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Sample records for 3-methylcholanthrene induces differential

  1. 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.

  2. 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.

  3. Aflatoxin B1 metabolism by 3-methylcholanthrene-induced hamster hepatic cytochrome P-450s.

    PubMed

    Lai, T S; Chiang, J Y

    1990-01-01

    We have studied the activation of aflatoxin B1 by hamster liver microsomes and purified hamster cytochrome P-450 isozymes using a umu mutagen test. The hamster liver microsomes or S-9 fractions were much more active than rat liver microsomes or S-9 fractions in the activation of umu gene expression by aflatoxin B1 metabolites. 3-Methyl-cholanthrene treatment increased aflatoxin B1 activation by hamster liver microsomes. Two major 3-methylcholanthrene-inducible cytochrome P-450 isozymes, P-450 MC1 (IIA) and P-450 MC4 (IA2), were purified from 3-methylcholanthrene-treated hamster liver microsomes, and the metabolism of aflatoxin B1 by these two cytochromes was studied. In the reconstituted enzyme system, both P-450 MC1 and P-450 MC4 were highly active in the activation of aflatoxin B1, and antibodies against these P-450s specifically inhibited these activities. Antibody against P-450 MC1 inhibited the activation of aflatoxin B1 by 20% in the presence of 3-methyl-cholanthrene-treated hamster liver microsomes. In contrast, antibody against P-450 MC4 stimulated the activity by 175%. These results indicated that hamster P-450 MC1 might convert aflatoxin B1 to more toxic metabolite(s), whereas P-450 MC4 might convert aflatoxin B1 to less toxic metabolite(s), than aflatoxin B1 in liver microsomes. The metabolite(s) produced by both hamster cytochrome P-450 MC1 and MC4 were genotoxic in the umu mutagen test. PMID:2126562

  4. Induction of UDP-glycosyltransferase family 1 genes in rat liver: different patterns of mRNA expression with two inducers, 3-methylcholanthrene and beta-naphthoflavone.

    PubMed

    Saarikoski, S T; Ikonen, T S; Oinonen, T; Lindros, K O; Ulmanen, I; Husgafvel-Pursiainen, K

    1998-09-01

    Uridine diphosphate (UDP)-glucuronosyltransferases (UGTs), presently called UDP-glycosyltransferases, catalyse the detoxification of many toxic and carcinogenic compounds. Glucuronidation is also a major metabolic pathway for numerous drugs. The UGT1A6 gene (formerly known as UGT1*06 and UGT1A1) has been suggested to belong to the aryl hydrocarbon (Ah) gene battery, which consists of several genes encoding for drug-metabolising enzymes regulated by dioxin and other ligands of the Ah receptor. In this study, we analysed the localisation of UGT1A6 expression in rat liver by in situ hybridisation to mRNA. Two different RNA probes were used, one which was specific to UGT1A6 and the other against the C terminal sequence shared by all UGT1 genes. In this study, no UGT1A6 mRNA was detected in the control animals. However, other gene(s) of the UGT1 family were expressed in the perivenous region surrounding the central veins as detected by hybridisation with the probe against the common region of the UGT1 genes. Treatment with the lower dose (5 mg/kg) of 3-methylcholanthrene (3MC) induced expression of UGT1A6 perivenously. Treatment with the higher dose (25 mg/kg) of 3-Methylcholanthrene resulted in a more panacinar expression pattern. In contrast to the perivenous induction observed with 3-methylcholanthrene, treatment with 15 mg/kg of beta-naphthoflavone (BNF) resulted in strong induction in the periportal region. The results reveal an inducer-specific pattern of UGT1A6 expression similar to that demonstrated earlier for other Ah battery genes, namely CYP1A1, CYP1A2, GSTYalpha and ALDH3. The finding further supports the notion that common factors regulate the regional hepatic expression of Ah battery genes. PMID:9783725

  5. 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

  6. 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

  7. 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

    2016-06-02

    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.

  8. Augmentation of 3-methylcholanthrene-induced bioactivation in the human hepatoma cell line HepG2 by the calcium channel blocker nicardipine.

    PubMed

    Hosaka, Takuomi; Sekimoto, Masashi; Nemoto, Kiyomitsu; Degawa, Masakuni

    2010-03-01

    The abilities of the dihydropyridine calcium channel blocker nicardipine (Nic) to induce cytochrome P450 1 family enzymes (CYP1s) and to enhance the 3-methylcholanthrene (MC)-mediated induction of CYP1s and formation of MC-DNA adduct were examined in the human hepatoma cell line HepG2. The results from real time RT-PCR analysis demonstrated that Nic could induce CYP1 mRNAs and enhance the MC-mediated induction of the CYP1 mRNAs. The luciferase-reporter gene assay using the HepG2-A10 cell line, which has been previously established for the screening of aryl hydrocarbon receptor (AhR) activators, also indicated the augmentation of MC-mediated activation of AhR (induction of luciferase) by Nic, although Nic showed limited capacity for the activation of AhR. Furthermore, the results from the Western blot analysis of CYP1s, the enzyme activity assay, and the assay for MC-DNA adduct formation indicated that Nic could enhance the MC-mediated induction of CYP1s, especially CYP1A1. Furthermore, the intracellular accumulation level of [(3)H]MC after treatment of HepG2 cells with [(3)H]MC significantly increased in the presence of Nic. The present findings demonstrate that Nic can enhance the MC-mediated induction of CYP1s, especially CYP1A1, and the formation of MC-DNA adduct in HepG2 cells. Furthermore, the augmentation of the MC-mediated bioactivation by Nic is demonstrated to occur mainly through an increase in intracellular accumulation of MC. PMID:20067464

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

    PubMed

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

    2012-11-15

    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 24h. 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.

  10. Kinetic isotope effects on cytochrome P-450-catalyzed oxidation reactions: full expression of the intrinsic isotope effect during the O-deethylation of 7-ethoxycoumarin by liver microsomes from 3-methylcholanthrene-induced hamsters

    SciTech Connect

    Miwa, G.T.; Harada, N.; Lu, A.Y.

    1985-05-15

    The intrinsic primary deuterium isotope effect for the O-deethylation of 7-ethoxycoumarin has been estimated by the Northrop method for the microsomal cytochrome P-448 system from 3-methylcholanthrene-induced hamster livers. The intrinsic isotope effect (Dk = 5.5) was found to be equivalent to the observed deuterium isotope effect, demonstrating that the isotope effect for this reaction was fully expressed by this cytochrome P-448 system. These data unequivocally demonstrate that C-H bond cleavage is the rate-limiting step in the overall reaction catalyzed by this system. The decrease in the rate of product formation, occurring as a consequence of deuterium substitution, resulted in a reduction in the quantity of substrate metabolized but was not accompanied by the change in regiospecificity observed in previous studies with a hepatic cytochrome P-448 isozyme purified from 3-methylcholanthrene-induced rats. These data demonstrate that the catalytic site of the hamster isozyme(s) offers more constraints to 7-ethoxycoumarin reorientation than does the catalytic site of the rat liver isozyme.

  11. 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

  12. Effects of combined butylated hydroxyanisole and 3-methylcholanthrene treatments on the expression of p-nitro-phenol UDP-glucuronyltransferase

    SciTech Connect

    Ananaba, G.A.; Stewart, J.

    1987-01-01

    UDP-glucuronyltransferase (UDP-GT) is a very important microsomal xenobiotic detoxicating enzyme. This enzyme has been shown to be induced by a variety of xenobiotics including the carcinogen, 3-methylcholanthrene (3-MC) and the phenolic antioxidant (BHA) which is a widely used food additive. Recently, our laboratory demonstrated that simultaneous administration of 3-MC and BHA to rats results to a synergistic induction of the biotransformation enzymes including UDP-GT. We have determined the elution profile of this enzyme and its multiple forms on ion-exchange and affinity chromatography columns. To further understand the expression of this particular enzyme, we have isolated poly (A/sup +/) RNA from BHA + 3-MC induced rat livers, and in vitro translated them to demonstrate the presence of a protein similar to the authentic UDP-GT in activity and molecular weight, as well as generated cDNA library from them. We hope to immunoprecipitate specific p-nitrophenol UDP-GT from the in vitro translated poly (A/sup +/) RNA and isolate specific mRNA by polysome immunoadsorption in order to generate cDNA to further characterize this unique and important enzyme.

  13. 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 ...

  14. 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

  15. Strain-dependent lung tumor formation in mice transplacentally exposed to 3-methylcholanthrene and post-natally exposed to butylated hydroxytoluene.

    PubMed

    Gressani, K M; Leone-Kabler, S; O'Sullivan, M G; Case, L D; Malkinson, A M; Miller, M S

    1999-11-01

    The carcinogenic effects of in utero exposure to 3-methylcholanthrene (MC) have been demonstrated in the tumor-resistant C57BL/6 (B6) and DBA (D2) strains of mice. In this study, we determined the effects of in utero exposure to MC in BALB/c mice, a strain which demonstrates greater susceptibility to lung tumor induction, and compared our findings with those previously found in [D2xB6D2F(1)]F(2) mice. In addition, we assessed the molecular pathogenesis of the chemically induced tumors and examined the effects of the putative lung tumor promoter butylated hydroxytoluene (BHT) in BALB/c mice. BALB/c mice were treated on day 17 of gestation with 5, 15 or 45 mg/kg MC and 6 weeks after birth with BHT for 6 consecutive weeks. Mice were killed at 6 months of age. Ki-ras, p16Ink4a and p19ARF gene loci were amplified from paraffin-embedded lung tumor tissue and screened for the presence of point mutations via allele-specific oligonucleotide hybridization and single strand conformation polymorphism (SSCP) analyses. Ki-ras point mutations were found in 56% (20/36) of BALB/c lung tumors, with 33% (2/6) of the hyperplasias, 58% (10/19) of the adenomas and 73% (8/11) of the carcinomas exhibiting point mutations at this gene locus. Similar incidences of Ki-ras mutations were previously found following transplacental exposure of [D2xB6D2F(1)]F(2) mice to MC and treatment of adult A/J mice with urethane. Interestingly, a strain-dependent difference was observed in the mutational spectrum. Sixty-two and 38% of the lung lesions in BALB/c mice exhibited G-->C and G-->T transversions, respectively, in contrast to the 13 and 84% incidences previously observed in [D2xB6D2F(1)]F(2) mice. SSCP analysis of the tumor suppressor gene p16Ink4a showed a 6% incidence of point mutations, consistent with that found in [D2xB6D2F(1)]F(2) mice. No mutations were found in exon 1beta of the p19ARF gene of either strain. BHT, a lung tumor promoter in adult mice, had no statistically significant effects

  16. 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.

  17. Transcriptional regulation by triiodothyronine of the UDP-glucuronosyltransferase family 1 gene complex in rat liver. Comparison with induction by 3-methylcholanthrene.

    PubMed

    Masmoudi, T; Hihi, A K; Vázquez, M; Artur, Y; Desvergne, B; Wahli, W; Goudonnet, H

    1997-07-01

    This study demonstrates that the expression of the phenol UDP-glucuronosyltransferase 1 gene (UGT1A1) is regulated at the transcriptional level by thyroid hormone in rat liver. Following 3,5, 3'-triiodo-L-thyronine (T3) stimulation in vivo, there is a gradual increase in the amount of UGT1A1 mRNA with maximum levels reached 24 h after treatment. In comparison, induction with the specific inducer, 3-methylcholanthrene (3-MC), results in maximal levels of UGT1A1 mRNA after 8 h of treatment. In primary hepatocyte cultures, the stimulatory effect of both T3 and 3-MC is also observed. This induction is suppressed by the RNA synthesis inhibitor actinomycin D, indicating that neither inducer acts at the level of mRNA stabilization. Indeed, nuclear run-on assays show a 3-fold increase in UGT1A1 transcription after T3 treatment and a 6-fold increase after 3-MC stimulation. This transcriptional induction by T3 is prevented by cycloheximide in primary hepatocyte cultures, while 3-MC stimulation is only partially affected after prolonged treatment with the protein synthesis inhibitor. Together, these data provide evidence for a transcriptional control of UGT1A1 synthesis and indicate that T3 and 3-MC use different activation mechanisms. Stimulation of the UGT1A1 gene by T3 requires de novo protein synthesis, while 3-MC-dependent activation is the result of a direct action of the compound, most likely via the aromatic hydrocarbon receptor complex. PMID:9202038

  18. 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.

  19. 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

  20. Demethylation of the pesticide methoxychlor in liver and intestine from untreated, methoxychlor-treated, and 3-methylcholanthrene-treated channel catfish (Ictalurus punctatus): evidence for roles of CYP1 and CYP3A family isozymes.

    PubMed

    Stuchal, Leah D; Kleinow, Kevin M; Stegeman, John J; James, Margaret O

    2006-06-01

    Exposure to the organochlorine pesticide methoxychlor (MXC) is associated with endocrine disruption in several species through biotransformation to mono-desmethyl-MXC (OH-MXC) and bis-desmethyl-MXC (HPTE), which interact with estrogen receptors. The biotransformation of [14C]methoxychlor was examined in channel catfish (Ictalurus punctatus), a freshwater species found in the southern United States. Hepatic microsomes formed OH-MXC and HPTE, assessed by comigration with authentic standards. The Km for OH-MXC formation by control liver microsomes was 3.8 +/- 1.3 microM (mean +/- S.D., n = 4), and Vmax was 131 +/- 53 pmol/min/mg protein. These values were similar to those of catfish pretreated with 2 mg/kg methoxychlor i.p. for 6 days (Km 3.3 +/- 0.8 microM and Vmax 99 +/- 17 pmol/min/mg) but less (p < 0.05) than the kinetic parameters for catfish treated with 3-methylcholanthrene (3-MC), which had Km of 6.0 +/- 1.1 microM and Vmax of 246 +/- 6 pmol/min/mg protein. Liver microsomes from 3-MC-treated fish produced significantly more of the secondary metabolite and more potent estrogen, HPTE. Intestinal microsomes formed OH-MXC at lower rates than liver. Methoxychlor pretreatment significantly reduced intestinal metabolite formation from 32 +/- 4 to 15 +/- 6 pmol/min/mg (mean +/- S.D., n = 4), whereas 3-MC treatment significantly increased OH-MXC production to 72 +/- 22 pmol/min/mg. Ketoconazole, clotrimazole, and alpha-naphthoflavone all decreased the production of OH-MXC in liver microsomes, whereas alpha-naphthoflavone stimulated HPTE formation, suggesting that CYP1 and CYP3 family isozymes demethylated methoxychlor. The results suggest that the formation of estrogenic metabolites from methoxychlor would be more rapid in catfish coexposed to CYP1 inducers.

  1. 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.

  2. Effects of chronic dietary exposure to a low-dose of Malathion, Aroclor-1254 and 3-methylcholanthrene on three biomarkers in male mice.

    PubMed

    Hackenberger, B K; Jarić, Davorka; Hackenberger, Dubravka; Stepić, Sandra

    2010-12-01

    The aim of this research was to examine the applicability of some chronic toxicological tests in the determination of exposure to xenobiotics present in concentrations below No Observed Adverse Effect Level (NOAEL) and below the detection limit of analytical instruments. In the present experiment tested chemicals (Malathion, Aroclor-1254 and 3-methylcholanthrene (3-MC)) were mixed with wheat grains and given to male mice as feed over a period of 12 months. 7-ethoxyresorufin-O-deethylase (EROD) activity with the 3-MC and Aroclor-1254 treatments reached the peak at 9th month of exposure (26.7 and 42.4 pmol⁻¹ mg(prot)-⁻¹, respectively), while malathion did not have significant influence. Glutathione (GSH) level depletion was highest after three months of exposure. Unexpectedly, acetylcholinesterase (AChE) activity increased after treatment with malathion, an organophosphorous insecticide. In conclusion, low-level concentrations chronically administered exert certain effects on the levels of selected enzymes, e.g. biomarkers.

  3. Glucuronidation and sulfonation, in vitro, of the major endocrine-active metabolites of methoxychlor in the channel catfish, Ictalurus punctatus, and induction following treatment with 3-methylcholanthrene

    PubMed Central

    James, Margaret O.; Stuchal, Leah D.; Nyagode, Beatrice A.

    2008-01-01

    The organochlorine pesticide, methoxychlor (MXC), is metabolized in animals to phenolic mono- and bis-demethylated metabolites (OH-MXC and HPTE respectively) that interact with estrogen receptors and may be endocrine disruptors. The phase II detoxication of these compounds will influence the duration of action of the estrogenic metabolites, but has not been investigated extensively. In this study, the glucuronidation and sulfonation of OH-MXC and HPTE were investigated in subcellular fractions of liver and intestine from untreated, MXC-treated and 3-methylcholanthrene (3-MC)-treated channel catfish, Ictalurus punctatus. MXC-treated fish were given i.p. injections of 2 mg MXC/kg daily for 6 days and sacrificed 24 hr after the last dose. The 3-MC treatment was a single 10 mg/kg i.p. dose 5 days prior to sacrifice. In hepatic microsomes from control fish, the Vmax value (mean ± S.D., n=4) for glucuronidation of OH-MXC was 270 ± 50 pmol/min/mg protein, higher than found for HPTE (110 ± 20 pmol/min/mg protein). For each substrate, the Vmax values observed in intestinal microsomes were approximately twice those found in the liver. The Km values for OH-MXC and HPTE glucuronidation in control liver were not significantly different and were 0.32 ± 0.04 mM for OH-MXC and 0.26 ± 0.06 mM for HPTE. The Km for the co-substrate, UDPGA, was higher in liver (0.28 ± 0.09 mM) than intestine (0.04 ± 0.02 mM). Treatment with 3-MC but not MXC increased the Vmax for glucuronidation in liver and intestine. Glucuronidation was a more efficient pathway than sulfonation for both substrates, in both tissues. The Vmax values for sulfonation of OH-MXC and HPTE respectively in liver cytosol were 7 ± 3 and 17 ± 4 pmol/min/mg protein and in intestinal cytosol were 13 ± 3 and 30 ± 5 pmol/min/mg protein. Treatment with 3-MC but not MXC increased rates of sulfonation of OH-MXC and HPTE and the model substrate, 3-hydroxy-benzo(a)pyrene in both intestine and liver. Comparison of the kinetics

  4. 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

  5. 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.

  6. Epilepsy-induced motility of differentiated neurons.

    PubMed

    Chai, Xuejun; Münzner, Gert; Zhao, Shanting; Tinnes, Stefanie; Kowalski, Janina; Häussler, Ute; Young, Christina; Haas, Carola A; Frotscher, Michael

    2014-08-01

    Neuronal ectopia, such as granule cell dispersion (GCD) in temporal lobe epilepsy (TLE), has been assumed to result from a migration defect during development. Indeed, recent studies reported that aberrant migration of neonatal-generated dentate granule cells (GCs) increased the risk to develop epilepsy later in life. On the contrary, in the present study, we show that fully differentiated GCs become motile following the induction of epileptiform activity, resulting in GCD. Hippocampal slice cultures from transgenic mice expressing green fluorescent protein in differentiated, but not in newly generated GCs, were incubated with the glutamate receptor agonist kainate (KA), which induced GC burst activity and GCD. Using real-time microscopy, we observed that KA-exposed, differentiated GCs translocated their cell bodies and changed their dendritic organization. As found in human TLE, KA application was associated with decreased expression of the extracellular matrix protein Reelin, particularly in hilar interneurons. Together these findings suggest that KA-induced motility of differentiated GCs contributes to the development of GCD and establish slice cultures as a model to study neuronal changes induced by epileptiform activity.

  7. Cisplatin Induces Differentiation of Breast Cancer Cells

    PubMed Central

    Prabhakaran, Praseetha; Hassiotou, Foteini; Blancafort, Pilar; Filgueira, Luis

    2013-01-01

    Breast tumors are heterogeneous including cells with stem cell properties and more differentiated cells. This heterogeneity is reflected into the molecular breast cancer subtypes. Breast cancer stem cells are resistant to chemotherapy, thus recent efforts are focusing on identifying treatments that shift them toward a more differentiated phenotype, making them more susceptible to chemotherapy. We examined whether the drug cisplatin induces differentiation in breast cancer cell lines that represent different breast cancer subtypes. We used three cell lines representing triple-negative breast cancers, BT-549 and MDA-MB-231 (claudin-low), and MDA-MB-468 (basal-like), along with estrogen and progesterone receptor positive MCF-7 cells (luminal). Cisplatin was applied at 2.5, 5, 10, and 20 μM, and cell viability and proliferation were measured using MTS and BrdU assays, respectively. The effect of cisplatin on the cellular hierarchy was examined by flow cytometry, immunofluorescence and qRT-PCR. Cisplatin treatment of 10 and 20 μM reduced cell viability by 36–51% and proliferation capacity by 36–67%. Treatment with cisplatin resulted in 12–67% down-regulation of stem cell markers (CD49f, SSEA4) and 10–130% up-regulation of differentiation markers (CK18, SMA, β-tubulin). At the mRNA level, CD49f was down-regulated whilst β-tubulin was up-regulated in the claudin-low cell lines. SSEA4 protein expression decreased upon cisplatin treatment, but SSEA4 mRNA expression increased indicating a differential regulation of cisplatin at the post-transcriptional level. It is concluded that cisplatin reduces breast cancer cell survival and induces differentiation of stem/progenitor cell subpopulations within breast cancer cell lines. These effects indicate the potential of this drug to target specific chemotherapy-resistant cells within a tumor. PMID:23761858

  8. 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

  9. 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.

  10. Royalactin induces queen differentiation in honeybees.

    PubMed

    Kamakura, Masaki

    2011-05-26

    The honeybee (Apis mellifera) forms two female castes: the queen and the worker. This dimorphism depends not on genetic differences, but on ingestion of royal jelly, although the mechanism through which royal jelly regulates caste differentiation has long remained unknown. Here I show that a 57-kDa protein in royal jelly, previously designated as royalactin, induces the differentiation of honeybee larvae into queens. Royalactin increased body size and ovary development and shortened developmental time in honeybees. Surprisingly, it also showed similar effects in the fruitfly (Drosophila melanogaster). Mechanistic studies revealed that royalactin activated p70 S6 kinase, which was responsible for the increase of body size, increased the activity of mitogen-activated protein kinase, which was involved in the decreased developmental time, and increased the titre of juvenile hormone, an essential hormone for ovary development. Knockdown of epidermal growth factor receptor (Egfr) expression in the fat body of honeybees and fruitflies resulted in a defect of all phenotypes induced by royalactin, showing that Egfr mediates these actions. These findings indicate that a specific factor in royal jelly, royalactin, drives queen development through an Egfr-mediated signalling pathway. PMID:21516106

  11. Thermospermine suppresses auxin-inducible xylem differentiation in Arabidopsis thaliana.

    PubMed

    Yoshimoto, Kaori; Noutoshi, Yoshiteru; Hayashi, Ken-ichiro; Shirasu, Ken; Takahashi, Taku; Motose, Hiroyasu

    2012-08-01

    Thermospermine, a structural isomer of spermine, is synthesized by a thermospermine synthase designated ACAULIS5 (ACL5). Thermospermine-deficient acl5 mutant of Arabidopsis thaliana shows severe dwarfism and excessive xylem differentiation. By screening for compounds that affect xylem differentiation in the acl5 mutant, we identified auxin analogs that remarkably enhanced xylem vessel differentiation in the acl5 mutant but not in the wild type. The xylem-inducing effect of auxin analogs was clearly suppressed by thermospermine, indicating that auxin-inducible xylem differentiation is normally limited by thermospermine. Here, we further characterized xylem-inducing effect of auxin analogs in various organs. Auxin analogs promoted protoxylem differentiation in roots and cotyledons in the acl5 mutant. Our results indicate that the opposite action between thermospermine and auxin in xylem differentiation is common in different organs and also suggest that thermospermine might be required for the suppression of protoxylem differentiation.

  12. Induction by phenobarbital in McA-RH7777 rat hepatoma cells of a polycyclic hydrocarbon inducible cytochrome P450

    SciTech Connect

    McManus, M.E.; Minchin, R.F.; Schwartz, D.M.; Wirth, P.J.; Huber, B.E.

    1986-05-29

    The metabolism of 2-acetylaminofluorene (AAF) to its six oxidative metabolites has been used to study cytochrome P-450 monooxygenase activity in two rat hepatoma cell lines, McA-RH7777 and Reuber H4-II-E. McA-RH7777 cells exhibited considerably higher basal activities than H4-II-E cells for all metabolic pathways studied. Phenobarbital induced AAF metabolite formation in McA-RH7777 cells to a similar extent as 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD), but was only a weak inducer of these activities in H4-II-E cells. Northern blot analysis utilizing specific phenobarbital or 3-methylcholanthrene inducible cytochrome P-450 cDNA probes indicated that there was at least a 10-fold increase in a 3-methylcholanthrene inducible cytochrome P-450 transcript in phenobarbital treated McA-RH7777 cells.

  13. 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

  14. 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.

  15. Chemical inducers and transcriptional markers of oligodendrocyte differentiation.

    PubMed

    Joubert, Lara; Foucault, Isabelle; Sagot, Yves; Bernasconi, Lilia; Duval, François; Alliod, Chantal; Frossard, Marie-José; Pescini Gobert, Rosanna; Curchod, Marie-Laure; Salvat, Catherine; Nichols, Anthony; Pouly, Sandrine; Rommel, Christian; Roach, Arthur; Hooft van Huijsduijnen, Rob

    2010-09-01

    Oligodendrocytes generate and maintain myelin, which is essential for axonal function and protection of the mammalian central nervous system. To advance our molecular understanding of differentiation by these cells, we screened libraries of pharmacologically active compounds and identified inducers of differentiation of Oli-neu, a stable cell line of mouse oligodendrocyte precursors (OPCs). We identified four broad classes of inducers, namely, forskolin/cAMP (protein kinase A activators), steroids (glucocorticoids and retinoic acid), ErbB2 inhibitors, and nucleoside analogs, and confirmed the activity of these compounds on rat primary oligodendrocyte precursors and mixed cortical cultures. We also analyzed transcriptional responses in the chemically induced mouse and rat OPC differentiation processes and compared these with earlier studies. We confirm the view that ErbB2 is a natural signaling component that is required for OPC proliferation, whereas ErbB2 inhibition or genetic knockdown results in OPC differentiation.

  16. Induction of cytochrome P450 1A2 by musk analogues and other inducing agents in rat liver.

    PubMed

    Iwata, N; Suzuki, K; Minegishi, K; Kawanishi, T; Hara, S; Endo, T; Takahashi, A

    1993-10-01

    We characterized the inducing effects of two musk analogues, musk xylene and musk ambrette, on phase I and phase II drug-metabolizing enzymes in rat liver and compared their effects with 3-methylcholanthrene, isosafrole and 2(3)-tertbutylhydroxyanisole (BHA) at 0.1 mmol/kg dose level. Musk xylene and isosafrole increased more efficiently the metabolic activation of 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1) to mutagen than that of benzo(a)pyrene. Musk ambrette increased both the activation of Glu-P-1 and benzo(a)pyrene to the same extent. Western blot analyses revealed that musk xylene, musk ambrette, isosafrole and BHA induced more strongly cytochrome P450 1A2 (CYP1A2) in microsomes than CYP1A1. 3-Methylcholanthrene induced CYP1A1 in preference to CYP1A2. On the other hand, all drugs except for 3-methylcholanthrene did not show remarkable increases in phase II enzyme activities, such as DT-diaphorase, glutathione S-transferase and UDP-glucuronyltransferase, at 0.1 mmol/kg dose level. These results show that musk xylene, musk ambrette, isosafrole and BHA at the dose level used in this study possess the potency to induce CYP1A2 without remarkable induction of CYP1A1 and phase II enzyme activities as observed for 3-methylcholanthrene, although they have been considered to induce both phase I and phase II drug-metabolizing enzymes at higher doses.

  17. 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

  18. X-radiation-induced differentiation of xenotransplanted human undifferentiated rhabdomyosarcoma

    SciTech Connect

    Takizawa, T.; Matsui, T.; Maeda, Y.; Okabe, S.; Mochizuki, M.; Tanaka, A.; Kawaguchi, K.; Fukayama, M.; Funata, N.; Koike, M.

    1989-01-01

    A serially xenotransplantable strain of undifferentiated embryonal rhabdomyosarcoma originating from the nasal cavity of a 42-year-old woman has been established in our laboratory. After radiotherapy for the tumor donor, distinct rhabdomyoblastic differentiation of the undifferentiated sarcoma cells appeared in the primary lesion, and it is a reasonable assumption that X-irradiation has a certain potentiality to induce morphologic differentiation of tumor cells. To study this possibility, tissue fragments of undifferentiated embryonal rhabdomyosarcoma that had grown to more than 10 mm after being transplanted to nude mice were selectively irradiated in situ. The degree of rhabdomyoblastic differentiation according to radiation dose was evaluated by light and electron microscopy and by immunostainability for myoglobin, creatine phosphokinase-MM, and desmin. Distinct morphologic differentiation of undifferentiated sarcoma cells could be induced by repeated X-irradiations at several-week intervals.

  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. Relaxin augments BMP-2-induced osteoblast differentiation and bone formation.

    PubMed

    Moon, Jung-Sun; Kim, Sun-Hun; Oh, Sin-Hye; Jeong, Yong-Wook; Kang, Jee-Hae; Park, Jong-Chun; Son, Hye-Ju; Bae, Suk; Park, Byung-Il; Kim, Min-Seok; Koh, Jeong-Tae; Ko, Hyun-Mi

    2014-07-01

    Relaxin (Rln), a polypeptide hormone of the insulin superfamily, is an ovarian peptide hormone that is involved in a diverse range of physiological and pathological reactions. In this study, we investigated the effect of Rln on bone morphogenetic protein 2 (BMP-2)-induced osteoblast differentiation and bone formation. Expression of Rln receptors was examined in the primary mouse bone marrow stem cells (BMSCs) and mouse embryonic fibroblast cell line C3H/10T1/2 cells by RT-PCR and Western blot during BMP-2-induced osteoblast differentiation. The effect of Rln on osteoblast differentiation and mineralization was evaluated by measuring the alkaline phosphatase activity, osteocalcin production, and Alizarin red S staining. For the in vivo evaluation, BMP-2 and/or Rln were administered with type I collagen into the back of mice, and after 3 weeks, bone formation was analyzed by micro-computed tomography (µCT). Western blot was performed to determine the effect of Rln on osteoblast differentiation-related signaling pathway. Expression of Rxfp 1 in BMSCs and C3H/10T1/2 cells was significantly increased by BMP-2. In vitro, Rln augmented BMP-2-induced alkaline phosphatase expression, osteocalcin production, and matrix mineralization in BMSCs and C3H/10T1/2 cells. In addition, in vivo administration of Rln enhanced BMP-2-induced bone formation in a dose-dependent manner. Interestingly, Rln synergistically increased and sustained BMP-2-induced Smad, p38, and transforming growth factor-β activated kinase (TAK) 1 phosphorylation. BMP-2-induced Runx 2 expression and activity were also significantly augmented by Rln. These results show that Rln enhanced synergistically BMP-2-induced osteoblast differentiation and bone formation through its receptor, Rxfp 1, by augmenting and sustaining BMP-2-induced Smad and p38 phosphorylation, which upregulate Runx 2 expression and activity. These results suggest that Rln might be useful for therapeutic application in destructive bone

  1. Contextual fear conditioning induces differential alternative splicing.

    PubMed

    Poplawski, Shane G; Peixoto, Lucia; Porcari, Giulia S; Wimmer, Mathieu E; McNally, Anna G; Mizuno, Keiko; Giese, K Peter; Chatterjee, Snehajyoti; Koberstein, John N; Risso, Davide; Speed, Terence P; Abel, Ted

    2016-10-01

    The process of memory consolidation requires transcription and translation to form long-term memories. Significant effort has been dedicated to understanding changes in hippocampal gene expression after contextual fear conditioning. However, alternative splicing by differential transcript regulation during this time period has received less attention. Here, we use RNA-seq to determine exon-level changes in expression after contextual fear conditioning and retrieval. Our work reveals that a short variant of Homer1, Ania-3, is regulated by contextual fear conditioning. The ribosome biogenesis regulator Las1l, small nucleolar RNA Snord14e, and the RNA-binding protein Rbm3 also change specific transcript usage after fear conditioning. The changes in Ania-3 and Las1l are specific to either the new context or the context-shock association, while the changes in Rbm3 occur after context or shock only. Our analysis revealed novel transcript regulation of previously undetected changes after learning, revealing the importance of high throughput sequencing approaches in the study of gene expression changes after learning. PMID:27451143

  2. 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

  3. 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...

  4. Differential expression of Ran GTPase during HMBA-induced differentiation in murine erythroleukemia cells.

    PubMed

    Vanegas, N; García-Sacristán, A; López-Fernández, L A; Párraga, M; del Mazo, J; Hernández, P; Schvartzman, J B; Krimer, D B

    2003-07-01

    Murine erythroleukemia (MEL) cells undergo erythroid differentiation in vitro when treated with hexamethylene bisacetamide (HMBA). To identify genes involved in the commitment of MEL cells to differentiate, we screened a cDNA library constructed from HMBA-induced cells by differential hybridization and isolated GTPase Ran as a down-regulated gene. We observed that Ran was expressed in a biphasic mode. Following a decrease in mRNA level during the initial hours of induction, Ran re-expressed at 24-48 h, and gradually declined again. To investigate the role of Ran during MEL differentiation we constructed MEL transfectants capable to express or block Ran mRNA production constitutively. No effects were observed on cell growth and proliferation. Blockage of Ran, however, interfered with MEL cell differentiation resulting in a decrease of cell survival in the committed population.

  5. IL-12 could induce monocytic tumor cells directional differentiation.

    PubMed

    Ma, Ting-Ting; Wu, Bi-Tao; Lin, Yan; Xiong, Hai-Yu; Wang, Qin; Li, Zi-Wei; Cheng, Feng; Tu, Zhi-Guang

    2015-04-01

    Interleukin-12 (IL-12), a member of interleukin family, plays a critical role in immune responses and anti-tumor activity. In this study, the effects of IL-12 on monocytic tumor cell lines differentiation to macrophagocyte and its likely mechanism was investigated. We examined the differentiation markers, morphological and functional changes, and possible mechanism in IL-12-treated THP-1 and U937 cells. It was found that IL-12 could up-regulated macrophage surface marker CD68 and CD11b expression in a time-dependent manner. Morphologically, after IL-12 treatment, THP-1 and U937 cells became round or irregular shape, even stretched many cell membrane protuberances; some cell nuclei became fuzzy or completely disappeared, and the chromatin appeared dense and cordlike. Furthermore, IL-12-induced monocytic tumor cell differentiation was accompanied by the growth arrest with G1-phase accumulation and S-phase reduction; apoptosis increased with anti-apoptosis protein Bcl-2 down-expression and pro-apoptosis protein Fas up-regulation, and enhanced phagocytosis function. The IL-12-induced macrophage differentiation of THP-1 and U937 cells was associated with the up-regulation of c-fms expression and the CSF-1R Tyr 809 site phosphorylation. These findings have revealed that IL-12 could induce monocytic tumor cells directional differentiation into macrophage-like cells, and its mechanism is possible connected with the up-regulation of c-fms expression and the phosphorylation of CSF-1R Tyr-809 site.

  6. 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

  7. Differentiated HL-60 promyelocytic leukaemia cells produce a factor inducing differentiation.

    PubMed

    Djulbegović, B; Christmas, S E; Moore, M

    1987-01-01

    The bipotential human promyelocytic leukaemia cell line HL-60 can be induced to differentiate into monocytic or granulocytic cells by treatment with 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) or dimethylsulphoxide (DMSO) respectively. Conditioned media (CM) from 1,25(OH)2D3- or DMSO-treated cells were able to induce monocytic differentiation in fresh HL-60 cells as measured by induction of non-specific esterase and macrophage surface markers. CM from 1,25(OH)2D3-treated cells also led to a dose dependent loss of proliferative capacity in soft agar colony assays. These effects were not due to a toxic effect of the CM or to residual inducer present in the CM. gamma-interferon and GM-CSF were apparently not responsible for these effects. CM from the human histiocytic lymphoma cell line U937 led to only a low level of induction of macrophage differentiation in fresh HL-60 cells. The defect in HL-60 leukaemic cells may therefore be at the level of induction of an autonomously-produced differentiation factor.

  8. Sambucus williamsii induced embryonic stem cells differentiated into neurons.

    PubMed

    Liu, Shih-Ping; Hsu, Chien-Yu; Fu, Ru-Huei; Huang, Yu-Chuen; Chen, Shih-Yin; Lin, Shinn-Zong; Shyu, Woei-Cherng

    2015-01-01

    The pluripotent stem cells, including embryonic stem cells (ESCs), are capable of self-renewal and differentiation into any cell type, thus making them the focus of many clinical application studies. However, the efficiency of ESCs differentiated into neurons needs to improve. In this study, we tried to increase efficiently to a neural fate in the presence of various transitional Chinese medicines through a three-step differentiation strategy. From extracts of 10 transitional Chinese medicine candidates, we determined that Sambucus williamsii (SW) extract triggers the up-regulation of Nestin and Tuj1 (neuron cells markers) gene expression levels. After determining the different concentrations of SW extract, the number of neurons in the 200 μg/ml SW extract group was higher than the control, 50, 100, and 400 μg/ml SW extract groups. In addition, the number of neurons in the 200 μg/ml SW extract group was higher and higher after each time passage (three times). We also detected the Oct4, Sox2 (stem cells markers), Tuj1, and Nestin genes expression levels by RT-PCR. In the differentiated process, Oct4 and Sox2 genes decreased while the Tuj1 and Nestin genes expression levels increased. In summary, we demonstrated that SW could induce pluripotent stem cells differentiated into neurons. Thus, SW might become a powerful material for neurons-differentiating strategies.

  9. 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.

  10. 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

  11. Shear stress induces osteogenic differentiation of human mesenchymal stem cells

    PubMed Central

    Yourek, Gregory; McCormick, Susan M; Mao, Jeremy J; Reilly, Gwendolen C

    2014-01-01

    Aim To determine whether fluid flow-induced shear stress affects the differentiation of bone marrow-derived human mesenchymal stem cells (hMSCs) into osteogenic cells. Materials & methods hMSCs cultured with or without osteogenic differentiation medium were exposed to fluid flow-induced shear stress and analyzed for alkaline phosphatase activity and expression of osteogenic genes. Results Immediately following shear stress, alkaline phosphatase activity in osteogenic medium was significantly increased. At days 4 and 8 of culture the mRNA expression of bone morphogenetic protein-2 and osteopontin was significantly higher in hMSCs subjected to shear stress than those cultured in static conditions. However, hMSCs cultured in osteogenic differentiation medium were less responsive in gene expression of alkaline phosphatase and bone morphogenetic protein-2. Conclusion These data demonstrate that shear stress stimulates hMSCs towards an osteoblastic phenotype in the absence of chemical induction, suggesting that certain mechanical stresses may serve as an alternative to chemical stimulation of stem cell differentiation. PMID:20868327

  12. 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

  13. 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

  14. Gelatin induces trophectoderm differentiation of mouse embryonic stem cells.

    PubMed

    Peng, Sha; Hua, Jinlian; Cao, Xuanhong; Wang, Huayan

    2011-06-01

    In this study, we selected gelatin as ECM (extracellular matrix) to support differentiation of mES (mouse embryonic stem) cells into TE (trophectoderm), as gelatin was less expensive and widely used. We found that 0.2% and 1.5% gelatin were the suitable concentrations to induce TE differentiation by means of detecting Cdx2 expression using real-time PCR. Moreover, about 15% cells were positive for Cdx2 staining after 6 days differentiation. We discovered that the expressions of specific markers for TE, such as Cdx2, Eomes, Hand1 and Esx1 were prominently increased after gelatin induction. Meanwhile, the expression of Oct4 was significantly decreased. We also found that inhibition of the BMP (bone morphogenetic protein) signalling by Noggin could promote mES cells differentiation into TE, whereas inhibition of the Wnt signalling by Dkk1 had the contrary effect. This could be used as a tool to study the differentiation and function of early trophoblasts as well as further elucidating the molecular mechanism during abnormal placental development.

  15. Inhibition of Rac1 promotes BMP-2-induced osteoblastic differentiation.

    PubMed

    Onishi, M; Fujita, Y; Yoshikawa, H; Yamashita, T

    2013-01-01

    Small G proteins of the Rho family are pivotal regulators of several signaling networks. The Ras homolog family (Rho) and one of its targets, Rho-associated protein kinase (ROCK), participate in a wide variety of biological processes, including bone formation. A previous study has demonstrated that the ROCK inhibitor Y-27632 enhanced bone formation induced by recombinant human bone morphogenetic protein-2 (BMP-2) in vivo and in vitro. However, the effect of other Rho family members, such as Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division cycle 42 (Cdc42), on bone formation remains unknown. In this study, we investigated whether Rac1 also participates in BMP-2-induced osteogenesis. Expression of a dominant-negative mutant of Rac1 enhanced BMP-2-induced osteoblastic differentiation in C2C12 cells, whereas a constitutively active mutant of Rac1 attenuated that effect. Knockdown of T-lymphoma invasion and metastasis 1 (Tiam1), a Rac-specific guanine nucleotide exchange factor, enhanced BMP-2-induced alkaline phosphatase activity. Further, we demonstrated that BMP-2 stimulated Rac1 activity. These results indicate that the activation of Rac1 attenuates osteoblastic differentiation in C2C12 cells.

  16. 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.

  17. 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.

  18. 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.

  19. Inducible growth mode switches influence Valonia rhizoid differentiation.

    PubMed

    Elvira, Paul Rommel; Sekida, Satoko; Okuda, Kazuo

    2013-02-01

    Cell differentiation and cell type commitment are an integral part of plant growth and development. Investigations on how environmental conditions affect the formation of shoots, roots, and rhizoids can help illustrate how plants determine cell fate and overall morphology. In this study, we evaluated the role of substratum and light on rhizoid differentiation in the coenocytic green alga, Valonia aegagropila. Elongating rhizoids displayed varying growth modes and cell shape upon exposure to different substrata and light conditions. It was found that soft substrata and dark incubation promoted rhizoid elongation via tip growth while subsequent exposure to light prevented tip growth and instead induced swelling in the apical region of rhizoids. Swelling was accompanied by the accumulation of protoplasm in the rhizoid tip through expansion of the cell wall and uninhibited cytoplasmic streaming. Subsequent diffuse growth led to the transformation from slender, rod-shaped rhizoids into spherical thallus-like structures that required photosynthesis. Further manipulation of light regimes caused vacillating cell growth redirections. An elongating V. aegagropila rhizoid cell thus appears capable of growth mode switching that is regulated by immediate environmental conditions thereby influencing ultimate cell shape and function. This is the first description of inducible, multiple growth mode shifts in a single intact plant cell that directly impact its differentiation.

  20. 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

  1. Multiple hormone-inducible enhancers as mediators of differential transcription.

    PubMed Central

    Toohey, M G; Morley, K L; Peterson, D O

    1986-01-01

    Sets of genes under a common regulatory control in a given cell type are often differentially transcribed. The possibility that this differential transcription can be modulated by the number or strength of cis-acting regulatory sequences associated with a given gene was tested by using the glucocorticoid-responsive enhancer element associated with the mouse mammary tumor virus promoter. Results indicate that differential levels of hormone-inducible gene expression can be modulated in an additive way by the number of glucocorticoid-responsive enhancers associated with this promoter. Realization of these effects shows little preference for position of the additional elements with respect to the promoter. When sequences that bind the glucocorticoid receptor in vitro with somewhat lower affinity than the enhancer were tested, these additive effects were not detected. The results support that differential transcription of genes subject to a common regulatory control can be mediated, at least in part, by the number or strength of their associated cis-acting regulatory sequences. Images PMID:3025659

  2. 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

  3. 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

  4. 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

  5. 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.

  6. Gene expression in TGFbeta-induced epithelial cell differentiation in a three-dimensional intestinal epithelial cell differentiation model

    PubMed Central

    Juuti-Uusitalo, Kati M; Kaukinen, Katri; Mäki, Markku; Tuimala, Jarno; Kainulainen, Heikki

    2006-01-01

    Background The TGFβ1-induced signal transduction processes involved in growth and differentiation are only partly known. The three-dimensional epithelial differentiation model, in which T84 epithelial cells are induced to differentiate either with TGFβ1 or IMR-90 mesenchymal cell-secreted soluble factors, is previously shown to model epithelial cell differentiation seen in intestine. That model has not been used for large scale gene expression studies, such as microarray method. Therefore the gene expression changes were studied in undifferentiated and differentiated three-dimensional T84 cultures with cDNA microarray method in order to study the molecular changes and find new players in epithelial cell differentiation. Results The expression of 372 genes out of 5188 arrayed sequences was significantly altered, and 47 of them were altered by both mediators. The data were validated and the altered genes are presented in ontology classes. For the genes tested the expressions in protein level were in accordance with the mRNA results. We also found 194 genes with no known function to be potentially important in epithelial cell differentiation. The mRNA expression changes induced by TGFβ1 were bigger than changes induced by soluble factors secreted by IMR-90 mesenchymal cells. The gene expression data was depicted in already known signaling pathway routes. Conclusion Our results reveal potential new signaling pathways and several new genes affected by TGFβ in epithelial cell differentiation. The differentiation induced by TGFβ1 appears to be more potent than the differentiation induced by mesenchymal cells. This study indicates that our cell culture model is a suitable tool in studying regulatory mechanisms during epithelial cell differentiation in intestine. Furthermore the present results indicate that our model is a good tool for finding new players acting in the differentiation of epithelial cells. PMID:17074098

  7. Stat3 and G-CSF-induced myeloid differentiation.

    PubMed

    Chakraborty, A; Tweardy, D J

    1998-08-01

    Granulocyte colony-stimulating factor (G-CSF) is the cytokine critical for directing neutrophilic granulocyte differentiation. Early G-CSF signaling events in myeloid cells involves activation of STATs, proteins that serve the dual function of signal transduction and activation of transcription, especially the activation of Stat3. A dominant-negative mutant construct of Stat3 inhibited G-CSF-mediated neutrophilic differentiation indicating that Stat3 is a essential component for driving the G-CSF-mediated differentiation program in myeloid cells. Three isoforms of Stat3 have been identified, alpha(p92), beta(p83) and gamma(p72) each derived from a single gene. Stat3alpha is the predominant isoform expressed in most cells. Stat3beta is derived from Stat3alpha by alternative RNA splicing. Stat3gamma is derived from Stat3alpha by limited proteolysis. Mapping of Stat3alpha and Stat3beta activation in M1 murine myeloid leukemia cells revealed that their optimal activation required G-CSFR constructs containing both Y704 and Y744. These amino acid residues has previously been demonstrated to be essential for G-CSF-induced differentiation in this cells. Phosphopeptide affinity and phosphopeptide inhibition studies indicate that Stat3alpha and Stat3beta are recruited to the G-CSF receptor complex through their interaction with the receptor at phosphotyrosines Y704 and Y744. Y744 is followed at the +3 position by Cys (C). This sequence YXXC, represents a novel motif implicated in the recruitment and activation of Stat3alpha, Stat3beta and Stat3gamma by the hG-CSFR. Structurally, Stat3alpha, Stat3beta and Stat3gamma differ from each other in their C-terminal transactivation domain. In the beta isoform, the Stat3alpha transactivation domain is replaced by 7 amino acid residues which enable Stat3beta to interact with c-Jun. In the gamma isoform, the Stat3alpha transactivation domain is removed by limited proteolysis creating a dominant negative isoform. In immature human

  8. Rejuvenation of Appalachian topography due to subsidence induced differential erosion

    NASA Astrophysics Data System (ADS)

    Liu, L.

    2014-12-01

    In ancient orogens, such as the Appalachian Mountains in the eastern United States, the difference between the high and low points—topographic relief—can continue to increase long after the tectonic forces that created the range have become inactive. Climatic forcing and mantle-induced dynamic uplift are proposed to drive formation of relief, but clear evidence is lacking in the Appalachian Mountains. Here I use a numerical simulation of dynamic topography in North America, combined with reconstructions of the sedimentation history from the Gulf of Mexico, to show that rejuvenation of topographic relief in the Appalachian Mountains since the Palaeogene period could have been caused by mantle-induced dynamic subsidence associated with sinking of the subducted Farallon slab. Specifically, I show that patterns of continental erosion and the eastward migration of sediment deposition centres in the Gulf of Mexico closely follow the locus of predicted dynamic subsidence. Furthermore, pulses of rapid sediment deposition in the Gulf of Mexico and western Atlantic correlate with enhanced erosion in the Appalachian Mountains during the Miocene epoch, caused by dynamic tilting of the continent. Calculations show that such subsidence-induced differential erosion caused flexural-isostatic adjustments of Appalachian topography that led to the development of both relief and elevation. I propose that dynamically induced continental tilting may provide a mechanism for topographic rejuvenation in ancient orogens.

  9. Small molecules induce efficient differentiation into insulin-producing cells from human induced pluripotent stem cells.

