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Sample records for 3-methylcholanthrene induces differential

  1. Differential metabolism of acetanilide versus ethoxycoumarin and benzo[a]pyrene by two 3-methylcholanthrene-inducible forms of rat liver cytochrome P-450.

    PubMed

    Sundheimer, D W; Caveness, M B; Goldstein, J A

    1983-10-15

    The present study compares the catalytic activities of two 3-methylcholanthrene (3-MC) inducible forms of cytochrome P-450. These isozymes (P-448HCB and P-448MC) were isolated from liver microsomes of rats treated with 3,4,5,3',4',5'-hexachlorobiphenyl (HCB) and 3-MC, respectively. Catalytic activities of the isozymes were compared in a reconstituted system and by antibody inhibition studies in microsomes. In a reconstituted system, P-448HCB had very little catalytic activity toward benzo[a]pyrene or ethoxycoumarin (substrates metabolized preferentially by P-448MC). In contrast, both isozymes had high turnover numbers for aniline and acetanilide. However, catalytic activities of the purified isozymes were affected dramatically by Emulgen 911, a nonionic detergent. Since nonionic detergents used in the purification of P-450 isozymes cannot be completely removed after purification, residual amounts of detergent probably affect turnover numbers in a reconstituted system. Therefore, specific antibodies to cytochromes P-448MC and P-448HCB were used to examine the contribution of these isozymes to microsomal metabolism. Antibody inhibition studies confirmed that the majority of benzo[a]pyrene and ethoxycoumarin metabolism in 3-MC-induced microsomes was catalyzed by cytochrome P-448MC. In contrast, P-448HCB accounted for the majority of the acetanilide hydroxylase activity in 3-MC- and HCB-induced microsomes. Neither isozyme contributed appreciably to metabolism of these substrates in control microsomes. PMID:6314905

  2. Regulation of nonmuscle myosin II during 3-methylcholanthrene induced dedifferentiation of C2C12 myotubes

    SciTech Connect

    Dey, Sumit K.; Saha, Shekhar; Das, Provas; Das, Mahua R.; Jana, Siddhartha S.

    2014-08-01

    3-Methylcholanthrene (3MC) induces tumor formation at the site of injection in the hind leg of mice within 110 days. Recent reports reveal that the transformation of normal muscle cells to atypical cells is one of the causes for tumor formation, however the molecular mechanism behind this process is not well understood. Here, we show in an in vitro study that 3MC induces fragmentation of multinucleate myotubes into viable mononucleates. These mononucleates form colonies when they are seeded into soft agar, indicative of cellular transformation. Immunoblot analysis reveals that phosphorylation of myosin regulatory light chain (RLC{sub 20}) is 5.6±0.5 fold reduced in 3MC treated myotubes in comparison to vehicle treated myotubes during the fragmentation of myotubes. In contrast, levels of myogenic factors such as MyoD, Myogenin and cell cycle regulators such as Cyclin D, Cyclin E1 remain unchanged as assessed by real-time PCR array and reverse transcriptase PCR analysis, respectively. Interestingly, addition of the myosin light chain kinase inhibitor, ML-7, enhances the fragmentation, whereas phosphatase inhibitor perturbs the 3MC induced fragmentation of myotubes. These results suggest that decrease in RLC{sub 20} phosphorylation may be associated with the fragmentation step of dedifferentiation. - Highlights: • 3-Methylcholanthrene induces fragmentation of C2C12-myotubes. • Dedifferentiation can be divided into two steps – fragmentation and proliferation. • Fragmentation is associated with rearrangement of nonmuscle myosin II. • Genes associated with differentiation and proliferation are not altered during fragmentation. • Phosphorylation of myosin regulatory light chain is reduced during fragmentation.

  3. Effect of 3-methylcholanthrene-induced increases in ascorbic acid levels on tissue. beta. -glucuronidase activity in rats

    SciTech Connect

    Calabrese, E.J.; Barrett, T.J.; Leonard, D.A.; Horton, H.M.; Kenyon, E.M.

    1988-01-01

    The interrelationship between tissue ascorbic acid levels and tissue ..beta..-glucuronidase activity was examined in rats injected with 3-methylcholanthrene, an agent which induces ascorbic acid synthesis in rats. Six Fisher 344 rats were dosed intraperitoneally (IP) with 30 mg/kg of 3-methylcholanthrene. Ascorbic acid levels and ..beta..-glucuronidase (..beta..-G) activity were determined for lung, liver and kidney tissues. In a follow-up study, rats were dosed for three consecutive days with 3-methylcholanthrene. Controls in both groups were dosed IP with Emulphor (EL-620). Animals were sacrificed one week after the final dosage and lung, liver and kidney tissues were examined.

  4. STRAIN-DEPENDENT SUSCEPTIBILITY TO TRANSPLACENTALLY-INDUCED MURINE LUNG TUMORS AND DNA ADDUCTS OF 3-METHYLCHOLANTHRENE

    EPA Science Inventory

    Strain-dependent susceptibility to transplacentally-induced murine lung tumors and DNA adducts of 3methylcholanthrene G B Nelson, J A Ross, J E Moore, M Xu, N D Kock, M S Miller Wake Forest University, Winston-Salem, NC and USEPA, Research Triangle Park, NC.

    It has been de...

  5. Inducible Monooxygenase Activities and 3-Methylcholanthrene-Initiated Tumorigenesis in Mouse Recombinant Inbred Sublines

    PubMed Central

    Atlas, Steven A.; Taylor, Benjamin A.; Diwan, Bhalchandra A.; Nebert, Daniel W.

    1976-01-01

    The induction of a certain group of hepatic monooxygenase activities by polycyclic aromatic compounds is regulated by the same locus or gene cluster controlling the formation of cytochrome P1–450 (P–448) in mice. Certain inbred strains of mice are "responsive" (Ahb) to such induction, whereas others are "nonresponsive" (Ahd). A pair of closely related sublines that differ with respect to the Ah locus (for aromatic hydrocarbon responsiveness) were used to identify or confirm the pleiotropic effects of this gene. The lines were derived by sibling-mating without selection from (C57L/J x AKR/J)F 2 mice; the two sublines were separated at the F12 generation. Ten microsomal monooxygenase activities and one cytosol enzyme activity known to be associated with the Ah locus were similarly associated with cytochrome P1–450 formation in these recombinant inbred sublines as well. Nine additional hepatic monooxygenase activities studied were found not to be associated with the Ah locus; certain of these activities were increased slightly, following treatment of nonresponsive as well as responsive mice with polycyclic aromatic compounds. The Ahb-containing subline was highly susceptible to 3-methylcholanthrene-induced subcutaneous sarcomas, whereas the Ah-d-containing subline was relatively resistant. These results emphasize the potential importance of this particular enzyme for the study of coordinated regulation in mammals. PMID:955403

  6. Modulating role of pheromonal cues from oestrus-specific urine on 3-methylcholanthrene-induced male reproductive toxicity.

    PubMed

    Suriyakalaa, U; Gayathiri, R; SankarGanesh, D; Kokilavani, P; Sukirtha, R; Ramachandran, R; Kamalakkannan, S; Achiraman, S

    2016-09-01

    Polyaromatic hydrocarbons (PAHs) are persistent organic pollutants that contribute to endocrine/gonadal disruption. This study was designed to investigate the endocrine modulating role of pheromones in alleviating the reproductive toxic effects of 3-MC (3-methylcholanthrene), one of the common PAHs, in rat model. The rats were injected intraperitoneally with 3-MC at a dose of 25 mg kg(-1) BW. The serum levels of testosterone and other biochemical parameters were altered to significant levels in 3-MC-treated rats and oestrus-specific urine exposure restored all these effects to near normal. Although testis weight did not indicate any significant change, sperm and spermatid counts were significantly reduced in 3-MC-treated rats, which became normal in oestrus-urine-exposed rats. Hence, this study suggests that oestrus-specific urinary pheromones have the potential to modulate the endocrine system and alleviate the male reproductive toxic effects produced by 3-MC. PMID:26715617

  7. Induction of 26S proteasome subunit PSMB5 by the bifunctional inducer 3-methylcholanthrene through the Nrf2-ARE, but not the AhR/Arnt-XRE, pathway

    SciTech Connect

    Kwak, Mi-Kyoung . E-mail: mkwak@yumail.ac.kr; Kensler, Thomas W.

    2006-07-14

    The 26S proteasome is responsible for degradation of abnormal intracellular proteins, including oxidatively damaged proteins and may play a role as a component of a cellular antioxidative system. However, little is known about regulation of proteasome expression. In the present study, regulation of proteasome expression by the bifunctional enzyme inducer and a specific signaling pathway for this regulation were investigated in murine neuroblastoma cells. Expression of catalytic core subunits including PSMB5 and peptidase activities of the proteasome were elevated following incubation with 3-methylcholanthrene (3-MC). Studies using reporter genes containing the murine Psmb5 promoter showed that transcriptional activity of this gene was enhanced by 3-MC. Overexpression of AhR/Arnt did not affect activation of the Pmsb5 promoter by 3-MC and deletion of the xenobiotic response elements (XREs) from this promoter exerted modest effects on inducibility in response to 3-MC. However, mutation of the proximal AREs of the Psmb5 promoter largely abrogated its inducibility by 3-MC. In addition, this promoter showed a blunted response toward 3-MC in the absence of nrf2; 3-MC incubation increased nuclear levels of Nrf2 only in wild-type cells. Collectively, these results indicate that expression of proteasome subunit PSMB5 is modulated by bifunctional enzyme inducers in a manner independent of the AhR/Arnt-XRE pathway but dependent upon the Nrf2-ARE pathway.

  8. Activation of liver X receptor inhibits the development of pulmonary carcinomas induced by 3-methylcholanthrene and butylated hydroxytoluene in BALB/c mice.

    PubMed

    Wang, Qixue; Sun, Lei; Yang, Xiaoxiao; Ma, Xingzhe; Li, Qi; Chen, Yuanli; Liu, Ying; Zhang, Di; Li, Xiaoju; Xiang, Rong; Wei, Yuquan; Han, Jihong; Duan, Yajun

    2016-01-01

    We previously reported that LXR ligand, T0901317, inhibited the growth of inoculated Lewis lung carcinoma in C57BL/6 mice by activating IFN-γ production. However, the effects of T0901317 on carcinogen-induced pulmonary carcinomas remain unknown. In this study, we initially conducted a statistical analysis on the data of human lung cancer samples extracted from the TCGA database, and determined that survival rate/time of lung cancer patients and grade of lung adenocarcinoma were positively and negatively related to lung IFN-γ levels, respectively. We then determined the inhibitory effects of T0901317 on mouse pulmonary carcinomas induced by 3-methylcholanthrene (MCA) and butylated hydroxytoluene (BHT) or urethane. We found that T0901317 reduced morbidity and mortality in MCA/BHT-injected BALB/c mice by inhibiting lung adenocarcinoma. T0901317 also protected C57BL/6 mice, but not IFN-γ deficient (IFN-γ(-/-), C57BL/6 background) mice, against MCA/BHT-induced lung hyperplasia/inflammation. In addition, we determined that T0901317 inhibited urethane-induced lung tumors in BABL/c mice. Furthermore, we determined that T0901317 prevented metastasis of 4T1 breast cancer cells in BALB/c mice. Administration of T0901317 substantially increased serum IFN-γ levels and lung IFN-γ expression in BABL/c and C57BL/6 mice. Taken together, our study demonstrates that LXR inhibits MCA/BHT-induced pulmonary carcinomas in BABL/c mice and the inhibition is associated with induction of IFN-γ production. PMID:27250582

  9. Activation of liver X receptor inhibits the development of pulmonary carcinomas induced by 3-methylcholanthrene and butylated hydroxytoluene in BALB/c mice

    PubMed Central

    Wang, Qixue; Sun, Lei; Yang, Xiaoxiao; Ma, Xingzhe; Li, Qi; Chen, Yuanli; Liu, Ying; Zhang, Di; Li, Xiaoju; Xiang, Rong; Wei, Yuquan; Han, Jihong; Duan, Yajun

    2016-01-01

    We previously reported that LXR ligand, T0901317, inhibited the growth of inoculated Lewis lung carcinoma in C57BL/6 mice by activating IFN-γ production. However, the effects of T0901317 on carcinogen-induced pulmonary carcinomas remain unknown. In this study, we initially conducted a statistical analysis on the data of human lung cancer samples extracted from the TCGA database, and determined that survival rate/time of lung cancer patients and grade of lung adenocarcinoma were positively and negatively related to lung IFN-γ levels, respectively. We then determined the inhibitory effects of T0901317 on mouse pulmonary carcinomas induced by 3-methylcholanthrene (MCA) and butylated hydroxytoluene (BHT) or urethane. We found that T0901317 reduced morbidity and mortality in MCA/BHT-injected BALB/c mice by inhibiting lung adenocarcinoma. T0901317 also protected C57BL/6 mice, but not IFN-γ deficient (IFN-γ−/−, C57BL/6 background) mice, against MCA/BHT-induced lung hyperplasia/inflammation. In addition, we determined that T0901317 inhibited urethane-induced lung tumors in BABL/c mice. Furthermore, we determined that T0901317 prevented metastasis of 4T1 breast cancer cells in BALB/c mice. Administration of T0901317 substantially increased serum IFN-γ levels and lung IFN-γ expression in BABL/c and C57BL/6 mice. Taken together, our study demonstrates that LXR inhibits MCA/BHT-induced pulmonary carcinomas in BABL/c mice and the inhibition is associated with induction of IFN-γ production. PMID:27250582

  10. Benzo[a]pyrene, 3-methylcholanthrene and beta-naphthoflavone induce oxidative stress in hepatoma hepa 1c1c7 Cells by an AHR-dependent pathway.

    PubMed

    Elbekai, Reem H; Korashy, Hesham M; Wills, Kelly; Gharavi, Negar; El-Kadi, Ayman O S

    2004-11-01

    Polycyclic aromatic hydrocarbons have been shown to cause oxidative stress in vitro and in vivo in various animal models but the mechanisms by which these compounds produce oxidative stress are unknown. In the current study we have investigated the role of the aryl hydrocarbon receptor (AHR) in the production of reactive oxygen species (ROS) by its cognate ligands and the consequent effect on cyp1a1 activity, mRNA and protein expressions. For this purpose, Hepa 1c1c7 cells wild-type (WT) and C12 mutant cells, which are AHR-deficient, were incubated with increasing concentrations of the AHR-ligands, benzo[a]pyrene (B[a]P, 0.25-25 microM), 3-methylcholanthrene (3MC, 0.1-10 microM) and beta-naphthoflavone (betaNF, 1-50 microM). The studied AHR-ligands dose-dependently increased lipid peroxidation in WT but not in C12 cells. However, only B[a]P and betaNF, at the highest concentrations tested, significantly increased H2O2 production in WT but not C12 cells. The increase in lipid peroxidation and H2O2 production by AHR-ligands were accompanied by a decrease in the cyp1a1 catalytic activity but not mRNA or protein expressions, which were significantly induced in a dose-dependent manner by all AHR-ligands, suggesting a post-translational mechanism is involved in the decrease of cyp1a1 activity. The AHR-ligand-mediated decrease in cyp1a1 activity was reversed by the antioxidant N-acetylcysteine. Our results show that the AHR-ligands induce oxidative stress by an AHR-dependent pathway. PMID:15621696

  11. Basal and 3-methylcholanthrene-induced expression of cytochrome P450 1A, 1B and 1C genes in the Brazilian guppy, Poecilia vivipara

    PubMed Central

    Dorrington, Tarquin; Zanette, Juliano; Zacchi, Flávia L.; Stegeman, John J.; Bainy, Afonso C.D.

    2015-01-01

    In fish there are four cytochrome P450 (CYP1) subfamilies: CYP1A, CYP1B, CYP1C, and CYP1D. Here we cloned Poecilia vivipara CYP1A, with an inferred amino acid sequence 91% identical to CYP1A from the killifish Fundulus heteroclitus, another member of the Cypriniformes, and an important model in ecotoxicology. In addition, we examined the expression of CYP1A, CYP1B1, and CYP1C1 by qPCR in liver, gill, and intestine of adult P. vivipara injected with 3-methylcholanthrene (3-MC) or held in clean water (control group) for 24 h. All three tissues examined showed basal expression of the three CYP1 genes. CYP1A was most strongly expressed in the liver, while CYP1B1, and CYP1C1 were most strongly expressed in the gill and intestine respectively. 3-MC induced CYP1A, CYP1B1, and CYP1C1 significantly (20–120-fold) in the three organs, consistent with the regulation of CYP1A, CYP1B1 and CYP1C1 via the aryl hydrocarbon receptor. Validation of CYP1 gene biomarkers in fish collected from a contaminated urban mangrove environment was confirmed with significant induction of CYP1A and CYP1C1 in gills (10–15-fold) and CYP1B1 in liver (23-fold), relative to fish from a control site. The responsiveness of these CYP1 genes indicates P. vivipara is suitable as a model for environmental toxicology studies and environmental assessment in Brazil. PMID:22940225

  12. BINDING OF POLYCHLORINATED BIPHENYLS CLASSIFIED AS EITHER PHENOBARBITONE-, 3-METHYLCHOLANTHRENE- OR MIXED-TYPE INDUCERS TO CYTOSOLIC AH RECEPTOR

    EPA Science Inventory

    It has been postulated that reversible, high-affinity binding of 3-methyl-cholanthrene (MC)-type inducers to a receptor protein (the Ah receptor) in hepatic cytosol is essential for induction of aryl hydrocarbon hydroxylase (AHH) enzymic activity. To test this postulate, the bind...

  13. Developmental regulation of the 3-methylcholanthrene- and dioxin-inducible CYP1A5 gene in chick embryo liver in vivo.

    PubMed

    Bentivegna, C S; Ihnat, M A; Baptiste, N S; Hamilton, J W

    1998-07-01

    The cDNA sequences for two dioxin-inducible cytochrome P450s in chicken, CYP1A4 and CYP1A5, have recently been reported which correspond to two dioxin-inducible forms of P450 previously designated as TCDDAHH and TCDDAA, respectively. The developmental expression of CYP1A4-associated aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH) activity and its association with expression of the Ah receptor had previously been characterized in chick embryo liver. The purpose of this study was to examine the developmental regulation of the second dioxin-inducible P450 gene, CYP1A5, in chick embryo liver. A partial gene sequence for CYP1A5 indicated that the intron/exon organization of this gene was identical to that of the CYP1A1 and CYP1A2 mammalian genes and was present in a single copy in the genome. CYP1A5 mRNA was expressed basally in chick embryo liver and was highly inducible by the Ah receptor ligands, 3-methylcholanthrene, beta-naphthoflavone, and 3,4,3', 4'-tetrachlorobiphenyl (TCB), but not by the phenobarbital analog, glutethimide. CYP1A5 mRNA levels were increased 40- to 50-fold within 5 h after a single TCB treatment, corresponding to a 30- to 40-fold increase in the transcription rate of the CYP1A5 gene at this time point. In contrast to a previous report that CYP1A5 mRNA expression was inducible by estradiol, we observed no effects of estradiol or dexamethasone on CYP1A5 mRNA expression, either alone or in combination with TCB. Basal and TCB-inducible CYP1A5 mRNA expression was maximal in liver at 8 days of development and remained high throughout the remainder of embryonic development. Thus, CYP1A5 appears to be regulated in a very similar manner to CYP1A4 in chick embryo liver. PMID:9705900

  14. Hyper- and Hypo- Induction of Cytochrome P450 activities with Aroclor 1254 and 3-Methylcholanthrene in Cyp1a2(−/−) mice

    PubMed Central

    Barker, Melissa L.; Hathaway, Laura B.; Arch, Dorinda D.; Westbroek, Mark L.; Kushner, James P.; Phillips, John D.; Franklin, Michael R.

    2009-01-01

    The response of hepatic mono-oxygenase activities to Aroclor 1254 or 3-methylcholanthrene was investigated in wild-type and Cyp1a2(−/−) mice. Cytochrome P450 concentrations were similar in naïve Cyp1a2(−/−) and wild-type mice. There was no difference between naïve wild-type and Cyp1a2(−/−) animals in 7-ethoxyresorufin and 7-ethoxy-4-trifluoromethylcoumarin dealkylase activities, nor was the induction response after 3-methylcholanthrene any different between the two genotypes. However, both activities were induced to a higher extent in Cyp1a2(−/−) mice after Aroclor 1254. In contrast, 7-pentoxyresorufin dealkylation activity was lower in Cyp1a2(−/−) mice and this differential was maintained during induction by both agents. 7-Methoxy- and 7-benzoxyresorufin dealkylation activities were also lower than wild-type in naïve Cyp1a2(−/−) animals and during 3-methylcholanthrene induction, but showed accelerated induction in Cyp1a2(−/−) mice with Aroclor 1254. Bufuralol 1′- and testosterone 6β-hydroxylation activities, and P450 characteristics were evaluated 48 hours after inducer administration. Bufuralol 1′-hydroxylation, a sexual dimorphic activity (female > male) showed no genotype differences in naïve animals. Activity changes varied across gender and genotype, with 3-methylcholanthrene and Aroclor 1254 inducing in male Cyp1a2(−/−), and Aroclor 1254 inducing in female wild-type. Testosterone 6β-hydroxylation activity was 16% higher in Cyp1a2(−/−) mice and neither 3-methylcholanthrene nor Aroclor 1254 elicited induction. After Aroclor 1254, a 24% increase in P450 concentration with a hypsochromic shift in the ferrous-CO maximum characteristic of CYP1A enzymes occurred in wild-type, compared to no change in either parameter in Cyp1a2(−/−) mice. Induction changes with 3-methylcholanthrene were greater in wild-type mice, a 60% increase in concentration and ~2 nm hypsochromic shift versus a 10% increase and ~1 nm hypsochromic

  15. Protective effect of Ocimum sanctum on 3-methylcholanthrene, 7,12-dimethylbenz(a)anthracene and aflatoxin B1 induced skin tumorigenesis in mice

    SciTech Connect

    Rastogi, Shipra; Shukla, Yogeshwer; Paul, Bhola N.; Chowdhuri, D. Kar; Khanna, Subhash K.; Das, Mukul

    2007-11-01

    A study on the protective effect of alcoholic extract of the leaves of Ocimum sanctum on 3-mthylcholanthrene (MCA), 7,12-dimethylbenzanthracene (DMBA) and aflatoxin B{sub 1} (AFB{sub 1}) induced skin tumorigenesis in a mouse model has been investigated. The study involved pretreatment of mice with the leaf extract prior to either MCA application or tetradecanoyl phorbol acetate (TPA) treatment in a two-stage tumor protocol viz a viz, DMBA/TPA and AFB1/TPA. The results of the present study indicate that the pretreatment with alcoholic extract of the leaves of O. sanctum decreased the number of tumors in MCA, DMBA/TPA and AFB1/TPA treated mice. The skin tumor induced animals pretreated with alcoholic extract led to a decrease in the expression of cutaneous {gamma}-glutamyl transpeptidase (GGT) and glutathione-S-transferase-P (GST-P) protein. The histopathological examination of skin tumors treated with leaf extract showed increased infiltration of polymorphonuclear, mononuclear and lymphocytic cells, decreased ornithine decarboxylase activity with concomitant enhancement of interleukin-1{beta} (IL-1{beta}) and tumor necrosis factor-{alpha} (TNF-{alpha}) in the serum, implying the in vivo antiproliferative and immunomodulatory activity of leaf extract. The decrease in cutaneous phase I enzymes and elevation of phase II enzymes in response to topical application of leaf extract prior to MCA, AFB1, DMBA/TPA and AFB1/TPA treatment indicate the possibility of impairment in reactive metabolite(s) formation and thereby reducing skin carcinogenicity. Furthermore, pretreatment of leaf extract in the carcinogen induced animals resulted in elevation of glutathione levels and decrease in lipid peroxidation along with heat shock protein expression, indicating a scavenging or antioxidant potential of the extract during chemical carcinogenesis. Thus it can be concluded that leaf extract of O. sanctum provides protection against chemical carcinogenesis in one or more of the

  16. Induction of hepatic cytochrome P-450 activity in wild cotton rats (Sigmodon hispidus) by phenobarbital and 3-methylcholanthrene

    SciTech Connect

    Elangbam, C.S.; Qualls, C.W.,Jr.; Bauduy, M. )

    1989-05-01

    Wild cotton rats (Sigmodon hispidus) are ubiquitous throughout the Southeast quadrant of the United States, easy to capture, have a generation interval of less than one year and a limited range of movement (less than one hectare). This species may prove to be an excellent model for monitoring environmental contamination. Traditionally, cytochrome P-450 inducing agents are grouped into two classes. One, represented by phenobarbital, induces P-450b and P-450e; the other, represented by 3-methylcholanthrene, induces P-450c and P-450d isoenzymes. The types and amounts of cytochrome P-450 vary among species, organs, health status, sex, and stress of the animal. If the levels of cytochrome P-450 of wild cotton rats are to be used in monitoring environmental pollution, it is necessary to characterize the inducibility and concentration of cytochrome P-450 in this species. This study was designed to determine the concentration and inducibility of cytochrome P-450 in the livers of cotton rats after intraperitoneal (ip) administration of phenobarbital and 3-methylcholanthrene.

  17. STRAIN-SPECIFIC SENSITIVITY TO INDUCTION OF MURINE LUNG TUMORS FOLLOWING IN UTERO EXPOSURE TO 3-METHYLCHOLANTHRENE

    EPA Science Inventory

    We previously demonstrated that different strains of fetal mice were more sensitive to lung tumor induction by 3-methylcholanthrene (MC) than were adults. Offspring from either a D2 x B6D2F1 backcross or from parental Balb/c mice exhibited a similar high incidence of lung tumors ...

  18. Consequences of 3-methylcholanthrene-type induction for the metabolism of 4-aminobiphenyl in isolated rat hepatocytes.

    PubMed

    Orzechowski, A; Schrenk, D; Schut, H A; Bock, K W

    1994-03-01

    Carcinogenic aromatic amines such as 4-aminobiphenyl (4-ABP) are extensively metabolized by both oxidative and conjugation reactions. Thus the burden of genotoxic metabolites of 4-ABP in a target organ is probably influenced by the balance of N-hydroxylation and alternative metabolic pathways in the hepatocyte. In freshly isolated rat hepatocytes, 4-ABP (at a substrate concentration of 10 microM) was mainly N-acetylated (54% of total metabolites), while 2% N-hydroxy-4-ABP-N-glucuronide and 21% of unconjugated N-hydroxylated metabolites were detectable. Ring-hydroxylated metabolites and the primary N-glucuronide of 4-ABP accounted for 8% and 4%, respectively. Pretreatment of rats with 3-methylcholanthrene (MC), a dioxin-type inducer of CYP1A isozymes and phenol UDP-glucuronosyltransferase (UGT1A1), led to a dramatic decrease of N-acetylated (2% of total metabolites) and an increase of N-hydroxylated (54% as free and glucuronidated compound) and ring-hydroxylated (35%) metabolites. Essentially similar effects were seen at a substrate concentration of 50 microM. Consistently, MC-type induction with beta-naphthoflavone resulted in a significant increase in the formation of DNA adducts of 4-ABP, detected by 32P-postlabeling of hepatocellular DNA. The results suggest that, similar to a previous study with 2-naphthylamine (2-NA), MC treatment leads to a marked shift from conjugation to N-oxidation. However, N-hydroxy-4-ABP (in contrast to N-hydroxy-2-NA) is mostly released from hepatocytes in the unconjugated form. PMID:8118934

  19. Hepatic mixed function oxidase system and effect of phenobarbital, 3-methylcholanthrene and 1, 1, 1-trichloro-2, 2-bis(p-chlorophenyl)ethane in developing chickens.

    PubMed

    Hatolkar, V S; Pawar, S S

    1992-05-01

    Contents of hepatic microsomal protein, aminopyrine N-demethylase, acetanilide hydroxylase, aniline hydroxylase, hydrogen peroxide formation, cytochrome-c-reductase, cytochrome b5 and cytochrome P-450 were examined in control, phenobarbital (PB), 3-methylcholanthrene (3-MC) and 1, 1, 1-trichloro-2, 2-bis(p-chlorophenyl)ethane (DDT) treated group of 1-28 days old chickens. Increase in aminopyrine N-demethylase, acetanilide hydroxylase, aniline hydroxylase, cytochrome-c-reductase, cytochrome b5 and cytochrome P-450 was noticed at all stages of development during administration of PB and 3-MC. But these enzyme activities were not always paralleled by increase in age. Aminopyrine N-demethylase was increased in early stages only during DDT administration, which indicates that the form of cytochrome P-450, responsible for aminopyrine N-demethylation is present in early stages only. However, acetanilide hydroxylase was decreased in all stages of development, in postnatal development the basal activities of the enzymes for various substrates do not exhibit identical pattern, the degree of inducibility by inducers varied in relation to age of animal. Hydrogen peroxide formation increased in all stages of developing chickens due to the administration of PB and DDT. It however decreased due to 3-MC administration which may be due to induction of high spin cytochrome P-450. PMID:1459619

  20. Modulation of 3-methylcholanthrene toxicity in cultured neoplastic keratinocytes by glucocorticoids and retinoids is not accounted for by macromolecular adduct formation

    SciTech Connect

    Rubin, A.L.; Rice, R.H. )

    1989-04-01

    3-Methylcholanthrene (3-MC) greatly inhibits the growth of two lines of human squamous carcinoma cells, SCC-9 and SCC-12B{sub 2}. The degree of 3-MC-mediated inhibition, however, was markedly alleviated by inclusion of retinoic acid and hydrocortisone or dexamethasone in the culture medium. These physiological effectors, which are known to have opposing actions on keratinocyte character in SCC cells, did not significantly alter either aryl hydrocarbon hydroxylase activity or macromolecular adduct formation. Further analysis of the cellular responses indicated that hydrocortisone and, in some experiments, retinoids increased the growth rate in 3-MC-exposed cultures, while 3-MC increased the saturation density in retinoic acid-exposed cultures, an example of interference with a physiological response of the cells. These results indicate that alteration of the differentiated state, regardless of the direction of the change, can alter the sensitivity of these cells to toxic stimuli. Further investigation of the bases of such toxic responses and their modulation by the microenvironment may enhance our understanding of the target cell specificity of polycyclic aromatic hydrocarbons.

  1. The effect of pyrazole, phenobarbital, ethanol and 3-methylcholanthrene pretreatment on the in vivo and in vitro genotoxicity of N-nitrosopyrrolidine.

    PubMed

    Gold, B; Brunk, G

    1988-06-01

    The in vitro genotoxicity of N-nitrosopyrrolidine (NPy) has been studied in Salmonella typhimurium strain TA1535 in the presence of untreated and pyrazole-, phenobarbital (PB)-, 4-day ethanol (EtOH)-, 10-day EtOH- and 3-methylcholanthrene (3-MC)-pretreated male Sprague-Dawley rat liver S-9 fractions. Unless stated otherwise, the last pretreatment exposure was 24 h prior to sacrifice and isolation of hepatic enzymes. Pyrazole and EtOH (10-day exposure) both effectively induced the conversion of NPy into a mutagen at doses as low as 500 microM. PB and EtOH (4-day exposure) had a modest enhancing effect on the number of revertants scored, while 3-MC and uninduced S-9 fractions gave results not significantly different from background (no NPy). The same pretreatment protocols were used to determine the in vivo genotoxicity of NPy in rat liver using the technique of alkaline elution. The inducing agents had the exact opposite effect in vivo with control, 3-MC- and 4-day EtOH-treated animals showing the highest level of DNA damage. Pyrazole and 10-day EtOH pretreatments gave DNA elution rate constants comparable to animals not treated with NPy. However, in 10-day EtOH-pretreated animals which were administered NPy without a 24-h interval between EtOH and NPy exposure, DNA damage was observed at the same high levels as was seen in uninduced and 3-MC treated rats. The results are discussed in terms of a detoxification role for microsomal proteins and that the observed in vivo DNA damage may be induced by enzymes associated with the nuclear compartment. PMID:3286025

  2. Flavin-containing monooxygenase-3: induction by 3-methylcholanthrene and complex regulation by xenobiotic chemicals in hepatoma cells and mouse liver1

    PubMed Central

    Celius, Trine; Pansoy, Andrea; Matthews, Jason; Okey, Allan B.; Henderson, Marilyn C.; Krueger, Sharon K.; Williams, David E.

    2010-01-01

    Flavin-containing monooxygenases often are thought not to be inducible but we recently demonstrated aryl hydrocarbon receptor (AHR)-dependent induction of FMO mRNAs in mouse liver by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Celius et al., Drug Metab Dispos 36:2499, 2008). We now evaluated FMO induction by other AHR ligands and xenobiotic chemicals in vivo and in mouse Hepa1c1c7 hepatoma cells (Hepa-1). In mouse liver, 3-methylcholanthrene (3MC) induced FMO3 mRNA 8-fold. In Hepa-1 cells, 3MC and benzo[a]pyrene (BaP) induced FMO3 mRNA >30-fold. Induction by 3MC and BaP was AHR-dependent but, surprisingly, the potent AHR agonist, TCDD, did not induce FMO3 mRNA in Hepa-1 cells nor did chromatin immunoprecipitation assays detect recruitment of AHR or ARNT to Fmo3 regulatory elements after exposure to 3MC in liver or in Hepa-1 cells. However, in Hepa-1, 3MC and BaP (but not TCDD) caused recruitment of p53 protein to a p53 response element in the 5'-flanking region of the Fmo3 gene. We tested the possibility that FMO3 induction in Hepa-1 cells might be mediated by Nrf2/antioxidant response pathways but agents known to activate Nrf2 or to induce oxidative stress did not affect FMO3 mRNA levels. The protein synthesis inhibitor, cycloheximide (which causes “superinduction” of CYP1A1 mRNA in TCDD-treated cells) by itself caused dramatic upregulation (>300-fold) of FMO3 mRNA in Hepa-1 suggesting that cycloheximide prevents synthesis of a labile protein that suppresses FMO3 expression. Although FMO3 mRNA is highly induced by 3MC or TCDD in mouse liver and in Hepa-1 cells, FMO protein levels and FMO catalytic function showed only modest elevation. PMID:20570689

  3. Flavin-containing monooxygenase-3: Induction by 3-methylcholanthrene and complex regulation by xenobiotic chemicals in hepatoma cells and mouse liver

    SciTech Connect

    Celius, Trine; Pansoy, Andrea; Matthews, Jason; Okey, Allan B.; Henderson, Marilyn C.; Krueger, Sharon K.; Williams, David E.

    2010-08-15

    Flavin-containing monooxygenases often are thought not to be inducible but we recently demonstrated aryl hydrocarbon receptor (AHR)-dependent induction of FMO mRNAs in mouse liver by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Celius et al., Drug Metab Dispos 36:2499, 2008). We now evaluated FMO induction by other AHR ligands and xenobiotic chemicals in vivo and in mouse Hepa1c1c7 hepatoma cells (Hepa-1). In mouse liver, 3-methylcholanthrene (3MC) induced FMO3 mRNA 8-fold. In Hepa-1 cells, 3MC and benzo[a]pyrene (BaP) induced FMO3 mRNA > 30-fold. Induction by 3MC and BaP was AHR dependent but, surprisingly, the potent AHR agonist, TCDD, did not induce FMO3 mRNA in Hepa-1 cells nor did chromatin immunoprecipitation assays detect recruitment of AHR or ARNT to Fmo3 regulatory elements after exposure to 3MC in liver or in Hepa-1 cells. However, in Hepa-1, 3MC and BaP (but not TCDD) caused recruitment of p53 protein to a p53 response element in the 5'-flanking region of the Fmo3 gene. We tested the possibility that FMO3 induction in Hepa-1 cells might be mediated by Nrf2/anti-oxidant response pathways, but agents known to activate Nrf2 or to induce oxidative stress did not affect FMO3 mRNA levels. The protein synthesis inhibitor, cycloheximide (which causes 'superinduction' of CYP1A1 mRNA in TCDD-treated cells), by itself caused dramatic upregulation (> 300-fold) of FMO3 mRNA in Hepa-1 suggesting that cycloheximide prevents synthesis of a labile protein that suppresses FMO3 expression. Although FMO3 mRNA is highly induced by 3MC or TCDD in mouse liver and in Hepa-1 cells, FMO protein levels and FMO catalytic function showed only modest elevation.

  4. Downregulation of Mouse Hepatic CYP3A Protein by 3-Methylcholanthrene Does Not Require Cytochrome P450-Dependent Metabolism

    PubMed Central

    Lee, Chunja; Ding, Xinxin

    2013-01-01

    The aryl hydrocarbon receptor (AHR)–dependent induction of cytochromes P450 (P450) such as CYP1A1 by 3-methylcholanthrene (MC) and related polycyclic aromatic hydrocarbons is well characterized. We reported previously that MC treatment triggers a pronounced downregulation, particularly at the protein level, of mouse hepatic Cyp3a11, a counterpart of the key human drug-metabolizing enzyme CYP3A4. To determine whether this effect of MC requires hepatic microsomal P450 activity, we studied liver Cpr-null (LCN) mice with hepatocyte-specific conditional deletion of the NADPH-cytochrome P450 oxidoreductase gene. In vehicle-treated animals, basal levels of CYP3A11 mRNA and CYP3A protein immunoreactivity were elevated by approximately 9-fold in LCN mice compared with wild-type (WT) mice, whereas CYP3A catalytic activity was profoundly compromised in LCN mice. MC treatment caused suppression of CYP3A11 mRNA, CYP3A protein immunoreactivity, and CYP3A catalytic activity in WT mice, and the MC effects at the mRNA and protein levels were maintained in LCN mice. Flavin-containing monooxygenase-3 (Fmo3) induction by MC was suggested previously to occur via an AHR-dependent mechanism requiring conversion of the parent compound to DNA-damaging reactive metabolites; however, hepatic FMO3 mRNA levels were dramatically increased by MC in both WT and LCN mice. MC did not function as a mechanism-based inactivator of CYP3A enzymes in hepatic microsomes prepared from untreated WT mice, under conditions in which 1-aminobenzotriazole caused marked NADPH-dependent loss of total P450 content and CYP3A catalytic activity. These results indicate that MC downregulates mouse hepatic CYP3A protein via a pretranslational mechanism that does not require hepatic microsomal P450-dependent activity. PMID:23846873

  5. Regulation of the CYP1A1 gene by 2,3,7,8-tetrachlorodibenzo-p-dioxin but not by beta-naphthoflavone or 3-methylcholanthrene is altered in hepatitis C virus replicon-expressing cells.

    PubMed

    Anderson, Garret R; Hasan, Aliya; Yin, Hao; Qadri, Ishtiaq; Quattrochi, Linda C

    2006-09-01

    Exposure to hepatitis C virus (HCV) can lead to the development of cirrhosis and hepatocellular carcinoma. To examine the effects of long-term HCV infection on hepatic cytochrome P450 1A1 (CYP1A1) expression and function, we used a human hepatoma cell line expressing the HCV subgenomic replicon (Huh.8) to evaluate CYP1A1 induction by the aryl hydrocarbon receptor (AhR) ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In this study, we demonstrate that the induction of CYP1A1 expression in Huh.8 cells by TCDD but not by beta-naphthoflavone or 3-methylcholanthrene was significantly diminished. TCDD exposure of Huh.8 cells resulted in greater than 55% suppression of CYP1A1 transcription compared with the parent cell line Huh7, whereas protein levels and enzyme activities were further diminished. Suppression of CYP1A1 mRNA expression in TCDD-treated Huh.8 cells was partially reversed after pretreatment with the antioxidants N-acetylcysteine and nordihydroguaiaretic acid, suggesting a role for oxidative stress. Induced CYP1A1 message, protein, and enzyme activity were partially restored in an Huh7 cell line expressing the HCV replicon containing a deletion in the nonstructural protein NS5A. Furthermore, adenoviral expression of NS5A in Huh7 partially suppressed TCDD-induced CYP1A1 protein and enzyme activity, implicating this protein in the mechanism of suppression. These findings demonstrate that TCDD-mediated AhR signaling is impaired in hepatocytes in which HCV is present and that NS5A alone or in the presence of other nonstructural proteins of the subgenomic replicon is in part responsible. PMID:16788090

  6. 3-Methylcholanthrene elicits DNA adduct formation in the CYP1A1 promoter region and attenuates reporter gene expression in rat H4IIE cells

    SciTech Connect

    Moorthy, Bhagavatula . E-mail: bmoorthy@bcm.tmc.edu; Muthiah, Kathirvel; Fazili, Inayat S.; Kondraganti, Sudha R.; Wang Lihua; Couroucli, Xanthi I.; Jiang Weiwu

    2007-03-23

    Cytochrome CYP1A (CYP1A) enzymes catalyze bioactivation of 3-methylcholanthrene (MC) to genotoxic metabolites. Here, we tested the hypothesis that CYP1A2 catalyzes formation of MC-DNA adducts that are preferentially formed in the promoter region of CYP1A1, resulting in modulation of CYP1A1 gene expression. MC bound covalently to plasmid DNA (50 {mu}g) containing human CYP1A1 promoter (pGL3-1A1), when incubated with wild-type (WT) liver microsomes (2 mg) and NAPPH 37 {sup o}C for 2 h, giving rise to 9 adducts, as determined by {sup 32}P-postlabeling. Eighty percent of adducts was located in the promoter region. Transient transfection of the adducted plasmids into rat hepatoma (H4IIE) cells for 16 h, followed by MC (1 {mu}M) treatment for 24 h inhibited reporter (luciferase) gene expression by 75%, compared to unadducted controls. Our results suggest that CYP1A2 plays a key role in sequence-specific MC-DNA adduct formation in the CYP1A1 promoter region, leading to attenuation of CYP1A1 gene expression.

  7. In vivo effects of 3-methylcholanthrene, phenobarbital, pyrethrum and 2,4,5-T isooctylester on liver, lung and kidney microsomal mixed-function oxidase system of guinea-pig: a comparative study.

    PubMed

    Işcan, M; Arinç, E; Vural, N; Işcan, M Y

    1984-01-01

    The optimum conditions (pH, microsomal protein amount and substrate concentration) of guinea-pig liver, lung and kidney microsomal aniline 4-hydroxylase, ethylmorphine N-demethylase and benzo[a]pyrene hydroxylase activities were determined. Male guinea-pigs weighing 500-700 g were administered 3-methylcholanthrene (25 mg/kg, i.p. 3 days), phenobarbital (75 mg/kg, i.p. 3 days), pyrethrum (120 mg/kg, i.p. 2 days) and 2,4,5-T isooctylester (200 mg/kg, i.p. 3 days). 3-Methylcholanthrene treatment caused significant increases in liver microsomal benzo[a]pyrene hydroxylase and kidney microsomal aniline 4-hydroxylase activities. However, with phenobarbital treatment the only significant increase was observed in liver microsomal ethylmorphine N-demethylase activity. Pyrethrum treatment decreased kidney microsomal ethylmorphine N-demethylase activity significantly. 2,4,5-T isooctylester treatment increased liver microsomal aniline 4-hydroxylase and lung microsomal ethylmorphine N-demethylase activities significantly. Liver microsomal NADPH-cytochrome c reductase activity was increased significantly by phenobarbital and pyrethrum treatment. The other treatments did not cause any significant changes in microsomal NADPH-cytochrome c reductase activities of liver, lung and kidney. Cytochrome P-450 content of guinea-pig liver microsomes were increased significantly about 2.5-fold and 2-fold by treatment with 3-methylcholanthrene and phenobarbital, respectively. 3-Methylcholanthrene also caused 1 nm spectral shift in the absorption maxima of CO difference spectrum of the dithionite-reduced liver microsomal cytochrome P-450, forming P-449. PMID:6141874

  8. Multiple forms of cytochrome P-450 purified from liver microsomes of phenobarbital- and 3-methylcholanthrene-pretreated rabbits. II. Spectral properties.

    PubMed

    Hashimoto-Yutsudo, C; Imai, Y; Sato, R

    1980-08-01

    The spectral properties of multiple forms of cytochrome P-450 purified or partially purified from liver microsomes of phenobarbital (PB)- and 3-methylcholanthrene (MC)-treated rabbits have been studied. Both optical absorption and EPR studies have shown that the oxidized forms of P-450(1), P-450(2) (from PB-treated animals), and P-450(3) (from MC-treated animals) are in the low spin state, having a Soret absorption peak at 417-418 nm. Oxidized P-448(1) (from both PB- and MC-treated animals), on the other hand, shows a Soret peak at 393 nm and a weak band at 646 nm. This and EPR evidence indicate that P-448(1) contains heme which is predominantly in the high spin state, though EPR studies at low temperature indicate the presence of a small amount of low spin ferric heme. The presence of tightly bound MC in P-448(1) purified from MM-treated animals is reflected by characteristic absorption peaks in the ultraviolet region, but this does not affect the absorption spectra in the Soret and visible regions. Emulgen 913, a nonionic detergent, causes the conversion of oxidized P-448(1) from the high to the low spin state, as evidenced by optical absorption and EPR results; bound MC inhibits this conversion in a noncompetitive way. Binding of ethyl isocyanide to reduced P-450(1) and P-448(1) results in the appearance of two Soret peaks in the 430 and 455 nm regions, the relative intensities of which are dependent on pH. At any pH the 455 nm peak of P-448(1) is always higher than that of P-450(1). Benzphetamine and aniline, added to oxidized P-450(1), cause Type I and Type II spectral changes, respectively, but the magnitudes of the changes are small in both cases. The Soret peak of oxidized P-448(1) at 393 nm is completely shifted to 420 nm on addition of aniline, resulting in a reverse Type I spectral change; acetanilide causes the conversion of the Soret peak to the low spin state to only a slight extent. The conversions caused by aniline and acetanilide are both inhibited

  9. Induction of Cyp1a1 and Cyp1b1 and formation of DNA adducts in C57BL/6, Balb/c, and F1 mice following in utero exposure to 3-methylcholanthrene

    SciTech Connect

    Xu Mian; Nelson, Garret B.; Moore, Joseph E.; McCoy, Thomas P.; Dai, Jian; Manderville, Richard A.; Ross, Jeffrey A.; Miller, Mark Steven . E-mail: msmiller@wfubmc.edu

    2005-11-15

    Fetal mice are more sensitive to chemical carcinogens than are adults. Previous studies from our laboratory demonstrated differences in the mutational spectrum induced in the Ki-ras gene from lung tumors isolated from [D2 x B6D2F1]F2 mice and Balb/c mice treated in utero with 3-methylcholanthrene (MC). We thus determined if differences in metabolism, adduct formation, or adduct repair influence strain-specific responses to transplacental MC exposure in C57BL/6 (B6), Balb/c (BC), and reciprocal F1 crosses between these two strains of mice. The induction of Cyp1a1 and Cyp1b1 in fetal lung and liver tissue was determined by quantitative fluorescent real-time PCR. MC treatment caused maximal induction of Cyp1a1 and Cyp1b1 RNA 2-8 h after injection in both organs. RNA levels for both genes then declined in both fetal organs, but a small biphasic, secondary increase in Cyp1a1 was observed specifically in the fetal lung 24-48 h after MC exposure in all four strains. Cyp1a1 induction by MC at 4 h was 2-5 times greater in fetal liver (7000- to 16,000-fold) than fetal lung (2000- to 6000-fold). Cyp1b1 induction in both fetal lung and liver was similar and much lower than that observed for Cyp1a1, with induction ratios of 8- to 18-fold in fetal lung and 10- to 20-fold in fetal liver. The overall kinetics and patterns of induction were thus very similar across the four strains of mice. The only significant strain-specific effect appeared to be the relatively poor induction of Cyp1b1 in the parental strain of B6 mice, especially in fetal lung tissue. We also measured the levels of MC adducts and their disappearance from lung tissue by the P{sup 32} post-labeling assay on gestation days 18 and 19 and postnatal days 1, 4, 11, and 18. Few differences were seen between the different strains of mice; the parental strain of B6 mice had nominally higher levels of DNA adducts 2 (gestation day 19) and 4 (postnatal day 1) days after injection, although this was not statistically

  10. Troglitazone induces differentiation in Trypanosoma brucei

    SciTech Connect

    Denninger, Viola; Figarella, Katherine; Schoenfeld, Caroline; Brems, Stefanie; Busold, Christian; Lang, Florian; Hoheisel, Joerg; Duszenko, Michael . E-mail: michael.duszenko@uni-tuebingen.de

    2007-05-15

    Trypanosoma brucei, a protozoan parasite causing sleeping sickness, is transmitted by the tsetse fly and undergoes a complex lifecycle including several defined stages within the insect vector and its mammalian host. In the latter, differentiation from the long slender to the short stumpy form is induced by a yet unknown factor of trypanosomal origin. Here we describe that some thiazolidinediones are also able to induce differentiation. In higher eukaryotes, thiazolidinediones are involved in metabolism and differentiation processes mainly by binding to the intracellular receptor peroxisome proliferator activated receptor {gamma}. Our studies focus on the effects of troglitazone on bloodstream form trypanosomes. Differentiation was monitored using mitochondrial markers (membrane potential, succinate dehydrogenase activity, inhibition of oxygen uptake by KCN, amount of cytochrome transcripts), morphological changes (Transmission EM and light microscopy), and transformation experiments (loss of the Variant Surface Glycoprotein coat and increase of dihydroliponamide dehydrogenase activity). To further investigate the mechanisms responsible for these changes, microarray analyses were performed, showing an upregulation of expression site associated gene 8 (ESAG8), a potential differentiation regulator.

