types of cells in rat brain, including neurons and astrocytes from cerebral cortex and meningeal cells from the leptomeninges/choroid plexus. T-kininogen mRNA was expressed by meningeal cells, but not by neurons and astroc... [more] Expression of kininogen mRNAs has been studied in cultures of three different types of cells in rat brain, including neurons and astrocytes from cerebral cortex and meningeal cells from the leptomeninges/choroid plexus. T-kininogen mRNA was expressed by meningeal cells, but not by neurons and astrocytes, and the expression in meningeal cells was enhanced by culture with prostaglandin E2 (PGE2) or dibutyryl cAMP (Bt2cAMP). Low-molecular-weight kininogen mRNA was not detected in these cultures of cells, even after treatment with PGE2. Although expression of high-molecular-weight kininogen mRNA was very low in these cultures of cells, PGE2 or Bt2cAMP markedly stimulated its expression in cultures of meningeal cells and slightly in neurons, but not in astrocytes. We also found that expression of plasma kallikrein mRNA was strong in cultures of meningeal cells and slight in astrocytes, but absent in neurons. These results suggest that cells in the leptomeninges/choroid plexus are major sources of kininogens in rat brain which may function as precursor proteins for kinins and/or potent cysteine proteinase inhibitors during cerebral inflammation.
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Sox6 overexpression causes cellular aggregation and the neuronal differentiation of P19 embryonic carcinoma cells in the absence of retinoic acid
FEBS Letters.
The Sox6 gene is a member of the Sox gene family that encodes transcription factors. Previous studies have suggested that Sox6 plays an important role in the development of the central nervous system. Aggregation of embryonic carcinoma P19 cells with retinoic acid (RA) results in the development of ... [more] The Sox6 gene is a member of the Sox gene family that encodes transcription factors. Previous studies have suggested that Sox6 plays an important role in the development of the central nervous system. Aggregation of embryonic carcinoma P19 cells with retinoic acid (RA) results in the development of neurons, glia and fibroblast-like cells. In this report, we have shown that Sox6 mRNA increased rapidly in P19 cells during RA induction and then decreased during the differentiation of P19 into neuronal cells. To explore the possible roles of Sox6 during this process, stably Sox6-overexpressing P19 cell lines (P19[Sox6]) were established. These P19[Sox6] had acquired both characteristics of the wild-type P19 induced by RA. First, P19[Sox6] cells showed a marked cellular aggregation in the absence of RA. Second, P19[Sox6] could differentiate into microtubule-associated protein 2 (MAP2)-expressing neuronal cells in the absence of RA. Sox6 expression could cause the activation of endogenous genes including the neuronal transcription factor Mash-1, the neuronal development-related gene Wnt-1, the neuron-specific cell adhesion molecule N-cadherin, and the neuron-specific protein MAP2, resulting in neurogenesis. Moreover, E-cadherin, a major cell adhesion molecule of wild-type P19, was strongly induced by Sox6, resulting in cellular aggregation without RA. Thus Sox6 may play a critical role in cellular aggregation and neuronal differentiation of P19 cells.
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