    PubMed

    Kunisada, Yuya; Tsubooka-Yamazoe, Noriko; Shoji, Masanobu; Hosoya, Masaki

    2012-03-01

    Human induced pluripotent stem (hiPS) cells have potential uses for drug discovery and cell therapy, including generation of pancreatic β-cells for diabetes research and treatment. In this study, we developed a simple protocol for generating insulin-producing cells from hiPS cells. Treatment with activin A and a GSK3β inhibitor enhanced efficient endodermal differentiation, and then combined treatment with retinoic acid, a bone morphogenic protein inhibitor, and a transforming growth factor-β (TGF-β) inhibitor induced efficient differentiation of pancreatic progenitor cells from definitive endoderm. Expression of the pancreatic progenitor markers PDX1 and NGN3 was significantly increased at this step and most cells were positive for anti-PDX1 antibody. Moreover, several compounds, including forskolin, dexamethasone, and a TGF-β inhibitor, were found to induce the differentiation of insulin-producing cells from pancreatic progenitor cells. By combined treatment with these compounds, more than 10% of the cells became insulin positive. The differentiated cells secreted human c-peptide in response to various insulin secretagogues. In addition, all five hiPS cell lines that we examined showed efficient differentiation into insulin-producing cells with this protocol.

  10. 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

  11. Mechanisms that uncouple growth and differentiation in myeloid leukemia cells: restoration of requirement for normal growth-inducing protein without restoring induction of differentiation-inducing protein.

    PubMed Central

    Lotem, J; Sachs, L

    1982-01-01

    There are different macrophage- and granulocyte-inducing (MGI) proteins. Normal myeloid precursors are induced to multiply by one form (MGI-1) and to differentiate by another form (MGI-2). There are clones of myeloid leukemia cells that no longer require MGI-1 for growth but can still be induced to differentiate by MGI-2. After induction of differentiation in these leukemia cells by adding MCI-2 or inducing endogenous production of MGI-2 by lipopolysaccharide, the differentiating leukemia cells, like normal cells, again required MGI-1 for growth. This growth requirement for MGI-1 could not be substituted for by adding other protein growth factors such as epidermal, fibroblast, or nerve growth factor or insulin. Induction of differentiation in these leukemia cells by dexamethasone, arabinonucleoside (cytosine arabinoside), or methotrexate instead of by MGI-2, did not restore the requirement of MGI-1 for growth. Mutant myeloid leukemia cells that could not be induced to differentiate by MGI-2 also did not show this restoration of the requirement of MGI-1 for growth. MGI-1 in normal cells induced cell growth and also induced MGI-2, so that the cells could then differentiate by the endogenously produced MGI-2. However, MGI-1 did not induce production of MGI-2 in the leukemia cells, even though they again required MGI-1 for growth, so that there was no induction of differentiation after adding MGI-1. This lack of induction of differentiation-inducing protein by growth-inducing protein has thus identified an effective mechanism for uncoupling of growth and differentiation in malignant cells. PMID:6981812

  12. Enhancement by retinoid of hemin-induced differentiation of human leukemia K562 cell line.

    PubMed

    Nakajima, O; Hashimoto, Y; Iwasaki, S

    1993-09-01

    The effect of retinoid on human leukemia K562 cell differentiation induced by hemin was examined. Retinoids (retinoic acid and synthetic retinoids [Am80 and Ch55]) dose-dependently enhanced hemin-induced erythroid differentiation of K562 cells, though these retinoids themselves did not induce the differentiation. Under optimal conditions, these retinoids caused a doubling of the population of hemin-induced differentiated cells. In addition, co-treatment of cells with hemin and retinoid led to longer maintenance of the differentiated state after the removal of hemin, which might imply acquisition of irreversibility of hemin-induced differentiation. These results suggest that the combination of retinoids with other differentiation inducers might be useful for leukemia therapy in cases where the leukemic cells are poorly responsive or unresponsive to retinoids, alone.

  13. Suppression of RANKL-induced osteoclast differentiation by cilostazol via SIRT1-induced RANK inhibition.

    PubMed

    Park, So Youn; Lee, Sung Won; Kim, Hye Young; Lee, Sang Yeob; Lee, Won Suk; Hong, Ki Whan; Kim, Chi Dae

    2015-10-01

    Osteoclasts are bone-specific multinucleated cells generated by differentiation of monocyte/macrophage hematopoietic lineages and degrade bone matrix by secretion of lytic enzymes. The regulation of osteoclast differentiation provides a potential strategy for treatment of bone-lytic damage. In this study, cilostazol, an inhibitor of type III phosphodiesterase, inhibited RANKL [receptor activator of nuclear factor kappa B (RANK) ligand]-induced RANK expression in bone marrow-derived monocyte/macrophage precursors (BMMs) and Raw 264.7 cells by inhibiting PU.1 via SIRT1 activation. RANKL-induced RANK expression was attenuated by cilostazol and rSIRT1 in Raw 264.7 cells, and these were blocked by sirtinol. In line with these, cilostazol elevated SIRT1 mRNA and protein levels in 12-24h and increased SIRT1 activity, and these effects were inhibited by sirtinol. Furthermore, the RANKL-induced nuclear expression of PU.1, a transcription factor required for macrophage differentiation, was suppressed by cilostazol. Additionally, marked RANKL-induced RANK immunofluorescence staining in Raw 264.7 cells was attenuated by cilostazol and rSIRT1, and both attenuations were prevented by sirtinol. Extensive RANK staining of knee synovial tissues in a mouse model of collagen-induced arthritis (CIA) was markedly reduced by cilostazol (30mg/kg/day). In line with these results, both RANKL- and M-CSF-induced differentiation of BMMs to multinucleated TRAP(+) giant cells and resorption pit formation were inhibited by cilostazol associated with a decrease in TRAP (a marker enzyme of osteoclasts) activity. In conclusion, cilostazol activates SIRT1, which suppresses the nuclear translocation of PU.1, and thus, inhibits RANKL-stimulated RANK expression and causes anti-osteoclast formation in BMMs in vitro and in their murine model of CIA.

  14. Bitter Taste Stimuli Induce Differential Neural Codes in Mouse Brain

    PubMed Central

    Wilson, David M.; Boughter, John D.; Lemon, Christian H.

    2012-01-01

    A growing literature suggests taste stimuli commonly classified as “bitter” induce heterogeneous neural and perceptual responses. Here, the central processing of bitter stimuli was studied in mice with genetically controlled bitter taste profiles. Using these mice removed genetic heterogeneity as a factor influencing gustatory neural codes for bitter stimuli. Electrophysiological activity (spikes) was recorded from single neurons in the nucleus tractus solitarius during oral delivery of taste solutions (26 total), including concentration series of the bitter tastants quinine, denatonium benzoate, cycloheximide, and sucrose octaacetate (SOA), presented to the whole mouth for 5 s. Seventy-nine neurons were sampled; in many cases multiple cells (2 to 5) were recorded from a mouse. Results showed bitter stimuli induced variable gustatory activity. For example, although some neurons responded robustly to quinine and cycloheximide, others displayed concentration-dependent activity (p<0.05) to quinine but not cycloheximide. Differential activity to bitter stimuli was observed across multiple neurons recorded from one animal in several mice. Across all cells, quinine and denatonium induced correlated spatial responses that differed (p<0.05) from those to cycloheximide and SOA. Modeling spatiotemporal neural ensemble activity revealed responses to quinine/denatonium and cycloheximide/SOA diverged during only an early, at least 1 s wide period of the taste response. Our findings highlight how temporal features of sensory processing contribute differences among bitter taste codes and build on data suggesting heterogeneity among “bitter” stimuli, data that challenge a strict monoguesia model for the bitter quality. PMID:22844505

  15. Laser-induced differential fluorescence for cancer diagnosis without biopsy

    SciTech Connect

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

    1997-01-01

    An optical diagnostic procedure based on laser-induced fluorescence was developed for direct {ital in vivo} cancer diagnosis without requiring biopsy. The methodology was applied in a clinical study involving over 100 patients in order to differentiate normal tissue from malignant tumors of the esophagus. Endogenous fluorescence of normal and malignant tissues was measured directly with the use of a fiber-optic probe inserted through an endoscope. The measurements were performed {ital in vivo} during routine endoscopy. Detection of the fluorescence signal from the tissue was performed with the use of laser excitation. This report describes the differential normalized fluorescence (DNF) procedure using the amplified spectral differences between the normalized fluorescence of malignant tissue and normal mucosa. The results of this DNF approach were compared with histopathology results of the biopsy samples and indicated excellent agreement in the classification of normal tissue and malignant tumors for the samples investigated. Data related to various grades of Barrett{close_quote}s esophagus are discussed. The DNF procedure could lead to the development of a rapid and cost-effective technique for cancer diagnosis. {copyright} {ital 1997} {ital Society for Applied Spectroscopy}

  16. Differential suppression of the aryl hydrocarbon receptor nuclear translocator-dependent function by an aryl hydrocarbon receptor PAS-A-derived inhibitory molecule

    PubMed Central

    Xie, Jinghang; Huang, Xin; Park, Miki S.; Pham, Hang M.; Chan, William K.

    2014-01-01

    The aryl hydrocarbon receptor (AhR) heterodimerizes with the aryl hydrocarbon receptor nuclear translocator (Arnt) for transcriptional regulation. We generated three N-terminal deletion constructs of the human AhR of 12–24 KDa in size – namely D1, D2, and D3 – to suppress the Arnt function. We observed that all three deletions interact with the human Arnt with similar affinities. D2, which contains part of the AhR PAS-A domain and interacts with the PAS-A domain of Arnt, inhibits the formation of the AhR gel shift complex. D2 suppresses the 3-methylcholanthrene-induced, dioxin response element (DRE)-driven luciferase activity in Hep3B cells and exogenous Arnt reverses this D2 suppression. D2 suppresses the induction of CYP1A1 at both the message and protein levels in Hep3B cells; however, the CYP1B1 induction is not affected. D2 suppresses the recruitment of Arnt to the cyp1a1 promoter but not to the cyp1b1 promoter, partly because the AhR/Arnt heterodimer binds better to the cyp1b1 DRE than to the cyp1a1 DRE. Interestingly, D2 has no effect on the cobalt chloride-induced, hypoxia inducible factor-1 (HIF-1)-dependent expression of vegf, aldolase c, and ldh-a messages. Our data reveal that the flanking sequences of the DRE contribute to the binding affinity of the AhR/Arnt heterodimer to its endogenous enhancers and the function of AhR and HIF-1 can be differentially suppressed by the D2 inhibitory molecule. PMID:24486526

  17. 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

  18. 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.

  19. 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

  20. 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

  1. 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

  2. Repression of HIP/RPL29 expression induces differentiation in colon cancer cells.

    PubMed

    Liu, Jian-Jun; Huang, Bao Hua; Zhang, Jinqiu; Carson, Daniel D; Hooi, Shing Chuan

    2006-05-01

    We had previously shown that the expression of heparin/heparan sulfate interacting protein/ribosomal protein L29 (HIP/RPL29) was upregulated in colon cancer tissues. The present study investigated the role of HIP/RPL29 in differentiation in colon cancer cells. Inducing cellular differentiation in HT-29 cells by both sodium butyrate and glucose deprivation resulted in a significant downregulation of HIP/RPL29 expression. The beta-catenin/Tcf-4 pathway is the most important pathway controlling the switch between cellular differentiation and proliferation in intestinal epithelial cells. Inducing differentiation by dominant-negative inhibition of the beta-catenin/Tcf-4 complexes in LS174T cells also resulted in downregulation of HIP/RPL29. To determine whether a lower expression of HIP/RPL29 could induce differentiation in cancer cells, small interfering RNA (siRNA) targeting HIP/RPL29 was transfected into LS174T cells. The resultant knockdown of HIP/RPL29 expression induced cellular differentiation, as shown by the increased expression of two known markers of differentiation in LS174T cells, galectin-4 and mucin-2. In addition, the differentiation process induced by repression of HIP/RPL29 expression was accompanied by the upregulation of p21 and p53. In conclusion, HIP/RPL29 plays a role in the cellular differentiation process in colon cancer cells. The differentiation process is at least partially mediated by the upregulation of p21 and p53 pathways. PMID:16475173

  3. 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.

  4. 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

  5. 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

  6. 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

  7. 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.

  8. Silybin from Silybum Marianum Seeds Inhibits Confluent-Induced Keratinocytes Differentiation as Effectively as Retinoic Acid without Inducing Inflammatory Cytokine.

    PubMed

    Kitajima, Seiji; Yamaguchi, Kohji

    2009-09-01

    Retinoic acid (RA) has been effective for improving wrinkles. However, it has also been reported that RA induces skin irritation. In this study, we explored new botanical compounds that show RA-like activity, but do not induce inflammation in vitro. Keratinocytes were maintained in a confluent condition and induced differentiation. Under this condition keratinocytes were treated with many botanical extracts and their morphological change were observed and compared with RA-treated. We found that silybin, which is a major flavonolignan from Silybum Marianum seeds, induced RA-like morphological change and prevented differentiation. We showed that silybin, like RA, reduced the expression of keratinocyte terminal differentiation markers and stimulated the expression of basement membrane component proteins. In contrast, silybin, unlike RA, did not stimulate the secretion of IL-1alpha, which is a skin irritation mediator. These results suggest that silybin has RA-like activity on keratinocytes and has the potential to improve winkle without inducing skin irritation.

  9. Studies on the differentiation inducers of myeloid leukemic cells from Citrus species.

    PubMed

    Sugiyama, S; Umehara, K; Kuroyanagi, M; Ueno, A; Taki, T

    1993-04-01

    An attempt was made to isolate differentiation inducers from Aurantii Nobilis Pericarpium and the fruit peel of Citrus reticulata Blanco (Rutaceae). Twenty-seven kinds of flavones, including five new flavones, were isolated after repeated chromatography from methanol extracts of these plants and their structures were established, from their physicochemical data, to be highly methoxylated flavones. Each compound, except for two flavone glucosides, showed the differentiation inducing activity toward mouse myeloid leukemia cells (M1), and the cells came to have phagocytic activity. Furthermore, differentiation inducing activity was tested using human acute promyelocytic leukemia cell line (HL-60). PMID:8508474

  10. 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

  11. Inhibition of chemically induced carcinogenesis by drugs used in homeopathic medicine.

    PubMed

    Kumar, K B Hari; Sunila, E S; Kuttan, Girija; Preethi, K C; Venugopal, C Nimita; Kuttan, Ramadasan

    2007-01-01

    Homeopathy is considered as one modality for cancer therapy. However, there are only very few clinical reports on the activity of the drugs, as well as in experimental animals. Presently we have evaluated the inhibitory effects of potentized homeopathic preparations against N'-nitrosodiethylamine (NDEA) induced hepatocellular carcinoma in rats as well as 3-methylcholanthrene-induced sarcomas in mice. We have used Ruta, Hydrastis, Lycopodium and Thuja, which are commonly employed in homeopathy for treating cancer. Administration of NDEA in rats resulted in tumor induction in the liver and elevated marker enzymes such as gamma-glutamyl transpeptidase, glutamate pyruvate transaminase, glutamate oxaloacetate transaminase and alkaline phosphatase in the serum and in liver. Concomitant administration of homeopathic drugs retarded the tumor growth and significantly reduced the elevated marker enzymes level as revealed by morphological, biochemical and histopathological evaluation. Out of the four drugs studied, Ruta 200c showed maximum inhibition of liver tumor development. Ruta 200c and phosphorus 1M were found to reduce the incidence of 3-methylcholanthrene-induced sarcomas and also increase the life span of mice harboring the tumours. These studies demonstrate that homeopathic drugs, at ultra low doses, may be able to decrease tumor induction by carcinogen administration. At present we do not know the mechanisms of action of these drugs useful against carcinogenesis. PMID:17477781

  12. 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

  13. 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.

  14. Directed differentiation of induced pluripotent stem cells towards T lymphocytes.

    PubMed

    Lei, Fengyang; Haque, Rizwanul; Xiong, Xiaofang; Song, Jianxun

    2012-05-14

    Adoptive cell transfer (ACT) of antigen-specific CD8(+) cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies (1). CTLs can recognize malignant cells by interacting tumor antigens with the T cell receptors (TCR), and release cytotoxins as well as cytokines to kill malignant cells. It is known that less-differentiated and central-memory-like (termed highly reactive) CTLs are the optimal population for ACT-based immunotherapy, because these CTLs have a high proliferative potential, are less prone to apoptosis than more differentiated cells and have a higher ability to respond to homeostatic cytokines (2-7). However, due to difficulties in obtaining a high number of such CTLs from patients, there is an urgent need to find a new approach to generate highly reactive Ag-specific CTLs for successful ACT-based therapies. TCR transduction of the self-renewable stem cells for immune reconstitution has a therapeutic potential for the treatment of diseases (8-10). However, the approach to obtain embryonic stem cells (ESCs) from patients is not feasible. Although the use of hematopoietic stem cells (HSCs) for therapeutic purposes has been widely applied in clinic (11-13), HSCs have reduced differentiation and proliferative capacities, and HSCs are difficult to expand in in vitro cell culture (14-16). Recent iPS cell technology and the development of an in vitro system for gene delivery are capable of generating iPS cells from patients without any surgical approach. In addition, like ESCs, iPS cells possess indefinite proliferative capacity in vitro, and have been shown to differentiate into hematopoietic cells. Thus, iPS cells have greater potential to be used in ACT-based immunotherapy compared to ESCs or HSCs. Here, we present methods for the generation of T lymphocytes from iPS cells in vitro, and in vivo programming of antigen-specific CTLs from iPS cells for promoting cancer immune surveillance. Stimulation in vitro with a Notch ligand

  15. Effect of inducers and inhibitors of glucuronidation on the biliary excretion and choleretic action of valproic acid in the rat.

    PubMed

    Watkins, J B; Klaassen, C D

    1982-02-01

    Valproic acid (VPA) induces an immediate choleresis in the rat which may be attributable to the osmotic properties of VPA-glucuronic acid conjugates in bile. The influence of inducers and inhibitors of glucuronidation of VPA on the biliary excretion and choleretic effect of VPA was studied. Hepatic UDP-glucuronyltransferase activity toward VPA was determined in vitro. Pretreatment with phenobarbital (75 mg/kg/day for 4 days) enhanced VPA glucuronidation; borneol (750 mg/kg) decreased VPA conjugation; 3-methylcholanthrene (20 mg/kg/day for 4 days) and galactosamine (600 mg/kg) had no effect on glucuronidation of VPA in vitro. Hepatic UDP-glucuronic acid content was decreased by borneol and galactosamine administration and was enhanced by phenobarbital and 3-methylcholanthrene pretreatment. The enzyme inducers increased the plasma disappearance of VPA in vivo but did not augment its biliary excretion or choleretic effect. Borneol and galactosamine, which inhibited the conjugation and plasma disappearance of VPA, decreased its biliary excretion and inhibited the VPA-induced increase in bile flow. Thus, the bile flow rate after VPA administration is closely related to the excretion of VPA-glucuronic acid. These data support the conclusion that the choleretic effect of VPA is due to the osmotic activity of VPA conjugates in bile.

  16. 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

  17. Interleukin-32 induces the differentiation of monocytes into macrophage-like cells

    PubMed Central

    Netea, Mihai G.; Lewis, Eli C.; Azam, Tania; Joosten, Leo A. B.; Jaekal, Jun; Bae, Su-Young; Dinarello, Charles A.; Kim, Soo-Hyun

    2008-01-01

    After emigration from the bone marrow to the peripheral blood, monocytes enter tissues and differentiate into macrophages, the prototype scavenger of the immune system. By ingesting and killing microorganisms and removing cellular debris, macrophages also process antigens as a first step in mounting a specific immune response. IL-32 is a cytokine inducing proinflammatory cytokines and chemokines via p38-MAPK and NF-κB. In the present study, we demonstrate that IL-32 induces differentiation of human blood monocytes as well as THP-1 leukemic cells into macrophage-like cells with functional phagocytic activity for live bacteria. Muramyl dipepide (MDP), the ligand for the intracellular nuclear oligomerization domain (NOD) 2 receptor, has no effect on differentiation alone but augments the monocyte-to-macrophage differentiation by IL-32. Unexpectedly, IL-32 reversed GM-CSF/IL-4-induced dendritic cell differentiation to macrophage-like cells. Whereas the induction of TNFα, IL-1β, and IL-6 by IL-32 is mediated by p38-MAPK, IL-32-induced monocyte-to-macrophage differentiation is mediated through nonapoptotic, caspase-3-dependent mechanisms. Thus, IL-32 not only contributes to host responses through the induction of proinflammatory cytokines but also directly affects specific immunity by differentiating monocytes into macrophage-like cells. PMID:18296636

  18. 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.

  19. Xestospongin C induces monocytic differentiation of HL60 cells through activation of the ERK pathway.

    PubMed

    Moon, Dong-Oh; Asami, Yukihiro; Kim, Mun-Ock; Jang, Jae-Hyuk; Kim, Bo Yeon; Ahn, Jong Seog; Kim, Gi-Young; Yun, Sung Gyu

    2013-05-01

    Xestospongin C (XC), which is a group of macrocyclic bis-1-oxaquinolizidines, is a potent inhibitor of sarcoendoplasmic reticulum calcium transport ATPase and IP3 receptor. Nevertheless, very less information is available regarding whether XC induces AML differentiation. We investigated the potential role of XC in the differentiation of human leukemia HL60 cells and mechanisms underlying XC actin. XC treatment inhibited proliferation by inducing G1-phase cell cycle arrest in the HL60 cells. In addition, XC induced differentiation of HL60 cells into the CD14(+) monocytic lineage, which was indicated by morphological changes, nitroblue tetrazolium reduction assay, and expressions of CD11b and CD14 surface antigens. Our results also showed that XC promotes phagocytic activity and granularity in HL60 cells, suggesting that the cells are functionally activated. Furthermore, XC enhanced tumor necrosis factor (TNF)-α-mediated cytotoxic effect by increasing the numbers of TNF receptors. Moreover, we showed that XC activates extracellular signal-regulated kinase (ERK) pathway in the differentiation stages. Inhibition of ERK activation using PD98059 significantly decreased NBT+HL60 cells induced by XC treatment. Taken together, the results show that XC promotes monocytic differentiation of HL60 cells via ERK pathway activation, suggesting that XC could be a candidate for use as a differentiation-inducing agent for AML treatment.

  20. An excitable gene regulatory circuit induces transient cellular differentiation

    NASA Astrophysics Data System (ADS)

    Süel, Gürol M.; Garcia-Ojalvo, Jordi; Liberman, Louisa M.; Elowitz, Michael B.

    2006-03-01

    Certain types of cellular differentiation are probabilistic and transient. In such systems individual cells can switch to an alternative state and, after some time, switch back again. In Bacillus subtilis, competence is an example of such a transiently differentiated state associated with the capability for DNA uptake from the environment. Individual genes and proteins underlying differentiation into the competent state have been identified, but it has been unclear how these genes interact dynamically in individual cells to control both spontaneous entry into competence and return to vegetative growth. Here we show that this behaviour can be understood in terms of excitability in the underlying genetic circuit. Using quantitative fluorescence time-lapse microscopy, we directly observed the activities of multiple circuit components simultaneously in individual cells, and analysed the resulting data in terms of a mathematical model. We find that an excitable core module containing positive and negative feedback loops can explain both entry into, and exit from, the competent state. We further tested this model by analysing initiation in sister cells, and by re-engineering the gene circuit to specifically block exit. Excitable dynamics driven by noise naturally generate stochastic and transient responses, thereby providing an ideal mechanism for competence regulation.

  1. Cardiomyocyte differentiation induced in cardiac progenitor cells by cardiac fibroblast-conditioned medium.

    PubMed

    Zhang, Xi; Shen, Man-Ru; Xu, Zhen-Dong; Hu, Zhe; Chen, Chao; Chi, Ya-Li; Kong, Zhen-Dong; Li, Zi-Fu; Li, Xiao-Tong; Guo, Shi-Lei; Xiong, Shao-Hu; Zhang, Chuan-Sen

    2014-05-01

    Our previous study showed that after being treated with 5-azacytidine, Nkx2.5(+) human cardiac progenitor cells (CPCs) derived from embryonic heart tubes could differentiate into cardiomyocytes. Although 5-azacytidine is a classical agent that induces myogenic differentiation in various types of cells, the drug is toxic and unspecific for myogenic differentiation. To investigate the possibility of inducing CPCs to differentiate into cardiomyocytes by a specific and non-toxic method, CPCs of passage 15 and mesenchymal stem cells (MSCs) were treated with cardiac ventricular fibroblast-conditioned medium (CVF-conditioned medium). Following this treatment, the Nkx2.5(+) CPCs underwent cardiomyogenic differentiation. Phase-contrast microscopy showed that the morphology of the treated CPCs gradually changed. Ultrastructural observation confirmed that the cells contained typical sarcomeres. The expression of cardiomyocyte-associated genes, such as alpha-cardiac actin, cardiac troponin T, and beta-myosin heavy chain (MHC), was increased in the CPCs that had undergone cardiomyogenic differentiation compared with untreated cells. In contrast, the MSCs did not exhibit changes in morphology or molecular expression after being treated with CVF-conditioned medium. The results indicated that Nkx2.5(+) CPCs treated with CVF-conditioned medium were capable of differentiating into a cardiac phenotype, whereas treated MSCs did not appear to undergo cardiomyogenic differentiation. Subsequently, following the addition of Dkk1 and the blocking of Wnt signaling pathway, CVF-conditioned medium-induced morphological changes and expression of cardiomyocyte-associated genes of Nkx2.5(+) CPCs were inhibited, which indicates that CVF-conditioned medium-induced cardiomyogenic differentiation of Nkx2.5(+) CPCs is associated with Wnt signaling pathway. In addition, we also found that the activation of Wnt signaling pathway was accompanied by higher expression of GATA-4 and the blocking of the

  2. IL-27 Induces Th17 Differentiation in the Absence of STAT1 Signaling.

    PubMed

    Peters, Anneli; Fowler, Kevin D; Chalmin, Fanny; Merkler, Doron; Kuchroo, Vijay K; Pot, Caroline

    2015-11-01

    It is known that differentiation of Th17 cells is promoted by activation of STAT3 and inhibited by activation of STAT1. Although both transcription factors are activated by several cytokines, including IL-6, IL-21, and IL-27, each of these cytokines has a very different effect on Th17 differentiation, ranging from strong induction (IL-6) to strong inhibition (IL-27). To determine the molecular basis for these differences, we measured STAT3 and STAT1 activation profiles for IL-6, IL-21, and IL-27, as well as for cytokine pairs over time. We found that the ratio of activated STAT3/activated STAT1 is crucial in determining whether cytokines promote or inhibit Th17 differentiation. IL-6 and IL-21 induced p-STAT3/p-STAT1 ratios > 1, leading to the promotion of Th17 differentiation, whereas IL-27 or IL-6+IL-27 induced p-STAT3/p-STAT1 ratios < 1, resulting in inhibition of Th17 differentiation. Consistent with these findings, we show that IL-27 induces sufficient p-STAT3 to promote Th17 differentiation in the absence of STAT1. Furthermore, IL-27-induced STAT1-deficient T cells were indistinguishable from bona fide highly proinflammatory Th17 cells because they induced severe experimental autoimmune encephalomyelitis upon adoptive transfer. Our results suggest that the ratio of p-STAT3/p-STAT1 induced by a cytokine or cytokine pairs can be used to predict whether they induce a competent Th17-differentiation program.

  3. 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

  4. Sensitivity of staurosporine-induced differentiated RGC-5 cells to homocysteine

    PubMed Central

    Ganapathy, Preethi S.; Dun, Ying; Ha, Yonju; Duplantier, Jennifer; Allen, John Bradley; Farooq, Amina; Bozard, B. Renee; Smith, Sylvia B.

    2011-01-01

    PURPOSE Homocysteine is implicated in ganglion cell death associated with glaucoma. To understand mechanisms of homocysteine-induced cell death, we analyzed the sensitivity of the RGC-5 cell line, differentiated using staurosporine, to physiologically-relevant levels of the excitotoxic amino acid homocysteine. METHODS RGC-5 cells were differentiated 24 h using 316 nM staurosporine and tested for expression of Thy 1.2 via immunodetection, RT-PCR and immunoblotting. The sensitivity of staurosporine-differentiated RGC-5 cells to physiological levels of homocysteine (50, 100, 250 µM) and to high levels of homocysteine (1 mM), glutamate (1 mM) and oxidative stress (25 µM:10 mU/ml xanthine:xanthine oxidase) was assessed by TUNEL assay and by immunodetection of cleaved caspase-3. The sensitivity of undifferentiated RGC-5 cells to high (1, 5, and 10 mM) homocysteine was also examined. RESULTS Undifferentiated RGC-5 cells express Thy 1.2 mRNA and protein. Staurosporine-differentiated RGC-5 cells extend neurite processes and express Thy 1.2 after 24 h differentiation; they express NF-L after 1 and 3 days differentiation. Treatment of staurosporine-differentiated RGC-5 cells with 50, 100 or 250µM homocysteine did not alter neurite processes nor induce cell death (detected by TUNEL and active caspase-3) to a level greater than that observed in non-homocysteine-treated, staurosporine-differentiated cells. The 1 mM dosage of homocysteine in staurosporine-differentiated RGC-5 cells also did not induce cell death above control levels, although 18 h treatment of non-differentiated RGC-5 cells with 5 mM homocysteine decreased survival by 50%. CONCLUSIONS RGC-5 cells differentiated for 24 h with 316 nM staurosporine project robust neurite processes and are positive for ganglion cell markers consistent with a more neuronal phenotype than non- staurosporine-differentiated RGC-5 cells. However, concentrations of homocysteine known to induce ganglion cell death in vivo and in primary

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

    PubMed

    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.

  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. 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.

  9. Differential genomic damage in different tumor lines induced by prodigiosin.

    PubMed

    Lins, Jeanne Cristina Lapenda; DE Melo, Maria Eliane Bezerra; DO Nascimento, Silene Carneiro; Adam, Monica Lucia

    2015-06-01

    Prodigiosin is a secondary metabolite produced by Serratia marcercens. As this pigment is suggested to be a cancer drug, genotoxicity studies are necessary. The aim of the present investigation was to evaluate the genotoxic effects of prodigiosin on tumoral and normal cell lines, NCIH-292, MCF-7 and HL-60. A normal line BGMK was used as control. Genomic damage induced by prodigiosin was observed in all tumor lines as well as the control line. The pigment induced the formation of micronuclei in tumor cells. The present data confirm the antitumor potential of prodigiosin. However, these findings also raise concerns regarding its target-specific action, as genotoxic effects on normal cells also occurred.

  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 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.

  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. Polarization of an electroactive functional film on titanium for inducing osteogenic differentiation

    PubMed Central

    Zhou, Zhengnan; Li, Weiping; He, Tianrui; Qian, Lei; Tan, Guoxin; Ning, Chengyun

    2016-01-01

    To enhance the surface bioactivity of titanium (Ti) prostheses, an electroactive polyvinylidene fluoride (PVDF) film was prepared on a Ti substrate to provide a mimetic of the electrical microenvironment, which facilitated the performance of cell functions. The results of cell proliferation and differentiation assays indicated that polarization of the PVDF-Ti (PTi) altered its surface charge, thus inducing adhesion, proliferation and osteogenic differentiation of cells. The polarized PVDF-Ti (PPTi) may therefore find applications in bone regeneration. PMID:27762318

  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. 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

  17. MicroRNA and DNA methylation alterations mediating retinoic acid induced neuroblastoma cell differentiation.

    PubMed

    Stallings, Raymond L; Foley, Niamh H; Bray, Isabella M; Das, Sudipto; Buckley, Patrick G

    2011-10-01

    Many neuroblastoma cell lines can be induced to differentiate into a mature neuronal cell type with retinoic acid and other compounds, providing an important model system for elucidating signalling pathways involved in this highly complex process. Recently, it has become apparent that miRNAs, which act as regulators of gene expression at a post-transcriptional level, are differentially expressed in differentiating cells and play important roles governing many aspects of this process. This includes the down-regulation of DNA methyltransferases that cause the de-methylation and transcriptional activation of numerous protein coding gene sequences. The purpose of this article is to review involvement of miRNAs and DNA methylation alterations in the process of neuroblastoma cell differentiation. A thorough understanding of miRNA and genetic pathways regulating neuroblastoma cell differentiation potentially could lead to targeted therapies for this disease.

  18. Hypoxia-inducible factor-1α induces ErbB4 signaling in the differentiating mammary gland.

    PubMed

    Paatero, Ilkka; Seagroves, Tiffany N; Vaparanta, Katri; Han, Wen; Jones, Frank E; Johnson, Randall S; Elenius, Klaus

    2014-08-01

    Conditional knock-out of Hif1a in the mouse mammary gland impairs lobuloalveolar differentiation during lactation. Here, we demonstrate that expression of ErbB4 was reduced in the lobulalveoli of mice with mammary gland-specific deletion of Hif1a. Erbb4 was not, however, a direct target gene for transcriptional regulation by HIF-1α in vitro. HIF-1α overexpression or HIF accumulating prolyl hydroxylase inhibitors reduced ErbB4 endocytosis, promoted transcriptional co-regulatory activity of ErbB4, and stimulated ErbB4-induced differentiation of mammary carcinoma cells. Consistently, RNA interference-mediated down-regulation of HIF-1α resulted in reduced ErbB4 protein amount and reduced mammary carcinoma cell differentiation. These findings indicate that HIF-1α is a physiologically relevant regulator of ErbB4 and that ErbB4 is involved in HIF-regulated differentiation of the mammary gland.

  19. 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

  20. 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.

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

    PubMed

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

    2015-10-14

    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. PMID:26372376

  2. Inhibition of the differentiation of human myeloid cell lines by redox changes induced through glutathione depletion.

    PubMed Central

    Esposito, F; Agosti, V; Morrone, G; Morra, F; Cuomo, C; Russo, T; Venuta, S; Cimino, F

    1994-01-01

    We have investigated the effect of redox changes in vivo on the differentiation of two human myeloid cell lines, HL-60 and KG-1. The glutathione-depleting agent diethyl maleate (DEM) prevented the development of differentiated features in response to phorbol esters, including adherence of the cells to plastic surfaces and repression of the myeloperoxidase and CD34 genes. Moreover, DEM abolished phorbol 12-myristate 13-acetate-induced activation of the transcription factors AP-1 and Egr-1, suggesting that inhibition of differentiation may be due, at least in part, to redox modifications of these proteins. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:7519845

  3. 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

  4. Hypoxia induces differential translation of enolase/MBP-1

    PubMed Central

    2010-01-01

    Background Hypoxic microenvironments in tumors contribute to transformation, which may alter metabolism, growth, and therapeutic responsiveness. The α-enolase gene encodes both a glycolytic enzyme (α-enolase) and a DNA-binding tumor suppressor protein, c-myc binding protein (MBP-1). These divergent α-enolase gene products play central roles in glucose metabolism and growth regulation and their differential regulation may be critical for tumor adaptation to hypoxia. We have previously shown that MBP-1 and its binding to the c-myc P2 promoter regulates the metabolic and cellular growth changes that occur in response to altered exogenous glucose concentrations. Results To examine the regulation of α-enolase and MBP-1 by a hypoxic microenvironment in breast cancer, MCF-7 cells were grown in low, physiologic, or high glucose under 1% oxygen. Our results demonstrate that adaptation to hypoxia involves attenuation of MBP-1 translation and loss of MBP-1-mediated regulation of c-myc transcription, evidenced by decreased MBP-1 binding to the c-myc P2 promoter. This allows for a robust increase in c-myc expression, "early c-myc response", which stimulates aerobic glycolysis resulting in tumor acclimation to oxidative stress. Increased α-enolase mRNA and preferential translation/post-translational modification may also allow for acclimatization to low oxygen, particularly under low glucose concentrations. Conclusions These results demonstrate that malignant cells adapt to hypoxia by modulating α-enolase/MBP-1 levels and suggest a mechanism for tumor cell induction of the hyperglycolytic state. This important "feedback" mechanism may help transformed cells to escape the apoptotic cascade, allowing for survival during limited glucose and oxygen availability. PMID:20412594

  5. Commensal microbe-derived butyrate induces the differentiation of colonic regulatory T cells.

    PubMed

    Furusawa, Yukihiro; Obata, Yuuki; Fukuda, Shinji; Endo, Takaho A; Nakato, Gaku; Takahashi, Daisuke; Nakanishi, Yumiko; Uetake, Chikako; Kato, Keiko; Kato, Tamotsu; Takahashi, Masumi; Fukuda, Noriko N; Murakami, Shinnosuke; Miyauchi, Eiji; Hino, Shingo; Atarashi, Koji; Onawa, Satoshi; Fujimura, Yumiko; Lockett, Trevor; Clarke, Julie M; Topping, David L; Tomita, Masaru; Hori, Shohei; Ohara, Osamu; Morita, Tatsuya; Koseki, Haruhiko; Kikuchi, Jun; Honda, Kenya; Hase, Koji; Ohno, Hiroshi

    2013-12-19

    Gut commensal microbes shape the mucosal immune system by regulating the differentiation and expansion of several types of T cell. Clostridia, a dominant class of commensal microbe, can induce colonic regulatory T (Treg) cells, which have a central role in the suppression of inflammatory and allergic responses. However, the molecular mechanisms by which commensal microbes induce colonic Treg cells have been unclear. Here we show that a large bowel microbial fermentation product, butyrate, induces the differentiation of colonic Treg cells in mice. A comparative NMR-based metabolome analysis suggests that the luminal concentrations of short-chain fatty acids positively correlates with the number of Treg cells in the colon. Among short-chain fatty acids, butyrate induced the differentiation of Treg cells in vitro and in vivo, and ameliorated the development of colitis induced by adoptive transfer of CD4(+) CD45RB(hi) T cells in Rag1(-/-) mice. Treatment of naive T cells under the Treg-cell-polarizing conditions with butyrate enhanced histone H3 acetylation in the promoter and conserved non-coding sequence regions of the Foxp3 locus, suggesting a possible mechanism for how microbial-derived butyrate regulates the differentiation of Treg cells. Our findings provide new insight into the mechanisms by which host-microbe interactions establish immunological homeostasis in the gut.

  6. Monocyte cell surface glycosaminoglycans positively modulate IL-4-induced differentiation toward dendritic cells.

    PubMed

    den Dekker, Els; Grefte, Sander; Huijs, Tonnie; ten Dam, Gerdy B; Versteeg, Elly M M; van den Berk, Lieke C J; Bladergroen, Bellinda A; van Kuppevelt, Toin H; Figdor, Carl G; Torensma, Ruurd

    2008-03-15

    IL-4 induces the differentiation of monocytes toward dendritic cells (DCs). The activity of many cytokines is modulated by glycosaminoglycans (GAGs). In this study, we explored the effect of GAGs on the IL-4-induced differentiation of monocytes toward DCs. IL-4 dose-dependently up-regulated the expression of DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN), CD80, CD206, and CD1a. Monocytes stained positive with Abs against heparan sulfate (HS) and chondroitin sulfate (CS) B (CSB; dermatan sulfate), but not with Abs that recognize CSA, CSC, and CSE. Inhibition of sulfation of monocyte/DC cell surface GAGs by sodium chlorate reduced the reactivity of sulfate-recognizing single-chain Abs. This correlated with hampered IL-4-induced DC differentiation as evidenced by lower expression of DC-SIGN and CD1a and a decreased DC-induced PBL proliferation, suggesting that sulfated monocyte cell surface GAGs support IL-4 activity. Furthermore, removal of cell surface chondroitin sulfates by chondroitinase ABC strongly impaired IL-4-induced STAT6 phosphorylation, whereas removal of HS by heparinase III had only a weak inhibitory effect. IL-4 bound to heparin and CSB, but not to HS, CSA, CSC, CSD, and CSE. Binding of IL-4 required iduronic acid, an N-sulfate group (heparin) and specific O sulfates (CSB and heparin). Together, these data demonstrate that monocyte cell surface chondroitin sulfates play an important role in the IL-4-driven differentiation of monocytes into DCs.

  7. 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

  8. 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

  9. 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.

  10. 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

  11. 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.

  12. Donor-dependent variations in hepatic differentiation from human-induced pluripotent stem cells.

    PubMed

    Kajiwara, Masatoshi; Aoi, Takashi; Okita, Keisuke; Takahashi, Ryosuke; Inoue, Haruhisa; Takayama, Naoya; Endo, Hiroshi; Eto, Koji; Toguchida, Junya; Uemoto, Shinji; Yamanaka, Shinya

    2012-07-31

    Hepatocytes generated from human induced pluripotent stem cells (hiPSCs) are unprecedented resources for pharmaceuticals and cell therapy. However, the in vitro directed differentiation of human pluripotent stem cells into mature hepatocytes remains challenging. Little attention has so far been paid to variations among hiPSC lines in terms of their hepatic differentiation. In the current study, we developed an improved hepatic differentiation protocol and compared 28 hiPSC lines originated from various somatic cells and derived using retroviruses, Sendai viruses, or episomal plasmids. This comparison indicated that the origins, but not the derivation methods, may be a major determinant of variation in hepatic differentiation. The hiPSC clones derived from peripheral blood cells consistently showed good differentiation efficiency, whereas many hiPSC clones from adult dermal fibroblasts showed poor differentiation. However, when we compared hiPSCs from peripheral blood and dermal fibroblasts from the same individuals, we found that variations in hepatic differentiation were largely attributable to donor differences, rather than to the types of the original cells. These data underscore the importance of donor differences when comparing the differentiation propensities of hiPSC clones.

  13. 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

  14. Differential changes in taste perception induced by benzoic acid prickling.

    PubMed

    Otero-Losada, M E

    2003-03-01

    Benzoic acid (Bz) is a prickling compound used to preserve foods. However, its effects on taste are unknown. This work examines Bz-taste interaction using psychophysical methods [magnitude estimation (ME) and paired comparison (PC)] to measure taste intensity in aqueous solutions of pure tastants (T) and their respective mixtures with 10 mM Bz (Mix). Prototypical tastants induced basic taste qualities (mM): sucrose [90-1440, sweetness (Sw)], citric acid [1-64, sourness (So)], NaCl [15-960, saltiness (Sa)], quinine [0.01-0.64, bitterness (Bitt)], KCl (12.5-400, Sa and Bitt). MEs were analysed using Steven's and Beidler's equations. Bz increased Sw (all concentrations) and ionic tastes (low concentrations) and Bz effects were reduced by concentration increase according with quality and tastant Bz reduced Bitt(Quinine) (high concentrations). Bz reduced taste slopes (percentage decrease): Sw 45% (P<.02), So 34% (P<.01), Sa 35% or 41% (NaCl or KCl, P<.03), Bitt 33% or 60% (quinine P<.01 or KCl P<.04). Bz reduced K(diss) (affinity(-1)) (percentage reduction): Sw 79% (P<.0002), So 40% (P<.03), Sa(NaCl) 63% (P<.005), Sa(KCl) 48% (P<.04), Bitt(KCl) 64% (P<.04). Bz reduced ME(max) (percentage reduction): Sw 31% (P<.004), Bitt(Quinine) 29% (P<.03). PCs confirmed taste increases by Bz (percentage of 'Mix(intensity)>T(intensity)' answers/total answers): Sw 79-69% (90-1440 mM sucrose), So 75% (1 mM citric acid) and 71% (2 mM citric acid), Sa 75-71% (15-120 mM NaCl). Negative concentration dependence of taste increases by Bz suggests different levels of interaction. Biophysical and neurophysiological changes are discussed in relation with Bz properties and mechanism of interaction with taste. PMID:12676277

  15. ATOH1 Can Regulate the Tumorigenicity of Gastric Cancer Cells by Inducing the Differentiation of Cancer Stem Cells

    PubMed Central

    Han, Myoung-Eun; Baek, Su-Jin; Kim, Seon-Young; Kang, Chi-Dug; Oh, Sae-Ock

    2015-01-01

    Cancer stem cells (CSCs) have been shown to mediate tumorigenicity, chemo-resistance, radio-resistance and metastasis, which suggest they be considered therapeutic targets. Because their differentiated daughter cells are no longer tumorigenic, to induce the differentiation of CSCs can be one of strategies which can eradicate CSCs. Here we show that ATOH1 can induce the differentiation of gastric cancer stem cells (GCSCs). Real time PCR and western blot analysis showed that ATOH1 was induced during the differentiation of GCSCs. Furthermore, the lentivirus-induced overexpression of ATOH1 in GCSCs and in gastric cancer cell lines significantly induced differentiation, reduced proliferation and sphere formation, and reduced in vivo tumor formation in the subcutaneous injection and liver metastasis xenograft models. These results suggest ATOH1 be considered for the development of a differentiation therapy for gastric cancer. PMID:25950549

  16. CSK negatively regulates nerve growth factor induced neural differentiation and augments AKT kinase activity

    SciTech Connect

    Dey, Nandini . E-mail: Don_Durden@oz.ped.emory.edu

    2005-07-01

    Src family kinases are involved in transducing growth factor signals for cellular differentiation and proliferation in a variety of cell types. The activity of all Src family kinases (SFKs) is controlled by phosphorylation at their C-terminal 527-tyrosine residue by C-terminal SRC kinase, CSK. There is a paucity of information regarding the role of CSK and/or specific Src family kinases in neuronal differentiation. Pretreatment of PC12 cells with the Src family kinase inhibitor, PP1, blocked NGF-induced activation of SFKs and obliterated neurite outgrowth. To confirm a role for CSK and specific isoforms of SFKs in neuronal differentiation, we overexpressed active and catalytically dead CSK in the rat pheochromocytoma cell line, PC12. CSK overexpression caused a profound inhibition of NGF-induced activation of FYN, YES, RAS, and ERK and inhibited neurite outgrowth, NGF-stimulated integrin-directed migration and blocked the NGF-induced conversion of GDP-RAC to its GTP-bound active state. CSK overexpression markedly augmented the activation state of AKT following NGF stimulation. In contrast, kinase-dead CSK augmented the activation of FYN, RAS, and ERK and increased neurite outgrowth. These data suggest a distinct requirement for CSK in the regulation of NGF/TrkA activation of RAS, RAC, ERK, and AKT via the differential control of SFKs in the orchestration of neuronal differentiation.

  17. Nerve growth factor induces survival and differentiation through two distinct signaling cascades in PC12 cells.

    PubMed

    Klesse, L J; Meyers, K A; Marshall, C J; Parada, L F

    1999-03-25

    Nerve growth factor induces differentiation and survival of rat PC12 pheochromocytoma cells. The activation of the erk cascade has been implicated in transducing the multitude of signals induced by NGF. In order to explore the role of this signaling cascade in NGF mediated survival, differentiation and proliferation, we generated recombinant adenoviruses which express the intermediates of the erk cascade in their wild type, dominant negative and constitutively activated forms. We show that differentiation of PC12 cells requires activity of the ras/erk pathway, whereas inhibition of this pathway had no effect on survival or proliferation. Constitutively active forms of ras, raf and mek induced PC12 cell differentiation, while dominant interfering forms inhibited differentiation. Survival of PC12 cells in serum-free medium did not require activity of the ras/erk pathway. Instead, PI3 Kinase signaling was necessary for PC12 cell survival. Interestingly, constitutively activated versions of raf and mek were able to promote survival, but again this was dependent on activation of PI3 Kinase. Therefore, at least two distinct signaling pathways are required in PC12 cells for mediation of NGF functions.

  18. 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

  19. Differentiation induced by physiological and pharmacological stimuli leads to increased antigenicity of human neuroblastoma cells.

    PubMed

    Carlson, Lena-Maria; Påhlman, Sven; De Geer, Anna; Kogner, Per; Levitskaya, Jelena

    2008-03-01

    Sympathetic neuronal differentiation is associated with favorable prognosis of neuroblastoma (NB), the most common extra-cranial solid tumor of early childhood. Differentiation agents have proved useful in clinical protocols of NB treatment, but using them as a sole treatment is not sufficient to induce tumor elimination in patients. Therefore, complementary approaches, such as immunotherapy, are warranted. Here we demonstrate that differentiation of NB cell lines and ex vivo isolated tumor cells in response to physiological or pharmacological stimuli is associated with acquisition of increased antigenicity. This manifests as increased expression of surface major histocompatibility class I complexes and ICAM-1 molecules and translates into increased sensitivity of NB cells to lysis by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. The latter is paralleled by enhanced ability of differentiated cells to form immune conjugates and bind increased amounts of granzyme B to the cell surface. We demonstrate, for the first time, that, regardless of the stimulus applied, the differentiation state in NBs is associated with increased tumor antigenicity that enables more efficient elimination of tumor cells by cytotoxic lymphocytes and paves the way for combined application of differentiation-inducing agents and immunotherapy as an auxiliary approach in NB patients.

  20. 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.

  1. Caffeine-induced arousal modulates somatomotor and autonomic differential classical conditioning in humans.

    PubMed

    Flaten, M A

    1998-01-01

    Two experiments (n = 48 and n = 45) investigated the effects of caffeine-induced arousal on differential classical conditioning of eyeblink (experiment 1) and autonomic (experiment 2) responses. Three groups of human subjects received double-blind administration of 0, 2, and 4 mg/kg oral caffeine (groups 0, 2, and 4, respectively). Twenty minutes after caffeine administration, a differential classical conditioning procedure was in effect. Physiological and subjective arousal was assessed by readings of blood pressure, skin conductance level, and a questionnaire, administered before caffeine administration, and after the conditioning procedure. The results showed increased indexes of physiological arousal in groups 2 and 4. In experiment 1, differential classical eyeblink conditioning was observed in groups 0 and 4, whereas no differential conditioning was seen in group 2. In experiment 2, differential classical conditioning was seen in group 0, whereas caffeine-induced arousal masked acquisition of conditioned skin conductance responses in group 4. This group displayed increased resistance to extinction compared to the other groups. Group 2, which had an intermediate level of arousal, did not display differential conditioning in either experiment. Taken together, the results indicate that small increases in arousal may be detrimental to learning, and larger increases in arousal may reverse this effect. PMID:9489937

  2. 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

  3. 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...