  11. Comparative in vitro transformation of the aromatic hydrocarbon receptor (AhR) by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3-methylcholanthrene (MC)

    SciTech Connect

    Riddick, D.S.; Harper, P.A.; Okey, A.B.; Riddick, D.S. )

    1992-02-26

    The induction of CYP1A1 by halogenated (e.g. TCDD) and nonhalogenated (e.g. MC) aromatic hydrocarbons is mediated by the AhR. In cytosol prepared from the mouse hepatoma cell line Hepa-1, AhR bound TCDD with 3 to 4-fold greater affinity than MC, whereas TCDD was 960-fold more potent than MC as an inducer of aryl hydrocarbon hydroxylase (AHH) activity in cultured Hepa-1 cells. The objective of this study was to compare the potency and efficacy of TCDD and MC with respect to transformation of the cytosolic AhR to its DNA-binding form. Following incubation of Hepa-1 cytosol with TCDD or MC at 30 C for 4 h, the extent of AhR transformation was assessed by measuring interaction of the AhR-ligand complex with a {sup 32}P-labeled 26-bp oligonucleotide containing a single dioxin-responsive element (DRE) consensus sequence in a gel retardation assay. Concentration-response studies indicated that TCDD and MC did not differ significantly in AhR transformation potency, but MC displayed only about 70% of the efficacy of TCDD. In vitro transformation efficacy appears to be a determinant of AHH induction efficacy, but the small difference between TCDD and MC in transformation potency does not seem adequate to explain quantitatively the large difference in AHH induction potency displayed by these ligands.

  12. Induced differentiation inhibits sphere formation in neuroblastoma.

    PubMed

    Craig, Brian T; Rellinger, Eric J; Alvarez, Alexandra L; Dusek, Haley L; Qiao, Jingbo; Chung, Dai H

    2016-08-19

    Neuroblastoma arises from the neural crest, the precursor cells of the sympathoadrenal axis, and differentiation status is a key prognostic factor used for clinical risk group stratification and treatment strategies. Neuroblastoma tumor-initiating cells have been successfully isolated from patient tumor samples and bone marrow using sphere culture, which is well established to promote growth of neural crest stem cells. However, accurate quantification of sphere-forming frequency of commonly used neuroblastoma cell lines has not been reported. Here, we show that MYCN-amplified neuroblastoma cell lines form spheres more frequently than non-MYCN-amplified cell lines. We also show that sphere formation is directly sensitive to cellular differentiation status. 13-cis-retinoic acid is a clinically used differentiating agent that induces a neuronal phenotype in neuroblastoma cells. Induced differentiation nearly completely blocked sphere formation. Furthermore, sphere formation was specifically FGF-responsive and did not respond to increasing doses of EGF. Taken together, these data suggest that sphere formation is an accurate method of quantifying the stemness phenotype in neuroblastoma. PMID:27297102

  13. The extinction differential induced virulence macroevolution

    NASA Astrophysics Data System (ADS)

    Zhang, Feng; Xu, Liufang; Wang, Jin

    2014-04-01

    We apply the potential-flux landscape theory to deal with the large fluctuation induced extinction phenomena. We quantify the most probable extinction pathway on the landscape and measure the extinction risk by the landscape topography. In this Letter, we investigate the disease extinction through an epidemic model described by a set of chemical reaction. We found the virulence-differential-dependent symbioses between mother and daughter pathogen species: mutualism and parasitism. The symbioses, whether mutualism or parasitism, benefit the higher virulence species. This implies that speciation towards lower virulence is an effective strategy for a pathogen species to reduce its extinction risk.

  14. Differential effects of mercury, lead and copper on the constitutive and inducible expression of aryl hydrocarbon receptor (AHR)-regulated genes in cultured hepatoma Hepa 1c1c7 cells.

    PubMed

    Korashy, Hesham M; El-Kadi, Ayman O S

    2004-09-01

    Both simultaneous and sequential exposure to heavy metals and aryl hydrocarbon receptor (AHR)-ligands potentially occur in human populations, yet there have been relatively few studies of combined effects of heavy metals and AHR-ligands on AHR-regulated genes. To investigate the effects of heavy metals on AHR-regulated genes; cytochrome P450 1a1 (cyp1a1), NAD(P)H:quinone oxidoreductase (QOR) and glutathione S-transferase Ya (GST Ya), murine hepatoma Hepa 1c1c7 cells were incubated with increasing concentrations of Hg2+ (2.5-10 microM), Pb2+ (10-100 microM), and Cu2+ (1-100 microM) alone or with the AHR-ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (0.1 nM), 3-methylcholanthrene (0.25 microM), beta-naphthoflavone (10 microM), or benzo[a]pyrene (1 microM). The results clearly showed that metals alone did not significantly alter the cyp1a1 activity and protein levels but increased its mRNA expression, whereas a significant reduction in AHR ligand-mediated induction of cyp1a1 activity was observed by all metals. The decrease in cyp1a1 activity was associated with an increase, no change, or decrease in cyp1a1 mRNA and protein levels by Hg2+, Pb2+ and Cu2+ respectively, suggesting pre- and post-transcription mechanisms are involved. With respect to QOR, the activity and mRNA levels were increased by all metals in the absence or presence of an AHR-ligand, with the exception of Cu2+ which significantly decreased the induction of QOR. Differently, GST Ya activity was significantly increased by Cu2+ and Pb2+ and inhibited by Hg2+, while its mRNA was increased by Hg2+ and Pb2+ and decreased by Cu2+. All metals significantly increased the expression of heme oxygenase-1, which coincided with the changes in the phase I and phase II enzyme activities. These results demonstrate that heavy metals differentially modulate the constitutive and the inducible expression of AHR-regulated genes. PMID:15297030

  15. Directed Myogenic Differentiation of Human Induced Pluripotent Stem Cells.

    PubMed

    Shoji, Emi; Woltjen, Knut; Sakurai, Hidetoshi

    2016-01-01

    Patient-derived induced pluripotent stem cells (iPSCs) have opened the door to recreating pathological conditions in vitro using differentiation into diseased cells corresponding to each target tissue. Yet for muscular diseases, a method for reproducible and efficient myogenic differentiation from human iPSCs is required for in vitro modeling. Here, we introduce a myogenic differentiation protocol mediated by inducible transcription factor expression that reproducibly and efficiently drives human iPSCs into myocytes. Delivering a tetracycline-inducible, myogenic differentiation 1 (MYOD1) piggyBac (PB) vector to human iPSCs enables the derivation of iPSCs that undergo uniform myogenic differentiation in a short period of time. This differentiation protocol yields a homogenous skeletal muscle cell population, reproducibly reaching efficiencies as high as 70-90 %. MYOD1-induced myocytes demonstrate characteristics of mature myocytes such as cell fusion and cell twitching in response to electric stimulation within 14 days of differentiation. This differentiation protocol can be applied widely in various types of patient-derived human iPSCs and has great prospects in disease modeling particularly with inherited diseases that require studies of early pathogenesis and drug screening. PMID:25971915

  16. Chemically induced bidirectional differentiation of embryonal carcinoma cells in vitro.

    PubMed Central

    Speers, W. C.; Birdwell, C. R.; Dixon, F. J.

    1979-01-01

    N,N-dimethylacetamide, hexamethylene bisacetamide, and Polybrene induced rapid and extensive differentiation in vitro in an otherwise slowly differentiating subline of embryonal carcinoma cells. The type of differentiated cell induced was dependent on the spatial organization of the stem cells during drug treatment. In monalayer culture "epithelial" cells were produced exclusively. However, treatment of aggregated suspension cultures yielded predominantly "fibroblast-like" cells. The undifferentiated embryonal carcinoma cells and the two differentiated cell types were morphologically distinct when examined by light microscopy, scanning electron microscopy, and transmission electron microscopy; and they had differences in cell surface antigens. Both differential cell types produced large amounts of fibronectin, whereas the embryonal carcinoma cells produced only minimal amounts. This system provides a convenient way to induce relatively synchronous differentiation of embryonal carcinoma cells into specific differentiated cell types. Images Figure 5 Figure 6 Figure 1 Figure 2 Figure 3 Figure 4 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 PMID:507191

  17. HEXIM1 Induces Differentiation of Human Pluripotent Stem Cells

    PubMed Central

    Ding, Vanessa; Lew, Qiao Jing; Chu, Kai Ling; Natarajan, Subaashini; Rajasegaran, Vikneswari; Gurumurthy, Meera; Choo, Andre B. H.; Chao, Sheng-Hao

    2013-01-01

    Hexamethylene bisacetamide inducible protein 1 (HEXIM1) is best known as the inhibitor of positive transcription elongation factor b (P-TEFb), which is composed of cyclin-dependent kinase 9 (CDK9)/cyclin T1. P-TEFb is an essential regulator for the transcriptional elongation by RNA polymerase II. A genome-wide study using human embryonic stem cells shows that most mRNA synthesis is regulated at the stage of transcription elongation, suggesting a possible role for P-TEFb/HEXIM1 in the gene regulation of stem cells. In this report, we detected a marked increase in HEXIM1 protein levels in the differentiated human pluripotent stem cells (hPSCs) induced by LY294002 treatment. Since no changes in CDK9 and cyclin T1 were observed in the LY294002-treated cells, increased levels of HEXIM1 might lead to inhibition of P-TEFb activity. However, treatment with a potent P-TEFb inhibiting compound, flavopiridol, failed to induce hPSC differentiation, ruling out the possible requirement for P-TEFb kinase activity in hPSC differentiation. Conversely, differentiation was observed when hPSCs were incubated with hexamethylene bisacetamide, a HEXIM1 inducing reagent. The involvement of HEXIM1 in the regulation of hPSCs was further supported when overexpression of HEXIM1 concomitantly induced hPSC differentiation. Collectively, our study demonstrates a novel role of HEXIM1 in regulating hPSC fate through a P-TEFb-independent pathway. PMID:23977357

  18. INDUCIBILITY OF HEPATIC CYP1A ENZYMES BY 3-METHYLCHOLANTHRENE AND ISOSAFROLE DIFFERS IN MALE RATS FED DIETS CONTAINING CASEIN, SOY PROTEIN ISOLATE OR WHEY FROM CONCEPTION TO ADULTHOOD

    Technology Transfer Automated Retrieval System (TEKTRAN)

    One possible mechanism by which diet may reduce cancer risk is through enhancement of metabolic systems that prevent activation of carcinogens or accelerate carcinogen inactivation. We studied the effects of diet and 7,12-dimethylbenz-(a)anthracene (DMBA) on hepatic and mammary gland CYP1A1, CYP1A2...

  19. Dexamethasone Suppresses Oxysterol-Induced Differentiation of Monocytic Cells

    PubMed Central

    Son, Yonghae; Kim, Bo-Young; Eo, Seong-Kug; Park, Young Chul; Kim, Koanhoi

    2016-01-01

    Oxysterol like 27-hydroxycholesterol (27OHChol) has been reported to induce differentiation of monocytic cells into a mature dendritic cell phenotype. We examined whether dexamethasone (Dx) affects 27OHChol-induced differentiation using THP-1 cells. Treatment of monocytic cells with Dx resulted in almost complete inhibition of transcription and surface expression of CD80, CD83, and CD88 induced by 27OHChol. Elevated surface levels of MHC class I and II molecules induced by 27OHChol were reduced to basal levels by treatment with Dx. A decreased endocytosis ability caused by 27OHChol was recovered by Dx. We also examined effects of Dx on expression of CD molecules involved in atherosclerosis. Increased levels of surface protein and transcription of CD105, CD137, and CD166 by treatment with 27OHChol were significantly inhibited by cotreatment with Dx. These results indicate that Dx inhibits 27OHChol-induced differentiation of monocytic cells into a mature dendritic cell phenotype and expression of CD molecules whose levels are associated with atherosclerosis. In addition, we examined phosphorylation of AKT induced by 27OHChol and effect of Dx, where cotreatment with Dx inhibited the phosphorylation of AKT. The current study reports that Dx regulates oxysterol-mediated dendritic cell differentiation of monocytic cells. PMID:27340507

  20. Biochemical changes induced by strontium ranelate in differentiating adipocytes.

    PubMed

    Vidal, Christopher; Gunaratnam, Krishanthi; Tong, Jessica; Duque, Gustavo

    2013-04-01

    Low bone formation in osteoporosis is associated with a shift from osteoblastic to adipogenic differentiation of mesenchymal stem cells (MSC) inducing a concomitant lipotoxic milieu within the bone marrow. Strontium ranelate (SrRN), a treatment for osteoporosis, has both anti-resorptive and anabolic effects on bone. The anabolic effect of SrRN has been associated with its effect on both osteoblastogenesis and adipogenesis. However, the effect of SrRN on the potentially lipotoxic factors produced by differentiating marrow adipocytes remains poorly understood. To expand the knowledge on the effect of SrRN treatment on the bone microenvironment, we assessed changes in adipogenic factors and adipokine expression in adipocytic differentiation of MSC in vitro. Primary human MSC were induced to differentiate in adipogenic conditions in the presence or absence of SrRN (1-2 mM). We tested the dose-dependent effects of SrRN on adipocyte differentiation including changes in the expression of adipogenic markers and adipokines. We report that adipogenesis was negatively affected in the presence of SrRN with a concomitant dose-dependent decrease in the expression of adipogenic markers and changes in adipokine profile. Taken together, our data suggests that SrRN induces biochemical changes in differentiating adipocytes that could generate a favorable osteogenic effect within the bone marrow milieu. PMID:23186800

  1. Gossypol-Induced Differentiation in Human Leukemia HL-60 Cells

    PubMed Central

    Wang, Wen-Qing; Li, Rong; Bai, Qing-Xian; Liu, Yu-Hong; Zhang, Wei-Ping; Wang, Juan-Hong; Wang, Zhe; Li, Yuan-Fei; Chen, Xie-Qun; Huang, Gao-Sheng

    2006-01-01

    The main treatment of leukemia is traditional radiochemotherapy, which is associated with serious side effects. In the past twenty years, differentiation was found as an important effective measure to treat leukemia with fewer side effects. Gossypol, a natural compound which has been used as an effective contraceptive drug, has been proposed to be a potent drug to treat leukemia, but the differentiation effect has not been studied. In the present study, we investigated the pro-differentiated effects, in vitro, of gossypol on the classic human myeloid leukemia HL-60 cell line. The effects of gossypol were investigated by using morphological changes, nitroblue tetrazolium (NBT) reduction, surface markers, cell-cycle analysis and Western blot analysis, etc. When HL-60 cells were incubated with low concentrations of gossypol (2-5μM) for 48hr, a prominent G0/G1 arrest was observed. At 96 hr of treatment, 90% of HL-60 cells differentiated, as evidenced by morphological changes, NBT reduction, and increase in cell surface expression of some molecules were detected. This study is the first to identify gossypol’s pro-differentiated effects on the leukemia cell line, and it induced differentiation through the PBK (PDZ-binding kinase)/TOPK (T-LAKcell-originated protein kinase) (PBK/TOPK) pathway. It is concluded that gossypol could induce differentiation in the leukemia HL-60 cells, and it may be a potential therapeutic agent, chemoprevention or chemotherapeutic adjuvant especially in combination drug therapy for leukemia. PMID:23675007

  2. SOCS3 induces neurite differentiation and promotes neuronal cell survival.

    PubMed

    Mishra, Kanchan Kumar; Gupta, Sakshi; Banerjee, Kakoli

    2016-06-01

    Cytokines and growth factors play an important role in neuronal survival as well as cell death. The family of suppressors of cytokine signalling (SOCS) proteins, which includes SOCS1-7 and cytokine-induced suppressor (CIS), has been shown to act as negative regulators of cytokine-induced signalling. In this report, we highlight the role of SOCS3 in regulating neuronal differentiation and survival. We observed increased SOCS3 expression upon differentiation of PC12 cells as well as neural stem cells. SOCS3 overexpression upregulated differentiation of both neural stem cells and PC12 cells even in the absence of NGF, as evidenced by enhanced neurite outgrowth and upregulation of GAP43, marker associated with neurite outgrowth. siRNA-mediated silencing of SOCS3 confirmed the potential role of SOCS3 in neuritogenesis. We observed that, SOCS3-induced neurite differentiation was mediated via the PI3 kinase pathway. Another interesting observation was that SOCS3 overexpression promoted neuronal cell survival under H2 O2 -mediated stress indicating its fundamental role in cell survival. In conclusion, our results indicate that SOCS3 promotes differentiation and survival of neural cells and could be potentially useful in future therapy for treatment of neurodegenerative disorders. © 2016 IUBMB Life, 68(6):468-476, 2016. PMID:27118613

  3. Cholesterol starvation induces differentiation of human leukemia HL-60 cells.

    PubMed

    Sánchez-Martín, Carolina C; Dávalos, Alberto; Martín-Sánchez, Covadonga; de la Peña, Gema; Fernández-Hernando, Carlos; Lasunción, Miguel A

    2007-04-01

    Cholesterol metabolism is particularly active in malignant, proliferative cells, whereas cholesterol starvation has been shown to inhibit cell proliferation. Inhibition of enzymes involved in cholesterol biosynthesis at steps before the formation of 7-dehydrocholesterol has been shown to selectively affect cell cycle progression from G(2) phase in human promyelocytic HL-60 cells. In the present work, we explored whether cholesterol starvation by culture in cholesterol-free medium and treatment with different distal cholesterol biosynthesis inhibitors induces differentiation of HL-60 cells. Treatment with SKF 104976, an inhibitor of lanosterol 14-alpha demethylase, or with zaragozic acid, which inhibits squalene synthase, caused morphologic changes alongside respiratory burst activity and expression of cluster of differentiation antigen 11c (CD11c) but not cluster of differentiation antigen 14. These effects were comparable to those produced by all-trans retinoic acid, which induces HL-60 cells to differentiate following a granulocyte lineage. In contrast, they differed from those produced by vitamin D(3), which promotes monocyte differentiation. The specificity of the response was confirmed by addition of cholesterol to the culture medium. Treatment with PD 98059, an inhibitor of extracellular signal-regulated kinase, abolished both the activation of NADPH oxidase and the expression of the CD11c marker. In sharp contrast, BM 15766, which inhibits sterol Delta(7)-reductase, failed to induce differentiation or arrest cell proliferation. These results show that changes in the sterol composition may trigger a differentiation response and highlight the potential of cholesterol pathway inhibition as a possible tool for use in cancer therapy. PMID:17409448

  4. Oligodendroglial differentiation induces mitochondrial genes and inhibition of mitochondrial function represses oligodendroglial differentiation

    PubMed Central

    Schoenfeld, Robert; Wong, Alice; Silva, Jillian; Li, Ming; Itoh, Aki; Horiuchi, Makoto; Itoh, Takayuki; Pleasure, David; Cortopassi, Gino

    2011-01-01

    Demyelination occurs in multiple inherited mitochondrial diseases. We studied which genes were induced as a consequence of differentiation in rodent and human oligodendroglia. Cholesterol, myelin and mitochondrial genes were significantly increased with oligodendroglial differentiation. Mitochondrial DNA content per cell and acetyl CoA-related transcripts increased significantly; thus, the large buildup of cholesterol necessary for myelination appears to require mitochondrial production of acetyl-CoA. Oligodendroglia were treated with low doses of the mitochondrial inhibitor rotenone to test the dependence of differentiation on mitochondrial function. Undifferentiated cells were resistant to rotenone, whereas differentiating cells were much more sensitive. Very low doses of rotenone that did not affect viability or ATP synthesis still inhibited differentiation, as measured by reduced levels of the myelin transcripts 2′,3′-Cyclic Nucleotide-3′-Phosphodiesterase and Myelin Basic Protein. Thus, mitochondrial transcripts and mtDNA are amplified during oligodendroglial differentiation, and differentiating oligodendroglia are especially sensitive to mitochondrial inhibition, suggesting mechanisms for demyelination observed in mitochondrial disease. PMID:20005986

  5. Serum-Induced Differentiation of Human Meibomian Gland Epithelial Cells

    PubMed Central

    Sullivan, David A.; Liu, Yang; Kam, Wendy R.; Ding, Juan; Green, Karin M.; Shaffer, Scott A.; Hatton, Mark P.; Liu, Shaohui

    2014-01-01

    Purpose. We hypothesize that culturing immortalized human meibomian gland epithelial cells in serum-containing medium will induce their differentiation. The purpose of this investigation was to begin to test our hypothesis, and explore the impact of serum on gene expression and lipid accumulation in human meibomian gland epithelial cells. Methods. Immortalized and primary human meibomian gland epithelial cells were cultured in the presence or absence of serum. Cells were evaluated for lysosome and lipid accumulation, polar and neutral lipid profiles, and gene expression. Results. Our results support our hypothesis that serum stimulates the differentiation of human meibomian gland epithelial cells. This serum-induced effect is associated with a significant increase in the expression of genes linked to cell differentiation, epithelium development, the endoplasmic reticulum, Golgi apparatus, vesicles, and lysosomes, and a significant decrease in gene activity related to the cell cycle, mitochondria, ribosomes, and translation. These cellular responses are accompanied by an accumulation of lipids within lysosomes, as well as alterations in the fatty acid content of polar and nonpolar lipids. Of particular importance, our results show that the molecular and biochemical changes of immortalized human meibomian gland epithelial cells during differentiation are analogous to those of primary cells. Conclusions. Overall, our findings indicate that immortalized human meibomian gland epithelial cells may serve as an ideal preclinical model to identify factors that control cellular differentiation in the meibomian gland. PMID:24867579

  6. Testicular acid phosphatase induces odontoblast differentiation and mineralization.

    PubMed

    Choi, Hwajung; Kim, Tak-Heun; Yun, Chi-Young; Kim, Jung-Wook; Cho, Eui-Sic

    2016-04-01

    Odontoblasts differentiate from dental mesenchyme during dentin formation and mineralization. However, the molecular mechanisms controlling odontoblast differentiation remain poorly understood. Here, we show that expression of testicular acid phosphatase (ACPT) is restricted in the early stage of odontoblast differentiation in proliferating dental mesenchymal cells and secretory odontoblasts. ACPT is expressed earlier than tissue-nonspecific alkaline phosphatase (TNAP) and partly overlaps with TNAP in differentiating odontoblasts. In MDPC-23 odontoblastic cells, expression of ACPT appears simultaneously with a decrease in β-catenin activity and is abolished with the expression of Phex and Dsp. Knockdown of ACPT in MDPC-23 cells stimulates cell proliferation together with an increase in active β-catenin and cyclin D1. In contrast, the overexpression of ACPT suppresses cell proliferation with a decrease in active β-catenin and cyclin D1. Expression of TNAP, Osx, Phex and Dsp is reduced by knockdown of ACPT but is enhanced by ACPT overexpression. When ACPT is blocked with IgG, alkaline phosphatase activity is inhibited but cell proliferation is unchanged regardless of ACPT expression. These findings suggest that ACPT inhibits cell proliferation through β-catenin-mediated signaling in dental mesenchyme but elicits odontoblast differentiation and mineralization by supplying phosphate during dentin formation. Thus, ACPT might be a novel candidate for inducing odontoblast differentiation and mineralization for dentin regeneration. PMID:26547858

  7. Nicotinic acetylcholine receptors mediate donepezil-induced oligodendrocyte differentiation.

    PubMed

    Imamura, Osamu; Arai, Masaaki; Dateki, Minori; Ogata, Toru; Uchida, Ryuji; Tomoda, Hiroshi; Takishima, Kunio

    2015-12-01

    Oligodendrocytes are the myelin-forming cells of the central nervous system (CNS). Failure of myelin development and oligodendrocyte loss results in serious human disorders, including multiple sclerosis. Here, we show that donepezil, an acetlycholinesterase inhibitor developed for the treatment of Alzheimer's disease, can stimulate oligodendrocyte differentiation and maturation of neural stem cell-derived oligodendrocyte progenitor cells without affecting proliferation or cell viability. Transcripts for essential myelin-associated genes, such as PLP, MAG, MBP, CNPase, and MOG, in addition to transcription factors that regulate oligodendrocyte differentiation and myelination, were rapidly increased after treatment with donepezil. Furthermore, luciferase assays confirmed that both MAG and MBP promoters display increased activity upon donepezil-induced oligodendrocytes differentiation, suggesting that donepezil increases myelin gene expression mainly through enhanced transcription. We also found that the increase in the number of oligodendrocytes observed following donepezil treatment was significantly inhibited by the nicotinic acetylcholine receptor (nAChR) antagonist mecamylamine, but not by the muscarinic acetylcholine receptor antagonist scopolamine. Moreover, donepezil-induced myelin-related gene expression was suppressed by mecamylamine at both the mRNA and protein level. These results suggest that donepezil stimulates oligodendrocyte differentiation and myelin-related gene expression via nAChRs in neural stem cell-derived oligodendrocyte progenitor cells. We show that donepezil, a drug for the treatment of Alzheimer disease, can stimulate oligodendrocyte differentiation and maturation of oligodendrocyte progenitor cells. Transcripts for essential myelin-associated genes, such as PLP, MAG, MBP, CNPase and MOG in addition to transcripton factors that regulate oligodendrocyte differentiation and myelination were rapidly increased after treatment with donepezil

  8. Magnetic field induced differential neutron phase contrast imaging

    SciTech Connect

    Strobl, M.; Treimer, W.; Walter, P.; Keil, S.; Manke, I.

    2007-12-17

    Besides the attenuation of a neutron beam penetrating an object, induced phase changes have been utilized to provide contrast in neutron and x-ray imaging. In analogy to differential phase contrast imaging of bulk samples, the refraction of neutrons by magnetic fields yields image contrast. Here, it will be reported how double crystal setups can provide quantitative tomographic images of magnetic fields. The use of magnetic air prisms adequate to split the neutron spin states enables a distinction of field induced phase shifts and these introduced by interaction with matter.

  9. Retinoic acid-induced neural differentiation of embryonal carcinoma cells.

    PubMed Central

    Jones-Villeneuve, E M; Rudnicki, M A; Harris, J F; McBurney, M W

    1983-01-01

    We have previously shown that the P19 line of embryonal carcinoma cells develops into neurons, astroglia, and fibroblasts after aggregation and exposure to retinoic acid. The neurons were initially identified by their morphology and by the presence of neurofilaments within their cytoplasm. We have more fully documented the neuronal nature of these cells by showing that their cell surfaces display tetanus toxin receptors, a neuronal cell marker, and that choline acetyl-transferase and acetyl cholinesterase activities appear coordinately in neuron-containing cultures. Several days before the appearance of neurons, there is a marked decrease in the amount of an embryonal carcinoma surface antigen, and at the same time there is a substantial decrease in the volumes of individual cells. Various retinoids were able to induce the development of neurons in cultures of aggregated P19 cells, but it did not appear that polyamine metabolism was involved in the effect. We have isolated a mutant clone which does not differentiate in the presence of any of the drugs which are normally effective in inducing differentiation of P19 cells. This mutant and others may help to elucidate the chain of events triggered by retinoic acid and other differentiation-inducing drugs. Images PMID:6656766

  10. INDUCTION OF CYP1A1 AD CYP1B1 AND FORMATION OF DNA ADDUCTS IN C57BL/6, BALB/C, AND F1 MICE FOLLOWING IN UTERO EXPOSURE TO 3-METHYLCHOLANTHRENE

    EPA Science Inventory

    Fetal mice are more sensitive to chemical carcinogens than are adults. Previous studies from our laboratory demonstrated differences in the mutational spectrum induced in the Ki-ras gene from lung tumors isolated from [D2 x B6D2F1]F2 mice and Balb/c mice treated in utero with 3�m...

  11. Insulin Cannot Induce Adipogenic Differentiation in Primary Cardiac Cultures.

    PubMed

    Parameswaran, Sreejit; Sharma, Rajendra K

    2016-09-01

    Cardiac tissue contains a heterogeneous population of cardiomyocytes and nonmyocyte population especially fibroblasts. Fibroblast differentiation into adipogenic lineage is important for fat accumulation around the heart which is important in cardiac pathology. The differentiation in fibroblast has been observed both spontaneously and due to increased insulin stimulation. The present study aims to observe the effect of insulin in adipogenic differentiation of cardiac cells present in primary murine cardiomyocyte cultures. Oil Red O (ORO) staining has been used for observing the lipid accumulations formed due to adipogenic differentiation in murine cardiomyocyte cultures. The accumulated lipids were quantified by ORO assay and normalized using protein estimation. The lipid accumulation in cardiac cultures did not increase in presence of insulin. However, addition of other growth factors like insulin-like growth factor 1 and epidermal growth factor promoted adipogenic differentiation even in the presence of insulin and other inhibitory molecules such as vitamins. Lipid accumulation also increased in cells grown in media without insulin after an initial exposure to insulin-containing growth media. The current study adds to the existing knowledge that the insulin by itself cannot induce adipogenic induction in the cardiac cultures. The data have significance in the understanding of cardiovascular health especially in diabetic patients. PMID:27574386

  12. Induced differentiation of adipose-derived stromal cells into myoblasts.

    PubMed

    Wu, Guizhu; Zheng, Xiu; Jiang, Zhongqing; Wang, Jinhua; Song, Yanfeng

    2010-06-01

    This study aimed to induce the differentiation of isolated and purified adipose-derived stromal cells (ADSCs) into myoblasts, which may provide a new strategy for tissue engineering in patients with stress urinary incontinence (SUI). ADSCs, isolated and cultured ex vivo, were identified by flow cytometry and induced to differentiate into myoblasts in the presence of an induction solution consisting of DMEM supplemented with 5-azacytidine (5-aza), 5% FBS, and 5% horse serum. Cellular morphology was observed under an inverted microscope. Ultrastructural changes occurring during the differentiation were observed by transmission electron microscopy and confocal laser scanning microscopy. Cellular immunohistochemical staining was applied to determine the expression of desmin protein in cells with and without induced differentiation. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were used to detect mRNA and protein expression, respectively, of sarcomeric and desmin smooth muscle proteins. The results showed that ADSCs were mainly of a spindle or polygon shape. Flow cytometry analysis revealed that ADSCs did not express CD34, CD45, and CD106 but high levels of CD44 and CD90, which confirmed that the cultured cells were indeed ADSCs. After induction with a 5-aza-containing solution, morphological changes in ADSCs, including irregular cell size, were observed. Cells gradually changed from long spindles to polygons and star-shaped cells with microvilli on the cell surface. Many organelles were observed and the cytoplasm was found to contain many mitochondria, rough endoplasmic reticulum (rER), and myofilament-like structures. Cell immunohistochemical staining revealed different levels of desmin expression in each phase of the induction process, with the highest expression level found on day 28 of induction. RT-PCR and Western blot results confirmed significantly higher desmin gene expression in induced cells compared with control cells, but no

  13. Particulate matter phagocytosis induces tissue factor in differentiating macrophages.

    PubMed

    Milano, M; Dongiovanni, P; Artoni, A; Gatti, S; Rosso, L; Colombo, F; Bollati, V; Maggioni, M; Mannucci, P M; Bertazzi, P A; Fargion, S; Valenti, L

    2016-01-01

    Airborne exposure to particulate matter with diameter < 10 mcM (PM10) has been linked to an increased risk of thromboembolic events, but the mechanisms are not completely understood. The aim of this study was to evaluate the effect of PM10 phagocytosis on the release of procoagulant molecules in human differentiating macrophages, and that of PM10 inhalation in an experimental model in rats. Human monocytes were separated from the peripheral blood by the lymphoprep method, differentiated in vitro and treated with standard PM10 or vehicle. Sprague-Dawley rats were instilled intratracheally with PM10 or vehicle alone. The outcome was expression of proinflammatory genes and of tissue factor (TF). In human differentiating macrophages, PM10 exposure upregulated inflammatory genes, but most consistently induced TF mRNA and protein levels, but not TF protein inhibitor, resulting in increased TF membrane expression and a procoagulant phenotype. Differentiation towards the anti-inflammatory M2 phenotype inhibited PM10 -mediated TF expression. TF induction required phagocytosis of PM10 , whereas phagocytosis of inert particles was less effective. PM10 phagocytosis was associated with a gene expression profile consistent with intracellular retention of iron, inducing oxidative stress. Both PM10 and iron activated the stress kinases ERK1/2 pathway, involved in the induction of TF expression. In rats, alveolar exposure to PM10 was associated with pulmonary recruitment of inflammatory cells and resulted in local, but not systemic, induction of TF expression, which was sufficient to increase circulating TF levels. In conclusion, TF induction by differentiating lung macrophages, activated following phagocytosis, contributes to the increased risk of thromboembolic complications associated with PM10 exposure. PMID:25858758

  14. Laser-induced differential normalized fluorescence method for cancer diagnosis

    DOEpatents

    Vo-Dinh, T.; Panjehpour, M.; Overholt, B.F.

    1996-12-03

    An apparatus and method for cancer diagnosis are disclosed. The diagnostic method includes the steps of irradiating a tissue sample with monochromatic excitation light, producing a laser-induced fluorescence spectrum from emission radiation generated by interaction of the excitation light with the tissue sample, and dividing the intensity at each wavelength of the laser-induced fluorescence spectrum by the integrated area under the laser-induced fluorescence spectrum to produce a normalized spectrum. A mathematical difference between the normalized spectrum and an average value of a reference set of normalized spectra which correspond to normal tissues is calculated, which provides for amplifying small changes in weak signals from malignant tissues for improved analysis. The calculated differential normalized spectrum is correlated to a specific condition of a tissue sample. 5 figs.

  15. Laser-induced differential normalized fluorescence method for cancer diagnosis

    DOEpatents

    Vo-Dinh, Tuan; Panjehpour, Masoud; Overholt, Bergein F.

    1996-01-01

    An apparatus and method for cancer diagnosis are disclosed. The diagnostic method includes the steps of irradiating a tissue sample with monochromatic excitation light, producing a laser-induced fluorescence spectrum from emission radiation generated by interaction of the excitation light with the tissue sample, and dividing the intensity at each wavelength of the laser-induced fluorescence spectrum by the integrated area under the laser-induced fluorescence spectrum to produce a normalized spectrum. A mathematical difference between the normalized spectrum and an average value of a reference set of normalized spectra which correspond to normal tissues is calculated, which provides for amplifying small changes in weak signals from malignant tissues for improved analysis. The calculated differential normalized spectrum is correlated to a specific condition of a tissue sample.

  16. Differential expression analysis of genes involved in high-temperature induced sex differentiation in Nile tilapia.

    PubMed

    Li, Chun Ge; Wang, Hui; Chen, Hong Ju; Zhao, Yan; Fu, Pei Sheng; Ji, Xiang Shan

    2014-01-01

    Nowadays, high temperature effects on the molecular pathways during sex differentiation in teleosts need to be deciphered. In this study, a systematic differential expression analysis of genes involved in high temperature-induced sex differentiation was done in the Nile tilapia gonad and brain. Our results showed that high temperature caused significant down-regulation of CYP19A1A in the gonad of both sexes in induction group, and FOXL2 in the ovary of the induction group. The expressions of GTHα, LHβ and ERα were also significantly down-regulated in the brain of both sexes in the induction and recovery groups. On the contrary, the expression of CYP11B2 was significantly up-regulated in the ovary, but not in the testis in both groups. Spearman rank correlation analysis showed that there are significant correlations between the expressions of CYP19A1A, FOXL2, or DMRT1 in the gonads and the expression of some genes in the brain. Another result in this study showed that high temperature up-regulated the expression level of DNMT1 in the testis of the induction group, and DNMT1 and DNMT3A in the female brain of both groups. The expression and correlation analysis of HSPs showed that high temperature action on tilapia HSPs might indirectly induce the expression changes of sex differentiation genes in the gonads. These findings provide new insights on TSD and suggest that sex differentiation related genes, heat shock proteins, and DNA methylation genes are new candidates for studying TSD in fish species. PMID:25199961

  17. Baicalin, a Flavone, Induces the Differentiation of Cultured Osteoblasts

    PubMed Central

    Guo, Ava J. Y.; Choi, Roy C. Y.; Cheung, Anna W. H.; Chen, Vicky P.; Xu, Sherry L.; Dong, Tina T. X.; Chen, Ji J.; Tsim, Karl W. K.

    2011-01-01

    Flavonoids, a group of natural compounds found in a variety of vegetables and herbal medicines, have been intensively reported on regarding their estrogen-like activities and particularly their ability to affect bone metabolism. Here, different subclasses of flavonoids were screened for their osteogenic properties by measuring alkaline phosphatase activity in cultured rat osteoblasts. The flavone baicalin derived mainly from the roots of Scutellaria baicalensis showed the strongest induction of alkaline phosphatase activity. In cultured osteoblasts, application of baicalin increased significantly the osteoblastic mineralization and the levels of mRNAs encoding the bone differentiation markers, including osteonectin, osteocalcin, and collagen type 1α1. Interestingly, the osteogenic effect of baicalin was not mediated by its estrogenic activity. In contrast, baicalin promoted osteoblastic differentiation via the activation of the Wnt/β-catenin signaling pathway; the activation resulted in the phosphorylation of glycogen synthase kinase 3β and, subsequently, induced the nuclear accumulation of the β-catenin, leading to the transcription activation of Wnt-targeted genes for osteogenesis. The baicalin-induced osteogenic effects were fully abolished by DKK-1, a blocker of Wnt/β-catenin receptor. Moreover, baicalin also enhanced the mRNA expression of osteoprotegerin, which could regulate indirectly the activation of osteoclasts. Taken together, our results suggested that baicalin could act via Wnt/β-catenin signaling to promote osteoblastic differentiation. The osteogenic flavonoids could be very useful in finding potential drugs, or food supplements, for treating post-menopausal osteoporosis. PMID:21652696

  18. Bile acids induce hepatic differentiation of mesenchymal stem cells

    PubMed Central

    Sawitza, Iris; Kordes, Claus; Götze, Silke; Herebian, Diran; Häussinger, Dieter

    2015-01-01

    Mesenchymal stem cells (MSC) have the potential to differentiate into multiple cell lineages and their therapeutic potential has become obvious. In the liver, MSC are represented by stellate cells which have the potential to differentiate into hepatocytes after stimulation with growth factors. Since bile acids can promote liver regeneration, their influence on liver-resident and bone marrow-derived MSC was investigated. Physiological concentrations of bile acids such as tauroursodeoxycholic acid were able to initiate hepatic differentiation of MSC via the farnesoid X receptor and transmembrane G-protein-coupled bile acid receptor 5 as investigated with knockout mice. Notch, hedgehog, transforming growth factor-β/bone morphogenic protein family and non-canonical Wnt signalling were also essential for bile acid-mediated differentiation, whereas β-catenin-dependent Wnt signalling was able to attenuate this process. Our findings reveal bile acid-mediated signalling as an alternative way to induce hepatic differentiaion of stem cells and highlight bile acids as important signalling molecules during liver regeneration. PMID:26304833

  19. Differential diagnosis of food protein-induced enterocolitis syndrome

    PubMed Central

    Fiocchi, Alessandro; Claps, Alessia; Dahdah, Lamia; Brindisi, Giulia; Dionisi-Vici, Carlo; Martelli, Alberto

    2014-01-01

    Purpose of review To assess all the possible differential diagnosis of food protein-induced enterocolitis syndrome (FPIES), both in acute and chronic presentation, reviewing the data reported in published studies. Recent findings There is an increase of reported cases of FPIES in recent years. As the disease presents with nonspecific symptoms, it can be misunderstood in many ways. The differential diagnosis includes, in acute presentations, the following: sepsis, other infectious diseases, acute gastrointestinal episodes, surgical emergencies, food allergies. In its chronic forms, FPIES may mimic malabsorption syndromes, metabolic disorders, primary immunodeficiencies, neurological conditions, coagulation defects, and other types of non-IgE-mediated food allergy. Summary A thorough clinical evaluation, including symptoms, signs, and laboratory findings, is necessary to lead the clinicians toward the diagnosis of FPIES. The major reason for delayed diagnosis appears to be the lack of knowledge of the disease. PMID:24739227

  20. Pattern formation induced by a differential Poiseuille flow

    NASA Astrophysics Data System (ADS)

    Stucchi, L.; Vasquez, D. A.

    2014-12-01

    Differential advection, where a reactant is advected while another one is immobilized, leads to instabilities in reaction-advection-diffusion systems. In particular, a homogeneous steady state looses stability for strong enough flows, leading to chemical patterns moving in the direction of the flow. In this paper we study the effects of differential advection due to a two-dimensional Poiseuille flow. We carry out a linear stability analysis on a homogeneous state using an activator-inhibitor reaction. We find that shear dispersion induced by the Poiseuille flow may lead to instabilities at slower flow rates. We find that contrary to the one-dimensional system, the instability depends on which substance is advected. We find a critical average flow speed for instability depending on tube size. Numerical solutions of the nonlinear reaction-advection-diffusion result in patterns of constant shape propagating along the tube.

  1. Acidic environments induce differentiation of Proteus mirabilis into swarmer morphotypes.

    PubMed

    Fujihara, Masatoshi; Obara, Hisato; Watanabe, Yusaku; Ono, Hisaya K; Sasaki, Jun; Goryo, Masanobu; Harasawa, Ryô

    2011-07-01

    Although swarmer morphotypes of Proteus mirabilis have long been considered to result from surfaced-induced differentiation, the present findings show that, in broth medium containing urea, acidic conditions transform some swimmer cells into elongated swarmer cells. This study has also demonstrates that P. mirabilis cells grown in acidic broth medium containing urea enhance virulence factors such as flagella production and cytotoxicity to human bladder carcinoma cell line T24, though no significant difference in urease activity under different pH conditions was found. Since there is little published data on the behavior of P. mirabilis at various hydrogen-ion concentrations, the present study may clarify aspects of cellular differentiation of P. mirabilis in patients at risk of struvite formation due to infection with urease-producing bacteria, as well as in some animals with acidic or alkaline urine. PMID:21707738

  2. SHP-1-dependent macrophage differentiation exacerbates virus-induced myositis

    PubMed Central

    Watson, Neva B.; Schneider, Karin M.; Massa, Paul T.

    2015-01-01

    Virus-induced myositis is an emerging global affliction that remains poorly characterized with few treatment options. Moreover, muscle-tropic viruses often spread to the central nervous system causing dramatically increased morbidity. Therefore, there is an urgent need to explore genetic factors involved in this class of human disease. This report investigates critical innate immune pathways affecting murine virus-induced myositis. Of particular importance, the key immune regulator SHP-1, which normally suppresses macrophage-mediated inflammation, is a major factor in promoting clinical disease in muscle. We show that Theiler’s murine encephalomyelitis virus infection of skeletal myofibers induces inflammation and subsequent dystrophic calcification with loss of ambulation in wild type mice. Surprisingly, although similar extensive myofiber infection and inflammation is observed in SHP-1-deficient (SHP-1−/−) mice, these mice neither accumulate dead calcified myofibers nor lose ambulation. Macrophages were the predominant effector cells infiltrating WT and SHP-1−/− muscle, and an increased infiltration of immature monocytes/macrophages correlated with absence of clinical disease in SHP-1−/− mice, while mature M1-like macrophages corresponded with increased myofiber degeneration in WT mice. Furthermore, blocking SHP-1 activation in WT macrophages blocked virus-induced myofiber degeneration, and pharmacologic ablation of macrophages inhibited muscle calcification in TMEV-infected WT animals. These data suggest that following TMEV infection of muscle, SHP-1 promotes M1 differentiation of infiltrating macrophages, and these inflammatory macrophages are likely involved in damaging muscle fibers. These findings reveal a pathological role for SHP-1 in promoting inflammatory macrophage differentiation and myofiber damage in virus-infected skeletal muscle, thus identifying SHP-1 and M1 macrophages as essential mediators of virus-induced myopathy. PMID:25681345

  3. Asenapine-Induced Restless Legs Syndrome: Differentiation from Akathisia

    PubMed Central

    McCall, W. Vaughn; Riley, Mary Anne; Hodges, Chelsea; McCloud, Laryssa; Phillips, Marjorie; Rosenquist, Peter B.

    2014-01-01

    Akathisia and restless legs syndrome (RLS) share some common clinical features and a common relationship with dopamine dysfunction. However, the underlying causes and appropriate treatments for akathisia and RLS are different. Herein we describe a case of RLS that was precipitated by a single dose of asenapine, which is an atypical antipsychotic, and dissect the features that support the contention that this was indeed a case of RLS and not akathisia. Citation: McCall WV, Riley MA, Hodges C, McCloud L, Phillips M, Rosenquist PB. Asenapine-induced restless legs syndrome: differentiation from akathisia. J Clin Sleep Med 2014;10(12):1341-1342. PMID:25325577

  4. Substrate Induced Osteoblast-Like Differentiation of Stromal Stem Cells

    NASA Astrophysics Data System (ADS)

    Belizar, Jacqueline; Glaser, Reena; Hung, Matthew; Simon, Marcia; Jurukovski, Vladimir; Rafailovich, Miriam; Shih, Alice

    2009-03-01

    We have demonstrated that Adipose-derived stem cells (ASCs) can be induced to biomineralize on a polybutadiene (PB) coated Si substrate. The cells began to generate calcium phosphate deposits after a five-day incubation period in the absence of dexamethasone. Control cells plated on tissue culture PS culture dish (TCP) did not biomineralize. In addition, the biomineralizing culture retained proliferative cells In order to determine whether the induction was transient, we transferred the cells exposed to polybutadiene after 14 and 28-day incubation periods to TCP dishes. These cells continued to biominerlize. Genetic testing is underway which will determine whether differentiation is maintained after transfer.

  5. Identifying factors inducing trophozoite differentiation into hypnospores in Perkinsus species.

    PubMed

    Casas, Sandra M; La Peyre, Jerome F

    2013-05-01

    Trophozoites of species of Perkinsus in host tissues readily differentiate into hypnospores when incubated in Ray's fluid thioglycollate medium (RFTM). In contrast, hypnospores have rarely been observed in vivo, and when reported they have been associated with dying hosts. The objective of this study was to determine what altered environmental conditions trigger the differentiation of Perkinsus trophozoites into hypnospores. In the first part of the study, cultured P. chesapeaki trophozoites were exposed to lowered oxygen, acidic pH, increased nutrient levels, heat shock, or osmotic shock conditions, and hypnospore density was measured. Acidic pH, lowered oxygen, or increased nutrient levels significantly increased P. chesapeaki hypnospore formation. In the second part of the study, P. olseni and P. marinus trophozoites were exposed to acidic pH, lowered oxygen, or increased nutrient levels resulting in hypnospore formation in P. olseni but not P. marinus. This study demonstrated that changes in environmental conditions consistent with changes expected in decaying tissues or with RFTM incubation induce trophozoite differentiation. The response of the cultured trophozoites varied between species and between isolates of the same species. PMID:22999495

  6. IL-4 Induces Cholinergic Differentiation of Retinal Cells In Vitro.

    PubMed

    Granja, Marcelo Gomes; Braga, Luis Eduardo Gomes; Carpi-Santos, Raul; de Araujo-Martins, Leandro; Nunes-Tavares, Nilson; Calaza, Karin C; Dos Santos, Aline Araujo; Giestal-de-Araujo, Elizabeth

    2015-07-01

    Interleukin-4 (IL-4) is a pleiotropic cytokine that regulates several phenomena, among them survival and differentiation of neuronal and glial cells. The aim of this work was to investigate the effect of IL-4 on the cholinergic differentiation of neonatal rat retinal cells in vitro, evaluating its effect on the levels of cholinergic markers (CHT1-high-affinity choline transporter; VAChT-vesicular acetylcholine transporter, ChAT-choline acetyltransferase, AChE-acetylcholinesterase), muscarinic receptors, and on the signaling pathways involved. Lister Hooded rat pups were used in postnatal days 0-2 (P0-P2). Our results show that IL-4 treatment (50 U/mL) for 48 h increases the levels of the cholinergic transporters VAChT and CHT1, the acetylcholinesterase activity, and the number of ChAT-positive cells. It also induces changes in muscarinic receptor levels, leading to a small decrease in M1 levels and a significant increase in M3 and M5 levels after 48 h of treatment. We also showed that IL-4 effect on M3 receptors is dependent on type I IL-4 receptor and on an increase in NFκB phosphorylation. These results indicate that IL-4 stimulates cholinergic differentiation of retinal cells. PMID:25682112

  7. Mineralized gelatin methacrylate-based matrices induce osteogenic differentiation of human induced pluripotent stem cells

    PubMed Central

    Kang, Heemin; Shih, Yu-Ru V.; Hwang, Yongsung; Wen, Cai; Rao, Vikram; Seo, Timothy; Varghese, Shyni

    2014-01-01

    Human induced pluripotent stem cells (hiPSCs) are a promising cell source with pluripotency and self-renewal properties. Design of simple and robust biomaterials with an innate ability to induce lineage-specificity of hiPSCs is desirable to realize their applications in regenerative medicine. In this study, we investigated the potential of biomaterials containing calcium phosphate minerals to induce osteogenic differentiation of hiPSCs. hiPSCs cultured using mineralized gelatin methacrylate-based matrices underwent osteogenic differentiation ex vivo, both in two- dimensional (2-D) and three-dimensional (3-D) cultures, in growth medium devoid of any osteogenic-inducing chemical components or growth factors. Our findings that osteogenic differentiation of hiPSCs can be achieved through biomaterial-based cues alone present new avenues for personalized regenerative medicine. Such biomaterials that could not only act as structural scaffolds, but could also provide tissue-specific functions such as directing stem cell differentiation commitment, have great potential in bone tissue engineering. PMID:25153779

  8. Dextran induces differentiation of circulating endothelial progenitor cells

    PubMed Central

    Obi, Syotaro; Masuda, Haruchika; Akimaru, Hiroshi; Shizuno, Tomoko; Yamamoto, Kimiko; Ando, Joji; Asahara, Takayuki

    2014-01-01

    Abstract Endothelial progenitor cells (EPCs) have been demonstrated to be effective for the treatment of cardiovascular diseases. However, the differentiation process from circulation to adhesion has not been clarified because circulating EPCs rarely attached to dishes in EPC cultures previously. Here we investigated whether immature circulating EPCs differentiate into mature adhesive EPCs in response to dextran. When floating‐circulating EPCs derived from ex vivo expanded human cord blood were cultured with 5% and 10% dextran, they attached to fibronectin‐coated dishes and grew exponentially. The bioactivities of adhesion, proliferation, migration, tube formation, and differentiated type of EPC colony formation increased in EPCs exposed to dextran. The surface protein expression rate of the endothelial markers vascular endothelial growth factor (VEGF)‐R1/2, VE‐cadherin, Tie2, ICAM1, VCAM1, and integrin αv/β3 increased in EPCs exposed to dextran. The mRNA levels of VEGF‐R1/2, VE‐cadherin, Tie2, endothelial nitric oxide synthase, MMP9, and VEGF increased in EPCs treated with dextran. Those of endothelium‐related transcription factors ID1/2, FOXM1, HEY1, SMAD1, FOSL1, NFkB1, NRF2, HIF1A, EPAS1 increased in dextran‐treated EPCs; however, those of hematopoietic‐ and antiangiogenic‐related transcription factors TAL1, RUNX1, c‐MYB, GATA1/2, ERG, FOXH1, HHEX, SMAD2/3 decreased in dextran‐exposed EPCs. Inhibitor analysis showed that PI3K/Akt, ERK1/2, JNK, and p38 signal transduction pathways are involved in the differentiation in response to dextran. In conclusion, dextran induces differentiation of circulating EPCs in terms of adhesion, migration, proliferation, and vasculogenesis. The differentiation mechanism in response to dextran is regulated by multiple signal transductions including PI3K/Akt, ERK1/2, JNK, and p38. These findings indicate that dextran is an effective treatment for EPCs in regenerative medicines. PMID:24760515

  9. Pathologic endoplasmic reticulum stress induced by glucotoxic insults inhibits adipocyte differentiation and induces an inflammatory phenotype.

    PubMed

    Longo, Michele; Spinelli, Rosa; D'Esposito, Vittoria; Zatterale, Federica; Fiory, Francesca; Nigro, Cecilia; Raciti, Gregory A; Miele, Claudia; Formisano, Pietro; Beguinot, Francesco; Di Jeso, Bruno

    2016-06-01

    Adipocyte differentiation is critical in obesity. By controlling new adipocyte recruitment, adipogenesis contrasts adipocyte hypertrophy and its adverse consequences, such as insulin resistance. Contrasting data are present in literature on the effect of endoplasmic reticulum (ER) stress and subsequent unfolded protein response (UPR) on adipocyte differentiation, being reported to be either necessary or inhibitory. In this study, we sought to clarify the effect of ER stress and UPR on adipocyte differentiation. We have used two different cell lines, the widely used pre-adipocyte 3T3-L1 cells and a murine multipotent mesenchymal cell line, W20-17 cells. A strong ER stress activator, thapsigargin, and a pathologically relevant inducer of ER stress, glucosamine (GlcN), induced ER stress and UPR above those occurring in the absence of perturbation and inhibited adipocyte differentiation. Very low concentrations of 4-phenyl butyric acid (PBA, a chemical chaperone) inhibited only the overactivation of ER stress and UPR elicited by GlcN, leaving unaltered the part physiologically activated during differentiation, and reversed the inhibitory effect of GlcN on differentiation. In addition, GlcN stimulated proinflammatory cytokine release and PBA prevented these effects. An inhibitor of NF-kB also reversed the effects of GlcN on cytokine release. These results indicate that while ER stress and UPR activation is "physiologically" activated during adipocyte differentiation, the "pathologic" part of ER stress activation, secondary to a glucotoxic insult, inhibits differentiation. In addition, such a metabolic insult, causes a shift of the preadipocyte/adipocyte population towards a proinflammatory phenotype. PMID:26940722

  10. Laboratory-induced hyperventilation differentiates female sexual arousal disorder subtypes.

    PubMed

    Brotto, Lori A; Klein, Carolin; Gorzalka, Boris B

    2009-08-01

    The effects of heightened sympathetic nervous system (SNS) activity via laboratory-induced hyperventilation (LIH) on subjective and physiological sexual arousal were examined in a heterogeneous group of women with Sexual Arousal Disorder (SAD; n = 60), as well as across subtypes of SAD, in comparison to a control group of women without sexual difficulties (n = 42). Participants took part in 2 min of rapid breathing, a technique previously found to increase SNS activity, immediately prior to viewing erotic stimuli. Physiological arousal (i.e., vaginal pulse amplitude; VPA) was measured via the vaginal photoplethysmograph and subjective arousal was measured via self-report questionnaires. LIH differentiated women with SAD from those in the control group, with LIH increasing VPA in the latter, but having no significant effect in the heterogeneous SAD group. However, among subtypes of SAD, LIH differentiated women with genital (n = 16) and subjective (n = 16) subtypes of SAD from women with combined SAD (n = 28) and women without sexual difficulties. Specifically, women in the control group and those with combined SAD had a significant increase in VPA whereas women with genital or subjective SAD had a significant decrease in VPA following LIH. There was no significant effect of LIH on any self-report measure of sexual arousal following erotic stimuli. Implications of the results for the conceptualization, diagnosis, and treatment of SAD are discussed. PMID:18343989

  11. Analysis of Differentially Expressed Genes Associated with Coronatine-Induced Laticifer Differentiation in the Rubber Tree by Subtractive Hybridization Suppression.

    PubMed

    Zhang, Shi-Xin; Wu, Shao-Hua; Chen, Yue-Yi; Tian, Wei-Min

    2015-01-01

    The secondary laticifer in the secondary phloem is differentiated from the vascular cambia of the rubber tree (Hevea brasiliensis Muell. Arg.). The number of secondary laticifers is closely related to the rubber yield potential of Hevea. Pharmacological data show that jasmonic acid and its precursor linolenic acid are effective in inducing secondary laticifer differentiation in epicormic shoots of the rubber tree. In the present study, an experimental system of coronatine-induced laticifer differentiation was developed to perform SSH identification of genes with differential expression. A total of 528 positive clones were obtained by blue-white screening, of which 248 clones came from the forward SSH library while 280 clones came from the reverse SSH library. Approximately 215 of the 248 clones and 171 of the 280 clones contained cDNA inserts by colony PCR screening. A total of 286 of the 386 ESTs were detected to be differentially expressed by reverse northern blot and sequenced. Approximately 147 unigenes with an average length of 497 bp from the forward and 109 unigenes with an average length of 514 bp from the reverse SSH libraries were assembled and annotated. The unigenes were associated with the stress/defense response, plant hormone signal transduction and structure development. It is suggested that Ca2+ signal transduction and redox seem to be involved in differentiation, while PGA and EIF are associated with the division of cambium initials for COR-induced secondary laticifer differentiation in the rubber tree. PMID:26147807

  12. Simvastatin induces osteogenic differentiation of murine embryonic stem cells.

    PubMed

    Pagkalos, Joseph; Cha, Jae Min; Kang, Yunyi; Heliotis, Manolis; Tsiridis, Eleftherios; Mantalaris, Athanasios

    2010-11-01

    Statins are potent inhibitors of cholesterol synthesis. Several statins are available with different molecular and pharmacokinetic properties. Simvastatin is more lipophilic than pravastatin and has a higher affinity to phospholipid membranes than atorvastatin, allowing its passive diffusion through the cell membrane. In vitro studies on bone marrow stromal cells, osteoblast-like cells, and embryonic stem cells have shown statins to have cholesterol-independent anabolic effects on bone metabolism; alas, statins were supplemented in osteogenic medium, which does not facilitate elucidation of their potential osteoinductive properties. Embryonic stem cells (ESCs), derived from the inner cell mass of the blastocyst, are unique in that they enjoy perpetual self-proliferation, are pluripotent, and are able to differentiate toward all the cellular lineages composing the body, including the osteogenic lineage. Consequently, ESCs represent a potentially potent cell source for future clinical cellular therapies of various bone diseases, even though there are several hurdles that still need to be overcome. Herein we demonstrate, for the first time to our knowledge, that simvastatin induces murine ESC (mESC) differentiation toward the osteogenic lineage in the absence of osteoinductive supplements. Specifically, we found that a simvastatin concentration in the micromolar range and higher was toxic to the cells and that an effective concentration for osteoinduction is 0.1 nM, as shown by increased alizarin red staining as well as increased osteocalcin and osetrix gene expression. These results suggest that in the future, lipophilic simvastatin may provide a novel pharmacologic agent for bone tissue engineering applications. PMID:20564244

  13. Retinoic acid‐induced glandular differentiation of the oesophagus

    PubMed Central

    Chang, Chih‐Long; Lao‐Sirieix, Pierre; Save, Vicki; De La Cueva Mendez, Guillermo; Laskey, Ron; Fitzgerald, Rebecca C

    2007-01-01

    Background Retinoic acid (RA) is a powerful differentiation agent. Barrett's oesophagus occurs when duodeno‐gastro‐oesophageal reflux causes squamous epithelium (SE) tissue to become columnar epithelium tissue by an unknown mechanism. The bile acid lithocholic acid (LCA) competes for the retinoid X receptor retinoid binding site. Hence, RA pathways may be implicated in Barrett's oesophagus. Methods RA activity in tissues and cell lines treated with all‐trans retinoic acid (ATRA) with or without LCA was assessed using a reporter. Expression of p21 was determined by real‐time PCR in Barrett's oesophagus cell lines with or without LCA. SE and Barrett's oesophagus biopsy specimens were exposed to 100 μM of ATRA or 20 mM of a RA inhibitor, citral, in organ culture for >72 h. Characteristics of treated specimens, compared with untreated controls, were analysed by immunohistochemical analysis (cytokeratins (CKs), vimentin) and RT‐PCR (CKs). Confocal microscopy assessed temporal changes in co‐localisation of CK8/18 and vimentin. Cell proliferation was assessed by bromo‐deoxyuridine incorporation and immunohistochemical analysis for Ki67 and p21. Results RA biosynthesis was increased in Barrett's oesophagus compared with SE (p<0.001). LCA and ATRA caused a synergistic increase in RA signalling as shown by increased p21 (p<0.01). Morphological and molecular analysis of SE exposed to ATRA showed columnar differentiation independent of proliferation. Metaplasia could be induced from the stromal compartment alone and vimentin expression co‐localised with CK8/18 at 24 h, which separated into CK8/18‐positive glands and vimentin‐positive stroma by 48 h. Citral‐treated Barrett's oesophagus led to phenotypic and immunohistochemical characteristics of SE, which was independent of proliferation. Conclusion RA activity is increased in Barrett's oesophagus and is induced by LCA. Under conditions of altered RA activity and an intact stroma, the

  14. Derivation, characterization and retinal differentiation of induced pluripotent stem cells.

    PubMed

    Mekala, Subba Rao; Vauhini, Vasundhara; Nagarajan, Usha; Maddileti, Savitri; Gaddipati, Subhash; Mariappan, Indumathi

    2013-03-01

    Millions of people world over suffer visual disability due to retinal dystrophies which can be age-related or a genetic disorder resulting in gradual degeneration of the retinal pigmented epithelial (RPE) cells and photoreceptors. Therefore, cell replacement therapy offers a great promise in treating such diseases. Since the adult retina does not harbour any stem cells, alternative stem cell sources like the embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) offer a great promise for generating different cell types of the retina. Here, we report the derivation of four iPSC lines from mouse embryonic fibroblasts (MEFs) using a cocktail of recombinant retroviruses carrying the genes for Oct4, Sox2, Klf4 and cMyc. The iPS clone MEF-4F3 was further characterized for stemness marker expression and stable reprogramming by immunocytochemistry, FACS and RT-PCR analysis. Methylation analysis of the nanog promoter confirmed the reprogrammed epigenetic state. Pluripotency was confirmed by embryoid body (EB) formation and lineage-specific marker expression. Also, upon retinal differentiation, patches of pigmented cells with typical cobble-stone phenotype similar to RPE cells are generated within 6 weeks and they expressed ZO-1 (tight junction protein), RPE65 and bestrophin (mature RPE markers) and showed phagocytic activity by the uptake of fluorescent latex beads. PMID:23385820

  15. Single Low-Dose Radiation Induced Regulation of Keratinocyte Differentiation in Calcium-Induced HaCaT Cells

    PubMed Central

    Hahn, Hyung Jin; Youn, Hae Jeong; Cha, Hwa Jun; Kim, Karam; An, Sungkwan

    2016-01-01

    Background We are continually exposed to low-dose radiation (LDR) in the range 0.1 Gy from natural sources, medical devices, nuclear energy plants, and other industrial sources of ionizing radiation. There are three models for the biological mechanism of LDR: the linear no-threshold model, the hormetic model, and the threshold model. Objective We used keratinocytes as a model system to investigate the molecular genetic effects of LDR on epidermal cell differentiation. Methods To identify keratinocyte differentiation, we performed western blots using a specific antibody for involucrin, which is a precursor protein of the keratinocyte cornified envelope and a marker for keratinocyte terminal differentiation. We also performed quantitative polymerase chain reaction. We examined whether LDR induces changes in involucrin messenger RNA (mRNA) and protein levels in calcium-induced keratinocyte differentiation. Results Exposure of HaCaT cells to LDR (0.1 Gy) induced p21 expression. p21 is a key regulator that induces growth arrest and represses stemness, which accelerates keratinocyte differentiation. We correlated involucrin expression with keratinocyte differentiation, and examined the effects of LDR on involucrin levels and keratinocyte development. LDR significantly increased involucrin mRNA and protein levels during calcium-induced keratinocyte differentiation. Conclusion These studies provide new evidence for the biological role of LDR, and identify the potential to utilize LDR to regulate or induce keratinocyte differentiation. PMID:27489424

  16. Identification of Pathways Mediating Growth Differentiation Factor5-Induced Tenogenic Differentiation in Human Bone Marrow Stromal Cells

    PubMed Central

    Tan, Sik-Loo; Ahmad, Tunku Sara; Ng, Wuey-Min; Azlina, Amir Abbas; Azhar, Mahmood Merican; Selvaratnam, Lakshmi; Kamarul, Tunku

    2015-01-01

    To date, the molecular signalling mechanisms which regulate growth factors-induced MSCs tenogenic differentiation remain largely unknown. Therefore, a study to determine the global gene expression profile of tenogenic differentiation in human bone marrow stromal cells (hMSCs) using growth differentiation factor 5 (GDF5) was conducted. Microarray analyses were conducted on hMSCs cultures supplemented with 100 ng/ml of GDF5 and compared to undifferentiated hMSCs and adult tenocytes. Results of QuantiGene® Plex assay support the use and interpretation of the inferred gene expression profiles and pathways information. From the 27,216 genes assessed, 873 genes (3.21% of the overall human transcriptome) were significantly altered during the tenogenic differentiation process (corrected p<0.05). The genes identified as potentially associated with tenogenic differentiation were ARHGAP29, CCL2, integrin alpha 8 and neurofilament medium polypeptides. These genes, were mainly associated with cytoskeleton reorganization (stress fibers formation) signaling. Pathway analysis demonstrated the potential molecular pathways involved in tenogenic differentiation were: cytoskeleton reorganization related i.e. keratin filament signaling and activin A signaling; cell adhesion related i.e. chemokine and adhesion signaling; and extracellular matrix related i.e. arachidonic acid production signaling. Further investigation using atomic force microscopy and confocal laser scanning microscopy demonstrated apparent cytoskeleton reorganization in GDF5-induced hMSCs suggesting that cytoskeleton reorganization signaling is an important event involved in tenogenic differentiation. Besides, a reduced nucleostemin expression observed suggested a lower cell proliferation rate in hMSCs undergoing tenogenic differentiation. Understanding and elucidating the tenogenic differentiation signalling pathways are important for future optimization of tenogenic hMSCs for functional tendon cell-based therapy and

  17. Phenazopyridine induces and synchronizes neuronal differentiation of embryonic stem cells.

    PubMed

    Suter, David M; Preynat-Seauve, Olivier; Tirefort, Diderik; Feki, Anis; Krause, Karl-Heinz

    2009-09-01

    Embryonic stem (ES) cells are powerful tools to understand mechanisms of neuronal differentiation and to engineer neurons for in vitro studies and cell therapy. We developed a screening approach to identify small organic molecules driving neuronal differentiation of ES cells. For this purpose, we used a lentivector carrying a dual luciferase reporter system to engineer an ES cell line which allowed us to screen for small organic molecules enhancing neuronal differentiation. One of them, phenazopyridine, was further analysed in human ES cells. Phenazopyridine: (i) enhanced neuronal differentiation, (ii) increased cell survival, (iii) decreased the amount of non-neuronal and undifferentiated cells and (iv) synchronized the cellular differentiation state. Phenazopyridine allowed the development of a differentiation protocol compatible with the generation of clinical grade neural precursors, which were able differentiate into different neuronal subtypes, astrocytes and oligodendrocytes. In summary, we describe a powerful approach to identify small molecules directing stem cell differentiation. This led to the establishment of a new application for an old drug and the development of a novel clinical grade protocol for neuronal differentiation of ES cells. PMID:20196783

  18. Identification of H7 as a novel peroxiredoxin I inhibitor to induce differentiation of leukemia cells

    PubMed Central

    Qin, Dongjun; Chen, Yingyi; Liu, Chuanxu; Xia, Li; Wang, Tongdan; Lei, Hu; Yu, Yun; Huang, Min; Tong, Yin; Xu, Hanzhang; Gao, Fenghou

    2016-01-01

    Identifying novel targets to enhance leukemia-cell differentiation is an urgent requirment. We have recently proposed that inhibiting the antioxidant enzyme peroxiredoxin I (Prdx I) may induce leukemia-cell differentiation. However, this concept remains to be confirmed. In this work, we identified H7 as a novel Prdx I inhibitor through virtual screening, in vitro activity assay, and surface plasmon resonance assay. Cellular thermal shift assay showed that H7 directly bound to Prdx I but not to Prdxs II–V in cells. H7 treatment also increased reactive oxygen species (ROS) level and cell differentiation in leukemia cells, as reflected by the upregulation of the cell surface differentiation marker CD11b/CD14 and the morphological maturation of cells. The differentiation-induction effect of H7 was further observed in some non-acute promyelocytic leukemia (APL) and primary leukemia cells apart from APL NB4 cells. Moreover, the ROS scavenger N-acetyl cysteine significantly reversed the H7-induced cell differentiation. We demonstrated as well that H7-induced cell differentiation was associated with the activation of the ROS-Erk1/2-C/EBPβ axis. Finally, we showed H7 treatment induced cell differentiation in an APL mouse model. All of these data confirmed that Prdx I was novel target for inducing leukemia-cell differentiation and that H7 was a novel lead compound for optimizing Prdx I inhibition. PMID:26716647

  19. MYC accelerates p21CIP-induced megakaryocytic differentiation involving early mitosis arrest in leukemia cells.

    PubMed

    Muñoz-Alonso, María J; Ceballos, Laura; Bretones, Gabriel; Frade, Pilar; León, Javier; Gandarillas, Alberto

    2012-05-01

    p21(CIP) is a potent cell cycle inhibitor often up-regulated in differentiation. Protooncogene MYC induces cell growth and proliferation, inhibits differentiation and represses p21(CIP). However, both molecules are involved in processes of polyploidisation, cell size increase, differentiation and senescence. It is unclear why MYC has a dual role in differentiation. We have previously shown that overexpression of p21(CIP) in K562 myeloid cells induces megakaryocytic differentiation with polyploidy. We have now investigated the requirements for p21(CIP) to block mitosis and induce differentiation in the presence of overactivated MYC. Silencing and over-expression studies showed that p21(CIP) is required to induce differentiation. However, the expression of p21(CIP) needs to be transient to irreversibly inhibit mitosis but not DNA replication, what leads to polyploidy. Transient overexpression of p21(CIP) caused early down-regulation of mitotic Cyclins and up-regulation of G1/S Cyclins D and E, changes typical of endoreplication. Interestingly, over-activation of MYC did not release the proliferative block imposed by p21(CIP) and instead, accelerated cell size increase, megakaryocytic differentiation and polyploidisation. Our data suggests that in some systems p21(CIP) takes part in a mitosis control driving MYC-induced cellular growth into differentiation. PMID:21769863

  20. Tetrandrine induces autophagy and differentiation by activating ROS and Notch1 signaling in leukemia cells.

    PubMed

    Liu, Ting; Men, Qiuxu; Wu, Guixian; Yu, Chunrong; Huang, Zan; Liu, Xin; Li, Wenhua

    2015-04-10

    All-trans retinoic acid (ATRA) is a differentiating agent for the treatment of acute promyelocytic leukemia (APL). However, the therapeutic efficacy of ATRA has limitations. Tetrandrine is a traditional Chinese medicinal herb extract with antitumor effects. In this study, we investigated the effects of tetrandrine on human PML-RARα-positive acute promyelocytic leukemia cells. Tetrandrine inhibited tumors in vivo. It induced autophagy and differentiation by triggering ROS generation and activating Notch1 signaling. Tetrandrine induced autophagy and differentiation in M5 type patient primary leukemia cells. The in vivo results indicated that low concentrations of tetrandrine inhibited leukemia cells proliferation and induced autophagy and then facilitated their differentiation, by activating ROS and Notch1 signaling. We suggest that tetrandrine is a potential agent for the treatment of APL by inducing differentiation of leukemia cells. PMID:25797266

  1. PI3K/AKT and ERK regulate retinoic acid-induced neuroblastoma cellular differentiation

    SciTech Connect

    Qiao, Jingbo; Paul, Pritha; Lee, Sora; Qiao, Lan; Josifi, Erlena; Tiao, Joshua R.; Chung, Dai H.

    2012-08-03

    Highlights: Black-Right-Pointing-Pointer Retinoic acid (RA) induces neuroblastoma cells differentiation, which is accompanied by G0/G1 cell cycle arrest. Black-Right-Pointing-Pointer RA resulted in neuroblastoma cell survival and inhibition of DNA fragmentation; this is regulated by PI3K pathway. Black-Right-Pointing-Pointer RA activates PI3K and ERK1/2 pathway; PI3K pathway mediates RA-induced neuroblastoma cell differentiation. Black-Right-Pointing-Pointer Upregulation of p21 is necessary for RA-induced neuroblastoma cell differentiation. -- Abstract: Neuroblastoma, the most common extra-cranial solid tumor in infants and children, is characterized by a high rate of spontaneous remissions in infancy. Retinoic acid (RA) has been known to induce neuroblastoma differentiation; however, the molecular mechanisms and signaling pathways that are responsible for RA-mediated neuroblastoma cell differentiation remain unclear. Here, we sought to determine the cell signaling processes involved in RA-induced cellular differentiation. Upon RA administration, human neuroblastoma cell lines, SK-N-SH and BE(2)-C, demonstrated neurite extensions, which is an indicator of neuronal cell differentiation. Moreover, cell cycle arrest occurred in G1/G0 phase. The protein levels of cyclin-dependent kinase inhibitors, p21 and p27{sup Kip}, which inhibit cell proliferation by blocking cell cycle progression at G1/S phase, increased after RA treatment. Interestingly, RA promoted cell survival during the differentiation process, hence suggesting a potential mechanism for neuroblastoma resistance to RA therapy. Importantly, we found that the PI3K/AKT pathway is required for RA-induced neuroblastoma cell differentiation. Our results elucidated the molecular mechanism of RA-induced neuroblastoma cellular differentiation, which may be important for developing novel therapeutic strategy against poorly differentiated neuroblastoma.

  2. Hypoxia induces adipocyte differentiation of adipose-derived stem cells by triggering reactive oxygen species generation.

    PubMed

    Kim, Ji Hye; Kim, Seok-Ho; Song, Seung Yong; Kim, Won-Serk; Song, Sun U; Yi, TacGhee; Jeon, Myung-Shin; Chung, Hyung-Min; Xia, Ying; Sung, Jong-Hyuk

    2014-01-01

    Generation of reactive oxygen species (ROS) by NADPH oxidase 4 (Nox4) induces the proliferation and migration of adipose-derived stem cells (ASCs). However, the functional role of mitochondrial ROS (mtROS) generation in ASCs is unknown. Therefore, we have investigated whether hypoxia induces the differentiation of ASCs via ROS generation. We also have tried to identify the cellular mechanisms of ROS generation underlying adipocyte differentiation. Hypoxia (2%) and ROS generators, such as antimycin and rotenone, induced adipocyte differentiation, which was attenuated by an ROS scavenger. Although Nox4 generates ROS and regulates proliferation of ASCs, Nox4 inhibition or Nox4 silencing did not inhibit adipocyte differentiation; indeed fluorescence intensity of mito-SOX increased in hypoxia, and treatment with mito-CP, a mtROS scavenger, significantly reduced hypoxia-induced adipocyte differentiation. Phosphorylation of Akt and mTOR was induced by hypoxia, while inhibition of these molecules prevented adipocyte differentiation. Thus hypoxia induces adipocyte differentiation by mtROS generation, and the PI3K/Akt/mTOR pathway is involved. PMID:23956071

  3. COMPOUND-INDUCED ALTERATIONS OF SEXUAL DIFFERENTIATION: A REVIEW OF EFFECTS IN HUMANS AND RODENTS

    EPA Science Inventory

    During sexual differentiation there are a number of critical periods when the reproductive system is uniquely susceptible to chemically-induced perturbations. At these times an inappropriate chemical signal can result in irreversible lesions that often result in infertility, wher...

  4. Polar/apolar compounds induce leukemia cell differentiation by modulating cell-surface potential.

    PubMed Central

    Arcangeli, A; Carlà, M; Del Bene, M R; Becchetti, A; Wanke, E; Olivotto, M

    1993-01-01

    The mechanism of action of polar/apolar inducers of cell differentiation, such as dimethyl sulfoxide and hexamethylene-bisacetamide, is still obscure. In this paper evidence is provided that their effects on murine erythroleukemia cells are modulated by various extracellular cations as a precise function of the cation effects on membrane surface potential. The interfacial effects of the inducers were directly measured on the charged electrode, showing that both dimethyl sulfoxide and hexamethylene-bisacetamide, at the effective concentrations for cell differentiation and within the physiological range of charge density, adsorb at the charged surface and produce a potential shift. A linear correlation was found between this shift and the inducer effects on cell differentiation. Besides offering a different interpretation of the mechanism of action of the inducers, these findings indicate that surface potential has a signaling function. They may also be relevant to cancer treatments based on tumor-cell commitment to terminal differentiation. Images Fig. 1 PMID:8516337

  5. Inducing endoderm differentiation by modulating mechanical properties of soft substrates.

    PubMed

    Jaramillo, Maria; Singh, Satish S; Velankar, Sachin; Kumta, Prashant N; Banerjee, Ipsita

    2015-01-01

    Early embryonic stem cell (ESC) differentiation is marked by the formation of three germ layers from which all tissues types arise. Conventionally, ESCs are differentiated by altering their chemical microenvironment. Recently however, it was established that a mechanical microenvironment can also contribute towards cellular phenotype commitment. In this study, we report how the cellular mechanical microenvironment of soft substrates affects the differentiation and phenotypic commitment of ESCs. Mouse ESCs were cultured in a fibrin hydrogel matrix in 2D and 3D cultures. The gelation characteristics of the substrates were modulated by systematically altering the fibrinogen concentration and the fibrinogen-thrombin crosslinking ratio. Analysis of the ESCs cultured on different substrate conditions clearly illustrated the strong influence that substrate physical characteristics assert on cellular behaviours. Specifically, it was found that ESCs had a higher proliferation rate in gels of lower stiffness. Early differentiation events were studied by analyzing the gene and protein expression levels of early germ layer markers. Our results revealed that lower substrate stiffness elicited stronger upregulation of endoderm related genes Sox17, Afp and Hnf4 compared to stiffer substrates. While both 2D and 3D cultures showed a similar response, the effects were much stronger in 3D culture. These results suggest that physical cues can be used to modulate ESC differentiation into clinically relevant tissues such as liver and pancreas. PMID:23008262

  6. Identification of differentially expressed genes in Dreissena polymorpha exposed to contaminants.

    PubMed

    Bultelle, F; Panchout, M; Leboulenger, F; Danger, J M

    2002-01-01

    Development of transcriptome analysis methods such as differential display PCR and construction of subtractive libraries now makes it possible to profile gene expression in response to xenobiotic exposure. As an example of application of these methods, zebra mussels (Dreissena polymorpha) were treated with various contaminants such as Aroclor 1254, 3-methylcholanthrene, chrysene and atrazine. A total of 242 mRNAs were identified as differentially expressed. Analysis of these mRNAs should provide valuable information regarding detoxification mechanisms in this bivalve species. In addition, the use of cDNA array technology applied to these gene products may constitute a multi-marker approach to monitor the effect of contamination on this aquatic species. PMID:12408591

  7. In vitro differentiation of rhabdomyosarcomas induced by nickel or by Moloney murine sarcoma virus.

    PubMed Central

    Nanni, P.; Azzarello, G.; Tessarollo, L.; De Giovanni, C.; Lollini, P. L.; Nicoletti, G.; Scotlandi, K.; Landuzzi, L.; Panozzo, M.; D'Andrea, E.

    1991-01-01

    In vitro cultures and clonal derivatives have been established from rat rhabdomyosarcomas induced by Moloney-Murine Sarcoma Virus (MSV) or by nickel sulfide; differentiation ability has been studied as expression of desmin, embryonic and adult myosin isoforms, alpha-actin isoforms and cellular fusion. The two rhabdomyosarcoma models showed different levels of myogenic differentiation. Multinucleated myotube-like structures were frequently observed in cultures derived from nickel-induced tumours. Desmin was present in 50-80% of cells and embryonic myosin in up to 10%. In MSV-tumour-derived cultures and in their metastases or clonal derivatives two cell types are present in different ratios: spindle-shaped cells, adherent to plastic surfaces, and rounded cells, loosely attached or floating free in the medium. These cultures showed features of myogenic differentiation (10-80% desmin-positive cells), but embryonic myosin expression and production of multinucleated myotube-like structures were very rare events. Cultures from autochthonous lymph node and lung metastatic cells showed similar patterns of differentiation. Retinoic acid increased differentiated features (myotube formation and embryonic myosin expression) only in nickel-induced rhabdomyosarcoma cells. The two models described here mimic the heterogeneity in differentiation pattern found among human rhabdomyosarcomas. Myogenic differentiation ability was retained at a good level by nickel-induced tumours, whereas it was strongly impaired in MSV-induced tumours. Images Figure 1 Figure 2 Figure 4 Figure 7 Figure 8 PMID:2039698

  8. Analogue modelling of salt diapirism induced by differential loading

    NASA Astrophysics Data System (ADS)

    Warsitzka, Michael; Kley, Jonas; Kukowski, Nina; Jähne, Fabian

    2010-05-01

    In salt tectonics, two general concepts exist to explain salt diapirism. First, the theory of active piercement by Trusheim (1960) states that salt rises up and pierces its overburden autonomously by buoyancy forces. Second, the theory of reactive piercement by Vendeville and Jackson (1992) considers a tectonic stress field responsible for initiation of salt uplift and has been tested in many analogue experiments. In this study, we investigated the hypothesis in which salt diapir formation is activated by sedimentary processes alone, i.e. without a tectonic trigger. Our models consisted of a viscous silicone layer simulating rock salt overlain by layers of sand that mimic brittle behaviour in natural overburden sediments. The experiments were monitored with a high-resolution strain analysis tool based on digital image correlation (particle image velocimetry, PIV). Deformation in the silicone was initiated by a lateral variation in the thickness or density of the overburden, which established a differential loading on the silicone layer. Subsequent sedimentation in certain time intervals forced the silicone to rise up and break through the initial sand layer by buoyancy forces. The model results support the hypothesis of active piercement of diapirs. Uplift of the silicone and creation of a pillow structure with a significant elevation can be achieved if the overburden does not exceed a critical thickness and if the load gradient in the overburden reaches a minimum value. Then, ongoing sedimentation in adjacent areas increases the lateral load gradient until the buoyancy force in the silicone is high enough to overcome the shear strength of the sand. Synkinematic sedimentation produces some typical strata geometries in the sand layer that can also be observed in nature, e.g. drag folds bordering the diapirs and layer thickening in the peripherical rim synclines. The creation of one diapir and its peripherical sinks induces a lateral migration of the deformation to

  9. Tinospora cordifolia Induces Differentiation and Senescence Pathways in Neuroblastoma Cells.

    PubMed

    Mishra, Rachana; Kaur, Gurcharan

    2015-08-01

    Children diagnosed with neuroblastomas often suffer from severe side as well as late effects of conventional treatments like chemotherapy and radiotherapy. Recent advances in understanding of molecular pathways involved in cellular differentiation and apoptosis have helped in the development of new therapeutic approach based on differentiation-based therapy of malignant tumours. Natural medicines with their holistic therapeutic approach are known to selectively eliminate cancer cells thus provide a better substitute for the conventional treatment modes. The current study was aimed to investigate the anti-cancer potential of aqueous ethanolic extract of Tinospora cordifolia (TCE) using IMR-32 human neuroblastoma cell line as a model system. TCE is highly recommended in Ayurveda for its general body and metal health-promoting properties. TCE treatment was seen to arrest the majority of cells in G0/G1 phase and modulated the expression of DNA clamp sliding protein (PCNA) and cyclin D1. Further, TCE-treated cells showed differentiation as revealed by their morphology and the expression of neuronal cell specific differentiation markers NF200, MAP-2 and NeuN in neuroblastoma cells. The differentiated phenotype was associated with induction of senescence and pro-apoptosis pathways by enhancing expression of senescence marker mortalin and Rel A subunit of nuclear factor kappa beta (NFkB) along with decreased expression of anti-apoptotic marker, Bcl-xl. TCE exhibited anti-metastatic activity and significantly reduced cell migration in the scratched area along with downregulation of neural cell adhesion molecule (NCAM) polysialylation and secretion of matrix metalloproteinases (MMPs). Our data suggest that crude extract or active phytochemicals from this plant may be a potential candidate for differentiation-based therapy of malignant neuroblastoma cells. PMID:25280667

  10. Graphene induces spontaneous cardiac differentiation in embryoid bodies

    NASA Astrophysics Data System (ADS)

    Ahadian, Samad; Zhou, Yuanshu; Yamada, Shukuyo; Estili, Mehdi; Liang, Xiaobin; Nakajima, Ken; Shiku, Hitoshi; Matsue, Tomokazu

    2016-03-01

    Graphene was embedded into the structure of mouse embryoid bodies (EBs) using the hanging drop technique. The inclusion of 0.2 mg per mL graphene in the EBs did not affect the viability of the stem cells. However, the graphene decreased the stem cell proliferation, probably by accelerating cell differentiation. The graphene also enhanced the mechanical properties and electrical conductivity of the EBs. Interestingly, the cardiac differentiation of the EB-graphene was significantly greater than that of the EBs at day 5 of culture, as confirmed by high-throughput gene analysis. Electrical stimulation (voltage, 4 V; frequency, 1 Hz; and duration, 10 ms for 2 continuous days) further enhanced the cardiac differentiation of the EBs, as demonstrated by analyses of the cardiac protein and gene expression and the beating activity of the EBs. Taken together, the results demonstrated that graphene played a major role in directing the cardiac differentiation of EBs, which has potential cell therapy and tissue regeneration applications.Graphene was embedded into the structure of mouse embryoid bodies (EBs) using the hanging drop technique. The inclusion of 0.2 mg per mL graphene in the EBs did not affect the viability of the stem cells. However, the graphene decreased the stem cell proliferation, probably by accelerating cell differentiation. The graphene also enhanced the mechanical properties and electrical conductivity of the EBs. Interestingly, the cardiac differentiation of the EB-graphene was significantly greater than that of the EBs at day 5 of culture, as confirmed by high-throughput gene analysis. Electrical stimulation (voltage, 4 V; frequency, 1 Hz; and duration, 10 ms for 2 continuous days) further enhanced the cardiac differentiation of the EBs, as demonstrated by analyses of the cardiac protein and gene expression and the beating activity of the EBs. Taken together, the results demonstrated that graphene played a major role in directing the cardiac

  11. Activation of PPAR{gamma} is not involved in butyrate-induced epithelial cell differentiation

    SciTech Connect

    Ulrich, S.; Waechtershaeuser, A.; Loitsch, S.; Knethen, A. von; Bruene, B.; Stein, J. . E-mail: j.stein@em.uni-frankfurt.de

    2005-10-15

    Histone deacetylase-inhibitors affect growth and differentiation of intestinal epithelial cells by inducing expression of several transcription factors, e.g. Peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) or vitamin D receptor (VDR). While activation of VDR by butyrate mainly seems to be responsible for cellular differentiation, the activation of PPAR{gamma} in intestinal cells remains to be elucidated. The aim of this study was to determine the role of PPAR{gamma} in butyrate-induced cell growth inhibition and differentiation induction in Caco-2 cells. Treatment with PPAR{gamma} ligands ciglitazone and BADGE (bisphenol A diglycidyl) enhanced butyrate-induced cell growth inhibition in a dose- and time-dependent manner, whereas cell differentiation was unaffected after treatment with PPAR{gamma} ligands rosiglitazone and MCC-555. Experiments were further performed in dominant-negative PPAR{gamma} mutant cells leading to an increase in cell growth whereas butyrate-induced cell differentiation was again unaffected. The present study clearly demonstrated that PPAR{gamma} is involved in butyrate-induced inhibition of cell growth, but seems not to play an essential role in butyrate-induced cell differentiation.