  4. 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.

  5. 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

  6. 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)

  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. 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

  9. 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.

  10. 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.

  11. Association of SIRT1 expression with shear stress induced endothelial progenitor cell differentiation.

    PubMed

    Cheng, Bin-Bin; Yan, Zhi-Qiang; Yao, Qing-Ping; Shen, Bao-Rong; Wang, Ji-Yao; Gao, Li-Zhi; Li, Yu-Qing; Yuan, Hai-Tao; Qi, Ying-Xin; Jiang, Zong-Lai

    2012-12-01

    Shear stress imposed by blood flow is crucial for differentiation of endothelial progenitor cells (EPCs). Histone deacetylase SIRT1 has been shown to play a pivotal role in many physiological processes. However, association of SIRT1 expression with shear stress-induced EPC differentiation remains to be elucidated. The present study was designed to determine the effect of SIRT1 on EPC differentiation induced by shear stress, and to seek the underlying mechanisms. Human umbilical cord blood-derived EPCs were exposed to laminar shear stress of 15 dyn/cm(2) by parallel plate flow chamber system. Shear stress enhanced EPC differentiation toward endothelial cells (ECs) while inhibited to smooth muscle cells (SMCs). The expressions of phospho-Akt, SIRT1 and histone H3 acetylation (Ac-H3) in EPCs were detected after exposure to shear stress for 2, 6, 12, and 24 h, respectively. Shear stress significantly activated Akt phosphorylation, augmented SIRT1 expression and downregulated Ac-H3. SIRT1 siRNA in EPCs diminished the expression of EC markers, but increased the expression of SMC markers, and resulted in upregulation of Ac-H3. Whereas, resveratrol, an activator of SIRT1, had the opposite effects on both EPC differentiation and histone H3 acetylation. Wortmannin, an inhibitor of PI3-kinase, suppressed endothelial differentiation of EPCs, decreased SIRT1, and upregulated Ac-H3 expression. In addition, SIRT1 promoted tube formation of EPCs in matrix gels. These results provided a mechanobiological basis of shear stress-induced EPC differentiation into ECs and suggest that PI3k/Akt-SIRT1-Ac-H3 pathway is crucial in such a process.

  12. Polysaccharides immobilized in polypyrrole matrices are able to induce osteogenic differentiation in mouse mesenchymal stem cells.

    PubMed

    Moreno, Judith Serra; Sabbieti, Maria Giovanna; Agas, Dimitrios; Marchetti, Luigi; Panero, Stefania

    2014-12-01

    Bone marrow mesenchymal stem cells (MSCs) have attracted considerable interest due to their ability to differentiate and contribute to the regeneration of mesenchymal tissues. The present study illustrates that the proper immobilization of heparin (Hep) and hyaluronic acid (HA) into a polypyrrole (PPy) matrix by electropolymerization results in an optimal interface for MSC differentiation towards osteoblast lineage. The obtained thin films showed good thermal stability, hydrophilicity and slow controlled polysaccharide release. The in vitro tests showed the main role of the interface chemical composition. Indeed, PPyHep and PPyHA thin films were able to induce osteogenic differentiation as determined by levels of specific early osteogenic markers (Runx2 and osterix) even in the absence of differentiating medium. Increased levels of ALP and Alizarin red staining, both indicating mineralization processes, confirmed the presence of mature osteoblasts.

  13. Substrate-induced PC12 cell differentiation without filopodial, lamellipodial activity or NGF stimulationa.

    PubMed

    Lamour, Guillaume; Souès, Sylvie; Hamraoui, Ahmed

    2015-03-01

    Nanoscale gradients in energy of adhesion are physical cues from the extracellular environment that can significantly affect cell functions and enhance the neuronal differentiation of PC12 cells. How such surface effects can trigger differentiation and initiate neurite outgrowth, remains to be elucidated. Here we used surface modification, atomic force microscopy and immunofluorescence to analyze PC12 cells. We studied the kinetics of neurites growth under cytochalasin-B treatment, known as an inhibitor of actin polymerization. We found that neither filopodia nor lamellipodia are involved in detecting the surface effects that induce the differentiation of PC12 cells. This finding suggests that the solution to this problem lies beyond identifying a precise cytoskeleton-associated cell-substrate intermediate. Thus, a more comprehensive model is probably required to identify the mechanism by which cell-substrate interactions are eventually translated into a differentiation signal. PMID:25350917

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

    PubMed

    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

  16. 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

  17. Human amnion epithelial cells can be induced to differentiate into functional insulin-producing cells.

    PubMed

    Hou, Yanan; Huang, Qin; Liu, Tianjin; Guo, Lihe

    2008-09-01

    Pancreatic islet transplantation has demonstrated that long-term insulin independence may be achieved in patients suffering from diabetes mellitus type 1. However, limited availability of islet tissue means that new sources of insulin-producing cells that are responsive to glucose are required. Here, we show that human amnion epithelial cells (HAEC) can be induced to differentiate into functional insulin-producing cells in vitro. After induction of differentiation, HAEC expressed multiple pancreatic beta-cell genes, including insulin, pancreas duodenum homeobox-1, paired box gene 6, NK2 transcription factor-related locus 2, Islet 1, glucokinase, and glucose transporter-2, and released C-peptide in a glucose-regulated manner in response to other extracellular stimulations. The transplantation of induced HAEC into streptozotocin-induced diabetic C57 mice reversed hyperglycemia, restored body weight, and maintained euglycemia for 30 d. These findings indicated that HAEC may be a new source for cell replacement therapy in type 1 diabetes.

  18. 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.

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

    PubMed Central

    Barbano, M. Flavia; Wang, Hui-Ling; Morales, Marisela

    2016-01-01

    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. SIGNIFICANCE STATEMENT Our study reports that the eating and the reward induced by optogenetic stimulation of a previously identified GABAergic projection from the lateral hypothalamus to the ventral tegmental area are differentially affected by low- and high-frequency stimulation, respectively. This suggests a way that stimulation of the same pathway can have very different motivational effects on behavior, inducing a drive state (usually thought to be aversive) under one condition and inducing the rewarding state under another. This offers an insight into what has been called the “drive-reward paradox”: why would an animal work for stimulation that established an apparent drive state? PMID:26961951

  20. 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

  1. 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.

  2. 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.

  3. Homogeneous and organized differentiation within embryoid bodies induced by microsphere-mediated delivery of small molecules

    PubMed Central

    Carpenedo, Richard L.; Bratt-Leal, Andrés M.; Marklein, Ross A.; Seaman, Scott A.; Bowen, Nathan J.; McDonald, John F.; McDevitt, Todd C.

    2010-01-01

    Cell specification and tissue formation during embryonic development are precisely controlled by the local concentration and temporal presentation of morphogenic factors. Similarly, pluripotent embryonic stem cells can be induced to differentiate in vitro into specific phenotypes in response to morphogen treatment. Embryonic stem cells (ESCs) are commonly differentiated as 3D spheroids referred to as embryoid bodies (EBs); however, differentiation of cells within EBs is typically heterogeneous and disordered. In this study, we demonstrate that in contrast to soluble morphogen treatment, delivery of morphogenic factors directly within EB microenvironments in a spatiotemporally controlled manner using polymer microspheres yields homogeneous, synchronous and organized ESC differentiation. Degradable PLGA microspheres releasing retinoic acid were incorporated directly within EBs and induced the formation of cystic spheroids uniquely resembling the phenotype and structure of early streak mouse embryos (E6.75), with an exterior of FOXA2+ visceral endoderm enveloping an epiblast-like layer of OCT4+ cells. These results demonstrate that controlled morphogen presentation to stem cells using degradable microspheres more efficiently directs cell differentiation and tissue formation than simple soluble delivery methods and presents a unique route to study the spatiotemporal effects of morphogenic factors on embryonic developmental processes in vitro. PMID:19162317

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. Wnt signaling pathways participate in Astragalus injection-induced differentiation of bone marrow mesenchymal stem cells.

    PubMed

    Zhong, Jingfei; Cao, Hui; Chen, Zhonghua; Zhou, Fei; Tan, Xiangling

    2013-10-11

    Bone marrow mesenchymal stem cells (MSCs) have the capacity for self-renewal and multi-directional differentiation, and MSCs can differentiate into neuron-like cells under certain conditions. In this study, we used the traditional Chinese medicine Astragalus as an inducer. After 7 days of induction, the expression of specific markers was detected in each induced group by immunocytochemical staining. The results of real-time quantitative PCR and Western blot confirmed the immunocytochemistry analysis. We also tested some key genes and proteins of the Wnt signaling pathway, and found that they were increased in Astragalus-treated groups. After treatment with lithium chloride (LiCl), the protein expression of phospho GSK-3β and β-catenin was increased in each group compared to the corresponding group without LiCl. These findings demonstrate that Astragalus injection can induce differentiation of MSCs into neuron-like cells and suggest that the process of differentiation might be mediated by activation of Wnt signaling pathways.

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

    PubMed

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

    2011-05-01

    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.

  12. Differentiation of human B lymphocyte subpopulations induced by an alloreactive helper T-cell clone

    SciTech Connect

    Anderson, S.J.; Hummell, D.S.; Lawton, A.R.

    1988-07-01

    We have used cloned alloreactive helper T cells to determine if direct T cell-B cell interaction can induce differentiation of human peripheral blood B cells which do not respond to pokeweed mitogen (PWM). T-cell clone 2F8 was derived from a one-way mixed lymphocyte reaction. 2F8 cells are T3+T4+T8-IL-2R+ and proliferate in response to irradiated stimulator cells, but not autologous cells, in the absence of exogenous interleukin-2. 2F8 cells provide allospecific help for polyclonal proliferation and differentiation of B cells in the absence of any other stimulus. The magnitude of this response is comparable to that of the response of the same B cells to PWM and fresh autologous T cells. 2F8 cells could also provide nonspecific help for unrelated donor B cells in the presence of PWM, with no requirement for costimulation by irradiated stimulator cells. Allospecific stimulation of B cells was completely inhibited by antibodies to class II major histocompatibility complex (MHC) framework determinants and was abrogated by 1000-rad irradiation. Cloned 2F8 T cells stimulated differentiation of both small, high-density B cells and larger B cells, generating up to 30% plasma cells with either fraction. B cells forming rosettes with mouse erythrocytes were also induced to differentiate by the helper T cell clone. As found previously, neither small, high-density B cells nor mouse rosette+ B cells responded well to PWM. Direct interaction with allospecific T cells induces differentiation of a broader spectrum of B cells than soluble growth and differentiation factors in conjunction with polyclonal activators such as PWM and protein A containing staphylococci.

  13. [Neural differentiation of pluripotent stem cells and application for metal-induced neural toxicity study].

    PubMed

    Taniguchi, Yuki; Tobe, Takao; Hayami, Hideaki; Okamoto, Yoshinori; Ueda, Koji; Takada, Tatsuyuki; Kojima, Nakao

    2014-01-01

    Metals are effectively used in biological systems under the strict regulation for exploiting their specific and broad reactivities. For example, manganese (Mn) can induce catecholamines-mediated oxidative biological damage in cooperation with iron (Fe) and/or copper (Cu). In children, the damage could induce developmental disorders such as attention deficit hyperactivity disorder (ADHD). We hypothesize that infant neurons are more labile to metals than adult ones due to the prematured protection systems and sensitive differentiating cells. An experimental system reconstituting neural differentiation is expected to assess the influences of endogenous/exogenous factors including metals. In this study, we investigated an impact of Mn together with Fe and dopamine (DA) on neural differentiation of mouse embryonic stem cells (mESCs). The differentiation of mESCs was initiated by embryoid bodies (EBs) formation in the presence of all-trans retinoic acid, and then EBs were treated with Mn, Fe and/or DA. Then, the mRNA levels of neural differentiation marker genes (Nestin, Emx2, Mtap2, Th, Olig2 and Gfap) were examined using realtime RT-PCR analysis. Mn or DA alone reduced Mtap2, Th and Olig2 expression levels and increased Nestin. Moreover, combined treatment of Mn and DA also increased Nestin expression level. On the other hand, Fe alone reduced Mtap2, Th and Olig2 expression levels, and increased Emx2. Combined treatments of Fe with Mn or DA also tended to increase Emx2 expression level. These effects emerged at about 100 times less concentration than that inducing cytotoxicity in human neuroblastoma. The present study showed that Mn inhibits neural development, and that our mESCs system can be a useful tool to elucidate the toxicity mechanism as well as to evaluate the effects of metals and chemicals on differentiating cells. PMID:24989467

  14. 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

  15. A new method to induce molecular low bias negative differential resistance with multi-peaks.

    PubMed

    Min, Y; Zhong, C G; Dong, Z C; Zhao, Z Y; Zhou, P X; Yao, K L

    2016-02-14

    According to a first-principles study of the transport properties of two thiolated anthracene-9,10-diono molecules sandwiching ethyl, a new method to induce molecular low bias negative differential resistance with multi-peaks for strong n- or p-type molecules is proposed. The anthracene-9,10-diono molecule shows strong n-type characteristics when in contact with Au and Ag electrodes via a thiolate. The multiple negative differential resistance effect originated from the molecule-electrode couple is different between Ag and Au electrodes. Our investigations may promise potential for applications in molecular devices with low power dissipation and multifunction in the future.

  16. Solvent-induced current-voltage hysteresis and negative differential resistance in molecular junctions

    NASA Astrophysics Data System (ADS)

    Dzhioev, Alan A.; Kosov, D. S.

    2012-01-01

    We consider a single molecule circuit embedded into solvent. The Born dielectric solvation model is combined with Keldysh nonequilibrium Green's functions to describe the electron-transport properties of the system. Depending on the dielectric constant, the solvent induces multiple nonequilibrium steady states with corresponding hysteresis in molecular current-voltage characteristics as well as negative differential resistance. We identify the physical range of solvent and molecular parameters where the effects are present. The position of the negative differential resistance peak can be controlled by the dielectric constant of the solvent.

  17. 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

  18. Differentiation of hepatocytes from induced pluripotent stem cells derived from human hair follicle mesenchymal stem cells.

    PubMed

    Shi, Xu; Lv, Shuang; He, Xia; Liu, Xiaomei; Sun, Meiyu; Li, Meiying; Chi, Guangfan; Li, Yulin

    2016-10-01

    Due to the limitations of organ donors and immune rejection in severe liver diseases, stem cell-based therapy presents a promising application for tissue repair and regeneration. As a novel cell source, mesenchymal stem cells separated from human hair follicles (HF-MSCs) are convenient to obtain and have no age limit. To date, the differentiation of HF-MSCs into hepatocytes has not been reported. In this study, we explored whether HF-MSCs and HF-MSC-derived-induced pluripotent stem cells (HF-iPS) could differentiate into hepatocytes in vitro. Flow cytometry, Oil Red O stain and Alizarin Red stain were used to identify the characteristics of HF-MSCs. The expression of liver-specific gene was detected by immunofluorescence and Quantitative Polymerase Chain Reaction. Periodic Acid-Schiff stain, Indocyanine Green stain and Low-Density Lipoprotein stain were performed to evaluate the functions of induced hepatocyte-like cells (HLCs). HF-MSCs were unable to differentiate into HLCs using previously reported procedures for MSCs from other tissues. However, HF-iPS efficiently induced the generation of HLCs that expressed hepatocyte markers and drug metabolism-related genes. HF-iPS can be used as novel and alternative cellular tools for inducing hepatocytes in vitro, simultaneously benefiting from utilizing HF-MSCs as a noninvasive and convenient cell source for reprogramming.

  19. Differentiation of hepatocytes from induced pluripotent stem cells derived from human hair follicle mesenchymal stem cells.

    PubMed

    Shi, Xu; Lv, Shuang; He, Xia; Liu, Xiaomei; Sun, Meiyu; Li, Meiying; Chi, Guangfan; Li, Yulin

    2016-10-01

    Due to the limitations of organ donors and immune rejection in severe liver diseases, stem cell-based therapy presents a promising application for tissue repair and regeneration. As a novel cell source, mesenchymal stem cells separated from human hair follicles (HF-MSCs) are convenient to obtain and have no age limit. To date, the differentiation of HF-MSCs into hepatocytes has not been reported. In this study, we explored whether HF-MSCs and HF-MSC-derived-induced pluripotent stem cells (HF-iPS) could differentiate into hepatocytes in vitro. Flow cytometry, Oil Red O stain and Alizarin Red stain were used to identify the characteristics of HF-MSCs. The expression of liver-specific gene was detected by immunofluorescence and Quantitative Polymerase Chain Reaction. Periodic Acid-Schiff stain, Indocyanine Green stain and Low-Density Lipoprotein stain were performed to evaluate the functions of induced hepatocyte-like cells (HLCs). HF-MSCs were unable to differentiate into HLCs using previously reported procedures for MSCs from other tissues. However, HF-iPS efficiently induced the generation of HLCs that expressed hepatocyte markers and drug metabolism-related genes. HF-iPS can be used as novel and alternative cellular tools for inducing hepatocytes in vitro, simultaneously benefiting from utilizing HF-MSCs as a noninvasive and convenient cell source for reprogramming. PMID:27053247

  20. Bisphenol A and Bisphenol S Induce Distinct Transcriptional Profiles in Differentiating Human Primary Preadipocytes

    PubMed Central

    Boucher, Jonathan G.; Gagné, Rémi; Rowan-Carroll, Andrea; Boudreau, Adèle; Yauk, Carole L.; Atlas, Ella

    2016-01-01

    Bisphenol S (BPS) is increasingly used as a replacement plasticizer for bisphenol A (BPA) but its effects on human health have not been thoroughly examined. Recent evidence indicates that both BPA and BPS induce adipogenesis, although the mechanisms leading to this effect are unclear. In an effort to identify common and distinct mechanisms of action in inducing adipogenesis, transcriptional profiles of differentiating human preadipocytes exposed to BPA or BPS were compared. Human subcutaneous primary preadipocytes were differentiated in the presence of either 25 μM BPA or BPS for 2 and 4 days. Poly-A RNA-sequencing was used to identify differentially expressed genes (DEGs). Functional analysis of DEGs was undertaken in Ingenuity Pathway Analysis. BPA-treatment resulted in 472 and 176 DEGs on days 2 and 4, respectively, affecting pathways such as liver X receptor (LXR)/retinoid X receptor (RXR) activation, hepatic fibrosis and cholestasis. BPS-treatment resulted in 195 and 51 DEGs on days 2 and 4, respectively, revealing enrichment of genes associated with adipogenesis and lipid metabolism including the adipogenesis pathway and cholesterol biosynthesis. Interestingly, the transcription repressor N-CoR was identified as a negative upstream regulator in both BPA- and BPS-treated cells. This study presents the first comparison of BPA- and BPS-induced transcriptional profiles in human differentiating preadipocytes. While we previously showed that BPA and BPS both induce adipogenesis, the results from this study show that BPS affects adipose specific transcriptional changes earlier than BPA, and alters the expression of genes specifically related to adipogenesis and lipid metabolism. The findings provide insight into potential BPS and BPA-mediated mechanisms of action in inducing adipogenesis in human primary preadipocytes. PMID:27685785

  1. Comparison of different protocols for neural differentiation of human induced pluripotent stem cells.

    PubMed

    Salimi, Ali; Nadri, Samad; Ghollasi, Marzieh; Khajeh, Khosro; Soleimani, Masoud

    2014-03-01

    Although embryonic stem cells (ESCs) have enormous potentials due to their pluripotency, their therapeutic use is limited by ethical, biological and safety issues. Compared to ESCs, induced pluripotent stem cells (iPSCs) can be obtained from mouse or human fibroblasts by reprogramming. Numerous studies have established many protocols for differentiation of human iPSCs (hiPSCs) into neural lineages. However, the low differentiation efficiency of such protocols motivates researchers to design new protocols for high yield differentiation. Herein, we compared neural differentiation potential of three induction media for conversion of hiPSCs into neural lineages. In this study, hiPSCs-derived embryoid bodies were plated on laminin coated dishes and were treated with three induction media including (1) bFGF, EGF (2) RA and (3) forskolin, IBMX. Immunofluorescence staining and quantitative real-time PCR (qPCR) analysis were used to detect the expression of neural genes and proteins. qPCR analysis showed that the expression of neural genes in differentiated hiPSCs in forskolin, IBMX supplemented media was significantly higher than undifferentiated cells and those in induction media containing bFGF, EGF or RA. In conclusion, our results indicated a successful establishment protocol with high efficiency for differentiation of hiPSCs into neural lineages.

  2. 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

  3. 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.

  4. SV40 enhancer activation during retinoic acid-induced differentiation of F9 embryonal carcinoma cells.

    PubMed Central

    Sleigh, M J; Lockett, T J

    1985-01-01

    The transient expression vector pSV2CAT, which carries the bacterial chloramphenicol acetyl transferase (CAT) gene under the control of the SV40 early promoter, was used to transfect the murine embryonal carcinoma cell line F9 at various times during the retinoic acid-induced differentiation of these cells. Expression of the CAT gene under SV40 promoter control was found to increase markedly on F9 cell differentiation, measured relative to expression from the thymidine kinase promoter in the same cells. A series of constructs was prepared to identify the features of the SV40 early promoter required for transcription in differentiated and undifferentiated cells, as well as the factors limiting transcription in each case. The increased transcription seen on F9 cell differentiation was not observed when cells were transfected with molecules lacking a functional enhancer. It appears that as embryonal carcinoma cells differentiate, increased SV40 transcription results from enhancer sequence activation. In both differentiated and undifferentiated cell types the level of transcription was found to be limited by the availability and/or activity of cellular factors necessary for enhancer function. Images Fig. 1. PMID:3004973

  5. 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.

  6. Concurrence of replicative senescence and elevated expression of p16(INK4A) with subculture-induced but not calcium-induced differentiation in normal human oral keratinocytes.

    PubMed

    Lee, G; Park, B S; Han, S E; Oh, J E; You, Y O; Baek, J H; Kim, G S; Min, B M

    2000-10-01

    Primary normal human oral keratinocytes (NHOKs) undergo differentiation in the presence of calcium concentrations higher than 0.15 mM in vitro, which is useful in investigating the mechanisms involved in the differentiation of epithelial cells. Serial subculture of NHOKs to the postmitotic stage also induces terminal differentiation. However, the detailed mechanisms of both differentiation processes remain substantially unknown. To investigate the molecular differences in these processes, NHOKs were induced to differentiate by exposure to 1.2 mM of calcium and by serial subculture to the postmitotic stage. To study whether the cells were induced to differentiate and to undergo replicative senescence, the amount of cellular involucrin and the expression of senescence-associated beta-galactosidase (SA-beta-gal) were measured respectively. The expression of replicative senescence-associated genes and the activity of telomerase from the differentiated cells were also determined. Both calcium treatment and serial subculture to the postmitotic stage notably elevated the cellular involucrin. The percentage of SA-beta-gal-positive cells was significantly elevated by the continued subculture, but such changes were not observed in keratinocytes exposed to calcium. The concentration of cellular p16(INK4A) protein was progressively increased by the continued subculture but was not changed by calcium treatment. On the other hand, the concentrations of cellular p53 were similar in both differentiation processes. However, telomerase activity was lost in NHOKs that had undergone differentiation by both calcium treatment and serial subculture. The results indicate that calcium-induced differentiation of NHOKs has similar characteristics to their serial subculture-induced differentiation, but that the differentiation processes are not identical, because calcium-induced differentiation does not concur with either replicative senescence or the gradually increased concentration of p16

  7. 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

  8. 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.

  9. N-acetylcysteine protects dental pulp stromal cells from HEMA-induced apoptosis by inducing differentiation of the cells.

    PubMed

    Paranjpe, Avina; Cacalano, Nicholas A; Hume, Wyatt R; Jewett, Anahid

    2007-11-15

    Resin-based materials are now widely used in dental restorations. Although the use of these materials is aesthetically appealing to patients, it carries the risk of local and systemic adverse effects. The potential risks are direct damage to the cells and induction of immune-based hypersensitivity reactions. Dental pulp stromal cells (DPSCs) and oral keratinocytes are the major cell types which may come in contact with dental resins such as 2-hydroxyethyl methacrylate (HEMA) after dental restorations. Here we show that N-acetylcysteine (NAC) inhibits HEMA-induced apoptotic cell death and restores the function of DPSCs and oral epithelial cells. NAC inhibits HEMA-mediated toxicity through induction of differentiation in DPSCs, because the genes for dentin sialoprotein, osteopontin (OPN), osteocalcin, and alkaline phosphatase, which are induced during differentiation, are also induced by NAC. Unlike NAC, vitamins E and C, which are known antioxidant compounds, failed to prevent either HEMA-mediated cell death or the decrease in VEGF secretion by human DPSCs. More importantly, when added either alone or in combination with HEMA, vitamin E and vitamin C did not increase the gene expression for OPN, and in addition vitamin E inhibited the protective effect of NAC on DPSCs. NAC inhibited the HEMA-mediated decrease in NF-kappaB activity, thus providing a survival mechanism for the cells. Overall, the studies reported in this paper indicate that undifferentiated DPSCs have exquisite sensitivity to HEMA-induced cell death, and their differentiation in response to NAC resulted in an increased NF-kappaB activity, which might have provided the basis for their increased protection from HEMA-mediated functional loss and cell death.

  10. 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

  11. 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

  12. N-Docosahexaenoylethanolamine ameliorates ethanol-induced impairment of neural stem cell neurogenic differentiation.

    PubMed

    Rashid, Mohammad Abdur; Kim, Hee-Yong

    2016-03-01

    Previous studies demonstrated that prenatal exposure to ethanol interferes with embryonic and fetal development, and causes abnormal neurodevelopment. Docosahexaenoic acid (DHA), an omega-3 polyunsaturated fatty acid highly enriched in the brain, was shown to be essential for proper brain development and function. Recently, we found that N-docosahexenoyethanolamine (synaptamide), an endogenous metabolite of DHA, is a potent PKA-dependent neurogenic factor for neural stem cell (NSC) differentiation. In this study, we demonstrate that ethanol at pharmacologically relevant concentrations downregulates cAMP signaling in NSC and impairs neurogenic differentiation. In contrast, synaptamide reverses ethanol-impaired NSC neurogenic differentiation through counter-acting on the cAMP production system. NSC exposure to ethanol (25-50 mM) for 4 days dose-dependently decreased the number of Tuj-1 positive neurons and PKA/CREB phosphorylation with a concomitant reduction of cellular cAMP. Ethanol-induced cAMP reduction was accompanied by the inhibition of G-protein activation and expression of adenylyl cyclase (AC) 7 and AC8, as well as PDE4 upregulation. In contrast to ethanol, synaptamide increased cAMP production, GTPγS binding, and expression of AC7 and AC8 isoforms in a cAMP-dependent manner, offsetting the ethanol-induced impairment in neurogenic differentiation. These results indicate that synaptamide can reduce ethanol-induced impairment of neuronal differentiation by counter-affecting shared targets in G-protein coupled receptor (GPCR)/cAMP signaling. The synaptamide-mediated mechanism observed in this study may offer a possible avenue for ameliorating the adverse impact of fetal alcohol exposure on neurodevelopment. PMID:26586023

  13. 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

  14. N-Docosahexaenoylethanolamine ameliorates ethanol-induced impairment of neural stem cell neurogenic differentiation.

    PubMed

    Rashid, Mohammad Abdur; Kim, Hee-Yong

    2016-03-01

    Previous studies demonstrated that prenatal exposure to ethanol interferes with embryonic and fetal development, and causes abnormal neurodevelopment. Docosahexaenoic acid (DHA), an omega-3 polyunsaturated fatty acid highly enriched in the brain, was shown to be essential for proper brain development and function. Recently, we found that N-docosahexenoyethanolamine (synaptamide), an endogenous metabolite of DHA, is a potent PKA-dependent neurogenic factor for neural stem cell (NSC) differentiation. In this study, we demonstrate that ethanol at pharmacologically relevant concentrations downregulates cAMP signaling in NSC and impairs neurogenic differentiation. In contrast, synaptamide reverses ethanol-impaired NSC neurogenic differentiation through counter-acting on the cAMP production system. NSC exposure to ethanol (25-50 mM) for 4 days dose-dependently decreased the number of Tuj-1 positive neurons and PKA/CREB phosphorylation with a concomitant reduction of cellular cAMP. Ethanol-induced cAMP reduction was accompanied by the inhibition of G-protein activation and expression of adenylyl cyclase (AC) 7 and AC8, as well as PDE4 upregulation. In contrast to ethanol, synaptamide increased cAMP production, GTPγS binding, and expression of AC7 and AC8 isoforms in a cAMP-dependent manner, offsetting the ethanol-induced impairment in neurogenic differentiation. These results indicate that synaptamide can reduce ethanol-induced impairment of neuronal differentiation by counter-affecting shared targets in G-protein coupled receptor (GPCR)/cAMP signaling. The synaptamide-mediated mechanism observed in this study may offer a possible avenue for ameliorating the adverse impact of fetal alcohol exposure on neurodevelopment.

  15. Linear relations among holomorphic quadratic differentials and induced Siegel's metric on g

    NASA Astrophysics Data System (ADS)

    Matone, Marco; Volpato, Roberto

    2011-10-01

    We find the explicit form of the volume form on the moduli space of non-hyperelliptic Riemann surfaces induced by the Siegel metric, a long-standing question in string theory. This question is related to the explicit form of the (g-2)(g-3)/2 linearly independent relations among the 2-fold products of holomorphic abelian differentials, that are provided in the case of canonical curves of genus g ⩾ 4. Such relations can be completely expressed in terms of determinants of the standard normalized holomorphic abelian differentials. Remarkably, it turns out that the induced volume form is the Kodaira-Spencer map of the square of the Bergman reproducing kernel.

  16. 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.

  17. Dynamic regulation of human endogenous retroviruses mediates factor-induced reprogramming and differentiation potential.

    PubMed

    Ohnuki, Mari; Tanabe, Koji; Sutou, Kenta; Teramoto, Ito; Sawamura, Yuka; Narita, Megumi; Nakamura, Michiko; Tokunaga, Yumie; Nakamura, Masahiro; Watanabe, Akira; Yamanaka, Shinya; Takahashi, Kazutoshi

    2014-08-26

    Pluripotency can be induced in somatic cells by overexpressing transcription factors, including POU class 5 homeobox 1 (OCT3/4), sex determining region Y-box 2 (SOX2), Krüppel-like factor 4 (KLF4), and myelocytomatosis oncogene (c-MYC). However, some induced pluripotent stem cells (iPSCs) exhibit defective differentiation and inappropriate maintenance of pluripotency features. Here we show that dynamic regulation of human endogenous retroviruses (HERVs) is important in the reprogramming process toward iPSCs, and in re-establishment of differentiation potential. During reprogramming, OCT3/4, SOX2, and KLF4 transiently hyperactivated LTR7s--the long-terminal repeats of HERV type-H (HERV-H)--to levels much higher than in embryonic stem cells by direct occupation of LTR7 sites genome-wide. Knocking down LTR7s or long intergenic non-protein coding RNA, regulator of reprogramming (lincRNA-RoR), a HERV-H-driven long noncoding RNA, early in reprogramming markedly reduced the efficiency of iPSC generation. KLF4 and LTR7 expression decreased to levels comparable with embryonic stem cells once reprogramming was complete, but failure to resuppress KLF4 and LTR7s resulted in defective differentiation. We also observed defective differentiation and LTR7 activation when iPSCs had forced expression of KLF4. However, when aberrantly expressed KLF4 or LTR7s were suppressed in defective iPSCs, normal differentiation was restored. Thus, a major mechanism by which OCT3/4, SOX2, and KLF4 promote human iPSC generation and reestablish potential for differentiation is by dynamically regulating HERV-H LTR7s.

  18. Matrix Stiffness–Induced Myofibroblast Differentiation Is Mediated by Intrinsic Mechanotransduction

    PubMed Central

    Huang, Xiangwei; Yang, Naiheng; Fiore, Vincent F.; Barker, Thomas H.; Sun, Yi; Morris, Stephan W.; Ding, Qiang; Thannickal, Victor J.

    2012-01-01

    The mechanical properties of the extracellular matrix have recently been shown to promote myofibroblast differentiation and lung fibrosis. Mechanisms by which matrix stiffness regulates myofibroblast differentiation are not fully understood. The goal of this study was to determine the intrinsic mechanisms of mechanotransduction in the regulation of matrix stiffness–induced myofibroblast differentiation. A well established polyacrylamide gel system with tunable substrate stiffness was used in this study. Megakaryoblastic leukemia factor-1 (MKL1) nuclear translocation was imaged by confocal immunofluorescent microscopy. The binding of MKL1 to the α-smooth muscle actin (α-SMA) gene promoter was quantified by quantitative chromatin immunoprecipitation assay. Normal human lung fibroblasts responded to matrix stiffening with changes in actin dynamics that favor filamentous actin polymerization. Actin polymerization resulted in nuclear translocation of MKL1, a serum response factor coactivator that plays a central role in regulating the expression of fibrotic genes, including α-SMA, a marker for myofibroblast differentiation. Mouse lung fibroblasts deficient in Mkl1 did not respond to matrix stiffening with increased α-SMA expression, whereas ectopic expression of human MKL1 cDNA restored the ability of Mkl1 null lung fibroblasts to express α-SMA. Furthermore, matrix stiffening promoted production and activation of the small GTPase RhoA, increased Rho kinase (ROCK) activity, and enhanced fibroblast contractility. Inhibition of RhoA/ROCK abrogated stiff matrix–induced actin cytoskeletal reorganization, MKL1 nuclear translocation, and myofibroblast differentiation. This study indicates that actin cytoskeletal remodeling–mediated activation of MKL1 transduces mechanical stimuli from the extracellular matrix to a fibrogenic program that promotes myofibroblast differentiation, suggesting an intrinsic mechanotransduction mechanism. PMID:22461426

  19. 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

  20. 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.

  1. 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

  2. Introduction of v-Ha-ras oncogene induces differentiation of cultured human medullary thyroid carcinoma cells

    SciTech Connect

    Nakagawa, T.; Mabry, M.; De Bustros, A.; Ihle, J.N.; Nelkin, B.D.; Baylin, S.B.

    1987-08-01

    Medullary thyroid carcinoma (MTC) is an endocrine tumor of the thyroid C cells that expresses high levels of the neuroendocrine peptide hormone calcitonin. During tumor progression in the host, there is an apparent loss of differentiation in MTC cells that involves a consistent decrease in calcitonin content of the tumor cells associated with decreased expression of the calcitonin gene and/or changes in a mRNA alternative-processing pattern away from that characteristic of the parent thyroid C cell. The authors now report that introduction of the viral Harvey ras (v-Ha-ras) oncogene into cultured human MTC cells can reverse such changes in gene expression and can induce endocrine differentiation of the tumor cells. The expression of v-Ha-ras is associated with decreased cellular proliferation and DNA synthesis. There is a marked increase in the number of cytoplasmic secretory granules that are a classic feature of differentiated thyroid C cells. v-Ha-ras expression induces increased expression of the calcitonin gene and the processing of the primary gene transcript is shifted to favor calcitonin mRNA rather than calcitonin-gene-related peptide (CGRP) mRNA production. These studies with cultured human MTC cells provide a model system to study the role of Ha-ras and related genes in neuroendocrine differentiation. The findings suggest an important approach for identifying genes in solid tumors whose altered expression may play a role in the impaired maturational capacity characteristic of cancer cells during tumor progression.

  3. Selecting antagonistic antibodies that control differentiation through inducible expression in embryonic stem cells

    PubMed Central

    Melidoni, Anna N.; Dyson, Michael R.; Wormald, Sam; McCafferty, John

    2013-01-01

    Antibodies that modulate receptor function have great untapped potential in the control of stem cell differentiation. In contrast to many natural ligands, antibodies are stable, exquisitely specific, and are unaffected by the regulatory mechanisms that act on natural ligands. Here we describe an innovative system for identifying such antibodies by introducing and expressing antibody gene populations in ES cells. Following induced antibody expression and secretion, changes in differentiation outcomes of individual antibody-expressing ES clones are monitored using lineage-specific gene expression to identify clones that encode and express signal-modifying antibodies. This in-cell expression and reporting system was exemplified by generating blocking antibodies to FGF4 and its receptor FGFR1β, identified through delayed onset of ES cell differentiation. Functionality of the selected antibodies was confirmed by addition of exogenous antibodies to three different ES reporter cell lines, where retained expression of pluripotency markers Oct4, Nanog, and Rex1 was observed. This work demonstrates the potential for discovery and utility of functional antibodies in stem cell differentiation. This work is also unique in constituting an example of ES cells carrying an inducible antibody that causes a functional protein “knock-down” and allows temporal control of stable signaling components at the protein level. PMID:24082130

  4. Directed neuronal differentiation of mouse embryonic and induced pluripotent stem cells and their gene expression profiles.

    PubMed

    Chen, Xuesong; Gu, Qi; Wang, Xiang; Ma, Qingwen; Tang, Huixiang; Yan, Xiaoshuang; Guo, Xinbing; Yan, Hao; Hao, Jie; Zeng, Fanyi

    2013-07-01

    Embryonic stem cells (ESCs) may be useful as a therapeutic source of cells for the production of healthy tissue; however, they are associated with certain challenges including immunorejection as well as ethical issues. Induced pluripotent stem cells (iPSCs) are a promising substitute since a patient's own adult cells would serve as tissue precursors. Ethical concerns prevent a full evaluation of the developmental potency of human ESCs and iPSCs, therefore, mouse iPSC models are required for protocol development and safety assessments. We used a modified culturing protocol to differentiate pluripotent cells from a mouse iPS cell line and two mouse ES cell lines into neurons. Our results indicated that all three pluripotent stem cell lines underwent nearly the same differentiation process when induced to form neurons in vitro. Genomic expression microarray profiling and single-cell RT-qPCR were used to analyze the neural lineage differentiation process, and more than one thousand differentially expressed genes involved in multiple molecular processes relevant to neural development were identified.

  5. 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

  6. 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

  7. 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

  8. Myocardin and pdx-1 synergistically induce hMSCs to differentiate into insulin secreting cells.

    PubMed

    Li, Jing-Ting; Sun, Fang-Xing

    2014-10-01

    Mesenchymal stem cells (MSCs) have been reported as an attractive source for the generation of transplantable surrogate β cells. The objective of this study was to investigate a new method to induce the differentiation of hMSCs into insulin secretion cells and to explore its molecular mechanisms. In this study, we investigated in vitro differentiation of hMSCs by overexpression of myocardin and pdx-1. Differentiated cells were evaluated by immunocytochemistry, reverse transcription-polymerase chain reaction (RT-PCR), quantificational real-time RT-PCR (qRT-PCR) and Western blotting. Furthermore, the molecular mechanisms were evaluated by chip assay, CO-IP and Luciferase assay. This study reported a new method to induce the differentiation of hMSCs into insulin secretion cells. The method is cotransduction of myocardin and pdx-1 for 7days. At the same time, we find myocardin and pdx-1 can form a complex to promote the transactivities of insulin by affecting the formation of the pdx-1/myocardin/SRF/CArG complex both in vitro and in vitro. The present study provided a simple and faithful in vitro model for further investigating the cell replacement therapy for diabetes.

  9. 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

  10. 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.

  11. 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.

  12. 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

  13. Depletion of glutamine enhances sodium butyrate-induced erythroid differentiation of K562 cells.

    PubMed

    Canh Hiep, Nguyen; Kinohira, Seiko; Furuyama, Kazumichi; Taketani, Shigeru

    2012-12-01

    Human erytholeukemia K562 cells are induced to differentiate along the erythroid lineage by a variety of chemical compounds, including hemin, sodium butyrate and 1-β-d-arabinofuranosylcytosine. We have investigated the induction of erythroid differentiation of K562 cells by glutamine depletion. When K562 cells were cultured in glutamine-minus medium, the induction of hemoglobin synthesis, accompanied by those of heme-biosynthetic enzymes and erythroid transcriptional factors, was observed. This induction was dependent on the temporally marked decrease of intracellular level of glutathione, followed by the marked activation of p38MAPK and SAPK/JNK, but not ERK. Under glutamine-deficient conditions, the treatment of K562 cells with sodium butyrate resulted in the marked enhancement of the induction of heme biosynthesis. Glutamine depletion also accelerated the expressions of erythroid-related factors including α-globin and heme-biosynthetic enzymes, GATA-1 and NF-E2, in sodium butyrate-induced K562 cells. The transcriptional activity of β-globin gene promoter-reporter was markedly enhanced by these treatments, indicating that glutamine deficiency in combination with sodium butyrate treatment gives high efficiency of chemical-induced differentiation in the hematopoiesis process.

  14. Human mesenchymal stromal cell-secreted lactate induces M2-macrophage differentiation by metabolic reprogramming

    PubMed Central

    Civini, Sara; Pacelli, Consiglia; Dieng, Mame Massar; Lemieux, William; Jin, Ping; Bazin, Renée; Patey, Natacha; Marincola, Francesco M.; Moldovan, Florina; Zaouter, Charlotte; Trudeau, Louis-Eric; Benabdhalla, Basma; Louis, Isabelle; Beauséjour, Christian; Stroncek, David; Le Deist, Françoise; Haddad, Elie

    2016-01-01

    Human mesenchymal stromal cells (MSC) have been shown to dampen immune response and promote tissue repair, but the underlying mechanisms are still under investigation. Herein, we demonstrate that umbilical cord-derived MSC (UC-MSC) alter the phenotype and function of monocyte-derived dendritic cells (DC) through lactate-mediated metabolic reprogramming. UC-MSC can secrete large quantities of lactate and, when present during monocyte-to-DC differentiation, induce instead the acquisition of M2-macrophage features in terms of morphology, surface markers, migratory properties and antigen presentation capacity. Microarray expression profiling indicates that UC-MSC modify the expression of metabolic-related genes and induce a M2-macrophage expression signature. Importantly, monocyte-derived DC obtained in presence of UC-MSC, polarize naïve allogeneic CD4+ T-cells into Th2 cells. Treatment of UC-MSC with an inhibitor of lactate dehydrogenase strongly decreases lactate concentration in culture supernatant and abrogates the effect on monocyte-to-DC differentiation. Metabolic analysis further revealed that UC-MSC decrease oxidative phosphorylation in differentiating monocytes while strongly increasing the spare respiratory capacity proportional to the amount of secreted lactate. Because both MSC and monocytes are recruited in vivo at the site of tissue damage and inflammation, we propose the local increase of lactate concentration induced by UC-MSC and the consequent enrichment in M2-macrophage generation as a mechanism to achieve immunomodulation. PMID:27070086

  15. 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

  16. Differential roles of hypoxia inducible factor subunits in multipotential stromal cells under hypoxic condition

    PubMed Central

    Tamama, Kenichi; Kawasaki, Haruhisa; Kerpedjieva, Svetoslava S.; Guan, Jianjun; Ganju, Ramesh K.; Sen, Chandan K.

    2014-01-01

    Cell therapy with bone marrow multipotential stromal cells (MSCs) represents a promising approach to promote wound healing and tissue regeneration. MSCs expanded in vitro lose early progenitors with differentiation and therapeutic potentials under normoxic condition, whereas hypoxic condition promotes MSC self-renewal through preserving colony forming early progenitors and maintaining undifferentiated phenotypes. Hypoxia inducible factor (HIF) pathway is a crucial signaling pathway activated in hypoxic condition. We evaluated the roles of HIFs in MSC differentiation, colony formation, and paracrine activity under hypoxic condition. Hypoxic condition reversibly decreased osteogenic and adipogenic differentiation. Decrease of osteogenic differentiation depended on HIF pathway; whereas decrease of adipogenic differentiation depended on the activation of unfolded protein response (UPR), but not HIFs. Hypoxia-mediated increase of MSC colony formation was not HIF-dependent also. Hypoxic exposure increased secretion of VEGF, HGF and basic FGF in a HIF-dependent manner. These findings suggest that HIF has a limited, but pivotal role in enhancing MSC self-renewal and growth factor secretions under hypoxic condition. PMID:21328454

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

    PubMed

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

    2015-08-13

    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 (Ca(2+)) 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. Ca(2+) response trace with Pb and KB-R7943 treatment did not drop down in the same recovery time as the control, which elevated intracellular Ca(2+) 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.

  18. Proteomic profiles of mesenchymal stem cells induced by a liver differentiation protocol.

    PubMed

    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.

  19. The aryl hydrocarbon receptor ligand ITE inhibits TGFβ1-induced human myofibroblast differentiation.

    PubMed

    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

    2011-04-01

    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.

  20. 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

  1. Control of thrombopoietin-induced megakaryocytic differentiation by the mitogen-activated protein kinase pathway.

    PubMed Central

    Rouyez, M C; Boucheron, C; Gisselbrecht, S; Dusanter-Fourt, I; Porteu, F

    1997-01-01

    Thrombopoietin (TPO) is the major regulator of both growth and differentiation of megakaryocytes. We previously showed that both functions can be generated by TPO in the megakaryoblastic cell line UT7, in which murine Mpl was introduced, and are independently controlled by distinct regions of the cytoplasmic domain of Mpl. Particularly, residues 71 to 94 of this domain (deleted in the mutant mpl delta3) were found to be required for megakaryocytic maturation but dispensable for proliferation. We show here that TPO-induced differentiation in UT7 cells is tightly dependent on a strong, long-lasting activation of the mitogen-activated protein kinase (MAPK) pathway. Indeed, (i) in UT7-mpl cells, TPO induced a strong activation of extracellular signal-regulated kinases (ERK) which was persistent until at least 4 days in TPO-containing medium; (ii) a specific MAPK kinase (MEK) inhibitor inhibited TPO-induced megakaryocytic gene expression; (iii) the Mpl mutant mpl delta3, which displayed no maturation activity, transduced only a weak and transient ERK activation in UT7 cells; and (iv) TPO-induced megakaryocytic differentiation in UT7-mpl delta3 cells was partially restored by expression of a constitutively activated mutant of MEK. The capacity of TPO to trigger a strong and prolonged MAPK signal depended on the cell in which Mpl was introduced. In BAF3-mpl cells, TPO triggered a weak and transient ERK activation, similar to that induced in UT7-mpl delta3 cells. In these cells, no difference in MAPK activation was found between normal Mpl and mpl delta3. Thus, depending on the cellular context, several distinct regions of the cytoplasmic domain of Mpl and signaling pathways may contribute to generate quantitative variations in MAPK activation. PMID:9271377

  2. Crocin suppresses tumor necrosis factor-alpha-induced cell death of neuronally differentiated PC-12 cells.

    PubMed

    Soeda, S; Ochiai, T; Paopong, L; Tanaka, H; Shoyama, Y; Shimeno, H

    2001-11-01

    Crocus sativus L. is used in Chinese traditional medicine to treat some disorders of the central nervous system. Crocin is an ethanol-extractable component of Crocus sativus L.; it is reported to prevent ethanol-induced impairment of learning and memory in mice. In this study, we demonstrate that crocin suppresses the effect of tumor necrosis factor (TNF)-alpha on neuronally differentiated PC-12 cells. PC-12 cells dead from exposure to TNF-alpha show apoptotic morphological changes and DNA fragmentation. These hallmark features of cell death did not appear in cells treated in the co-presence of 10 microM crocin. Moreover, crocin suppressed the TNF-alpha-induced expression of Bcl-Xs and LICE mRNAs and simultaneously restored the cytokine-induced reduction of Bcl-X(L) mRNA expression. The modulating effects of crocin on the expression of Bcl-2 family proteins led to a marked reduction of a TNF-alpha-induced release of cytochrome c from the mitochondria. Crocin also blocked the cytochrome c-induced activation of caspase-3. To learn how crocin exhibits these anti-apoptotic actions in PC-12 cells, we tested the effect of crocin on PC-12 cell death induced by daunorubicin. We found that crocin inhibited the effect of daunorubicin as well. Our findings suggest that crocin inhibits neuronal cell death induced by both internal and external apoptotic stimuli.

  3. Uremic toxins enhance statin-induced cytotoxicity in differentiated human rhabdomyosarcoma cells.

    PubMed

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

    2014-09-03

    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.

  4. Uremic toxins enhance statin-induced cytotoxicity in differentiated human rhabdomyosarcoma cells.

    PubMed

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

    2014-09-01

    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

  5. 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

  6. 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

  7. Corticosteroids reverse cytokine-induced block of survival and differentiation of oligodendrocyte progenitor cells from rats

    PubMed Central

    Mann, Stefan A; Versmold, Beatrix; Marx, Romy; Stahlhofen, Sabine; Dietzel, Irmgard D; Heumann, Rolf; Berger, Richard

    2008-01-01

    Background Periventricular leukomalacia (PVL) is a frequent complication of preterm delivery. Proinflammatory cytokines, such as interferon-γ (IFN-γ) and tumor necrosis factor α (TNF-α) released from astrocytes and microglia activated by infection or ischemia have previously been shown to impair survival and maturation of oligodendrocyte progenitors and could thus be considered as potential factors contributing to the generation of this disease. The first goal of the present study was to investigate whether exposure of oligodendrocyte precursors to these cytokines arrests the maturation of ion currents in parallel to its effects on myelin proteins and morphological maturation. Secondly, in the search for agents, that can protect differentiating oligodendrocyte precursor cells from cytokine-induced damage we investigated effects of coapplications of corticosteroids with proinflammatory cytokines on the subsequent survival and differentiation of oligodendrocyte progenitor cells. Methods To exclude influences from factors released from other cell types purified cultures of oligodendrocyte precursors were exposed to cytokines and/or steroids and allowed to differentiate for further 6 days in culture. Changes in membrane surface were investigated with capacitance recordings and Scanning Ion Conductance Microscopy. Na+- and K+- currents were investigated using whole cell patch clamp recordings. The expression of myelin specific proteins was investigated using western blots and the precursor cells were identified using immunostaining with A2B5 antibodies. Results Surviving IFN-γ and TNF-α treated cells continued to maintain voltage-activated Na+- and K+ currents characteristic for the immature cells after 6 days in differentiation medium. Corticosterone, dihydrocorticosterone and, most prominently dexamethasone, counteracted the deleterious effects of IFN-γ and TNF-α on cell survival, A2B5-immunostaining and expression of myelin basic protein. The most potent

  8. 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

  9. 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.