  12. Induced myelomonocytic differentiation in leukemia cells is accompanied by noncanonical transcription factor expression

    PubMed Central

    Jensen, Holly A.; Yourish, Harmony B.; Bunaciu, Rodica P.; Varner, Jeffrey D.; Yen, Andrew

    2015-01-01

    Transcription factors that drive non-neoplastic myelomonocytic differentiation are well characterized but have not been systematically analyzed in the leukemic context. We investigated widely used, patient-derived myeloid leukemia cell lines with proclivity for differentiation into granulocytes by retinoic acid (RA) and/or monocytes by 1,25-dihyrdroxyvitamin D3 (D3). Using K562 (FAB M1), HL60 (FAB M2), RA-resistant HL60 sublines, NB4 (FAB M3), and U937 (FAB M5), we correlated nuclear transcription factor expression to immunophenotype, G1/G0 cell cycle arrest and functional inducible oxidative metabolism. We found that myelomonocytic transcription factors are aberrantly expressed in these cell lines. Monocytic-lineage factor EGR1 was not induced by D3 (the monocytic inducer) but instead by RA (the granulocytic inducer) in lineage bipotent myeloblastic HL60. In promyelocytic NB4 cells, EGR1 levels were increased by D3, while Gfi-1 expression (which promotes the granulocytic lineage) was upregulated during D3-induced monocytic differentiation in HL60, and by RA treatment in monocytic U937 cells. Furthermore, RARα and VDR expression were not strongly correlated to differentiation. In response to different differentiation inducers, U937 exhibited the most distinct transcription factor expression profile, while similarly mature NB4 and HL60 were better coupled. Overall, the differentiation induction agents RA and D3 elicited cell-specific responses across these common FAB M1-M5 cell lines. PMID:26566473

  13. Polycan suppresses osteoclast differentiation and titanium particle-induced osteolysis in mice.

    PubMed

    Lee, Young-Eun; Park, Kwang-Soo; Park, Eui-Kyun; Im, Sang-Uk; Choi, Youn-Hee; Song, Keun-Bae

    2016-08-01

    Particle-induced osteolysis is a major issue, and it is most likely the result of enhanced osteoclast activation in the pathogenesis of various skeletal diseases. This study investigated whether the inhibitory effect that Polycan has on osteoclast differentiation can be used to treat osteolysis induced by titanium (Ti) particles. To this end, the effects of Polycan were examined in terms of the cytotoxicity, osteoclast differentiation, cytokine expression, and Ti-induced calvarial osteolysis. Polycan had no significant cytotoxic effects on bone marrow macrophages (BMMs) but instead increased BMM proliferation. High levels of interleukin (IL)-6, IL-12, and macrophage colony-stimulating factor (M-CSF) were expressed in BMM cells in the presence of Polycan, suggesting that Polycan drives the differentiation of BMMs into M1 macrophages. Polycan significantly inhibited osteoclast differentiation induced by M-CSF and the receptor activator of nuclear factor kappa-B ligand (RANKL). The expression levels of the osteoclast marker genes significantly decreased, and Polycan induced and maintained the expression of IL-12, which suppressed osteoclast differentiation. In contrast, the RANKL signaling pathway was not inhibited by Polycan. An in vivo calvarial osteolysis model revealed that Polycan significantly decreased the osteoclast numbers and suppressed osteolysis. Our results suggest that the natural compound Polycan is a good candidate for therapeutic intervention against enhanced osteoclast differentiation and Ti particle-induced osteolysis. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1170-1175, 2016. PMID:26097144

  14. EGF Inhibits Wnt/β-Catenin-Induced Osteoblast Differentiation by Promoting β-Catenin Degradation.

    PubMed

    Boonanantanasarn, Kanitsak; Lee, Hye-Lim; Baek, Kyunghwa; Woo, Kyung Mi; Ryoo, Hyun-Mo; Baek, Jeong-Hwa; Kim, Gwan-Shik

    2015-12-01

    Bone morphogenetic protein (BMP) and canonical Wnts are representative developmental signals that enhance osteoblast differentiation and bone formation. Previously, we demonstrated that epidermal growth factor (EGF) inhibits BMP2-induced osteoblast differentiation by inducing Smurf1 expression. However, the regulatory role of EGF in Wnt/β-catenin-induced osteoblast differentiation has not been elucidated. In this study, we investigated the effect of EGF on Wnt/β-catenin signaling-induced osteoblast differentiation using the C2C12 cell line. EGF significantly suppressed the expression of osteoblast marker genes, which were induced by Wnt3a and a GSK-3β inhibitor. EGF increased the expression levels of Smurf1 mRNA and protein. Smurf1 knockdown rescued Wnt/β-catenin-induced osteogenic marker gene expression in the presence of EGF. EGF treatment or Smurf1 overexpression did not affect β-catenin mRNA expression levels, but reduced β-catenin protein levels and TOP-Flash activity. EGF and Smurf1 promoted β-catenin ubiquitination. Co-immunoprecipitation and GST pull-down assays showed that Smurf1 associates with β-catenin. These results suggest that EGF/Smurf1 inhibits Wnt/β-catenin-induced osteogenic differentiation and that Smurf1 downregulates Wnt/β-catenin signaling by enhancing proteasomal degradation of β-catenin. PMID:26015066

  15. Hypoxia induced the differentiation of Tbx18-positive epicardial cells to CoSMCs

    PubMed Central

    Jing, Xiaodong; Gao, Yulin; Xiao, Songlin; Qin, Qin; Wei, Xiaoming; Yan, Yuling; Wu, Ling; Deng, Songbai; Du, Jianlin; Liu, Yajie; She, Qiang

    2016-01-01

    Understanding the origin and differentiation mechanism of coronary vascular smooth muscle cells (CoSMCs) is very important to cardiovascular biology. The early cardiovascular system is formed in a hypoxic microenvironment, and Tbx18-positive epicardial cells are a source of CoSMCs. However, the effects of hypoxia on the differentiation of Tbx18-positive epicardial cells to CoSMCs and the primary regulatory mechanism are insufficiently understood. Using Tbx18:Cre/R26REYFP/LacZ fate-tracing mice, we cultured highly purified Tbx18-positive epicardial cells. We further showed that hypoxia induced Tbx18-positive epicardial cells to differentiate into CoSMCs and promoted the epithelial-mesenchymal transition (EMT) process of the cells in vitro. The induction of differentiation was primarily achieved via the hypoxia inducible factor-1α (HIF-1α)-mediated effects exerted on Snail. Using a cell migration assay, we showed that hypoxia enhanced the motility of Tbx18-positive epicardial cells. By constructing a hypoxic model of the embryonic epicardium in vivo, we showed that hypoxia led to premature in situ differentiation of Tbx18-positive epicardial cells to CoSMCs. Furthermore, hypoxia was sufficient to induce Snail expression in Tbx18-positive epicardial cells in vivo. Our study suggests that hypoxia intervention was sufficient to induce the differentiation of Tbx18-positive epicardial cells to CoSMCs. Furthermore, this differentiation was achieved primarily via HIF-1α-mediated regulation of Snail. PMID:27456656

  16. Hypoxia induced the differentiation of Tbx18-positive epicardial cells to CoSMCs.

    PubMed

    Jing, Xiaodong; Gao, Yulin; Xiao, Songlin; Qin, Qin; Wei, Xiaoming; Yan, Yuling; Wu, Ling; Deng, Songbai; Du, Jianlin; Liu, Yajie; She, Qiang

    2016-01-01

    Understanding the origin and differentiation mechanism of coronary vascular smooth muscle cells (CoSMCs) is very important to cardiovascular biology. The early cardiovascular system is formed in a hypoxic microenvironment, and Tbx18-positive epicardial cells are a source of CoSMCs. However, the effects of hypoxia on the differentiation of Tbx18-positive epicardial cells to CoSMCs and the primary regulatory mechanism are insufficiently understood. Using Tbx18:Cre/R26R(EYFP/LacZ) fate-tracing mice, we cultured highly purified Tbx18-positive epicardial cells. We further showed that hypoxia induced Tbx18-positive epicardial cells to differentiate into CoSMCs and promoted the epithelial-mesenchymal transition (EMT) process of the cells in vitro. The induction of differentiation was primarily achieved via the hypoxia inducible factor-1α (HIF-1α)-mediated effects exerted on Snail. Using a cell migration assay, we showed that hypoxia enhanced the motility of Tbx18-positive epicardial cells. By constructing a hypoxic model of the embryonic epicardium in vivo, we showed that hypoxia led to premature in situ differentiation of Tbx18-positive epicardial cells to CoSMCs. Furthermore, hypoxia was sufficient to induce Snail expression in Tbx18-positive epicardial cells in vivo. Our study suggests that hypoxia intervention was sufficient to induce the differentiation of Tbx18-positive epicardial cells to CoSMCs. Furthermore, this differentiation was achieved primarily via HIF-1α-mediated regulation of Snail. PMID:27456656

  17. Wnt signaling pathway participates in valproic acid-induced neuronal differentiation of neural stem cells

    PubMed Central

    Wang, Li; Liu, Yuan; Li, Sen; Long, Zai-Yun; Wu, Ya-Min

    2015-01-01

    Neural stem cells (NSCs) are multipotent cells that have the capacity for differentiation into the major cell types of the nervous system, i.e. neurons, astrocytes and oligodendrocytes. Valproic acid (VPA) is a widely prescribed drug for seizures and bipolar disorder in clinic. Previously, a number of researches have been shown that VPA has differential effects on growth, proliferation and differentiation in many types of cells. However, whether VPA can induce NSCs from embryonic cerebral cortex differentiate into neurons and its possible molecular mechanism is also not clear. Wnt signaling is implicated in the control of cell growth and differentiation during CNS development in animal model, but its action at the cellular level has been poorly understood. In this experiment, we examined neuronal differentiation of NSCs induced by VPA culture media using vitro immunochemistry assay. The neuronal differentiation of NSCs was examined after treated with 0.75 mM VPA for three, seven and ten days. RT-PCR assay was employed to examine the level of Wnt-3α and β-catenin. The results indicated that there were more β-tublin III positive cells in NSCs treated with VPA medium compared to the control group. The expression of Wnt-3α and β-catenin in NSCs treated with VPA medium was significantly greater compared to that of control media. In conclusion, these findings indicated that VPA could induce neuronal differentiation of NSCs by activating Wnt signal pathway. PMID:25755748

  18. High glucose induces adipogenic differentiation of muscle-derived stem cells

    PubMed Central

    Aguiari, Paola; Leo, Sara; Zavan, Barbara; Vindigni, Vincenzo; Rimessi, Alessandro; Bianchi, Katiuscia; Franzin, Chiara; Cortivo, Roberta; Rossato, Marco; Vettor, Roberto; Abatangelo, Giovanni; Pozzan, Tullio; Pinton, Paolo; Rizzuto, Rosario

    2008-01-01

    Regeneration of mesenchymal tissues depends on a resident stem cell population, that in most cases remains elusive in terms of cellular identity and differentiation signals. We here show that primary cell cultures derived from adipose tissue or skeletal muscle differentiate into adipocytes when cultured in high glucose. High glucose induces ROS production and PKCβ activation. These two events appear crucial steps in this differentiation process that can be directly induced by oxidizing agents and inhibited by PKCβ siRNA silencing. The differentiated adipocytes, when implanted in vivo, form viable and vascularized adipose tissue. Overall, the data highlight a previously uncharacterized differentiation route triggered by high glucose that drives not only resident stem cells of the adipose tissue but also uncommitted precursors present in muscle cells to form adipose depots. This process may represent a feed-forward cycle between the regional increase in adiposity and insulin resistance that plays a key role in the pathogenesis of diabetes mellitus. PMID:18212116

  19. Versican V1 Isoform Induces Neuronal Differentiation and Promotes Neurite Outgrowth

    PubMed Central

    Wu, Yaojiong; Sheng, Wang; Chen, Liwen; Dong, Haiheng; Lee, Vivian; Lu, Fred; Wong, C. Shun; Lu, Wei-Yang; Yang, Burton B.

    2004-01-01

    The chondroitin sulfate proteoglycan versican is one of the major extracellular components in the developing and adult brain. Here, we show that isoforms of versican play different roles in neuronal differentiation and neurite outgrowth. Expression of versican V1 isoform in PC12 cells induced complete differentiation, whereas expression of V2 induced an aborted differentiation accompanied by apoptosis. V1 promoted neurite outgrowth of hippocampal neurons, but V2 failed to do so. V1 transfection enhanced expression of epidermal growth factor receptor and integrins, and facilitated sustained extracellular signal-regulated kinase/MAPK phosphorylation. Blockade of the epidermal growth factor receptor, β1 integrin, or Src significantly inhibited neuronal differentiation. Finally, we demonstrated that versican V1 isoform also promoted differentiation of neural stem cells into neurons. Our results have implications for understanding how versican regulates neuronal development, function, and repair. PMID:14978219

  20. Natural Product Vibsanin A Induces Differentiation of Myeloid Leukemia Cells through PKC Activation.

    PubMed

    Yu, Zu-Yin; Xiao, He; Wang, Li-Mei; Shen, Xing; Jing, Yu; Wang, Lin; Sun, Wen-Feng; Zhang, Yan-Feng; Cui, Yu; Shan, Ya-Jun; Zhou, Wen-Bing; Xing, Shuang; Xiong, Guo-Lin; Liu, Xiao-Lan; Dong, Bo; Feng, Jian-Nan; Wang, Li-Sheng; Luo, Qing-Liang; Zhao, Qin-Shi; Cong, Yu-Wen

    2016-05-01

    All-trans retinoic acid (ATRA)-based cell differentiation therapy has been successful in treating acute promyelocytic leukemia, a unique subtype of acute myeloid leukemia (AML). However, other subtypes of AML display resistance to ATRA-based treatment. In this study, we screened natural, plant-derived vibsane-type diterpenoids for their ability to induce differentiation of myeloid leukemia cells, discovering that vibsanin A potently induced differentiation of AML cell lines and primary blasts. The differentiation-inducing activity of vibsanin A was mediated through direct interaction with and activation of protein kinase C (PKC). Consistent with these findings, pharmacological blockade of PKC activity suppressed vibsanin A-induced differentiation. Mechanistically, vibsanin A-mediated activation of PKC led to induction of the ERK pathway and decreased c-Myc expression. In mouse xenograft models of AML, vibsanin A administration prolonged host survival and inhibited PKC-mediated inflammatory responses correlated with promotion of skin tumors in mice. Collectively, our results offer a preclinical proof of concept for vibsanin A as a myeloid differentiation-inducing compound, with potential application as an antileukemic agent. Cancer Res; 76(9); 2698-709. ©2016 AACR. PMID:26984756

  1. Erythroid differentiation of human induced pluripotent stem cells is independent of donor cell type of origin

    PubMed Central

    Dorn, Isabel; Klich, Katharina; Arauzo-Bravo, Marcos J.; Radstaak, Martina; Santourlidis, Simeon; Ghanjati, Foued; Radke, Teja F.; Psathaki, Olympia E.; Hargus, Gunnar; Kramer, Jan; Einhaus, Martin; Kim, Jeong Beom; Kögler, Gesine; Wernet, Peter; Schöler, Hans R.; Schlenke, Peter; Zaehres, Holm

    2015-01-01

    Epigenetic memory in induced pluripotent stem cells, which is related to the somatic cell type of origin of the stem cells, might lead to variations in the differentiation capacities of the pluripotent stem cells. In this context, induced pluripotent stem cells from human CD34+ hematopoietic stem cells might be more suitable for hematopoietic differentiation than the commonly used fibroblast-derived induced pluripotent stem cells. To investigate the influence of an epigenetic memory on the ex vivo expansion of induced pluripotent stem cells into erythroid cells, we compared induced pluripotent stem cells from human neural stem cells and human cord blood-derived CD34+ hematopoietic stem cells and evaluated their potential for differentiation into hematopoietic progenitor and mature red blood cells. Although genome-wide DNA methylation profiling at all promoter regions demonstrates that the epigenetic memory of induced pluripotent stem cells is influenced by the somatic cell type of origin of the stem cells, we found a similar hematopoietic induction potential and erythroid differentiation pattern of induced pluripotent stem cells of different somatic cell origin. All human induced pluripotent stem cell lines showed terminal maturation into normoblasts and enucleated reticulocytes, producing predominantly fetal hemoglobin. Differences were only observed in the growth rate of erythroid cells, which was slightly higher in the induced pluripotent stem cells derived from CD34+ hematopoietic stem cells. More detailed methylation analysis of the hematopoietic and erythroid promoters identified similar CpG methylation levels in the induced pluripotent stem cell lines derived from CD34+ cells and those derived from neural stem cells, which confirms their comparable erythroid differentiation potential. PMID:25326431

  2. Azithromycin differentially affects the IL-13-induced expression profile in human bronchial epithelial cells.

    PubMed

    Mertens, Tinne C J; Hiemstra, Pieter S; Taube, Christian

    2016-08-01

    The T helper 2 (Th2) cytokine interleukin(IL)-13 is a central regulator in goblet cell metaplasia and induces the recently described Th2 gene signature consisting of periostin (POSTN), chloride channel regulator 1 (CLCA1) and serpin B2 (SERPINB2) in airway epithelial cells. This Th2 gene signature has been proposed as a biomarker to classify asthma into Th2-high and Th2-low phenotypes. Clinical studies have shown that the macrolide antibiotic azithromycin reduced clinical symptoms in neutrophilic asthma, but not in the classical Th2-mediated asthma despite the ability of azithromycin to reduce IL-13-induced mucus production. We therefore hypothesize that azithromycin differentially affects the IL-13-induced expression profile. To investigate this, we focus on IL-13-induced mucin and Th2-signature expression in human bronchial epithelial cells and how this combined expression profile is affected by azithromycin treatment. Primary bronchial epithelial cells were differentiated at air liquid interface in presence of IL-13 with or without azithromycin. Azithromycin inhibited IL-13-induced MUC5AC, which was accompanied by inhibition of IL-13-induced CLCA1 and SERPINB2 expression. In contrast, IL-13-induced expression of POSTN was further increased in cells treated with azithromycin. This indicates that azithromycin has a differential effect on the IL-13-induced Th2 gene signature. Furthermore, the ability of azithromycin to decrease IL-13-induced MUC5AC expression may be mediated by a reduction in CLCA1. PMID:27246785

  3. Interleukin-34 induces monocytic-like differentiation in leukemia cell lines

    PubMed Central

    Booker, Burthia E; Clark, Ryan S; Pellom, Samuel T; Adunyah, Samuel E

    2015-01-01

    Interleukin-34 (IL-34) is a cytokine consisting of a 39kD homodimer, shown to be a ligand for both the Macrophage Colony Stimulating Factor (M-CSF/CSF-1) receptor and the Receptor-like protein tyrosine phosphatase-zeta (RPTP-ƺ). IL-34 has been shown to promote monocyte viability and proliferation as well as the differentiation of bone marrow cells into macrophage progenitors. Published work on IL-34 involves its effects on normal hematopoietic and osteoclast progenitors. However, it is not known whether IL-34 has biologic effects in cancer, including leukemia. Here we report that the biological effects of IL-34 include induction of differential expression of Interleukins-1α and -1β as well as induction of differentiation of U937, HL-60 and THP-1 leukemia cell lines demonstrating monocyte-like characteristics. The ability of IL-34 to induce monocytic-like differentiation is supported by strong morphological and functional evidence. Cell surface markers of myeloid lineage, CD64 and CD86, remain constant while the levels of CD11b and CD71 decline with IL-34 treatment. IL-34 also induced increases in CD14 and CD68 expression, further supporting maturation toward monocytic character. IL-34-induced differentiated U937 and THP-1 cell lines exhibited biological functions such as endocytosis and respiratory burst activities. Collectively, we conclude that while IL-34 does not induce cell growth or proliferation, it is able to induce differentiation of leukemia cell lines from monoblastic precursor cells towards monocyte- and macrophage-like cells, mediated through the JAK/STAT and PI3K/Akt pathways. To our knowledge, this is the first report that IL-34 induces differentiation in human leukemic cells, let alone any cancer model. PMID:26045972

  4. SKW 6.4 cell differentiation induced by interleukin 6 is stimulated by butyrate.

    PubMed

    Kawamoto, T; Gohda, E; Iji, H; Fujiwara, M; Yamamoto, I

    1998-08-01

    We investigated if sodium butyrate (NaBu), an inhibitor of histone deacetylase, and its analogs modulate cytokine-induced differentiation of the human B cell line SKW 6.4 transformed by the Epstein-Barr virus. NaBu markedly enhanced interleukin (IL)-6-induced IgM production with an accompanying increase in the level of histone H4 acetylation and augmented IgM production induced by IL-4 and phorbol 12-myristate 13-acetate. From both the enhancing effect of cell differentiation and the effect of inducing histone hyperacetylation in SKW 6.4 cells, other histone deacetylase inhibitors and NaBu analogs were divided into three groups: those that increased both IL-6-induced antibody production and histone acetylation, those that caused histone hyperacetylation, but failed to induce the differentiation, and those that were ineffective at inducing either activity. No agent that enhanced IgM production without inducing histone hyperacetylation was found among the inhibitors and analogs we tested. These results suggest that the increase in the histone acetylation is necessary, but it is insufficient to augment differentiation of SKW 6.4 cells. Thus another activity of NaBu in addition to the inhibition of histone deacetylase may be involved in promoting IL-6-induced differentiation. Our results also suggest that fatty acids that have a straight chain of four carbon atoms or are branched with four and five carbon atoms, which contain no hydrophilic substituents, or those with similar structures, show this other activity. PMID:9826026

  5. [Transcriptomics and proteomics in studies of induced differentiation of leukemia cells].

    PubMed

    Novikova, S E; Zgoda, V G

    2015-01-01

    Induced differentiation of leukemia cells is in the focus of basic and applied biomedical studies medicine and biology for more than 30 years. During this period specific regulatory molecules involved in the maturation process have been identified by biochemical and molecular biological methods. Recent developments of high-throughput transcriptomic and proteomic techniques made it possible to analyze large sets of mRNA and proteins; this resulted in identification of functionally important signal transduction pathways and networks of molecular interactions, and thus extent existing knowledge on the molecular mechanisms of induced differentiation. Despite significant advances in mechanisms of induced differentiation, many problems related to the molecular mechanism of cell maturation, a phenomenon of therapeutic resistance of leukemic cells need better understanding and thus require further detailed study. Transcriptomics and proteomics methods provide a suitable methodological platform for the implementation of such studies. This review highlights the use of transcriptomic and proteomic methods in studies aimed at various aspects of the induced differentiation. Special attention is paid to the employment of the systems approach for investigation of various aspects of cell maturation. The use of the systems approach in studies of induced differentiation is an important step for the transition from the formal data accumulation on expression of mRNA and proteins towards creating models of biological processes in silico. PMID:26539862

  6. Forced expression of Hnf4a induces hepatic gene activation through directed differentiation.

    PubMed

    Yahoo, Neda; Pournasr, Behshad; Rostamzadeh, Jalal; Fathi, Fardin

    2016-08-01

    Embryonic stem (ES) cells are capable of unlimited self-renewal and have a diverse differentiation potential. These unique features make ES cells as an attractive source for developmental biology studies. Having the mature hepatocyte in the lab with functional activities is valuable in drug discovery studies. Overexpression of hepatocyte lineage-specific transcription factors (TFs) becomes a promising approach in pluripotent cell differentiation toward liver cells. Many studies generate transgenic ES cell lines to examine the effects of specific TFs overexpression in cell differentiation. In the present report, we have addressed whether a suspension or adherent model of differentiation is an appropriate way to study the role of Hnf4a overexpression. We generated ES cells that carried a doxycycline (Dox)-inducible Hnf4a using lentiviral vectors. The transduced cells were subjected to induced Hnf4a overexpression through both spontaneous and directed differentiation methods. Gene expression analysis showed substantially increased expression of hepatic gene markers, particularly Ttr and endogenous Hnf4a, in transduced cells differentiated by the directed approach. These results demonstrated that forced expression of TFs during directed differentiation would be an appropriate way to study relevant gene activation and the effects of overexpression in the context of hepatic differentiation. PMID:27233607

  7. Inhibition of KDM6 activity during murine ESC differentiation induces DNA damage.

    PubMed

    Hofstetter, Christine; Kampka, Justyna M; Huppertz, Sascha; Weber, Heike; Schlosser, Andreas; Müller, Albrecht M; Becker, Matthias

    2016-02-15

    Pluripotent embryonic stem cells (ESCs) are characterised by their capacity to self-renew indefinitely while maintaining the potential to differentiate into all cell types of an adult organism. Both the undifferentiated and differentiated states are defined by specific gene expression programs that are regulated at the chromatin level. Here, we have analysed the contribution of the H3K27me2- and H3K27me23-specific demethylases KDM6A and KDM6B to murine ESC differentiation by employing the GSK-J4 inhibitor, which is specific for KDM6 proteins, and by targeted gene knockout (KO) and knockdown. We observe that inhibition of the H3K27 demethylase activity induces DNA damage along with activation of the DNA damage response (DDR) and cell death in differentiating but not in undifferentiated ESCs. Laser microirradiation experiments revealed that the H3K27me3 mark, but not the KDM6B protein, colocalise with γH2AX-positive sites of DNA damage in differentiating ESCs. Lack of H3K27me3 attenuates the GSK-J4-induced DDR in differentiating Eed-KO ESCs. Collectively, our findings indicate that differentiating ESCs depend on KDM6 and that the H3K27me3 demethylase activity is crucially involved in DDR and survival of differentiating ESCs. PMID:26759175

  8. The AhR agonist VAF347 augments retinoic acid-induced differentiation in leukemia cells.

    PubMed

    Ibabao, Christopher N; Bunaciu, Rodica P; Schaefer, Deanna M W; Yen, Andrew

    2015-01-01

    In binary cell-fate decisions, driving one lineage and suppressing the other are conjoined. We have previously reported that aryl hydrocarbon receptor (AhR) promotes retinoic acid (RA)-induced granulocytic differentiation of lineage bipotent HL-60 myeloblastic leukemia cells. VAF347, an AhR agonist, impairs the development of CD14(+)CD11b(+) monocytes from granulo-monocytic (GM) stage precursors. We thus hypothesized that VAF347 propels RA-induced granulocytic differentiation and impairs D3-induced monocytic differentiation of HL-60 cells. Our results show that VAF347 enhanced RA-induced cell cycle arrest, CD11b integrin expression and neutrophil respiratory burst. Granulocytic differentiation is known to be driven by MAPK signaling events regulated by Fgr and Lyn Src-family kinases, the CD38 cell membrane receptor, the Vav1 GEF, the c-Cbl adaptor, as well as AhR, all of which are embodied in a putative signalsome. We found that the VAF347 AhR ligand regulates the signalsome. VAF347 augments RA-induced expression of AhR, Lyn, Vav1, and c-Cbl as well as p47(phox). Several interactions of partners in the signalsome appear to be enhanced: Fgr interaction with c-Cbl, CD38, and with pS259c-Raf and AhR interaction with c-Cbl and Lyn. Thus, we report that, while VAF347 impedes monocytic differentiation induced by 1,25-dihydroxyvitamin D3, VAF347 promotes RA-induced differentiation. This effect seems to involve but not to be limited to Lyn, Vav1, c-Cbl, AhR, and Fgr. PMID:25941627

  9. Differentiation of murine embryonic stem and induced pluripotent stem cells to renal lineage in vitro

    SciTech Connect

    Morizane, Ryuji; Monkawa, Toshiaki; Itoh, Hiroshi

    2009-12-25

    Embryonic stem (ES) cells which have the unlimited proliferative capacity and extensive differentiation potency can be an attractive source for kidney regeneration therapies. Recent breakthroughs in the generation of induced pluripotent stem (iPS) cells have provided with another potential source for the artificially-generated kidney. The purpose of this study is to know how to differentiate mouse ES and iPS cells into renal lineage. We used iPS cells from mouse fibroblasts by transfection of four transcription factors, namely Oct4, Sox2, c-Myc and Klf4. Real-time PCR showed that renal lineage markers were expressed in both ES and iPS cells after the induction of differentiation. It also showed that a tubular specific marker, KSP progressively increased to day 18, although the differentiation of iPS cells was slower than ES cells. The results indicated that renal lineage cells can be differentiated from both murine ES and iPS cells. Several inducing factors were tested whether they influenced on cell differentiation. In ES cells, both of GDNF and BMP7 enhanced the differentiation to metanephric mesenchyme, and Activin enhanced the differentiation of ES cells to tubular cells. Activin also enhanced the differentiation of iPS cells to tubular cells, although the enhancement was lower than in ES cells. ES and iPS cells have a potential to differentiate to renal lineage cells, and they will be an attractive resource of kidney regeneration therapy. This differentiation is enhanced by Activin in both ES and iPS cells.

  10. SRY alone can induce normal male sexual differentiation

    SciTech Connect

    Lopez, M.; Torres, L.; Cervantes, A.

    1995-01-30

    Most individuals with the rare 46,XX male {open_quotes}syndrome{close_quotes} arise due to an unequal interchange between Xp and Yp termini during paternal meiosis. The pattern of Y-sequences in these patients varies considerably, but very few cases have been reported showing only SRY. The phenotype in these patients is also variable ranging from severe impairment of the external genitalia through hypospadias and/or cryptorchidism to occasional normal male phenotype. We report a Mexican 46,XX male patient without genital ambiguities in whom DNA analysis showed the presence of SRY and the absence of ZFY. We conclude that in this case SRY alone was enough for complete male sexual differentiation. 25 refs., 1 fig.

  11. Differentiation of Isomeric Ginsenosides by Using Electron-Induced Dissociation Mass Spectrometry.

    PubMed

    Wong, Y-L Elaine; Chen, Xiangfeng; Li, Wan; Wang, Ze; Hung, Y-L Winnie; Wu, Ri; Chan, T-W Dominic

    2016-06-01

    Current phytochemical research on ginsengs focuses on the structural characterization and isomer differentiation of ginsenosides. In this Letter, electron-induced dissociation (EID) was initially investigated by analyzing isomeric ginsenosides. EID provided more structural information on their differentiation than collision-induced dissociation (CID) did. Glycosyl group migration previously observed in the CID of oligosaccharide ions could also be found in the EID of protonated Rg1. This rearrangement reaction would show substantial ambiguities in differentiating Rg1 from Rf. Although other charge carriers could alleviate this problem, the use of EID in dissociating deprotonated ginsenoside ions was superior to other techniques in terms of eliminating glycosyl group migration and generating diagnostic fragment ions for the differentiation of structural isomers. This study demonstrates a potential method to analyze natural products and thus help discover and evaluate novel compounds. PMID:27181402

  12. Recombinant human bone morphogenetic protein-9 potently induces osteogenic differentiation of human periodontal ligament fibroblasts.

    PubMed

    Fuchigami, Sawako; Nakamura, Toshiaki; Furue, Kirara; Sena, Kotaro; Shinohara, Yukiya; Noguchi, Kazuyuki

    2016-04-01

    To accomplish effective periodontal regeneration for periodontal defects, several regenerative methods using growth and differentiation factors, including bone morphogenetic proteins (BMPs), have been developed. Bone morphogenetic protein-9 exhibits the most potent osteogenic activity of this growth factor family. However, it is unclear whether exogenous BMP-9 can induce osteogenic differentiation in human periodontal ligament (PDL) fibroblasts. Here, we examined the effects of recombinant human (rh) BMP-9 on osteoblastic differentiation in human PDL fibroblasts in vitro, compared with rhBMP-2. Recombinant human BMP-9 potently induced alkaline phosphatase (ALP) activity, mineralization, and increased expression of runt-related transcription factor-2/core binding factor alpha 1 (RUNX2/CBFA1), osterix, inhibitor of DNA binding/differentiation-1 (ID1), osteopontin, and bone sialoprotein genes, compared with rhBMP-2. The levels of rhBMP-9-induced osterix and ALP mRNA were significantly reduced in activin receptor-like kinase-1 and -2 small interfering RNA (siRNA)-transfected human PDL fibroblasts. Recombinant human BMP-9-induced ALP activity was not inhibited by noggin, in contrast to rhBMP-2 induced ALP activity, which was. Phosphorylation of SMAD1/5/8 in human PDL fibroblasts was induced by addition of rhBMP-9. Recombinant human BMP-9-induced ALP activity was suppressed by SB203580, SP600125, and U0126, which are inhibitors of p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase 1/2 (ERK1/2), respectively. Our data suggest that rhBMP-9 is a potent inducer of the differentiation of human PDL fibroblasts into osteoblast-like cells and that this may be mediated by the SMAD and mitogen-activated protein kinase (p38, ERK1/2, and JNK) pathways. PMID:26879145

  13. Metformin induces differentiation in acute promyelocytic leukemia by activating the MEK/ERK signaling pathway

    SciTech Connect

    Huai, Lei; Wang, Cuicui; Zhang, Cuiping; Li, Qihui; Chen, Yirui; Jia, Yujiao; Li, Yan; Xing, Haiyan; Tian, Zheng; Rao, Qing; Wang, Min; Wang, Jianxiang

    2012-06-08

    Highlights: Black-Right-Pointing-Pointer Metformin induces differentiation in NB4 and primary APL cells. Black-Right-Pointing-Pointer Metformin induces activation of the MEK/ERK signaling pathway in APL cells. Black-Right-Pointing-Pointer Metformin synergizes with ATRA to trigger maturation of NB4 and primary APL cells. Black-Right-Pointing-Pointer Metformin induces the relocalization and degradation of the PML-RAR{alpha} fusion protein. Black-Right-Pointing-Pointer The study may be applicable for new differentiation therapy in cancer treatment. -- Abstract: Recent studies have shown that metformin, a widely used antidiabetic agent, may reduce the risk of cancer development. In this study, we investigated the antitumoral effect of metformin on both acute myeloid leukemia (AML) and acute promyelocytic leukemia (APL) cells. Metformin induced apoptosis with partial differentiation in an APL cell line, NB4, but only displayed a proapoptotic effect on several non-M3 AML cell lines. Further analysis revealed that a strong synergistic effect existed between metformin and all-trans retinoic acid (ATRA) during APL cell maturation and that metformin induced the hyperphosphorylation of extracellular signal-regulated kinase (ERK) in APL cells. U0126, a specific MEK/ERK activation inhibitor, abrogated metformin-induced differentiation. Finally, we found that metformin induced the degradation of the oncoproteins PML-RAR{alpha} and c-Myc and activated caspase-3. In conclusion, these results suggest that metformin treatment may contribute to the enhancement of ATRA-induced differentiation in APL, which may deepen the understanding of APL maturation and thus provide insight for new therapy strategies.

  14. Effects and mechanisms of melatonin on neural differentiation of induced pluripotent stem cells.

    PubMed

    Shu, Tao; Wu, Tao; Pang, Mao; Liu, Chang; Wang, Xuan; Wang, Juan; Liu, Bin; Rong, Limin

    2016-06-01

    Melatonin, a lipophilic molecule mainly synthesized in the pineal gland, has properties of antioxidation, anti-inflammation, and antiapoptosis to improve neuroprotective functions. Here, we investigate effects and mechanisms of melatonin on neural differentiation of induced pluripotent stem cells (iPSCs). iPSCs were induced into neural stem cells (NSCs), then further differentiated into neurons in medium with or without melatonin, melatonin receptor antagonist (Luzindole) or Phosphatidylinositide 3 kinase (PI3K) inhibitor (LY294002). Melatonin significantly promoted the number of neurospheres and cell viability. In addition, Melatonin markedly up-regulated gene and protein expression of Nestin and MAP2. However, Luzindole or LY294002 attenuated these increase. The expression of pAKT/AKT were increased by Melatonin, while Luzindole or LY294002 declined these melatonin-induced increase. These results suggest that melatonin significantly increased neural differentiation of iPSCs via activating PI3K/AKT signaling pathway through melatonin receptor. PMID:27130826

  15. Direct hepatic differentiation of mouse embryonic stem cells induced by valproic acid and cytokines

    PubMed Central

    Dong, Xue-Jun; Zhang, Guo-Rong; Zhou, Qing-Jun; Pan, Ruo-Lang; Chen, Ye; Xiang, Li-Xin; Shao, Jian-Zhong

    2009-01-01

    AIM: To develop a protocol for direct hepatic lineage differentiation from early developmental progenitors to a population of mature hepatocytes. METHODS: Hepatic progenitor cells and then mature hepatocytes from mouse embryonic stem (ES) cells were obtained in a sequential manner, induced by valproic acid (VPA) and cytokines (hepatocyte growth factor, epidermal growth factor and insulin). Morphological changes of the differentiated cells were examined by phase-contrast microscopy and electron microscopy. Reverse transcription polymerase chain reaction and immunocytochemical analyses were used to evaluate the gene expression profiles of the VPA-induced hepatic progenitors and the hepatic progenitor-derived hepatocytes. Glycogen storage, cytochrome P450 activity, transplantation assay, differentiation of bile duct-like structures and tumorigenic analyses were performed for the functional identification of the differentiated cells. Furthermore, FACS and electron microscopy were used for the analyses of cell cycle profile and apoptosis in VPA-induced hepatic differentiated cells. RESULTS: Based on the combination of VPA and cytokines, mouse ES cells differentiated into a uniform and homogeneous cell population of hepatic progenitor cells and then matured into functional hepatocytes. The progenitor population shared several characteristics with ES cells and hepatic stem/progenitor cells, and represented a novel progenitor cell between ES and hepatic oval cells in embryonic development. The differentiated hepatocytes from progenitor cells shared typical characteristics with mature hepatocytes, including the patterns of gene expression, immunological markers, in vitro hepatocyte functions and in vivo capacity to restore acute-damaged liver function. In addition, the differentiation of hepatic progenitor cells from ES cells was accompanied by significant cell cycle arrest and selective survival of differentiating cells towards hepatic lineages. CONCLUSION: Hepatic cells

  16. Autocrine fibroblast growth factor 18 mediates dexamethasone-induced osteogenic differentiation of murine mesenchymal stem cells.

    PubMed

    Hamidouche, Zahia; Fromigué, Olivia; Nuber, Ulrike; Vaudin, Pascal; Pages, Jean-Christophe; Ebert, Regina; Jakob, Franz; Miraoui, Hichem; Marie, Pierre J

    2010-08-01

    The potential of mesenchymal stem cells (MSC) to differentiate into functional bone forming cells provides an important tool for bone regeneration. The identification of factors capable of promoting osteoblast differentiation in MSCs is therefore critical to enhance the osteogenic potential of MSCs. Using microarray analysis combined with biochemical and molecular approach, we found that FGF18, a member of the FGF family, is upregulated during osteoblast differentiation induced by dexamethasone in murine MSCs. We showed that overexpression of FGF18 by lentiviral (LV) infection, or treatment of MSCs with recombinant human (rh)FGF18 increased the expression of the osteoblast specific transcription factor Runx2, and enhanced osteoblast phenotypic marker gene expression and in vitro osteogenesis. Molecular silencing using lentiviral shRNA demonstrated that downregulation of FGFR1 or FGFR2 abrogated osteoblast gene expression induced by either LV-FGF18 or rhFGF18, indicating that FGF18 enhances osteoblast differentiation in MSCs via activation of FGFR1 or FGFR2 signaling. Biochemical and pharmacological analyses showed that the induction of phenotypic osteoblast markers by LV-FGF18 is mediated by activation of ERK1/2-MAPKs and PI3K signaling in MSCs. These results reveal that FGF18 is an essential autocrine positive regulator of the osteogenic differentiation program in murine MSCs and indicate that osteogenic differentiation induced by FGF18 in MSCs is triggered by FGFR1/FGFR2-mediated ERK1/2-MAPKs and PI3K signaling. PMID:20432451

  17. A Cell-Based High-Throughput Screening for Inducers of Myeloid Differentiation

    PubMed Central

    Radomska, Hanna S.; Jernigan, Finith; Nakayama, Sohei; Jorge, Susan E.; Sun, Lijun; Tenen, Daniel G.; Kobayashi, Susumu S.

    2015-01-01

    Recent progress of genetic studies has dramatically unveiled pathogenesis of acute myeloid leukemia (AML). However, overall survival of AML still remains unsatisfactory and development of novel therapeutics is required. CCAAT/Enhancer Binding Protein α (C/EBPα) is one of crucial transcription factors that induce granulocytic differentiation and its activity is perturbed in human myeloid leukemias. As its re-expression can induce differentiation and subsequent apoptosis of leukemic cells in vitro, we hypothesized that chemical compounds that restore C/EBPα expression and/or activity would lead to myeloid differentiation of leukemic cells. Using a cell-based high-throughput screening, we identified 2-[(E)-2-(3,4-dihydroxyphenyl)vinyl]-3-(2-methoxyphenyl)-4(3H)-quinazolinone as a potent inducer of C/EBPα and myeloid differentiation. Leukemia cell lines and primary blast cells isolated from human AML patients treated with ICCB280 demonstrated evidence of morphological and functional differentiation, as well as massive apoptosis. We performed conformational analyses of the high-throughput screening hit compounds to postulate the spatial requirements for high potency. Our results warrant a development of novel differentiation therapies and significantly impact care of AML patients with unfavorable prognosis in the near future. PMID:26109609

  18. A Cell-Based High-Throughput Screening for Inducers of Myeloid Differentiation.

    PubMed

    Radomska, Hanna S; Jernigan, Finith; Nakayama, Sohei; Jorge, Susan E; Sun, Lijun; Tenen, Daniel G; Kobayashi, Susumu S

    2015-10-01

    Recent progress of genetic studies has dramatically unveiled pathogenesis of acute myeloid leukemia (AML). However, overall survival of AML still remains unsatisfactory, and development of novel therapeutics is required. CCAAT/enhancer binding protein α (C/EBPα) is one of the crucial transcription factors that induce granulocytic differentiation, and its activity is perturbed in human myeloid leukemias. As its reexpression can induce differentiation and subsequent apoptosis of leukemic cells in vitro, we hypothesized that chemical compounds that restore C/EBPα expression and/or activity would lead to myeloid differentiation of leukemic cells. Using a cell-based high-throughput screening, we identified 2-[(E)-2-(3,4-dihydroxyphenyl)vinyl]-3-(2-methoxyphenyl)-4(3H)-quinazolinone as a potent inducer of C/EBPα and myeloid differentiation. Leukemia cell lines and primary blast cells isolated from human patients with AML treated with ICCB280 demonstrated evidence of morphological and functional differentiation, as well as massive apoptosis. We performed conformational analyses of the high-throughput screening hit compounds to postulate the spatial requirements for high potency. Our results warrant a development of novel differentiation therapies and significantly affect care of patients with AML with unfavorable prognosis in the near future. PMID:26109609

  19. Derivation, expansion and differentiation of induced pluripotent stem cells in continuous suspension cultures

    PubMed Central

    Fluri, David A.; Tonge, Peter D.; Song, Hannah; Baptista, Ricardo P.; Shakiba, Nika; Shukla, Shreya; Clarke, Geoffrey; Nagy, Andras; Zandstra, Peter W.

    2016-01-01

    We demonstrate derivation of induced pluripotent stem cells (iPSCs) from terminally differentiated mouse cells in serum- and feeder-free stirred suspension cultures. Temporal analysis of global gene expression revealed high correlations between cells reprogrammed in suspension and cells reprogrammed in adhesion-dependent conditions. Suspension (S) reprogrammed iPSCs (SiPSCs) could be differentiated into all three germ layers in vitro and contributed to chimeric embryos in vivo. SiPSC generation allowed for efficient selection of reprogramming factor expressing cells based on their differential survival and proliferation in suspension. Seamless integration of SiPSC reprogramming and directed differentiation enabled the scalable production of functionally and phenotypically defined cardiac cells in a continuous single cell- and small aggregate-based process. This method is an important step towards the development of a robust PSC generation, expansion and differentiation technology. PMID:22447133

  20. Blocking p55PIK signaling inhibits proliferation and induces differentiation of leukemia cells.

    PubMed

    Wang, G; Deng, Y; Cao, X; Lai, S; Tong, Y; Luo, X; Feng, Y; Xia, X; Gong, J; Hu, J

    2012-11-01

    p55PIK, a regulatory subunit of phosphatidylinositol 3-kinases, promotes cell cycle progression by interacting with cell cycle modulators such as retinoblastoma protein (Rb) via its unique amino-terminal 24 amino-acid residue (N24). Overexpression of N24 specifically inhibits these interactions and leads to cell cycle arrest. Herein, we describe the generation of a fusion protein (Tat transactivator protein (TAT)-N24) that contains the protein transduction domain and N24, and examined its effects on the proliferation and differentiation of leukemia cells. TAT-N24 not only blocks cell proliferation but remarkably induces differentiation of leukemia cells in vitro and in vivo. Systemically administered TAT-N24 also significantly decreases growth of leukemia cell tumors in animal models. Furthermore, overexpression of p55PIK in leukemia cells leads to increased proliferation; however, TAT-N24 blocks this effect and concomitantly induces differentiation. There is significant upregulation of p55PIK mRNA and protein expression in leukemia cells from patients. TAT-N24 inhibits cell cycle progression and induces differentiation of bone marrow cells derived from patients with several different types of leukemia. These results show that cell-permeable N24 peptide induces leukemia cell differentiation and suggest that p55PIK may be a novel drug target for the treatment of hematopoetic malignancies. PMID:22722333

  1. Silver nanoparticles impede phorbol myristate acetate-induced monocyte-macrophage differentiation and autophagy

    NASA Astrophysics Data System (ADS)

    Xu, Yingying; Wang, Liming; Bai, Ru; Zhang, Tianlu; Chen, Chunying

    2015-09-01

    Monocytes/macrophages are important constituents of the innate immune system. Monocyte-macrophage differentiation is not only crucial for innate immune responses, but is also related to some cardiovascular diseases. Silver nanoparticles (AgNPs) are one of the most widely used nanomaterials because of their broad-spectrum antimicrobial properties. However, the effect of AgNPs on the functions of blood monocytes is scarcely reported. Here, we report the impedance effect of AgNPs on THP-1 monocyte differentiation, and that this effect was mediated by autophagy blockade and lysosomal impairment. Firstly, AgNPs inhibit phorbol 12-myristate 13-acetate (PMA)-induced monocyte differentiation by down-regulating both expression of surface marker CD11b and response to lipopolysaccharide (LPS) stimulation. Secondly, autophagy is activated during PMA-induced THP-1 monocyte differentiation, and the autophagy inhibitor chloroquine (CQ) can inhibit this process. Thirdly, AgNPs block the degradation of the autophagy substrate p62 and induce autophagosome accumulation, which demonstrates the blockade of autophagic flux. Fourthly, lysosomal impairments including alkalization and decrease of lysosomal membrane stability were observed in AgNP-treated THP-1 cells. In conclusion, we demonstrate that the impedance of monocyte-macrophage differentiation by AgNPs is mediated by autophagy blockade and lysosomal dysfunction. Our results suggest that crosstalk exists in different biological effects induced by AgNPs.

  2. Efficient differentiation of neural stem cells induced by the rat bone marrow stromal cells

    PubMed Central

    Gu, Ping; Qiu, Fu-Cheng; Han, Rui; Zhang, Zhong-Xia; Dong, Ci; Zhang, Li-Na; Wang, Yan-Yong; Ma, Qing-Ying; Yan, Bao-Yong

    2015-01-01

    Neural stem cells (NSCs) are valuable self-renewing cells that can maintain the capacity to differentiate into specific brain cell types. NSCs may repair and even replace the brain tissue, and ultimatley promoting the central nervous system regeneration. Therefore, it is important, for scientists and pjysicians, to study the method for efficient culture and differentiation of NSCs. Our previous study demonstrated that Bone Marrow Stromal Cells (BMSCs) can directly regulate the differentiation of NSCs into neurons, and soluble molecules excreted by BMSCs played a key role in this process. Hereby, we further identified the BMSCs-induced neurons could form the synapses, convey dopamine and express voltage-depend and receptor-depend calcium channels. Moreover, the extracellular signal-regulated protein kinase ERK1/2 pathway was founded to be involved in the process of neuron differentiation and proliferation by the in vitro experiments. Finally, by using protein array, we, for the first time, found that the cytokine-induced neutrophil chemoattractant-3 (CINC-3, a small molecule cytokine) can promote the leukocytes invasion into the inflammation site, and have the ability to induce mesencephal NSCs into neurons. Consequently, these positive findings suggested that our BMSCs-induced culture system could provide a useful tool to investigate the molecular mechanisms of neural differentiation of NSCs, which may be benifical for neurodegenerative diseases in the near future. PMID:26221209

  3. Field-induced negative differential spin lifetime in silicon

    NASA Astrophysics Data System (ADS)

    Li, Jing; Qing, Lan; Dery, Hanan; Appelbaum, Ian

    2012-02-01

    Using experimental measurements of spin transport in undoped silicon, we show that the electric field-induced thermal asymmetry between the electron and lattice systems substantially impacts the identity of the dominant spin relaxation mechanism. In contrast to the Elliott-Yafet theory where intraband phonon absorption leads to spin relaxation, here we induce phonon emission during which electrons are scattered between conduction band valleys that reside on different crystal axes. This leads to anomalous behavior, where reduction of the transit time between spin-injector and spin-detector with larger electric field is accompanied by a counterintuitive reduction in spin polarization and an apparent negative spin lifetime.[4pt] Work at UMD is supported by the Office of Naval Research and the National Science Foundation. We acknowledge the support of the Center for Nanophysics and Advanced Materials and Maryland NanoCenter and its FabLab. Work at UR is supported by AFOSR and NSF (No. FA9550-09-1-0493 and No. DMR 1124601).

  4. Hypoxia promotes thyroid differentiation of native murine induced pluripotent stem cells.

    PubMed

    Yang, Yipeng; Lu, Yunshu; Chen, Tong; Zhang, Shenglai; Chu, Bingfeng; Gong, Yurong; Zhao, Weixin; Zhu, Jian; Liu, Yingbin

    2016-01-01

    Hypothyroidism is a very common hormonal deficiency and the stem cell technology which developed in the recent years may offer a therapeutic strategy for treating this disorder. Hypoxia has been demonstrated to play an important role in embryonic formation and development and to modulate stem cell differentiation. However, the influence of oxygen tension on thyroid differentiation has not been studied. In this study, we used murine induced pluripotent stem (iPS) cells for thyroid cell differentiation under normoxic and hypoxic conditions and compared differentiation efficiency in morphology, function, gene and protein expression under both conditions. We found that hypoxia promoted adhesion and outgrowth of embryoid bodies (EBs) derived from murine iPS cells. Expression of endodermal markers (Foxa2 and Gata4) and thyroid transcription factors (Pax8 and Nkx2.1) was increased by hypoxia at both gene and protein levels during early-mid differentiation stages (p<0.05). And so were the thyroid specific markers NIS and TSHR at the end of the experiment (p<0.05). In addition, functional iodide uptake by differentiated cells was also increased after hypoxia. Thyroid differentiation from iPS cells is enhanced under hypoxia and this may involve hypoxia inducible factors (HIFs) and their downstream gene FGF2. Our data offer a foundation for understanding thyroid development and provide a potentially more efficient way to use cell therapy for treating thyroid deficiency. PMID:27389981

  5. Field-Induced Negative Differential Spin Lifetime in Silicon

    NASA Astrophysics Data System (ADS)

    Li, Jing; Qing, Lan; Dery, Hanan; Appelbaum, Ian

    2012-04-01

    We show that the electric-field-induced thermal asymmetry between the electron and lattice systems in pure silicon substantially impacts the identity of the dominant spin relaxation mechanism. Comparison of empirical results from long-distance spin transport devices with detailed Monte Carlo simulations confirms a strong spin depolarization beyond what is expected from the standard Elliott-Yafet theory even at low temperatures. The enhanced spin-flip mechanism is attributed to phonon emission processes during which electrons are scattered between conduction band valleys that reside on different crystal axes. This leads to anomalous behavior, where (beyond a critical field) reduction of the transit time between spin-injector and spin-detector is accompanied by a counterintuitive reduction in spin polarization and an apparent negative spin lifetime.

  6. Differential Tomato Transcriptomic Responses Induced by Pepino Mosaic Virus Isolates with Differential Aggressiveness1[W][OA

    PubMed Central

    Hanssen, Inge M.; Peter van Esse, H.; Ballester, Ana-Rosa; Hogewoning, Sander W.; Parra, Nelia Ortega; Paeleman, Anneleen; Lievens, Bart; Bovy, Arnaud G.; Thomma, Bart P.H.J.

    2011-01-01

    Pepino mosaic virus (PepMV) is a highly infectious potexvirus and a major disease of greenhouse tomato (Solanum lycopersicum) crops worldwide. Damage and economic losses caused by PepMV vary greatly and can be attributed to differential symptomatology caused by different PepMV isolates. Here, we used a custom-designed Affymetrix tomato GeneChip array with probe sets to interrogate over 22,000 tomato transcripts to study transcriptional changes in response to inoculation of tomato seedlings with a mild and an aggressive PepMV isolate that share 99.4% nucleotide sequence identity. The two isolates induced a different transcriptomic response, despite accumulating to similar viral titers. PepMV inoculation resulted in repression of photosynthesis. In addition, defense responses were stronger upon inoculation with the aggressive isolate, in both cases mediated by salicylic acid signaling rather than by jasmonate signaling. Our results furthermore show that PepMV differentially regulates the RNA silencing pathway, suggesting a role for a PepMV-encoded silencing suppressor. Finally, perturbation of pigment biosynthesis, as shown by differential regulation of the flavonoid and lycopene biosynthesis pathways, was monitored. Metabolite analyses on mature fruits of PepMV-infected tomato plants, which showed typical fruit marbling, revealed a decrease in carotenoids, likely responsible for the marbled phenotype, and an increase in alkaloids and phenylpropanoids that are associated with pathogen defense in the yellow sectors of the fruit. PMID:21427280

  7. Differentiation of traumatic and heat-induced dental tissue fractures via SEM analysis.

    PubMed

    Campbell, Miranda N; Fairgrieve, Scott I

    2011-05-01

    Previous studies have examined the effects of heating on teeth; however, none have identified characteristics that allow analysts to differentiate traumatic from heat-induced fractures. This study examined our ability to discern notable differences in preincineration traumatic fractures and heat-induced fractures in postincineration dentition. Twelve anterior dental specimens were subjected to blunt force trauma while a second set were not. All 24 samples were then incinerated in a muffle furnace at a peak temperature (900°C) consistent with house fires. The specimens were subsequently examined with a scanning electron microscope to identify and compare heat-induced and traumatic fractures. The results obtained during examination yielded no differences between the features displayed by specimens that had been inflicted with preincineration trauma and those that did not. Unlike bone, distinguishing features for the differentiation of traumatic and heat-induced fractures could not be compiled. PMID:21521217

  8. BMP4 and FGF strongly induce differentiation of mouse ES cells into oral ectoderm.

    PubMed

    Ochiai, Hiroshi; Suga, Hidetaka; Yamada, Tomiko; Sakakibara, Mayu; Kasai, Takatoshi; Ozone, Chikafumi; Ogawa, Koichiro; Goto, Motomitsu; Banno, Ryoichi; Tsunekawa, Shin; Sugimura, Yoshihisa; Arima, Hiroshi; Oiso, Yutaka

    2015-09-01

    During embryonic development, oral ectoderm differentiates into the adenohypophysis, dental epithelia, salivary glands, and nasal pit. Few reports exist concerning the induction of oral ectoderm from embryonic stem (ES) cells. Generally, any lot differences in fetal bovine serum (FBS) and serum replacer may affect the induction of ES cell-differentiation. Using a previously established culture strategy for differentiation, the proportion of cell aggregates containing Pitx1+ oral ectoderm varied widely between 9-36% when several different lots of FBS or serum replacer were used. We therefore tried to enhance the differentiation method. We found that bone morphogenetic protein (BMP) 4 and fibroblast growth factor (FGF) treatments improved oral ectoderm induction. Such treatment also improved the differentiation of oral ectoderm into the adenohypophysis. Furthermore, increased BMP4 treatment induced dental epithelium and mesenchyme. Such differentiation suggests that the Pitx1+ layer displays similar properties to oral ectoderm, as found in vivo. Differentiation of ES cells into oral ectoderm using different lots of FBS and serum replacer increased 78-90% after treatment with BMP4 and FGF. In summary, we have established a robust strategy for the induction of oral ectoderm differentiation from mouse ES cells. PMID:26209816

  9. Differentiation-specific increase in ALA-induced protoporphyrin IX accumulation in primary mouse keratinocytes.

    PubMed Central

    Ortel, B.; Chen, N.; Brissette, J.; Dotto, G. P.; Maytin, E.; Hasan, T.

    1998-01-01

    A treatment regimen that takes advantage of the induction of intracellular porphyrins such as protoporphyrin IX (PPIX) by exposure to exogenous 5-amino-laevulinic acid (ALA) followed by localized exposure to visible light represents a promising new approach to photodynamic therapy (PDT). Acting upon the suggestion that the effectiveness of ALA-dependent PDT may depend upon the state of cellular differentiation, we investigated the effect of terminal differentiation upon ALA-induced synthesis of and the subsequent phototoxicity attributable to PPIX in primary mouse keratinocytes. Induction of keratinocyte differentiation augmented intracellular PPIX accumulation in cells treated with ALA. These elevated PPIX levels resulted in an enhanced lethal photodynamic sensitization of differentiated cells. The differentiation-dependent increase in cellular PPIX levels resulted from several factors including: (a) increased ALA uptake, (b) enhanced PPIX production and (c) decreased PPIX export into the culture media. Simultaneously, steady-state levels of coproporphyrinogen oxidase mRNA increased but aminolaevulinic acid dehydratase mRNA levels remained unchanged. From experiments using 12-o-tetradecanoylphorbol-13-acetate, transforming growth factor beta 1 and calcimycin we demonstrated that the increase in PPIX concentration in terminally differentiating keratinocytes is calcium- and differentiation specific. Stimulation of the haem synthetic capacity is seen in primary keratinocytes, but not in PAM 212 cells that fail to undergo differentiation. Interestingly, increased PPIX formation and elevated coproporphyrinogen oxidase mRNA levels are not limited to differentiating keratinocytes; these were also elevated in the C2C12 myoblast and the PC12 adrenal cell lines upon induction of differentiation. Overall, the therapeutic implications of these results are that the effectiveness of ALA-dependent PDT depends on the differentiation status of the cell and that this may enable

  10. Ameloblasts serum-free conditioned medium: bone morphogenic protein 4-induced odontogenic differentiation of mouse induced pluripotent stem cells.

    PubMed

    Liu, Li; Liu, Ying-Feng; Zhang, Jing; Duan, Yin-Zhong; Jin, Yan

    2016-06-01

    Induced pluripotent stem (iPS) cells possess the ability of self-renewal and can differentiate into cells of the three germ layers, both in vitro and in vivo. Here we report a new method to efficiently induce differentiation of mouse iPS cells into the odontogenic lineage. Using ameloblasts serum-free conditioned medium (ASF-CM), we successfully generated ameloblast-like cells from mouse iPS cells. Importantly, culturing mouse iPS cells in ASF-CM supplemented with BMP4 (ASF-BMP4) promoted odontogenic differentiation, which was evident by the upregulation of ameloblast-specific as well as odontoblast-specific genes. On the other hand, culturing mouse iPS cells in ASF-CM supplemented with noggin (ASF-noggin), an inhibitor of BMP4, abrogated this effect. These results suggest that mouse iPS cells can be induced by ASF-BMP4 to differentiate into ameloblast-like and odontoblast-like cells. The results of our study raise the possibility of using patient-specific iPS cells for tooth regeneration in the future. Copyright © 2016 John Wiley & Sons, Ltd. PMID:23606575

  11. Differential sensitivity of mouse oocytes to colchicine-induced aneuploidy

    SciTech Connect

    Mailhes, J.B.; Yuan, Z.P.

    1987-01-01

    Unpublished results from our laboratory showed that colchicine increased the incidence of hyperploid mouse metaphase II (MII) oocytes when injected at the same time as human chorionic gonadotrophin (HCG). The objective of the present study was to determine whether the time of administering colchicine influenced the incidence of aneuploidy in MII oocytes. CD-1 mice were given pregnant mare's serum (PMS) and, 48 hr later, HCG. An intraperitoneal injection of 0.2 mg/kg colchicine was given at +4, +2, 0, -2, or -4 hr relative to HCG. Oocytes were collected 17 hr post-HCG and processed, and chromosomes were subsequently C-banded. The percentage of hyperploid oocytes was 0.77, 2.56, 5.71, 7.79, 3.54, and 2.70 for control, +4, +2, 0, -2, or -4 hr pre/post-HCG, respectively. Chi-square analyses of these data demonstrated that colchicine significantly increases the proportion of aneuploid oocytes, and that the relative sensitivity of colchicine-induced aneuploidy depends upon the time that this drug is administered relative to HCG.

  12. Murine bone cell lines as models for spaceflight induced effects on differentiation and gene expression

    NASA Astrophysics Data System (ADS)

    Lau, P.; Hellweg, C. E.; Baumstark-Khan, C.; Reitz, G.

    Critical health factors for space crews especially on long-term missions are radiation exposure and the absence of gravity DNA double strand breaks DSB are presumed to be the most deleterious DNA lesions after radiation as they disrupt both DNA strands in close proximity Besides radiation risk the absence of gravity influences the complex skeletal apparatus concerning muscle and especially bone remodelling which results from mechanical forces exerting on the body Bone is a dynamic tissue which is life-long remodelled by cells from the osteoblast and osteoclast lineage Any imbalance of this system leads to pathological conditions such as osteoporosis or osteopetrosis Osteoblastic cells play a crucial role in bone matrix synthesis and differentiate either into bone-lining cells or into osteocytes Premature terminal differentiation has been reported to be induced by a number of DNA damaging or cell stress inducing agents including ionising and ultraviolet radiation as well as treatment with mitomycin C In the present study we compare the effects of sequential differentiation by adding osteoinductive substances ss -glycerophosphate and ascorbic acid Radiation-induced premature differentiation was investigated regarding the biosynthesis of specific osteogenic marker molecules and the differentiation dependent expression of marker genes The bone cell model established in our laboratory consists of the osteocyte cell line MLO-Y4 the osteoblast cell line OCT-1 and the subclones 4 and 24 of the osteoblast cell line MC3T3-E1 expressing several

  13. Abscisic-acid-induced cellular apoptosis and differentiation in glioma via the retinoid acid signaling pathway.

    PubMed

    Zhou, Nan; Yao, Yu; Ye, Hongxing; Zhu, Wei; Chen, Liang; Mao, Ying

    2016-04-15

    Retinoid acid (RA) plays critical roles in regulating differentiation and apoptosis in a variety of cancer cells. Abscisic acid (ABA) and RA are direct derivatives of carotenoids and share structural similarities. Here we proposed that ABA may also play a role in cellular differentiation and apoptosis by sharing a similar signaling pathway with RA that may be involved in glioma pathogenesis. We reported for the first time that the ABA levels were twofold higher in low-grade gliomas compared with high-grade gliomas. In glioma tissues, there was a positive correlation between the ABA levels and the transcription of cellular retinoic acid-binding protein 2 (CRABP2) and a negative correlation between the ABA levels and transcription of fatty acid-binding protein 5 (FABP5). ABA treatment induced a significant increase in the expression of CRABP2 and a decrease in the expression of peroxisome proliferator-activated receptor (PPAR) in glioblastoma cells. Remarkably, both cellular apoptosis and differentiation were increased in the glioblastoma cells after ABA treatment. ABA-induced cellular apoptosis and differentiation were significantly reduced by selectively silencing RAR-α, while RAR-α overexpression exaggerated the ABA-induced effects. These results suggest that ABA may play a role in the pathogenesis of glioma by promoting cellular apoptosis and differentiation through the RA signaling pathway. PMID:26594836

  14. Lignin Induces ES Cells to Differentiate into Neuroectodermal Cells through Mediation of the Wnt Signaling Pathway

    PubMed Central

    Inoue, Yu; Hasegawa, Seiji; Yamada, Takaaki; Date, Yasushi; Mizutani, Hiroshi; Nakata, Satoru; Akamatsu, Hirohiko

    2013-01-01

    Embryonic stem cells (ES cells) are characterized by their pluripotency and infinite proliferation potential. Ever since ES cells were first established in 1981, there have been a growing number of studies aimed at clinical applications of ES cells. In recent years, various types of differentiation inducement systems using ES cells have been established. Further studies have been conducted to utilize differentiation inducement systems in the field of regenerative medicine. For cellular treatments using stem cells including ES cells, differentiation induction should be performed in a sufficient manner to obtain the intended cell lineages. Lignin is a high-molecular amorphous material that forms plants together with cellulose and hemicelluloses, in which phenylpropane fundamental units are complexly condensed. Lignin derivatives have been shown to have several bioactive functions. In spite of these findings, few studies have focused on the effects of lignin on stem cells. Our study aimed to develop a novel technology using lignin to effectively induce ES cells to differentiate into neuroectodermal cells including ocular cells and neural cells. Since lignin can be produced at a relatively low cost in large volumes, its utilization is expected for more convenient differentiation induction technologies and in the field of regenerative medicine in the future. PMID:23805217

  15. Differential Sympathetic Vasomotor Activation Induced by Liver Cirrhosis in Rats

    PubMed Central

    Bergamaschi, Cássia T.; Campos, Ruy R.

    2016-01-01

    We tested the hypothesis that there is a topographical sympathetic activation in rats submitted to experimental cirrhosis. Baseline renal (rSNA) and splanchnic (sSNA) sympathetic nerve activities were evaluated in anesthetized rats. In addition, we evaluated main arterial pressure (MAP), heart rate (HR), and baroreceptor reflex sensitivity (BRS). Cirrhotic Wistar rats were obtained by bile duct ligation (BDL). MAP and HR were measured in conscious rats, and cardiac BRS was assessed by changes in blood pressure induced by increasing doses of phenylephrine or sodium nitroprusside. The BRS and baseline for the control of sSNA and rSNA were also evaluated in urethane-anesthetized rats. Cirrhotic rats had increased baseline sSNA (BDL, 102 vs control, 58 spikes/s; p<0.05), but no baseline changes in the rSNA compared to controls. These data were accompanied by increased splanchnic BRS (p<0.05) and decreased cardiac (p<0.05) and renal BRS (p<0.05). Furthermore, BDL rats had reduced basal MAP (BDL, 93 vs control, 101 mmHg; p<0.05) accompanied by increased HR (BDL, 378 vs control, 356; p<0.05). Our data have shown topographical sympathetic activation in rats submitted to experimental cirrhosis. The BDL group had increased baseline sSNA, independent of dysfunction in the BRS and no changes in baseline rSNA. However, an impairment of rSNA and HR control by arterial baroreceptor was noted. We suggest that arterial baroreceptor impairment of rSNA and HR is an early marker of cardiovascular dysfunction related to liver cirrhosis and probably a major mechanism leading to sympathoexcitation in decompensated phase. PMID:27055088

  16. Nicotinamide induces differentiation of embryonic stem cells into insulin-secreting cells

    SciTech Connect

    Vaca, Pilar; Berna, Genoveva; Araujo, Raquel; Carneiro, Everardo M.; Bedoya, Francisco J.; Soria, Bernat; Martin, Franz

    2008-03-10

    The poly(ADP-ribose) polymerase (PARP) inhibitor, nicotinamide, induces differentiation and maturation of fetal pancreatic cells. In addition, we have previously reported evidence that nicotinamide increases the insulin content of cells differentiated from embryonic stem (ES) cells, but the possibility of nicotinamide acting as a differentiating agent on its own has never been completely explored. Islet cell differentiation was studied by: (i) X-gal staining after neomycin selection; (ii) BrdU studies; (iii) single and double immunohistochemistry for insulin, C-peptide and Glut-2; (iv) insulin and C-peptide content and secretion assays; and (v) transplantation of differentiated cells, under the kidney capsule, into streptozotocin (STZ)-diabetic mice. Here we show that undifferentiated mouse ES cells treated with nicotinamide: (i) showed an 80% decrease in cell proliferation; (ii) co-expressed insulin, C-peptide and Glut-2; (iii) had values of insulin and C-peptide corresponding to 10% of normal mouse islets; (iv) released insulin and C-peptide in response to stimulatory glucose concentrations; and (v) after transplantation into diabetic mice, normalized blood glucose levels over 7 weeks. Our data indicate that nicotinamide decreases ES cell proliferation and induces differentiation into insulin-secreting cells. Both aspects are very important when thinking about cell therapy for the treatment of diabetes based on ES cells.

  17. Measurement of doubly differential electron distributions induced by atomic collisions: Apparatus and related instrumental effects

    NASA Astrophysics Data System (ADS)

    Bernardi, G.; Suárez, S.; Fregenal, D.; Focke, P.; Meckbach, W.

    1996-05-01

    We describe the experimental setup at Centro Atómico Bariloche for the measurement of doubly differential electron distributions, in energy and angle of emission, induced by atomic collisions. We present detailed information about the performance of the equipment including a discussion of instrumental effects that could have affected the measured spectra.

  18. Differentiation-induced skin cancer suppression by FOS, p53, and TACE/ADAM17.

    PubMed

    Guinea-Viniegra, Juan; Zenz, Rainer; Scheuch, Harald; Jiménez, María; Bakiri, Latifa; Petzelbauer, Peter; Wagner, Erwin F

    2012-08-01

    Squamous cell carcinomas (SCCs) are heterogeneous and aggressive skin tumors for which innovative, targeted therapies are needed. Here, we identify a p53/TACE pathway that is negatively regulated by FOS and show that the FOS/p53/TACE axis suppresses SCC by inducing differentiation. We found that epidermal Fos deletion in mouse tumor models or pharmacological FOS/AP-1 inhibition in human SCC cell lines induced p53 expression. Epidermal cell differentiation and skin tumor suppression were caused by a p53-dependent transcriptional activation of the metalloprotease TACE/ADAM17 (TNF-α-converting enzyme), a previously unknown p53 target gene that was required for NOTCH1 activation. Although half of cutaneous human SCCs display p53-inactivating mutations, restoring p53/TACE activity in mouse and human skin SCCs induced tumor cell differentiation independently of the p53 status. We propose FOS/AP-1 inhibition or p53/TACE reactivating strategies as differentiation-inducing therapies for SCCs. PMID:22772468

  19. Differentiating stress to wheat fields induced by Diuraphis noxia from other stress causing factors

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this study was to develop a method to differentiate two categories of stress to wheat fields, stress induced by the Russian wheat aphid, Diuraphis noxia (Mordvilko), and stress caused by other factors. The study used a set of 11 spatial pattern metrics derived from multispectral im...

  20. Differentiation-induced skin cancer suppression by FOS, p53, and TACE/ADAM17

    PubMed Central

    Guinea-Viniegra, Juan; Zenz, Rainer; Scheuch, Harald; Jiménez, María; Bakiri, Latifa; Petzelbauer, Peter; Wagner, Erwin F.

    2012-01-01

    Squamous cell carcinomas (SCCs) are heterogeneous and aggressive skin tumors for which innovative, targeted therapies are needed. Here, we identify a p53/TACE pathway that is negatively regulated by FOS and show that the FOS/p53/TACE axis suppresses SCC by inducing differentiation. We found that epidermal Fos deletion in mouse tumor models or pharmacological FOS/AP-1 inhibition in human SCC cell lines induced p53 expression. Epidermal cell differentiation and skin tumor suppression were caused by a p53-dependent transcriptional activation of the metalloprotease TACE/ADAM17 (TNF-α–converting enzyme), a previously unknown p53 target gene that was required for NOTCH1 activation. Although half of cutaneous human SCCs display p53-inactivating mutations, restoring p53/TACE activity in mouse and human skin SCCs induced tumor cell differentiation independently of the p53 status. We propose FOS/AP-1 inhibition or p53/TACE reactivating strategies as differentiation-inducing therapies for SCCs. PMID:22772468

  1. Am80 induces neuronal differentiation in a human neuroblastoma NH-12 cell line.

    PubMed

    Shiohira, Hideo; Kitaoka, Akira; Shirasawa, Hiromi; Enjoji, Munechika; Nakashima, Manabu

    2010-09-01

    Retinoids including natural vitamin A, its derivatives and synthetic compounds work as transcription factors through the retinoic acid receptors (RAR, RXR). All-trans retinoic acid (ATRA), a family of retinoids, is an internal ligand of RAR and well known as a useful differentiation inducer to treat acute promyelocytic leukemia (APL). ATRA therapy is now established as an initial treatment for APL. Recently, to improve therapeutic potency and reduce adverse effects of ATRA, a novel synthetic selective agonist for RARalpha and beta, Am80, was developed and applied to APL treatment. In this study, we tested whether Am80 was capable of inducing neuronal differentiation in a human neuroblastoma cell line, NH-12 and compared the differentiation effects between Am80 and ATRA. Morphological studies demonstrated that Am80 induced more potent neurite outgrowth and also proved lesser cell toxicity than ATRA. Am80 up-regulated the expression of tropomyosin-related kinase B as well as ATRA. Moreover, Am80 increased the expression of the neuronal marker, growth-associated protein 43. These findings suggest that Am80 induces neuronal differentiation to a greater extent than ATRA and thus may help establishing therapeutic strategies against neuronal degenerative disorders such as Parkinson's disease. PMID:20664956

  2. Local Probing of Electrochemically Induced Negative Differential Resistance in TiO2 Memristive Materials

    SciTech Connect

    Kim, Yunseok; Jesse, Stephen; Kalinin, Sergei V

    2013-01-01

    Early stage of electroforming in TiO2 was observed by deliberately combining conductive atomic force microscopy and electrochemical strain microscopy. The negative differential resistance and the corresponding surface deformation were observed below electroforming voltages. The surface deformations induced by surface oxidation are thermodynamically stable, reversibly controlled by applying voltage bias of different polarities, and electrochemically less active.

  3. How-to-Do-It: Cytokinin Induced Cell Division & Differentiation Using Intact Plants.

    ERIC Educational Resources Information Center

    Bohnsack, Charles W.

    1989-01-01

    Presents a procedure by which cytokinins are used to induce a population of dividing and differentiating cells on the cut surface of the roots of an intact plant. Includes the method used, results, and suggestions for a variety of variables that may be tested. (RT)

  4. Secreted Cyclic Di-GMP Induces Stalk Cell Differentiation in the Eukaryote Dictyostelium discoideum.

    PubMed

    Chen, Zhi-hui; Schaap, Pauline

    2016-01-01

    Cyclic di-GMP (c-di-GMP) is currently recognized as the most widely used intracellular signal molecule in prokaryotes, but roles in eukaryotes were only recently discovered. In the social amoeba Dictyostelium discoideum, c-di-GMP, produced by a prokaryote-type diguanylate cyclase, induces the differentiation of stalk cells, thereby enabling the formation of spore-bearing fruiting bodies. In this review, we summarize the currently known mechanisms that control the major life cycle transitions of Dictyostelium and focus particularly on the role of c-di-GMP in stalk formation. Stalk cell differentiation has characteristics of autophagic cell death, a process that also occurs in higher eukaryotes. We discuss the respective roles of c-di-GMP and of another signal molecule, differentiation-inducing factor 1, in autophagic cell death in vitro and in stalk formation in vivo. PMID:26013485

  5. Maintenance and neuronal differentiation of chicken induced pluripotent stem-like cells.

    PubMed

    Dai, Rui; Rossello, Ricardo; Chen, Chun-Chun; Kessler, Joeran; Davison, Ian; Hochgeschwender, Ute; Jarvis, Erich D

    2014-01-01

    Pluripotent stem cells have the potential to become any cell in the adult body, including neurons and glia. Avian stem cells could be used to study questions, like vocal learning, that would be difficult to examine with traditional mouse models. Induced pluripotent stem cells (iPSCs) are differentiated cells that have been reprogrammed to a pluripotent stem cell state, usually using inducing genes or other molecules. We recently succeeded in generating avian iPSC-like cells using mammalian genes, overcoming a limitation in the generation and use of iPSCs in nonmammalian species (Rosselló et al., 2013). However, there were no established optimal cell culture conditions for avian iPSCs to establish long-term cell lines and thus to study neuronal differentiation in vitro. Here we present an efficient method of maintaining chicken iPSC-like cells and for differentiating them into action potential generating neurons. PMID:25610469

  6. Maintenance and Neuronal Differentiation of Chicken Induced Pluripotent Stem-Like Cells

    PubMed Central

    Rossello, Ricardo; Chen, Chun-chun; Kessler, Joeran; Davison, Ian; Jarvis, Erich D.

    2014-01-01

    Pluripotent stem cells have the potential to become any cell in the adult body, including neurons and glia. Avian stem cells could be used to study questions, like vocal learning, that would be difficult to examine with traditional mouse models. Induced pluripotent stem cells (iPSCs) are differentiated cells that have been reprogrammed to a pluripotent stem cell state, usually using inducing genes or other molecules. We recently succeeded in generating avian iPSC-like cells using mammalian genes, overcoming a limitation in the generation and use of iPSCs in nonmammalian species (Rosselló et al., 2013). However, there were no established optimal cell culture conditions for avian iPSCs to establish long-term cell lines and thus to study neuronal differentiation in vitro. Here we present an efficient method of maintaining chicken iPSC-like cells and for differentiating them into action potential generating neurons. PMID:25610469

  7. Secreted Cyclic Di-GMP Induces Stalk Cell Differentiation in the Eukaryote Dictyostelium discoideum

    PubMed Central

    Chen, Zhi-hui

    2015-01-01

    Cyclic di-GMP (c-di-GMP) is currently recognized as the most widely used intracellular signal molecule in prokaryotes, but roles in eukaryotes were only recently discovered. In the social amoeba Dictyostelium discoideum, c-di-GMP, produced by a prokaryote-type diguanylate cyclase, induces the differentiation of stalk cells, thereby enabling the formation of spore-bearing fruiting bodies. In this review, we summarize the currently known mechanisms that control the major life cycle transitions of Dictyostelium and focus particularly on the role of c-di-GMP in stalk formation. Stalk cell differentiation has characteristics of autophagic cell death, a process that also occurs in higher eukaryotes. We discuss the respective roles of c-di-GMP and of another signal molecule, differentiation-inducing factor 1, in autophagic cell death in vitro and in stalk formation in vivo. PMID:26013485

  8. Strain Gauges Indicate Differential-CTE-Induced Failures

    NASA Technical Reports Server (NTRS)

    Harris, Brian

    2007-01-01

    A method of detecting mechanical failure induced by variation in temperature at an adhesive bond between two materials that have different coefficients of thermal expansion (CTEs) involves monitoring of strain-gauge readings. This method can be regarded as an exploitation of the prior observation that the readings of strain gauges commonly used in tensile and compressive testing of material specimens include features indicative of incremental failures in the specimens. In this method, one or more strain gauges are bonded to either or both of the two materials near the bond between the materials. (The adhesive used to bond the strain gauges would not ordinarily be the same as the one used to bond the two materials). Then strain-gauge readings are recorded as the temperature of the materials is varied through a range of interest. Any significant discontinuity in the slope of the resulting strain-versus-temperature curve(s) is taken to be a qualitative indication of a failure of the bond between the two materials and/or a failure within one of the materials in the vicinity of the bond. The method has been demonstrated in experiments on specimens consisting of polyacrylonitrile-fiber/epoxy-matrix laminated composite plates bonded by epoxy to smaller plates made, variously, of aluminum, titanium, and a low-CTE nickel/iron alloy. In preparation for each experiment, strain gauges were bonded, by use of cryogenic-rated adhesives, to the composite plate near the corners of the metal plate (see Figure 1). In each experiment, strain-gauge and temperature readings were taken as the specimen was cooled from room temperature to 20 K. The specimens were then returned to room temperature and ultrasonically inspected for damage in the bond region. No failure events were detectable in the strain-gauge readings from the composite/ titanium and composite/low-thermalexpansion- alloy specimens, and ultrasonic inspection of these specimens revealed no damage. However, failure events were

  9. Smurf1 plays a role in EGF inhibition of BMP2-induced osteogenic differentiation

    SciTech Connect

    Lee, Hye-Lim; Park, Hyun-Jung; Kwon, Arang; Baek, Kyunghwa; Woo, Kyung Mi; Ryoo, Hyun-Mo; Kim, Gwan-Shik; Baek, Jeong-Hwa

    2014-05-01

    It has been demonstrated that epidermal growth factor (EGF) plays a role in supporting the proliferation of bone marrow stromal cells in bone but inhibits their osteogenic differentiation. However, the mechanism underlying EGF inhibition of osteoblast differentiation remains unclear. Smurf1 is an E3 ubiquitin ligase that targets Smad1/5 and Runx2, which are critical transcription factors for bone morphogenetic protein 2 (BMP2)-induced osteoblast differentiation. In this study, we investigated the effect of EGF on the expression of Smurf1, and the role of Smurf1 in EGF inhibition of osteogenic differentiation using C2C12 cells, a murine myoblast cell line. EGF increased Smurf1 expression, which was blocked by inhibiting the activity of either JNK or ERK. Chromatin immunoprecipitation and Smurf1 promoter assays demonstrated that c-Jun and Runx2 play roles in the EGF induction of Smurf1 transcription. EGF suppressed BMP2-induced expression of osteogenic marker genes, which were rescued by Smurf1 knockdown. EGF downregulated the protein levels of Runx2 and Smad1 in a proteasome-dependent manner. EGF decreased the transcriptional activity of Runx2 and Smurf1, which was partially rescued by Smurf1 silencing. Taken together, these results suggest that EGF increases Smurf1 expression via the activation of JNK and ERK and the subsequent binding of c-Jun and Runx2 to the Smurf1 promoter and that Smurf1 mediates the inhibitory effect of EGF on BMP2-induced osteoblast differentiation. - Highlights: • EGF increases the expression level of Smurf1 in mesenchymal precursor cells. • EGF reduces the protein levels and transcriptional activity of Runx2 and Smad1. • EGF suppresses BMP2-induced osteogenic differentiation, which is rescued by Smurf1 knockdown.

  10. Tryptanthrin induces growth inhibition and neuronal differentiation in the human neuroblastoma LA-N-1 cells.

    PubMed

    Liao, Xuemei; Leung, Kwok Nam

    2013-04-25

    Neuroblastoma is one of the most common extracranial solid cancers found in young children. The prognosis of neuroblastoma patients in advanced stages having N-myc amplification remains poor despite intensive multimodal therapy. Agents that trigger neuroblastoma cells to undergo cellular differentiation and thereby stop proliferation have attracted considerable interest as an alternative therapy. Tryptanthrin (12-dihydro-6,12-dioxoindolo-(2,1-b)-quinazoline) is a weakly basic alkaloid isolated from the dried roots of medicinal indigo plants known as Banlangen. It has been shown to possess various biological activities, such as anti-microbial, anti-inflammatory and anti-tumor activities. However, its effects and mechanism(s) of action on human neuroblastoma cells remain poorly understood. Therefore, the objective of this study is to investigate the effects of tryptanthrin on the growth and differentiation of human neuroblastoma LA-N-1 cells with N-myc amplification. Our results show that tryptanthrin inhibited the growth of the human neuroblastoma cells in a dose- and time-dependent manner. Mechanistic studies indicated that tryptanthrin induced cell cycle arrest of the human neuroblastoma LA-N-1 cells at the G0/G1 phase. Tryptanthrin also induced neuronal differentiation of LA-N-1 cells, as assessed by morphological criteria, enhancement of acetylcholine esterase activity and up-regulation of various differentiation markers. Moreover, tryptanthrin treatment led to the significant reduction of N-myc expression in LA-N-1 cells while siRNA directed against N-myc induced morphological differentiation of LA-N-1 cells. These results, when taken together, suggest that tryptanthrin suppressed the growth and induced neuronal differentiation in the human neuroblastoma LA-N-1 cells and might be exploited as a potential therapeutic candidate for the treatment of high-risk neuroblastomas with N-myc-amplification. PMID:23500671

  11. Differentiation patterns of mouse embryonic stem cells and induced pluripotent stem cells into neurons.

    PubMed

    Nakamura, Mai; Kamishibahara, Yu; Kitazawa, Ayako; Kawaguchi, Hideo; Shimizu, Norio

    2016-05-01

    Mouse embryonic stem (ES) cells and induced pluripotent stem (iPS) cells have the ability to differentiate in vitro into various cell lineages including neurons. The differentiation of these cells into neurons has potential applications in regenerative medicine. Previously, we reported that a chick dorsal root ganglion (DRG)-conditioned medium (CM) promoted the differentiation of mouse ES and iPS cells into neurons. Here, we used real-time PCR to investigate the differentiation patterns of ES and iPS cells into neurons when DRG-CM was added. DRG-CM promoted the expression levels of βIII-tubulin gene (a marker of postmitotic neurons) in ES and iPS cells. ES cells differentiated into neurons faster than iPS cells, and the maximum peaks of gene expression involved in motor, sensory, and dopaminergic neurons were different. Rho kinase (ROCK) inhibitors could be very valuable at numerous stages in the production and use of stem cells in basic research and eventual cell-based therapies. Thus, we investigated whether the addition of a ROCK inhibitor Y-27632 and DRG-CM on the basis of the differentiation patterns promotes the neuronal differentiation of ES cells. When the ROCK inhibitor was added to the culture medium at the initial stages of cultivation, it stimulated the neuronal differentiation of ES cells more strongly than that stimulated by DRG-CM. Moreover, the combination of the ROCK inhibitor and DRG-CM promoted the neuronal differentiation of ES cells when the ROCK inhibitor was added to the culture medium at day 3. The ROCK inhibitor may be useful for promoting neuronal differentiation of ES cells. PMID:25354731

  12. BMP-2 Induced Expression of PLCβ1 That is a Positive Regulator of Osteoblast Differentiation.

    PubMed

    Ramazzotti, Giulia; Bavelloni, Alberto; Blalock, William; Piazzi, Manuela; Cocco, Lucio; Faenza, Irene

    2016-03-01

    Bone morphogenetic protein 2 (BMP-2) is a critical growth factor that directs osteoblast differentiation and bone formation. Phosphoinositide-phospholipase Cβ 1 (PLCβ1) plays a crucial role in the initiation of the genetic program responsible for muscle differentiation. Differentiation of C2C12 mouse myoblasts in response to insulin stimulation is characterized by a marked increase in nuclear PLCβ1. Here, the function of PLCβ1 in the osteogenic differentiation was investigated. Briefly, in C2C12 cells treated with BMP-2 we assist to a remarkable increase in PLCβ1 protein and mRNA expression. The data regarding the influence on differentiation demonstrated that PLCβ1 promotes osteogenic differentiation by up-regulating alkaline phosphatase (ALP). Moreover, PLCβ1 is present in the nuclear compartment of these cells and overexpression of a cytosolic-PLCβ1mutant (cyt-PLCβ1), which lacks a nuclear localization sequence, prevented the differentiation of C2C12 cells into osteocytes. Recent evidence indicates that miRNAs act as important post transcriptional regulators in a large number of processes, including osteoblast differentiation. Since miR-214 is a regulator of Osterix (Osx) which is an osteoblast-specific transcription factor that is needful for osteoblast differentiation and bone formation, we further investigated whether PLCβ1 could be a potential target of miR-214 in the control of osteogenic differentiation by gain- and loss- of function experiment. The results indicated that inhibition of miR-214 in C2C12 cells significantly enhances the protein level of PLCβ1 and promotes C2C12 BMP-2-induced osteogenesis by targeting PLCβ1. PMID:26217938

  13. Inducer-mediated commitment of murine erythroleukemia cells to differentiation: a multistep process.

    PubMed Central

    Chen, Z; Banks, J; Rifkind, R A; Marks, P A

    1982-01-01

    There are a number of agents which, when added to cultures of murine erythroleukemia cells (MELC), markedly increase the probability of commitment to express the characteristics of terminal erythroid differentiation, including loss of proliferative capacity and increased accumulation of globin mRNA and hemoglobin. Some characteristics of inducer-mediated commitment of MELC to terminal erythroid differentiation were examined by determining the effects of dexamethasone (an inhibitor of inducer-mediated MELC differentiation) and of hemin (an inducer of globin mRNA accumulation). Previously, it was shown that exposure of MELC to hexamethylene-bisacetamide (HMBA) leads to commitment, detectable within 12 hr. MELC cultured with both HMBA and dexamethasone do not express commitment. MELC transferred from culture with HMBA and dexamethasone to cloning medium without these agents express commitment to terminal erythroid differentiation, indicating that MELC retain a "memory" for some early HMBA-mediated changes leading to commitment which occur even in the presence of the inhibitory steroid. The kinetics of commitment in experiments in which exposure to HMBA is interrupted, or dexamethasone is added to the culture in HMBA, suggest that there is a rate-limiting step early in the commitment process. The memory for this step persists for more than one cell cycle. Addition of hemin to cultures with HMBA and dexamethasone initiated accumulation of globin mRNA but does not reverse the steroid-mediated inhibition of terminal cell division (that is, the cells retain their proliferative capacity). Inducer-mediated MELC commitment is associated with accumulation of the chromatin protein IP25; dexamethasone does not inhibit this accumulation. Accumulation of IP25 may be inducer-related, but it is not sufficient to cause expression of terminal erythroid differentiation. Images PMID:6952199

  14. Phosphoprotein Phosphatase 1 Is Required for Extracellular Calcium-Induced Keratinocyte Differentiation

    PubMed Central

    Fan, Hong; Zeng, Qin; Pennypacker, Sally D.; Xie, Zhongjian

    2016-01-01

    Extracellular calcium is a major regulator of keratinocyte differentiation in vitro and appears to play that role in vivo, but the mechanism is unclear. We have previously demonstrated that, following calcium stimulation, PIP5K1α is recruited by the E-cadherin-β-catenin complex to the plasma membrane where it provides the substrate PIP2 for both PI3K and PLC-γ1. This signaling pathway is critical for calcium-induced generation of second messengers including IP3 and intracellular calcium and keratinocyte differentiation. In this study, we explored the upstream regulatory mechanism by which calcium activates PIP5K1α and the role of this activation in calcium-induced keratinocyte differentiation. We found that treatment of human keratinocytes in culture with calcium resulted in an increase in serine dephosphorylation and PIP5K1α activation. PP1 knockdown blocked extracellular calcium-induced increase in serine dephosphorylation and activity of PIP5K1α and induction of keratinocyte differentiation markers. Knockdown of PLC-γ1, the downstream effector of PIP5K1α, blocked upstream dephosphorylation and PIP5K1α activation induced by calcium. Coimmunoprecipitation revealed calcium induced recruitment of PP1 to the E-cadherin-catenin-PIP5K1α complex in the plasma membrane. These results indicate that PP1 is recruited to the extracellular calcium-dependent E-cadherin-catenin-PIP5K1α complex in the plasma membrane to activate PIP5K1α, which is required for PLC-γ1 activation leading to keratinocyte differentiation. PMID:27340655

  15. Phosphoprotein Phosphatase 1 Is Required for Extracellular Calcium-Induced Keratinocyte Differentiation.

    PubMed

    Shrestha, Chandrama; Tang, Yuanyuan; Fan, Hong; Li, Lusha; Zeng, Qin; Pennypacker, Sally D; Bikle, Daniel D; Xie, Zhongjian

    2016-01-01

    Extracellular calcium is a major regulator of keratinocyte differentiation in vitro and appears to play that role in vivo, but the mechanism is unclear. We have previously demonstrated that, following calcium stimulation, PIP5K1α is recruited by the E-cadherin-β-catenin complex to the plasma membrane where it provides the substrate PIP2 for both PI3K and PLC-γ1. This signaling pathway is critical for calcium-induced generation of second messengers including IP3 and intracellular calcium and keratinocyte differentiation. In this study, we explored the upstream regulatory mechanism by which calcium activates PIP5K1α and the role of this activation in calcium-induced keratinocyte differentiation. We found that treatment of human keratinocytes in culture with calcium resulted in an increase in serine dephosphorylation and PIP5K1α activation. PP1 knockdown blocked extracellular calcium-induced increase in serine dephosphorylation and activity of PIP5K1α and induction of keratinocyte differentiation markers. Knockdown of PLC-γ1, the downstream effector of PIP5K1α, blocked upstream dephosphorylation and PIP5K1α activation induced by calcium. Coimmunoprecipitation revealed calcium induced recruitment of PP1 to the E-cadherin-catenin-PIP5K1α complex in the plasma membrane. These results indicate that PP1 is recruited to the extracellular calcium-dependent E-cadherin-catenin-PIP5K1α complex in the plasma membrane to activate PIP5K1α, which is required for PLC-γ1 activation leading to keratinocyte differentiation. PMID:27340655

  16. Glucocorticoid-induced differentiation of fetal rat calvarial osteoblasts is mediated by bone morphogenetic protein-6.

    PubMed

    Boden, S D; Hair, G; Titus, L; Racine, M; McCuaig, K; Wozney, J M; Nanes, M S

    1997-07-01

    Glucocorticoids (GCs) at physiological concentrations promote osteoblast differentiation from fetal calvarial cells, calvarial organ cultures, and bone marrow stromal cells; however, the cellular pathways involved are not known. Bone morphogenetic proteins (BMPs) are recognized as important mediators of osteoblast differentiation. Specific roles for individual BMPs during postembryonic membranous bone formation have yet to be determined. We recently reported that GC potentiated the osteoblast differentiation effects of BMP-2 and BMP-4, but not of BMP-6, which, by itself, was the most potent of the three. In the present study, we used fetal rat secondary calvarial cultures to study the role of BMP-6 during early osteoblast differentiation. Treatment with the GC triamcinolone (10(-9) M) resulted in a 5- to 8-fold increase in BMP-6 steady-state messenger RNA levels, peaking at 12 h. In contrast, BMPs -2, -4, -5, -7, and transforming growth factor (TGF)-beta1 messenger RNA levels increased by less than 2-fold, after GC treatment, compared with untreated control cultures at 24 h. BMP-6 protein secretion increased 6- to 7-fold by 12 h and 12-fold (from 7.5 to 90 ng/ml) by 24 h, as measured by quantitative Western analysis. Treatment of cells with oligodeoxynucleotides antisense to BMP-6 diminished secretion of BMP-6 protein and significantly inhibited the GC-induced differentiation, as determined by a 10-fold decrease in the number of mineralized bone nodules, compared with controls that were treated with sense oligonucleotides or no oligonucleotides (ANOVA, P < 0.05). The antisense oligonucleotide inhibition of differentiation was rescued by treatment with exogenous recombinant human BMP-6. We conclude that GC-induced differentiation of osteoblasts from the pluripotent precursors is mediated, in part, by BMP-6. These results suggest that BMP-6 has an important and unique role during early osteoblast differentiation. PMID:9202223

  17. Severe Global DNA Hypomethylation Blocks Differentiation and Induces Histone Hyperacetylation in Embryonic Stem Cells

    PubMed Central

    Jackson, Melany; Krassowska, Anna; Gilbert, Nick; Chevassut, Timothy; Forrester, Lesley; Ansell, John; Ramsahoye, Bernard

    2004-01-01

    It has been reported that DNA methyltransferase 1-deficient (Dnmt1−/−) embryonic stem (ES) cells are hypomethylated (20% CpG methylation) and die through apoptosis when induced to differentiate. Here, we show that Dnmt[3a−/−,3b−/−] ES cells with just 0.6% of their CpG dinucleotides behave differently: the majority of cells within the culture are partially or completely blocked in their ability to initiate differentiation, remaining viable while retaining the stem cell characteristics of alkaline phosphatase and Oct4 expression. Restoration of DNA methylation levels rescues these defects. Severely hypomethylated Dnmt[3a−/−,3b−/−] ES cells have increased histone acetylation levels, and those cells that can differentiate aberrantly express extraembryonic markers of differentiation. Dnmt[3a−/−,3b−/−] ES cells with >10% CpG methylation are able to terminally differentiate, whereas Dnmt1−/− ES cells with 20% of the CpG methylated cannot differentiate. This demonstrates that successful terminal differentiation is not dependent simply on adequate methylation levels. There is an absolute requirement that the methylation be delivered by the maintenance enzyme Dnmt1. PMID:15456861

  18. Severe global DNA hypomethylation blocks differentiation and induces histone hyperacetylation in embryonic stem cells.

    PubMed

    Jackson, Melany; Krassowska, Anna; Gilbert, Nick; Chevassut, Timothy; Forrester, Lesley; Ansell, John; Ramsahoye, Bernard

    2004-10-01

    It has been reported that DNA methyltransferase 1-deficient (Dnmt1-/-) embryonic stem (ES) cells are hypomethylated (20% CpG methylation) and die through apoptosis when induced to differentiate. Here, we show that Dnmt[3a-/-,3b-/-] ES cells with just 0.6% of their CpG dinucleotides behave differently: the majority of cells within the culture are partially or completely blocked in their ability to initiate differentiation, remaining viable while retaining the stem cell characteristics of alkaline phosphatase and Oct4 expression. Restoration of DNA methylation levels rescues these defects. Severely hypomethylated Dnmt[3a-/-,3b-/-] ES cells have increased histone acetylation levels, and those cells that can differentiate aberrantly express extraembryonic markers of differentiation. Dnmt[3a-/-,3b-/-] ES cells with >10% CpG methylation are able to terminally differentiate, whereas Dnmt1-/- ES cells with 20% of the CpG methylated cannot differentiate. This demonstrates that successful terminal differentiation is not dependent simply on adequate methylation levels. There is an absolute requirement that the methylation be delivered by the maintenance enzyme Dnmt1. PMID:15456861

  19. Cellular Zinc Homeostasis Contributes to Neuronal Differentiation in Human Induced Pluripotent Stem Cells

    PubMed Central

    Pfaender, Stefanie; Föhr, Karl; Lutz, Anne-Kathrin; Putz, Stefan; Achberger, Kevin; Linta, Leonhard; Liebau, Stefan; Boeckers, Tobias M.; Grabrucker, Andreas M.