  10. Dendritic cells enhance UHMWPE wear particle-induced osteoclast differentiation of macrophages.

    PubMed

    Cang, Dingwei; Guo, Kaijin; Zhao, Fengchao

    2015-10-01

    Ultra-high molecular weight polyethylene (UHMWPE) has been widely used in large joint replacement. Osteolysis induced by the UHMWPE wear particles is one of the main causes of replacement failure. This study aims to elucidate whether dendritic cells play a role in UHMWPE particle-induced osteolysis. An in vitro Raw 264.7 and DC 2.4 coculture system was employed to examine the effects of dendritic cells on the inflammatory and osteoclastogenic responses of Raw 264.7 toward UHMWPE particles. The expression of cytokines, NF-κB, and osteoclast marker genes was analyzed by ELISA, western blot, or quantitative PCR. The osteoclast differentiation was measured by TRAP staining and flow cytometry. UHMWPE particles induced Raw 264.7 cells to differentiate into osteoclasts, which was enhanced by coculturing with DC 2.4 cells. DC 2.4 cells augmented UHMWPE particle-elicited activation of NF-κB signaling, higher levels of TNF-α and MCP-1, and an increased expression of MMP-9, Calcr, and Ctsk, though DC 2.4 coculture alone did not significantly cause the aforementioned changes. These results suggest that dendritic cells, among other immune cells recruited by UHMWPE particle induced inflammation, could further exacerbate inflammation and osteolysis.

  11. Bradykinin-induced inhibition of proliferation rate during neurosphere differentiation: consequence or cause of neuronal enrichment?

    PubMed

    Pillat, Micheli M; Cheffer, Arquimedes; de Andrade, Cinthia M; Morsch, Vera M; Schetinger, Maria R C; Ulrich, Henning

    2015-10-01

    Neural stem cells proliferate and differentiate into neurons and glial cells, being responsible for embryonic and postnatal development of the central nervous system (CNS) as well as for regeneration in the adult brain. These cells also play a key role in maintaining the physiological integrity of the CNS in face of injury or disease. The previous study has demonstrated that bradykinin (BK) treatment simultaneously induces neuronal enrichment (indicating that BK contributes to neurogenesis) and reduced proliferation rates during in vitro differentiation of rat embryonic telencephalon neural precursor cells (NPCs). Here, we provide a mechanism for the unresolved question whether (i) the low rate of proliferation is owed to enhanced neurogenesis or, conversely, (ii) the alteration of the population ratio could result from low proliferation of NPCs and glial cells. In agreement with the previous study, BK promoted neuron-specific β3-tubulin and MAP2 expression in differentiating embryonic mouse neurospheres, whereas glial protein expression and global proliferation rates decreased. Furthermore, BK augmented the global frequency of cells in G0 -phase of cell cycle after differentiation. Heterogeneous cell populations were observed at this stage, including neurons that always remaining a quiescent state (G0 -phase). It is noteworthy that BK did not interfere with proliferation of any particular cell type, evidenced by coimmunostaining for nestin, β3-tubulin, glial fibrillary acidic protein (GFAP), and 5-ethynyl-2'-deoxyuridine (EdU). Thus, we conclude that neuronal enrichment is owing only to the fostering of neurogenesis, and that the low proliferation rate on the seventh day of differentiation is a consequence and not the cause of BK-induced neuronal enrichment.

  12. Cytosine arabinoside induces ectoderm and inhibits mesoderm expression in human embryonic stem cells during multilineage differentiation

    PubMed Central

    Jagtap, S; Meganathan, K; Gaspar, J; Wagh, V; Winkler, J; Hescheler, J; Sachinidis, A

    2011-01-01

    BACKGROUND AND PURPOSE Teratogenic substances induce adverse effects during the development of the embryo. Multilineage differentiation of human embryonic stem cells (hESCs) mimics the development of the embryo in vitro. Here, we propose a transcriptomic approach in hESCs for monitoring specific toxic effects of compounds as an alternative to traditional time-consuming and cost-intensive in vivo tests requiring large numbers of animals. This study was undertaken to explore the adverse effects of cytosine arabinoside (Ara-C) on randomly differentiated hESCs. EXPERIMENTAL APPROACH Human embryonic stem cells were used to investigate the effects of a developmental toxicant Ara-C. Sublethal concentrations of Ara-C were given for two time points, day 7 and day 14 during the differentiation. Gene expression was assessed with microarrays to determine the dysregulated transcripts in presence of Ara-C. KEY RESULTS Randomly differentiated hESCs were able to generate the multilineage markers. The low concentration of Ara-C (1 nM) induced the ectoderm and inhibited the mesoderm at day 14. The induction of ectodermal markers such as MAP2, TUBB III, PAX6, TH and NESTIN was observed with an inhibition of mesodermal markers such as HAND2, PITX2, GATA5, MYL4, TNNT2, COL1A1 and COL1A2. In addition, no induction of apoptosis was observed. Gene ontology revealed unique dysregulated biological process related to neuronal differentiation and mesoderm development. Pathway analysis showed the axon guidance pathway to be dysregulated. CONCLUSIONS AND IMPLICATIONS Our results suggest that hESCs in combination with toxicogenomics offer a sensitive in vitro developmental toxicity model as an alternative to traditional animal experiments. PMID:21198554

  13. Coupled Global and Targeted Proteomics of Human Embryonic Stem Cells during Induced Differentiation*S⃞

    PubMed Central

    Yocum, Anastasia K.; Gratsch, Theresa E.; Leff, Nancy; Strahler, John R.; Hunter, Christie L.; Walker, Angela K.; Michailidis, George; Omenn, Gilbert S.; O'Shea, K. Sue; Andrews, Philip C.

    2008-01-01

    Elucidating the complex combinations of growth factors and signaling molecules that maintain pluripotency or, alternatively, promote the controlled differentiation of human embryonic stem cells (hESCs) has important implications for the fundamental understanding of human development, devising cell replacement therapies, and cancer cell biology. hESCs are commonly grown on irradiated mouse embryonic fibroblasts (MEFs) or in conditioned medium from MEFs. These culture conditions interfere with many experimental conclusions and limit the ability to perform conclusive proteomics studies. The current investigation avoided the use of MEFs or MEF-conditioned medium for hESC culture, allowing global proteomics analysis without these confounding conditions, and elucidated neural cell-specific signaling pathways involved in noggin-induced hESC differentiation. Based on these analyses, we propose the following early markers of hESC neural differentiation: collapsin response mediator proteins 2 and 4 and the nuclear autoantigenic sperm protein as a marker of pluripotent hESCs. We then developed a directed mass spectrometry assay using multiple reaction monitoring (MRM) to identify and quantify these markers and in addition the epidermal ectoderm marker cytokeratin-8. Analysis of global proteomics, quantitative RT-PCR, and MRM data led to testing the isoform interference hypothesis where redundant peptides dilute quantification measurements of homologous proteins. These results show that targeted MRM analysis on non-redundant peptides provides more exact quantification of homologous proteins. This study describes the facile transition from discovery proteomics to targeted MRM analysis and allowed us to identify and verify several potential biomarkers for hESCs during noggin-induced neural and BMP4-induced epidermal ectoderm differentiation. PMID:18304949

  14. 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

  15. 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

  16. 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.

  17. Cobalt protoporphyrin induces differentiation of monocytic THP-1 cells through regulation of cytoplasmic Ref-1-related NADPH oxidase activity.

    PubMed

    Song, Ju Dong; Lee, Sang Kwon; Park, Si Eun; Kim, Kang Mi; Kim, Koanhoi; Park, Yeong Min; Park, Young Chul

    2011-11-01

    Cobalt protoporphyrin (CoPP) is a potent and effective metalloporphyrin inducer of heme oxygenase-1 (HO-1) activity in many tissues. Here, we report that CoPP induces differentiation of monocytic THP-1 cells into macrophage-like cells. CoPP induced a marked growth inhibition with a slight reduction in viability, and increased adhesion and spreading of THP-1 cells. However, other protoporphyrins did not. CoPP also resulted in expression of CD11b, MMP9, MSR1, CD14 and ICAM-1, which are differentiation markers for macrophages. Interestingly, we observed a decrease of cytoplasmic redox factor-1 (Ref-1) levels in the process of CoPP-induced differentiation of THP-1 cells. In addition, knockdown of Ref-1 by siRNA enhanced cell adhesion induced by CoPP. Furthermore, an inhibitor of NADPH oxidase, diphenyleneiodonium (DPI), completely abolished CoPP-induced adhesion of Ref-1-deficient cells using an siRNA. A cytosolic factor for NADPH oxidase activity, p47phox, was significantly increased in THP-1 cells by CoPP treatment. Κnockdown of Ref-1 increased CoPP-induced p47phox expression in THP-1 cells. Taken together, these results suggest that CoPP induces differentiation of monocytic THP-1 cells, and that the CoPP-induced differentiation is associated with cytoplasmic Ref-1-related NADPH oxidase activity.

  18. 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

  19. 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.

  20. Mechanism of HIV protein induced modulation of mesenchymal stem cell osteogenic differentiation

    PubMed Central

    Cotter, Eoin J; Ip, Herbert Shi Ming; Powderly, William G; Doran, Peter P

    2008-01-01

    Background A high incidence of decreased bone mineral density (BMD) has been associated with HIV infection. Normal skeletal homeostasis is controlled, at least in part, by the maturation and activity of mature osteoblasts. Previous studies by our group have demonstrated the ability of HIV proteins to perturb osteoblast function, and the degree of osteogenesis in differentiating mesenchymal stem cells (MSCs). This study attempts to further dissect the dynamics of this effect. Methods MSCs were cultured under both osteogenic (cultured in commercially available differentiation media) and quiescent (cultured in basal medium) conditions. Both cell populations were exposed to HIV p55-gag and HIV rev (100 ng/ml). Time points were taken at 3, 6, 9, and 15 days for osteogenic conditions, while quiescent cells were treated for 1 week. Cell function (alkaline phosphatase [ALP] activity, calcium deposition, and lipid levels) and the activity of the key MSC transcription factors, RUNX-2 and PPARgamma were determined post-exposure. Also, in cells cultured in differentiating conditions, cellular levels of connective tissue growth factor (CTGF) were analysed using whole cell ELISA, while BMP-2 secretion was also examined. Results In differentiating MSCs, exposure to HIV proteins caused significant changes in both the timing and magnitude of key osteogenic events and signals. Treatment with REV increased the overall rate of mineralization, and induced earlier increases in CTGF levels, RUNX-2 activity and BMP-2 secretion, than those observed in the normal course of differntiation. In contrast, p55-gag reduced the overall level of osteogenesis, and reduced BMP-2 secretion, RUNX-2 activity, CTGF levels and ALP activity at many of the timepoints examined. Finally, in cells cultured in basal conditions, treatment with HIV proteins did not in and of itself induce a significant degree of differentiation over the time period examined. Conclusion These data demonstrate that the effect of

  1. 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

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

    PubMed Central

    TAO, LE; ZENG, YIGANG; WANG, JUN; LIU, ZHIHONG; SHEN, BING; GE, JIFU; LIU, YONG; GUO, YIFENG; QIU, JIANXIN

    2015-01-01

    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

  3. Hypoxic stress induces, but cannot sustain trophoblast stem cell differentiation to labyrinthine placenta due to mitochondrial insufficiency

    PubMed Central

    Xie, Yufen; Zhou, Sichang; Jiang, Zhongliang; Dai, Jing; Puscheck, Elizabeth E; Lee, Icksoo; Parker, Graham; Hüttemann, Maik; Rappolee, Daniel A

    2014-01-01

    Dysfunctional stem cell differentiation into placental lineages is associated with gestational diseases. Of the differentiated lineages available to trophoblast stem cells (TSC), elevated O2 and mitochondrial function are necessary to placental lineages at the maternal-placental surface and important in the etiology of preeclampsia. TSC lineage imbalance leads to embryonic failure during uterine implantation. Stress at implantation exacerbates stem cell depletion by decreasing proliferation and increasing differentiation. Implantation site O2 is normally ~2%. In culture, exposure to 2% O2 and fibroblast growth factor (FGF)4 enabled highest mouse TSC multipotency and proliferation. In contrast, hypoxic stress (0.5% O2) initiated the most TSC differentiation after 24 hr despite FGF4. However, hypoxic stress supported differentiation poorly after 4–7 days, despite FGF4 removal. At all tested O2 levels, FGF4 maintained Warburg metabolism; mitochondrial inactivity and aerobic glycolysis. However, hypoxic stress suppressed mitochondrial membrane potential, maintained low mitochondrial cytochrome c oxidase (oxidative phosphorylation/OxPhos), and high pyruvate kinase M2 (glycolysis) despite FGF4 removal. Inhibiting OxPhos inhibited differentiation at the differentiation optimum at 20% O2. Moreover, adding differentiation-inducing hyperosmolar stress failed to induce differentiation during hypoxia. Thus, differentiation depended on OxPhos at 20% O2; hypoxic and hyperosmolar stresses did not induce differentiation at 0.5% O2. Hypoxia-limited differentiation and mitochondrial inhibition and activation suggest that differentiation into two lineages of the labyrinthine placenta requires O2>0.5–2% and mitochondrial function. Stress-activated protein kinase increases an early lineage and suppresses later lineages in proportion to the deviation from optimal O2 for multipotency, thus it is the first enzyme reported to prioritize differentiation. PMID:25239494

  4. Interleukin-24 induces neuroblastoma SH-SY5Y cell differentiation, growth inhibition, and apoptosis by promoting ROS production.

    PubMed

    Li, Yuan; Zhang, Hongwei; Zhu, Xiaoyu; Feng, Dongchuan; Gong, Jinchao; Han, Tao

    2013-11-01

    Neuroblastoma is among the most aggressive tumors that occur in childhood and infancy. The clinical prognosis of children with advanced-stage neuroblastoma is still poor. Interleukin-24 (IL-24) is emerging as a new cytokine involved in tumor cellular proliferation, differentiation, and apoptosis and has been widely studied as a tumor inhibitor. However, little is known about this cytokine's role in neuroblastoma. In this study, we investigated the possible effects of IL-24 on inducing neuroblastoma cell differentiation, growth inhibition, and apoptosis in vitro. Our data show that IL-24 promotes neuroblastoma SH-SY5Y cell differentiation, growth inhibition, and apoptosis. Furthermore, we found that the differentiation- and apoptosis-inducing action of IL-24 depends on the accumulation of reactive oxygen species (ROS). These results suggest that IL-24 can induce neuroblastoma cell differentiation and apoptosis and may be a potential therapeutic agent for neuroblastoma.

  5. M. tuberculosis Secretory Protein ESAT-6 Induces Metabolic Flux Perturbations to Drive Foamy Macrophage Differentiation.

    PubMed

    Singh, Varshneya; Kaur, Charanpreet; Chaudhary, Vijay K; Rao, Kanury V S; Chatterjee, Samrat

    2015-01-01

    The Foamy Macrophage (FM) differentiation forms a major component of the host dependent survival axis of M. tuberculosis. The FM which are characterized by the intracellular accumulation of lipid bodies (LBs), ensure a privileged existence for the bacilli through ready provision of nutrients and by conferring protection against bactericidal pathways. The mycobacterial secretory protein ESAT-6 has been identified as the molecular mediator of the FM differentiation process although little is known about the mechanism through which it induces this process. In the present study, we show that ESAT-6 induces GLUT-1 mediated enhanced glucose uptake by macrophages which is coupled to metabolic flux perturbations in the glycolytic pathway caused by differential rates of reaction at several steps in the pathway. Two major changes identified were the simultaneous buildup of DHAP (for Triglyceride synthesis) and AcCoA (for synthesis of 3-HB, ligand for the anti-lipolytic GPR109A). We also show that part of the observed effects involve protein- protein interactions between ESAT-6 and the macrophage glycolytic enzymes, Enolase1 and Phosphoglycerate kinase1. PMID:26250836

  6. 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

  7. miR-142-3p prevents macrophage differentiation during cancer-induced myelopoiesis.

    PubMed

    Sonda, Nada; Simonato, Francesca; Peranzoni, Elisa; Calì, Bianca; Bortoluzzi, Stefania; Bisognin, Andrea; Wang, Ena; Marincola, Francesco M; Naldini, Luigi; Gentner, Bernhard; Trautwein, Christian; Sackett, Sara Dutton; Zanovello, Paola; Molon, Barbara; Bronte, Vincenzo

    2013-06-27

    Tumor progression is accompanied by an altered myelopoiesis causing the accumulation of immunosuppressive cells. Here, we showed that miR-142-3p downregulation promoted macrophage differentiation and determined the acquisition of their immunosuppressive function in tumor. Tumor-released cytokines signaling through gp130, the common subunit of the interleukin-6 cytokine receptor family, induced the LAP∗ isoform of C/EBPβ transcription factor, promoting macrophage generation. miR-142-3p downregulated gp130 by canonical binding to its messenger RNA (mRNA) 3' UTR and repressed C/EBPβ LAP∗ by noncanonical binding to its 5' mRNA coding sequence. Enforced miR expression impaired macrophage differentiation both in vitro and in vivo. Mice constitutively expressing miR-142-3p in the bone marrow showed a marked increase in survival following immunotherapy with tumor-specific T lymphocytes. By modulating a specific miR in bone marrow precursors, we thus demonstrated the feasibility of altering tumor-induced macrophage differentiation as a potent tool to improve the efficacy of cancer immunotherapy.

  8. Discovery of novel inducers of cellular differentiation using HL-60 promyelocytic cells.

    PubMed

    Mata-Greenwood, E; Ito, A; Westenburg, H; Cui, B; Mehta, R G; Kinghorn, A D; Pezzuto, J M

    2001-01-01

    Non-physiological inducers of terminal differentiation have been used as novel therapies for the prevention and therapy of cancer. We have used cultured HL-60 promyelocytic cells to monitor differentiation, proliferation and cell death events as induced by a large set of extracts derived from plants. Screening of more than 1400 extracts led to the discovery of 34 with potent activity (ED50 <8 mg/ml). Bioassay-guided fractionation led to the isolation of zapotin and 2',5,6-trimethoxyflavone as active principles from Casimiroa edulis, dibenzyltrisulfide and 2-[(phenylmethyl)dithio]ethanol as active principles from Petiveria alliacea, and desmethylrocaglamide from Aglaia ponapensis. Zapotin demonstrated the most favorable biological profile in that induction of differentiation correlated with proliferation arrest, and a lack of cytotoxicity. We conclude that the HL-60 cell model is a useful system for the discovery of novel pharmacophores with potential to suppress the process of carcinogenesis, and that flavonoids may be especially useful in this capacity.

  9. Effect of amorphous silica nanoparticles on in vitro RANKL-induced osteoclast differentiation in murine macrophages

    NASA Astrophysics Data System (ADS)

    Nabeshi, Hiromi; Yoshikawa, Tomoaki; Akase, Takanori; Yoshida, Tokuyuki; Tochigi, Saeko; Hirai, Toshiro; Uji, Miyuki; Ichihashi, Ko-Ichi; Yamashita, Takuya; Higashisaka, Kazuma; Morishita, Yuki; Nagano, Kazuya; Abe, Yasuhiro; Kamada, Haruhiko; Tsunoda, Shin-Ichi; Itoh, Norio; Yoshioka, Yasuo; Tsutsumi, Yasuo

    2011-07-01

    Amorphous silica nanoparticles (nSP) have been used as a polishing agent and/or as a remineralization promoter for teeth in the oral care field. The present study investigates the effects of nSP on osteoclast differentiation and the relationship between particle size and these effects. Our results revealed that nSP exerted higher cytotoxicity in macrophage cells compared with submicron-sized silica particles. However, tartrate-resistant acid phosphatase (TRAP) activity and the number of osteoclast cells (TRAP-positive multinucleated cells) were not changed by nSP treatment in the presence of receptor activator of nuclear factor κB ligand (RANKL) at doses that did not induce cytotoxicity by silica particles. These results indicated that nSP did not cause differentiation of osteoclasts. Collectively, the results suggested that nanosilica exerts no effect on RANKL-induced osteoclast differentiation of RAW264.7 cells, although a detailed mechanistic examination of the nSP70-mediated cytotoxic effect is needed.

  10. 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.

  11. 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

  12. Structurally distinct polycyclic aromatic hydrocarbons induce differential transcriptional responses in developing zebrafish

    SciTech Connect

    Goodale, Britton C.; Tilton, Susan C.; Corvi, Margaret M.; Wilson, Glenn R.; Janszen, Derek B.; Anderson, Kim A.; Waters, Katrina M.; Tanguay, Robert L.

    2013-11-01

    Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in the environment as components of fossil fuels and by-products of combustion. These multi-ring chemicals differentially activate the aryl hydrocarbon receptor (AHR) in a structurally dependent manner, and induce toxicity via both AHR-dependent and -independent mechanisms. PAH exposure is known to induce developmental malformations in zebrafish embryos, and recent studies have shown cardiac toxicity induced by compounds with low AHR affinity. Unraveling the potentially diverse molecular mechanisms of PAH toxicity is essential for understanding the hazard posed by complex PAH mixtures present in the environment. We analyzed transcriptional responses to PAH exposure in zebrafish embryos exposed to benz(a)anthracene (BAA), dibenzothiophene (DBT) and pyrene (PYR) at concentrations that induced developmental malformations by 120 h post-fertilization (hpf). Whole genome microarray analysis of mRNA expression at 24 and 48 hpf identified genes that were differentially regulated over time and in response to the three PAH structures. PAH body burdens were analyzed at both time points using GC–MS, and demonstrated differences in PAH uptake into the embryos. This was important for discerning dose-related differences from those that represented unique molecular mechanisms. While BAA misregulated the least number of transcripts, it caused strong induction of cyp1a and other genes known to be downstream of the AHR, which were not induced by the other two PAHs. Analysis of functional roles of misregulated genes and their predicted regulatory transcription factors also distinguished the BAA response from regulatory networks disrupted by DBT and PYR exposure. These results indicate that systems approaches can be used to classify the toxicity of PAHs based on the networks perturbed following exposure, and may provide a path for unraveling the toxicity of complex PAH mixtures. - Highlights: • Defined global mRNA expression

  13. Epigenetic Modulation of Human Induced Pluripotent Stem Cell Differentiation to Oligodendrocytes

    PubMed Central

    Douvaras, Panagiotis; Rusielewicz, Tomasz; Kim, Kwi Hye; Haines, Jeffery D.; Casaccia, Patrizia; Fossati, Valentina

    2016-01-01

    Pluripotent stem cells provide an invaluable tool for generating human, disease-relevant cells. Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system, characterized by myelin damage. Oligodendrocytes are the myelinating cells of the central nervous system (CNS); they differentiate from progenitor cells, and their membranes ensheath axons, providing trophic support and allowing fast conduction velocity. The current understanding of oligodendrocyte biology was founded by rodent studies, where the establishment of repressive epigenetic marks on histone proteins, followed by activation of myelin genes, leads to lineage progression. To assess whether this epigenetic regulation is conserved across species, we differentiated human embryonic and induced pluripotent stem cells to oligodendrocytes and asked whether similar histone marks and relative enzymatic activities could be detected. The transcriptional levels of enzymes responsible for methylation and acetylation of histone marks were analyzed during oligodendrocyte differentiation, and the post-translational modifications on histones were detected using immunofluorescence. These studies showed that also in human cells, differentiation along the oligodendrocyte lineage is characterized by the acquisition of multiple repressive histone marks, including deacetylation of lysine residues on histone H3 and trimethylation of residues K9 and K27. These data suggest that the epigenetic modulation of oligodendrocyte identity is highly conserved across species. PMID:27110779

  14. Differentiation of Human Induced-Pluripotent Stem Cells into Smooth-Muscle Cells: Two Novel Protocols.

    PubMed

    Yang, Libang; Geng, Zhaohui; Nickel, Thomas; Johnson, Caitlin; Gao, Lin; Dutton, James; Hou, Cody; Zhang, Jianyi

    2016-01-01

    Conventional protocols for differentiating human induced-pluripotent stem cells (hiPSCs) into smooth-muscle cells (SMCs) can be inefficient and generally fail to yield cells with a specific SMC phenotype (i.e., contractile or synthetic SMCs). Here, we present two novel hiPSC-SMC differentiation protocols that yield SMCs with predominantly contractile or synthetic phenotypes. Flow cytometry analyses of smooth-muscle actin (SMA) expression indicated that ~45% of the cells obtained with each protocol assumed an SMC phenotype, and that the populations could be purified to ~95% via metabolic selection. Assessments of cellular mRNA and/or protein levels indicated that SMA, myosin heavy chain II, collagen 1, calponin, transgelin, connexin 43, and vimentin expression in the SMCs obtained via the Contractile SMC protocol and in SMCs differentiated via a traditional protocol were similar, while SMCs produced via the Sythetic SMC protocol expressed less calponin, more collagen 1, and more connexin 43. Differences were also observed in functional assessments of the two SMC populations: the two-dimensional surface area of Contractile SMCs declined more extensively (to 12% versus 44% of original size) in response to carbachol treatment, while quantification of cell migration and proliferation were greater in Synthetic SMCs. Collectively, these data demonstrate that our novel differentiation protocols can efficiently generate SMCs from hiPSCs. PMID:26771193

  15. Laminin 411 and 511 promote the cholangiocyte differentiation of human induced pluripotent stem cells.

    PubMed

    Takayama, Kazuo; Mitani, Seiji; Nagamoto, Yasuhito; Sakurai, Fuminori; Tachibana, Masashi; Taniguchi, Yukimasa; Sekiguchi, Kiyotoshi; Mizuguchi, Hiroyuki

    2016-05-20

    The drug discovery research for cholestatic liver diseases has been hampered by the lack of a well-established human cholangiocyte model. Functional cholangiocyte-like cells differentiated from human induced pluripotent stem (iPS) cells are expected to be a promising candidate for such research, but there remains no well-established method for differentiating cholangiocytes from human iPS cells. In this study, we searched for a suitable extracellular matrix to promote cholangiocyte differentiation from human iPS cells, and found that both laminin 411 and laminin 511 were suitable for this purpose. The gene expression levels of the cholangiocyte markers, aquaporin 1 (AQP1), SRY-box 9 (SOX9), cystic fibrosis transmembrane conductance regulator (CFTR), G protein-coupled bile acid receptor 1 (GPBAR1), Jagged 1 (JAG1), secretin receptor (SCTR), and γ-glutamyl transferase (GGT1) were increased by using laminin 411 or laminin 511 as a matrix. In addition, the percentage of AQP1-positive cells was increased from 61.8% to 92.5% by using laminin 411 or laminin 511. Furthermore, the diameter and number of cysts consisted of cholangiocyte-like cells were increased when using either matrix. We believe that the human iPS cell-derived cholangiocyte-like cells, which were generated by using our differentiation technology, would be useful for the drug discovery research of cholestatic liver diseases. PMID:27103433

  16. Lack of immune response to differentiated cells derived from syngeneic induced pluripotent stem cells.

    PubMed

    Guha, Prajna; Morgan, John W; Mostoslavsky, Gustavo; Rodrigues, Neil P; Boyd, Ashleigh S

    2013-04-01

    The prospects for using autologous induced pluripotent stem cells (iPSCs) in cell replacement therapy have been tempered by evidence that undifferentiated, syngeneic mouse iPSCs are immunogenic upon transplantation. However, the immunogenicity of more therapeutically relevant differentiated cells remains unexplored. Here, we differentiated mouse iPSCs into embryoid bodies (EBs) or representative cell types spanning the three embryonic germ layers and assessed their immunogenicity in vitro and after their transplantation into syngeneic recipients. We found no evidence of increased T cell proliferation in vitro, rejection of syngeneic iPSC-derived EBs/tissue-specific cells (TSCs) after transplantation, or an antigen-specific secondary immune response. Thus, differentiated cells derived from syngeneic iPSCs do not appear to be rejected after transplantation. We also found little evidence of an immune response to undifferentiated, syngeneic iPSCs. Our data support the idea that differentiated cells generated from autologous iPSCs could be applied for cell replacement therapy without eliciting immune rejection.

  17. Small Molecule-Induced Complement Factor D (Adipsin) Promotes Lipid Accumulation and Adipocyte Differentiation.

    PubMed

    Song, No-Joon; Kim, Suji; Jang, Byung-Hyun; Chang, Seo-Hyuk; Yun, Ui Jeong; Park, Ki-Moon; Waki, Hironori; Li, Dean Y; Tontonoz, Peter; Park, Kye Won

    2016-01-01

    Adipocytes are differentiated by various transcriptional cascades integrated on the master regulator, Pparγ. To discover new genes involved in adipocyte differentiation, preadipocytes were treated with three newly identified pro-adipogenic small molecules and GW7845 (a Pparγ agonist) for 24 hours and transcriptional profiling was analyzed. Four genes, Peroxisome proliferator-activated receptor γ (Pparγ), human complement factor D homolog (Cfd), Chemokine (C-C motif) ligand 9 (Ccl9), and GIPC PDZ Domain Containing Family Member 2 (Gipc2) were induced by at least two different small molecules but not by GW7845. Cfd and Ccl9 expressions were specific to adipocytes and they were altered in obese mice. Small hairpin RNA (shRNA) mediated knockdown of Cfd in preadipocytes inhibited lipid accumulation and expression of adipocyte markers during adipocyte differentiation. Overexpression of Cfd promoted adipocyte differentiation, increased C3a production, and led to induction of C3a receptor (C3aR) target gene expression. Similarly, treatments with C3a or C3aR agonist (C4494) also promoted adipogenesis. C3aR knockdown suppressed adipogenesis and impaired the pro-adipogenic effects of Cfd, further suggesting the necessity for C3aR signaling in Cfd-mediated pro-adipogenic axis. Together, these data show the action of Cfd in adipogenesis and underscore the application of small molecules to identify genes in adipocytes. PMID:27611793

  18. Wnt/beta-catenin pathway activation and myogenic differentiation are induced by cholesterol depletion.

    PubMed

    Mermelstein, Cláudia S; Portilho, Débora M; Mendes, Fábio A; Costa, Manoel L; Abreu, José Garcia

    2007-03-01

    Myogenic differentiation is a multistep process that begins with the commitment of mononucleated precursors that withdraw from cell cycle. These myoblasts elongate while aligning to each other, guided by the recognition between their membranes. This step is followed by cell fusion and the formation of long and striated multinucleated myotubes. We have recently shown that cholesterol depletion by methyl-beta-cyclodextrin (MbetaCD) induces myogenic differentiation by enhancing myoblast recognition and fusion. Here, we further studied the signaling pathways responsible for early steps of myogenesis. As it is known that Wnt plays a role in muscle differentiation, we used the chemical MbetaCD to deplete membrane cholesterol and investigate the involvement of the Wnt/beta-catenin pathway during myogenesis. We show that cholesterol depletion promoted a significant increase in expression of beta-catenin, its nuclear translocation and activation of the Wnt pathway. Moreover, we show that the activation of the Wnt pathway after cholesterol depletion can be inhibited by the soluble protein Frzb-1. Our data suggest that membrane cholesterol is involved in Wnt/beta-catenin signaling in the early steps of myogenic differentiation.

  19. Small Molecule-Induced Complement Factor D (Adipsin) Promotes Lipid Accumulation and Adipocyte Differentiation

    PubMed Central

    Jang, Byung-Hyun; Chang, Seo-Hyuk; Yun, Ui Jeong; Park, Ki-Moon; Waki, Hironori; Li, Dean Y.; Tontonoz, Peter; Park, Kye Won

    2016-01-01

    Adipocytes are differentiated by various transcriptional cascades integrated on the master regulator, Pparγ. To discover new genes involved in adipocyte differentiation, preadipocytes were treated with three newly identified pro-adipogenic small molecules and GW7845 (a Pparγ agonist) for 24 hours and transcriptional profiling was analyzed. Four genes, Peroxisome proliferator-activated receptor γ (Pparγ), human complement factor D homolog (Cfd), Chemokine (C-C motif) ligand 9 (Ccl9), and GIPC PDZ Domain Containing Family Member 2 (Gipc2) were induced by at least two different small molecules but not by GW7845. Cfd and Ccl9 expressions were specific to adipocytes and they were altered in obese mice. Small hairpin RNA (shRNA) mediated knockdown of Cfd in preadipocytes inhibited lipid accumulation and expression of adipocyte markers during adipocyte differentiation. Overexpression of Cfd promoted adipocyte differentiation, increased C3a production, and led to induction of C3a receptor (C3aR) target gene expression. Similarly, treatments with C3a or C3aR agonist (C4494) also promoted adipogenesis. C3aR knockdown suppressed adipogenesis and impaired the pro-adipogenic effects of Cfd, further suggesting the necessity for C3aR signaling in Cfd-mediated pro-adipogenic axis. Together, these data show the action of Cfd in adipogenesis and underscore the application of small molecules to identify genes in adipocytes. PMID:27611793

  20. PRELIMINARY OBSERVATIONS OF ATRAZINE-INDUCED EFFECTS UPON GONADAL DIFFERENTIATION IN RIVULUS MARMORATUS, A NATURALLY HERMAPHRODITIC FISH

    EPA Science Inventory

    The commonly used agricultural herbicide atrazine has been recognized as an endocrine disrupting chemical. In amphibians and reptiles, atrazine has been reported to alter sexual differentiation and induce secondary sexual characteristics that have been attributed to enhanced arom...

  1. Wnt/{beta}-catenin signaling changes C2C12 myoblast proliferation and differentiation by inducing Id3 expression

    SciTech Connect

    Zhang, Long; Shi, Songting; Zhang, Juan; Zhou, Fangfang; Dijke, Peter ten

    2012-03-02

    Highlights: Black-Right-Pointing-Pointer Expression of Id3 but not Id1 is induced by Wnt3a stimulation in C2C12 cells. Black-Right-Pointing-Pointer Wnt3a induces Id3 expression via canonical Wnt/{beta}-catenin pathway. Black-Right-Pointing-Pointer Wnt3a-induced Id3 expression does not depend on BMP signaling activation. Black-Right-Pointing-Pointer Induction of Id3 expression is critical determinant in Wnt3a-induced cell proliferation and differentiation. -- Abstract: Canonical Wnt signaling plays important roles in regulating cell proliferation and differentiation. In this study, we report that inhibitor of differentiation (Id)3 is a Wnt-inducible gene in mouse C2C12 myoblasts. Wnt3a induced Id3 expression in a {beta}-catenin-dependent manner. Bone morphogenetic protein (BMP) also potently induced Id3 expression. However, Wnt-induced Id3 expression occurred independent of the BMP/Smad pathway. Functional studies showed that Id3 depletion in C2C12 cells impaired Wnt3a-induced cell proliferation and alkaline phosphatase activity, an early marker of osteoblast cells. Id3 depletion elevated myogenin induction during myogenic differentiation and partially impaired Wnt3a suppressed myogenin expression in C2C12 cells. These results suggest that Id3 is an important Wnt/{beta}-catenin induced gene in myoblast cell fate determination.

  2. Differentiation of cultured epithelial cells: Response to toxic agents

    SciTech Connect

    Rice, R.H.; LaMontagne, A.D.; Petito, C.T.; Rong, Xianhui )

    1989-03-01

    Cell culture systems are instrumental in elucidating regulation of normal function and mechanisms of its perturbation by toxic substances. To this end, three applications of epithelial cells cultured with 3T3 feeder layer support are described. First, treatment of the premalignant human epidermal keratinocyte line SCC-12F2 with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate suppressed cell growth and differentiation. This agent produced a biphasic growth response greatly inhibiting cell growth at 1 to 10 nM, but much less above 100 nM. Expression of the differentiated functions involucrin and transglutaminase was found to be inhibited markedly at concentrations above 10 nM. Second, 3-methylcholanthrene toxicity was surveyed in a variety of rat epithelial cell types. The two most sensitive to growth inhibition were epidermal and mammary epithelial cells, while those from bladder, prostate, thyroid, and endometrium were insensitive to growth inhibition. Finally, expression of estrogen receptors in rat endometrial cells was shown to be stimulated by the cAmP-elevating agent forskolin. Maximal stimulation of 3- to 6-fold occurred in 6 hr, compatible with a requirement for protein synthesis. Pursuit of such results will aid in understanding differences in response among cell types and species, in elucidating mechanisms of action of known toxic substances and, ultimately, in predicting toxicity of less well understood agents.

  3. Algal Toxin Azaspiracid-1 Induces Early Neuronal Differentiation and Alters Peripherin Isoform Stoichiometry

    PubMed Central

    Hjørnevik, Linda V.; Frøyset, Ann K.; Grønset, Toril A.; Rungruangsak-Torrissen, Krisna; Fladmark, Kari E.

    2015-01-01

    Azaspiracid-1 is an algal toxin that accumulates in edible mussels, and ingestion may result in human illness as manifested by vomiting and diarrhoea. When injected into mice, it causes neurotoxicological symptoms and death. Although it is well known that azaspiracid-1 is toxic to most cells and cell lines, little is known about its biological target(s). A rat PC12 cell line, commonly used as a model for the peripheral nervous system, was used to study the neurotoxicological effects of azaspiracid-1. Azaspiracid-1 induced differentiation-related morphological changes followed by a latter cell death. The differentiated phenotype showed peripherin-labelled neurite-like processes simultaneously as a specific isoform of peripherin was down-regulated. The precise mechanism behind this down-regulation remains uncertain. However, this study provides new insights into the neurological effects of azaspiracid-1 and into the biological significance of specific isoforms of peripherin. PMID:26694421

  4. The prelamin A pre-peptide induces cardiac and skeletal myoblast differentiation

    SciTech Connect

    Brodsky, Gary L. . E-mail: Gary.Brodsky@uchsc.edu; Bowersox, Jeffrey A.; Fitzgerald-Miller, Lisa; Miller, Leslie A.; Maclean, Kenneth N.

    2007-05-18

    Prelamin A processing is unique amongst mammalian proteins and results in the production of a farnesylated and carboxymethylated peptide. We examined the effect of pathogenic LMNA mutations on prelamin A processing, and of the covalently modified peptide on cardiac and skeletal myoblast differentiation. Here we report a mutation associated with dilated cardiomyopathy prevents prelamin A peptide production. In addition, topical application of the covalently modified C-terminal peptide to proliferating skeletal and cardiac myoblasts induced myotube and striated tissue formation, respectively. Western blot analysis revealed that skeletal and cardiac myoblasts are the first cell lines examined to contain unprocessed prelamin A, and immunostaining of peptide-treated cells revealed a previously unidentified role for prelamin A in cytoskeleton formation and intercellular organization. These results demonstrate a direct role for prelamin A in myoblast differentiation and indicate the prelamin A peptide may have therapeutic potential.

  5. Milk-derived ribonuclease 5 preparations induce myogenic differentiation in vitro and muscle growth in vivo.

    PubMed

    Knight, Matthew I; Tester, Angus M; McDonagh, Matthew B; Brown, Andrew; Cottrell, Jeremy; Wang, Jianghui; Hobman, Peter; Cocks, Benjamin G

    2014-12-01

    Ribonuclease 5, also known as angiogenin, is a stable and abundant ribonuclease in milk whey protein, which is able to regulate several cellular functions, including capillary formation, neuron survival, and epithelial cell growth. Ribonuclease 5 is important for protein synthesis directly stimulating rRNA synthesis in the nucleolus. Here, we show that biologically active RNase5 can be purified from bovine milk. Furthermore, we show that milk-derived RNase5 directly stimulates muscle cell differentiation in vitro, inducing C2C12 cell differentiation and myogenesis. When supplemented into the diet of healthy adult mice, milk-derived RNase5 preparations promoted muscle weight gain and grip strength. Collectively, these data indicate that milk-derived RNase5 preparations exhibit a novel role in skeletal muscle cell function. PMID:25282415

  6. Valproic acid defines a novel class of HDAC inhibitors inducing differentiation of transformed cells.

    PubMed

    Göttlicher, M; Minucci, S; Zhu, P; Krämer, O H; Schimpf, A; Giavara, S; Sleeman, J P; Lo Coco, F; Nervi, C; Pelicci, P G; Heinzel, T

    2001-12-17

    Histone deacetylases (HDACs) play important roles in transcriptional regulation and pathogenesis of cancer. Thus, HDAC inhibitors are candidate drugs for differentiation therapy of cancer. Here, we show that the well-tolerated antiepileptic drug valproic acid is a powerful HDAC inhibitor. Valproic acid relieves HDAC-dependent transcriptional repression and causes hyperacetylation of histones in cultured cells and in vivo. Valproic acid inhibits HDAC activity in vitro, most probably by binding to the catalytic center of HDACs. Most importantly, valproic acid induces differentiation of carcinoma cells, transformed hematopoietic progenitor cells and leukemic blasts from acute myeloid leukemia patients. More over, tumor growth and metastasis formation are significantly reduced in animal experiments. Therefore, valproic acid might serve as an effective drug for cancer therapy. PMID:11742974

  7. Valproic acid defines a novel class of HDAC inhibitors inducing differentiation of transformed cells

    PubMed Central

    Göttlicher, Martin; Minucci, Saverio; Zhu, Ping; Krämer, Oliver H.; Schimpf, Annemarie; Giavara, Sabrina; Sleeman, Jonathan P.; Lo Coco, Francesco; Nervi, Clara; Pelicci, Pier Giuseppe; Heinzel, Thorsten

    2001-01-01

    Histone deacetylases (HDACs) play important roles in transcriptional regulation and pathogenesis of cancer. Thus, HDAC inhibitors are candidate drugs for differentiation therapy of cancer. Here, we show that the well-tolerated antiepileptic drug valproic acid is a powerful HDAC inhibitor. Valproic acid relieves HDAC-dependent transcriptional repression and causes hyperacetylation of histones in cultured cells and in vivo. Valproic acid inhibits HDAC activity in vitro, most probably by binding to the catalytic center of HDACs. Most importantly, valproic acid induces differentiation of carcinoma cells, transformed hematopoietic progenitor cells and leukemic blasts from acute myeloid leukemia patients. More over, tumor growth and metastasis formation are significantly reduced in animal experiments. Therefore, valproic acid might serve as an effective drug for cancer therapy. PMID:11742974

  8. Milk-derived ribonuclease 5 preparations induce myogenic differentiation in vitro and muscle growth in vivo.

    PubMed

    Knight, Matthew I; Tester, Angus M; McDonagh, Matthew B; Brown, Andrew; Cottrell, Jeremy; Wang, Jianghui; Hobman, Peter; Cocks, Benjamin G

    2014-12-01

    Ribonuclease 5, also known as angiogenin, is a stable and abundant ribonuclease in milk whey protein, which is able to regulate several cellular functions, including capillary formation, neuron survival, and epithelial cell growth. Ribonuclease 5 is important for protein synthesis directly stimulating rRNA synthesis in the nucleolus. Here, we show that biologically active RNase5 can be purified from bovine milk. Furthermore, we show that milk-derived RNase5 directly stimulates muscle cell differentiation in vitro, inducing C2C12 cell differentiation and myogenesis. When supplemented into the diet of healthy adult mice, milk-derived RNase5 preparations promoted muscle weight gain and grip strength. Collectively, these data indicate that milk-derived RNase5 preparations exhibit a novel role in skeletal muscle cell function.

  9. Preadipocyte factor 1 induces pancreatic ductal cell differentiation into insulin-producing cells.

    PubMed

    Rhee, Marie; Lee, Seung-Hwan; Kim, Ji-Won; Ham, Dong-Sik; Park, Heon-Seok; Yang, Hae Kyung; Shin, Ju-Young; Cho, Jae-Hyoung; Kim, Young-Bum; Youn, Byung-Soo; Sul, Hei Sook; Yoon, Kun-Ho

    2016-01-01

    The preadipocyte factor 1 (Pref-1) is involved in the proliferation and differentiation of various precursor cells. However, the intracellular signaling pathways that control these processes and the role of Pref-1 in the pancreas remain poorly understood. Here, we showed that Pref-1 induces insulin synthesis and secretion via two independent pathways. The overexpression of Pref-1 activated MAPK signaling, which induced nucleocytoplasmic translocation of FOXO1 and PDX1 and led to the differentiation of human pancreatic ductal cells into β-like cells and an increase in insulin synthesis. Concurrently, Pref-1 activated Akt signaling and facilitated insulin secretion. A proteomics analysis identified the Rab43 GTPase-activating protein as a downstream target of Akt. A serial activation of both proteins induced various granular protein syntheses which led to enhanced glucose-stimulated insulin secretion. In a pancreatectomised diabetic animal model, exogenous Pref-1 improved glucose homeostasis by accelerating pancreatic ductal and β-cell regeneration after injury. These data establish a novel role for Pref-1, opening the possibility of applying this molecule to the treatment of diabetes. PMID:27044861

  10. Proteomics unveil corticoid-induced S100A11 shuttling in keratinocyte differentiation

    SciTech Connect

    Dezitter, Xavier; Hammoudi, Fatma; Belverge, Nicolas; Deloulme, Jean-Christophe; Drobecq, Herve; Masselot, Bernadette; Formstecher, Pierre; Mendy, Denise; Idziorek, Thierry . E-mail: thierry.idziorek@lille.inserm.fr

    2007-08-31

    Unlike classical protein extraction techniques, proteomic mapping using a selective subcellular extraction kit revealed S100A11 as a new member of the S100 protein family modulated by glucocorticoids in keratinocytes. Glucocorticoids (GC)-induced S100A11 redistribution in the 'organelles and membranes' compartment. Microscopic examination indicated that glucocorticoids specifically routed cytoplasmic S100A11 toward perinuclear compartment. Calcium, a key component of skin terminal differentiation, directed S100A11 to the plasma membrane as previously reported. When calcium was added to glucocorticoids, minor change was observed at the proteomic level while confocal microscopy revealed a rapid and dramatic translocation of S100A11 toward plasma membrane. This effect was accompanied by strong nuclear condensation, loss of mitochondrial potential and DNA content, and increased high molecular weight S100A11 immunoreactivity, suggesting corticoids accelerate calcium-induced terminal differentiation. Finally, our results suggest GC-induced S100A11 relocalization could be a key step in both keratinocyte homeostasis and glucocorticoids side effects in human epidermis.

  11. Safrole oxide induced neuronal differentiation of rat bone-marrow mesenchymal stem cells by elevating Hsp70.

    PubMed

    Zhao, YanChun; Xin, Jie; Sun, ChunHui; Zhao, BaoXiang; Zhao, Jing; Su, Le

    2012-11-01

    In a previous study, we found that at low concentrations, safrole oxide (SFO) could induce vascular endothelial cell (VEC) transdifferentiation into neuron-like cells; however, whether SFO could induce bone-marrow mesenchymal stem cell (BMSC) neural differentiation was unknown. Here, we found that SFO could effectively induce BMSC neural differentiation in the presence of serum and fibroblast growth factor 2 and did not affect cell viability at low concentrations. The levels of neuron-specific enolase and neurofilament-L were increased greatly, but that of glial fibrillary acidic protein was absent with SFO treatment for 48h. Furthermore, SFO could increase the level of heat shock protein 70 (Hsp70), an important factor in neuronal differentiation. Knockdown of Hsp70 by its small interfering RNA blocked SFO-induced BMSC differentiation. Thus, SFO is a novel inducer of BMSC differentiation to neuron-like cells and Hsp70 is implicated in the differentiation process. We provide a new tool for obtaining neuron-like cells from BMSCs and for further investigating the new effect of Hsp70 on BMSC neuronal differentiation.

  12. Histone Deacetylase Inhibitor Valproic Acid Promotes the Differentiation of Human Induced Pluripotent Stem Cells into Hepatocyte-Like Cells

    PubMed Central

    Kondo, Yuki; Iwao, Takahiro; Yoshihashi, Sachimi; Mimori, Kayo; Ogihara, Ruri; Nagata, Kiyoshi; Kurose, Kouichi; Saito, Masayoshi; Niwa, Takuro; Suzuki, Takayoshi; Miyata, Naoki; Ohmori, Shigeru; Nakamura, Katsunori; Matsunaga, Tamihide

    2014-01-01

    In this study, we aimed to elucidate the effects and mechanism of action of valproic acid on hepatic differentiation from human induced pluripotent stem cell-derived hepatic progenitor cells. Human induced pluripotent stem cells were differentiated into endodermal cells in the presence of activin A and then into hepatic progenitor cells using dimethyl sulfoxide. Hepatic progenitor cells were matured in the presence of hepatocyte growth factor, oncostatin M, and dexamethasone with valproic acid that was added during the maturation process. After 25 days of differentiation, cells expressed hepatic marker genes and drug-metabolizing enzymes and exhibited drug-metabolizing enzyme activities. These expression levels and activities were increased by treatment with valproic acid, the timing and duration of which were important parameters to promote differentiation from human induced pluripotent stem cell-derived hepatic progenitor cells into hepatocytes. Valproic acid inhibited histone deacetylase activity during differentiation of human induced pluripotent stem cells, and other histone deacetylase inhibitors also enhanced differentiation into hepatocytes. In conclusion, histone deacetylase inhibitors such as valproic acid can be used to promote hepatic differentiation from human induced pluripotent stem cell-derived hepatic progenitor cells. PMID:25084468

  13. Leptin changes differentiation fate and induces senescence in chondrogenic progenitor cells

    PubMed Central

    Zhao, X; Dong, Y; Zhang, J; Li, D; Hu, G; Yao, J; Li, Y; Huang, P; Zhang, M; Zhang, J; Huang, Z; Zhang, Y; Miao, Y; Xu, Q; Li, H

    2016-01-01

    Body weight is a component of the mechanical theory of OA (osteoarthritis) pathogenesis. Obesity was also found to be a risk factor for digital OA involving non-weight-bearing joints, which suggested that metabolism influences the occurrence and progression of OA. The metabolic origin of OA has been partially attributed to the involvement of adipokines, such as leptin, the levels of which are significantly and positively correlated with cartilage degeneration in OA patients. However, the mechanisms by which leptin-induced cartilage degeneration occurs are poorly understood. The discovery of chondrogenic progenitor cells (CPCs) opened up new opportunities for investigation. Investigating the effects of leptin on differentiation and proliferation in CPCs would increase our understanding of the roles played by leptin in the aetiology and development of OA. Here, CPCs were harvested using single-cell sorting from rat cartilage tissues to obtain mesenchymal stem-like cells, which possess clonogenicity, proliferation and stemness. High doses of leptin decreased the ability of the CPCs to migrate, inhibited their chondrogenic potential and increased their osteogenic potential, suggesting that leptin changes differentiation fates in CPCs. High doses of leptin induced cell cycle arrest and senescence in CPCs by activating the p53/p21 pathway and inhibiting the Sirt1 pathway. Inhibiting the Sirt1 pathway accelerated cartilage senescence in knockout (KO) mice. Activating the leptin pathway induced higher Ob-Rb expression and was significantly correlated with cartilage degeneration (lower levels of Coll-2) and tissue senescence (higher levels of p53/p21 and lower levels of Sirt1) in OA patients, suggesting that leptin-induced CPCs senescence contributes to the development of OA. Taken together, our results reveal new links between obesity and cartilage damage that are induced by leptin-mediated effects on cell behaviour and senescence. PMID:27077804

  14. Structurally Distinct Polycyclic Aromatic Hydrocarbons Induce Differential Transcriptional Responses in Developing Zebrafish

    SciTech Connect

    Goodale, Britton; Tilton, Susan C.; Corvi, Margaret M.; Wilson, Glenn V.; Janszen, Derek B.; Anderson, Kim A.; Waters, Katrina M.; Tanguay, Robert

    2013-11-01

    Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in the environment as components of fossil fuels and by-products of combustion. These multi-ring chemicals differentially activate the aryl hydrocarbon receptor (AHR) in a structurally dependent manner, and induce toxicity via both AHR-dependent and -independent mechanisms. PAH exposure is known to induce developmental malformations in zebrafish embryos, and recent studies have shown cardiac toxicity induced by compounds with low AHR affinity. Unraveling the potentially diverse molecular mechanisms of PAH toxicity is essential for understanding the hazard posed by complex PAH mixtures present in the environment. We analyzed transcriptional responses to PAH exposure in zebrafish embryos exposed to benz(a)anthracene (BAA), dibenzothiophene (DBT) and pyrene (PYR) at concentrations that induced developmental malformations by 120 h post-fertilization (hpf). Whole genome microarray analysis of mRNA expression at 24 and 48 hpf identified genes that were differentially regulated over time and in response to the three PAH structures. PAH body burdens were analyzed at both time points using GC-MS, and demonstrated differences in PAH uptake into the embryos. This was important for discerning dose-related differences from those that represented unique molecular mechanisms. While BAA misregulated the least number of transcripts, it caused strong induction of cyp1a and other genes known to be downstream of the AHR, which were not induced by the other two PAHs. Analysis of functional roles of misregulated genes and their predicted regulatory transcription factors also distinguished the BAA response from regulatory networks disrupted by DBT and PYR exposure. These results indicate that systems approaches can be used to classify the toxicity of PAHs based on the networks perturbed following exposure, and may provide a path for unraveling the toxicity of complex PAH mixtures.

  15. EGF induces the progeny of subventricular zone type B cells to migrate and differentiate into oligodendrocytes

    PubMed Central

    Gonzalez-Perez, Oscar; Romero-Rodriguez, Ricardo; Soriano-Navarro, Mario; Garcia-Verdugo, Jose Manuel; Alvarez-Buylla, Arturo

    2012-01-01

    New neurons and oligodendrocytes are continuously produced in the subventricular zone (SVZ) of adult mammalian brains. Under normal conditions, the SVZ primary precursors (type B1 cells) generate type C cells, the majority of which differentiate into neurons, with a small sub-population giving rise to oligodendrocytes. Epidermal growth factor (EGF) signaling induces dramatic proliferation and migration of SVZ progenitors, a process that could have therapeutic applications. However, the fate of cells derived from adult neural stem cells after EGF stimulation remains unknown. Here, we specifically labeled SVZ B1 cells and followed their progeny after a 7-day intraventricular infusion of EGF. Cells derived from SVZ B1 cells invaded the parenchyma around the SVZ into striatum, septum, corpus callosum, and fimbria-fornix. The majority of these B1-derived cells gave rise to cells in the oligodendrocyte lineage including local NG2+ progenitors, pre-myelinating and myelinating oligodendrocytes. SVZ B1 cells also gave rise to a population of highly branched S100β+/GFAP+ cells in the striatum and septum, but no neuronal differentiation was observed. Interestingly, when demyelination was induced in the corpus callosum by a local injection of lysolecithin, increased number of cells derived from SVZ B1 cells and stimulated to migrate and proliferate by EGF infusion, differentiated into oligodendrocytes at the lesion site. This work indicates that EGF infusion can greatly expand the number of progenitors derived from the SVZ primary progenitors, which migrate and differentiate into oligodendroglial cells. This expanded population could be used for the repair of white matter lesions. PMID:19544429

  16. Neurogenic differentiation factor NeuroD confers protection against radiation-induced intestinal injury in mice

    PubMed Central

    Li, Ming; Du, Aonan; Xu, Jing; Ma, Yanchao; Cao, Han; Yang, Chao; Yang, Xiao-Dong; Xing, Chun-Gen; Chen, Ming; Zhu, Wei; Zhang, Shuyu; Cao, Jianping

    2016-01-01

    The gastrointestinal tract, especially the small intestine, is particularly sensitive to radiation, and is prone to radiation-induced injury as a result. Neurogenic differentiation factor (NeuroD) is an evolutionarily-conserved basic helix-loop-helix (bHLH) transcription factor. NeuroD contains a protein transduction domain (PTD), which allows it to be exogenously delivered across the membrane of mammalian cells, whereupon its transcription activity can be unleashed. Whether NeuroD has therapeutic effects for radiation-induced injury remains unclear. In the present study, we prepared a NeuroD-EGFP recombinant protein, and explored its protective effects on the survival and intestinal damage induced by ionizing radiation. Our results showed that NeuroD-EGFP could be transduced into small intestine epithelial cells and tissues. NeuroD-EGFP administration significantly increased overall survival of mice exposed to lethal total body irradiation (TBI). This recombinant NeuroD also reduced radiation-induced intestinal mucosal injury and apoptosis, and improved crypt survival. Expression profiling of NeuroD-EGFP-treated mice revealed upregulation of tissue inhibitor of metalloproteinase 1 (TIMP-1), a known inhibitor of apoptosis in mammalian cells. In conclusion, NeuroD confers protection against radiation-induced intestinal injury, and provides a novel therapeutic clinical option for the prevention of intestinal side effects of radiotherapy and the treatment of victims of incidental exposure. PMID:27436572

  17. Effect of Lycium bararum polysaccharides on methylmercury-induced abnormal differentiation of hippocampal stem cells

    PubMed Central

    Tian, Jian-Ying; Chen, Wei-Wei; Cui, Jing; Wang, Hao; Chao, Ci; Lu, Zhi-Yan; Bi, Yong-Yi

    2016-01-01

    The aim of the present study was to observe the effects of a general extract of Lycium bararum polysaccharides (LBPs) on methylmercury (MeHg)-induced damage in hippocampus neural stem cells (hNSCs). The hippocampal tissues of embryonic day 16 Sprague-Dawley rats were extracted for the isolation, purification and cloning of hNSCs. Following passage and proliferation for 10 days, the cells were allocated at random into the following groups: Control, LBPs, MeHg and MeHg + LBPs. MTT and microtubule-associated protein 2 (MAP-2)/glial fibrillary acidic protein/Hoechst immunofluorescence tests were performed to detect the differentiation and growth of hNSCs in the various groups. The differentiation rate of MeHg-treated hNSCs and the perimeter of MAP-2-positive neurons were 3.632±0.63% and 62.36±5.58 µm, respectively, significantly lower compared with the control group values of 6.500±0.81% and 166±8.16 µm (P<0.05). Furthermore, the differentiation rate and the perimeter of MAP-2-positive neurons in LBPs groups cells was 7.75±0.59% and 253.3±11.21 µm, respectively, significantly higher compared with the control group (P<0.05). The same parameters in the MeHg + LBPs group were 5.92±0.98% and 111.9±6.07 µm, respectively, significantly higher than the MeHg group (P<0.05). The astrocyte differentiation rates in the MeHg and MeHg + LBPs group were 41.19±2.14 and 34.58±1.70, respectively (P<0.05). These results suggest that LBPs may promote the generation and development of new neurons and inhibit the MeHg-induced abnormal differentiation of astrocytes. Thus, LBPs may be considered to be a potential new treatment for MeHg-induced neurotoxicity in hNSCs. PMID:27446261

  18. The endocrine disruptor diethylstilbestrol induces adipocyte differentiation and promotes obesity in mice

    SciTech Connect

    Hao, Chan-Juan; Cheng, Xue-Jia; Xia, Hong-Fei Ma, Xu

    2012-08-15

    Epidemiology studies indicate that exposure to endocrine disruptors during developmental “window” contributes to adipogenesis and the development of obesity. Implication of endocrine disruptor such as diethylstilbestrol (DES) on adipose tissue development has been poorly investigated. Here we evaluated the effects of DES on adipocyte differentiation in vitro and in vivo, and explored potential mechanism involved in its action. DES induced 3T3-L1 preadipocyte differentiation in a dose-dependent manner, and activated the expression of estrogen receptor (ER) and peroxisome proliferator-acivated receptor (PPAR) γ as well as its target genes required for adipogenesis in vitro. ER mediated the enhancement of DES-induced PPARγ activity. Moreover, DES perturbed key regulators of adipogenesis and lipogenic pathway in vivo. In utero exposure to low dose of DES significantly increased body weight, liver weight and fat mass in female offspring at postnatal day (PND) 60. In addition, serum triglyceride and glucose levels were also significantly elevated. These results suggest that perinatal exposure to DES may be expected to increase the incidence of obesity in a sex-dependent manner and can act as a potential chemical stressor for obesity and obesity-related disorders. -- Highlights: ► DES induced adipocyte differentiation in a dose-dependent manner in 3T3-L1 cells. ► DES activated adipogenic critical regulators and markers in vitro and in vivo. ► Perinatal exposure to DES led to the obese phenotype in female offspring. ► DES might be a potential chemical stressor for obesity and obesity-related disorders.

  19. Leishmania donovani Infection Induces Anemia in Hamsters by Differentially Altering Erythropoiesis in Bone Marrow and Spleen

    PubMed Central

    Lafuse, William P.; Story, Ryan; Mahylis, Jocelyn; Gupta, Gaurav; Varikuti, Sanjay; Steinkamp, Heidi; Oghumu, Steve; Satoskar, Abhay R.

    2013-01-01

    Leishmania donovani is a parasite that causes visceral leishmaniasis by infecting and replicating in macrophages of the bone marrow, spleen, and liver. Severe anemia and leucopenia is associated with the disease. Although immune defense mechanisms against the parasite have been studied, we have a limited understanding of how L. donovani alters hematopoiesis. In this study, we used Syrian golden hamsters to investigate effects of L. donovani infection on erythropoiesis. Infection resulted in severe anemia and leucopenia by 8 weeks post-infection. Anemia was associated with increased levels of serum erythropoietin, which indicates the hamsters respond to the anemia by producing erythropoietin. We found that infection also increased numbers of BFU-E and CFU-E progenitor populations in the spleen and bone marrow and differentially altered erythroid gene expression in these organs. In the bone marrow, the mRNA expression of erythroid differentiation genes (α-globin, β-globin, ALAS2) were inhibited by 50%, but mRNA levels of erythroid receptor (c-kit, EpoR) and transcription factors (GATA1, GATA2, FOG1) were not affected by the infection. This suggests that infection has a negative effect on differentiation of erythroblasts. In the spleen, erythroid gene expression was enhanced by infection, indicating that the anemia activates a stress erythropoiesis response in the spleen. Analysis of cytokine mRNA levels in spleen and bone marrow found that IFN-γ mRNA is highly increased by L. donovani infection. Expression of the IFN-γ inducible cytokine, TNF-related apoptosis-inducing ligand (TRAIL), was also up-regulated. Since TRAIL induces erythroblasts apoptosis, apoptosis of bone marrow erythroblasts from infected hamsters was examined by flow cytometry. Percentage of erythroblasts that were apoptotic was significantly increased by L. donovani infection. Together, our results suggest that L. donovani infection inhibits erythropoiesis in the bone marrow by cytokine

  20. Prenatal exposure of ethanol induces increased glutamatergic neuronal differentiation of neural progenitor cells

    PubMed Central

    2010-01-01

    Background Prenatal ethanol exposure during pregnancy induces a spectrum of mental and physical disorders called fetal alcohol spectrum disorder (FASD). The central nervous system is the main organ influenced by FASD, and neurological symptoms include mental retardation, learning abnormalities, hyperactivity and seizure susceptibility in childhood along with the microcephaly. In this study, we examined whether ethanol exposure adversely affects the proliferation of NPC and de-regulates the normal ratio between glutamatergic and GABAergic neuronal differentiation using primary neural progenitor culture (NPC) and in vivo FASD models. Methods Neural progenitor cells were cultured from E14 embryo brain of Sprague-Dawley rat. Pregnant mice and rats were treated with ethanol (2 or 4 g/kg/day) diluted with normal saline from E7 to E16 for in vivo FASD animal models. Expression level of proteins was investigated by western blot analysis and immunocytochemical assays. MTT was used for cell viability. Proliferative activity of NPCs was identified by BrdU incorporation, immunocytochemistry and FACS analysis. Results Reduced proliferation of NPCs by ethanol was demonstrated using BrdU incorporation, immunocytochemistry and FACS analysis. In addition, ethanol induced the imbalance between glutamatergic and GABAergic neuronal differentiation via transient increase in the expression of Pax6, Ngn2 and NeuroD with concomitant decrease in the expression of Mash1. Similar pattern of expression of those transcription factors was observed using an in vivo model of FASD as well as the increased expression of PSD-95 and decreased expression of GAD67. Conclusions These results suggest that ethanol induces hyper-differentiation of glutamatergic neuron through Pax6 pathway, which may underlie the hyper-excitability phenotype such as hyperactivity or seizure susceptibility in FASD patients. PMID:21073715

  1. Ethylene-induced differential gene expression during abscission of citrus leaves

    PubMed Central

    Merelo, Paz; Cercós, Manuel; Tadeo, Francisco R.; Talón, Manuel

    2008-01-01

    The main objective of this work was to identify and classify genes involved in the process of leaf abscission in Clementina de Nules (Citrus clementina Hort. Ex Tan.). A 7 K unigene citrus cDNA microarray containing 12 K spots was used to characterize the transcriptome of the ethylene-induced abscission process in laminar abscission zone-enriched tissues and the petiole of debladed leaf explants. In these conditions, ethylene induced 100% leaf explant abscission in 72 h while, in air-treated samples, the abscission period started later and took 240 h. Gene expression monitored during the first 36 h of ethylene treatment showed that out of the 12 672 cDNA microarray probes, ethylene differentially induced 725 probes distributed as follows: 216 (29.8%) probes in the laminar abscission zone and 509 (70.2%) in the petiole. Functional MIPS classification and manual annotation of differentially expressed genes highlighted key processes regulating the activation and progress of the cell separation that brings about abscission. These included cell-wall modification, lipid transport, protein biosynthesis and degradation, and differential activation of signal transduction and transcription control pathways. Expression data associated with the petiole indicated the occurrence of a double defensive strategy mediated by the activation of a biochemical programme including scavenging ROS, defence and PR genes, and a physical response mostly based on lignin biosynthesis and deposition. This work identifies new genes probably involved in the onset and development of the leaf abscission process and suggests a different but co-ordinated and complementary role for the laminar abscission zone and the petiole during the process of abscission. PMID:18515267

  2. Nanoparticle-induced negative differential resistance and memory effect in polymer bistable light-emitting device

    NASA Astrophysics Data System (ADS)

    Tseng, Ricky J.; Ouyang, Jianyong; Chu, Chih-Wei; Huang, Jinsong; Yang, Yang

    2006-03-01

    Recently, electrical bistability was demonstrated in polymer thin films incorporated with metal nanoparticles [J. Ouyang, C. W. Chu, C. R. Szmanda, L. P. Ma, and Y. Yang, Nat. Mater. 3, 918 (2004)]. In this letter, we show the evidence that electrons are the dominant charge carriers in these bistable devices. Direct integration of bistable polymer layer with a light-emitting polymer layer shows a unique light-emitting property modulated by the electrical bistability. A unique negative differential resistance induced by the charged gold nanoparticles is observed due to the charge trapping effect from the nanoparticles when interfaced with the light-emitting layer.

  3. Pressure Measurement in Supersonic Air Flow by Differential Absorptive Laser-Induced Thermal Acoustics

    NASA Technical Reports Server (NTRS)

    Hart, Roger C.; Herring, Gregory C.; Balla, Robert J.

    2007-01-01

    Nonintrusive, off-body flow barometry in Mach-2 airflow has been demonstrated in a large-scale supersonic wind tunnel using seedless laser-induced thermal acoustics (LITA). The static pressure of the gas flow is determined with a novel differential absorption measurement of the ultrasonic sound produced by the LITA pump process. Simultaneously, stream-wise velocity and static gas temperature of the same spatially-resolved sample volume were measured with this nonresonant time-averaged LITA technique. Mach number, temperature and pressure have 0.2%, 0.4%, and 4% rms agreement, respectively, in comparison with known free-stream conditions.

  4. Aggregates-induced dynamic negative differential resistance in conducting organic films

    NASA Astrophysics Data System (ADS)

    Xie, Xian Ning; Wang, Junzhong; Loh, Kian Ping; Wee, Andrew Thye Shen

    2009-11-01

    This letter reports the negative differential resistance (NDR) behavior of perylene-3,4,9,10-tetracarboxylic-3,4,9,10-dianhydride films induced by aggregate formation in the film. It is observed that aggregate-states in the energy gap can by-pass the common charge conduction mode, and electron injection, trapping, and conduction through these states lead to the NDR characteristic. The rate-dependence of NDR is discussed in terms of the transit time and lifetime of the aggregates-states electrons. The quenching of NDR by photoillumination is also observed, and is attributed to the saturation of aggregates-states by photoelectrons.

  5. Carbon nanotube array inducing osteogenic differentiation of human mesenchymal stem cells.

    PubMed

    Xu, Baiyao; Ju, Yang; Cui, Yanbin; Song, Guanbin

    2015-06-01

    Carbon nanotubes (CNTs) are a kind of nanomaterials which have been shown a promising application for biomedicine. There are a lot of studies to use CNTs to induce the differentiation of mesenchymal stem cells (MSCs). However, the cellular behavior of MSCs on the top layer of CNT array was still not well understood. In this study, we evaluated the morphology, the gene expressions of the osteogenic differentiation related markers, and the gene expressions of collagen type II (Col II, a marker of chondrogenesis), PPARγ (a marker of adipogenesis) and scleraxis (SCX, a marker of tenogenesis) in human mesenchymal stem cells (hMSCs) cultured on multi-walled carbon nanotube (MWCNT) array. The effect of MWCNT array on the mineralization of hMSCs which were cultured in osteogenic differentiation medium (ODM) was further assayed. Our results showed that the hMSCs cultured on MWCNT array spread well, formed numerous spiral shaped cell colons and showed perinuclear morphology. Compared to hMSCs cultured on dish, the gene expression of osteocalcin (OCN) was increased while the gene expressions of collagen type II (Col II), PPARγ and scleraxis (SCX) were decreased in hMSCs which were cultured on MWCNT array without any differentiation factors. Furthermore, compared with hMSCs on dish, the gene expressions of collagen type I (Col I), osteocalcin (OCN), osteopontin (OPN) and RUNX2, and the mineralization of hMSCs on MWCNT array were enhanced when they were cultured in osteogenic differentiation medium (ODM). Our results indicated that MWCNT array was able to promote the osteogenesis of hMSCs.

  6. 1,25-dihydroxycholecalciferol-induced differentiation of myelomonocytic leukemic cells unresponsive to colony stimulating factors and phorbol esters

    SciTech Connect

    Bettens, F.; Schlick, E.; Farrar, W.; Ruscetti, F.

    1986-12-01

    The murine myelomonocytic leukemia cell line WEHI-3B D/sup +/, which differentiates in response to granulocyte colony stimulating factor (G-CSF), can also be induced to differentiate into monocyte-macrophages by phorbol myristate acetate (PMA) treatment, whereas the WEHI-3B D/sup -/ subline, which is unresponsive to G-CSF and PMA, can be induced to differentiate to granulocytes as well as monocytes by 1,25-dihydroxycholecalciferol (1,25-(OH)/sub 2/ D3), the biologically active metabolite of vitamin D3. A newly developed variant of the WEHI-3B D/sup +/ line, named WEHI-3B D/sup +/G, which was responsive to G-CSF but not to PMA, was also differentiated to granulocytes by 1,25-(OH)/sub 2/ D3. Although vitamin D3 has been reported to induce macrophage differentiation in responsive tumor cells, this is the first demonstration that 1,25-(OH)/sub 2/ D3 can induce granulocyte differentiation. In both differentiation pathways, cessation of cellular proliferation accompanies changes in morphologic and cytochemical properties of the cells. This suggests that leukemic cell lines unresponsive to differentiation agents acting at the cell surface retain their ability to differentiate in response to agents that do not act via the plasma membrane such as 1,25-(OH)/sub 2/ D3, which has cytosolic/nuclear receptors. These results suggest that low doses of 1,25-(OH)/sub 2/ D3 may be useful in combination with hemopoietic growth factors (CSFs) as therapeutic agent to induce leukemic cell differentiation in vivo.

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

    PubMed

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

    2013-02-01

    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. PMID:23291166

  8. Ganoderma lucidum Polysaccharides Induce Macrophage-Like Differentiation in Human Leukemia THP-1 Cells via Caspase and p53 Activation.

    PubMed

    Hsu, Jia-Wei; Huang, Hsuan-Cheng; Chen, Shui-Tein; Wong, Chi-Huey; Juan, Hsueh-Fen

    2011-01-01

    Differentiation therapy by induction of tumor cells is an important method in the treatment of hematological cancers such as leukemia. Tumor cell differentiation ends cancer cells' immortality, thus stopping cell growth and proliferation. In our previous study, we found that fucose-containing polysaccharide fraction F3 extracted from Ganoderma lucidum can bring about cytokine secretion and cell death in human leukemia THP-1 cells. This prompted us to further investigate on how F3 induces the differentiation in human leukemia cells. We integrated time-course microarray analysis and network modeling to study the F3-induced effects on THP-1 cells. In addition, we determined the differentiation effect using Liu's staining, nitroblue tetrazolium (NBT) reduction assay, flow cytometer, western blotting and Q-PCR. We also examined the modulation and regulation by F3 during the differentiation process. Dynamic gene expression profiles showed that cell differentiation was induced in F3-treated THP-1 cells. Furthermore, F3-treated THP-1 cells exhibited enhanced macrophage differentiation, as demonstrated by changes in cell adherence, cell cycle arrest, NBT reduction and expression of differentiation markers including CD11b, CD14, CD68, matrix metalloproteinase-9 and myeloperoxidase. In addition, caspase cleavage and p53 activation were found to be significantly enhanced in F3-treated THP-1 cells. We unraveled the role of caspases and p53 in F3-induced THP-1 cells differentiation into macrophages. Our results provide a molecular explanation for the differentiation effect of F3 on human leukemia THP-1 cells and offer a prospect for a potential leukemia differentiation therapy.

  9. Depletion of white adipocyte progenitors induces beige adipocyte differentiation and suppresses obesity development

    PubMed Central

    Daquinag, A C; Tseng, C; Salameh, A; Zhang, Y; Amaya-Manzanares, F; Dadbin, A; Florez, F; Xu, Y; Tong, Q; Kolonin, M G

    2015-01-01

    Overgrowth of white adipose tissue (WAT) in obesity occurs as a result of adipocyte hypertrophy and hyperplasia. Expansion and renewal of adipocytes relies on proliferation and differentiation of white adipocyte progenitors (WAP); however, the requirement of WAP for obesity development has not been proven. Here, we investigate whether depletion of WAP can be used to prevent WAT expansion. We test this approach by using a hunter-killer peptide designed to induce apoptosis selectively in WAP. We show that targeted WAP cytoablation results in a long-term WAT growth suppression despite increased caloric intake in a mouse diet-induced obesity model. Our data indicate that WAP depletion results in a compensatory population of adipose tissue with beige adipocytes. Consistent with reported thermogenic capacity of beige adipose tissue, WAP-depleted mice display increased energy expenditure. We conclude that targeting of white adipocyte progenitors could be developed as a strategy to sustained modulation of WAT metabolic activity. PMID:25342467

  10. How the differential load induced by normal fault scarps controls the distribution of monogenic volcanism

    NASA Astrophysics Data System (ADS)

    Maccaferri, Francesco; Acocella, Valerio; Rivalta, Eleonora

    2016-04-01

    Understanding shallow magma transfer and the related vent distribution is crucial for volcanic hazard. In the present study we investigate the link between the stress induced by topographic scarps and the distribution of monogenic volcanoes at divergent plate boundaries. With a numerical model of dyke propagation we show that vertical dykes beneath a normal fault scarp tend to deflect towards the footwall side of the scarp. This effect increases with the scarp height, is stronger for dykes propagating underneath the hanging wall side, and decreases with the distance from the scarp. A comparison to the East African Rift System, Afar and Iceland shows that: 1) the inner rift structure, which shapes the topography, controls shallow dyke propagation; 2) differential loading due to mass redistribution affects magma propagation over a broad scale range (100 - 105 m). Our results find application to any volcanic field with tectonics- or erosion-induced topographic variations.

  11. How the differential load induced by normal fault scarps controls the distribution of monogenic volcanism

    NASA Astrophysics Data System (ADS)

    Maccaferri, F.; Acocella, V.; Rivalta, E.

    2015-09-01

    Understanding shallow magma transfer and the related vent distribution is crucial for volcanic hazard. Here we investigate how the stress induced by topographic scarps linked to normal faults affects the distribution of monogenic volcanoes at divergent plate boundaries. Our numerical models of dyke propagation below a fault scarp show that the dykes tend to propagate toward and erupt on the footwall side. This effect, increasing with the scarp height, is stronger for dykes propagating underneath the hanging wall side and decreases with the distance from the scarp. A comparison to the East African Rift System, Afar and Iceland shows that (1) the inner rift structure, which shapes the topography, controls shallow dyke propagation; (2) differential loading due to mass redistribution affects magma propagation over a broad scale range (100-105 m). Our results find application to any volcanic field with tectonics- or erosion-induced topographic variations and should be considered in any volcanic hazard assessment.

  12. Free fatty acids induce cell differentiation to infective forms in Trypanosoma cruzi.

    PubMed Central

    Wainszelbaum, Marisa J; Belaunzarán, María L; Lammel, Estela M; Florin-Christensen, Mónica; Florin-Christensen, Jorge; Isola, Elvira L D

    2003-01-01

    Intestinal extracts of Triatoma infestans induce cell differentiation of Trypanosoma cruzi epimastigotes into the infective metacyclic form. Part of this effect can be explained by the presence of haemoglobin fragments, which stimulate trypanosomal adenylate cyclase. In this work we examined the metacyclogenic activity of lipids present in this intestinal extract. We found that lipid extracts of the intestinal extract have significant stimulatory effects that reside with the free-fatty-acid fraction, especially oleic acid. These compounds stimulate de novo diacylglycerol formation and protein kinase C activity in the parasite. Moreover, metacyclogenesis is stimulated by phorbol esters and cell-permeant diacylglycerol, while protein kinase C down-regulation or incubation with inhibitors of this kinase abrogates this effect. These results indicate that free fatty acids are a novel signal, inducing metacyclogenesis, acting through a pathway involving diacylglycerol biosynthesis and protein kinase C activation. PMID:12887332

  13. Mechanisms of manganese-induced rat pheochromocytoma (PC12) cell death and cell differentiation.

    PubMed

    Roth, Jerome A; Horbinski, Craig; Higgins, Dennis; Lein, Pamela; Garrick, Michael D

    2002-07-01

    Mn is a neurotoxin that leads to a syndrome resembling Parkinson's disease after prolonged exposure to high concentrations. Our laboratory has been investigating the mechanism by which Mn induces neuronal cell death. To accomplish this, we have utilized rat pheochromocytoma (PC12) cells as a model since they possess much of the biochemical machinery associated with dopaminergic neurons. Mn, like nerve growth factor (NGF), can induce neuronal differentiation of PC12 cells but Mn-induced cell differentiation is dependent on its interaction with the cell surface integrin receptors and basement membrane proteins, vitronectin or fibronectin. Similar to NGF, Mn-induced neurite outgrowth is dependent on the phosphorylation and activation of the MAP kinases, ERK1 and 2 (p44/42). Unlike NGF, Mn is also cytotoxic having an IC50 value of approximately 600 microM. Although many apoptotic signals are turned on by Mn, cell death is caused ultimately by disruption of mitochondrial function leading to loss of ATP. RT-PCR and immunoblotting studies suggest that some uptake of Mn into PC12 cells depends on the divalent metal transporter 1 (DMT1). DMT1 exists in two isoforms resulting from alternate splicing of a single gene product with one of the two mRNA species containing an iron response element (IRE) motif downstream from the stop codon. The presence of the IRE provides a binding site for the iron response proteins (IRP1 and 2); binding of either of these proteins could stabilize DMT1 mRNA and would increase expression of the +IRE form of the transporter. Iron and Mn compete for transport into PC12 cells via DMT1, so removal of iron from the culture media enhances Mn toxicity. The two isoforms of DMT1 (+/-IRE) are distributed in different subcellular compartments with the -IRE species selectively present in the nucleus of neuronal and neuronal-like cells. PMID:12224755

  14. Differentiation-inducing factor-1 induces cyclin D1 degradation through the phosphorylation of Thr{sup 286} in squamous cell carcinoma

    SciTech Connect

    Mori, Jun; Takahashi-Yanaga, Fumi . E-mail: yanaga@clipharm.med.kyushu-u.ac.jp; Miwa, Yoshikazu; Watanabe, Yutaka; Hirata, Masato; Morimoto, Sachio; Shirasuna, Kanemitsu; Sasaguri, Toshiyuki

    2005-11-01

    Differentiation-inducing factors (DIFs) are morphogens which induce cell differentiation in Dictyostelium. We reported that DIF-1 and DIF-3 inhibit proliferation and induce differentiation in mammalian cells. In this study, we investigated the effect of DIF-1 on oral squamous cell carcinoma cell lines NA and SAS, well differentiated and poorly differentiated cell lines, respectively. Although DIF-1 did not induce the expression of cell differentiation makers in these cell lines, it inhibited the proliferation of NA and SAS in a dose-dependent manner by restricting the cell cycle in the G{sub 0}/G{sub 1} phase. DIF-1 induced cyclin D1 degradation, but this effect was prevented by treatment with lithium chloride and SB216763, the inhibitors of glycogen synthase kinase-3{beta} (GSK-3{beta}). Depletion of endogenous GSK-3{beta} by RNA interference also attenuated the effect of DIF-1 on cyclin D1 degradation. Therefore, we investigated the effect of DIF-1 on GSK-3{beta} and found that DIF-1 dephosphorylated GSK-3{beta} on Ser{sup 9} and induced the nuclear translocation of GSK-3{beta}, suggesting that DIF-1 activated GSK-3{beta}. Then, we examined the effect of DIF-1 on cyclin D1 mutants (Thr286Ala, Thr288Ala, and Thr286/288Ala). We revealed that Thr286Ala and Thr286/288Ala mutants were highly resistant to DIF-1-induced degradation compared with wild-type cyclin D1, indicating that the phosphorylation of Thr{sup 286} was critical for cyclin D1 degradation induced by DIF-1. These results suggest that DIF-1 induces degradation of cyclin D1 through the GSK-3{beta}-mediated phosphorylation of Thr{sup 286}.

  15. IL-21 induces inhibitor of differentiation 2 and leads to complete abrogation of anaphylaxis in mice.

    PubMed

    Kishida, Tsunao; Hiromura, Yayoi; Shin-Ya, Masaharu; Asada, Hidetsugu; Kuriyama, Hiroko; Sugai, Manabu; Shimizu, Akira; Yokota, Yoshifumi; Hama, Takemitsu; Imanishi, Jiro; Hisa, Yasuo; Mazda, Osam

    2007-12-15

    IL-21 exerts pleiotrophic immunomodulatory activities on a variety of target cells including B cells that undergo class switch recombination (CSR) to IgE. In this study, we examined whether IgE-mediated systemic anaphylaxis was controlled by in vivo administration of IL-21 using the peanut allergy model in mice and investigated the molecular mechanisms underlying the IL-21-induced regulation of IgE. The anaphylactic reaction was completely abolished by the administration of recombinant mouse IL-21 or an IL-21 expression plasmid in terms of the change of body temperature and anaphylactic symptoms. The recombinant mouse IL-21 treatment remarkably suppressed IgE CSR in splenic B cells, resulting in significant decrease in serum concentrations of total as well as allergen-specific IgE. In the meanwhile, IL-21 provoked B cells in normal as well as allergic mice to express the inhibitor of differentiation 2 (Id2) gene that was shown to be crucially involved in the regulation of the activation-induced cytidine deaminase and IgE CSR. Moreover, mice genetically deficient for Id2 were completely unsusceptible to IL-21-induced prevention of IgE CSR and anaphylaxis. The present study strongly suggests that IL-21 is capable of regulating systemic allergic reactions by inducing the transcriptional regulator Id2, and the cytokine may be useful for clinical intervention for allergic diseases including anaphylaxis.

  16. Potato suberin induces differentiation and secondary metabolism in the genus Streptomyces.

    PubMed

    Lerat, Sylvain; Forest, Martin; Lauzier, Annie; Grondin, Gilles; Lacelle, Serge; Beaulieu, Carole

    2012-01-01

    Bacteria of the genus Streptomyces are soil microorganisms with a saprophytic life cycle. Previous studies have revealed that the phytopathogenic agent S. scabiei undergoes metabolic and morphological modifications in the presence of suberin, a complex plant polymer. This paper investigates morphological changes induced by the presence of potato suberin in five species of the genus Streptomyces, with emphasis on S. scabiei. Streptomyces scabiei, S. acidiscabies, S. avermitilis, S. coelicolor and S. melanosporofaciens were grown both in the presence and absence of suberin. In all species tested, the presence of the plant polymer induced the production of aerial hyphae and enhanced resistance to mechanical lysis. The presence of suberin in liquid minimal medium also induced the synthesis of typical secondary metabolites in S. scabiei and S. acidiscabies (thaxtomin A), S. coelicolor (actinorhodin) and S. melanosporofaciens (geldanamycin). In S. scabiei, the presence of suberin modified the fatty acid composition of the bacterial membrane, which translated into higher membrane fluidity. Moreover, suberin also induced thickening of the bacterial cell wall. The present data indicate that suberin hastens cellular differentiation and triggers the onset of secondary metabolism in the genus Streptomyces. PMID:22129602

  17. Testosterone-induced male traits in female ruffs (Philomachus pugnax): autosomal inheritance and gender differentiation

    PubMed Central

    Lank, D. B.; Coupe, M.; Wynne-Edwards, K. E.

    1999-01-01

    A balanced polymorphism in male mating behaviour exists in male ruffs, with no obvious parallel expression in females. Pedigree data of male phenotypes support an autosomal model of inheritance, in contrast to sex-linked inheritance patterns found in other taxa with sex-limited alternative mating strategy polymorphisms. We tested this model by inducing male courtship behaviour in gonad-intact female ruffs, using subcutaneous testosterone implants that produced physiological concentrations of testosterone. The implants rapidly induced in females both types of male mating behaviour, an increase in body mass typical of pre-breeding males, and the growth of normally male-limited breeding plumage. As predicted under an autosomal model, the distributions of induced male behaviour types in females paralleled those of their brothers and half-brothers, and were inconsistent with sex-linked models. Effects were reversible, and experimental females bred normally in subsequent years. Our results show that genotype-specific male characteristics can be induced by testosterone in female adults that have presumably not undergone neural organization for them early in life, showing direct use of genetic information in intra- and intersexual differentiation.

  18. Potato Suberin Induces Differentiation and Secondary Metabolism in the Genus Streptomyces

    PubMed Central

    Lerat, Sylvain; Forest, Martin; Lauzier, Annie; Grondin, Gilles; Lacelle, Serge; Beaulieu, Carole

    2012-01-01

    Bacteria of the genus Streptomyces are soil microorganisms with a saprophytic life cycle. Previous studies have revealed that the phytopathogenic agent S. scabiei undergoes metabolic and morphological modifications in the presence of suberin, a complex plant polymer. This paper investigates morphological changes induced by the presence of potato suberin in five species of the genus Streptomyces, with emphasis on S. scabiei. Streptomyces scabiei, S. acidiscabies, S. avermitilis, S. coelicolor and S. melanosporofaciens were grown both in the presence and absence of suberin. In all species tested, the presence of the plant polymer induced the production of aerial hyphae and enhanced resistance to mechanical lysis. The presence of suberin in liquid minimal medium also induced the synthesis of typical secondary metabolites in S. scabiei and S. acidiscabies (thaxtomin A), S. coelicolor (actinorhodin) and S. melanosporofaciens (geldanamycin). In S. scabiei, the presence of suberin modified the fatty acid composition of the bacterial membrane, which translated into higher membrane fluidity. Moreover, suberin also induced thickening of the bacterial cell wall. The present data indicate that suberin hastens cellular differentiation and triggers the onset of secondary metabolism in the genus Streptomyces. PMID:22129602

  19. Donor Dependent Variations in Hematopoietic Differentiation among Embryonic and Induced Pluripotent Stem Cell Lines.

    PubMed

    Féraud, Olivier; Valogne, Yannick; Melkus, Michael W; Zhang, Yanyan; Oudrhiri, Noufissa; Haddad, Rima; Daury, Aurélie; Rocher, Corinne; Larbi, Aniya; Duquesnoy, Philippe; Divers, Dominique; Gobbo, Emilie; Brunet de la Grange, Philippe; Louache, Fawzia; Bennaceur-Griscelli, Annelise; Mitjavila-Garcia, Maria Teresa

    2016-01-01

    Hematopoiesis generated from human embryonic stem cells (ES) and induced pluripotent stem cells (iPS) are unprecedented resources for cell therapy. We compared hematopoietic differentiation potentials from ES and iPS cell lines originated from various donors and derived them using integrative and non-integrative vectors. Significant differences in differentiation toward hematopoietic lineage were observed among ES and iPS. The ability of engraftment of iPS or ES-derived cells in NOG mice varied among the lines with low levels of chimerism. iPS generated from ES cell-derived mesenchymal stem cells (MSC) reproduce a similar hematopoietic outcome compared to their parental ES cell line. We were not able to identify any specific hematopoietic transcription factors that allow to distinguish between good versus poor hematopoiesis in undifferentiated ES or iPS cell lines. There is a relatively unpredictable variation in hematopoietic differentiation between ES and iPS cell lines that could not be predicted based on phenotype or gene expression of the undifferentiated cells. These results demonstrate the influence of genetic background in variation of hematopoietic potential rather than the reprogramming process.

  20. Adipocyte differentiation induced using nonspecific siRNA controls in cultured human mesenchymal stem cells

    PubMed Central

    Xu, Yunhe; Mirmalek-Sani, Sayed-Hadi; Lin, Feng; Zhang, Junlong; Oreffo, Richard O.C.

    2007-01-01

    RNA interference (RNAi) is gene silencing induced by double-stranded RNA of 21–23 nucleotides in length, termed small interfering RNA, or siRNA. RNAi-based techniques have been widely applied to elucidate gene function, identify drug targets, and used in trials as a promising adjunct to silence disease-causing genes. However, emerging evidence suggests unexpected changes in expression of untargeted genes as a consequence of an off-target effect by RNAi in mammalian cells. To date, our understanding of such effects on stem cells is limited. We transfected human fetal femur-derived mesenchymal stem cells using commercially available nonspecific siRNA controls and examined adipocyte differentiation in the cells using morphology, histochemistry, and quantitative real-time PCR to examine the expression of key genes for adipogenic or osteogenic differentiation. We report here the induction of adipocyte differentiation in human mesenchymal stem cells using nonspecific siRNAs raising concerns as to the specificity of RNAi in stem cells and, critically, a need to understand and delineate the rules governing the specificity of RNAi. PMID:17556710

  1. The mineralocorticoid receptor mediates aldosterone-induced differentiation of T37i cells into brown adipocytes.

    PubMed

    Penfornis, P; Viengchareun, S; Le Menuet, D; Cluzeaud, F; Zennaro, M C; Lombès, M

    2000-08-01

    By use of targeted oncogenesis, a brown adipocyte cell line was derived from a hibernoma of a transgenic mouse carrying the proximal promoter of the human mineralocorticoid receptor (MR) linked to the SV40 large T antigen. T37i cells remain capable of differentiating into brown adipocytes upon insulin and triiodothyronine treatment as judged by their ability to express uncoupling protein 1 and maintain MR expression. Aldosterone treatment of undifferentiated cells induced accumulation of intracytoplasmic lipid droplets and mitochondria. This effect was accompanied by a significant and dose-dependent increase in intracellular triglyceride content (half-maximally effective dose 10(-9) M) and involved MR, because it was unaffected by RU-38486 treatment but was totally abolished in the presence of aldosterone antagonists (spironolactone, RU-26752). The expression of early adipogenic gene markers, such as lipoprotein lipase, peroxisome proliferator-activated receptor-gamma, and adipocyte-specific fatty acid binding protein 2, was enhanced by aldosterone, confirming activation of the differentiation process. We demonstrate that, in the T37i cell line, aldosterone participates in the very early induction of brown adipocyte differentiation. Our findings may have a broader biological significance and suggest that MR is not only implicated in maintaining electrolyte homeostasis but could also play a role in metabolism and energy balance.

  2. Marmoset induced pluripotent stem cells: Robust neural differentiation following pretreatment with dimethyl sulfoxide.

    PubMed

    Qiu, Zhifang; Mishra, Anuja; Li, Miao; Farnsworth, Steven L; Guerra, Bernadette; Lanford, Robert E; Hornsby, Peter J

    2015-07-01

    The marmoset is an important nonhuman primate model for regenerative medicine. For experimental autologous cell therapy based on induced pluripotent (iPS) cells in the marmoset, cells must be able to undergo robust and reliable directed differentiation that will not require customization for each specific iPS cell clone. When marmoset iPS cells were aggregated in a hanging drop format for 3 days, followed by exposure to dual SMAD inhibitors and retinoic acid in monolayer culture for 3 days, we found substantial variability in the response of different iPS cell clones. However, when clones were pretreated with 0.05-2% dimethyl sulfoxide (DMSO) for 24 hours, all clones showed a very similar maximal response to the directed differentiation scheme. Peak responses were observed at 0.5% DMSO in two clones and at 1% DMSO in a third clone. When patterns of gene expression were examined by microarray analysis, hierarchical clustering showed very similar responses in all 3 clones when they were pretreated with optimal DMSO concentrations. The change in phenotype following exposure to DMSO and the 6 day hanging drop/monolayer treatment was confirmed by immunocytochemistry. Analysis of DNA content in DMSO-exposed cells indicated that it is unlikely that DMSO acts by causing cells to exit from the cell cycle. This approach should be generally valuable in the directed neural differentiation of pluripotent cells for experimental cell therapy. PMID:26070112

  3. Differentiation induced by Achyrocline satureioides (Lam) infusion in PC12 cells.

    PubMed

    Blasina, M F; Vaamonde, L; Morquio, A; Echeverry, C; Arredondo, F; Dajas, F

    2009-09-01

    Epidemiological studies have shown that flavonoid-rich plants induce beneficial health effects that are likely beyond their potent antioxidant capacity. Thus, the mechanisms by which Achyrocline satureioides (AS), a popular South American medicinal plant, protects cells and neurons in culture, are still unclear. In this sense, a recently described trophic capacity for flavonoids, similar to that evoked by growth factors, could be one of the mechanisms involved in AS cellular protection. Since this trophic activity causes differentiation of PC12 cells, the cell differentiation capacity of AS and some of its flavonoids were evaluated. PC12 cells were treated with AS infusion (10 or 20 microg/mL of total polyphenols), quercetin (Q) (12.5 or 25 microm), luteolin (L) (25 microm), Q + L (12.5 microm each one) or nerve growth factor (NGF) for 3 days. Four morphological parameters (percentage of cells with neurites longer than one cell body diameter, percentage of cells with neurites, average number of neurites per cell and percentage of fusiform cells) were explored. The AS infusion showed differentiation capacity on all parameters with similar potency when compared with NGF. Besides, AS was more potent than some of its constituent flavonoids: Q, L or their combination.

  4. Glyphosate Inhibits PPAR Gamma Induction and Differentiation of Preadipocytes and is able to Induce Oxidative Stress.

    PubMed

    Martini, Claudia N; Gabrielli, Matías; Brandani, Javier N; Vila, María Del C

    2016-08-01

    Glyphosate-based herbicides (GF) are extensively used for weed control. Thus, it is important to investigate their putative toxic effects. We have reported that GF at subagriculture concentrations inhibits proliferation and differentiation to adipocytes of 3T3-L1 fibroblasts. In this investigation, we evaluated the effect of GF on genes upregulated during adipogenesis. GF was able to inhibit the induction of PPAR gamma, the master gene in adipogenesis but not C/EBP beta, which precedes PPAR gamma activation. GF also inhibited differentiation and proliferation of another model of preadipocyte: mouse embryonic fibroblasts. In exponentially growing 3T3-L1 cells, GF increased lipid peroxidation and the activity of the antioxidant enzyme, superoxide dismutase. We also found that proliferation was inhibited with lower concentrations of GF when time of exposure was extended. Thus, GF was able to inhibit proliferation and differentiation of preadipocytes and to induce oxidative stress, which is indicative of its ability to alter cellular physiology. PMID:27044015

  5. Donor Dependent Variations in Hematopoietic Differentiation among Embryonic and Induced Pluripotent Stem Cell Lines

    PubMed Central

    Féraud, Olivier; Valogne, Yannick; Melkus, Michael W.; Zhang, Yanyan; Oudrhiri, Noufissa; Haddad, Rima; Daury, Aurélie; Rocher, Corinne; Larbi, Aniya; Duquesnoy, Philippe; Divers, Dominique; Gobbo, Emilie; Brunet de la Grange, Philippe; Louache, Fawzia; Bennaceur-Griscelli, Annelise; Mitjavila-Garcia, Maria Teresa

    2016-01-01

    Hematopoiesis generated from human embryonic stem cells (ES) and induced pluripotent stem cells (iPS) are unprecedented resources for cell therapy. We compared hematopoietic differentiation potentials from ES and iPS cell lines originated from various donors and derived them using integrative and non-integrative vectors. Significant differences in differentiation toward hematopoietic lineage were observed among ES and iPS. The ability of engraftment of iPS or ES-derived cells in NOG mice varied among the lines with low levels of chimerism. iPS generated from ES cell-derived mesenchymal stem cells (MSC) reproduce a similar hematopoietic outcome compared to their parental ES cell line. We were not able to identify any specific hematopoietic transcription factors that allow to distinguish between good versus poor hematopoiesis in undifferentiated ES or iPS cell lines. There is a relatively unpredictable variation in hematopoietic differentiation between ES and iPS cell lines that could not be predicted based on phenotype or gene expression of the undifferentiated cells. These results demonstrate the influence of genetic background in variation of hematopoietic potential rather than the reprogramming process. PMID:26938212

  6. Sox15 enhances trophoblast giant cell differentiation induced by Hand1 in mouse placenta.

    PubMed

    Yamada, Kayo; Kanda, Hiromi; Tanaka, Satoshi; Takamatsu, Nobuhiko; Shiba, Tadayoshi; Ito, Michihiko

    2006-06-01

    Some members of the Sry-type HMG box (Sox) protein family play important roles in embryogenesis as transcription factors. Here, we report that Sox15 transcripts were much more abundant in mouse placenta than in the fetus, the yolk sac, or several adult tissues. In situ hybridization analysis of the mouse E8.0 conceptus indicated that Sox15 mRNA was predominantly expressed in the trophoblast giant cells of the placenta. We also observed that the amount of Sox15 mRNA dramatically increased during the differentiation of mouse trophoblast stem cells. Ectopic expression of Sox15 in Rat choriocarcinoma cells enhanced the giant cell differentiation induced by a bHLH transcription factor, Hand1. Binding experiments in cotransfected 293 T cells and in vitro revealed that Sox15 interacted with Hand1. We next examined the effects of this interaction on the transcriptional activity of Hand1 and Sox15 using the luciferase reporter assay. Overexpression of Hand1 repressed the Sox15-driven reporter expression, but Sox15 enhanced the Hand1-driven transcription. This enhancement required both the Hand1-binding region and the transactivation domain of Sox15. These results may suggest that the increased transcriptional activity of Hand1 caused by Sox15 might promote the transcription of the target gene resulting in the trophoblast giant cell differentiation in the mouse placenta.