    2016-01-01

    Disturbances in neuronal differentiation and function are an underlying factor of many brain disorders. Zinc homeostasis and signaling are important mediators for a normal brain development and function, given that zinc deficiency was shown to result in cognitive and emotional deficits in animal models that might be associated with neurodevelopmental disorders. One underlying mechanism of the observed detrimental effects of zinc deficiency on the brain might be impaired proliferation and differentiation of stem cells participating in neurogenesis. Thus, to examine the molecular mechanisms regulating zinc metabolism and signaling in differentiating neurons, using a protocol for motor neuron differentiation, we characterized the expression of zinc homeostasis genes during neurogenesis using human induced pluripotent stem cells (hiPSCs) and evaluated the influence of altered zinc levels on the expression of zinc homeostasis genes, cell survival, cell fate, and neuronal function. Our results show that zinc transporters are highly regulated genes during neuronal differentiation and that low zinc levels are associated with decreased cell survival, altered neuronal differentiation, and, in particular, synaptic function. We conclude that zinc deficiency in a critical time window during brain development might influence brain function by modulating neuronal differentiation. PMID:27247802

  20. Detection of Explosives Using Differential Laser-Induced Perturbation Spectroscopy with a Raman-based Probe.

    PubMed

    Oztekin, Erman K; Burton, Dallas J; Hahn, David W

    2016-04-01

    Explosives detection is carried out with a novel spectral analysis technique referred to as differential laser-induced perturbation spectroscopy (DLIPS) on thin films of TNT, RDX, HMX, and PETN. The utility of Raman spectroscopy for detection of explosives is enhanced by inducing deep ultraviolet laser perturbation on molecular structures in combination with a differential Raman sensing scheme. Principal components analysis (PCA) is used to quantify the DLIPS method as benchmarked against a traditional Raman scattering probe, and the related photo-induced effects on the molecular structure of the targeted explosives are discussed in detail. Finally, unique detection is observed with TNT samples deposited on commonly available background substrates of nylon and polyester. Overall, the data support DLIPS as a noninvasive method that is promising for screening explosives in real-world environments and backgrounds. PMID:26865581

  1. A new approach to chemotherapy: drug-induced differentiation kills African trypanosomes

    PubMed Central

    Wenzler, Tanja; Schumann Burkard, Gabriela; S. Schmidt, Remo; Mäser, Pascal; Bergner, Andreas; Roditi, Isabel; Brun, Reto

    2016-01-01

    Human African trypanosomiasis (sleeping sickness) is a neglected tropical disease caused by Trypanosoma brucei spp. The parasites are transmitted by tsetse flies and adapt to their different hosts and environments by undergoing a series of developmental changes. During differentiation, the trypanosome alters its protein coat. Bloodstream form trypanosomes in humans have a coat of variant surface glycoprotein (VSG) that shields them from the immune system. The procyclic form, the first life-cycle stage to develop in the tsetse fly, replaces the VSG coat by procyclins; these proteins do not protect the parasite from lysis by serum components. Our study exploits the parasite-specific process of differentiation from bloodstream to procyclic forms to screen for potential drug candidates. Using transgenic trypanosomes with a reporter gene in a procyclin locus, we established a whole-cell assay for differentiation in a medium-throughput format. We screened 7,495 drug-like compounds and identified 28 hits that induced expression of the reporter and loss of VSG at concentrations in the low micromolar range. Small molecules that induce differentiation to procyclic forms could facilitate studies on the regulation of differentiation as well as serving as scaffolds for medicinal chemistry for new treatments for sleeping sickness. PMID:26931380

  2. A new approach to chemotherapy: drug-induced differentiation kills African trypanosomes.

    PubMed

    Wenzler, Tanja; Schumann Burkard, Gabriela; Schmidt, Remo S; Mäser, Pascal; Bergner, Andreas; Roditi, Isabel; Brun, Reto

    2016-01-01

    Human African trypanosomiasis (sleeping sickness) is a neglected tropical disease caused by Trypanosoma brucei spp. The parasites are transmitted by tsetse flies and adapt to their different hosts and environments by undergoing a series of developmental changes. During differentiation, the trypanosome alters its protein coat. Bloodstream form trypanosomes in humans have a coat of variant surface glycoprotein (VSG) that shields them from the immune system. The procyclic form, the first life-cycle stage to develop in the tsetse fly, replaces the VSG coat by procyclins; these proteins do not protect the parasite from lysis by serum components. Our study exploits the parasite-specific process of differentiation from bloodstream to procyclic forms to screen for potential drug candidates. Using transgenic trypanosomes with a reporter gene in a procyclin locus, we established a whole-cell assay for differentiation in a medium-throughput format. We screened 7,495 drug-like compounds and identified 28 hits that induced expression of the reporter and loss of VSG at concentrations in the low micromolar range. Small molecules that induce differentiation to procyclic forms could facilitate studies on the regulation of differentiation as well as serving as scaffolds for medicinal chemistry for new treatments for sleeping sickness. PMID:26931380

  3. Sphingomyelin metabolism is involved in the differentiation of MDCK cells induced by environmental hypertonicity

    PubMed Central

    Favale, Nicolás Octavio; Santacreu, Bruno Jaime; Pescio, Lucila Gisele; Marquez, Maria Gabriela; Sterin-Speziale, Norma Beatriz

    2015-01-01

    Sphingolipids (SLs) are relevant lipid components of eukaryotic cells. Besides regulating various cellular processes, SLs provide the structural framework for plasma membrane organization. Particularly, SM is associated with detergent-resistant microdomains. We have previously shown that the adherens junction (AJ) complex, the relevant cell-cell adhesion structure involved in cell differentiation and tissue organization, is located in an SM-rich membrane lipid domain. We have also demonstrated that under hypertonic conditions, Madin-Darby canine kidney (MDCK) cells acquire a differentiated phenotype with changes in SL metabolism. For these reasons, we decided to evaluate whether SM metabolism is involved in the acquisition of the differentiated phenotype of MDCK cells. We found that SM synthesis mediated by SM synthase 1 is involved in hypertonicity-induced formation of mature AJs, necessary for correct epithelial cell differentiation. Inhibition of SM synthesis impaired the acquisition of mature AJs, evoking a disintegration-like process reflected by the dissipation of E-cadherin and β- and α-catenins from the AJ complex. As a consequence, MDCK cells did not develop the hypertonicity-induced differentiated epithelial cell phenotype. PMID:25670801

  4. Histone deacetylase inhibitors induce growth arrest and differentiation in uveal melanoma

    PubMed Central

    Landreville, Solange; Agapova, Olga A.; Matatall, Katie A.; Kneass, Zachary T.; Onken, Michael D.; Lee, Ryan S.; Bowcock, Anne M.; Harbour, J. William

    2011-01-01

    Purpose Metastasis is responsible for the death of most cancer patients, yet few therapeutic agents are available which specifically target the molecular events that lead to metastasis. We recently showed that inactivating mutations in the tumor suppressor gene BAP1 are closely associated with loss of melanocytic differentiation in uveal melanoma and metastasis (UM). The purpose of this study was to identify therapeutic agents that reverse the phenotypic effects of BAP1 loss in UM. Experimental Design In silico screens were performed to identify therapeutic compounds predicted to differentiate UM cells using Gene Set Enrichment Analysis and Connectivity Map databases. Valproic acid, trichostatin A, LBH-589 and suberoylanilide hydroxamic acid were evaluated for their effects on UM cells using morphologic evaluation, MTS viability assays, BrdU incorporation, flow cytometry, clonogenic assays, gene expression profiling, histone acetylation and ubiquitination assays, and a murine xenograft tumorigenicity model. Results HDAC inhibitors induced morphologic differentiation, cell cycle exit, and a shift to a differentiated, melanocytic gene expression profile in cultured UM cells. Valproic acid inhibited the growth of UM tumors in vivo. Conclusions These findings suggest that HDAC inhibitors may have therapeutic potential for inducing differentiation and prolonged dormancy of micrometastatic disease in UM. PMID:22038994

  5. Isoliquiritigenin-Induced Differentiation in Mouse Melanoma B16F0 Cell Line

    PubMed Central

    Chen, Xiaoyu; Zhang, Bo; Yuan, Xuan; Yang, Fan; Liu, Jinglei; Zhao, Hong; Liu, Liangliang; Wang, Yanming; Wang, Zhenhua; Zheng, Qiusheng

    2012-01-01

    The chemotherapeutical treatment is very limited for malignant melanoma, a highly lethal disease occurs globally. Natural products derived from traditional Chinese medicine licorice are attractive in quest new treatments due to their anti-tumor activities. A new dietary flavonoid isoliquiritigenin (ISL) were thus investigated to indentify its anti-melanoma activities on mouse melanoma B16F0 cells in present study. Using biochemical and free radical biological experiments in vitro, we identified the pro-differentiated profiles of ISL and evaluated the role of reactive oxygen species (ROS) during B16F0 cell differentiation. The data showed a strong dose-response relationship between ISL exposure and the characteristics of B16F0 differentiation in terms of morphology changes and melanogenesis. The accumulated intercellular ROS during exposure are necessary to support ISL-induced differentiation, which was proven by additional redox modulators. It was confirmed further by the relative activities of enzymes and genes modulated melanogenesis in ISL-treatments with or without ROS modulators. The tumorigenicity of ISL-treated cells was limited significantly by using the colony formation assay in vitro and an animal model assay in vivo respectively. Our research demonstrated that isoliquiritigenin is a differentiation-inducing agent, and its mechanisms involve ROS accumulation facilitating melanogenesis. PMID:23304254

  6. Genistein as an inducer of tumor cell differentiation : possible mechanisms of action.

    SciTech Connect

    Constantinou, A.; Huberman, E.; Center for Mechanistic Biology and Biotechnology; Univ. of Illinois at Chicago

    1995-01-01

    Decreased activity of either topoisomerases or tyrosine kinases has been implicated in the differentiation of a number of cell types. It is therefore conceivable that genistein, because of its reported ability to inhibit these activities in vitro, may be an inducer of cellular differentiation. We investigated this possibility in human promyelocytic HL-60 and erythroid K-562 leukemia cells and in human SK-MEL-131 melanoma cells. Our results indicated that genistein, in a dose-dependent manner, inhibited cell multiplication and induced cell differentiation. The maturing HL-60 cells acquired granulocytic and monocytic markers. The differentiating K-562 cells stained positively with benzidine, which indicates the production of hemoglobin, an erythroid marker. Following genistein treatment, maturing SK-MEL-131 melanoma cells formed dendrite-like structures and exhibited increased tyrosinase activity and melanin content. Experiments were designed to identify the molecular mechanism of genistein's action. Data from our laboratory suggest that this isoflavone triggers the pathway that leads to cellular differentiation by stabilizing protein-linked DNA strand breakage. Other possible mechanisms reported in the literature are discussed.

  7. Gold nanorod delivery of LSD1 siRNA induces human mesenchymal stem cell differentiation.

    PubMed

    Zhao, Xiongfei; Huang, Qianying; Jin, Yiqiang

    2015-09-01

    Over the past decade, theranostic nanoparticles with microsize and multifunctional ability have emerged as a new platform in biomedical field, such as cancer therapy, optical imaging and gene therapy. Gene therapy has been recently shown as a promising tool for tissue engineering as safe and effective nanotechnology-based delivery methods are developed. Controlling adhesion and differentiation of stem cells is critical for tissue regeneration. In this study, we have developed poly-sodium 4-styrenesulfonate (PSS) and poly-allylamine hydrochloride (PAH) coated AuNR-based nanocarriers, which are capable of delivering small interfering RNA (siRNA) against LSD1 to induce the differentiation of human mesenchymal stem cells. To further study the mechanism, we tested the stemness and differentiation genes and found that they have been changed with LSD1 down-regulation. In addition, with the hepatocyte growth factor (HGF), LSD1 siRNA delivery by AuNRs could promote the differentiation of the human mesenchymal stem cells (human MSCs) into a hepatocyte lineage in vitro. Our results suggest for the first time use of AuNRs as nanocarriers of delivery LSD1 siRNA to induce the differentiation of human MSCs into a hepatocyte lineage, and envision the potential application of nanotechnology in tissue remodeling (such as liver and bone) in vivo, eventually translating to clinical applications. PMID:26046277

  8. Material-induced chondrogenic differentiation of mesenchymal stem cells is material-dependent.

    PubMed

    Zheng, Li; Yang, Jinsong; Fan, Hongsong; Zhang, Xingdong

    2014-05-01

    Certain materials may mimic natural cartilage to provide an amenable cellular microenvironment for the chondrogenic differentiation of mesenchymal stem cells. The chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) has been demonstrated to be induced by collagen-based hydrogels in vivo, but whether the induction is material-driven or self-differentiation has not been elucidated. In the present study, BMSCs were encapsulated in porous materials, namely, a biphasic calcium phosphate ceramic (BCP), silk fibroin protein matrix (SFP) and collagen sponge (CS), to further study the chondrogenic effects of various materials. Diffusion chambers that allow the body fluid to permeate and deter the host cells from invasion were also loaded with the cell-scaffold constructs. Chambers containing the scaffold-BMSC composites were implanted subcutaneously in the dorsa of rabbits. The specimens in the chamber were harvested for histological and immunohistochemical analyses eight weeks after implantation. The results showed that no chondrogenic differentiation of the BMSCs occurred when the BMSCs were encapsulated in BCP, SFP and CS, indicating that chondrogenesis induced by materials is material-dependent and that these particular porous materials are not suitable for inducing chondrogenesis. However, the diffusion chamber was effective in preventing host immune rejection, host cell invasion and vascular invasion. The results are likely to serve as a valuable clinical reference when selecting an appropriate scaffold for cartilage repair. PMID:24940401

  9. A Receptor Tyrosine Kinase Inhibitor, Dovitinib (TKI-258), Enhances BMP-2-Induced Osteoblast Differentiation In Vitro.

    PubMed

    Lee, Yura; Bae, Kyoung Jun; Chon, Hae Jung; Kim, Seong Hwan; Kim, Soon Ae; Kim, Jiyeon

    2016-05-31

    Dovitinib (TKI258) is a small molecule multi-kinase inhibitor currently in clinical phase I/II/III development for the treatment of various types of cancers. This drug has a safe and effective pharmacokinetic/pharmacodynamic profile. Although dovitinib can bind several kinases at nanomolar concentrations, there are no reports relating to osteoporosis or osteoblast differentiation. Herein, we investigated the effect of dovitinib on human recombinant bone morphogenetic protein (BMP)-2-induced osteoblast differentiation in a cell culture model. Dovitinib enhanced the BMP-2-induced alkaline phosphatase (ALP) induction, which is a representative marker of osteoblast differentiation. Dovitinib also stimulated the translocation of phosphorylated Smad1/5/8 into the nucleus and phosphorylation of mitogen-activated protein kinases, including ERK1/2 and p38. In addition, the mRNA expression of BMP-4, BMP-7, ALP, and OCN increased with dovitinib treatment. Our results suggest that dovitinib has a potent stimulating effect on BMP-2-induced osteoblast differentiation and this existing drug has potential for repositioning in the treatment of bone-related disorders. PMID:27025387

  10. Trans-Differentiation of Neural Stem Cells: A Therapeutic Mechanism Against the Radiation Induced Brain Damage

    PubMed Central

    Kang, Bong Gu; Lee, Se Jeong; Kim, Kang Ho; Yang, Heekyoung; Lee, Young-Ae; Cho, Yu Jin; Im, Yong-Seok; Lee, Dong-Sup; Lim, Do-Hoon; Kim, Dong Hyun; Um, Hong-Duck; Lee, Sang-Hun; Lee, Jung-II; Nam, Do-Hyun

    2012-01-01

    Radiation therapy is an indispensable therapeutic modality for various brain diseases. Though endogenous neural stem cells (NSCs) would provide regenerative potential, many patients nevertheless suffer from radiation-induced brain damage. Accordingly, we tested beneficial effects of exogenous NSC supplementation using in vivo mouse models that received whole brain irradiation. Systemic supplementation of primarily cultured mouse fetal NSCs inhibited radiation-induced brain atrophy and thereby preserved brain functions such as short-term memory. Transplanted NSCs migrated to the irradiated brain and differentiated into neurons, astrocytes, or oligodendrocytes. In addition, neurotrophic factors such as NGF were significantly increased in the brain by NSCs, indicating that both paracrine and replacement effects could be the therapeutic mechanisms of NSCs. Interestingly, NSCs also differentiated into brain endothelial cells, which was accompanied by the restoration the cerebral blood flow that was reduced from the irradiation. Inhibition of the VEGF signaling reduced the migration and trans-differentiation of NSCs. Therefore, trans-differentiation of NSCs into brain endothelial cells by the VEGF signaling and the consequential restoration of the cerebral blood flow would also be one of the therapeutic mechanisms of NSCs. In summary, our data demonstrate that exogenous NSC supplementation could prevent radiation-induced functional loss of the brain. Therefore, successful combination of brain radiation therapy and NSC supplementation would provide a highly promising therapeutic option for patients with various brain diseases. PMID:22347993

  11. Titanium With Nanotopography Induces Osteoblast Differentiation by Regulating Endogenous Bone Morphogenetic Protein Expression and Signaling Pathway.

    PubMed

    M S Castro-Raucci, Larissa; S Francischini, Marcelo; N Teixeira, Lucas; P Ferraz, Emanuela; B Lopes, Helena; T de Oliveira, Paulo; Hassan, Mohammad Q; Losa, Adalberto L; Beloti, Marcio M

    2016-07-01

    We aimed at evaluating the effect of titanium (Ti) with nanotopography (Nano) on the endogenous expression of BMP-2 and BMP-4 and the relevance of this process to the nanotopography-induced osteoblast differentiation. MC3T3-E1 cells were grown on Nano and machined (Machined) Ti surfaces and the endogenous BMP-2/4 expression and the effect of BMP receptor BMPR1A silencing in both osteoblast differentiation and expression of genes related to TGF-β/BMP signaling were evaluated. Nano supported higher BMP-2 gene and protein expression and upregulated the osteoblast differentiation compared with Machined Ti surface. The BMPR1A silencing inhibited the osteogenic potential induced by Nano Ti surface as indicated by reduced alkaline phosphatase (ALP), osteocalcin and RUNX2 gene expression, RUNX2 protein expression and ALP activity. In addition, the expression of genes related to TGF-β/BMP signaling was deeply affected by BMPR1A-silenced cells grown on Nano Ti surface. In conclusion, we have demonstrated for the first time that nanotopography induces osteoblast differentiation, at least in part, by upregulating the endogenous production of BMP-2 and modulating BMP signaling pathway. J. Cell. Biochem. 117: 1718-1726, 2016. © 2015 Wiley Periodicals, Inc. PMID:26681207

  12. The benzophenanthridine alkaloid fagaronine induces erythroleukemic cell differentiation by gene activation.

    PubMed

    Dupont, Claude; Couillerot, Eric; Gillet, Reynald; Caron, Catherine; Zeches-Hanrot, Monique; Riou, Jean-François; Trentesaux, Chantal

    2005-06-01

    Fagaronine, a benzophenanthridine alkaloid from Fagara zanthoxyloides Lam. (Rutaceae), has been tested on the erythroleukemic cell line K562 in order to explain some previous results on cell differentiation. In this study we showed that fagaronine induces a significant hemoglobinization of the human erythroleukemic cell line K562. This hemoglobin synthesis was accompanied by a strong increase of erythroid mRNA expression such as gamma- and alpha-globin, and PBGD, an enzyme of heme synthesis. In addition, the Epo-R transcripts were also stimulated indicating that cells are engaged in a maturation process. Both transcription factors GATA-1 and NF-E2, which play an important role in the regulation of genes involved in the erythroid differentiation, were also transcriptionally up-regulated. To elucidate the possible role of GATA-1 in the FAG-induced differentiation of K562 cells, we transfected reporter constructs containing regulatory regions of erythroid genes encompassing GATA-1 binding sites. After 48 hours of treatment, FAG stimulated the EPO-R and gamma-globin promoters by 2- to 3-fold and the promoter/enhancer region of GATA-1 gene by 3.2-fold. A mutation within the GATA-1 binding sites strongly decreased the promoter activation induced by FAG. Taken together, our results represent a demonstration that FAG exerts its differentiating activity by a specific activation of the regulating GATA-1 regions of genes involved in the erythroid phenotype expression. PMID:15971117

  13. A Receptor Tyrosine Kinase Inhibitor, Dovitinib (TKI-258), Enhances BMP-2-Induced Osteoblast Differentiation In Vitro

    PubMed Central

    Lee, Yura; Bae, Kyoung Jun; Chon, Hae Jung; Kim, Seong Hwan; Kim, Soon Ae; Kim, Jiyeon

    2016-01-01

    Dovitinib (TKI258) is a small molecule multi-kinase inhibitor currently in clinical phase I/II/III development for the treatment of various types of cancers. This drug has a safe and effective pharmacokinetic/pharmacodynamic profile. Although dovitinib can bind several kinases at nanomolar concentrations, there are no reports relating to osteoporosis or osteoblast differentiation. Herein, we investigated the effect of dovitinib on human recombinant bone morphogenetic protein (BMP)-2-induced osteoblast differentiation in a cell culture model. Dovitinib enhanced the BMP-2-induced alkaline phosphatase (ALP) induction, which is a representative marker of osteoblast differentiation. Dovitinib also stimulated the translocation of phosphorylated Smad1/5/8 into the nucleus and phosphorylation of mitogen-activated protein kinases, including ERK1/2 and p38. In addition, the mRNA expression of BMP-4, BMP-7, ALP, and OCN increased with dovitinib treatment. Our results suggest that dovitinib has a potent stimulating effect on BMP-2-induced osteoblast differentiation and this existing drug has potential for repositioning in the treatment of bone-related disorders. PMID:27025387

  14. Identification of Centella asiatica's Effective Ingredients for Inducing the Neuronal Differentiation

    PubMed Central

    Jiang, Hui; Zheng, Guoshuai; Lv, Junwei; Chen, Heyu; Lin, Jinjin; Li, Yiyang; Fan, Guorong

    2016-01-01

    Centella asiatica, commonly known as Gotu kola, has been widely used as a traditional herb for decades. Yet, the study on which compounds or compound combinations actually lead to its brain benefits remains scarce. To study the neuroprotection effects of Centella asiatica, neuronal differentiation of PC12 cells was applied. In our pilot study, we isolated 45 Centella asiatica fractions and tested their abilities for inducing neuronal differentiation on PC12 cells. The most effective fraction showed robust induction in neurite outgrowth and neurofilament expression. LC-MS fingerprint analysis of this fraction revealed asiatic acid and madecassic acid as the dominant components. A further investigation on the pure combination of these two compounds indicated that the combination of these two compounds extensively promoted nerve differentiation in vitro. Application of PD98059, a protein MEK inhibitor, attenuated combination-induced neurofilament expression, indicating the combination-induced nerve differentiation through activation of MEK signaling pathway. Our results support the use of combination of asiatic acid and madecassic acid as an effective mean to intervene neurodegenerative diseases in which neurotrophin deficiency is involved. PMID:27446228

  15. Auxin Deprivation Induces Synchronous Golgi Differentiation in Suspension-Cultured Tobacco BY-2 Cells1

    PubMed Central

    Winicur, Zev M.; Feng Zhang, Guo; Andrew Staehelin, L.

    1998-01-01

    To date, the lack of a method for inducing plant cells and their Golgi stacks to differentiate in a synchronous manner has made it difficult to characterize the nature and extent of Golgi retailoring in biochemical terms. Here we report that auxin deprivation can be used to induce a uniform population of suspension-cultured tobacco (Nicotiana tabacum cv BY-2) cells to differentiate synchronously during a 4-d period. Upon removal of auxin, the cells stop dividing, undergo elongation, and differentiate in a manner that mimics the formation of slime-secreting epidermal and peripheral root-cap cells. The morphological changes to the Golgi apparatus include a proportional increase in the number of trans-Golgi cisternae, a switch to larger-sized secretory vesicles that bud from the trans-Golgi cisternae, and an increase in osmium staining of the secretory products. Biochemical alterations include an increase in large, fucosylated, mucin-type glycoproteins, changes in the types of secreted arabinogalactan proteins, and an increase in the amounts and types of molecules containing the peripheral root-cap-cell-specific epitope JIM 13. Taken together, these findings support the hypothesis that auxin deprivation can be used to induce tobacco BY-2 cells to differentiate synchronously into mucilage-secreting cells. PMID:9625703

  16. Choline kinase alpha expression during RA-induced neuronal differentiation: role of C/EBPβ.

    PubMed

    Domizi, Pablo; Aoyama, Chieko; Banchio, Claudia

    2014-04-01

    Neuronal differentiation is a complex process characterized by a halt in proliferation and extension of neurites from the cell body. This process is accompanied by changes in gene expression that mediate the redirection leading to neurite formation and function. Acceleration of membrane phospholipids synthesis is associated with neurite elongation, and phosphatidylcholine (PtdCho) is the major membrane phospholipid in mammalian cells. The transcription of two genes in particular encoding key enzymes in the CDP-choline pathway for PtdCho biosynthesis are stimulated; the Chka gene for choline kinase (CK) alpha isoform and the Pcyt1a gene for the CTP:phosphocholine cytidylyltransferase (CCT) alpha isoform. We report that the stimulation of CKα expression during retinoic acid (RA) induced differentiation depends on a promoter region that contains two CCAAT/Enhancer-binding Protein-β (C/EBPβ) sites. We demonstrate that during neuronal differentiation of Neuro-2a cells, RA induces Chka expression by a mechanism that involves ERK1/2 activation which triggers C/EBPβ expression. Elevated levels of C/EBPβ bind to the Chka proximal promoter (Box1) inducing CKα expression. In addition we identified a downstream sequence named Box2 which together with Box1 is required for the promoter to reach the full induction. This is the first elucidation of the mechanism by which the expression of Chka is coordinately regulated during neuronal differentiation. PMID:24440820

  17. Modeling and analysis of retinoic acid induced differentiation of uncommitted precursor cells†

    PubMed Central

    Tasseff, Ryan; Nayak, Satyaprakash; Song, Sang Ok; Yen, Andrew; Varner, Jeffrey D.

    2013-01-01

    Manipulation of differentiation programs has therapeutic potential in a spectrum of human cancers and neurodegenerative disorders. In this study, we integrated computational and experimental methods to unravel the response of a lineage uncommitted precursor cell-line, HL-60, to Retinoic Acid (RA). HL-60 is a human myeloblastic leukemia cell-line used extensively to study human differentiation programs. Initially, we focused on the role of the BLR1 receptor in RA-induced differentiation and G1/0-arrest in HL-60. BLR1, a putative G protein-coupled receptor expressed following RA exposure, is required for RA-induced cell-cycle arrest and differentiation and causes persistent MAPK signaling. A mathematical model of RA-induced cell-cycle arrest and differentiation was formulated and tested against BLR1 wild-type (wt) knock-out and knock-in HL-60 cell-lines with and without RA. The current model described the dynamics of 729 proteins and protein complexes interconnected by 1356 interactions. An ensemble strategy was used to compensate for uncertain model parameters. The ensemble of HL-60 models recapitulated the positive feedback between BLR1 and MAPK signaling. The ensemble of models also correctly predicted Rb and p47phox regulation and the correlation between p21-CDK4-cyclin D formation and G1/0-arrest following exposure to RA. Finally, we investigated the robustness of the HL-60 network architecture to structural perturbations and generated experimentally testable hypotheses for future study. Taken together, the model presented here was a first step toward a systematic framework for analysis of programmed differentiation. These studies also demonstrated that mechanistic network modeling can help prioritize experimental directions by generating falsifiable hypotheses despite uncertainty. PMID:21437295

  18. Cisplatin induces resistance by triggering differentiation of testicular embryonal carcinoma cells.

    PubMed

    Abada, Paolo B; Howell, Stephen B

    2014-01-01

    Although testicular germ cell tumors are generally quite responsive to treatment with cisplatin, a small fraction of them acquire resistance during therapy. Even when cisplatin treatment is successful the patient is often left with a residual teratoma at the site of the primary tumor suggesting that cisplatin may trigger differentiation in some tumors. Using the human embryonal carcinoma cell line NTera2/D1, we confirmed that exposure to the differentiating agent retinoic acid produced a reduction in pluripotency markers NANOG and POU5F1 (Oct3/4) and an acute concentration-dependent increase in resistance to both cisplatin and paclitaxel that reached as high as 18-fold for cisplatin and 61-fold for paclitaxel within four days. A two day exposure to cisplatin also produced a concentration-dependent decrease in the expression of the NANOG and POU5F1 and increased expression of three markers whose levels increase with differentiation including Nestin, SCG10 and Fibronectin. In parallel, exposure to cisplatin induced up to 6.2-fold resistance to itself and 104-fold resistance to paclitaxel. Paclitaxel did not induce differentiation or resistance to either itself or cisplatin. Neither retinoic acid nor cisplatin induced resistance in cervical or prostate cancer cell lines or other germ cell tumor lines in which they failed to alter the expression of NANOG and POU5F1. Forced expression of NANOG prevented the induction of resistance to cisplatin by retinoic acid. We conclude that cisplatin can acutely induce resistance to itself and paclitaxel by triggering a differentiation response in pluripotent germ cell tumor cells. PMID:24475288

  19. Inhibition of the NAD-Dependent Protein Deacetylase SIRT2 Induces Granulocytic Differentiation in Human Leukemia Cells

    PubMed Central

    Sunami, Yoshitaka; Araki, Marito; Hironaka, Yumi; Morishita, Soji; Kobayashi, Masaki; Liew, Ei Leen; Edahiro, Yoko; Tsutsui, Miyuki; Ohsaka, Akimichi; Komatsu, Norio

    2013-01-01

    Sirtuins, NAD-dependent protein deacetylases, play important roles in cellular functions such as metabolism and differentiation. Whether sirtuins function in tumorigenesis is still controversial, but sirtuins are aberrantly expressed in tumors, which may keep cancerous cells undifferentiated. Therefore, we investigated whether the inhibition of sirtuin family proteins induces cellular differentiation in leukemic cells. The sirtuin inhibitors tenovin-6 and BML-266 induce granulocytic differentiation in the acute promyelocytic leukemia (APL) cell line NB4. This differentiation is likely caused by an inhibition of SIRT2 deacetylase activity, judging from the accumulation of acetylated α-tubulin, a major SIRT2 substrate. Unlike the clinically used differentiation inducer all-trans retinoic acid, tenovin-6 shows limited effects on promyelocytic leukemia–retinoic acid receptor α (PML-RAR-α) stability and promyelocytic leukemia nuclear body formation in NB4 cells, suggesting that tenovin-6 does not directly target PML-RAR-α activity. In agreement with this, tenovin-6 induces cellular differentiation in the non-APL cell line HL-60, where PML-RAR-α does not exist. Knocking down SIRT2 by shRNA induces granulocytic differentiation in NB4 cells, which demonstrates that the inhibition of SIRT2 activity is sufficient to induce cell differentiation in NB4 cells. The overexpression of SIRT2 in NB4 cells decreases the level of granulocytic differentiation induced by tenovin-6, which indicates that tenovin-6 induces granulocytic differentiation by inhibiting SIRT2 activity. Taken together, our data suggest that targeting SIRT2 is a viable strategy to induce leukemic cell differentiation. PMID:23460888

  20. Adipose differentiation-related protein is not involved in hypoxia inducible factor-1-induced lipid accumulation under hypoxia

    PubMed Central

    SHEN, GUOMIN; NING, NING; ZHAO, XINGSHENG; LIU, XI; WANG, GUANGYU; WANG, TIANZHEN; ZHAO, RAN; YANG, CHAO; WANG, DONGMEI; GONG, PINGYUAN; SHEN, YAN; SUN, YONGJIAN; ZHAO, XIAO; JIN, YINJI; YANG, WEIWEI; HE, YAN; ZHANG, LEI; JIN, XIAOMING; LI, XIAOBO

    2015-01-01

    Increasing evidence has showed that hypoxia inducible factor-1 (HIF1) has an important role in hypoxia-induced lipid accumulation, a common feature of solid tumors; however, its role remains to be fully elucidated. Adipose differentiation-related protein (ADRP), a structural protein of lipid droplets, is found to be upregulated under hypoxic conditions. In the present study, an MCF7 breast cancer cell line was used to study the role of ADRP in hypoxia-induced lipid accumulation. It was demonstrated that hypoxia induced the gene expression of ADRP in a HIF1-dependent manner. Increases in the mRNA and protein levels of ADRP was accompanied by increased HIF1A activity. In addition, a significant decrease in the mRNA and protein levels of ADRP were detected in presence of siRNA targeting HIF1A. Using a dual-luciferase reporting experiment and chromatin immunoprecipitation assay, the present study demonstrated that ADRP is a direct target gene of HIF1, and identified a functional hypoxia response element localized 33 bp upstream of the transcriptional start site of the ADRP gene. Furthermore, the present study demonstrated the role of ADRP in low density liporotein (LDL) and very-LDL uptake-induced lipid accumulation under hypoxia. The knockdown of ADRP did not reduce HIF1-induced lipid accumulation under hypoxia. Together, these results showed that ADRP may be not involved in HIF1-induced lipid accumulation. PMID:26498183

  1. Sphingosine 1-phosphate receptor activation enhances BMP-2-induced osteoblast differentiation

    SciTech Connect

    Sato, Chieri; Iwasaki, Tsuyoshi; Kitano, Sachie; Tsunemi, Sachi; Sano, Hajime

    2012-06-22

    Highlights: Black-Right-Pointing-Pointer We investigated the role of S1P signaling for osteoblast differentiation. Black-Right-Pointing-Pointer Both S1P and FTY enhanced BMP-2-stimulated osteoblast differentiation by C2C12 cells. Black-Right-Pointing-Pointer S1P signaling enhanced BMP-2-stimulated Smad and ERK phosphorylation by C2C12 cells. Black-Right-Pointing-Pointer MEK/ERK signaling is a pathway underlying S1P signaling for osteoblast differentiation. -- Abstract: We previously demonstrated that sphingosine 1-phosphate (S1P) receptor-mediated signaling induced proliferation and prostaglandin productions by synovial cells from rheumatoid arthritis (RA) patients. In the present study we investigated the role of S1P receptor-mediated signaling for osteoblast differentiation. We investigated osteoblast differentiation using C2C12 myoblasts, a cell line derived from murine satellite cells. Osteoblast differentiation was induced by the treatment of bone morphogenic protein (BMP)-2 in the presence or absence of either S1P or FTY720 (FTY), a high-affinity agonist of S1P receptors. Osteoblast differentiation was determined by osteoblast-specific transcription factor, Runx2 mRNA expression, alkaline phosphatase (ALP) activity and osteocalcin production by the cells. Smad1/5/8 and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation was examined by Western blotting. Osteocalcin production by C2C12 cells were determined by ELISA. Runx2 expression and ALP activity by BMP-2-stimulated C2C12 cells were enhanced by addition of either S1P or FTY. Both S1P and FTY enhanced BMP-2-induced ERK1/2 and Smad1/5/8 phosphorylation. The effect of FTY was stronger than that of S1P. S1P receptor-mediated signaling on osteoblast differentiation was inhibited by addition of mitogen-activated protein kinase/ERK kinase (MEK) 1/2 inhibitor, indicating that the S1P receptor-mediated MEK1/2-ERK1/2 signaling pathway enhanced BMP-2-Smad signaling. These results indicate that S1P

  2. A-Raf and C-Raf differentially regulate mechanobiological response of osteoblasts to guide mechanical stress-induced differentiation.

    PubMed

    Zhang, Qi; Matsui, Hiroyuki; Horiuchi, Hisanori; Liang, Xing; Sasaki, Keiichi

    2016-08-01

    Regulation of osteoblast activity by mechanical stress is important for bone remodeling. However, the precise mechanotransduction mechanism that triggers the anabolic reaction of osteoblasts is largely unknown. In this study, we performed RNA interference (RNAi) screening to identify the signaling molecules upstream of ERK, which was responsible for osteogenesis. Of twenty-two mitogen-activated protein kinase (MAPK) kinase kinases (MAP3Ks), we identified A-Raf and C-Raf as upstream MAP3Ks of the mechanical stretch-activated ERK pathway. Subsequently we screened the mechanosensitive cation channel, and identified P2X7 as an upstream molecule of the ERK pathway. Intriguingly, P2X7 functioned as an upstream activator of A-Raf but not of C-Raf. Furthermore, A-Raf contributed to mechanical stretch-induced osteoblast differentiation. In contrast, C-Raf but not A-Raf protected osteoblasts from mechanical stretch-induced apoptosis. These results suggested that A-Raf and C-Raf were involved in mechanobiological osteogenesis in a distinct way: A-Raf was responsible for osteogenesis while C-Raf for anti-apoptotic protection and promotion. PMID:27240957

  3. Alpha-lipoic acid enhances DMSO-induced cardiomyogenic differentiation of P19 cells.

    PubMed

    Shen, Xinghua; Yang, Qinghui; Jin, Peng; Li, Xueqi

    2014-09-01

    Alpha-lipoic acid (α-LA) is a potent antioxidant that acts as an essential cofactor in mitochondrial dehydrogenase reactions. α-LA has been shown to possess anti-inflammatory and cytoprotective properties, and is used to improve symptoms of diabetic neuropathy. However, the role of α-LA in stem cell differentiation and the underlying molecular mechanisms remain unknown. In the present study, we showed that α-LA significantly promoted dimethyl sulfoxide (DMSO)-induced cardiomyogenic differentiation of mouse embryonic carcinoma P19 cells. α-LA dose dependently increased beating embryonic body (EB) percentages of DMSO-differentiated P19 cells. The expressions of cardiac specific genes TNNT2, Nkx2.5, GATA4, MEF2C, and MLC2V and cardiac isoform of troponin T (cTnT)-positively stained cell population were significantly up-regulated by the addition of α-LA. We also demonstrated that the differentiation time after EB formation was critical for α-LA to take effect. Interestingly, without DMSO treatment, α-LA did not stimulate the cardiomyogenic differentiation of P19 cells. Further investigation indicated that collagen synthesis-enhancing activity, instead of the antioxidative property, plays a significant role in the cardiomyogenic differentiation-promoting function of α-LA. These findings highlight the potential use of α-LA for regenerative therapies in heart diseases. PMID:25112287

  4. Retinoic acid induces nuclear accumulation of Raf1 during differentiation of HL-60 cells

    SciTech Connect

    Smith, James; Bunaciu, Rodica P.; Reiterer, Gudrun; Coder, David; George, Thaddeus; Asaly, Michael; Yen, Andrew

    2009-08-01

    All trans-retinoic acid (RA) is a standard therapeutic agent used in differentiation induction therapy treatment of acute promyelocytic leukemia (APL). RA and its metabolites use a diverse set of signal transduction pathways during the differentiation program. In addition to the direct transcriptional targets of the nuclear RAR and RXR receptors, signals derived from membrane receptors and the Raf-MEK-ERK pathway are required. Raf1 phosphorylation and the prolonged activation of Raf1 persisting during the entire differentiation process are required for RA-dependent differentiation of HL-60 cells. Here we identify a nuclear redistribution of Raf1 during the RA-induced differentiation of HL-60 cells. In addition, the nuclear accumulation of Raf1 correlates with an increase in Raf1 phosphorylated at serine 621. The serine 621 phosphorylated Raf1 is predominantly localized in the nucleus. The RA-dependent nuclear accumulation of Raf1 suggests a novel nuclear role for Raf1 during the differentiation process.

  5. Poly-L-lactic acid/hydroxyapatite electrospun nanocomposites induce chondrogenic differentiation of human MSC.

    PubMed

    Spadaccio, Cristiano; Rainer, Alberto; Trombetta, Marcella; Vadalá, Gianluca; Chello, Massimo; Covino, Elvio; Denaro, Vincenzo; Toyoda, Yoshiya; Genovese, Jorge A

    2009-07-01

    Cartilage and bone tissue engineering has been widely investigated but is still hampered by cell differentiation and transplant integration issues within the constructs. Scaffolds represent the pivotal structure of the engineered tissue and establish an environment for neo-extracellular matrix synthesis. They can be associated to signals to modulate cell activity. In this study, considering the well reported role of hydroxyapatite (HA) in cartilage repair, we focused on the putative chondrogenic differentiation of human mesenchymal stem cells (hMSCs) following culture on membranes of electrospun fibers of poly-L-lactic acid (PLLA) loaded with nanoparticles of HA. hMSCs were seeded on PLLA/HA and bare PLLA membranes and cultured in basal medium, using chondrogenic differentiation medium as a positive control. After 14 days of culture, SOX-9 positive cells could be detected in the PLLA/HA group. Cartilage specific proteoglycan immunostain confirmed the presence of neo-extracellular-matrix production. Co-expression of CD29, a typical surface marker of MSCs and SOX-9, suggested different degrees in the differentiation process. We developed a hydroxyapatite functionalized scaffold with the aim to recapitulate the native histoarchitecture and the molecular signaling of osteochondral tissue to facilitate cell differentiation toward chondrocyte. PLLA/HA nanocomposites induced differentiation of hMSCs in a chondrocyte-like phenotype with generation of a proteoglycan based matrix. This nanocomposite could be an amenable alternative scaffold for cartilage tissue engineering using hMSCs. PMID:19418224

  6. A cost-effective system for differentiation of intestinal epithelium from human induced pluripotent stem cells

    PubMed Central

    Ogaki, Soichiro; Morooka, Mayu; Otera, Kaito; Kume, Shoen

    2015-01-01

    The human intestinal epithelium is a useful model for pharmacological studies of absorption, metabolism, drug interactions, and toxicology, as well as for studies of developmental biology. We established a rapid and cost effective system for differentiation of human induced pluripotent stem (iPS) cells into definitive endoderm (DE) cells. In the presence of dimethyl sulfoxide (DMSO), a low concentration of Activin at 6.25 ng/ml is sufficient to give a similar differentiation efficiency with that using Activin at 100 ng/ml at the presence of Wnt activator. In the presence of DMSO, Activin at low concentration triggered hiPS cells to undergo differentiation through G1 arrest, reduce apoptosis, and potentiate activation of downstream targets, such as SMAD2 phosphorylation and SOX17 expression. This increased differentiation into CDX2 + SOX17 + DE cells. The present differentiation procedure therefore permits rapid and efficient derivation of DE cells, capable of differentiating into intestinal epithelium upon BIO and DAPT treatment and of giving rise to functional cells, such as enterocytes. PMID:26616277

  7. Dasatinib inhibits TGFβ-induced myofibroblast differentiation through Src-SRF Pathway.

    PubMed

    Abdalla, Maha; Thompson, LeeAnn; Gurley, Erin; Burke, Samantha; Ujjin, Jessica; Newsome, Robert; Somanath, Payaningal R

    2015-12-15

    Persistent myofibroblast differentiation is a hallmark of fibrotic diseases. Myofibroblasts are characterized by de novo expression of alpha smooth muscle actin (αSMA) and excess fibronectin assembly. Recent studies provide conflicting reports on the effects of tyrosine kinase inhibitor dasatinib on myofibroblast differentiation and fibrosis. Also, it is not fully understood whether dasatinib modulates myofibroblast differentiation by targeting Src kinase. Herein, we investigated the effect of dasatinib on cSrc and transforming growth factor-β (TGFβ)-induced myofibroblast differentiation in vitro. Our results indicated that selective Src kinase inhibition using PP2 mimicked the effect of dasatinib in attenuating myofibroblast differentiation as evident by blunted αSMA expression and modest, but significant inhibition of fibronectin assembly in both NIH 3T3 and fibrotic human lung fibroblasts. Mechanistically, our data showed that dasatinib modulates αSMA synthesis through Src kinase-mediated modulation of serum response factor expression. Collectively, our results demonstrate that dasatinib modulates myofibroblast differentiation through Src-SRF pathway. Thus, dasatinib could potentially be a therapeutic option in fibrotic diseases. PMID:26548624

  8. A cost-effective system for differentiation of intestinal epithelium from human induced pluripotent stem cells.

    PubMed

    Ogaki, Soichiro; Morooka, Mayu; Otera, Kaito; Kume, Shoen

    2015-01-01

    The human intestinal epithelium is a useful model for pharmacological studies of absorption, metabolism, drug interactions, and toxicology, as well as for studies of developmental biology. We established a rapid and cost effective system for differentiation of human induced pluripotent stem (iPS) cells into definitive endoderm (DE) cells. In the presence of dimethyl sulfoxide (DMSO), a low concentration of Activin at 6.25 ng/ml is sufficient to give a similar differentiation efficiency with that using Activin at 100 ng/ml at the presence of Wnt activator. In the presence of DMSO, Activin at low concentration triggered hiPS cells to undergo differentiation through G1 arrest, reduce apoptosis, and potentiate activation of downstream targets, such as SMAD2 phosphorylation and SOX17 expression. This increased differentiation into CDX2 + SOX17 + DE cells. The present differentiation procedure therefore permits rapid and efficient derivation of DE cells, capable of differentiating into intestinal epithelium upon BIO and DAPT treatment and of giving rise to functional cells, such as enterocytes. PMID:26616277

  9. microRNA-21 Mediates Stretch-Induced Osteogenic Differentiation in Human Periodontal Ligament Stem Cells

    PubMed Central

    Liu, Dongxu; Feng, Cheng; Zhang, Fan; Yang, Shuangyan; Hu, Yijun; Ding, Gang

    2015-01-01

    microRNAs (miRNAs) are short 20- to 22-nucleotide noncoding RNAs that negatively regulate the expression of target genes at the post-transcriptional level. The expression of specific miRNAs and their roles in the osteogenic differentiation of human periodontal ligament stem cells (PDLSCs) exposed to mechanical stretch remain unclear. Here, we found that stretch induced both osteogenic differentiation and the differential expression of miR-21 in PDLSCs. Furthermore, we identified activin receptor type IIB (ACVR2B) as a target gene of miR-21. Luciferase reporter assays showed that miR-21 interacts directly with the 3′-untranslated repeat sequence of ACVR2B mRNA. Mechanical stretch suppressed ACVR2B protein levels in PDLSCs, and this suppressive effect was modulated when endogenous miR-21 levels were either enhanced or inhibited. Both stretch and the expression of miR-21 altered endogenous ACVR2B protein levels and thus the osteogenic differentiation of PDLSCs. In addition, gain- and loss of function of ACVR2B mediated the osteogenic differentiation of PDLSCs. This study demonstrates that miR-21 is a mechanosensitive gene that plays an important role in the osteogenic differentiation of PDLSCs exposed to stretch. PMID:25203845

  10. Mechanical wounding-induced laticifer differentiation in rubber tree: An indicative role of dehydration, hydrogen peroxide, and jasmonates.

    PubMed

    Tian, Wei-Min; Yang, Shu-Guang; Shi, Min-Jing; Zhang, Shi-Xin; Wu, Ji-Lin

    2015-06-15

    The secondary laticifer in the secondary phloem of rubber tree are a specific tissue differentiating from vascular cambia. The number of the secondary laticifers is closely related to the rubber productivity of Hevea. Factors involved in the mechanical wounding-induced laticifer differentiation were analyzed by using paraffin section, gas chromatography-mass spectrometry (GC-MS), and Northern-blot techniques. Dehydration of the wounded bark tissues triggered a burst of hydrogen peroxide, abscisic acid, and jasmonates and up-regulated the expression of HbAOSa, which was associated with the secondary laticifer differentiation strictly limited to the wounded area. Application of exogenous hydrogen peroxide, methyl jasmonate, and polyethylene glycol 6000 (PEG6000) could induce the secondary laticifer differentiation, respectively. Moreover, 6-Benzylaminopurine, a synthetic cytokinin, enhanced the methyl jasmonate-induced secondary laticifer differentiation. However, the dehydration-induced secondary laticifer differentiation was inhibited by exogenous abscisic acid. Diphenyleneiodonium chloride (DPI), a specific inhibitor of NADPH oxidase, was effective in inhibiting the accumulation of hydrogen peroxide as well as of jasmonates upon dehydration. It blocked the dehydration-induced but not the methyl jasmonate-induced secondary laticifer differentiation. The results suggested a stress signal pathway mediating the wound-induced secondary laticifer differentiation in rubber tree. PMID:26070085

  11. Maintenance of in vivo induced cytochrome P-450s in hepatocyte monolayers at non freezing temperatures.

    PubMed

    Evans, Peter J

    2015-04-01

    Cytochrome P450s (CYPs) induced in rats by 3-methylcholanthrene (3-MC), phenobarbital (PB) and dexamethasone (Dex) were investigated. The inducers had no effect on hepatocyte yield, viability, attachment or spreading on collagen. 3-MC induced ethoxyresorufin deethylase (EROD). Under normothermic conditions the activity fell in culture. However, it was maintained when cells were preserved at 10°C under a gelatin gel. Upon reactivation the activity mirrored that of freshly isolated cells at 37°C. Induced levels were stable for at least 6h , the time to form a confluent monolayer. The investigation was extended to other CYPs by looking at patterns of testosterone metabolism. Phenobarbital had the greatest influence in terms of the quantity and number of metabolites. Culture at 37°C decreased the peaks dramatically within 24 h. All 7 peaks were maintained in the preservation system. PMID:25666303

  12. Effect of anthocyanidins on myogenic differentiation in induced and non-induced primary myoblasts from rainbow trout (Oncorhynchus mykiss).