  7. Calcium phosphate-bearing matrices induce osteogenic differentiation of stem cells through adenosine signaling

    PubMed Central

    Shih, Yu-Ru V.; Hwang, YongSung; Phadke, Ameya; Kang, Heemin; Hwang, Nathaniel S.; Caro, Eduardo J.; Nguyen, Steven; Siu, Michael; Theodorakis, Emmanuel A.; Gianneschi, Nathan C.; Vecchio, Kenneth S.; Chien, Shu; Lee, Oscar K.; Varghese, Shyni

    2014-01-01

    Synthetic matrices emulating the physicochemical properties of tissue-specific ECMs are being developed at a rapid pace to regulate stem cell fate. Biomaterials containing calcium phosphate (CaP) moieties have been shown to support osteogenic differentiation of stem and progenitor cells and bone tissue formation. By using a mineralized synthetic matrix mimicking a CaP-rich bone microenvironment, we examine a molecular mechanism through which CaP minerals induce osteogenesis of human mesenchymal stem cells with an emphasis on phosphate metabolism. Our studies show that extracellular phosphate uptake through solute carrier family 20 (phosphate transporter), member 1 (SLC20a1) supports osteogenic differentiation of human mesenchymal stem cells via adenosine, an ATP metabolite, which acts as an autocrine/paracrine signaling molecule through A2b adenosine receptor. Perturbation of SLC20a1 abrogates osteogenic differentiation by decreasing intramitochondrial phosphate and ATP synthesis. Collectively, this study offers the demonstration of a previously unknown mechanism for the beneficial role of CaP biomaterials in bone repair and the role of phosphate ions in bone physiology and regeneration. These findings also begin to shed light on the role of ATP metabolism in bone homeostasis, which may be exploited to treat bone metabolic diseases. PMID:24395775

  8. Differential protein expression in the midgut of Culex quinquefasciatus mosquitoes induced by the insecticide temephos.

    PubMed

    Games, P D; Alves, S N; Katz, B B; Tomich, J M; Serrão, J E

    2016-09-01

    Mosquitoes are vectors for pathogens of malaria, lymphatic filariasis, dengue, chikungunya, yellow fever and Japanese encephalitis. Culex quinquefasciatus Say, 1823 (Diptera: Culicidae) is a known vector of lymphatic filariasis. Its control in Brazil has been managed using the organophosphate temephos. Studies examining the proteins of Cx. quinquefasciatus that are differentially expressed in response to temephos further understanding of the modes of action of the insecticide and may potentially identify resistance factors in the mosquito. In the present study, a comparative proteomic analysis, using 2-dimensional electrophoresis coupled with matrix-assisted laser desorption/ionization (MALDI) time of flight (TOF)/TOF mass spectrometry, and bioinformatics analyses were performed to identify midgut proteins in Cx. quinquefasciatus larvae that were differentially expressed in response to exposure to temephos relative to those in untreated controls. A total of 91 protein spots were differentially expressed; 40 were upregulated and 51 were downregulated by temephos. A total of 22 proteins, predominantly upregulated, were identified as known to play a role in the immune response, whereas the downregulated proteins were involved in energy and protein catabolism. This is the first proteome study of the midgut of Cx. quinquefasciatus and it provides insights into the molecular mechanisms of insecticide-induced responses in the mosquito.

  9. Retinoic acid inducible gene-I and melanoma differentiation-associated gene 5 are induced but not essential for dengue virus induced type I interferon response.

    PubMed

    Qin, Cheng-Feng; Zhao, Hui; Liu, Zhong-Yu; Jiang, Tao; Deng, Yong-Qiang; Yu, Xu-Dong; Yu, Man; Qin, E-De

    2011-08-01

    Dengue viruses (DENVs) are important human pathogens that cause mild dengue fever, and severe dengue hemorrhagic fever/dengue shock syndrome, and no vaccine or antiviral therapy are currently available. At the initial stage of DENV infection, host pattern recognition receptors are responsible for sensing viral proteins or nucleic acids and initiating innate antiviral responses, including the activation of type I interferon (IFN) and proinflammatory cytokines. Two RNA helicases, retinoic acid inducible gene-I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5), are recently identified as cytoplasmic PPRs for virus infection. Here, in this study the involvement of RIG-I and MDA5 in DENV-induced IFN-β response A549 cells were investigated. DENV infection readily up-regulated RIG-I expression, activated IRF-3 and RIG-I mRNA transcription, and induced the production of IFN-β in A549 cells in a strain- and serotype-independent manner. While gene silencing of RIG-I by small interfering RNAs failed to significantly inhibit IFN-β production induced by DENV infection. Further experiments demonstrated that MDA5 was also induced by DENV infection, and MDA5 knockout did not block DENV induced IFN-β production in A549 cells. Our results demonstrated that both RIG-I and MDA5 were induced but neither of the two was essential for DENV induced IFN IFN-β response in A549 cells. These findings suggest that innate immune pathway are involved in the recognition of DENV by human non-immune cells, and provide insights for the understanding of the molecular mechanism for DENV-induced antiviral response.

  10. Current-induced forces: a new mechanism to induce negative differential resistance and current-switching effect in molecular junctions

    NASA Astrophysics Data System (ADS)

    Gu, Lei; Fu, Hua-Hua

    2015-12-01

    Current-induced forces can excite molecules, polymers and other low-dimensional materials, which in turn leads to an effective gate voltage through Holstein interaction. Here, by taking a short asymmetric DNA junction as an example, and using the Langevin approach, we find that when suppression of charge transport by the effective gate voltage surpasses the current increase from an elevated voltage bias, the current-voltage (I-V) curves display strong negative differential resistance (NDR) and perfect current-switching characteristics. The asymmetric DNA chain differs in mechanical stability under inverse voltages and the I-V curve is asymmetric about inverse biases, which can be used to understand recent transport experiments on DNA chains, and meanwhile provides a new strategy to realize NDR in molecular junctions and other low-dimensional quantum systems.

  11. The inhibitory effect of vitamin K on RANKL-induced osteoclast differentiation and bone resorption.

    PubMed

    Wu, Wei-Jie; Kim, Min Seuk; Ahn, Byung-Yong

    2015-10-01

    To further understand the correlation between vitamin K and bone metabolism, the effects of vitamins K1, menaquinone-4 (MK-4), and menaquinone-7 (MK-7) on RANKL-induced osteoclast differentiation and bone resorption were comparatively investigated. Vitamin K2 groups (MK-4 and MK-7) were found to significantly inhibit RANKL-medicated osteoclast cell formation of bone marrow macrophages (BMMs) in a dose-dependent manner, without any evidence of cytotoxicity. The mRNA expression of specific osteoclast differentiation markers, such as c-Fos, NFATc1, OSCAR, and TRAP, as well as NFATc1 protein expression and TRAP activity in RANKL-treated BMMs were inhibited by vitamin K2, although MK-4 exhibited a significantly greater efficiency compared to MK-7. In contrast, the same dose of vitamin K1 had no inhibitory effect on RANKL-induced osteoclast cell formation, but increased the expression of major osteoclastogenic genes. Interestingly, vitamins K1, MK-4 and MK-7 all strongly inhibited osteoclastic bone resorption (p < 0.01) in a dose dependent manner. These results suggest that vitamins K1, MK-4 and MK-7 have anti-osteoporotic properties, while their regulation effects on osteoclastogenesis are somewhat different.

  12. A Sphingolipid Inhibitor Induces a Cytokinesis Arrest and Blocks Stage Differentiation in Giardia lamblia▿

    PubMed Central

    Sonda, Sabrina; Štefanić, Saša; Hehl, Adrian B.

    2008-01-01

    Sphingolipid biosynthesis pathways have recently emerged as a promising target for therapeutic intervention against pathogens, including parasites. A key step in the synthesis of complex sphingolipids is the glucosylation of ceramide, mediated by glucosylceramide (GlcCer) synthase, whose activity can be inhibited by PPMP (1-phenyl-2-palmitoylamino-3-morpholino-1-propanol). In this study, we investigated whether PPMP inhibits the proliferation and differentiation of the pathogenic parasite Giardia lamblia, the major cause of parasite-induced diarrhea worldwide. PPMP was found to block in vitro parasite replication in a dose-dependent manner, with a 50% inhibitory concentration of 3.5 μM. The inhibition of parasite replication was irreversible at 10 μM PPMP, a concentration that did not affect mammalian cell metabolism. Importantly, PPMP inhibited the completion of cell division at a specific stage in late cytokinesis. Microscopic analysis of cells incubated with PPMP revealed the aberrant accumulation of cellular membranes belonging to the endoplasmic reticulum network in the caudal area of the parasites. Finally, PPMP induced a 90% reduction in G. lamblia differentiation into cysts, the parasite stage responsible for the transmission of the disease. These results show that PPMP is a powerful inhibitor of G. lamblia in vitro and that as-yet-uncharacterized sphingolipid biosynthetic pathways are potential targets for the development of anti-G. lamblia agents. PMID:18086854

  13. A sphingolipid inhibitor induces a cytokinesis arrest and blocks stage differentiation in Giardia lamblia.

    PubMed

    Sonda, Sabrina; Stefanic, Sasa; Hehl, Adrian B

    2008-02-01

    Sphingolipid biosynthesis pathways have recently emerged as a promising target for therapeutic intervention against pathogens, including parasites. A key step in the synthesis of complex sphingolipids is the glucosylation of ceramide, mediated by glucosylceramide (GlcCer) synthase, whose activity can be inhibited by PPMP (1-phenyl-2-palmitoylamino-3-morpholino-1-propanol). In this study, we investigated whether PPMP inhibits the proliferation and differentiation of the pathogenic parasite Giardia lamblia, the major cause of parasite-induced diarrhea worldwide. PPMP was found to block in vitro parasite replication in a dose-dependent manner, with a 50% inhibitory concentration of 3.5 muM. The inhibition of parasite replication was irreversible at 10 muM PPMP, a concentration that did not affect mammalian cell metabolism. Importantly, PPMP inhibited the completion of cell division at a specific stage in late cytokinesis. Microscopic analysis of cells incubated with PPMP revealed the aberrant accumulation of cellular membranes belonging to the endoplasmic reticulum network in the caudal area of the parasites. Finally, PPMP induced a 90% reduction in G. lamblia differentiation into cysts, the parasite stage responsible for the transmission of the disease. These results show that PPMP is a powerful inhibitor of G. lamblia in vitro and that as-yet-uncharacterized sphingolipid biosynthetic pathways are potential targets for the development of anti-G. lamblia agents. PMID:18086854

  14. Induced nitric oxide synthetase and peroxiredoxin expression in intramucosal poorly differentiated gastric cancer of young patients.

    PubMed

    Hirahashi, Minako; Koga, Yutaka; Kumagai, Reiko; Aishima, Shinichi; Taguchi, Kenichi; Oda, Yoshinao

    2014-04-01

    To investigate the relationship between oxidative stress and gastric carcinogenesis of poorly differentiated adenocarcinoma in young patients, we analyzed the surgically resected specimens of 22 young patients (21-30 years) and 29 older patients (41-72 years) with intramucosal gastric cancer of the poorly differentiated type. We used immunohistochemical staining to evaluate the expression of 8-hydroxydeoxyguanosine (8OHdG), induced nitric oxide synthetase (iNOS), and antioxidant enzymes (thioredoxin [TRX] and peroxiredoxin [PRDX1, 2 and 3]). We assessed these proteins in the cancer, noncancerous gastric foveolar epithelium and noncancerous mucosal neck. In both the young and older patient groups, the 8OHdG and TRX expressions were gradually increased in cancer cells compared with the noncancerous foveolar epithelial cells and the noncancerous mucosal neck cells (P < 0.001). Although the iNOS and PRDXs expressions were increased in the noncancerous mucosal neck cells compared with the noncancerous foveolar epithelial cells, regardless of age (P < 0.001), the iNOS and PRDX2 expression in the cancer cells were significantly reduced in the young patients compared with the older patients (P < 0.001, P < 0.05). In conclusion, the reduced expression of iNOS or PRDX2 may play an important role in the carcinogenesis of gastric cancer associated with Helicobacter pylori-induced chronic active gastritis in young patients.

  15. FGF8 regulates myogenesis and induces Runx2 expression and osteoblast differentiation in cultured cells.

    PubMed

    Omoteyama, Kazuki; Takagi, Minoru

    2009-03-01

    In the current study, treatment of the rat osteogenic cell line ROB-C26 cells with fibroblast growth factor 8 (FGF8) stimulated alkaline phosphatase (ALP) activity, and also induced the expression of the Runx2 transcription factor, and increased the activity of a luciferase reporter gene containing the osteocalcin (OCN) promoter and six copies of the osteoblast specific cis-acting element 2 (OSE2) response element. In contrast, FGF8 treatment of the mouse myoblast cell line C2C12 inhibited the expression of desmin and the synthesis of myotubes. The expression of MyoD, Myogenin, Foxc2, and Hand1 was also decreased by FGF8. Transient expression of Foxc2 in C2C12 cells induced the expression of Hand1, and chromatin immunoprecipitation (ChIP) analysis indicated that Foxc2 binds to the promoter region of the Hand1 gene. These results indicated that Foxc2 is directly involved in the regulation of Hand1 expression. The results of the current study indicate that FGF8 regulates myoblast differentiation through the regulation of MyoD expression, and that this regulation is independent of Hand1 in cultured cells. Conversely, FGF8 supports bone development and cell differentiation though the induction of Runx2 expression.

  16. Spirulina phycocyanin induces differential protein expression and apoptosis in SKOV-3 cells.

    PubMed

    Pan, Ruowang; Lu, Rongmao; Zhang, Ying; Zhu, Mei; Zhu, Wen; Yang, Rongrong; Zhang, Enyong; Ying, Jun; Xu, Teng; Yi, Huiguang; Li, Jinsong; Shi, Mengru; Zhou, Li; Xu, Zuyuan; Li, Peizhen; Bao, Qiyu

    2015-11-01

    The present study was designed to determine the effects of phycocyanin (PC) on Human ovarian cancer SKOV-3 cells and the underlying molecular mechanisms of action. The inhibitory effects of PC on the cell proliferation were detected by MTT assay. The IC50 values of PC were 182.0μM and 133.6μM for 24h and 48h exposure, respectively. PC induced apoptosis in SKOV-3 cells was observed by electron microscopy and flow cytometry. The apoptosis rate was increased from 1.6% to 19.8% after PC exposure. The fluorescence intensity of ROS and the activities of Caspase-3, Caspase-8, and Caspase-9 were increased. Differentiated expression protein spots were selected and identified using proteomic techniques. There were 698±73 and 683±79 protein spots resolved in untreated and PC-treated cells, respectively. Forty five differential protein spots were analyzed by MALDI-TOF-MS, including mtSSB, PSME3, and nucleolin. The mRNA expression profiles determined by RT-PCR were consistent with that of the two-dimensional electrophoresis. The decreased proteins such as HSP60, nucleolin, PPase, peroxiredoxin-4 and the increased protein (mtSSB) were identified in SKOV-3 cells after PC treatment, indicating that the effects of PC on tumor cell apoptosis may be relate to multiple target proteins. And the mitochondrial pathway may be the main pathway for PC-induced apoptosis. PMID:26410814

  17. Nanotopography Promotes Pancreatic Differentiation of Human Embryonic Stem Cells and Induced Pluripotent Stem Cells.

    PubMed

    Kim, Jong Hyun; Kim, Hyung Woo; Cha, Kyoung Je; Han, Jiyou; Jang, Yu Jin; Kim, Dong Sung; Kim, Jong-Hoon

    2016-03-22

    Although previous studies suggest that nanotopographical features influence properties and behaviors of stem cells, only a few studies have attempted to derive clinically useful somatic cells from human pluripotent stem cells using nanopatterned surfaces. In the present study, we report that polystyrene nanopore-patterned surfaces significantly promote the pancreatic differentiation of human embryonic and induced pluripotent stem cells. We compared different diameters of nanopores and showed that 200 nm nanopore-patterned surfaces highly upregulated the expression of PDX1, a critical transcription factor for pancreatic development, leading to an approximately 3-fold increase in the percentage of differentiating PDX1(+) pancreatic progenitors compared with control flat surfaces. Furthermore, in the presence of biochemical factors, 200 nm nanopore-patterned surfaces profoundly enhanced the derivation of pancreatic endocrine cells producing insulin, glucagon, or somatostatin. We also demonstrate that nanopore-patterned surface-induced upregulation of PDX1 is associated with downregulation of TAZ, suggesting the potential role of TAZ in nanopore-patterned surface-mediated mechanotransduction. Our study suggests that appropriate cytokine treatments combined with nanotopographical stimulation could be a powerful tool for deriving a high purity of desired cells from human pluripotent stem cells. PMID:26900863

  18. Pinoresinol inhibits proliferation and induces differentiation on human HL60 leukemia cells.

    PubMed

    Sepporta, Maria Vittoria; Mazza, Teresa; Morozzi, Guido; Fabiani, Roberto

    2013-01-01

    Pinoresinol (PIN), one of the simplest lignans, is the precursor of other dietary lignans that are present in whole-grain cereals, legumes, fruits, and other vegetables. Several experimental and epidemiological evidences suggest that lignans may prevent human cancer in different organs. In this study we investigated the chemopreventive properties of PIN on cell lines derived from different sites either expressing or not the functional tumor suppressor protein p53. It was found that PIN inhibited the proliferation of p53 wild type colon and prostate tumor cells (HCT116 and LNCaP) while in breast cells the inhibition of growth was observed only in p53 mutant cells (MDA-MB-231). A potent antiproliferative activity of PIN was also observed on p53 null cells HL60 (IC50% 8 μM), their multidrug resistant variant HL60R (IC50% 32 μM) and K562. On HL60 cells, PIN caused a block of cell cycle in the G0/G1 phase, induced a weak proapoptotic effect but it was a good trigger of differentiation (NBT reduction and CD11b expression). PIN caused an upregulation of the CDK inhibitor p21(WAF1/Cip1) both at mRNA and protein levels so suggesting that this could be a mechanism by which PIN reduced proliferation and induced differentiation on HL60 cells.

  19. Nanotopography Promotes Pancreatic Differentiation of Human Embryonic Stem Cells and Induced Pluripotent Stem Cells.

    PubMed

    Kim, Jong Hyun; Kim, Hyung Woo; Cha, Kyoung Je; Han, Jiyou; Jang, Yu Jin; Kim, Dong Sung; Kim, Jong-Hoon

    2016-03-22

    Although previous studies suggest that nanotopographical features influence properties and behaviors of stem cells, only a few studies have attempted to derive clinically useful somatic cells from human pluripotent stem cells using nanopatterned surfaces. In the present study, we report that polystyrene nanopore-patterned surfaces significantly promote the pancreatic differentiation of human embryonic and induced pluripotent stem cells. We compared different diameters of nanopores and showed that 200 nm nanopore-patterned surfaces highly upregulated the expression of PDX1, a critical transcription factor for pancreatic development, leading to an approximately 3-fold increase in the percentage of differentiating PDX1(+) pancreatic progenitors compared with control flat surfaces. Furthermore, in the presence of biochemical factors, 200 nm nanopore-patterned surfaces profoundly enhanced the derivation of pancreatic endocrine cells producing insulin, glucagon, or somatostatin. We also demonstrate that nanopore-patterned surface-induced upregulation of PDX1 is associated with downregulation of TAZ, suggesting the potential role of TAZ in nanopore-patterned surface-mediated mechanotransduction. Our study suggests that appropriate cytokine treatments combined with nanotopographical stimulation could be a powerful tool for deriving a high purity of desired cells from human pluripotent stem cells.

  20. Blockade of LFA-1 augments in vitro differentiation of antigen-induced Foxp3+ Treg cells

    PubMed Central

    Verhagen, Johan; Wraith, David C.

    2014-01-01

    Adoptive transfer of antigen-specific, in vitro-induced Foxp3+ Treg (iTreg) cells protects against autoimmune disease. To generate antigen-specific iTreg cells at high purity, however, remains a challenge. Whereas polyclonal T cell stimulation with anti-CD3 and anti-CD28 antibody yields Foxp3+ iTreg cells at a purity of 90–95%, antigen-induced iTreg cells typically do not exceed a purity of 65–75%, even in a TCR-transgenic model. In a similar vein to thymic Treg cell selection, iTreg cell differentiation is influenced not only by antigen recognition and the availability of TGF-β but also by co-factors including costimulation and adhesion molecules. In this study, we demonstrate that blockade of the T cell integrin Leukocyte Function-associated Antigen-1 (LFA-1) during antigen-mediated iTreg cell differentiation augments Foxp3 induction, leading to approximately 90% purity of Foxp3+ iTreg cells. This increased efficacy not only boosts the yield of Foxp3+ iTreg cells, it also reduces contamination with activated effector T cells, thus improving the safety of adoptive transfer immunotherapy. PMID:25108241

  1. Directed Differentiation of Human-Induced Pluripotent Stem Cells to Mesenchymal Stem Cells.

    PubMed

    Lian, Qizhou; Zhang, Yuelin; Liang, Xiaoting; Gao, Fei; Tse, Hung-Fat

    2016-01-01

    Multipotent stromal cells, also known as mesenchymal stem cells (MSCs), possess great potential to generate a wide range of cell types including endothelial cells, smooth muscle cells, bone, cartilage, and lipid cells. This protocol describes in detail how to perform highly efficient, lineage-specific differentiation of human-induced pluripotent stem cells (iPSCs) with an MSCs fate. The approach uses a clinically compliant protocol with chemically defined media, feeder-free conditions, and a CD105 positive and CD24 negative selection to achieve a single cell-based MSCs derivation from differentiating human pluripotent cells in approximately 20 days. Cells generated with this protocol express typical MSCs surface markers and undergo adipogenesis, osteogenesis, and chondrogenesis similar to adult bone marrow-derived MSCs (BM-MSCs). Nonetheless, compared with adult BM-MSCs, iPSC-MSCs display a higher proliferative capacity, up to 120 passages, without obvious loss of self-renewal potential and constitutively express MSCs surface antigens. MSCs generated with this protocol have numerous applications, including expansion to large scale cell numbers for tissue engineering and the development of cellular therapeutics. This approach has been used to rescue limb ischemia, allergic disorders, and cigarette smoke-induced lung damage and to model mesenchymal and vascular disorders of Hutchinson-Gilford progeria syndrome (HGPS). PMID:27236679

  2. Biochemical correlates of thiazolidinedione-induced adipocyte differentiation by high-resolution magic angle spinning NMR spectroscopy.

    PubMed

    Chen, Jin-Hong; Enloe, Brian M; Weybright, Patrick; Campbell, Natalee; Dorfman, David; Fletcher, Christopher D; Cory, D G; Singer, Samuel

    2002-10-01

    Thiazolidinediones, a class of synthetic ligands to the peroxisome proliferator-activated receptor-gamma, induce terminal adipocyte differentiation of 3T3 F442A cells, and have already been used as alternative therapeutic agents for the treatment of liposarcoma in clinical trials. The biochemical changes occurring in the 3T3 F442A cell line and well-differentiated liposarcoma following induction of adipocyte differentiation with the thiazolidinedione troglitazone were measured using high-resolution magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy. 3T3 F442A cell differentiation was characterized by a large accumulation of intracellular triglyceride and withdrawal from the cell cycle. Phosphatidylcholine (PTC), phosphocholine (PC), myo-inositol, and glycerol were found to be possible biochemical markers for adipocyte differentiation induced by thiazolidenediones. The molar ratio of PTC to PC increased fourfold in differentiated 3T3 F442A cells compared to undifferentiated cells, suggesting a substantial increase in CTP:phosphocholine cytidylyltransferase activity with differentiation. A 2.8-fold increase in the PTC:PC ratio was observed in the lipoma-like well-differentiated liposarcoma of three patients who were treated with troglitazone when compared to liposarcoma from patients not treated with this drug. Thus, this ratio may be an NMR-detectable marker of troglitazone efficacy and response to differentiation therapy for liposarcoma.

  3. miR-21 induces myofibroblast differentiation and promotes the malignant progression of breast phyllodes tumors.

    PubMed

    Gong, Chang; Nie, Yan; Qu, Shaohua; Liao, Jian-You; Cui, Xiuying; Yao, Herui; Zeng, Yunjie; Su, Fengxi; Song, Erwei; Liu, Qiang

    2014-08-15

    Phyllodes tumors of breast, even histologically diagnosed as benign, can recur locally and have metastatic potential. Histologic markers only have limited value in predicting the clinical behavior of phyllodes tumors. It remains unknown what drives the malignant progression of phyllodes tumors. We found that the expression of myofibroblast markers, α-smooth muscle actin (α-SMA), fibroblast activation protein (FAP), and stromal cell-derived factor-1 (SDF-1), is progressively increased in the malignant progression of phyllodes tumors. Microarray showed that miR-21 was one of the most significantly upregulated microRNAs in malignant phyllodes tumors compared with benign phyllodes tumors. In addition, increased miR-21 expression was primarily localized to α-SMA-positive myofibroblasts. More importantly, α-SMA and miR-21 are independent predictors of recurrence and metastasis, with their predictive value of recurrence better than histologic grading. Furthermore, miR-21 mimics promoted, whereas miR-21 antisense oligos inhibited, the expression of α-SMA, FAP, and SDF-1, as well as the proliferation and invasion of primary stromal cells of phyllodes tumors. The ability of miR-21 to induce myofibroblast differentiation was mediated by its regulation on Smad7 and PTEN, which regulate the migration and proliferation, respectively. In breast phyllodes tumor xenografts, miR-21 accelerated tumor growth, induced myofibroblast differentiation, and promoted metastasis. This study suggests an important role of myofibroblast differentiation in the malignant progression of phyllodes tumors that is driven by increased miR-21.

  4. High and Low LET Radiation Differentially Induce Normal Tissue Damage Signals

    SciTech Connect

    Niemantsverdriet, Maarten; Goethem, Marc-Jan van; Bron, Reinier; Hogewerf, Wytse; Brandenburg, Sytze; Langendijk, Johannes A.; Luijk, Peter van; Coppes, Robert P.

    2012-07-15

    Purpose: Radiotherapy using high linear energy transfer (LET) radiation is aimed at efficiently killing tumor cells while minimizing dose (biological effective) to normal tissues to prevent toxicity. It is well established that high LET radiation results in lower cell survival per absorbed dose than low LET radiation. However, whether various mechanisms involved in the development of normal tissue damage may be regulated differentially is not known. Therefore the aim of this study was to investigate whether two actions related to normal tissue toxicity, p53-induced apoptosis and expression of the profibrotic gene PAI-1 (plasminogen activator inhibitor 1), are differentially induced by high and low LET radiation. Methods and Materials: Cells were irradiated with high LET carbon ions or low LET photons. Cell survival assays were performed, profibrotic PAI-1 expression was monitored by quantitative polymerase chain reaction, and apoptosis was assayed by annexin V staining. Activation of p53 by phosphorylation at serine 315 and serine 37 was monitored by Western blotting. Transfections of plasmids expressing p53 mutated at serines 315 and 37 were used to test the requirement of these residues for apoptosis and expression of PAI-1. Results: As expected, cell survival was lower and induction of apoptosis was higher in high -LET irradiated cells. Interestingly, induction of the profibrotic PAI-1 gene was similar with high and low LET radiation. In agreement with this finding, phosphorylation of p53 at serine 315 involved in PAI-1 expression was similar with high and low LET radiation, whereas phosphorylation of p53 at serine 37, involved in apoptosis induction, was much higher after high LET irradiation. Conclusions: Our results indicate that diverse mechanisms involved in the development of normal tissue damage may be differentially affected by high and low LET radiation. This may have consequences for the development and manifestation of normal tissue damage.

  5. Low intensity lasers differently induce primary human osteoblast proliferation and differentiation.

    PubMed

    Oliveira, Flávia A; Matos, Adriana A; Santesso, Mariana R; Tokuhara, Cintia K; Leite, Aline L; Bagnato, Vanderley S; Machado, Maria A A M; Peres-Buzalaf, Camila; Oliveira, Rodrigo C

    2016-10-01

    Among various compounds used in research and clinic for degenerative bone diseases, low level laser therapy (LLLT), comprising low level lasers (LLL) and light emitting diodes (LEDs), has been investigated regarding its effects on bone metabolism. They have specific wavelengths but in general act as a cellular biomodulator, and as a therapeutic agent, rebalancing and normalizing their activity. However, they are not standardized yet, since their parameters of use are relevant for the effects and mechanisms of action. Therefore, the aim of this study was to compare the influence of two spectrums of LLL and LED phototherapy, at the same energy densities (10 and 50J/cm(2)), on human osteoblasts proliferation and differentiation. The involvement of ERK signaling on proliferation was also investigated by evaluating its activation during proliferation under different phototherapies by western blotting and CFSE-based osteoblast proliferation was measured in a presence or absence of the ERK-specific inhibitor. Osteogenic differentiation was evaluated through in vitro mineralization and gene expression of type I collagen (COL1A1) and osteonectin (SPARC) by Real Time- PCR. Increases in viable cells and proliferation were obtained after irradiation, regardless of LLLT type. However, only red at 10J/cm(2) and infrared at both doses, but not LED, induced ERK1/2 activation. In the presence of ERK inhibitor, the LLL-induced proliferation was prevented. In addition, while COL1A1 gene expression was upregulated by red laser, SPARC does so by infrared stimulation. However, LED, at both doses, increased both COL1A1 and SPARC expression. All LLLT increased mineralization, dependent on the dose and time. Thus, LLL and LED differently modulated the metabolism of human osteoblasts, increasing proliferation by mechanism dependent or not of ERK signaling activation and osteogenic differentiation markers.

  6. Jagged1-selective notch signaling induces smooth muscle differentiation via a RBP-Jkappa-dependent pathway.

    PubMed

    Doi, Hiroshi; Iso, Tatsuya; Sato, Hiroko; Yamazaki, Miki; Matsui, Hiroki; Tanaka, Toru; Manabe, Ichiro; Arai, Masashi; Nagai, Ryozo; Kurabayashi, Masahiko

    2006-09-29

    The Notch signaling pathway plays a crucial role in specifying cellular fates by interaction between cellular neighbors; however, the molecular mechanism underlying smooth muscle cell (SMC) differentiation by Notch signaling has not been well characterized. Here we demonstrate that Jagged1-Notch signaling promotes SMC differentiation from mesenchymal cells. Overexpression of the Notch intracellular domain, an activated form of Notch, up-regulates the expression of multiple SMC marker genes including SMC-myosin heavy chain (Sm-mhc) in mesenchymal 10T1/2 cells, but not in non-mesenchymal cells. Physiological Notch stimulation by its ligand Jagged1, but not Dll4, directly induces Sm-mhc expression in 10T1/2 cells without de novo protein synthesis, indicative of a ligand-selective effect. Jagged1-induced expression of SM-MHC was blocked bygamma-secretase inhibitor, N-(N-(3,5-difluorophenyl)-l-alanyl)-S-phenylglycine t-butyl ester, which impedes Notch signaling. Using Rbp-jkappa-deficient cells and site-specific mutagenesis of the SM-MHC gene, we show that such an induction is independent of the myocardin-serum response factor-CArG complex, but absolutely dependent on RBP-Jkappa, a major mediator of Notch signaling, and its cognate binding sequence. Of importance, Notch signaling and myocardin synergistically activate SM-MHC gene expression. Taken together, these data suggest that the Jagged1-Notch pathway constitutes an instructive signal for SMC differentiation through an RBP-Jkappa-dependent mechanism and augments gene expression mediated by the myocardin-SRF-CArG complex. Given that Notch pathway components are expressed in vascular SMC during normal development and disease, Notch signaling is likely to play a pivotal role in such situations to modulate the vascular smooth muscle cell phenotype. PMID:16867989

  7. Low intensity lasers differently induce primary human osteoblast proliferation and differentiation.

    PubMed

    Oliveira, Flávia A; Matos, Adriana A; Santesso, Mariana R; Tokuhara, Cintia K; Leite, Aline L; Bagnato, Vanderley S; Machado, Maria A A M; Peres-Buzalaf, Camila; Oliveira, Rodrigo C

    2016-10-01

    Among various compounds used in research and clinic for degenerative bone diseases, low level laser therapy (LLLT), comprising low level lasers (LLL) and light emitting diodes (LEDs), has been investigated regarding its effects on bone metabolism. They have specific wavelengths but in general act as a cellular biomodulator, and as a therapeutic agent, rebalancing and normalizing their activity. However, they are not standardized yet, since their parameters of use are relevant for the effects and mechanisms of action. Therefore, the aim of this study was to compare the influence of two spectrums of LLL and LED phototherapy, at the same energy densities (10 and 50J/cm(2)), on human osteoblasts proliferation and differentiation. The involvement of ERK signaling on proliferation was also investigated by evaluating its activation during proliferation under different phototherapies by western blotting and CFSE-based osteoblast proliferation was measured in a presence or absence of the ERK-specific inhibitor. Osteogenic differentiation was evaluated through in vitro mineralization and gene expression of type I collagen (COL1A1) and osteonectin (SPARC) by Real Time- PCR. Increases in viable cells and proliferation were obtained after irradiation, regardless of LLLT type. However, only red at 10J/cm(2) and infrared at both doses, but not LED, induced ERK1/2 activation. In the presence of ERK inhibitor, the LLL-induced proliferation was prevented. In addition, while COL1A1 gene expression was upregulated by red laser, SPARC does so by infrared stimulation. However, LED, at both doses, increased both COL1A1 and SPARC expression. All LLLT increased mineralization, dependent on the dose and time. Thus, LLL and LED differently modulated the metabolism of human osteoblasts, increasing proliferation by mechanism dependent or not of ERK signaling activation and osteogenic differentiation markers. PMID:27521889

  8. Mechanism study for hypoxia induced differentiation of insulin-producing cells from umbilical cord blood-derived mesenchymal stem cells.

    PubMed

    Sun, Bo; Meng, Xian-Hui; Liu, Rui; Yan, Shancheng; Xiao, Zhong-Dang

    2015-10-23

    Recently, we have successfully obtained functional IPCs efficiently from umbilical cord blood-derived mesenchymal stem cells by using hypoxia treatment. In this study, we further elaborated that the improved function and viability of IPCs are the result of the interaction β cell development pathway and c-Met/HGF axis induced by hypoxia. We found that hypoxia induced c-MET elevation is efficiently initiated the early stage differentiation IPCs from MSCs, and HGF improved the fully differentiation of IPCs by inducing the expression of NGN3. This finding may contribute to understanding β cell development and the development of stem cell therapy for diabetes.

  9. Effects of cellular origin on differentiation of human induced pluripotent stem cell–derived endothelial cells

    PubMed Central

    Zhao, Ming-Tao; Jahanbani, Fereshteh; Lee, Won Hee; Snyder, Michael P.

    2016-01-01

    Human induced pluripotent stem cells (iPSCs) can be derived from various types of somatic cells by transient overexpression of 4 Yamanaka factors (OCT4, SOX2, C-MYC, and KLF4). Patient-specific iPSC derivatives (e.g., neuronal, cardiac, hepatic, muscular, and endothelial cells [ECs]) hold great promise in drug discovery and regenerative medicine. In this study, we aimed to evaluate whether the cellular origin can affect the differentiation, in vivo behavior, and single-cell gene expression signatures of human iPSC–derived ECs. We derived human iPSCs from 3 types of somatic cells of the same individuals: fibroblasts (FB-iPSCs), ECs (EC-iPSCs), and cardiac progenitor cells (CPC-iPSCs). We then differentiated them into ECs by sequential administration of Activin, BMP4, bFGF, and VEGF. EC-iPSCs at early passage (10 < P < 20) showed higher EC differentiation propensity and gene expression of EC-specific markers (PECAM1 and NOS3) than FB-iPSCs and CPC-iPSCs. In vivo transplanted EC-iPSC–ECs were recovered with a higher percentage of CD31+ population and expressed higher EC-specific gene expression markers (PECAM1, KDR, and ICAM) as revealed by microfluidic single-cell quantitative PCR (qPCR). In vitro EC-iPSC–ECs maintained a higher CD31+ population than FB-iPSC–ECs and CPC-iPSC–ECs with long-term culturing and passaging. These results indicate that cellular origin may influence lineage differentiation propensity of human iPSCs; hence, the somatic memory carried by early passage iPSCs should be carefully considered before clinical translation. PMID:27398408

  10. Protein Malnutrition Induces Bone Marrow Mesenchymal Stem Cells Commitment to Adipogenic Differentiation Leading to Hematopoietic Failure

    PubMed Central

    Cunha, Mayara Caldas Ramos; Lima, Fabiana da Silva; Vinolo, Marco Aurélio Ramirez; Hastreiter, Araceli; Curi, Rui; Borelli, Primavera; Fock, Ricardo Ambrósio

    2013-01-01

    Protein malnutrition (PM) results in pathological changes that are associated with peripheral leukopenia, bone marrow (BM) hypoplasia and alterations in the BM microenvironment leading to hematopoietic failure; however, the mechanisms involved are poorly understood. In this context, the BM mesenchymal stem cells (MSCs) are cells intimately related to the formation of the BM microenvironment, and their differentiation into adipocytes is important because adipocytes are cells that have the capability to negatively modulate hematopoiesis. Two-month-old male Balb/c mice were subjected to protein-energy malnutrition with a low-protein diet containing 2% protein, whereas control animals were fed a diet containing 12% protein. The hematopoietic parameters and the expression of CD45 and CD117 positive cells in the BM were evaluated. MSCs were isolated from BM, and their capability to produce SCF, IL-3, G-CSF and GM-CSF were analyzed. The expression of PPAR-γ and C/EBP-α as well as the expression of PPAR-γ and SREBP mRNAs were evaluated in MSCs together with their capability to differentiate into adipocytes in vitro. The malnourished animals had anemia and leukopenia as well as spleen and bone marrow hypoplasia and a reduction in the expression of CD45 and CD117 positive cells from BM. The MSCs of the malnourished mice presented an increased capability to produce SCF and reduced production of G-CSF and GM-CSF. The MSCs from the malnourished animals showed increased expression of PPAR-γ protein and PPAR-γ mRNA associated with an increased capability to differentiate into adipocytes. The alterations found in the malnourished animals allowed us to conclude that malnutrition committed MSC differentiation leading to adipocyte decision and compromised their capacity for cytokine production, contributing to an impaired hematopoietic microenvironment and inducing the bone marrow failure commonly observed in protein malnutrition states. PMID:23516566

  11. Icariin promotes angiogenic differentiation and prevents oxidative stress-induced autophagy in endothelial progenitor cells.

    PubMed

    Tang, Yubo; Jacobi, Angela; Vater, Corina; Zou, Lijin; Zou, Xuenong; Stiehler, Maik

    2015-06-01

    Reduced tissue levels of endothelial progenitor cells (EPCs) and functional impairment of endothelium are frequently observed in patients with diabetes and cardiovascular disease. The vascular endothelium is specifically sensitive to oxidative stress, and this is one of the mechanisms that causes widespread endothelial dysfunction in most cardiovascular diseases and disorders. Hence attention has increasingly been paid to enhance mobilization and differentiation of EPCs for therapeutic purposes. The aim of this study was to investigate whether Icariin, a natural bioactive component known from traditional Chinese Medicine, can induce angiogenic differentiation and inhibit oxidative stress-induced cell dysfunction in bone marrow-derived EPCs (BM-EPCs), and, if so, through what mechanisms. We observed that treatment of BM-EPCs with Icariin significantly promoted cell migration and capillary tube formation, substantially abrogated hydrogen peroxide (H2 O2 )-induced apoptotic and autophagic programmed cell death that was linked to the reduced intracellular reactive oxygen species levels and restored mitochondrial membrane potential. Icariin downregulated endothelial nitric oxide synthase 3, as well as nicotinamide-adenine dinucleotide phosphate-oxidase expression upon H2 O2 induction. These antiapoptotic and antiautophagic effects of Icariin are possibly mediated by restoring the loss of mammalian target of rapamycin /p70S6K/4EBP1 phosphorylation as well as attenuation of ATF2 and ERK1/2 protein levels after H2 O2 treatment. In summary, favorable modulation of the angiogenesis and redox states in BM-EPCs make Icariin a promising proangiogenic agent both enhancing vasculogenesis and protecting against endothelial dysfunction.

  12. Neutrophils are required for 3-methylcholanthrene-initiated, butylated hydroxytoluene-promoted lung carcinogenesis.

    PubMed

    Vikis, Haris G; Gelman, Andrew E; Franklin, Andrew; Stein, Lauren; Rymaszewski, Amy; Zhu, Jihong; Liu, Pengyuan; Tichelaar, Jay W; Krupnick, Alexander S; You, Ming

    2012-12-01

    Multiple studies have shown a link between chronic inflammation and lung tumorigenesis. Inbred mouse strains vary in their susceptibility to methylcholanthrene (MCA)-initiated butylated hydroxytoluene (BHT)-promoted lung carcinogenesis. In the present study we investigated whether neutrophils play a role in strain dependent differences in susceptibility to lung tumor promotion. We observed a significant elevation in homeostatic levels of neutrophils in the lungs of tumor-susceptible BALB/cByJ (BALB) mice compared to tumor-resistant C57BL/6J (B6) mice. Additionally, BHT treatment further elevated neutrophil numbers as well as neutrophil chemoattractant keratinocyte-derived cytokine (KC)/chemokine (C-X-C motif) ligand 1 (Cxcl1) levels in BALB lung airways. Lung CD11c+ cells were a major source of KC expression and depletion of neutrophils in BALB mice resulted in a 71% decrease in tumor multiplicity. However, tumor multiplicity did not depend on the presence of T cells, despite the accumulation of T cells following BHT treatment. These data demonstrate that neutrophils are essential to promote tumor growth in the MCA/BHT two-step lung carcinogenesis model.

  13. FACTORS INFLUENCING AGE AND STRAIN-RELATED SUSCEPTIBILITY TO 3-METHYLCHOLANTHRENE CARCINOGENICITY

    EPA Science Inventory

    Fetal mice are more sensitive to chemical carcinogens than are adults. Further, some strains of mice are more susceptible to chemical carcinogens than others. We have been conducting studies to understand the interactions between age and genetic background underlying these suscep...

  14. Increased cyclooxygenase-2 and thromboxane synthase expression is implicated in diosgenin-induced megakaryocytic differentiation in human erythroleukemia cells.

    PubMed

    Cailleteau, C; Liagre, B; Battu, S; Jayat-Vignoles, C; Beneytout, J L

    2008-09-01

    Differentiation induction as a therapeutic strategy has, so far, the greatest impact in hematopoietic malignancies, most notably leukemia. Diosgenin is a very interesting natural product because, depending on the specific dose used, its biological effect is very different in HEL (human erythroleukemia) cells. For example, at 10 microM, diosgenin induced megakaryocytic differentiation, in contrast to 40 microM diosgenin, which induced apoptosis in HEL cells previously demonstrated using sedimentation field-flow fractionation (SdFFF). The goal of this work focused on the correlation between cyclooxygenase-2 (COX-2) and thromboxane synthase (TxS) and megakaryocytic differentiation induced by diosgenin in HEL cells. Furthermore, the technique of SdFFF, having been validated in our models, was used in this new study as an analytical tool that provided us with more or less enriched differentiated cell fractions that could then be used for further analyses of enzyme protein expression and activity for the first time. In our study, we showed the implication of COX-2 and TxS in diosgenin-induced megakaryocytic differentiation in HEL cells. Furthermore, we showed that the analytical technique of SdFFF may be used as a tool to confirm our results as a function of the degree of cell differentiation.

  15. Simvastatin induces differentiation of rabbit articular chondrocytes via the ERK-1/2 and p38 kinase pathways.

    PubMed

    Han, Yohan; Kim, Song Ja

    2016-08-15

    Statins are competitive inhibitors of hydroxy-methyl-glutaryl Coenzyme A (HMG-CoA) reductase, a key enzyme involved in the conversion of HMG-CoA to the cholesterol precursor mevalonate. Some statins, such as simvastatin (simvastatin), have been shown to have anti-cancer and anti-inflammatory effects, reducing cartilage degradation in osteoarthritic rabbits in vivo. However, the regulatory mechanisms undergirding simvastatin mediated chondrocyte differentiation have not been well elucidated. Thus, we investigated the action and mechanism of simvastatin on differentiation of rabbit articular chondrocytes through western blot analyses, RT-PCR, and immunohistochemical (IHC) and immunofluorescence (IF) staining. Simvastatin treatment was found to induce type II collagen expression and sulfated-proteoglycan synthesis in a dose- and time-dependent manner. Indeed, RT-PCR revealed increased expression of type II collagen on treatment with simvastatin. Both IHC and IF staining indicated differentiation of chondrocytes. Simvastatin treatment reduced activation of ERK-1/2 and stimulated activation of p38 kinase. Inhibition of ERK-1/2 with PD98059 enhanced simvastatin induced differentiation, whereas inhibition of p38 kinase with SB203580 inhibited simvastatin induced differentiation. Simvastatin treatment also inhibits loss of type II collagen in serial monolayer culture. Collectively, our results indicate that ERK-1/2 and p38 kinase regulate simvastatin-induced differentiation of chondrocytes in opposing manners. Thus, these findings suggest that simvastatin may be a potential therapeutic drug for osteoarthritis.

  16. Ascorbic acid inhibits TPA-induced HL-60 cell differentiation by decreasing cellular H₂O₂ and ERK phosphorylation.

    PubMed

    Yiang, Giou-Teng; Chen, Jen-Ni; Wu, Tsai-Kun; Wang, Hsueh-Fang; Hung, Yu-Ting; Chang, Wei-Jung; Chen, Chinshuh; Wei, Chyou-Wei; Yu, Yung-Luen

    2015-10-01

    Retinoic acid (RA), vitamin D and 12-O‑tetradecanoyl phorbol-13-acetate (TPA) can induce HL-60 cells to differentiate into granulocytes, monocytes and macrophages, respectively. Similar to RA and vitamin D, ascorbic acid also belongs to the vitamin family. High‑dose ascorbic acid (>100 µM) induces HL‑60 cell apoptosis and induces a small fraction of HL‑60 cells to express the granulocyte marker, CD66b. In addition, ascorbic acid exerts an anti‑oxidative stress function. Oxidative stress is required for HL‑60 cell differentiation following treatment with TPA, however, the effect of ascorbic acid on HL‑60 cell differentiation in combination with TPA treatment remains to be fully elucidated. The aim of the present study was to investigate the cellular effects of ascorbic acid treatment on TPA-differentiated HL-60 cells. TPA-differentiated HL-60 cells were used for this investigation, this study and the levels of cellular hydrogen peroxide (H2O2), caspase activity and ERK phosphorylation were determined following combined treatment with TPA and ascorbic acid. The results demonstrated that low‑dose ascorbic acid (5 µM) reduced the cellular levels of H2O2 and inhibited the differentiation of HL‑60 cells into macrophages following treatment with TPA. In addition, the results of the present study further demonstrated that low‑dose ascorbic acid inactivates the ERK phosphorylation pathway, which inhibited HL‑60 cell differentiation following treatment with TPA.

  17. Ascorbic acid delivered by mesoporous silica nanoparticles induces the differentiation of human embryonic stem cells into cardiomyocytes.

    PubMed

    Ren, Mingming; Han, Zhen; Li, Jinglai; Feng, Gang; Ouyang, Shuyuan

    2015-11-01

    Embryonic stem (ES) cells offer the potential to generate all cell types in the body, which provide a promising approach to repair tissue damage or dysfunction. In the past decade, great efforts have been made to induce the differentiation of ES cells into numerous types of cells, such as adipocytes, neurocytes and cardiomyocytes. However, the low differentiated efficiency and successful rate limit the development of induction of the differentiation of stem cells for tissue engineering. Here, we utilize ascorbic acid (AA)-loaded fluorescent TRITC-mesoporous silica nanoparticles (TMSN-AA) as a potential tool to induce the differentiation of human ES cells into cardiomyocytes. The treatment of human ES cells by TMSN-AA nanoplex arrests cell cycle at G1 phase and decreases the expression of stemness genes octamer-binding transcription factor 4 (OCT4) and sex determining region Y-box 2 (SOX2), which exhibits more significant induction efficiency of stem cell differentiation than the treatment by AA alone. Furthermore, we have tested the myocardial marker genes cardiac Troponin I (cTnI) and fetal liver kinase 1 (FLK-1), and found these genes are up-regulated by TMSN-AA nanoplex. Importantly, this work demonstrates the more efficient induction efficiency of human ES cells differentiation by the nanoparticle-drug formulation. Our studies reveal a novel approach based on MSNs as nanocarriers to induce the differentiation of human ES cells into cardiomyocytes efficiently and feasibly, and offer the potential perspectives for tissue engineering, eventually in clinical applications.