    PubMed

    Villasante, Alejandro; Powell, Madison S; Murdoch, Gordon K; Overturf, Ken; Cain, Kenneth; Wacyk, Jurij; Hardy, Ronald W

    2016-01-01

    A study was conducted to test whether an anthocyanidin mixture (peonidin, cyanidin and pelargonidin chloride) modulates myogenesis in both induced and non-induced myogenic cells from juvenile rainbow trout (Oncorhynchus mykiss). We evaluated three different anthocyanidin concentrations (1×, 2.5× and 10×) at two sampling times (24 and 36h). To test for treatment effects, we analyzed the expression of myoD and pax7 as well as two target genes of the Notch signaling pathway, hey2 and her6. In induced myogenic cells, the lowest and middle anthocyanidin doses caused significantly greater expression of myoD after 24h of treatment compared to control. A significantly higher expression of pax7 in cells exposed to either anthocyanidin treatment during 36h compared was observed. Similarly, the pax7/myoD ratio was significantly lower in cells exposed to the lowest anthocyanidin doses during 24h compared to control. No significant effect of anthocyanidin treatments on the expression of hey2 and her6 at either sampling point was detected. In non-induced cells, we observed no effect of anthocyanidins on myoD expression and significant down-regulation on pax7 expression in cells exposed to either anthocyanidin mixture concentrations after 24 and 36h of treatment compared to control. Further, the pax7/myoD ratio was significantly lower in cells exposed to either anthocyanidin doses at both sampling time. In non-induced cells, the highest anthocyanidin dose provoked significantly greater expression of hey2 after 24h of treatment compared to control. We detected no such effect in non-induced cells exposed to the lowest and middle anthocyanidin doses during 24h of treatment. The expression of her6 was unaffected by anthocyanidin treatments at either sampling time or doses compared to control. Collectively, these findings provide evidence that anthocyanidins modulate specific components of the myogenic programming in fish, thereby potentially affecting somatic growth in fish fed

  13. Equine Induced Pluripotent Stem Cells have a Reduced Tendon Differentiation Capacity Compared to Embryonic Stem Cells

    PubMed Central

    Bavin, Emma P.; Smith, Olivia; Baird, Arabella E. G.; Smith, Lawrence C.; Guest, Deborah J.

    2015-01-01

    Tendon injuries occur commonly in horses and their repair through scar tissue formation predisposes horses to a high rate of re-injury. Pluripotent stem cells may provide a cell replacement therapy to improve tendon tissue regeneration and lower the frequency of re-injury. We have previously demonstrated that equine embryonic stem cells (ESCs) differentiate into the tendon cell lineage upon injection into the damaged horse tendon and can differentiate into functional tendon cells in vitro to generate artificial tendons. Induced pluripotent stem cells (iPSCs) have now been derived from horses but, to date, there are no reports on their ability to differentiate into tendon cells. As iPSCs can be produced from adult cell types, they provide a more accessible source of cells than ESCs, which require the use of horse embryos. The aim of this study was to compare tendon differentiation by ESCs and iPSCs produced through two independent methods. In two-dimensional differentiation assays, the iPSCs expressed tendon-associated genes and proteins, which were enhanced by the presence of transforming growth factor-β3. However, in three-dimensional (3D) differentiation assays, the iPSCs failed to differentiate into functional tendon cells and generate artificial tendons. These results demonstrate the utility of the 3D in vitro tendon assay for measuring tendon differentiation and the need for more detailed studies to be performed on equine iPSCs to identify and understand their epigenetic differences from pluripotent ESCs prior to their clinical application. PMID:26664982

  14. Steroid sex hormone dynamics during estradiol-17β induced gonadal differentiation in Paralichthys olivaceus (Teleostei)

    NASA Astrophysics Data System (ADS)

    Sun, Peng; You, Feng; Liu, Mengxia; Wu, Zhihao; Wen, Aiyun; Li, Jun; Xu, Yongli; Zhang, Peijun

    2010-03-01

    Steroid sex hormones, such as estradiol-17β (E2) and testosterone (T), are important regulators of sex change in fish. In this study, we examined the effects of E2 treatment on the dynamics of E2 and T during gonadal differentiation in the olive flounder Paralichthys olivaceus using histology and radioimmunoassay (RIA). Flounder larvae were divided into five groups (G0-G4), and fed with 0 (control), 0.2, 2, 20 and 100 mg E2/kg feed from 35 to 110 day post hatching (dph). Fish growth in the G1 and G2 groups was not significantly different from that of the control group ( P>0.05), while fish in the G3 and G4 groups were less active and showed growth depression and high mortality. The gonads of fish in the G3 and G4 groups were smaller and surrounded by hyperplastic connective tissue. The frequency of females in the G0-G4 groups was 54.5%, 75.0%, 100%, 100% and 93.3%, respectively. The RIA analyses of E2 and T showed that T levels decreased during gonadal differentiation, and increased slightly at the onset of ovarian differentiation, while E2 levels increased gradually and peaked at the onset of ovarian differentiation in the control group. In the E2-treated groups, T levels decreased before the onset of ovarian differentiation. E2 levels were high on the 48 dph, but declined to a lower level on the 54 dph, and then increased gradually during gonadal differentiation. And a sharp increase of E2 levels were observed in all E2-treated groups at the onset of ovarian differentiation. The data suggest that T and E2 play important roles during gonadal differentiation, and an E2 dose of 2 mg/kg feed could induce sex reversal in P. olivaceus.

  15. Optimizing neuronal differentiation from induced pluripotent stem cells to model ASD

    PubMed Central

    Kim, Dae-Sung; Ross, P. Joel; Zaslavsky, Kirill; Ellis, James

    2014-01-01

    Autism spectrum disorder (ASD) is an early-onset neurodevelopmental disorder characterized by deficits in social communication, and restricted and repetitive patterns of behavior. Despite its high prevalence, discovery of pathophysiological mechanisms underlying ASD has lagged due to a lack of appropriate model systems. Recent advances in induced pluripotent stem cell (iPSC) technology and neural differentiation techniques allow for detailed functional analyses of neurons generated from living individuals with ASD. Refinement of cortical neuron differentiation methods from iPSCs will enable mechanistic studies of specific neuronal subpopulations that may be preferentially impaired in ASD. In this review, we summarize recent accomplishments in differentiation of cortical neurons from human pluripotent stems cells and efforts to establish in vitro model systems to study ASD using personalized neurons. PMID:24782713

  16. Cell-Penetrating Peptide as a Means of Directing the Differentiation of Induced Pluripotent Stem Cells

    PubMed Central

    Kaitsuka, Taku; Tomizawa, Kazuhito

    2015-01-01

    Protein transduction using cell-penetrating peptides (CPPs) is useful for the delivery of large protein molecules, including some transcription factors. This method is safer than gene transfection methods with a viral vector because there is no risk of genomic integration of the exogenous DNA. Recently, this method was reported as a means for the induction of induced pluripotent stem (iPS) cells, directing the differentiation into specific cell types and supporting gene editing/correction. Furthermore, we developed a direct differentiation method to obtain a pancreatic lineage from mouse and human pluripotent stem cells via the protein transduction of three transcription factors, Pdx1, NeuroD, and MafA. Here, we discuss the possibility of using CPPs as a means of directing the differentiation of iPS cells and other stem cell technologies. PMID:26561805

  17. B-Raf inhibitors induce epithelial differentiation in BRAF-mutant colorectal cancer cells.

    PubMed

    Herr, Ricarda; Köhler, Martin; Andrlová, Hana; Weinberg, Florian; Möller, Yvonne; Halbach, Sebastian; Lutz, Lisa; Mastroianni, Justin; Klose, Martin; Bittermann, Nicola; Kowar, Silke; Zeiser, Robert; Olayioye, Monilola A; Lassmann, Silke; Busch, Hauke; Boerries, Melanie; Brummer, Tilman

    2015-01-01

    BRAF mutations are associated with aggressive, less-differentiated and therapy-resistant colorectal carcinoma. However, the underlying mechanisms for these correlations remain unknown. To understand how oncogenic B-Raf contributes to carcinogenesis, in particular to aspects other than cellular proliferation and survival, we generated three isogenic human colorectal carcinoma cell line models in which we can dynamically modulate the expression of the B-Raf(V600E) oncoprotein. Doxycyclin-inducible knockdown of endogenous B-Raf(V600E) decreases cellular motility and invasion in conventional and three-dimensional (3D) culture, whereas it promotes cell-cell contacts and induces various hallmarks of differentiated epithelia. Importantly, all these effects are recapitulated by B-Raf (PLX4720, vemurafenib, and dabrafenib) or MEK inhibitors (trametinib). Surprisingly, loss of B-Raf(V600E) in HT29 xenografts does not only stall tumor growth, but also induces glandular structures with marked expression of CDX2, a tumor-suppressor and master transcription factor of intestinal differentiation. By performing the first transcriptome profiles of PLX4720-treated 3D cultures of HT29 and Colo-205 cells, we identify several upregulated genes linked to epithelial differentiation and effector functions, such as claudin-1, a Cdx-2 target gene encoding a critical tight junction component. Thereby, we provide a mechanism for the clinically observed correlation between mutant BRAF and the loss of Cdx-2 and claudin-1. PLX4720 also suppressed several metastasis-associated transcripts that have not been implicated as targets, effectors or potential biomarkers of oncogenic B-Raf signaling so far. Together, we identify a novel facet of clinically applied B-Raf or MEK inhibitors by showing that they promote cellular adhesion and differentiation of colorectal carcinoma cells. PMID:25381152

  18. Differentiation-Inducing Factor-1 and -2 Function also as Modulators for Dictyostelium Chemotaxis

    PubMed Central

    Kuwayama, Hidekazu; Kubohara, Yuzuru

    2009-01-01

    Background In the early stages of development of the cellular slime mold Dictyostelium discoideum, chemotaxis toward cAMP plays a pivotal role in organizing discrete cells into a multicellular structure. In this process, a series of signaling molecules, such as G-protein-coupled cell surface receptors for cAMP, phosphatidylinositol metabolites, and cyclic nucleotides, function as the signal transducers for controlling dynamics of cytoskeleton. Differentiation-inducing factor-1 and -2 (DIF-1 and DIF-2) were originally identified as the factors (chlorinated alkylphenones) that induce Dictyostelium stalk cell differentiation, but it remained unknown whether the DIFs had any other physiologic functions. Methodology/Principal Findings To further elucidate the functions of DIFs, in the present study we investigated their effects on chemotaxis under various conditions. Quite interestingly, in shallow cAMP gradients, DIF-1 suppressed chemotaxis whereas DIF-2 promoted it greatly. Analyses with various mutants revealed that DIF-1 may inhibit chemotaxis, at least in part, via GbpB (a phosphodiesterase) and a decrease in the intracellular cGMP concentration ([cGMP]i). DIF-2, by contrast, may enhance chemotaxis, at least in part, via RegA (another phosphodiesterase) and an increase in [cGMP]i. Using null mutants for DimA and DimB, the transcription factors that are required for DIF-dependent prestalk differentiation, we also showed that the mechanisms for the modulation of chemotaxis by DIFs differ from those for the induction of cell differentiation by DIFs, at least in part. Conclusions/Significance Our findings indicate that DIF-1 and DIF-2 function as negative and positive modulators for Dictyostelium chemotaxis, respectively. To our knowledge, this is the first report in any organism of physiologic modulators (small molecules) for chemotaxis having differentiation-inducing activity. PMID:19684855

  19. Differential Costs of Two Distinct Resistance Mechanisms Induced by Different Herbivore Species in Arabidopsis.

    PubMed

    Onkokesung, Nawaporn; Reichelt, Michael; van Doorn, Arjen; Schuurink, Robert C; Dicke, Marcel

    2016-02-01

    Plants respond to herbivory with the induction of resistance, mediated by distinct phytohormonal signaling pathways and their interactions. Phloem feeders are known to induce plant resistance via the salicylic acid pathway, whereas biting-chewing herbivores induce plant resistance mainly via the jasmonate pathway. Here, we show that a specialist caterpillar (biting-chewing herbivore) and a specialist aphid (phloem feeder) differentially induce resistance against Pieris brassicae caterpillars in Arabidopsis (Arabidopsis thaliana) plants. Caterpillar feeding induces resistance through the jasmonate signaling pathway that is associated with the induction of kaempferol 3,7-dirhamnoside, whereas aphid feeding induces resistance via a novel mechanism involving sinapoyl malate. The role of sinapoyl malate is confirmed through the use of a mutant compromised in the biosynthesis of this compound. Caterpillar-induced resistance is associated with a lower cost in terms of plant growth reduction than aphid-induced resistance. A strong constitutive resistance against P. brassicae caterpillars in combination with a strong growth attenuation in plants of a transfer DNA (T-DNA) insertion mutant of WRKY70 (wrky70) suggest that the WRKY70 transcription factor, a regulator of downstream responses mediated by jasmonate-salicylic acid signaling cross talk, is involved in the negative regulation of caterpillar resistance and in the tradeoff between growth and defense. In conclusion, different mechanisms of herbivore-induced resistance come with different costs, and a functional WRKY70 transcription factor is required for the induction of low-cost resistance. PMID:26603653

  20. NF1 loss induces senescence during human melanocyte differentiation in an iPSC-based model.

    PubMed

    Larribere, Lionel; Wu, Huizi; Novak, Daniel; Galach, Marta; Bernhardt, Mathias; Orouji, Elias; Weina, Kasia; Knappe, Nathalie; Sachpekidis, Christos; Umansky, Ludmila; Beckhove, Philipp; Umansky, Viktor; De Schepper, Sofie; Kaufmann, Dieter; Ballotti, Robert; Bertolotto, Corine; Utikal, Jochen

    2015-07-01

    Neurofibromatosis type 1 (NF1) is a frequent genetic disease leading to the development of Schwann cell-derived neurofibromas or melanocytic lesions called café-au-lait macules (CALMs). The molecular mechanisms involved in CALMs formation remain largely unknown. In this report, we show for the first time pathophysiological mechanisms of abnormal melanocyte differentiation in a human NF1(+/-) -induced pluripotent stem cell (iPSC)-based model. We demonstrate that NF1 patient-derived fibroblasts can be successfully reprogrammed in NF1(+/-) iPSCs with active RAS signaling and that NF1 loss induces senescence during melanocyte differentiation as well as in patient's-derived CALMs, revealing a new role for NF1 in the melanocyte lineage. PMID:25824590

  1. Myostatin acts as an autocrine/paracrine negative regulator in myoblast differentiation from human induced pluripotent stem cells

    SciTech Connect

    Gao, Fei; Kishida, Tsunao; Ejima, Akika; Gojo, Satoshi; Mazda, Osam

    2013-02-08

    Highlights: ► iPS-derived cells express myostatin and its receptor upon myoblast differentiation. ► Myostatin inhibits myoblast differentiation by inhibiting MyoD and Myo5a induction. ► Silencing of myostatin promotes differentiation of human iPS cells into myoblasts. -- Abstract: Myostatin, also known as growth differentiation factor (GDF-8), regulates proliferation of muscle satellite cells, and suppresses differentiation of myoblasts into myotubes via down-regulation of key myogenic differentiation factors including MyoD. Recent advances in stem cell biology have enabled generation of myoblasts from pluripotent stem cells, but it remains to be clarified whether myostatin is also involved in regulation of artificial differentiation of myoblasts from pluripotent stem cells. Here we show that the human induced pluripotent stem (iPS) cell-derived cells that were induced to differentiate into myoblasts expressed myostatin and its receptor during the differentiation. An addition of recombinant human myostatin (rhMyostatin) suppressed induction of MyoD and Myo5a, resulting in significant suppression of myoblast differentiation. The rhMyostatin treatment also inhibited proliferation of the cells at a later phase of differentiation. RNAi-mediated silencing of myostatin promoted differentiation of human iPS-derived embryoid body (EB) cells into myoblasts. These results strongly suggest that myostatin plays an important role in regulation of myoblast differentiation from iPS cells of human origin. The present findings also have significant implications for potential regenerative medicine for muscular diseases.

  2. Rspo1-activated signalling molecules are sufficient to induce ovarian differentiation in XY medaka (Oryzias latipes).

    PubMed

    Zhou, Linyan; Charkraborty, Tapas; Zhou, Qian; Mohapatra, Sipra; Nagahama, Yoshitaka; Zhang, Yueguang

    2016-01-01

    In contrast to our understanding of testicular differentiation, ovarian differentiation is less well understood in vertebrates. In mammals, R-spondin1 (Rspo1), an activator of Wnt/β-catenin signaling pathway, is located upstream of the female sex determination pathway. However, the functions of Rspo1 in ovarian differentiation remain unclear in non-mammalian species. In order to elucidate the detailed functions of Rspo/Wnt signaling pathway in fish sex determination/differentiation, the ectopic expression of the Rspo1 gene was performed in XY medaka (Oryzias latipes). The results obtained demonstrated that the gain of Rspo1 function induced femininity in XY fish. The overexpression of Rspo1 enhanced Wnt4b and β-catenin transcription, and completely suppressed the expression of male-biased genes (Dmy, Gsdf, Sox9a2 and Dmrt1) as well as testicular differentiation. Gonadal reprograming of Rspo1-over-expressed-XY (Rspo1-OV-XY) fish, induced the production of female-biased genes (Cyp19a1a and Foxl2), estradiol-17β production and further female type secondary sexuality. Moreover, Rspo1-OV-XY females were fertile and produced successive generations. Promoter analyses showed that Rspo1 transcription was directly regulated by DM domain genes (Dmy, the sex-determining gene, and Dmrt1) and remained unresponsive to Foxl2. Taken together, our results strongly suggest that Rspo1 is sufficient to activate ovarian development and plays a decisive role in the ovarian differentiation in medaka. PMID:26782368

  3. Rspo1-activated signalling molecules are sufficient to induce ovarian differentiation in XY medaka (Oryzias latipes)

    PubMed Central

    Zhou, Linyan; Charkraborty, Tapas; Zhou, Qian; Mohapatra, Sipra; Nagahama, Yoshitaka; Zhang, Yueguang

    2016-01-01

    In contrast to our understanding of testicular differentiation, ovarian differentiation is less well understood in vertebrates. In mammals, R-spondin1 (Rspo1), an activator of Wnt/β-catenin signaling pathway, is located upstream of the female sex determination pathway. However, the functions of Rspo1 in ovarian differentiation remain unclear in non-mammalian species. In order to elucidate the detailed functions of Rspo/Wnt signaling pathway in fish sex determination/differentiation, the ectopic expression of the Rspo1 gene was performed in XY medaka (Oryzias latipes). The results obtained demonstrated that the gain of Rspo1 function induced femininity in XY fish. The overexpression of Rspo1 enhanced Wnt4b and β-catenin transcription, and completely suppressed the expression of male-biased genes (Dmy, Gsdf, Sox9a2 and Dmrt1) as well as testicular differentiation. Gonadal reprograming of Rspo1-over-expressed-XY (Rspo1-OV-XY) fish, induced the production of female-biased genes (Cyp19a1a and Foxl2), estradiol-17β production and further female type secondary sexuality. Moreover, Rspo1-OV-XY females were fertile and produced successive generations. Promoter analyses showed that Rspo1 transcription was directly regulated by DM domain genes (Dmy, the sex-determining gene, and Dmrt1) and remained unresponsive to Foxl2. Taken together, our results strongly suggest that Rspo1 is sufficient to activate ovarian development and plays a decisive role in the ovarian differentiation in medaka. PMID:26782368

  4. Feeding and Reward Are Differentially Induced by Activating GABAergic Lateral Hypothalamic Projections to VTA.

    PubMed

    Barbano, M Flavia; Wang, Hui-Ling; Morales, Marisela; Wise, Roy A

    2016-03-01

    Electrical stimulation of the lateral hypothalamus (LH) has two motivational effects: long trains of stimulation induce drive-like effects such as eating, and short trains are rewarding. It has not been clear whether a single set of activated fibers subserves the two effects. Previous optogenetic stimulation studies have confirmed that reinforcement and induction of feeding can each be induced by selective stimulation of GABAergic fibers originating in the bed nucleus of the LH and projecting to the ventral tegmental area (VTA). In the present study we determined the optimal stimulation parameters for each of the two optogenetically induced effects in food-sated mice. Stimulation-induced eating was strongest with 5 Hz and progressively weaker with 10 and 20 Hz. Stimulation-induced reward was strongest with 40 Hz and progressively weaker with lower or higher frequencies. Mean preferred duration for continuous 40 Hz stimulation was 61.6 s in a "real-time" place preference task; mean preferred duration for 5 Hz stimulation was 45.6 s. The differential effects of high- and low-frequency stimulation of this pathway seem most likely to be due to differential effects on downstream targets. PMID:26961951

  5. Intercellular Communication between Keratinocytes and Fibroblasts Induces Local Osteoclast Differentiation: a Mechanism Underlying Cholesteatoma-Induced Bone Destruction.

    PubMed

    Iwamoto, Yoriko; Nishikawa, Keizo; Imai, Ryusuke; Furuya, Masayuki; Uenaka, Maki; Ohta, Yumi; Morihana, Tetsuo; Itoi-Ochi, Saori; Penninger, Josef M; Katayama, Ichiro; Inohara, Hidenori; Ishii, Masaru

    2016-06-01

    Bone homeostasis is maintained by a balance in activity between bone-resorbing osteoclasts and bone-forming osteoblasts. Shifting the balance toward bone resorption causes osteolytic bone diseases such as rheumatoid arthritis and periodontitis. Osteoclast differentiation is regulated by receptor activator of nuclear factor κB ligand (RANKL), which, under some pathological conditions, is produced by T and B lymphocytes and synoviocytes. However, the mechanism underlying bone destruction in other diseases is little understood. Bone destruction caused by cholesteatoma, an epidermal cyst in the middle ear resulting from hyperproliferation of keratinizing squamous epithelium, can lead to lethal complications. In this study, we succeeded in generating a model for cholesteatoma, epidermal cyst-like tissue, which has the potential for inducing osteoclastogenesis in mice. Furthermore, an in vitro coculture system composed of keratinocytes, fibroblasts, and osteoclast precursors was used to demonstrate that keratinocytes stimulate osteoclast differentiation through the induction of RANKL in fibroblasts. Thus, this study demonstrates that intercellular communication between keratinocytes and fibroblasts is involved in the differentiation and function of osteoclasts, which may provide the molecular basis of a new therapeutic strategy for cholesteatoma-induced bone destruction. PMID:27001307

  6. Pulsed DC Electric Field-Induced Differentiation of Cortical Neural Precursor Cells.

    PubMed

    Chang, Hui-Fang; Lee, Ying-Shan; Tang, Tang K; Cheng, Ji-Yen

    2016-01-01

    We report the differentiation of neural stem and progenitor cells solely induced by direct current (DC) pulses stimulation. Neural stem and progenitor cells in the adult mammalian brain are promising candidates for the development of therapeutic neuroregeneration strategies. The differentiation of neural stem and progenitor cells depends on various in vivo environmental factors, such as nerve growth factor and endogenous EF. In this study, we demonstrated that the morphologic and phenotypic changes of mouse neural stem and progenitor cells (mNPCs) could be induced solely by exposure to square-wave DC pulses (magnitude 300 mV/mm at frequency of 100-Hz). The DC pulse stimulation was conducted for 48 h, and the morphologic changes of mNPCs were monitored continuously. The length of primary processes and the amount of branching significantly increased after stimulation by DC pulses for 48 h. After DC pulse treatment, the mNPCs differentiated into neurons, astrocytes, and oligodendrocytes simultaneously in stem cell maintenance medium. Our results suggest that simple DC pulse treatment could control the fate of NPCs. With further studies, DC pulses may be applied to manipulate NPC differentiation and may be used for the development of therapeutic strategies that employ NPCs to treat nervous system disorders. PMID:27352251

  7. Regulation of acid phosphatase activity in human promyelocytic leukemic cells induced to differentiate in culture

    PubMed Central

    1979-01-01

    Induction of differentiation of a human promyelocytic leukemic cell line (HL60) in culture is accompanied by changes in acid phosphatase (Acpase) activity. The increase in activity is less than twofold when the leukemic cells are stimulated by dimethylsulfoxide (DMSO) to differentiate into metamyelocytes and granulocytes but is eightfold when the cells are stimulated by the tumor-promoting agent 12-0- tetradecanoylphorbol 13-acetate (TPA) to differentiate into macrophage- like cells. Five different isozymes of Acpase were separated by acrylamide gel electrophoresis. Isozyme 1, the most anodal isozyme, was found to be present in undifferentiated, DMSO-treated and TPA-treated cells; isozyme 2 was a very faint band observed both in DMSO- and TPA- treated cells, the isoenzymes 3a and 3b were present only in TPA- induced cells; and isozyme 4, the most cathodal isozyme, was present both in TPA- and DMSO-induced cells. A time sequence study on the appearance of the various forms after TPA treatment indicated that the expression of the isozymes is regulated in an uncoordinated fashion. Acpase activity has been shown by ultrastructural cytochemistry to be localized in the entire rough endoplasmic reticulum (RER) and in areas of the smooth endoplasmic reticulum (SER) located near the Golgi complex in differentiating cells but to be extremely weak, if at all detectable, in undifferentiated promyelocytes. PMID:291600

  8. Pulsed DC Electric Field–Induced Differentiation of Cortical Neural Precursor Cells

    PubMed Central

    Chang, Hui-Fang; Lee, Ying-Shan; Tang, Tang K.; Cheng, Ji-Yen

    2016-01-01

    We report the differentiation of neural stem and progenitor cells solely induced by direct current (DC) pulses stimulation. Neural stem and progenitor cells in the adult mammalian brain are promising candidates for the development of therapeutic neuroregeneration strategies. The differentiation of neural stem and progenitor cells depends on various in vivo environmental factors, such as nerve growth factor and endogenous EF. In this study, we demonstrated that the morphologic and phenotypic changes of mouse neural stem and progenitor cells (mNPCs) could be induced solely by exposure to square-wave DC pulses (magnitude 300 mV/mm at frequency of 100-Hz). The DC pulse stimulation was conducted for 48 h, and the morphologic changes of mNPCs were monitored continuously. The length of primary processes and the amount of branching significantly increased after stimulation by DC pulses for 48 h. After DC pulse treatment, the mNPCs differentiated into neurons, astrocytes, and oligodendrocytes simultaneously in stem cell maintenance medium. Our results suggest that simple DC pulse treatment could control the fate of NPCs. With further studies, DC pulses may be applied to manipulate NPC differentiation and may be used for the development of therapeutic strategies that employ NPCs to treat nervous system disorders. PMID:27352251

  9. Retinoic acid induces Sertoli cell paracrine signals for spermatogonia differentiation but cell autonomously drives spermatocyte meiosis

    PubMed Central

    Raverdeau, Mathilde; Gely-Pernot, Aurore; Féret, Betty; Dennefeld, Christine; Benoit, Gérard; Davidson, Irwin; Chambon, Pierre; Mark, Manuel; Ghyselinck, Norbert B.

    2012-01-01

    Direct evidence for a role of endogenous retinoic acid (RA), the active metabolite of vitamin A in the initial differentiation and meiotic entry of spermatogonia, and thus in the initiation of spermatogenesis is still lacking. RA is synthesized by dedicated enzymes, the retinaldehyde dehydrogenases (RALDH), and binds to and activates nuclear RA receptors (RARA, RARB, and RARG) either within the RA-synthesizing cells or in the neighboring cells. In the present study, we have used a combination of somatic genetic ablations and pharmacological approaches in vivo to show that during the first, prepubertal, spermatogenic cycle (i) RALDH-dependent synthesis of RA by Sertoli cells (SC), the supporting cells of the germ cell (GC) lineage, is indispensable to initiate differentiation of A aligned into A1 spermatogonia; (ii) RARA in SC mediates the effects of RA, possibly through activating Mafb expression, a gene whose Drosophila homolog is mandatory to GC differentiation; (iii) RA synthesized by premeiotic spermatocytes cell autonomously induces meiotic initiation through controlling the RAR-dependent expression of Stra8. Furthermore, we show that RA of SC origin is no longer necessary for the subsequent spermatogenic cycles but essential to spermiation. Altogether, our data establish that the effects of RA in vivo on spermatogonia differentiation are indirect, via SC, but direct on meiotic initiation in spermatocytes, supporting thereby the notion that, contrary to the situation in the female, RA is necessary to induce meiosis in the male. PMID:23012458

  10. Activated Wnt signaling induces myofibroblast differentiation of mesenchymal stem cells, contributing to pulmonary fibrosis.

    PubMed

    Sun, Zhaorui; Wang, Cong; Shi, Chaowen; Sun, Fangfang; Xu, Xiaomeng; Qian, Weiping; Nie, Shinan; Han, Xiaodong

    2014-05-01

    Acute lung injury may lead to fibrogenesis. However, no treatment is currently available. This study was conducted to determine the effects of bone marrow-derived mesenchymal stem cells (MSCs) in a model of HCl-induced acute lung injury in Sprague-Dawley (SD) rats. Stromal cell-derived factor (SDF)-1 and its receptor CXC chemokine receptor (CXCR)4 have been shown to participate in mobilizing MSCs. Adenovirus carrying the CXCR4 gene was used to transfect MSCs in order to increase the engraftment numbers of MSCs at injured sites. Histological examination data demonstrated that the engraftment of MSCs did not attenuate lung injury and pulmonary fibrosis. The results showed that engraftment of MSCs almost differentiated into myofibroblasts, but rarely differentiated into lung epithelial cells. Additionally, it was demonstrated that activated canonical Wnt/β-catenin signaling in injured lung tissue regulated the myofibroblast differentiation of MSCs in vivo. The in vitro study results demonstrated that activation of the Wnt/β-catenin signaling stimulated MSCs to express myofibroblast markers; however, this process was attenuated by Wnt antagonist DKK1. Therefore, the results demonstrated that the aberrant activation of Wnt signaling induces the myofibroblast differentiation of engrafted MSCs, thus contributing to pulmonary fibrosis following lung injury. PMID:24573542

  11. Extracellular matrix components induce endocrine differentiation in vitro in NCI-H716 cells.

    PubMed Central

    de Bruïne, A. P.; Dinjens, W. N.; van der Linden, E. P.; Pijls, M. M.; Moerkerk, P. T.; Bosman, F. T.

    1993-01-01

    Endocrine cells occur in +/- 30% of colorectal adenocarcinomas. The significance of this phenomenon in terms of tumor behavior is still controversial. Endocrine differentiation in colorectal cancer cell lines is almost confined to tumor xenografts in vivo, suggesting that endocrine differentiation might be regulated by epithelial-stromal interactions. This hypothesis was studied in the cecal adenocarcinoma-derived cell line NCI-H716 by comparing the expression of chromogranin A protein and messenger RNA in vivo and in vitro and by attempts to induce differentiation in vitro. We found that chromogranin A expression, which was strongest in vivo, could be significantly enhanced in vitro by culturing tumor cells in the presence of native extracellular matrix, on fibroblast feeder layers, and in a defined medium with basic fibroblast growth factor. The results suggest that the extracellular matrix induces endocrine differentiation through factors (e.g., basic fibroblast-growth factor) that may be produced by stromal cells and after secretion bind to the extracellular matrix. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 6 PMID:8456938

  12. Impaired Neural Differentiation of Induced Pluripotent Stem Cells Generated from a Mouse Model of Sandhoff Disease

    PubMed Central

    Ogawa, Yasuhiro; Tanaka, Makoto; Tanabe, Miho; Suzuki, Toshihiro; Togawa, Tadayasu; Fukushige, Tomoko; Kanekura, Takuro; Sakuraba, Hitoshi; Oishi, Kazuhiko

    2013-01-01

    Sandhoff disease (SD) is a glycosphingolipid storage disease that arises from mutations in the Hexb gene and the resultant deficiency in β-hexosaminidase activity. This deficiency results in aberrant lysosomal accumulation of the ganglioside GM2 and related glycolipids, and progressive deterioration of the central nervous system. Dysfunctional glycolipid storage causes severe neurodegeneration through a poorly understood pathogenic mechanism. Induced pluripotent stem cell (iPSC) technology offers new opportunities for both elucidation of the pathogenesis of diseases and the development of stem cell-based therapies. Here, we report the generation of disease-specific iPSCs from a mouse model of SD. These mouse model-derived iPSCs (SD-iPSCs) exhibited pluripotent stem cell properties and significant accumulation of GM2 ganglioside. In lineage-directed differentiation studies using the stromal cell-derived inducing activity method, SD-iPSCs showed an impaired ability to differentiate into early stage neural precursors. Moreover, fewer neurons differentiated from neural precursors in SD-iPSCs than in the case of the wild type. Recovery of the Hexb gene in SD-iPSCs improved this impairment of neuronal differentiation. These results provide new insights as to understanding the complex pathogenic mechanisms of SD. PMID:23383290

  13. Synthesis and evaluation of synthetic retinoid derivatives as inducers of stem cell differentiation.

    PubMed

    Christie, Victoria B; Barnard, Jonathan H; Batsanov, Andrei S; Bridgens, Caroline E; Cartmell, Emily B; Collings, Jonathan C; Maltman, Daniel J; Redfern, Christopher P F; Marder, Todd B; Przyborski, Stefan; Whiting, Andrew

    2008-10-01

    All-trans-retinoic acid (ATRA) and its associated analogues are important mediators of cell differentiation and function during the development of the nervous system. It is well known that ATRA can induce the differentiation of neural tissues from human pluripotent stem cells. However, it is not always appreciated that ATRA is highly susceptible to isomerisation when in solution, which can influence the effective concentration of ATRA and subsequently its biological activity. To address this source of variability, synthetic retinoid analogues have been designed and synthesised that retain stability during use and maintain biological function in comparison to ATRA. It is also shown that subtle modifications to the structure of the synthetic retinoid compound impacts significantly on biological activity, as when exposed to cultured human pluripotent stem cells, synthetic retinoid 4-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-ylethynyl)benzoic acid, 4a (para-isomer), induces neural differentiation similarly to ATRA. In contrast, stem cells exposed to synthetic retinoid 3-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-ylethynyl)benzoic acid, 4b (meta-isomer), produce very few neurons and large numbers of epithelial-like cells. This type of structure-activity-relationship information for such synthetic retinoid compounds will further the ability to design more targeted systems capable of mediating robust and reproducible tissue differentiation. PMID:19082150

  14. Chronic heat-shock treatment driven differentiation induces apoptosis in Leishmania donovani.

    PubMed

    Raina, Puneet; Kaur, Sukhbir

    2006-09-01

    The present study investigates the role of apoptosis in the regulation of cell numbers of Leishmania donovani during the in vitro differentiation of promastigote stage to amastigote stage in axenic conditions. We report that apoptosis is induced in Leishmania donovani due to chronic heat-shock treatment of 37 ( degrees )C that also mediates the differentiation of promastigotes to amastigotes. This is characterized by the fragmentation of DNA, blebbing in the parasite cell membrane, nuclear condensation, formation of preapoptotic bodies and involvement of Ca(++) in the apoptotic process. The flowcytometric analysis shows an early and steep rise in percentage apoptotic nuclei till 48-hour stage of differentiation and then a gradual decline, suggesting synergistic action of Ca(++) ATPase and probably Hsp70. Hsp70 might be rescuing cells from apoptosis in the death signaling pathway. Incubation of the culture with Ca(++) chelator EGTA (1 mM) brings down the percentage of apoptotic nuclei considerably showing thereby that calcium is needed for the process of cell death here that occurs by apoptosis. The survival of the infective individuals appears to be decided by the parasite in the early stages of its differentiation. Our studies show the potential of the physiological temperature of 37 ( degrees )C in inducing apoptosis in Leishmania donovani and the therapeutic use it can be put to. PMID:16718376

  15. BMS-777607 promotes megakaryocytic differentiation and induces polyploidization in the CHRF-288-11 cells.

    PubMed

    Nurhayati, Retno Wahyu; Ojima, Yoshihiro; Taya, Masahito

    2015-04-01

    Introduction of a polyploidy inducer is a promising strategy to achieve a high level of polyploidization during megakaryocytic (MK) differentiation. Here, we report that a multi-kinase inhibitor, BMS-777607, is a potent polyploidy inducer for elevating high ploidy cell formation in the MK-differentiated CHRF-288-11 (CHRF) cells. Our result showed that BMS-777607 strongly inhibited cell division without affecting cell viability when detected at day 1 after treatment. As a consequence, the high ploidy (≥8N) cells were accumulated in culture for 8 days, with an increase from 16.2 to 75.2 % of the total cell population. The elevated polyploidization was accompanied by the increased expression level of MK marker, CD41 (platelet glycoprotein IIb/IIIa, GPIIb/IIIa), suggesting that BMS-777607 promoted both polyploidization and commitment of MK-differentiated CHRF cells. Platelet-like fragments (PFs) were released by mature CHRF cells. Based on a flow cytometry assay, it was found that the PFs produced from BMS-777607-treated cells tended to have larger size and higher expression of GPIIb/IIIa, a receptor for platelet adhesion. Taken together, these results suggested that BMS-777607 promoted MK differentiation of CHRF cells and increased the functional property of platelet-like fragments. PMID:25304900

  16. Differentiation of bone mesenchymal stem cells into hepatocyte-like cells induced by liver tissue homogenate.

    PubMed

    Xing, X K; Feng, H G; Yuan, Z Q

    2016-01-01

    This study investigated the efficacy and feasibility of inducing the differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) into hepatocyte-like cells in vitro using Sprague Dawley rats, as a model of hepatocyte generation for cell transplantation. BMSCs were isolated and grown using the adherent method and exposed to 5 or 10% liver tissue homogenate, before being collected for analysis after 0, 7, 14, and 21 days. Immunofluorescence and western blotting were employed to detect the liver-specific markers a-fetoprotein (AFP) and albumin (ALB). Supernatant urea content was also measured to verify that differentiation had been induced. After 7 days in the presence of 10% liver tissue homogenate, BMSCs demonstrated hepatocyte-like morphological characteristics, and with prolonged culture time, liver-specific markers were gradually produced at levels indicating cell maturation. AFP expression peaked at 14 days then began to decrease, while both urea and ALB levels increased with induction time. Overall, marker expression in the 5% homogenate group was less than or equal to the 10% group at each time point. Thus, in a rat model, liver tissue homogenate obtained from partial hepatectomy can induce the differentiation of BMSCs into hepatocyte-like cells. This method is simple, feasible, and has remarkable real-world application potential. PMID:27525848

  17. Tumor necrosis factor-{alpha} enhances IL-15-induced natural killer cell differentiation

    SciTech Connect

    Lee, Jiwon; Lee, Suk Hyung; Shin, Nara; Jeong, Mira; Kim, Mi Sun; Kim, Mi Jeong; Yoon, Suk Ran; Chung, Jin Woong; Kim, Tae-Don; Choi, Inpyo

    2009-09-04

    The differentiation of natural killer (NK) cells is regulated by various factors including soluble growth factors and transcription factors. Here, we have demonstrated that tumor necrosis factor-{alpha} (TNF-{alpha}) is a positive regulator of NK cell differentiation. TNF-{alpha} augmented the IL-15-induced expression of NK1.1 and CD122 in mature NK cells, and TNF-{alpha} alone also induced NK cell maturation as well as IL-15. TNF-{alpha} also increased IFN-{gamma} production in NK cells in the presence of IL-15. Meanwhile, mRNA expression of several transcription factors, including T-bet and GATA-3, was increased by the addition of TNF-{alpha} and IL-15. In addition, TNF-{alpha} increased nuclear factor-kappa B (NF-{kappa}B) activity in NK cells and inhibition of NF-{kappa}B impeded TNF-{alpha}-enhanced NK cell maturation. Overall, these data suggest that TNF-{alpha} significantly increased IL-15-driven NK cell differentiation by increasing the expression of transcription factors that play crucial roles in NK cell maturation and inducing the NF-{kappa}B activity.

  18. Latexin is involved in bone morphogenetic protein-2-induced chondrocyte differentiation

    SciTech Connect

    Kadouchi, Ichiro; Sakamoto, Kei; Tangjiao, Liu; Murakami, Takashi; Kobayashi, Eiji; Hoshino, Yuichi; Yamaguchi, Akira

    2009-01-16

    Latexin is the only known carboxypeptidase A inhibitor in mammals. We previously demonstrated that BMP-2 significantly induced latexin expression in Runx2-deficient mesenchymal cells (RD-C6 cells), during chondrocyte and osteoblast differentiation. In this study, we investigated latexin expression in the skeleton and its role in chondrocyte differentiation. Immunohistochemical studies revealed that proliferating and prehypertrophic chondrocytes expressed latexin during skeletogenesis and bone fracture repair. In the early phase of bone fracture, latexin mRNA expression was dramatically upregulated. BMP-2 upregulated the expression of the mRNAs of latexin, Col2a1, and the gene encoding aggrecan (Agc1) in a micromass culture of C3H10T1/2 cells. Overexpression of latexin additively stimulated the BMP-2-induced expression of the mRNAs of Col2a, Agc1, and Col10a1. BMP-2 treatment upregulated Sox9 expression, and Sox9 stimulated the promoter activity of latexin. These results indicate that latexin is involved in BMP-2-induced chondrocyte differentiation and plays an important role in skeletogenesis and skeletal regeneration.

  19. JAK2 and MPL protein levels determine TPO-induced megakaryocyte proliferation vs differentiation

    PubMed Central

    Besancenot, Rodolphe; Roos-Weil, Damien; Tonetti, Carole; Abdelouahab, Hadjer; Lacout, Catherine; Pasquier, Florence; Willekens, Christophe; Rameau, Philippe; Lecluse, Yann; Micol, Jean-Baptiste; Constantinescu, Stefan N.; Vainchenker, William; Solary, Eric

    2014-01-01

    Megakaryopoiesis is a 2-step differentiation process, regulated by thrombopoietin (TPO), on binding to its cognate receptor myeloproliferative leukemia (MPL). This receptor associates with intracytoplasmic tyrosine kinases, essentially janus kinase 2 (JAK2), which regulates MPL stability and cell-surface expression, and mediates TPO-induced signal transduction. We demonstrate that JAK2 and MPL mediate TPO-induced proliferation arrest and megakaryocytic differentiation of the human megakaryoblastic leukemia cell line UT7-MPL. A decrease in JAK2 or MPL protein expression, and JAK2 chemical inhibition, suppress this antiproliferative action of TPO. The expression of JAK2 and MPL, which progressively increases along normal human megakaryopoiesis, is decreased in platelets of patients diagnosed with JAK2- or MPL-mutated essential thrombocytemia and primary myelofibrosis, 2 myeloproliferative neoplasms in which megakaryocytes (MKs) proliferate excessively. Finally, low doses of JAK2 chemical inhibitors are shown to induce a paradoxical increase in MK production, both in vitro and in vivo. We propose that JAK2 and MPL expression levels regulate megakaryocytic proliferation vs differentiation in both normal and pathological conditions, and that JAK2 chemical inhibitors could promote a paradoxical thrombocytosis when used at suboptimal doses. PMID:25143485

  20. Induced neural-type differentiation in the cleavage-arrested blastomere isolated from early ascidian embryos.

    PubMed Central

    Okado, H; Takahashi, K

    1990-01-01

    1. Isolated blastomeres and pairs of blastomeres from 8-cell embryos of Halocynthia roretzi and Halocynthia aurantium were cleavage-arrested with cytochalasin B and cultured. Their differentiation was examined in terms of membrane excitability, immunoreactivity to an epidermis-specific monoclonal antibody (2C5), and the presence of acetylcholinesterase. 2. The blastomeres that showed epidermal-type differentiation had Ca2(+)-dependent action potentials and membrane currents, and immunoreactivity to 2C5. The blastomeres that showed neural-type differentiation had Na(+)-, Ca2(+)- and TEA-sensitive delayed K+ channels, and lacked immunoreactivity to 2C5. 3. Cleavage-arrested anterior-animal blastomeres, a4-2, when cultured in isolation from an 8-cell embryo, differentiated exclusively into epidermal-type cells. However, when cultured in contact with anterior-vegetal blastomeres, A4-1, they mostly showed neural-type differentiation (seventeen out of twenty-four cells in H. roretzi). 4. Reduction of the cytochalasin B concentration enhanced neural-type development of a4-2 blastomeres in contact with A4-1 blastomeres in H. aurantium, possibly by tightening the physical contact between the blastomeres. 5. When a cleavage-arrested and isolated a4-2 blastomere was treated with 2% pronase at 10 degrees C for 15 min at the time when sister control embryos reached the 32-cell stage, the blastomere underwent neural-type differentiation in a manner identical to that of a4-2 blastomeres contacted by A4-1 cells. 6. The period during which neural-type differentiation of a4-2 blastomeres could be induced by treatment with pronase was from the 8-cell to the 110-cell stage. At the late gastrula stage neural-type differentiation of a4-2 blastomeres was not induced by pronase. The effective period for neural-type differentiation of a4-2 blastomeres in contact with A4-1 cells was between the 64-cell stage and late gastrula stage. Competence of the a4-2 blastomere to undergo neural

  1. MyoD synergizes with the E-protein HEB beta to induce myogenic differentiation.

    PubMed

    Parker, Maura H; Perry, Robert L S; Fauteux, Mélanie C; Berkes, Charlotte A; Rudnicki, Michael A

    2006-08-01

    The MyoD family of basic helix-loop-helix transcription factors function as heterodimers with members of the E-protein family to induce myogenic gene activation. The E-protein HEB is alternatively spliced to generate alpha and beta isoforms. While the function of these molecules has been studied in other cell types, questions persist regarding the molecular functions of HEB proteins in skeletal muscle. Our data demonstrate that HEB alpha expression remains unchanged in both myoblasts and myotubes, whereas HEB beta is upregulated during the early phases of terminal differentiation. Upon induction of differentiation, a MyoD-HEB beta complex bound the E1 E-box of the myogenin promoter leading to transcriptional activation. Importantly, forced expression of HEB beta with MyoD synergistically lead to precocious myogenin expression in proliferating myoblasts. However, after differentiation, HEB alpha and HEB beta synergized with myogenin, but not MyoD, to activate the myogenin promoter. Specific knockdown of HEB beta by small interfering RNA in myoblasts blocked differentiation and inhibited induction of myogenin transcription. Therefore, HEB alpha and HEB beta play novel and central roles in orchestrating the regulation of myogenic factor activity through myogenic differentiation. PMID:16847330

  2. Overexpression of the beta 1 thyroid receptor induces differentiation in neuro-2a cells.

    PubMed Central

    Lebel, J M; Dussault, J H; Puymirat, J

    1994-01-01

    To determine the functions of the alpha 1 and beta 1 thyroid hormone receptors (TRs) in neural differentiation, we have established stable transfected neuronal cell lines (Neuro-2a) that overexpress either TR alpha 1 or TR beta 1. 3,5,3'-Triiodothyronine (T3) treatment of cells that overexpress TR beta 1 blocks proliferation by an arrest of cells in G0/G1 and induces morphological and functional differentiation of Neuro-2a cells as indicated by the marked increase in the number of perisomatal filopodia-like neurites and in acetylcholinesterase (AChE) activity. The effect on AChE activity was dose-dependent, and the time-course analysis reveals that this effect occurs after 24 hr of T3 treatment, with a maximal increase occurring after 48 hr of treatment. The increase of AChE activity is paralleled by an increase of AChE mRNAs. Last, we present evidence that shows that the effects of T3 on differentiation are independent of its effect on proliferation. T3 had no effect on the differentiation of Neuro-2a cells that overexpressed TR alpha 1. Our results indicate that TR beta 1 may play a key role in the effects of T3 in neuroblastoma cell differentiation. Images PMID:8146169

  3. Proteomic Profiles of Mesenchymal Stem Cells Induced by a Liver Differentiation Protocol

    PubMed Central

    Leelawat, Kawin; Narong, Siriluck; Chaijan, Suthidarak; Sa-ngiamsuntorn, Khanit; Disthabanchong, Sinee; Wongkajornsilp, Adisak; Hongeng, Suradej

    2010-01-01

    The replacement of disease hepatocytes and the stimulation of endogenous or exogenous regeneration by human mesenchymal stem cells (MSCs) are promising candidates for liver-directed cell therapy. In this study, we isolated MSCs from adult bone marrow by plastic adhesion and induced differentiation with a liver differentiation protocol. Western blot analyses were used to assess the expression of liver-specific markers. Next, MSC-specific proteins were analyzed with two-dimensional (2D) gel electrophoresis and peptide mass fingerprinting matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF)-mass spectrometry (MS). To confirm the results from the proteomic study, semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analyses were performed. We demonstrated that MSCs treated with the liver differentiation protocol expressed significantly more albumin, CK19 and CK20, than did undifferentiated cells. In addition the results of proteomic study demonstrated increases expression of FEM1B, PSMC2 and disulfide-isomerase A3 in MSCs treated with the liver differentiation protocol. These results from proteomic profiling will not only provide insight into the global responses of MSCs to hepatocyte differentiation, but will also lead to in-depth studies on the mechanisms of proteomic changes in MSCs. PMID:21614181

  4. The Aryl Hydrocarbon Receptor Ligand ITE Inhibits TGFβ1-Induced Human Myofibroblast Differentiation

    PubMed Central

    Lehmann, Geniece M.; Xi, Xia; Kulkarni, Ajit A.; Olsen, Keith C.; Pollock, Stephen J.; Baglole, Carolyn J.; Gupta, Shikha; Casey, Ann E.; Huxlin, Krystel R.; Sime, Patricia J.; Feldon, Steven E.; Phipps, Richard P.