  18. p38alpha and p38gamma mediate oncogenic ras-induced senescence through differential mechanisms.

    PubMed

    Kwong, Jinny; Hong, Lixin; Liao, Rong; Deng, Qingdong; Han, Jiahuai; Sun, Peiqing

    2009-04-24

    Oncogene-induced senescence is a tumor-suppressive defense mechanism triggered upon activation of certain oncogenes in normal cells. Recently, the senescence response to oncogene activation has been shown to act as a bona fide barrier to cancer development in vivo. Multiple previous studies have implicated the importance of the p38 MAPK pathway in oncogene-induced senescence. However, the contribution of each of the four p38 isoforms (encoded by different genes) to senescence induction is unclear. In the current study, we demonstrated that p38alpha and p38gamma, but not p38beta, play an essential role in oncogenic ras-induced senescence. Both p38alpha and p38gamma are expressed in primary human fibroblasts and are activated upon transduction of oncogenic ras. Small hairpin RNA-mediated silencing of p38alpha or p38gamma expression abrogated ras-induced senescence, whereas constitutive activation of p38alpha and p38gamma caused premature senescence. Furthermore, upon activation by oncogenic ras, p38gamma stimulated the transcriptional activity of p53 by phosphorylating p53 at Ser(33), suggesting that the ability of p38gamma to mediate senescence is at least partly achieved through p53. However, p38alpha contributed to ras-inducted senescence via a p53-indepdendent mechanism in cells by mediating ras-induced expression of p16(INK4A), another key senescence effector. These findings have identified p38alpha and p38gamma as essential components of the signaling pathway that regulates the tumor-suppressing senescence response, providing insights into the molecular mechanisms underlying the differential involvement of the p38 isoforms in senescence induction.

  19. Differential Gene Expression Patterns in Chicken Cardiomyocytes during Hydrogen Peroxide-Induced Apoptosis

    PubMed Central

    Li, Youwen; Guo, Dingzong

    2016-01-01

    Hydrogen peroxide (H2O2) is both an exogenous and endogenous cytotoxic agent that can reliably induce apoptosis in numerous cell types for studies on apoptosis signaling pathways. However, little is known of these apoptotic processes in myocardial cells of chicken, a species prone to progressive heart failure. Sequencing of mRNA transcripts (RNA-Seq) allows for the identification of differentially expressed genes under various physiological and pathological conditions to elucidate the molecular pathways involved, including cellular responses to exogenous and endogenous toxins. We used RNA-seq to examine genes differentially expressed during H2O2-induced apoptosis in primary cultures of embryonic chicken cardiomyocytes. Following control or H2O2 treatment, RNA was extracted and sequencing performed to identify novel transcripts up- or downregulated in the H2O2 treatment group and construct protein−protein interaction networks. Of the 19,268 known and 2,160 novel transcripts identified in both control and H2O2 treatment groups, 4,650 showed significant differential expression. Among them, 55.63% were upregulated and 44.37% downregulated. Initiation of apoptosis by H2O2 was associated with upregulation of caspase-8, caspase-9, and caspase-3, and downregulation of anti-apoptotic genes API5 and TRIA1. Many other differentially expressed genes were associated with metabolic pathways (including ‘Fatty acid metabolism’, ‘Alanine, aspartate, and glutamate metabolism’, and ‘Biosynthesis of unsaturated fatty acids’) and cell signaling pathways (including ‘PPAR signaling pathway’, ‘Adipocytokine signaling pathway’, ‘TGF-beta signaling pathway’, ‘MAPK signaling pathway’, and ‘p53 signaling pathway’). In chicken cardiomyocytes, H2O2 alters the expression of numerous genes linked to cell signaling and metabolism as well as genes directly associated with apoptosis. In particular, H2O2 also affects the biosynthesis and processing of proteins and

  20. NGF induces adult stem Leydig cells to proliferate and differentiate during Leydig cell regeneration

    SciTech Connect

    Zhang, Lei; Wang, Huaxi; Yang, Yan; Liu, Hui; Zhang, Qihao; Xiang, Qi; Ge, Renshan; Su, Zhijian; Huang, Yadong

    2013-06-28

    Highlights: •Nerve growth factor has shown significant changes on mRNA levels during Adult Leydig cells regeneration. •We established the organ culture model of rat seminiferous tubules with ethane dimethyl sulphonate (EDS) treatment. •Nerve growth factor has shown proliferation and differentiation-promoting effects on Adult stem Leydig cells. •Nerve growth factor induces progenitor Leydig cells to proliferate and differentiate and immature Leydig cells to proliferate. -- Abstract: Nerve growth factor (NGF) has been reported to be involved in male reproductive physiology. However, few reports have described the activity of NGF during Leydig cell development. The objective of the present study was to examine the role of NGF during stem-Leydig-cell (SLC) regeneration. We investigated the effects of NGF on Leydig-cell (LC) regeneration by measuring mRNA levels in the adult rat testis after ethane dimethanesulfonate (EDS) treatment. Furthermore, we used the established organ culture model of rat seminiferous tubules to examine the regulation of NGF during SLC proliferation and differentiation using EdU staining, real-time PCR and western blotting. Progenitor Leydig cells (PLCs) and immature Leydig cells (ILCs) were also used to investigate the effects of NGF on LCs at different developmental stages. NGF mRNA levels changed significantly during Leydig-cell regeneration in vivo. In vitro, NGF significantly promoted the proliferation of stem Leydig cells and also induced steroidogenic enzyme gene expression and 3β-HSD protein expression. The data from PLCs and ILCs showed that NGF could increase Cyclin D1 and Hsd 17b3 mRNA levels in PLCs and Cyclin D1 mRNA levels in ILCs. These results indicate that NGF may play an important role during LC regeneration by regulating the proliferation and differentiation of LCs at different developmental stages, from SLCs to PLCs and from PLCs to ILCs. The discovery of this effect of NGF on Leydig cells will provide useful

  1. Sulfuretin induces osteoblast differentiation through activation of TGF-β signaling.

    PubMed

    Song, No-Joon; Kwon, So-Mi; Kim, Suji; Yoon, Hyang-Jin; Seo, Cho-Rong; Jang, Byunghyun; Chang, Seo-Hyuk; Ku, Jin-Mo; Lee, Jeong-Soo; Park, Ki-Moon; Hong, Joung-Woo; Kim, Geun Hyung; Park, Kye Won

    2015-12-01

    The identification and examination of potential determinants controlling the progression of cell fate toward osteoblasts can be intriguing subjects. In this study, the effects of sulfuretin, a major compound isolated from Rhus verniciflua Stokes, on osteoblast differentiation were investigated. Treatments of sulfuretin induced alkaline phosphatase (ALP) activity in mesenchymal C3H10T1/2 cells and mineralization in preosteoblast MC3T3-E1 cells. Pro-osteogenic effects of sulfuretin were consistently observed in freshly isolated primary bone marrow cells. In mechanical studies, sulfuretin specifically induced expression of TGF-β target genes, such as SMAD7 and PAI-1, but not other signaling pathway-related genes. Similar to the results of gene expression analysis, reporter assays further demonstrated TGF-β-specific induction by sulfuretin. Furthermore, disruption of TGF-β signaling using treatment with TGF-β-specific inhibitor, SB-431542, and introduction of SMAD2/3 small interfering RNA impaired the effects of sulfuretin in inducing ALP activity and expression of ALP mRNA. Together, these data indicate that the pro-osteogenic effects of sulfuretin are mediated through activation of TGF-β signaling, further supporting the potential of sulfuretin in the prevention of bone-related diseases such as bone fracture and osteoporosis.

  2. Glycoantigens Induce Human Peripheral Tr1 Cell Differentiation with Gut-homing Specialization*

    PubMed Central

    Kreisman, Lori S. C.; Cobb, Brian A.

    2011-01-01

    The carbohydrate antigen (glycoantigen) PSA from an intestinal commensal bacteria is able to down-regulate inflammatory bowel disease in model mice, suggesting that stimulation with PSA results in regulatory T cell (Treg) generation. However, mechanisms of how peripheral human T cells respond and home in response to commensal antigens are still not understood. Here, we demonstrate that a single exposure to PSA induces differentiation of human peripheral CD4+ T cells into type-Tr1 Tregs. This is in contrast to mouse models where PSA induced the production of Foxp3+ iTregs. The human PSA-induced Tr1 cells are profoundly anergic and exhibit nonspecific bystander suppression mediated by IL-10 secretion. Most surprisingly, glycoantigen exposure provoked expression of gut homing receptors on their surface. These findings reveal a mechanism for immune homeostasis in the gut whereby exposure to commensal glycoantigens provides the requisite information to responding T cells for proper tissue localization (gut) and function (anti-inflammatory/regulatory). PMID:21228275

  3. Retinoic acid alleviates Con A-induced hepatitis and differentially regulates effector production in NKT cells.

    PubMed

    Lee, Kyoo-A; Song, You Chan; Kim, Ga-Young; Choi, Gyeyoung; Lee, Yoon-Sook; Lee, Jung-Mi; Kang, Chang-Yuil

    2012-07-01

    Retinoic acid (RA) is a diverse regulator of immune responses. Although RA promotes natural killer T (NKT) cell activation in vitro by increasing CD1d expression on antigen-presenting cells (APCs), the direct effects of RA on NKT-cell responses in vivo are not known. In the present study, we demonstrated the effect of RA on the severity of Con A-induced hepatitis and molecular changes of NKT cells. First, we demonstrated that Con A-induced liver damage was ameliorated by RA. In correlation with cytokine levels in serum, RA regulated the production of IFN-γ and IL-4 but not TNF-α by NKT cells without influencing the NKT-cell activation status. However, RA did not alleviate α-GalCer-induced liver injury, even though it reduced IFN-γ and IL-4 but not TNF-α levels in serum. This regulation was also detected when liver mononuclear cells (MNCs) or NKT hybridoma cells were treated with RA in vitro. The regulatory effect of RA on NKT cells was mediated by RAR-α, and RA reduced the phosphorylation of MAPK. These results suggest that RA differentially modulates the production of effector cytokines by NKT cells in hepatitis, and the suppressive effect of RA on hepatitis varies with the pathogenic mechanism of liver injury.

  4. IL-22 inhibits epidermal differentiation and induces proinflammatory gene expression and migration of human keratinocytes.

    PubMed

    Boniface, Katia; Bernard, François-Xavier; Garcia, Martine; Gurney, Austin L; Lecron, Jean-Claude; Morel, Franck

    2005-03-15

    IL-22 belongs to a family of cytokines structurally related to IL-10, including IL-19, IL-20, IL-24, and IL-26. In contrast to IL-10, IL-22 has proinflammatory activities. IL-22 signals through a class II cytokine receptor composed of an IL-22-binding chain, IL-22RA1, and the IL-10RB subunit, which is shared with the IL-10R. In the present study, we show that short-term cultured human epidermal keratinocytes express a functional IL-22R but no IL-10R. Accordingly, IL-22 but not IL-10 induces STAT3 activation in keratinocytes. Using a cDNA array screening approach, real-time RT-PCR, and Western blot analysis, we demonstrate that IL-22 up-regulates, in a dose-dependent manner, the expression of S100A7, S100A8, S100A9, a group of proinflammatory molecules belonging to the S100 family of calcium-binding proteins, as well as the matrix metalloproteinase 3, the platelet-derived growth factor A, and the CXCL5 chemokine. In addition, IL-22 induces keratinocyte migration in an in vitro injury model and down-regulates the expression of at least seven genes associated with keratinocyte differentiation. Finally, we show that IL-22 strongly induces hyperplasia of reconstituted human epidermis. Taken together, these results suggest that IL-22 plays an important role in skin inflammatory processes and wound healing.

  5. Successful differentiation to T cells, but unsuccessful B-cell generation, from B-cell-derived induced pluripotent stem cells.

    PubMed

    Wada, Haruka; Kojo, Satoshi; Kusama, Chie; Okamoto, Naoki; Sato, Yorino; Ishizuka, Bunpei; Seino, Ken-ichiro

    2011-01-01

    Forced expression of certain transcription factors in somatic cells results in generation of induced pluripotent stem (iPS) cells, which differentiate into various cell types. We investigated T-cell and B-cell lineage differentiation from iPS cells in vitro. To evaluate the impact of iPS cell source, murine splenic B-cell-derived iPS (B-iPS) cells were generated after retroviral transduction of four transcription factors (Oct4, Sox2, Klf4 and c-Myc). B-iPS cells were identical to embryonic stem (ES) cells and mouse embryonic fibroblast (MEF)-derived iPS cells in morphology, ES cell marker expression as well as teratoma and chimera mouse formation. Both B-iPS and MEF-derived iPS cells differentiated into lymphocytes in OP9 co-culture systems. Both efficiently differentiated into T-cell lineage that produced IFN-γ on T-cell receptor stimulation. However, iPS cells including B-iPS cells were relatively resistant to B-cell lineage differentiation. One of the reasons of the failure of B-cell lineage differentiation seemed due to a defect of Pax5 expression in the differentiated cells. Therefore, current in vitro differentiation systems using iPS cells are sufficient for inducing T-cell but not B-cell lineage. PMID:21135032

  6. Differentiation inducing factor-1 (DIF-1) induces gene and protein expression of the Dictyostelium nuclear calmodulin-binding protein nucleomorphin.

    PubMed

    O'Day, Danton H; Poloz, Yekaterina; Myre, Michael A

    2009-02-01

    The nucleomorphin gene numA1 from Dictyostelium codes for a multi-domain, calmodulin binding protein that regulates nuclear number. To gain insight into the regulation of numA, we assessed the effects of the stalk cell differentiation inducing factor-1 (DIF-1), an extracellular signalling molecule, on the expression of numA1 RNA and protein. For comparison, the extracellular signalling molecules cAMP (mediates chemotaxis, prestalk and prespore differentiation) and ammonia (NH(3)/NH(4)(+); antagonizes DIF) were also studied. Starvation, which is a signal for multicellular development, results in a greater than 80% decrease in numA1 mRNA expression within 4 h. Treatment with ammonium chloride led to a greater than 90% inhibition of numA1 RNA expression within 2 h. In contrast, the addition of DIF-1 completely blocked the decrease in numA1 gene expression caused by starvation. Treatment of vegetative cells with cAMP led to decreases in numA1 RNA expression that were equivalent to those seen with starvation. Western blotting after various morphogen treatments showed that the maintenance of vegetative levels of numA1 RNA by DIF-1 in starved cells was reflected in significantly increased numA1 protein levels. Treatment with cAMP and/or ammonia led to decreased protein expression and each of these morphogens suppressed the stimulatory effects of DIF-1. Protein expression levels of CBP4a, a calcium-dependent binding partner of numA1, were regulated in the same manner as numA1 suggesting this potential co-regulation may be related to their functional relationship. NumA1 is the first calmodulin binding protein shown to be regulated by developmental morphogens in Dictyostelium being upregulated by DIF-1 and down-regulated by cAMP and ammonia. PMID:19000924

  7. Interaction with RXR is necessary for NPM-RAR-induced myeloid differentiation blockade.

    PubMed

    Rush, Elizabeth A; Pollock, Sheri L; Abecassis, Irina; Redner, Robert L

    2013-12-01

    The t(5;17)(q35;q21) APL variant results in expression of a fusion protein linking the N-terminus of nucleophosmin (NPM) to the C-terminus of the retinoic acid receptor alpha (RAR). We have previously shown that NPM-RAR is capable of binding to DNA either as a homodimer or heterodimer with RXR. To determine the biological significance of NPM-RAR/RXR interaction, we developed two mutants of NPM-RAR that showed markedly diminished ability to bind RXR. U937 subclones expressing the NPM-RAR mutants showed significantly less inhibition of vitamin D3/TGFbeta-induced differentiation, compared with NPM-RAR. These results support the hypothesis that RXR interaction is necessary for NPM-RAR-mediated myeloid maturation arrest.

  8. Chiral Differentiation of Amino Acids by In-Source Collision-Induced Dissociation Mass Spectrometry.

    PubMed

    Kong, Xianglei; Huo, Zhaiyi; Zhai, Wei

    2014-01-01

    Chiral recognition of d- and l-amino acids is achieved by a method combining electrospray ionization (ESI) and in-source collision-induced dissociation (CID) mass spectrometry (MS). Trimeric cluster ions [Cu(II)(A)(ref)2-H](+) are formed by ESI of mixtures of d- or l-analyte amino acid (A), chiral reference (ref) and CuSO4. By increasing the applied voltage in the ESI source region, the trimeric ions become unstable and dissociate progressively. Thus chiral differentiation of the analyte can be achieved by comparing the dependence of their relative intensities to a reference ion on applied voltages. The method does not need MS/MS technique, thus can be readily performed on single-stage MS instruments by turning the voltage of sampling cone.

  9. Differential gene expression and lipid metabolism in fatty liver induced by acute ethanol treatment in mice

    SciTech Connect

    Yin Huquan; Kim, Mingoo; Kim, Ju-Han; Kong, Gu; Kang, Kyung-Sun; Kim, Hyung-Lae; Yoon, Byung-IL; Lee, Mi-Ock; Lee, Byung-Hoon

    2007-09-15

    Ethanol induces cumulative liver damage including steatosis, steatohepatitis and cirrhosis. The aim of this study is to investigate the global intrahepatic gene expression profile in the mouse liver treated with ethanol. A single oral dose of 0.5 or 5 g/kg ethanol was administered to male ICR mice, and liver samples were obtained after 6, 24 and 72 h. Histopathological evaluation showed typical fatty livers in the high-dose group at 24 h. Microarray analysis identified 28 genes as being ethanol responsive (two-way ANOVA; p < 0.05), after adjustment by the Benjamini-Hochberg multiple testing correction; these genes displayed {>=} 2-fold induction or repression. The expression of genes that are known to be involved in fatty acid synthesis was examined. The transcript for lipogenic transcription factor, sterol regulatory element (SRE)-binding factor 1 (Srebf1), was upregulated by acute ethanol exposure. Of the genes known to contain SRE or SRE-like sequences and to be regulated by SRE-binding protein 1 (SREBP1), those encoding malic enzyme (Mod1), ATP-citrate lyase (Acly), fatty acid synthase (Fasn) and stearyl-CoA desaturase (Scd1) were induced by ethanol. Quantitative real-time PCR confirmed the changes in the expression levels of the selected genes. The change in the Srebf1 mRNA level correlates well with that of the SREBP1 protein expression as well as its binding to the promoters of the target genes. The present study identifies differentially expressed genes that can be applied to the biomarkers for alcohol-binge-induced fatty liver. These results support the hypothesis by which ethanol-induced steatosis in mice is mediated by the fatty acid synthetic pathway regulated by SREBP1.

  10. S. macrurus myogenic regulatory factors induce mammalian skeletal muscle differentiation: Evidence for functional conservation of MRFs

    PubMed Central

    Kim, Hyun-Jung; Güth, Robert; Jonsson, Colleen B.; Unguez, Graciela A.

    2009-01-01

    The current-producing cells of the electric organ (EO), i.e., electrocytes, in Sternopygus macrurus derive from skeletal muscle fibers. Mature electrocytes are not contractile but they do retain some muscle proteins, are multinucleated, and receive cholinergic innervation. Electrocytes express the myogenic regulatory factors (MRFs) MyoD, myogenin, Myf5 and MRF4 despite their incomplete muscle phenotype. Although S. macrurus MRFs share functional domains that are highly conserved and their expression is confined to the myogenic lineage, their capability to induce the muscle phenotype has not been determined. To test the functional conservation of S. macrurus MRFs to transcriptionally activate skeletal muscle gene expression and induce the myogenic program, we transiently over-expressed S. macrurus MyoD (SmMyoD) and myogenin (SmMyoG) in mouse C3H/10T1/2 and NIH3T3 embryonic cells. RT-PCR and immunolabeling studies showed that SmMyoD and SmMyoG efficiently can convert these two cell lines into multinucleated myotubes that expressed differentiated muscle markers. The levels of myogenic induction by SmMyoD and SmMyoG were comparable to those obtained with mouse MRF homologs. Furthermore, SmMyoD and SmMyoG proteins were able to induce mouse MyoD and myogenin in C3H/10T1/2 cells. We conclude that S. macrurus MRFs are functionally conserved as they can transcriptionally activate skeletal muscle gene expression and induce the myogenic program in mammalian non-muscle cells. Hence, these data suggest that the partial muscle phenotype of electrocytes is not likely due to differences in the MRF-dependent transcriptional program between skeletal muscle and electric organ. PMID:19598116

  11. Early osteoblastic differentiation induced by dexamethasone enhances adenoviral gene delivery to marrow stromal cells.

    PubMed

    Blum, Jeremy S; Parrott, M Brandon; Mikos, Antonios G; Barry, Michael A

    2004-03-01

    We investigated the implications of induced osteogenic differentiation on gene delivery in multipotent rat marrow stromal cells (MSCs). Prior to genetic manipulation cells were cultured with or without osteogenic supplements (5x10(-8) M dexamethasone, 160 microM l-ascorbic acid 2-phosphate, and 10 mM beta-glycerophosphate). Comparison of liposome, retroviral, and adenoviral vectors demonstrated that all three vectors could mediate gene delivery to primary rat MSCs. When these vectors were applied in the absence or presence of osteogenic supplements, we found that MSCs differentiated prior to transduction with adenovirus type 5 vectors produced a 300% increase in transgene expression compared to MSCs that were not exposed to osteogenic supplements. This differentiation effect appeared specific to adenoviral mediated gene delivery, since there was minimal increase in retroviral gene delivery and no increase in liposome gene delivery when MSCs were treated with osteogenic supplements. In addition, we also determined this increase in transgene production to occur at a higher concentration of dexamethasone (5x10(-8) M) in the culture medium of MSCs prior to adenoviral transduction. We found that this increased transgene production could be extended to the osteogenic protein, human bone morphogenetic protein 2 (hBMP-2). When delivered by an adenoviral vector, hBMP-2 transgene production could be increased from 1.4 ng/10(5) cells/3 days to 4.3 ng/10(5) cells/3 days by culture of MSCs with osteogenic supplements prior to transduction. These results indicate that the utility of MSCs as a therapeutic protein delivery mechanism through genetic manipulation can be enhanced by pre-culture of these cells with dexamethasone. PMID:15013104

  12. Demineralized Dentin Matrix Induces Odontoblastic Differentiation of Dental Pulp Stem Cells.

    PubMed

    Liu, Guolin; Xu, Guoquan; Gao, Zhenhua; Liu, Zhenhai; Xu, Junji; Wang, Jinsong; Zhang, Chunmei; Wang, Songlin

    2016-01-01

    The aim of this study was to investigate the effect of demineralized dentin matrix (DDM) on dental pulp stem cells (DPSCs) and the potential of complexes with DPSCs and DDM for mineralized tissue formation. Stem cells derived from the dental pulp of healthy pigs aged 18 months were isolated and cultured. DPSCs were incubated with alpha-minimum essential medium treated with DDM extract at 1 mg/ml (DDM1) or 10 mg/ml (DDM10). The concentrations of 3 growth factors in DDM extract was measured by enzyme-linked immunosorbent assay. Adhesion of DPSCs on DDM and hydroxyapatite-tricalcium phosphate (HA-TCP) surfaces was observed using scanning electron microscopy. Cell proliferation was evaluated with cell counting kit-8 and migration by Transwell migration assays. Odontoblastic differentiation was assessed by alkaline phosphatase (ALP) and alizarin red staining, ALP activity and real-time polymerase chain reaction analysis of markers of ALP, runt-related transcription factor 2, type I collagen, dentin matrix acidic phosphoprotein-1, osteonectin and dentin sialophosphoprotein (DSPP). Finally, DPSCs were combined with DDM and placed subcutaneously in nude mice for 12 weeks; DPSCs combined with HA-TCP and DDM alone served as controls. DDM could promote DPSC adhesion, migration and odontoblastic differentiation. Mineralized tissue formation was observed with the DPSC and DDM combination and the DPSC and HA-TCP combination. The mineralized tissue of the DPSC + DDM combination stained positive for DSPP, similar to the dentin tissue. These results indicate that DDM induces DPSC odontoblastic differentiation, suggesting applications for dentin regeneration.

  13. A screen of suitable inducers for germline differentiation of chicken embryonic stem cells.

    PubMed

    Shi, Qing-Qing; Sun, Min; Zhang, Zhen-Tao; Zhang, Ya-Ni; Elsayed, Ahmed Kamel; Zhang, Lei; Huang, Xiao-Mei; Li, Bi-Chun

    2014-06-10

    Differentiation of germ cells from embryonic stem cells in vitro could have great application for treating infertility and provide an excellent model for uncovering molecular mechanisms of germline generation. In this study, we aim to screen the suitable inducers that may prove the efficiency of driving chicken embryonic stem cells (ES cells) toward germ cells. The male ES cells were separeted into different groups: single retinoic acid (RA) treatment, co-cultured with sertoli cell feeder with RA induction, cultured on matrix proteins (fibronectin, laminin and collagen) with RA treatment, cultured on fibronectin with sertoli cell feeder and RA induction, and single bone morphogenetic protein 4 (BMP4) treatment. Quantitative RT-PCR and immunoourescence were performed to characterize the ES cells differentiation process. The results showed that spermatogonial stem cells (SSCs)-like were not detected in single RA and RA with collagen groups, but were observed in the other groups. The expression of ES specific genes (Nanog and Sox2) was decreased while SSCs marker genes (Dazl, Stra8, integrin α6, integrinβ1 and C-kit) was remarkably increased. The multiple comparsion results showed that the expression of SSCs marker genes in RA with sertoli cells group was significantly higher than the other groups(P<0.05). Collectively, our results suggested that chicken ES cells possess the potency to differentiate into SSCs-like cells in vitro through RA, matrix proteins, sertoli cells and BMP4 induction, of which co-cultured with sertoli cell feeder with RA induction was proved to be the best.

  14. Olig2-expressing progenitor cells preferentially differentiate into oligodendrocytes in cuprizone-induced demyelinated lesions.

    PubMed

    Islam, Mohammad Shyful; Tatsumi, Kouko; Okuda, Hiroaki; Shiosaka, Sadao; Wanaka, Akio

    2009-01-01

    Many oligodendrocyte progenitor cells (OPCs) are found in acute or chronic demyelinated area, but not all of them differentiate efficiently into mature oligodendrocytes in the demyelinated central nervous system (CNS). Recent studies have shown that the basic helix-loop-helix transcription factor Olig2, which stimulates OPCs to differentiate into oligodendrocyte, is strongly up-regulated in many pathological conditions including acute or chronic demyelinating lesions in the adult CNS. Despite their potential role in the treatment of demyelinating diseases, the long-term fate of these up-regulated Olig2 cells has not been identified due to the lack of stable labeling methods. To trace their fate we have used double-transgenic mice, in which we were able to label Olig2-positive cells conditionally with green fluorescent protein (GFP). Demyelination was induced in these mice by feeding cuprizone, a copper chelator. After 6 weeks of cuprizone exposure, GFP-positive (GFP(+)) cells were processed for a second labeling with antibodies to major neural cell markers APC (mature oligodendrocyte marker), GFAP (astrocyte marker), NeuN (neuron marker), Iba1 (microglia marker) and NG2 proteoglycan (oligodendrocyte progenitor marker). More than half of the GFP(+) cells in the external capsule showed co-localization with NG2 proteoglycan. While the percentages of NG2-positive (NG2(+)) and APC-positive (APC(+)) oligodendrocyte lineage cells in cuprizone-treated mice were significantly higher than those in the normal diet group, no significant difference was observed for GFAP-positive (GFAP(+)) astrocytic lineage cells. Our data therefore provide direct evidence that proliferation and differentiation of local and/or recruited Olig2 progenitors contribute to remyelination in demyelinated lesions. PMID:19070638

  15. Differential Incorporation of β-actin as A Component of RNA Polymerase II into Regulatory Regions of Stemness/Differentiation Genes in Retinoic Acid-Induced Differentiated Human Embryonic Carcinoma Cells

    PubMed Central

    Falahzadeh, Khadijeh; Shahhoseini, Maryam; Afsharian, Parvaneh

    2016-01-01

    Objective Nuclear actin is involved in transcription regulation by recruitment of histone modifiers and chromatin remodelers to the regulatory regions of active genes. In recent years, further attention has been focused on the role of actin as a nuclear protein in transcriptional processes. In the current study, the epigenetic role of nuclear actin on transcription regulation of two stemness (OCT4 and NANOG) and two differentiation) NESTIN and PAX6) marker genes was evaluated in a human embryonal carcinoma cell line (NT2) before and after differentiation induction. Materials and Methods In this experimental study, differentiation of embryonal cells was induced by retinoic acid (RA), and quantitative real-time polymerase chain reaction (PCR) was used to evaluate differential expression of marker genes before and 3 days after RA- induced differentiation. Chromatin immunoprecipitation (ChIP) coupled with real-time PCR was then undertaken to monitor the incorporation of β-actin, as a functional component of RNA polymerase II, in the regulatory regions of marker genes. Results Data showed significant change in nuclear actin incorporation into the promoter regions of NESTIN and PAX6 after RA-induction. Conclusion We emphasize the dynamic functional role of nuclear actin in differentiation of embryonal cells and its role as a subunit of RNA polymerase II. PMID:27540526

  16. Sodium butyrate induces differentiation of gastric cancer cells to intestinal cells via the PTEN/phosphoinositide 3-kinase pathway.

    PubMed

    Bai, Zhigang; Zhang, Zhongtao; Ye, Yingjiang; Wang, Shan

    2010-12-01

    NaB (sodium butyrate) inhibits cell proliferation and induces differentiation in a variety of tumour cells. In this study, we aimed to determine whether NaB induced differentiation and regulated the expression of the mucosal factor MUC2 through the PTEN/PI3K (phosphoinositide 3-kinase) pathway. BGC823 cells treated with NaB for 24-72 h showed marked inhibition of cell proliferation and alteration in cellular morphology. NaB treatment markedly increased the expression of PTEN and MUC2, but it decreased the expression of PI3K. These effects were enhanced by intervention with PI3K inhibitors and were reduced by intervention with PTEN siRNA. Hence, we conclude that NaB increased PTEN expression, promoted the expression of MUC2 and induced the differentiation of gastric cancer cells through the PTEN/PI3K signalling pathway.

  17. Understanding Cell Shape Phenotypes Associated with Stem Cell Differentiation Induced by Topographical Cues of Nanofiber Microenvironment

    NASA Astrophysics Data System (ADS)

    Chen, Desu; Sarkar, Sumona; Losert, Wolfgang

    It is increasingly important to understand cell responses to bioinspired material structures and topographies designed to guide cell functional alterations. In this study, we investigated association between early stage cell morphological response and osteogenic differentiation of human bone marrow stromal cells (hBMSCs) induced by poly(ɛ-caprolactone) (PCL) nanofiber scaffolds (PCL-NF). Accounting for both multi-parametric complexity and biological heterogeneity, we developed an analysis framework based on support vector machines and a multi-cell level averaging method (supercell) to determine the most pronounced cell shape features describing shape phenotypes of cells in PCL-NF compared to cells on flat PCL films. We found that smaller size and more dendritic shape were the major morphological responses of hBMSCs to PCL-NF on day 1 of cell culture. Further, we investigated the shape phenotypes of hBMSCs in PCL-NF of different fiber densities to monitor the transition between 2-D and 3-D topographies. We tracked the genotypic, phenotypic and morphological responses of hBMSCs to different fiber densities at multiple time points to identify correlations between hBMSCs differentiation and early stage morphology in PCL-NF scaffolds.

  18. Differentiation of human induced pluripotent stem cells to mature functional Purkinje neurons.

    PubMed

    Wang, Shuyan; Wang, Bin; Pan, Na; Fu, Linlin; Wang, Chaodong; Song, Gongru; An, Jing; Liu, Zhongfeng; Zhu, Wanwan; Guan, Yunqian; Xu, Zhi-Qing David; Chan, Piu; Chen, Zhiguo; Zhang, Y Alex

    2015-01-01

    It remains a challenge to differentiate human induced pluripotent stem cells (iPSCs) or embryonic stem (ES) cells to Purkinje cells. In this study, we derived iPSCs from human fibroblasts and directed the specification of iPSCs first to Purkinje progenitors, by adding Fgf2 and insulin to the embryoid bodies (EBs) in a time-sensitive manner, which activates the endogenous production of Wnt1 and Fgf8 from EBs that further patterned the cells towards a midbrain-hindbrain-boundary tissue identity. Neph3-positive human Purkinje progenitors were sorted out by using flow cytometry and cultured either alone or with granule cell precursors, in a 2-dimensional or 3-dimensional environment. However, Purkinje progenitors failed to mature further under above conditions. By co-culturing human Purkinje progenitors with rat cerebellar slices, we observed mature Purkinje-like cells with right morphology and marker expression patterns, which yet showed no appropriate membrane properties. Co-culture with human fetal cerebellar slices drove the progenitors to not only morphologically correct but also electrophysiologically functional Purkinje neurons. Neph3-posotive human cells could also survive transplantation into the cerebellum of newborn immunodeficient mice and differentiate to L7- and Calbindin-positive neurons. Obtaining mature human Purkinje cells in vitro has significant implications in studying the mechanisms of spinocerebellar ataxias and other cerebellar diseases. PMID:25782665

  19. Differentiation of human induced pluripotent stem cells to mature functional Purkinje neurons

    PubMed Central

    Wang, Shuyan; Wang, Bin; Pan, Na; Fu, Linlin; Wang, Chaodong; Song, Gongru; An, Jing; Liu, Zhongfeng; Zhu, Wanwan; Guan, Yunqian; Xu, Zhi-Qing David; Chan, Piu; Chen, Zhiguo; Zhang, Y. Alex

    2015-01-01

    It remains a challenge to differentiate human induced pluripotent stem cells (iPSCs) or embryonic stem (ES) cells to Purkinje cells. In this study, we derived iPSCs from human fibroblasts and directed the specification of iPSCs first to Purkinje progenitors, by adding Fgf2 and insulin to the embryoid bodies (EBs) in a time-sensitive manner, which activates the endogenous production of Wnt1 and Fgf8 from EBs that further patterned the cells towards a midbrain-hindbrain-boundary tissue identity. Neph3-positive human Purkinje progenitors were sorted out by using flow cytometry and cultured either alone or with granule cell precursors, in a 2-dimensional or 3-dimensional environment. However, Purkinje progenitors failed to mature further under above conditions. By co-culturing human Purkinje progenitors with rat cerebellar slices, we observed mature Purkinje-like cells with right morphology and marker expression patterns, which yet showed no appropriate membrane properties. Co-culture with human fetal cerebellar slices drove the progenitors to not only morphologically correct but also electrophysiologically functional Purkinje neurons. Neph3-posotive human cells could also survive transplantation into the cerebellum of newborn immunodeficient mice and differentiate to L7- and Calbindin-positive neurons. Obtaining mature human Purkinje cells in vitro has significant implications in studying the mechanisms of spinocerebellar ataxias and other cerebellar diseases. PMID:25782665

  20. The receptor for advanced glycation end products (RAGE) affects T cell differentiation in OVA induced asthma.

    PubMed

    Akirav, Eitan M; Henegariu, Octavian; Preston-Hurlburt, Paula; Schmidt, Ann Marie; Clynes, Raphael; Herold, Kevan C

    2014-01-01

    The receptor for glycation end products (RAGE) has been previously implicated in shaping the adaptive immune response. RAGE is expressed in T cells after activation and constitutively in T cells from patients with diabetes. The effects of RAGE on adaptive immune responses are not clear: Previous reports show that RAGE blockade affects Th1 responses. To clarify the role of RAGE in adaptive immune responses and the mechanisms of its effects, we examined whether RAGE plays a role in T cell activation in a Th2 response involving ovalbumin (OVA)-induced asthma in mice. WT and RAGE deficient wild-type and OT-II mice, expressing a T cell receptor specific for OVA, were immunized intranasally with OVA. Lung cellular infiltration and T cell responses were analyzed by immunostaining, FACS, and multiplex bead analyses for cytokines. RAGE deficient mice showed reduced cellular infiltration in the bronchial alveolar lavage fluid and impaired T cell activation in the mediastinal lymph nodes when compared with WT mice. In addition, RAGE deficiency resulted in reduced OT-II T cell infiltration of the lung and impaired IFNγ and IL-5 production when compared with WT mice and reduced infiltration when transferred into WT hosts. When cultured under conditions favoring the differentiation of T cells subsets, RAGE deficient T cells showed reduced production of IFNγ but increased production of IL-17. Our data show a stimulatory role for RAGE in T activation in OVA-induced asthma. This role is largely mediated by the effects of RAGE on T cell proliferation and differentiation. These findings suggest that RAGE may play a regulatory role in T cell responses following immune activation.

  1. Docosahexaenoic acid ester of phloridzin inhibit lipopolysaccharide-induced inflammation in THP-1 differentiated macrophages.

    PubMed

    Sekhon-Loodu, Satvir; Ziaullah; Rupasinghe, H P Vasantha

    2015-03-01

    Phloridzin or phlorizin (PZ) is a predominant phenolic compound found in apple and also used in various natural health products. Phloridzin shows poor absorption and cellular uptake due to its hydrophilic nature. The aim was to investigate and compare the effect of docosahexaenoic acid (DHA) ester of PZ (PZ-DHA) and its parent compounds (phloridzin and DHA), phloretin (the aglycone of PZ) and cyclooxygenase inhibitory drugs (diclofenac and nimesulide) on production of pro-inflammatory biomarkers in inflammation-induced macrophages by lipopolysaccharide (LPS)-stimulation. Human THP-1 monocytes were seeded in 24-well plates (5×10(5)/well) and treated with phorbol 12-myristate 13-acetate (PMA, 0.1μg/mL) for 48h to induce macrophage differentiation. After 48h, the differentiated macrophages were washed with Hank's buffer and treated with various concentrations of test compounds for 4h, followed by the LPS-stimulation (18h). Pre-exposure of PZ-DHA ester was more effective in reducing tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and cyclooxygenase-2 (COX-2) protein levels compared to DHA and nimesulide. However, diclofenac was the most effective in reducing prostaglandin (PGE2) level by depicting a dose-dependent response. However, PZ-DHA ester and DHA were the most effective in inhibiting the activation of nuclear factor-kappa B (NF-κB) among other test compounds. Our results suggest that PZ-DHA ester might possess potential therapeutic activity to treat inflammation related disorders such as type 2 diabetes, asthma, atherosclerosis and inflammatory bowel disease. PMID:25637769

  2. Multiple Differential Networks Strategy Reveals Carboplatin and Melphalan-Induced Dynamic Module Changes in Retinoblastoma.

    PubMed

    Chen, Cui; Ma, Feng-Wei; Du, Cui-Yun; Wang, Ping

    2016-01-01

    BACKGROUND Retinoblastoma (RB) is the most common malignant tumor of the eye in childhood. The objective of this paper was to investigate carboplatin (CAR)- and melphalan (MEL)-induced dynamic module changes in RB based on multiple (M) differential networks, and to generate systems-level insights into RB progression. MATERIAL AND METHODS To achieve this goal, we constructed M-differential co-expression networks (DCNs), assigned a weight to each edge, and identified seed genes in M DCNs by ranking genes based on their topological features. Starting with seed genes, a module search was performed to explore candidate modules in CAR and MEL condition. M-DMs were detected according to significance evaluations of M-modules, which originated from refinement of candidate modules. Further, we revealed dynamic changes in M-DM activity and connectivity on the basis of significance of Module Connectivity Dynamic Score (MCDS). RESULTS In the present study, M=2, a total of 21 seed genes were obtained. By assessing module search, refinement, and evaluation, we gained 18 2-DMs. Moreover, 3 significant 2-DMs (Module 1, Module 2, and Module 3) with dynamic changes across CAR and MEL condition were determined, and we denoted them as dynamic modules. Module 1 had 27 nodes of which 6 were seed genes and 56 edges. Module 2 was composed of 28 nodes and 54 edges. A total of 28 nodes interacted with 45 edges presented in Module 3. CONCLUSIONS We have identified 3 dynamic modules with changes induced by CAR and MEL in RB, which might give insights in revealing molecular mechanism for RB therapy. PMID:27144687

  3. Differentially expressed proteins in the pancreas of diet-induced diabetic mice.

    PubMed

    Qiu, Linghua; List, Edward O; Kopchick, John J

    2005-09-01

    The pancreas is a heterogeneous organ mixed with both exocrine and endocrine cells. The pancreas is involved in metabolic activities with the endocrine cells participating in the regulation of blood glucose, while the exocrine portion provides a compatible environment for the pancreatic islets and is responsible for secretion of digestive enzymes. The purpose of this study was to identify pancreatic proteins that are differentially expressed in normal mice and those with diet-induced type 2 diabetes (T2DM). In this study, C57BL/6J male mice fed a high fat diet became obese and developed T2DM. The pancreatic protein profiles were compared between control and diabetic mice using two-dimensional gel electrophoresis. Differentially expressed protein "spots" were identified by mass spectrometry. REG1 and REG2 proteins, which may be involved in the proliferation of pancreatic beta cells, were up-regulated very early in the progression of obese mice to T2DM. Glutathione peroxidase, which functions in the clearance of reactive oxidative species, was found to be down-regulated in the diabetic mice at later stages. The RNA levels encoding REG2 and glutathione peroxidase were compared by Northern blot analysis and were consistent to the changes in protein levels between diabetic and control mice. The up-regulation of REG1 and REG2 suggests the effort of the pancreas in trying to ameliorate the hyperglycemic condition by stimulating the proliferation of pancreatic beta cells and enhancing the subsequent insulin secretion. The down-regulation of glutathione peroxidase in pancreas could contribute to the progressive deterioration of beta cell function due to the hyperglycemia-induced oxidative stress. PMID:15961380

  4. Bilobalide induces neuronal differentiation of P19 embryonic carcinoma cells via activating Wnt/β-catenin pathway.

    PubMed

    Liu, Mei; Guo, Jingjing; Wang, Juan; Zhang, Luyong; Pang, Tao; Liao, Hong

    2014-08-01

    Bilobalide, a natural product extracted from Ginkgo biloba leaf, is known to exhibit a number of pharmacological activities. So far, whether it could affect embryonic stem cell differentiation is still unknown. The main aim of this study was to investigate the effect of bilobalide on P19 embryonic carcinoma cells differentiation and the underlying mechanisms. Our results showed that bilobalide induced P19 cells differentiation into neurons in a concentration- and time-dependent manner. We also found that bilobalide promoted neuronal differentiation through activation of Wnt/β-catenin signaling pathway. Exposure to bilobalide increased inactive GSK-3β phosphorylation, further induced the nuclear accumulation of β-catenin, and also up-regulated the expression of Wnt ligands Wnt1 and Wnt7a. Neuronal differentiation induced by bilobalide was totally abolished by XAV939, an inhibitor of Wnt/β-catenin pathway. These results revealed a novel role of bilobalide in neuronal differentiation from P19 embryonic cells acting through Wnt/β-catenin signaling pathway, which would provide a better insight into the beneficial effects of bilobalide in brain diseases.

  5. An ester extract of Cochinchina momordica seeds induces differentiation of melanoma B16 F1 cells via MAPKs signaling.

    PubMed

    Zhao, Lian-Mei; Han, Li-Na; Ren, Feng-Zhi; Chen, Shu-Hong; Liu, Li-Hua; Wang, Ming-Xia; Sang, Mei-Xiang; Shan, Bao-En

    2012-01-01

    Cochinchina momordica seeds (CMS) have been widely used due to antitumor activity by Mongolian tribes of China. However, the details of the underlying mechanisms remain unknown. In the present study, we found that an EtOAc (ethyl ester) extract of CMS (CMSEE) induced differentiation and caused growth inhibition of melanoma B16 F1 cells. CMSEE at the concentration of 5-200 μg/ml exhibited strongest anti-proliferative effects on B16 F1 cells among other CMS fractions (water or petroleum ether). Moreover, CMSEE induced melanoma B16 F1 cell differentiation, characterized by dendrite-like outgrowth, increasing melanogenesis production, as well as enhancing tyrosinase activity. Western blot analysis showed that sustained phosphorylation of p38 MAP accompanied by decrease in ERK1/2 and JNK dephosphorylation were involved in CMSEE-induced B16 F1 cell differentiation. Notably, 6 compounds that were isolated and identified may be responsible for inducing differentiation of CMSEE. These results indicated that CMSEE contributes to the differentiation of B16 F1 cells through modulating MAPKs activity, which may throw some light on the development of potentially therapeutic strategies for melanoma treatment. PMID:23098473

  6. Purple Sweet Potato Leaf Extract Induces Apoptosis and Reduces Inflammatory Adipokine Expression in 3T3-L1 Differentiated Adipocytes.

    PubMed

    Lee, Shou-Lun; Chin, Ting-Yu; Tu, Ssu-Chieh; Wang, Yu-Jie; Hsu, Ya-Ting; Kao, Ming-Ching; Wu, Yang-Chang

    2015-01-01

    Background. Purple sweet potato leaves (PSPL) are widely grown and are considered a healthy vegetable in Taiwan. PSPL contain a high content of flavonoids, and the boiling water-extracted PSPL (PSPLE) is believed to prevent metabolic syndrome. However, its efficacy has not yet been verified. Therefore, we investigated the effect of PSPLE on adipocytes. Methods. The differentiated 3T3-L1 cells used in this study were derived from preadipocytes that were differentiated into adipocytes using an adipogenic agent (insulin, dexamethasone, and 3-isobutyl-1-methylxanthine); approximately 90% of the cells were differentiated using this method. Results. Treating the differentiated 3T3-L1 cells with PSPLE caused a dose-dependent decrease in the number of adipocytes rather than preadipocytes. In addition, treatment with PSPLE resulted in apoptosis of the differentiated 3T3-L1 cells as determined by DAPI analysis and flow cytometry. PSPLE also increased the expression of cleaved caspase-3 and poly ADP-ribose polymerase (PARP). Furthermore, PSPLE induced downregulation of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) gene expression in the differentiated 3T3-L1 cells. Conclusions. These results suggest that PSPLE not only induced apoptosis but also downregulated inflammation-associated genes in the differentiated 3T3-L1 cells. PMID:26170870

  7. Co-expression analysis of differentially expressed genes in hepatitis C virus-induced hepatocellular carcinoma.

    PubMed

    Song, Qingfeng; Zhao, Chang; Ou, Shengqiu; Meng, Zhibin; Kang, Ping; Fan, Liwei; Qi, Feng; Ma, Yilong

    2015-01-01

    The aim of the current study was to investigate the molecular mechanisms underlying hepatitis C virus (HCV)-induced hepatocellular carcinoma (HCC) using the expression profiles of HCV-infected Huh7 cells at different time points. The differentially expressed genes (DEGs) were identified with the Samr package in R software once the data were normalized. Functional and pathway enrichment analysis of the identified DEGs was also performed. Subsequently, MCODE in Cytoscape software was applied to conduct module analysis of the constructed co-expression networks. A total of 1,100 DEGs were identified between the HCV-infected and control samples at 12, 18, 24 and 48 h post-infection. DEGs at 24 and 48 h were involved in the same signaling pathways and biological processes, including sterol biosynthetic processes and tRNA amino-acylation. There were 22 time series genes which were clustered into 3 expression patterns, and the demarcation point of the 2 expression patterns that 401 overlapping DEGs at 24 and 48 h clustered into was 24 h post-infection. tRNA synthesis-related biological processes emerged at 24 and 48 h. Replication and assembly of HCV in HCV-infected Huh7 cells occurred mainly at 24 h post-infection. In view of this, the screened time series genes have the potential to become candidate target molecules for monitoring, diagnosing and treating HCV-induced HCC. PMID:25339452

  8. UV-induced DNA excision repair in rat fibroblasts during immortalization and terminal differentiation in vitro

    SciTech Connect

    Vijg, J.; Mullaart, E.; Berends, F.; Lohman, P.H.; Knook, D.L.

    1986-12-01

    UV-induced DNA excision repair was studied as DNA repair synthesis and dimer removal in rat fibroblast cultures, initiated from either dense or sparse inocula of primary cells grown from skin biopsies. During passaging in vitro an initial increase in DNA repair synthesis, determined both autoradiographically as unscheduled DNA synthesis (UDS) and by means of the BrdU photolysis assay as the number and average size of repair patches, was found to be associated with a morphological shift from small spindle-shaped to large pleiomorphic cells observed over the first twenty generations. In cell populations in growth crisis, a situation exclusively associated with thin-inoculum cultures in which the population predominantly consisted of large pleiomorphic cells, UDS was found to occur at a low level. After development of secondary cultures into immortal cell lines, both repair synthesis and morphology appeared to be the same as in the original primary spindle-shaped cells. At all passages the capacity to remove UV-induced pyrimidine dimers was found to be low, as indicated by the persistence of Micrococcus luteus UV endonuclease-sensitive sites. These results are discussed in the context of terminal differentiation and immortalization of rat fibroblasts upon establishment in vitro.