    2011-01-01

    Fibrosis can occur in any human tissue when the normal wound healing response is amplified. Such amplification results in fibroblast proliferation, myofibroblast differentiation, and excessive extracellular matrix deposition. Occurrence of these sequelae in organs such as the eye or lung can result in severe consequences to health. Unfortunately, medical treatment of fibrosis is limited by a lack of safe and effective therapies. These therapies may be developed by identifying agents that inhibit critical steps in fibrotic progression; one such step is myofibroblast differentiation triggered by transforming growth factor-β1 (TGFβ1). In this study, we demonstrate that TGFβ1-induced myofibroblast differentiation is blocked in human fibroblasts by a candidate endogenous aryl hydrocarbon receptor (AhR) ligand 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE). Our data show that ITE disrupts TGFβ1 signaling by inhibiting the nuclear translocation of Smad2/3/4. Although ITE functions as an AhR agonist, and biologically persistent AhR agonists, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, cause severe toxic effects, ITE exhibits no toxicity. Interestingly, ITE effectively inhibits TGFβ1-driven myofibroblast differentiation in AhR−/− fibroblasts: Its ability to inhibit TGFβ1 signaling is AhR independent. As supported by the results of this study, the small molecule ITE inhibits myofibroblast differentiation and may be useful clinically as an antiscarring agent. PMID:21406171

  5. Lead Poisoning Disturbs Oligodendrocytes Differentiation Involved in Decreased Expression of NCX3 Inducing Intracellular Calcium Overload

    PubMed Central

    Ma, Teng; Wu, Xiyan; Cai, Qiyan; Wang, Yun; Xiao, Lan; Tian, Yanping; Li, Hongli

    2015-01-01

    Lead (Pb) poisoning has always been a serious health concern, as it permanently damages the central nervous system. Chronic Pb accumulation in the human body disturbs oligodendrocytes (OLs) differentiation, resulting in dysmyelination, but the molecular mechanism remains unknown. In this study, Pb at 1 μM inhibits OLs precursor cells (OPCs) differentiation via decreasing the expression of Olig 2, CNPase proteins in vitro. Moreover, Pb treatment inhibits the sodium/calcium exchanger 3 (NCX3) mRNA expression, one of the major means of calcium (Ca2+) extrusion at the plasma membrane during OPCs differentiation. Also addition of KB-R7943, NCX3 inhibitor, to simulate Pb toxicity, resulted in decreased myelin basic protein (MBP) expression and cell branching. Ca2+ response trace with Pb and KB-R7943 treatment did not drop down in the same recovery time as the control, which elevated intracellular Ca2+ concentration reducing MBP expression. In contrast, over-expression of NCX3 in Pb exposed OPCs displayed significant increase MBP fluorescence signal in positive regions and CNPase expression, which recovered OPCs differentiation to counterbalance Pb toxicity. In conclusion, Pb exposure disturbs OLs differentiation via affecting the function of NCX3 by inducing intracellular calcium overload. PMID:26287169

  6. Resveratrol-induced SIRT1 activation promotes neuronal differentiation of human bone marrow mesenchymal stem cells.

    PubMed

    Joe, I-Seul; Jeong, Sin-Gu; Cho, Goang-Won

    2015-01-01

    Resveratrol-3,4',5-trihydroxy-trans-stillbene (resveratrol; RSV), a natural non-flavonoid polyphenol compound, provides protection against stress injury, excessive sunlight, ultraviolet radiation, infections, and invading fungi. There is increasing evidence that resveratrol, a sirtuin1 activator, plays a pivotal role in neuroprotection and neuronal differentiation. In this study, we investigated whether resveratrol induces neuronal differentiation of human bone marrow-mesenchymal stem cells (hBM-MSCs). Quantitative PCR results showed that resveratrol-treated MSCs (RSV-MSCs) had significantly increased expression of the neuroprogenitor markers Nestin, Musashi, CD133, and GFAP. When RSV-MSCs were differentiated with neuronal induction media (RSV-dMSCs), they exhibited a cell body and dendritic morphology similar to neurons. The number and neurite length of these RSV-dMSCs were significantly increased compared to differentiated MSCs (dMSCs). The RSV-dMSCs and dMSCs had significantly increased expression of the neuronal-specific marker genes Nestin, Musashi, CD133, GFAP, NF-M, MAP-2, and KCNH1. The RSV-dMSCs also showed a higher expression of the neuronal marker proteins, Nestin and NF-M, based on immunocytochemical staining and immunoblot analysis. This effect was abolished by the treatment of sirtuin1 inhibitor EX527. Therefore, we have shown that resveratrol treatment, along with the use of neuronal induction media, effectively stimulates neuronal cell differentiation of hBM-MSCs. PMID:25459285

  7. Differential interferometry for measurement of density fluctuations and fluctuation-induced transport (invited)

    SciTech Connect

    Lin, L.; Ding, W. X.; Brower, D. L.; Bergerson, W. F.; Yates, T. F.

    2010-10-15

    Differential interferometry employs two parallel laser beams with a small spatial offset (less than beam width) and frequency difference (1-2 MHz) using common optics and a single mixer for a heterodyne detection. The differential approach allows measurement of the electron density gradient, its fluctuations, as well as the equilibrium density distribution. This novel interferometry technique is immune to fringe skip errors and is particularly useful in harsh plasma environments. Accurate calibration of the beam spatial offset, accomplished by use of a rotating dielectric wedge, is required to enable broad application of this approach. Differential interferometry has been successfully used on the Madison Symmetric Torus reversed-field pinch plasma to directly measure fluctuation-induced transport along with equilibrium density profile evolution during pellet injection. In addition, by combining differential and conventional interferometry, both linear and nonlinear terms of the electron density fluctuation energy equation can be determined, thereby allowing quantitative investigation of the origin of the density fluctuations. The concept, calibration, and application of differential interferometry are presented.

  8. Short-Term Differentiation of Glioblastoma Stem Cells Induces Hypoxia Tolerance.

    PubMed

    Skjellegrind, Håvard K; Fayzullin, Artem; Johnsen, Erik O; Eide, Lars; Langmoen, Iver A; Moe, Morten C; Vik-Mo, Einar O

    2016-07-01

    Glioblastoma is the most common and malignant brain cancer. In spite of surgical removal, radiation and chemotherapy, this cancer recurs within short time and median survival after diagnosis is less than a year. Glioblastoma stem cells (GSCs) left in the brain after surgery is thought to explain the inevitable recurrence of the tumor. Although hypoxia is a prime factor contributing to treatment resistance in many cancers, its effect on GSC has been little studied. Especially how differentiation influences the tolerance to acute hypoxia in GSCs is not well explored. We cultured GSCs from three patient biopsies and exposed these and their differentiated (1- and 4-weeks) progeny to acute hypoxia while monitoring intracellular calcium and mitochondrial membrane potential (ΔΨm). Undifferentiated GSCs were not hypoxia tolerant, showing both calcium overload and mitochondrial depolarization. One week differentiated cells were the most tolerant to hypoxia, preserving intracellular calcium stability and ΔΨm during 15 min of acute hypoxia. After 4 weeks of differentiation, mitochondrial mass was significantly reduced. In these cells calcium homeostasis was maintained during hypoxia, although the mitochondria were depolarized, suggesting a reduced mitochondrial dependency. Basal metabolic rate increased by differentiation, however, low oxygen consumption and high ΔΨm in undifferentiated GSCs did not provide hypoxia tolerance. The results suggest that undifferentiated GSCs are oxygen dependent, and that limited differentiation induces relative hypoxia tolerance. Hypoxia tolerance may be a factor involved in high-grade malignancy. This warrants a careful approach to differentiation as a glioblastoma treatment strategy. PMID:26915110

  9. The Dictyostelium prestalk inducer differentiation-inducing factor-1 (DIF-1) triggers unexpectedly complex global phosphorylation changes

    PubMed Central

    Sugden, Chris; Urbaniak, Michael D.; Araki, Tsuyoshi; Williams, Jeffrey G.

    2015-01-01

    Differentiation-inducing factor-1 (DIF-1) is a polyketide that induces Dictyostelium amoebae to differentiate as prestalk cells. We performed a global quantitative screen for phosphorylation changes that occur within the first minutes after addition of DIF-1, using a triple-label SILAC approach. This revealed a new world of DIF-1–controlled signaling, with changes in components of the MAPK and protein kinase B signaling pathways, components of the actinomyosin cytoskeletal signaling networks, and a broad range of small GTPases and their regulators. The results also provide evidence that the Ca2+/calmodulin–dependent phosphatase calcineurin plays a role in DIF-1 signaling to the DimB prestalk transcription factor. At the global level, DIF-1 causes a major shift in the phosphorylation/dephosphorylation equilibrium toward net dephosphorylation. Of interest, many of the sites that are dephosphorylated in response to DIF-1 are phosphorylated in response to extracellular cAMP signaling. This accords with studies that suggest an antagonism between the two inducers and also with the rapid dephosphorylation of the cAMP receptor that we observe in response to DIF-1 and with the known inhibitory effect of DIF-1 on chemotaxis to cAMP. All MS data are available via ProteomeXchange with identifier PXD001555. PMID:25518940

  10. Differential effects of chronic alcohol administration to rats on the activation of aromatic amines to mutagens in the Ames test.

    PubMed

    Steele, C M; Ioannides, C

    1986-05-01

    Male Wistar albino rats were maintained on alcohol-containing liquid diets for 4 weeks. Hepatic post-mitochondrial preparations derived from these animals were more efficient than control in activating 4-aminobiphenyl and 2-aminofluorene to mutagens in the Ames test. The alcohol-induced enhancement in mutagenicity was not inhibited by dimethylsulphoxide indicating that the generation of hydroxyl radicals is not involved. The activation of 2-naphthylamine was not affected by the treatment with alcohol but the mutagenicities of 2-aminoanthracene, benzo[a]pyrene and 3-methylcholanthrene were inhibited. The same treatment markedly increased hepatic microsomal aniline p-hydroxylase and ethoxyresorufin O-de-ethylase activities and to a lesser extent benzphetamine N-demethylase and microsomal levels of total cytochromes P-450. It is concluded that chronic alcohol administration to rats modulates the metabolic activation of pre-carcinogens to their reactive intermediates presumably by causing the redistribution of cytochrome P-450 isozymes. PMID:3009048

  11. Vanadium pentoxide induces pulmonary inflammation and tumor promotion in a strain-dependent manner

    PubMed Central

    2010-01-01

    Background Elevated levels of air pollution are associated with increased risk of lung cancer. Particulate matter (PM) contains transition metals that may potentiate neoplastic development through the induction of oxidative stress and inflammation, a lung cancer risk factor. Vanadium pentoxide (V2O5) is a component of PM derived from fuel combustion as well as a source of occupational exposure in humans. In the current investigation we examined the influence of genetic background on susceptibility to V2O5-induced inflammation and evaluated whether V2O5 functions as a tumor promoter using a 2-stage (initiation-promotion) model of pulmonary neoplasia in mice. Results A/J, BALB/cJ (BALB), and C57BL/6J (B6) mice were treated either with the initiator 3-methylcholanthrene (MCA; 10 μg/g; i.p.) or corn oil followed by 5 weekly aspirations of V2O5 or PBS and pulmonary tumors were enumerated 20 weeks following MCA treatment. Susceptibility to V2O5-induced pulmonary inflammation was assessed in bronchoalveolar lavage fluid (BALF), and chemokines, transcription factor activity, and MAPK signaling were quantified in lung homogenates. We found that treatment of animals with MCA followed by V2O5 promoted lung tumors in both A/J (10.3 ± 0.9 tumors/mouse) and BALB (2.2 ± 0.36) mice significantly above that observed with MCA/PBS or V2O5 alone (P < 0.05). No tumors were observed in the B6 mice in any of the experimental groups. Mice sensitive to tumor promotion by V2O5 were also found to be more susceptible to V2O5-induced pulmonary inflammation and hyperpermeability (A/J>BALB>B6). Differential strain responses in inflammation were positively associated with elevated levels of the chemokines KC and MCP-1, higher NFκB and c-Fos binding activity, as well as sustained ERK1/2 activation in lung tissue. Conclusions In this study we demonstrate that V2O5, an occupational and environmentally relevant metal oxide, functions as an in vivo lung tumor promoter among different inbred

  12. Uremic Toxins Enhance Statin-Induced Cytotoxicity in Differentiated Human Rhabdomyosarcoma Cells

    PubMed Central

    Uchiyama, Hitoshi; Tsujimoto, Masayuki; Shinmoto, Tadakazu; Ogino, Hitomi; Oda, Tomoko; Yoshida, Takuya; Furukubo, Taku; Izumi, Satoshi; Yamakawa, Tomoyuki; Tachiki, Hidehisa; Minegaki, Tetsuya; Nishiguchi, Kohshi

    2014-01-01

    The risk of myopathy and rhabdomyolysis is considerably increased in statin users with end-stage renal failure (ESRF). Uremic toxins, which accumulate in patients with ESRF, exert cytotoxic effects that are mediated by various mechanisms. Therefore, accumulation of uremic toxins might increase statin-induced cytotoxicity. The purpose of this study was to determine the effect of four uremic toxins—hippuric acid, 3-carboxy-4-methyl-5-propyl-2-furanpropionate, indole-3-acetic acid, and 3-indoxyl sulfate—on statin-induced myopathy. Differentiated rhabdomyosarcoma cells were pre-treated with the uremic toxins for seven days, and then the cells were treated with pravastatin or simvastatin. Cell viability and apoptosis were assessed by viability assays and flow cytometry. Pre-treatment with uremic toxins increased statin- but not cisplatin-induced cytotoxicity (p < 0.05 vs. untreated). In addition, the pre-treatment increased statin-induced apoptosis, which is one of the cytotoxic factors (p < 0.05 vs. untreated). However, mevalonate, farnesol, and geranylgeraniol reversed the effects of uremic toxins and lowered statin-induced cytotoxicity (p < 0.05 vs. untreated). These results demonstrate that uremic toxins enhance statin-induced apoptosis and cytotoxicity. The mechanism underlying this effect might be associated with small G-protein geranylgeranylation. In conclusion, the increased severity of statin-induced rhabdomyolysis in patients with ESRF is likely due to the accumulation of uremic toxins. PMID:25192420

  13. Hepatic Differentiation of Human Induced Pluripotent Stem Cells in a Perfused Three-Dimensional Multicompartment Bioreactor

    PubMed Central

    Freyer, Nora; Knöspel, Fanny; Strahl, Nadja; Amini, Leila; Schrade, Petra; Bachmann, Sebastian; Damm, Georg; Seehofer, Daniel; Jacobs, Frank; Monshouwer, Mario; Zeilinger, Katrin

    2016-01-01

    Abstract The hepatic differentiation of human induced pluripotent stem cells (hiPSC) holds great potential for application in regenerative medicine, pharmacological drug screening, and toxicity testing. However, full maturation of hiPSC into functional hepatocytes has not yet been achieved. In this study, we investigated the potential of a dynamic three-dimensional (3D) hollow fiber membrane bioreactor technology to improve the hepatic differentiation of hiPSC in comparison to static two-dimensional (2D) cultures. A total of 100 × 106 hiPSC were seeded into each 3D bioreactor (n = 3). Differentiation into definitive endoderm (DE) was induced by adding activin A, Wnt3a, and sodium butyrate to the culture medium. For further maturation, hepatocyte growth factor and oncostatin M were added. The same differentiation protocol was applied to hiPSC maintained in 2D cultures. Secretion of alpha-fetoprotein (AFP), a marker for DE, was significantly (p < 0.05) higher in 2D cultures, while secretion of albumin, a typical characteristic for mature hepatocytes, was higher after hepatic differentiation of hiPSC in 3D bioreactors. Functional analysis of multiple cytochrome P450 (CYP) isoenzymes showed activity of CYP1A2, CYP2B6, and CYP3A4 in both groups, although at a lower level compared to primary human hepatocytes (PHH). CYP2B6 activities were significantly (p < 0.05) higher in 3D bioreactors compared with 2D cultures, which is in line with results from gene expression. Immunofluorescence staining showed that the majority of cells was positive for albumin, cytokeratin 18 (CK18), and hepatocyte nuclear factor 4-alpha (HNF4A) at the end of the differentiation process. In addition, cytokeratin 19 (CK19) staining revealed the formation of bile duct-like structures in 3D bioreactors similar to native liver tissue. The results indicate a better maturation of hiPSC in the 3D bioreactor system compared to 2D cultures and emphasize the potential of dynamic 3D culture

  14. Hepatic Differentiation of Human Induced Pluripotent Stem Cells in a Perfused Three-Dimensional Multicompartment Bioreactor.

    PubMed

    Freyer, Nora; Knöspel, Fanny; Strahl, Nadja; Amini, Leila; Schrade, Petra; Bachmann, Sebastian; Damm, Georg; Seehofer, Daniel; Jacobs, Frank; Monshouwer, Mario; Zeilinger, Katrin

    2016-01-01

    The hepatic differentiation of human induced pluripotent stem cells (hiPSC) holds great potential for application in regenerative medicine, pharmacological drug screening, and toxicity testing. However, full maturation of hiPSC into functional hepatocytes has not yet been achieved. In this study, we investigated the potential of a dynamic three-dimensional (3D) hollow fiber membrane bioreactor technology to improve the hepatic differentiation of hiPSC in comparison to static two-dimensional (2D) cultures. A total of 100 × 10(6) hiPSC were seeded into each 3D bioreactor (n = 3). Differentiation into definitive endoderm (DE) was induced by adding activin A, Wnt3a, and sodium butyrate to the culture medium. For further maturation, hepatocyte growth factor and oncostatin M were added. The same differentiation protocol was applied to hiPSC maintained in 2D cultures. Secretion of alpha-fetoprotein (AFP), a marker for DE, was significantly (p < 0.05) higher in 2D cultures, while secretion of albumin, a typical characteristic for mature hepatocytes, was higher after hepatic differentiation of hiPSC in 3D bioreactors. Functional analysis of multiple cytochrome P450 (CYP) isoenzymes showed activity of CYP1A2, CYP2B6, and CYP3A4 in both groups, although at a lower level compared to primary human hepatocytes (PHH). CYP2B6 activities were significantly (p < 0.05) higher in 3D bioreactors compared with 2D cultures, which is in line with results from gene expression. Immunofluorescence staining showed that the majority of cells was positive for albumin, cytokeratin 18 (CK18), and hepatocyte nuclear factor 4-alpha (HNF4A) at the end of the differentiation process. In addition, cytokeratin 19 (CK19) staining revealed the formation of bile duct-like structures in 3D bioreactors similar to native liver tissue. The results indicate a better maturation of hiPSC in the 3D bioreactor system compared to 2D cultures and emphasize the potential of dynamic 3D culture systems

  15. Eriodicyol inhibits osteoclast differentiation and ovariectomy-induced bone loss in vivo.

    PubMed

    Lee, Juhyun; Noh, A Long Sae Mi; Zheng, Ting; Kang, Ju-hee; Yim, Mijung

    2015-12-10

    Osteoclasts are responsible for bone erosion in diseases such as osteoporosis and rheumatoid arthritis. In the present study, we investigate the effects of eriodictyol, a flavonoid found naturally in citrus fruits, on the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation using mouse bone marrow macrophages (BMMs). Eriodictyol inhibited RANKL-induced osteoclast formation in a dose-dependent manner without cytotoxicity. In addition, eriodictyol suppressed bone resorption activity of differentiated osteoclasts. The inhibitory effect of eriodictyol was associated with impaired activation of multiple signaling events downstream of RANK, including extracellular signal-regulated kinase, p38, and c-Jun terminal kinase phosphorylation, followed by decreased nuclear factor of activated T cells (NFAT)c1 expression. Ectopic overexpression of a constitutively active form of NFATc1 completely rescued the anti-osteoclastogenic effect of eriodictyol, suggesting that the anti-osteoclastogenic effect was mainly attributed to the reduction in NFATc1 expression. Consistent with the in vitro anti-osteoclastogenic effect, eriodictyol suppressed lipopolysaccharide-induced osteoclast formation in the calvarial model and ovariectomy-induced bone loss in vivo. Taken together, our data demonstrate that eriodictyol is a new therapeutic agent with the potential to prevent bone destructive diseases by reducing both osteoclast differentiation and function. PMID:26450448

  16. Scoparone attenuates RANKL-induced osteoclastic differentiation through controlling reactive oxygen species production and scavenging

    SciTech Connect

    Lee, Sang-Hyun; Jang, Hae-Dong

    2015-02-15

    Scoparone, one of the bioactive components of Artemisia capillaris Thunb, has various biological properties including immunosuppressive, hepatoprotective, anti-allergic, anti-inflammatory, and antioxidant effects. This study aims at evaluating the anti-osteoporotic effect of scoparone and its underlying mechanism in vitro. Scoparone demonstrated potent cellular antioxidant capacity. It was also found that scoparone inhibited the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation and suppressed cathepsin K and tartrate-resistant acid phosphatase (TRAP) expression via c-jun N-terminal kinase (JNK)/extracellular signal-regulated kinase (ERK)/p38-mediated c-Fos–nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) signaling pathway. During osteoclast differentiation, the production of general reactive oxygen species (ROS) and superoxide anions was dose-dependently attenuated by scoparone. In addition, scoparone diminished NADPH (nicotinamide adenine dinucleotide phosphate) oxidase 1 (Nox1) expression and activation via the tumor necrosis factor receptor-associated factor 6 (TRAF6)–cSrc–phosphatidylinositol 3-kinase (PI3k) signaling pathway and prevented the disruption of mitochondrial electron transport chain system. Furthermore, scoparone augmented the expression of superoxide dismutase 1 (SOD1) and catalase (CAT). The overall results indicate that the inhibitory effect of scoparone on RANKL-induced osteoclast differentiation is attributed to the suppressive effect on ROS and superoxide anion production by inhibiting Nox1 expression and activation and protecting the mitochondrial electron transport chain system and the scavenging effect of ROS resulting from elevated SOD1 and CAT expression. - Highlights: • Scoparone dose-dependently inhibited RANKL-induced osteoclast differentiation. • Scoparone diminished general ROS and superoxide anions in a dose-dependent manner. • Scoparone inhibited Nox1 expression and

  17. Bone marrow stromal cells as an inducer for cardiomyocyte differentiation from mouse embryonic stem cells.

    PubMed

    Yue, Fengming; Johkura, Kohei; Tomotsune, Daihachiro; Shirasawa, Sakiko; Yokoyama, Tadayuki; Nagai, Mika; Sasaki, Katsunori

    2010-09-20

    Bone marrow stromal cells (BMSCs) secrete soluble factors and display varied cell-biological functions. To confirm the ability and efficiency of BMSCs to induce embryonic stem cells (ESCs) into cardiomyocytes, mouse embryoid bodies (EBs) were co-cultured with rat BMSCs. After about 10 days, areas of rhythmically contracting cells in more solid aggregates became evident with bundle-like structures formed along borders between EB outgrowth and BMSC layer. ESC-derived cardiomyocytes exhibited sarcomeric striations when stained with troponin I (Trop I), organized in separated bundles. Besides, the staining for connexin 43 was detected in cell-cell junctions, which demonstrated that ESC-derived cardiomyocytes were coupled by gap junction in culture. The related genes of cardiomyocytes were found in these beating and no-beating EBs co-cultured with BMSCs. In addition, an improved efficiency of cardiomyocyte differentiation from ESC-BMSC co-culture was found in the serum-free medium: 5-fold up-regulation in the number of beating area compared with the serum medium. Effective cardiac differentiation was also recognized in transfer filter assay and in condition medium obtained from BMSC culture. A clear increase in the expression of cardiac genes and TropI protein confirmed further cardiac differentiation by BMP4 and Retinoic Acid (RA) treatment. These results demonstrate that BMSCs can induce cardiomyocyte differentiation from ESCs through soluble factors and enhance it with BMP4 or RA treatment. Serum-free ESC-BMSC co-culture represents a defined in vitro model for identifying the cardiomyocyte-inducing activity from BMSCs and, in addition, a straightforward experimental system for assessing clinical applications. PMID:20801009

  18. A new magnetorheological damper with improved displacement differential self-induced ability

    NASA Astrophysics Data System (ADS)

    Hu, Guoliang; Zhou, Wei; Li, Weihua

    2015-08-01

    This work is an extension of our previous study on the development of a linear variable differential sensor (LVDS)-based magnetorheological (MR) damper with self-sensing capability, where a new MR damper integrated with LVDS technology was developed and prototyped, then its self-induced performance under static and dynamic working conditions was experimentally evaluated. The results of the static and dynamic experiments indicated that the self-induced voltage was proportional to the displacement of the damper. Moreover, the damping performance of this new MR damper was also evaluated through an experimental study. Compared with our previous study, the new MR damper performed better in terms of its self-induced sensing ability and damping capacity.

  19. μ- and κ-Opioids Induce the Differentiation of Embryonic Stem Cells to Neural Progenitors*

    PubMed Central

    Kim, Eunhae; Clark, Amy L.; Kiss, Alexi; Hahn, Jason W.; Wesselschmidt, Robin; Coscia, Carmine J.; Belcheva, Mariana M.

    2008-01-01

    Growth factors, hormones, and neurotransmitters have been implicated in the regulation of stem cell fate. Since various neural precursors express functional neurotransmitter receptors, which include G protein-coupled receptors, it is anticipated that they are involved in cell fate decisions. We detected μ-opioid receptor (MOR-1) and κ-opioid receptor (KOR-1) expression and immunoreactivity in embryonic stem (ES) cells and in retinoic acid-induced ES cell-derived, nestin-positive, neural progenitors. Moreover, these G protein-coupled receptors are functional, since [D-Ala2,MePhe4,Gly-ol5]enkephalin, a MOR-selective agonist, and U69,593, a KOR-selective agonist, induce a sustained activation of extracellular signal-regulated kinase (ERK) signaling throughout a 24-h treatment period in undifferentiated, self-renewing ES cells. Both opioids promote limited proliferation of undifferentiated ES cells via the ERK/MAP kinase signaling pathway. Importantly, biochemical and immunofluorescence data suggest that [D-Ala2,MePhe4,Gly-ol5]enkephalin and U69,593 divert ES cells from self-renewal and coax the cells to differentiate. In retinoic acid-differentiated ES cells, opioid-induced signaling features a biphasic ERK activation profile and an opioid-induced, ERK-independent inhibition of proliferation in these neural progenitors. Collectively, the data suggest that opioids may have opposite effects on ES cell self-renewal and ES cell differentiation and that ERK activation is only required by the latter. Finally, opioid modulation of ERK activity may play an important role in ES cell fate decisions by directing the cells to specific lineages. PMID:16954126

  20. Mu- and kappa-opioids induce the differentiation of embryonic stem cells to neural progenitors.

    PubMed

    Kim, Eunhae; Clark, Amy L; Kiss, Alexi; Hahn, Jason W; Wesselschmidt, Robin; Coscia, Carmine J; Belcheva, Mariana M

    2006-11-01

    Growth factors, hormones, and neurotransmitters have been implicated in the regulation of stem cell fate. Since various neural precursors express functional neurotransmitter receptors, which include G protein-coupled receptors, it is anticipated that they are involved in cell fate decisions. We detected mu-opioid receptor (MOR-1) and kappa-opioid receptor (KOR-1) expression and immunoreactivity in embryonic stem (ES) cells and in retinoic acid-induced ES cell-derived, nestin-positive, neural progenitors. Moreover, these G protein-coupled receptors are functional, since [D-Ala(2),MePhe(4),Gly-ol(5)]enkephalin, a MOR-selective agonist, and U69,593, a KOR-selective agonist, induce a sustained activation of extracellular signal-regulated kinase (ERK) signaling throughout a 24-h treatment period in undifferentiated, self-renewing ES cells. Both opioids promote limited proliferation of undifferentiated ES cells via the ERK/MAP kinase signaling pathway. Importantly, biochemical and immunofluorescence data suggest that [D-Ala(2),MePhe(4),Gly-ol(5)]enkephalin and U69,593 divert ES cells from self-renewal and coax the cells to differentiate. In retinoic acid-differentiated ES cells, opioid-induced signaling features a biphasic ERK activation profile and an opioid-induced, ERK-independent inhibition of proliferation in these neural progenitors. Collectively, the data suggest that opioids may have opposite effects on ES cell self-renewal and ES cell differentiation and that ERK activation is only required by the latter. Finally, opioid modulation of ERK activity may play an important role in ES cell fate decisions by directing the cells to specific lineages. PMID:16954126

  1. Differential regulation of peripheral IL-1β-induced mechanical allodynia and thermal hyperalgesia in rats.

    PubMed

    Kim, Min J; Lee, Sang Y; Yang, Kui Y; Nam, Soon H; Kim, Hyun J; Kim, Young J; Bae, Yong C; Ahn, Dong K

    2014-04-01

    This study examined the differential mechanisms of mechanical allodynia and thermal hyperalgesia after injection of interleukin (IL) 1β into the orofacial area of male Sprague-Dawley rats. The subcutaneous administration of IL-1β produced both mechanical allodynia and thermal hyperalgesia. Although a pretreatment with iodoresiniferatoxin (IRTX), a transient receptor potential vanilloid 1 (TRPV1) antagonist, did not affect IL-1β-induced mechanical allodynia, it significantly abolished IL-1β-induced thermal hyperalgesia. On the other hand, a pretreatment with D-AP5, an N-methyl-d-aspartate (NMDA) receptor antagonist, and NBQX, an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist, blocked IL-1β-induced mechanical allodynia. Pretreatment with H89, a protein kinase A (PKA) inhibitor, blocked IL-1β-induced mechanical allodynia but not thermal hyperalgesia. In contrast, pretreatment with chelerythrine, a protein kinase C (PKC) inhibitor, inhibited IL-1β-induced thermal hyperalgesia. Subcutaneous injections of 2% lidocaine, a local anesthetic agent, blocked IL-1β-induced thermal hyperalgesia but not IL-1β-induced mechanical allodynia. In the resiniferatoxin (RTX)-pretreated rats, a subcutaneous injection of IL-1β did not produce thermal hyperalgesia due to the depletion of TRPV1 in the primary afferent fibers. Double immunofluorescence revealed the colocalization of PKA with neurofilament 200 (NF200) and of PKC with the calcitonin gene-related peptide (CGRP) in the trigeminal ganglion. Furthermore, NMDA receptor 1 (NR1) and TRPV1 predominantly colocalize with PKA and PKC, respectively, in the trigeminal ganglion. These results suggest that IL-1β-induced mechanical allodynia is mediated by sensitized peripheral NMDA/AMPA receptors through PKA-mediated signaling in the large-diameter primary afferent nerve fibers, whereas IL-1β-induced thermal hyperalgesia is mediated by sensitized peripheral TRPV1 receptors through PKC

  2. Tissue transglutaminase is involved in mechanical load-induced osteogenic differentiation of human ligamentum flavum cells.

    PubMed

    Chao, Yuan-Hung; Huang, Shih-Yung; Yang, Ruei-Cheng; Sun, Jui-Sheng

    2016-07-01

    Mechanical load-induced osteogenic differentiation might be the key cellular event in the calcification and ossification of ligamentum flavum. The aim of this study was to investigate the influence of tissue transglutaminase (TGM2) on mechanical load-induced osteogenesis of ligamentum flavum cells. Human ligamentum flavum cells were obtained from 12 patients undergoing lumbar spine surgery. Osteogenic phenotypes of ligamentum flavum cells, such as alkaline phosphatase (ALP), Alizarin red-S stain, and gene expression of osteogenic makers were evaluated following the administration of mechanical load and BMP-2 treatment. The expression of TGM2 was evaluated by real-time PCR, Western blotting, and enzyme-linked immunosorbent assay (ELISA) analysis. Our results showed that mechanical load in combination with BMP-2 enhanced calcium deposition and ALP activity. Mechanical load significantly increased ALP and OC gene expression on day 3, whereas BMP-2 significantly increased ALP, OPN, and Runx2 on day 7. Mechanical load significantly induced TGM2 gene expression and enzyme activity in human ligamentum flavum cells. Exogenous TGM2 increased ALP and OC gene expression; while, inhibited TG activity significantly attenuated mechanical load-induced and TGM2-induced ALP activity. In summary, mechanical load-induced TGM2 expression and enzyme activity is involved in the progression of the calcification of ligamentum flavum. PMID:27115725

  3. Differential microRNA expression in aristolochic acid-induced upper urothelial tract cancers ex vivo.

    PubMed

    Tao, Le; Zeng, Yigang; Wang, Jun; Liu, Zhihong; Shen, Bing; Ge, Jifu; Liu, Yong; Guo, Yifeng; Qiu, Jianxin

    2015-11-01

    Aristolochic acid (AA) is a carcinogenic, mutagenic and nephrotoxic compound commonly isolated from members of the plant family of Aristolochiaceae (such as Aristolochia and Asarum) and used in Chinese herbal medicine. Use of AA and AA‑containing plants causes chronic kidney disease (CKD) and upper urinary tract carcinoma (UUC); however, the underlying mechanism remains to be defined. miRNAs regulate a number of biological processes, including cell proliferation, differentiation and metabolism. This study explored differentially expressed miRNAs between AA‑induced upper urothelial tract cancer (AAN‑UUC) and non‑AAN‑UUC tissues. Patients with AAN‑UUC and non‑AAN‑UUC (n=20/group) were recruited in the present study. Five tissue samples from each group were used for miRNA microarray profiling and the rest of the tissue samples were subjected to reverse transcription-quantitative polymerase chain reaction analysis including seven selected miRNAs for confirmation. A total of 29 miRNAs were differentially expressed between AAN‑UUC and non‑AAN‑UUC tissues (P<0.05). TargenScan and Gene ontology analyses predicted the functions and targeted genes of these differentially expressed miRNAs, i.e. Akt3, FGFR3, PSEN1, VEGFa and AR. Subsequently, expression of the selected differentially expressed miRNAs (Hsa‑miR‑4795‑5p, Hsa‑miR‑488, Hsa‑miR‑4784, Hsa‑miR‑330, Hsa‑miR‑3916, Hsa‑miR‑4274 and Hsa‑miR‑181c) was validated in another set of tissue samples. A total of 29 miRNAs were identified to be differentially expressed between AAN‑UUC and non‑AAN‑UUC tissues and these miRNA target genes in FGFR3 and Akt pathways, which regulate cell growth and tumor progression, respectively. PMID:26397152

  4. A hybrid microfluidic system for regulation of neural differentiation in induced pluripotent stem cells.

    PubMed

    Hesari, Zahra; Soleimani, Massoud; Atyabi, Fatemeh; Sharifdini, Meysam; Nadri, Samad; Warkiani, Majid Ebrahimi; Zare, Mehrak; Dinarvand, Rassoul

    2016-06-01

    Controlling cellular orientation, proliferation, and differentiation is valuable in designing organ replacements and directing tissue regeneration. In the present study, we developed a hybrid microfluidic system to produce a dynamic microenvironment by placing aligned PDMS microgrooves on surface of biodegradable polymers as physical guidance cues for controlling the neural differentiation of human induced pluripotent stem cells (hiPSCs). The neuronal differentiation capacity of cultured hiPSCs in the microfluidic system and other control groups was investigated using quantitative real time PCR (qPCR) and immunocytochemistry. The functionally of differentiated hiPSCs inside hybrid system's scaffolds was also evaluated on the rat hemisected spinal cord in acute phase. Implanted cell's fate was examined using tissue freeze section and the functional recovery was evaluated according to the Basso, Beattie, and Bresnahan (BBB) locomotor rating scale. Our results confirmed the differentiation of hiPSCs to neuronal cells on the microfluidic device where the expression of neuronal-specific genes was significantly higher compared to those cultured on the other systems such as plain tissue culture dishes and scaffolds without fluidic channels. Although survival and integration of implanted hiPSCs did not lead to a significant functional recovery, we believe that combination of fluidic channels with nanofiber scaffolds provides a great microenvironment for neural tissue engineering, and can be used as a powerful tool for in situ monitoring of differentiation potential of various kinds of stem cells. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1534-1543, 2016. PMID:26914600

  5. Statins activate GATA-6 and induce differentiated vascular smooth muscle cells

    SciTech Connect

    Wada, Hiromichi Abe, Mitsuru; Ono, Koh; Morimoto, Tatsuya; Kawamura, Teruhisa; Takaya, Tomohide; Satoh, Noriko; Fujita, Masatoshi; Kita, Toru; Shimatsu, Akira; Hasegawa, Koji

    2008-10-03

    The beneficial effects of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) beyond cholesterol lowering involve their direct actions on vascular smooth muscle cells (VSMCs). However, the effects of statins on phenotypic modulation of VSMCs are unknown. We herein show that simvastatin (Sm) and atorvastatin (At) inhibited DNA synthesis in human aortic VSMCs dose-dependently, while cell toxicity was not observed below the concentration of 1 {mu}M of Sm or 100 nM of At. Stimulating proliferative VSMCs with Sm or At induced the expression of SM-{alpha}-actin and SM-MHC, highly specific markers of differentiated phenotype. Sm up-regulated the binding activity of GATA-6 to SM-MHC GATA site and activated the transfected SM-MHC promoter in proliferative VSMCs, while mutating the GATA-6 binding site abolished this activation. Geranylgeranylpyrophosphate (10 {mu}M), an inhibitor of Rho family proteins, abolished the statin-mediated induction of the differentiated phenotype in VSMCs. These findings suggest that statins activate GATA-6 and induce differentiated VSMCs.

  6. Monophasic and Biphasic Electrical Stimulation Induces a Precardiac Differentiation in Progenitor Cells Isolated from Human Heart

    PubMed Central

    Pietronave, Stefano; Zamperone, Andrea; Oltolina, Francesca; Colangelo, Donato; Follenzi, Antonia; Novelli, Eugenio; Diena, Marco; Pavesi, Andrea; Consolo, Filippo; Fiore, Gianfranco Beniamino; Soncini, Monica

    2014-01-01

    Electrical stimulation (ES) of cells has been shown to induce a variety of responses, such as cytoskeleton rearrangements, migration, proliferation, and differentiation. In this study, we have investigated whether monophasic and biphasic pulsed ES could exert any effect on the proliferation and differentiation of human cardiac progenitor cells (hCPCs) isolated from human heart fragments. Cells were cultured under continuous exposure to monophasic or biphasic ES with fixed cycles for 1 or 3 days. Results indicate that neither stimulation protocol affected cell viability, while the cell shape became more elongated and reoriented more perpendicular to the electric field direction. Moreover, the biphasic ES clearly induced the upregulation of early cardiac transcription factors, MEF2D, GATA-4, and Nkx2.5, as well as the de novo expression of the late cardiac sarcomeric proteins, troponin T, cardiac alpha actinin, and SERCA 2a. Both treatments increased the expression of connexin 43 and its relocation to the cell membrane, but biphasic ES was faster and more effective. Finally, when hCPCs were exposed to both monophasic and biphasic ES, they expressed de novo the mRNA of the voltage-dependent calcium channel Cav 3.1(α1G) subunit, which is peculiar of the developing heart. Taken together, these results show that ES alone is able to set the conditions for early differentiation of adult hCPCs toward a cardiac phenotype. PMID:24328510

  7. MCM2 mediates progesterone-induced endometrial stromal cell proliferation and differentiation in mice.

    PubMed

    Kong, Shuangbo; Han, Xue; Cui, Tongtong; Zhou, Chan; Jiang, Yufei; Zhang, Hangxiao; Wang, Bingyan; Wang, Haibin; Zhang, Shuang

    2016-08-01

    Uterine decidualization characterized by stromal cell proliferation and differentiation is critical to the establishment of pregnancy in many species. Progesterone is a key factor in regulating endometrial cell decidualization, however, the molecular basis involved in mediating the effects of progesterone during decidualization remains largely unknown. We report here that the DNA replication licensing factor MCM2, one of the conserved set of six-related proteins (MCM complex: MCM2-7) essential for eukaryotic DNA replication, is dynamically expressed in both proliferative and differentiated stromal cells during mouse periimplantation uterus. Applying PR-knockout mouse model and pharmacological strategy, we further found that the expression of Mcm2 is induced by progesterone action in the mouse uterine stroma. Employing a primary cell culture system, we further demonstrated that siRNA-mediated silencing of MCM2 arrests the cell cycle at G1-S transition during stromal cell proliferation. Moreover, the downregulation of Mcm2 could also compromise stromal cell differentiation. Collectively, our studies uncovered the role of a unique DNA replication licensing molecule MCM2 in mediating Progesterone-induced stromal cell decidualization in mouse uterus. PMID:26910396

  8. Collagen-Hydroxyapatite Scaffolds Induce Human Adipose Derived Stem Cells Osteogenic Differentiation In Vitro.

    PubMed

    Calabrese, Giovanna; Giuffrida, Raffaella; Fabbi, Claudia; Figallo, Elisa; Lo Furno, Debora; Gulino, Rosario; Colarossi, Cristina; Fullone, Francesco; Giuffrida, Rosario; Parenti, Rosalba; Memeo, Lorenzo; Forte, Stefano

    2016-01-01

    Mesenchymal stem cells (MSCs) play a crucial role in regulating normal skeletal homeostasis and, in case of injury, in bone healing and reestablishment of skeletal integrity. Recent scientific literature is focused on the development of bone regeneration models where MSCs are combined with biomimetic three-dimensional scaffolds able to direct MSC osteogenesis. In this work the osteogenic potential of human MSCs isolated from adipose tissue (hADSCs) has been evaluated in vitro in combination with collagen/Mg doped hydroxyapatite scaffolds. Results demonstrate the high osteogenic potential of hADSCs when cultured in specific differentiation induction medium, as revealed by the Alizarin Red S staining and gene expression profile analysis. In combination with collagen/hydroxyapatite scaffold, hADSCs differentiate into mature osteoblasts even in the absence of specific inducing factors; nevertheless, the supplement of the factors markedly accelerates the osteogenic process, as confirmed by the expression of specific markers of pre-osteoblast and mature osteoblast stages, such as osterix, osteopontin (also known as bone sialoprotein I), osteocalcin and specific markers of extracellular matrix maturation and mineralization stages, such as ALPL and osteonectin. Hence, the present work demonstrates that the scaffold per se is able to induce hADSCs differentiation, while the addition of osteo-inductive factors produces a significant acceleration of the osteogenic process. This observation makes the use of our model potentially interesting in the field of regenerative medicine for the treatment of bone defects. PMID:26982592

  9. Dissecting the retinoid-induced differentiation of F9 embryonal stem cells by integrative genomics

    PubMed Central

    Mendoza-Parra, Marco A; Walia, Mannu; Sankar, Martial; Gronemeyer, Hinrich

    2011-01-01

    Retinoic acid (RA) triggers physiological processes by activating heterodimeric transcription factors (TFs) comprising retinoic acid receptor (RARα, β, γ) and retinoid X receptor (RXRα, β, γ). How a single signal induces highly complex temporally controlled networks that ultimately orchestrate physiological processes is unclear. Using an RA-inducible differentiation model, we defined the temporal changes in the genome-wide binding patterns of RARγ and RXRα and correlated them with transcription regulation. Unexpectedly, both receptors displayed a highly dynamic binding, with different RXRα heterodimers targeting identical loci. Comparison of RARγ and RXRα co-binding at RA-regulated genes identified putative RXRα–RARγ target genes that were validated with subtype-selective agonists. Gene-regulatory decisions during differentiation were inferred from TF-target gene information and temporal gene expression. This analysis revealed six distinct co-expression paths of which RXRα–RARγ is associated with transcription activation, while Sox2 and Egr1 were predicted to regulate repression. Finally, RXRα–RARγ regulatory networks were reconstructed through integration of functional co-citations. Our analysis provides a dynamic view of RA signalling during cell differentiation, reveals RAR heterodimer dynamics and promiscuity, and predicts decisions that diversify the RA signal into distinct gene-regulatory programs. PMID:21988834

  10. Collagen-Hydroxyapatite Scaffolds Induce Human Adipose Derived Stem Cells Osteogenic Differentiation In Vitro

    PubMed Central

    Fabbi, Claudia; Figallo, Elisa; Lo Furno, Debora; Gulino, Rosario; Colarossi, Cristina; Fullone, Francesco; Giuffrida, Rosario; Parenti, Rosalba; Memeo, Lorenzo; Forte, Stefano

    2016-01-01

    Mesenchymal stem cells (MSCs) play a crucial role in regulating normal skeletal homeostasis and, in case of injury, in bone healing and reestablishment of skeletal integrity. Recent scientific literature is focused on the development of bone regeneration models where MSCs are combined with biomimetic three-dimensional scaffolds able to direct MSC osteogenesis. In this work the osteogenic potential of human MSCs isolated from adipose tissue (hADSCs) has been evaluated in vitro in combination with collagen/Mg doped hydroxyapatite scaffolds. Results demonstrate the high osteogenic potential of hADSCs when cultured in specific differentiation induction medium, as revealed by the Alizarin Red S staining and gene expression profile analysis. In combination with collagen/hydroxyapatite scaffold, hADSCs differentiate into mature osteoblasts even in the absence of specific inducing factors; nevertheless, the supplement of the factors markedly accelerates the osteogenic process, as confirmed by the expression of specific markers of pre-osteoblast and mature osteoblast stages, such as osterix, osteopontin (also known as bone sialoprotein I), osteocalcin and specific markers of extracellular matrix maturation and mineralization stages, such as ALPL and osteonectin. Hence, the present work demonstrates that the scaffold per se is able to induce hADSCs differentiation, while the addition of osteo-inductive factors produces a significant acceleration of the osteogenic process. This observation makes the use of our model potentially interesting in the field of regenerative medicine for the treatment of bone defects. PMID:26982592