  9. Carnosine protects against Abeta42-induced neurotoxicity in differentiated rat PC12 cells.

    PubMed

    Fu, Qiuli; Dai, Haibin; Hu, Weiwei; Fan, Yanying; Shen, Yao; Zhang, Weiping; Chen, Zhong

    2008-02-01

    (1) The present study was designed to investigate whether histamine is involved in the protective effect of carnosine on Abeta42-induced impairment in differentiated PC12 cells. (2) PC12 cells were exposed to Abeta42 (5 muM) for 24 h after carnosine (5 mM) applied for 18 h. Histamine receptor antagonists (diphenhydramine, zolantidine, thioperamide, clobenpropit) or histidine decarboxylase inhibitor (alpha-fluoromethylhistidine) were added 15 min before carnosine. Cell viability, glutamate release or cell surface expression of NMDA receptor was examined. (3) Abeta42 caused a concentration-dependent reduction of viability in PC12 cells and pretreatment with carnosine ameliorated this impairment. This amelioration was reversed by the H(3) receptor antagonists thioperamide and clobenpropit, but not by either the H(1) receptor antagonist diphenhydramine or the H(2) receptor antagonist zolantidine. Further, alpha-fluoromethylhistidine, an irreversible inhibitor of histidine decarboxylase, also had no effect. In the presence of Abeta42, carnosine significantly decreased glutamate release and carnosine increased the surface expression of NMDA receptor. (4) These results indicate that the mechanism by which carnosine attenuates Abeta42-induced neurotoxicity is independent of the carnosine-histidine-histamine pathway, but may act through regulation of glutamate release and NMDA receptor trafficking.

  10. Inhibition of inducible nitric oxide synthase and osteoclastic differentiation by Atractylodis Rhizoma Alba extract

    PubMed Central

    Choi, Sung-Ho; Kim, Sung-Jin

    2014-01-01

    Background: Atractylodis Rhizoma Alba (ARA) has been used in Korean folk medicine for constipation, dizziness, and anticancer agent. In the present study, we performed to test whether the methanolic extract of ARA has antioxidant and antiosteoclastogenesis activity in RAW 264.7 macrophage cells. Materials and Methods: Antioxidant capacities were tested by measuring free radical scavenging activity, nitric oxide (NO) levels, reducing power, and inducible nitric oxide synthase (iNOS) expression in response to lipopolysaccharides (LPS). Antiosteoclastogenesis activity was evaluated by performing tartrate-resistant acid phosphatase assay in RAW 264.7 macrophage cells. Results: The extract exerted significant 1,1-diphenyl-2-picrylhydrazyl and NO radical scavenging activity, and it exerted dramatic reducing power. Induction of iNOS and NO by LPS in RAW 264.7 cells was significantly inhibited by the extract, suggesting that the ARA extract inhibits NO production by suppressing iNOS expression. Strikingly, the ARA extracts substantially inhibited the receptor activator of NF-κB ligand-induced osteclastic differentiation of LPS-activated RAW 264.7 cells. The ARA extract contains a significant amount of antioxidant components, including phenolics, flavonoids and anthocyanins. Conclusion: These results suggest that the methanolic extract of ARA exerts significant antioxidant activities potentially via inhibiting free radicals and iNOS induction, thereby leading to the inhibition of osteoclastogenesis. PMID:25298665

  11. Hepatocyte growth factor induces proliferation and differentiation of multipotent and erythroid hemopoietic progenitors

    PubMed Central

    1994-01-01

    Hepatocyte growth factor (HGF) is a mesenchymal derived growth factor known to induce proliferation and "scattering" of epithelial and endothelial cells. Its receptor is the tyrosine kinase encoded by the c- MET protooncogene. Here we show that highly purified recombinant HGF stimulates hemopoietic progenitors to form colonies in vitro. In the presence of erythropoietin, picomolar concentrations of HGF induced the formation of erythroid burst-forming unit colonies from CD34-positive cells purified from human bone marrow, peripheral blood, or umbilical cord blood. The growth stimulatory activity was restricted to the erythroid lineage. HGF also stimulated the formation of multipotent CFU- GEMM colonies. This effect is synergized by stem cell factor, the ligand of the tyrosine kinase receptor encoded by the c-KIT protooncogene, which is active on early hemopoietic progenitors. By flow cytometry analysis, the receptor for HGF was found to be expressed on the cell surface in a fraction of CD34+ progenitors. Moreover, in situ hybridization experiments showed that HGF receptor mRNA is highly expressed in embryonic erythroid cells (megaloblasts). HGF mRNA was also found to be produced in the embryonal liver. These data show that HGF plays a direct role in the control of proliferation and differentiation of erythroid progenitors, and they suggest that it may be one of the long-sought mediators of paracrine interactions between stromal and hemopoietic cells within the hemopoietic microenvironment. PMID:7528222

  12. Differentiation-inducing factor-1 suppresses gene expression of cyclin D1 in tumor cells

    SciTech Connect

    Yasmin, Tania; Takahashi-Yanaga, Fumi . E-mail: yanaga@clipharm.med.kyushu-u.ac.jp; Mori, Jun; Miwa, Yoshikazu; Hirata, Masato; Watanabe, Yutaka; Morimoto, Sachio; Sasaguri, Toshiyuki

    2005-12-16

    To determine the mechanism by which differentiation-inducing factor-1 (DIF-1), a morphogen of Dictyostelium discoideum, inhibits tumor cell proliferation, we examined the effect of DIF-1 on the gene expression of cyclin D1. DIF-1 strongly reduced the expression of cyclin D1 mRNA and correspondingly decreased the amount of {beta}-catenin in HeLa cells and squamous cell carcinoma cells. DIF-1 activated glycogen synthase kinase-3{beta} (GSK-3{beta}) and inhibition of GSK-3{beta} attenuated the DIF-1-induced {beta}-catenin degradation, indicating the involvement of GSK-3{beta} in this effect. Moreover, DIF-1 reduced the activities of T-cell factor (TCF)/lymphoid enhancer factor (LEF) reporter plasmid and a reporter gene driven by the human cyclin D1 promoter. Eliminating the TCF/LEF consensus site from the cyclin D1 promoter diminished the effect of DIF-1. These results suggest that DIF-1 inhibits Wnt/{beta}-catenin signaling, resulting in the suppression of cyclin D1 promoter activity.

  13. Ionizing radiation induces senescence and differentiation of human dental pulp stem cells.

    PubMed

    Havelek, R; Soukup, T; Ćmielová, J; Seifrtová, M; Suchánek, J; Vávrová, J; Mokrý, J; Muthná, D; Řezáčová, M

    2013-01-01

    Head and neck cancer is one of the most common cancers in Europe. Many current anti-cancer treatments, including ionizing radiation, induce apoptosis via DNA damage. Unfortunately, such treatments are non-selective to cancer cells and produce similar toxicity in normal cells, including adult stem cells. One of the fundamental properties of an adult stem cell is that it does not have any tissue-specific structures that allow it to perform specialized functions. However, under certain stimuli, unspecialized adult stem cells can give rise to specialized cells to generate replacements for cells that are lost during one's life or due to injury or disease. Nevertheless, specialization of stem cells must be controlled by specific milieu and also initiated at the proper time, making the entire process beneficial for tissue recovery and maintaining it for a long time. In this paper we assess whether irradiated dental pulp stem cells have maintained open their options to mature into specialized cells, or whether they have lost their unspecialized (immature) state following irradiation. Our findings showed radiation-induced premature differentiation of dental pulp stem cells towards odonto-/osteoblast lineages in vitro. Matrix calcification was visualized from Day 6 or Day 9 following irradiation of cells expressing low or high levels of CD146, respectively.

  14. Fluoxetine Induces Proliferation and Inhibits Differentiation of Hypothalamic Neuroprogenitor Cells In Vitro

    PubMed Central

    Sousa-Ferreira, Lígia; Aveleira, Célia; Botelho, Mariana; Álvaro, Ana Rita; Pereira de Almeida, Luís; Cavadas, Cláudia

    2014-01-01

    A significant number of children undergo maternal exposure to antidepressants and they often present low birth weight. Therefore, it is important to understand how selective serotonin reuptake inhibitors (SSRIs) affect the development of the hypothalamus, the key center for metabolism regulation. In this study we investigated the proliferative actions of fluoxetine in fetal hypothalamic neuroprogenitor cells and demonstrate that fluoxetine induces the proliferation of these cells, as shown by increased neurospheres size and number of proliferative cells (Ki-67+ cells). Moreover, fluoxetine inhibits the differentiation of hypothalamic neuroprogenitor cells, as demonstrated by decreased number of mature neurons (Neu-N+ cells) and increased number of undifferentiated cells (SOX-2+ cells). Additionally, fluoxetine-induced proliferation and maintenance of hypothalamic neuroprogenitor cells leads to changes in the mRNA levels of appetite regulator neuropeptides, including Neuropeptide Y (NPY) and Cocaine-and-Amphetamine-Regulated-Transcript (CART). This study provides the first evidence that SSRIs affect the development of hypothalamic neuroprogenitor cells in vitro with consequent alterations on appetite neuropeptides. PMID:24598761

  15. Ram ion scattering caused by Space Shuttle v x B induced differential charging

    NASA Technical Reports Server (NTRS)

    Katz, I.; Davis, V. A.

    1987-01-01

    Observations of secondary, high-inclination ions streams have been reported in the literature. The authors of these previous papers attributed the source of the secondary ions to a disturbed region in the plasma about 10 m from the Space Shuttle Orbiter. A new theory has been developed which shows how v x B induced differential charging on the plasma diagnostics package (PDP) can scatter the ram ion flux. Some of these ions are reflected back to the PDP and may be the sorce of the observed ion distributions. The effect is unique to large spacecraft; it occurs only when the magnitude of the induced v x B potentials are much larger than the electron thermal energy and of the order of the ion ram energy. That the ion streams observed at large angles must have been reflected from the PDP surface is demonstrated with three-dimensional sheath and particle trajectory calculations using the low earth orbit version of the NASA Charging Analyzer Program (NASCAP/LEO).

  16. Differential Effects of Olanzapine and Haloperidol on MK-801-induced Memory Impairment in Mice

    PubMed Central

    Song, Jae Chun; Seo, Mi Kyoung; Park, Sung Woo; Lee, Jung Goo; Kim, Young Hoon

    2016-01-01

    Objective We investigated the differential effects of the antipsychotic drugs olanzapine and haloperidol on MK-801-induced memory impairment and neurogenesis in mice. Methods MK-801 (0.1 mg/kg) was administered 20 minutes prior to behavioral testing over 9 days. Beginning on the sixth day of MK-801 treatment, either olanzapine (0.05 mg/kg) or haloperidol (0.05 mg/kg) was administered 40 minutes prior to MK-801 for the final 4 days. Spatial memory performance was measured using a Morris water maze (MWM) test for 9 days (four trials/day). Immunohistochemistry with bromodeoxyuridine (BrdU) was used to identify newborn cells labeled in tissue sections from the dentate gyrus of the hippocampus. Results MK-801 administration over 9 days significantly impaired memory performance in the MWM test compared to untreated controls (p<0.05) and these deficits were blocked by treatment with olanzapine (p<0.05) but not haloperidol. The administration of MK-801 also resulted in a decrease in the number of BrdU-labeled cells in the dentate gyrus (28.6%; p<0.01), which was prevented by treatment with olanzapine (p<0.05) but not haloperidol. Conclusion These results suggest that olanzapine has a protective effect against cognitive impairments induced by MK-801 in mice via the stimulating effects of neurogenesis. PMID:27489382

  17. Insulin-like 3-induced rat preantral follicular growth is mediated by growth differentiation factor 9.

    PubMed

    Xue, Kai; Kim, Ji Young; Liu, Jia-yin; Tsang, Benjamin K

    2014-01-01

    The communication of somatic cells and oocytes by intrafollicular paracrine factors is essential for follicular growth in the ovary. Insulin-like 3 (INSL3) is a theca cell-secreted paracrine factor. Androgens and growth differentiation factor 9 (GDF9), an oocyte-derived growth factor, are essential for follicular development. Using a rat preantral follicle culture model, we examined in the present study the influence of INSL3 on preantral follicular growth and the molecular mechanisms involved. We have observed that the receptor for INSL3, relaxin/insulin-like family peptide receptor 2 (RXFP2), was exclusively expressed in oocytes. Recombinant INSL3 stimulated Gdf9 expression, preantral follicular growth, and testosterone synthesis in vitro. Inhibition of the cAMP/protein kinase A signaling pathway (with cAMP antagonist, 8-bromoadenosine 3',5'-cyclic monophosphorothioate, Rp-isomer) attenuated INSL3-induced Gdf9 expression and preantral follicular growth. Moreover, knocking down Gdf9 expression (with small interfering RNA) or inhibiting GDF9 signaling (with SB431542, an activin receptor-like kinase receptor 5 inhibitor, or specific inhibitor of mothers against decapentaplegic homolog 3) or androgen action (with flutamide, an androgen receptor antagonist) suppressed INSL3-induced preantral follicular growth. In addition, LH and DHT regulated the expression of Insl3 mRNA in preantral follicles. These observations suggest that INSL3 is a key theca cell-derived growth factor for preantral follicle and that its action is mediated by GDF9.

  18. Nanopit-induced osteoprogenitor cell differentiation: The effect of nanopit depth

    PubMed Central

    Davison, Martin J; McMurray, Rebecca J; Smith, Carol-Anne; Dalby, Matthew J; Meek, RM Dominic

    2016-01-01

    We aimed to assess osteogenesis in osteoprogenitor cells by nanopits and to assess optimal feature depth. Topographies of depth 80, 220 and 333 nm were embossed onto polycaprolactone discs. Bone marrow–derived mesenchymal stromal cells were seeded onto polycaprolactone discs, suspended in media and incubated. Samples were fixed after 3 and 28 days. Cells were stained for the adhesion molecule vinculin and the osteogenic transcription factor RUNX2 after 3 days. Adhesion was lowest on planar controls and it was the shallowest, and 80-nm-deep pits supported optimal adhesion formation. Deep pits (80 and 220 nm) induced most RUNX2 accumulation. After 28 days, osteocalcin and osteopontin expression were used as markers of osteoblastic differentiation. Deep pits (220 nm) produced cells with the highest concentrations of osteopontin and osteocalcin. All topographies induced higher expression levels than controls. We demonstrated stimulation of osteogenesis in a heterogeneous population of mesenchymal stromal cells. All nanopit depths gave promising results with an optimum depth of 220 nm after 28 days. Nanoscale modification of implant surfaces could optimise fracture union or osteointegration. PMID:27298716

  19. Mechanical load induces sarcoplasmic wounding and FGF release in differentiated human skeletal muscle cultures

    NASA Technical Reports Server (NTRS)

    Clarke, M. S.; Feeback, D. L.

    1996-01-01

    The transduction mechanism (or mechanisms) responsible for converting a mechanical load into a skeletal muscle growth response are unclear. In this study we have used a mechanically active tissue culture model of differentiated human skeletal muscle cells to investigate the relationship between mechanical load, sarcolemma wounding, fibroblast growth factor release, and skeletal muscle cell growth. Using the Flexcell Strain Unit we demonstrate that as mechanical load increases, so too does the amount of sarcolemma wounding. A similar relationship was also observed between the level of mechanical load inflicted on the cells and the amount of bFGF (FGF2) released into the surrounding medium. In addition, we demonstrate that the muscle cell growth response induced by chronic mechanical loading in culture can be inhibited by the presence of an antibody capable of neutralizing the biological activity of FGF. This study provides direct evidence that mechanically induced, sarcolemma wound-mediated FGF release is an important autocrine mechanism for transducing the stimulus of mechanical load into a skeletal muscle growth response.

  20. Inhibitors of differentiation-1 promotes nitrosopyrrolidine-induced transformation of HPV 16-immortalized cervical epithelial cell

    PubMed Central

    Xie, Lingxia; Li, Jinke; Zhang, Yi; Liu, Bao; Peng, Xue; Lin, Yong; Xu, Wenming; Hu, Lina

    2014-01-01

    Our previous study implied a correlation between inhibitors of differentiation-1 (Id-1) and cervical cancer development. However, how Id-1 contributes to cervical carcinogenesis is unknown. In the present study, we used an in vitro transformation model to investigate the role of Id-1 in the transformation of cervical cells. Human papillomavirus (HPV)-immortalized cervical epithelial cells (H8) were successfully transformed by exposure to the carcinogen N-nitrosopyrrolidine (NPYR). The expression of both Id-1 RNA and protein was significantly increased in transformed H8 cells, suggesting a possible role of Id-1 in cervical cell transformation. Ectopic expression of Id-1 in H8 cells potentiated NPYR-induced cell transformation. In contrast, silencing of Id-1 suppressed NPYR-induced H8 cell transformation. In addition, the expression of HPV E6 and E7 oncoproteins was upregulated while that of the tumor suppressors p53 and pRb was suppressed after H8 cell transformation. Our results suggest that Id-1 plays an oncogenic role in HPV-related cervical carcinogenesis, which sheds light on cervical cancer development mechanisms and implies that Id-1 is a potential target for cervical cancer prevention and therapy. PMID:24628854

  1. ARHGEF3 controls HDACi-induced differentiation via RhoA-dependent pathways in acute myeloid leukemias.

    PubMed

    D'Amato, Loredana; Dell'Aversana, Carmela; Conte, Mariarosaria; Ciotta, Alfonso; Scisciola, Lucia; Carissimo, Annamaria; Nebbioso, Angela; Altucci, Lucia

    2015-01-01

    Altered expression and activity of histone deacetylases (HDACs) have been correlated with tumorigenesis. Inhibitors of HDACs (HDACi) induce acetylation of histone and non-histone proteins affecting gene expression, cell cycle progression, cell migration, terminal differentiation and cell death. Here, we analyzed the regulation of ARHGEF3, a RhoA-specific guanine nucleotide exchange factor, by the HDACi MS275 (entinostat). MS275 is a well-known benzamide-based HDACi, which induces differentiation of the monoblastic-like human histiocytic lymphoma cell line U937 to monocytes/macrophages. Incubation of U937 cells with MS275 resulted in an up regulation of ARHGEF3, followed by a significant enhancement of the marker of macrophage differentiation CD68. ARHGEF3 protein is primarily nuclear, but MS275 treatment rapidly induced its translocation into the cytoplasm. ARHGEF3 cytoplasmic localization is associated with activation of the RhoA/Rho-associated Kinase (ROCK) pathway. In addition to cytoskeletal rearrangements orchestrated by RhoA, we showed that ARHGEF3/RhoA-dependent signals involve activation of SAPK/JNK and then Elk1 transcription factor. Importantly, MS275-induced CD68 expression was blocked by exposure of U937 cells to exoenzyme C3 transferase and Y27632, inhibitors of Rho and ROCK respectively. Moreover, ARHGEF3 silencing prevented RhoA activation leading to a reduction in SAPK/JNK phosphorylation, Elk1 activation and CD68 expression, suggesting a crucial role for ARHGEF3 in myeloid differentiation. Taken together, our results demonstrate that ARHGEF3 modulates acute myeloid leukemia differentiation through activation of RhoA and pathways directly controlled by small GTPase family proteins. The finding that GEF protein modulation by HDAC inhibition impacts on cell differentiation may be important for understanding the antitumor mechanism(s) by which HDACi treatment stimulates differentiation in cancer.

  2. Testosterone differentially alters cocaine-induced ambulatory and rearing behavioral responses in adult and adolescent rats

    PubMed Central

    Minerly, AnaChristina E.; Wu, Hui Bing K.; Weierstall, Karen M.; Niyomchai, Tipyamol; Kemen, Lynne; Jenab, Shirzad; Quinones-Jenab, Vanya

    2016-01-01

    Little is known about the physiological and behavioral effects of testosterone when co-administered with cocaine during adolescence. The present study aimed to determine whether exogenous testosterone administration differentially alters psychomotor responses to cocaine in adolescent and adult male rats. To this end, intact adolescent (30-days-old) and adult (60-day-old) male Fisher rats were pretreated with vehicle (sesame oil) or testosterone (5 or 10 mg/kg) 45 minutes prior to saline or cocaine (20 mg/kg) administration. Behavioral responses were monitored 1 hour after drug treatment, and serum testosterone levels were determined. Serum testosterone levels were affected by age: saline- and cocaine-treated adults in the vehicle groups had higher serum testosterone levels than adolescents rats, but after co-administration of testosterone the adolescent rats had higher serum testosterone levels than the adults. Pretreatment with testosterone affected baseline activity in adolescent rats: 5 mg/kg of testosterone increased both rearing and ambulatory behaviors in saline-treated adolescent rats. After normalizing data to % saline, an interaction between hormone administration and cocaine-induced behavioral responses was observed; 5 mg/kg of testosterone decreased both ambulatory and rearing behaviors among adolescents whereas 10 mg/kg of testosterone decreased only rearing behaviors. Testosterone pretreatment did not alter cocaine-induced behavioral responses in adult rats. These findings suggest that adolescents are more sensitive than adults to an interaction between testosterone and cocaine, and, indirectly, suggest that androgen abuse may lessen cocaine-induced behavioral responses in younger cocaine users. PMID:19822170

  3. Highly Efficient Differentiation of Functional Hepatocytes From Human Induced Pluripotent Stem Cells

    PubMed Central

    Ma, Xiaocui; Tschudy-Seney, Benjamin; Roll, Garrett; Behbahan, Iman Saramipoor; Ahuja, Tijess P.; Tolstikov, Vladimir; Wang, Charles; McGee, Jeannine; Khoobyari, Shiva; Nolta, Jan A.; Willenbring, Holger

    2013-01-01

    Human induced pluripotent stem cells (hiPSCs) hold great potential for use in regenerative medicine, novel drug development, and disease progression/developmental studies. Here, we report highly efficient differentiation of hiPSCs toward a relatively homogeneous population of functional hepatocytes. hiPSC-derived hepatocytes (hiHs) not only showed a high expression of hepatocyte-specific proteins and liver-specific functions, but they also developed a functional biotransformation system including phase I and II metabolizing enzymes and phase III transporters. Nuclear receptors, which are critical for regulating the expression of metabolizing enzymes, were also expressed in hiHs. hiHs also responded to different compounds/inducers of cytochrome P450 as mature hepatocytes do. To follow up on this observation, we analyzed the drug metabolizing capacity of hiHs in real time using a novel ultraperformance liquid chromatography-tandem mass spectrometry. We found that, like freshly isolated primary human hepatocytes, the seven major metabolic pathways of the drug bufuralol were found in hiHs. In addition, transplanted hiHs engrafted, integrated, and proliferated in livers of an immune-deficient mouse model, and secreted human albumin, indicating that hiHs also function in vivo. In conclusion, we have generated a method for the efficient generation of hepatocytes from induced pluripotent stem cells in vitro and in vivo, and it appears that the cells function similarly to primary human hepatocytes, including developing a complete metabolic function. These results represent a significant step toward using patient/disease-specific hepatocytes for cell-based therapeutics as well as for pharmacology and toxicology studies. PMID:23681950

  4. Lapatinib induces autophagic cell death and differentiation in acute myeloblastic leukemia

    PubMed Central

    Chen, Yu-Jen; Fang, Li-Wen; Su, Wen-Chi; Hsu, Wen-Yi; Yang, Kai-Chien; Huang, Huey-Lan

    2016-01-01

    Lapatinib is an oral-form dual tyrosine kinase inhibitor of epidermal growth factor receptor (EGFR or ErbB/Her) superfamily members with anticancer activity. In this study, we examined the effects and mechanism of action of lapatinib on several human leukemia cells lines, including acute myeloid leukemia (AML), chronic myeloid leukemia (CML), and acute lymphoblastic leukemia (ALL) cells. We found that lapatinib inhibited the growth of human AML U937, HL-60, NB4, CML KU812, MEG-01, and ALL Jurkat T cells. Among these leukemia cell lines, lapatinib induced apoptosis in HL-60, NB4, and Jurkat cells, but induced nonapoptotic cell death in U937, K562, and MEG-01 cells. Moreover, lapatinib treatment caused autophagic cell death as shown by positive acridine orange staining, the massive formation of vacuoles as seen by electronic microscopy, and the upregulation of LC3-II, ATG5, and ATG7 in AML U937 cells. Furthermore, autophagy inhibitor 3-methyladenine and knockdown of ATG5, ATG7, and Beclin-1 using short hairpin RNA (shRNA) partially rescued lapatinib-induced cell death. In addition, the induction of phagocytosis and ROS production as well as the upregulation of surface markers CD14 and CD68 was detected in lapatinib-treated U937 cells, suggesting the induction of macrophagic differentiation in AML U937 cells by lapatinib. We also noted the synergistic effects of the use of lapatinib and cytotoxic drugs in U937 leukemia cells. These results indicate that lapatinib may have potential for development as a novel antileukemia agent. PMID:27499639

  5. ERβ induces the differentiation of cultured osteoblasts by both Wnt/β-catenin signaling pathway and estrogen signaling pathways

    SciTech Connect

    Yin, Xinhua; Wang, Xiaoyuan; Hu, Xiongke; Chen, Yong; Zeng, Kefeng; Zhang, Hongqi

    2015-07-01

    Although 17β-estradial (E2) is known to stimulate bone formation, the underlying mechanisms are not fully understood. Recent studies have implicated the Wnt/β-catenin pathway as a major signaling cascade in bone biology. The interactions between Wnt/β-catenin signaling pathway and estrogen signaling pathways have been reported in many tissues. In this study, E2 significantly increased the expression of β-catenin by inducing phosphorylations of GSK3β at serine 9. ERβ siRNAs were transfected into MC3T3-E1 cells and revealed that ERβ involved E2-induced osteoblasts proliferation and differentiation via Wnt/β-catenin signaling. The osteoblast differentiation genes (BGP, ALP and OPN) and proliferation related gene (cyclin D1) expression were significantly induced by E2-mediated ERβ. Furthermore immunofluorescence and immunoprecipitation analysis demonstrated that E2 induced the accumulation of β-catenin protein in the nucleus which leads to interaction with T-cell-specific transcription factor/lymphoid enhancer binding factor (TCF/LEF) transcription factors. Taken together, these findings suggest that E2 promotes osteoblastic proliferation and differentiation by inducing proliferation-related and differentiation-related gene expression via ERβ/GSK-3β-dependent Wnt/β-catenin signaling pathway. Our findings provide novel insights into the mechanisms of action of E2 in osteoblastogenesis. - Highlights: • 17β-estradial (E2) promotes GSK3-β phosphorylation. • E2 activates the Wnt/β-catenin signaling pathway. • The Wnt/β-catenin signaling pathway interacts with estrogen signaling pathways. • E2-mediated ER induced osteoblast differentiation and proliferation related genes expression.

  6. Cooperation of myocardin and Smad2 in inducing differentiation of mesenchymal stem cells into smooth muscle cells.

    PubMed

    Wang, Nan; Ren, Guang-Da; Zhou, Zhen; Xu, Yao; Qin, Tao; Yu, Ru-Fa; Zhang, Tong-Cun

    2012-04-01

    Several reports demonstrated that mesenchymal stem cells (MSCs) might differentiate into smooth muscle cells (SMCs) in vitro and in vivo. It has been shown that myocardin protein is a strong inducer of smooth muscle genes and MSCs can differentiate into SMCs in response to transforming growth factor-β (TGF-β). However, the relationship or link between myocardin and TGF-β3-induced MSC differentiation has not been fully elucidated. Here, we demonstrated that both myocardin and TGF-β3 were able to induce differentiation of rat bone marrow-derived MSCs toward smooth-muscle-like cell types, as evidenced by increasing expression of SMC-specific genes. Of note, myocardin cooperated with Smad2 to synergistically activate SM22α promoter and significantly enhance the expression of SM22α. Report assays with site-direct mutation analysis of SM22α promoter demonstrated that myocardin and Smad2 coactivated SM22α promoter mainly depending on CArG box and less on smad binding elements (SBE) sites as well. These findings reveal the cooperation of myocardin and Smad2 in process of MSC differentiation into SMCs. PMID:22362485

  7. An EWS-FLI1-Induced Osteosarcoma Model Unveiled a Crucial Role of Impaired Osteogenic Differentiation on Osteosarcoma Development

    PubMed Central

    Komura, Shingo; Semi, Katsunori; Itakura, Fumiaki; Shibata, Hirofumi; Ohno, Takatoshi; Hotta, Akitsu; Woltjen, Knut; Yamamoto, Takuya; Akiyama, Haruhiko; Yamada, Yasuhiro

    2016-01-01

    Summary EWS-FLI1, a multi-functional fusion oncogene, is exclusively detected in Ewing sarcomas. However, previous studies reported that rare varieties of osteosarcomas also harbor EWS-ETS family fusion. Here, using the doxycycline-inducible EWS-FLI1 system, we established an EWS-FLI1-dependent osteosarcoma model from murine bone marrow stromal cells. We revealed that the withdrawal of EWS-FLI1 expression enhances the osteogenic differentiation of sarcoma cells, leading to mature bone formation. Taking advantage of induced pluripotent stem cell (iPSC) technology, we also show that sarcoma-derived iPSCs with cancer-related genetic abnormalities exhibited an impaired differentiation program of osteogenic lineage irrespective of the EWS-FLI1 expression. Finally, we demonstrate that EWS-FLI1 contributed to secondary sarcoma development from the sarcoma iPSCs after osteogenic differentiation. These findings demonstrate that modulating cellular differentiation is a fundamental principle of EWS-FLI1-induced osteosarcoma development. This in vitro cancer model using sarcoma iPSCs should provide a unique platform for dissecting relationships between the cancer genome and cellular differentiation. PMID:26997645

  8. The histamine H2-receptor antagonist, cimetidine, inhibits the articular osteopenia in rats with adjuvant-induced arthritis by suppressing the osteoclast differentiation induced by histamine.

    PubMed

    Yamaura, Katsunori; Yonekawa, Taeko; Nakamura, Tomonori; Yano, Shingo; Ueno, Koichi

    2003-05-01

    The effects of cimetidine on rat adjuvant arthritis (AA) and rat osteoclast differentiation were studied. For the in vivo experiments, AA was induced by injections of Mycobacterium tuberculosis H37RA either subcutaneously into the base of the tail or into the right hind paw. The osteoclast differentiation was assessed by estimating the number of tartrate-resistant acid phosphatase-positive multinuclear cells in the bone marrow culture. Cimetidine, at the dose of 25 mg/kg body weight, reduced the paw swelling by 70% (P<0.01). Cimetidine, at 10 microM concentration, inhibited 1,25-dihydroxyvitamin D(3) (1,25[OH](2)D(3)) and histamine mediated osteoclast differentiations by 40% (P<0.01) and 60% (P<0.001), respectively. Dimaprit, at 0.3 microM, stimulated the cell differentiation by 100% (P<0.01). Mepyramine reduced osteoclast differentiation, but the reduction was not statistically significant. Measurements of bone mineral density of the femur indicated that 5 mg/kg of cimetidine treated animals had 30% (P<0.01) higher mineral density in comparison with that of the AA control group that received no cimetidine. These results suggest that histamine is a potent inducer of osteoclast differentiation, at least in part, through the histamine H(2)-receptor, and cimetidine has a preventive effect on articular destruction and accompanying inflammation in arthritic rats. These observations may provide critical insights into the pathogenesis of the bone pathology seen in patients with RA.

  9. Laminin 411 acts as a potent inducer of umbilical cord mesenchymal stem cell differentiation into insulin-producing cells

    PubMed Central

    2014-01-01

    Background Diabetes mellitus (DM) is an incurable metabolic disease constituting a major threat to human health. Insulin-producing cells (IPCs) differentiated from mesenchymal stem cells (MSCs) hold great promise in the treatment of DM. The development of an efficient IPC induction system is a crucial step for the clinical application of IPCs for DM. Laminin 411 is a key component of the basement membrane and is involved in the regulation of cell differentiation; however, little is known about a role of laminin 411 in the regulation of IPC differentiation from human MSCs. Methods MSCs were isolated from human umbilical cord (UC-MSCs) and expanded in an in vitro culture system. UC-MSCs were then cultured in the IPC induction and differentiation medium in the presence of laminin 411. Flow cytometry, Quantitative realtime PCR, immunofluorescence staining, ELISA, Western blotting and other techniques were applied to determine IPC generation, insulin expression and related mechanisms. To evaluate potential therapeutic efficacy of IPCs induced from UC-MSCs, a type-1 diabetes (T1DM) rat model was generated using streptozotocin. Blood glucose, insulin levels, and survival of rats were monitored periodically following intravenous injection of the tested cells. Results Laminin 411 markedly induced the expression of the genes Foxa2 and Sox17, markers for pancreatic precursor cells, efficiently induced IPC differentiation from MSCs, and up-regulated insulin expression at both mRNA and protein levels. Furthermore, the expression of the genes known to govern insulin expression including Pdx1 and Ngn3 was markedly induced by laminin 411, which suggests that Pdx1 and Ngn3 signaling pathways are involved in laminin 411 induced-insulin expression machinery. More importantly, administration of laminin 411-induced IPCs rapidly and significantly down-regulated fasting blood glucose levels, significantly reduced the HbA1c concentration and markedly improved the symptoms and survival of

  10. Development of a rapid culture method to induce adipocyte differentiation of human bone marrow-derived mesenchymal stem cells

    SciTech Connect

    Ninomiya, Yuichi; Sugahara-Yamashita, Yzumi; Nakachi, Yutaka; Tokuzawa, Yoshimi; Okazaki, Yasushi; Nishiyama, Masahiko

    2010-04-02

    Human mesenchymal stem cells (hMSCs) derived from bone marrow are multipotent stem cells that can regenerate mesenchymal tissues such as adipose, bone or muscle. It is thought that hMSCs can be utilized as a cell resource for tissue engineering and as human models to study cell differentiation mechanisms, such as adipogenesis, osteoblastogenesis and so on. Since it takes 2-3 weeks for hMSCs to differentiate into adipocytes using conventional culture methods, the development of methods to induce faster differentiation into adipocytes is required. In this study we optimized the culture conditions for adipocyte induction to achieve a shorter cultivation time for the induction of adipocyte differentiation in bone marrow-derived hMSCs. Briefly, we used a cocktail of dexamethasone, insulin, methylisobutylxanthine (DIM) plus a peroxisome proliferator-activated receptor {gamma} agonist, rosiglitazone (DIMRo) as a new adipogenic differentiation medium. We successfully shortened the period of cultivation to 7-8 days from 2-3 weeks. We also found that rosiglitazone alone was unable to induce adipocyte differentiation from hMSCs in vitro. However, rosiglitazone appears to enhance hMSC adipogenesis in the presence of other hormones and/or compounds, such as DIM. Furthermore, the inhibitory activity of TGF-{beta}1 on adipogenesis could be investigated using DIMRo-treated hMSCs. We conclude that our rapid new culture method is very useful in measuring the effect of molecules that affect adipogenesis in hMSCs.

  11. Proliferation and differentiation of oligodendrocyte progenitor cells induced from rat embryonic neural precursor cells followed by flow cytometry.

    PubMed

    Lü, He-Zuo; Wang, Yan-Xia; Li, Ying; Fu, Sai-Li; Hang, Qin; Lu, Pei-Hua

    2008-08-01

    Previous studies have shown that a cell-intrinsic timer might determine when oligodendrocyte progenitor cells (OPCs) isolated from the central nervous system (CNS) stop dividing and initiate differentiation in a defined environment. In this report, the proliferation and differentiation of OPCs induced from neural precursor cells (NPCs) were analyzed by flow cytometry combined with carboxyfluorescein diacetate succinimidyl ester labeling and propidium iodide staining, respectively. When OPCs were cultured in OPC-medium, more than 30% of cells were in S- and G2/M-phases, and continuously self-renewed without differentiation. After exposure to thyroid hormone, there was an obvious decrease in the fraction of cells in both S- and G2/M-phases (<10%). Furthermore, the OPCs no longer proliferated, but differentiated into oligodendrocytes. The dynamic proliferation and differentiation characteristics of OPCs induced from NPCs and analyzed by flow cytometry were similar to those of OPCs isolated from the CNS and analyzed by other methods. These studies indicated that the proliferation and differentiation of OPCs can be followed simply and rapidly by flow cytometry. PMID:18473382

  12. Inhibitory effect of leptin on rosiglitazone-induced differentiation of primary adipocytes prepared from TallyHO/Jng mice

    SciTech Connect

    Kim, Ki Young; Kim, Joo Young; Sung, Yoon-Young; Jung, Won Hoon; Kim, Hee-Youn; Park, Ji Seon; Cheon, Hyae Gyeong; Rhee, Sang Dal

    2011-03-25

    Research highlights: {yields} In this study, we investigated the effects of leptin on adipocyte differentiation prepared from subcutaneous fat of TallyHo mice. {yields} Leptin inhibited the adipocytes differentiation at physiological concentration via inhibition of PPAR{gamma} expression. {yields} Inhibitors of ERK and STAT1 restored the leptin's inhibitory activity both in vitro and in vivo. -- Abstract: The effects of leptin on rosiglitazone-induced adipocyte differentiation were investigated in the primary adipocytes prepared from subcutaneous fat of TallyHO/Jng (TallyHO) mouse, a recently developed model animal for type 2 diabetes mellitus (T2DM). The treatment of leptin inhibited the rosiglitazone-induced adipocyte differentiation with a decreased expression of peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) a key adipogenic transcription factor, both in mRNA and protein levels. Leptin (10 nM) was sufficient to inhibit the adipocyte differentiation, which seemed to come from increased expression of leptin receptor genes in the fat of TallyHO mice. The inhibition of adipogenesis by leptin was restored by the treatment of inhibitors for extracellular-signal-regulated kinase (ERK) (PD98059) and signal transducer and activator of transcription-1 (STAT1) (fludarabine). Furthermore, in vivo intraperitoneal administration of PD98059 and fludarabine increased the PPAR{gamma} expression in the subcutaneous fat of TallyHO mice. These data suggest that leptin could inhibit the PPAR{gamma} expression and adipocyte differentiation in its physiological concentration in TallyHO mice.

  13. Taurine Protected Against the Impairments of Neural Stem Cell Differentiated Neurons Induced by Oxygen-Glucose Deprivation.

    PubMed

    Xiao, Bo; Liu, Huazhen; Gu, Zeyun; Liu, Sining; Ji, Cheng

    2015-11-01

    Cell transplantation of neural stem cells (NSCs) is a promising approach for neurological recovery both structurally and functionally. However, one big obstacle is to promote differentiation of NSCs into neurons and the followed maturation. In the present study, we aimed to investigate the protective effect of taurine on the differentiation of NSCs and subsequent maturation of their neuronal lineage, when exposed to oxygen-glucose deprivation (OGD). The results suggested that taurine (5-20 mM) promoted the viability and proliferation of NSCs, and it protected against 8 h of OGD induced impairments. Furthermore, 20 mM taurine promoted NSCs to differentiate into neurons after 7 days of culture, and it also protected against the suppressive impairments of 8 h of OGD. Consistently, taurine (20 mM) promoted the neurite sprouting and outgrowth of the NSC differentiated neurons after 14 days of differentiation, which were significantly inhibited by OGD (8 h). At D21, the mushroom spines and spine density were promoted or restored by 20 mM taurine. Taken together, the enhanced viability and proliferation of NSCs, more differentiated neurons and the promoted maturation of neurons by 20 mM taurine support its therapeutic application during stem cell therapy to enhance neurological recovery. Moreover, it protected against the impairments induced by OGD, which may highlight its role for a more direct therapeutic application especially in an ischemic stroke environment. PMID:26415593

  14. Inducing Differentiation of Premalignant Hepatic Cells as a Novel Therapeutic Strategy in Hepatocarcinoma.

    PubMed

    Wolf, Benita; Krieg, Kathrin; Falk, Christine; Breuhahn, Kai; Keppeler, Hildegard; Biedermann, Tilo; Schmid, Evi; Warmann, Steven; Fuchs, Joerg; Vetter, Silvia; Thiele, Dennis; Nieser, Maike; Avci-Adali, Meltem; Skokowa, Yulia; Schöls, Ludger; Hauser, Stefan; Ringelhan, Marc; Yevsa, Tetyana; Heikenwalder, Mathias; Kossatz-Boehlert, Uta

    2016-09-15

    Hepatocellular carcinoma (HCC) represents the second leading cause of cancer-related deaths and is reported to be resistant to chemotherapy caused by tumor-initiating cells. These tumor-initiating cells express stem cell markers. An accumulation of tumor-initiating cells can be found in 2% to 50% of all HCC and is correlated with a poor prognosis. Mechanisms that mediate chemoresistance include drug export, increased metabolism, and quiescence. Importantly, the mechanisms that regulate quiescence in tumor-initiating cells have not been analyzed in detail so far. In this research we have developed a single cell tracking method to follow up the fate of tumor-initiating cells during chemotherapy. Thereby, we were able to demonstrate that mCXCL1 exerts cellular state-specific effects regulating the resistance to chemotherapeutics. mCXCL1 is the mouse homolog of the human IL8, a chemokine that correlates with poor prognosis in HCC patients. We found that mCXCL1 blocks differentiation of premalignant cells and activates quiescence in tumor-initiating cells. This process depends on the activation of the mTORC1 kinase. Blocking of the mTORC1 kinase induces differentiation of tumor-initiating cells and allows their subsequent depletion using the chemotherapeutic drug doxorubicin. Our work deciphers the mCXCL1-mTORC1 pathway as crucial in liver cancer stem cell maintenance and highlights it as a novel target in combination with conventional chemotherapy. Cancer Res; 76(18); 5550-61. ©2016 AACR. PMID:27488521

  15. Domesticated transposase Kat1 and its fossil imprints induce sexual differentiation in yeast

    PubMed Central

    Rajaei, Naghmeh; Chiruvella, Kishore K.; Lin, Feng; Åström, Stefan U.

    2014-01-01

    Transposable elements (TEs) have had a major influence on shaping both prokaryotic and eukaryotic genomes, largely through stochastic events following random or near-random insertions. In the mammalian immune system, the recombination activation genes1/2 (Rag1/2) recombinase has evolved from a transposase gene, demonstrating that TEs can be domesticated by the host. In this study, we uncovered a domesticated transposase, Kluyveromyces lactis hobo/Activator/Tam3 (hAT) transposase 1 (Kat1), operating at the fossil imprints of an ancient transposon, that catalyzes the differentiation of cell type. Kat1 induces mating-type switching from mating type a (MATa) to MATα in the yeast K. lactis. Kat1 activates switching by introducing two hairpin-capped DNA double-strand breaks (DSBs) in the MATa1–MATa2 intergenic region, as we demonstrate both in vivo and in vitro. The DSBs stimulate homologous recombination with the cryptic hidden MAT left alpha (HMLα) locus resulting in a switch of the cell type. The sites where Kat1 acts in the MATa locus most likely are ancient remnants of terminal inverted repeats from a long-lost TE. The KAT1 gene is annotated as a pseudogene because it contains two overlapping ORFs. We demonstrate that translation of full-length Kat1 requires a programmed −1 frameshift. The frameshift limited Kat1 activity, because restoring the zero frame causes switching to the MATα genotype. Kat1 also was transcriptionally activated by nutrient limitation via the transcription factor mating type switch 1 (Mts1). A phylogenetic analysis indicated that KAT1 was domesticated specifically in the Kluyveromyces clade of the budding yeasts. We conclude that Kat1 is a highly regulated transposase-derived endonuclease vital for sexual differentiation. PMID:25313032

  16. Regional Differentiation of Retinoic Acid-Induced Human Pluripotent Embryonic Carcinoma Stem Cell Neurons

    PubMed Central

    Coyle, Dennis E.; Li, Jie; Baccei, Mark

    2011-01-01

    The NTERA2 cl D1 (NT2) cell line, derived from human teratocarcinoma, exhibits similar properties as embryonic stem (ES) cells or very early neuroepitheial progenitors. NT2 cells can be induced to become postmitotic central nervous system neurons (NT2N) with retinoic acid. Although neurons derived from pluripotent cells, such as NT2N, have been characterized for their neurotransmitter phenotypes, their potential suitability as a donor source for neural transplantation also depends on their ability to respond to localized environmental cues from a specific region of the CNS. Therefore, our study aimed to characterize the regional transcription factors that define the rostocaudal and dorsoventral identity of NT2N derived from a monolayer differentiation paradigm using quantitative PCR (qPCR). Purified NT2N mainly expressed both GABAergic and glutamatergic phenotypes and were electrically active but did not form functional synapses. The presence of immature astrocytes and possible radial glial cells was noted. The NT2N expressed a regional transcription factor code consistent with forebrain, hindbrain and spinal cord neural progenitors but showed minimal expression of midbrain phenotypes. In the dorsoventral plane NT2N expressed both dorsal and ventral neural progenitors. Of major interest was that even under the influence of retinoic acid, a known caudalization factor, the NT2N population maintained a rostral phenotype subpopulation which expressed cortical regional transcription factors. It is proposed that understanding the regional differentiation bias of neurons derived from pluripotent stem cells will facilitate their successful integration into existing neuronal networks within the CNS. PMID:21283767

  17. Different types of exercise induce differential effects on neuronal adaptations and memory performance.

    PubMed

    Lin, Tzu-Wei; Chen, Shean-Jen; Huang, Tung-Yi; Chang, Chia-Yuan; Chuang, Jih-Ing; Wu, Fong-Sen; Kuo, Yu-Min; Jen, Chauying J

    2012-01-01

    Different exercise paradigms show differential effects on various forms of memory. We hypothesize that the differential effects of exercises on memory performance are caused by different neuroplasticity changes in relevant brain regions in response to different exercise trainings. We examined the effects of treadmill running (TR) and wheel running (WR) on the Pavlovian fear conditioning task that assesses learning and memory performance associated with the amygdala (cued conditioning) and both the amygdala and hippocampus (contextual conditioning). The skeletal muscle citrate synthase activity, an indicator of aerobic capacity, was elevated in rats received 4 w of TR, but not WR. While both TR and WR elevated the contextual conditional response, only TR facilitated the cued conditional response. Using a single-neuron labeling technique, we found that while both TR and MR enlarged the dendritic field and increased the spine density in hippocampal CA3 neurons, only TR showed these effects in basolateral amygdalar neurons. Moreover, both types of exercise upregulated synaptic proteins (i.e., TrkB and SNAP-25) in the hippocampus; however only TR showed similar effects in the amygdala. Injection of K252a, a TrkB kinase inhibitor, in the dorsal hippocampus or basolateral amygdala abolished the exercise-facilitated contextual or cued fear learning and memory performance, respectively, regardless of the types of exercise. In summary, our results supported that different types of exercise affect the performance of learning and memory via BDNF-TrkB signaling and neuroplasticity in specific brain regions. The brain region-specific neuronal adaptations are possibly induced by various levels of intensity/stress elicited by different types of exercise.

  18. Differentiation of Odontoblast-Like Cells From Mouse Induced Pluripotent Stem Cells by Pax9 and Bmp4 Transfection

    PubMed Central

    Seki, Daisuke; Takeshita, Nobuo; Oyanagi, Toshihito; Sasaki, Shutaro; Takano, Ikuko; Hasegawa, Masakazu

    2015-01-01

    The field of tooth regeneration has progressed in recent years, and human tooth regeneration could become viable in the future. Because induced pluripotent stem (iPS) cells can differentiate into odontogenic cells given appropriate conditions, iPS cells are a potential cell source for tooth regeneration. However, a definitive method to induce iPS cell-derived odontogenic cells has not been established. We describe a novel method of odontoblast differentiation from iPS cells using gene transfection. We generated mouse iPS cell-derived neural crest-like cells (iNCLCs), which exhibited neural crest markers. Next, we differentiated iNCLCs into odontoblast-like cells by transfection of Pax9 and Bmp4 expression plasmids. Exogenous Pax9 upregulated expression of Msx1 and dentin matrix protein 1 (Dmp1) in iNCLCs but not bone morphogenetic protein 4 (Bmp4) or dentin sialophosphoprotein (Dspp). Exogenous Bmp4 upregulated expression of Msx1, Dmp1, and Dspp in iNCLCs, but not Pax9. Moreover, cotransfection of Pax9 and Bmp4 plasmids in iNCLCs revealed a higher expression of Pax9 than when Pax9 plasmid was used alone. In contrast, exogenous Pax9 downregulated Bmp4 overexpression. Cotransfection of Pax9 and Bmp4 synergistically upregulated Dmp1 expression; however, Pax9 overexpression downregulated exogenous Bmp4-induced Dspp expression. Together, these findings suggest that an interaction between exogenous Pax9- and Bmp4-induced signaling modulated Dmp1 and Dspp expression. In conclusion, transfection of Pax9 and Bmp4 expression plasmids in iNCLCs induced gene expression associated with odontoblast differentiation, suggesting that iNCLCs differentiated into odontoblast-like cells. The iPS cell-derived odontoblast-like cells could be a useful cell source for tooth regeneration. Significance It has been reported that induced pluripotent stem (iPS) cells differentiate into odontogenic cells by administration of recombinant growth factors and coculture with odontogenic cells

  19. Theoretical and experimental quantification of doubly and singly differential cross sections for electron-induced ionization of isolated tetrahydrofuran molecules

    DOE PAGES

    Champion, Christophe; Quinto, Michele A.; Bug, Marion U.; Baek, Woon Y.; Weck, Philippe F.

    2014-07-29

    Electron-induced ionization of the commonly used surrogate of the DNA sugar-phosphate backbone, namely, the tetrahydrofuran molecule, is here theoretically described within the 1st Born approximation by means of quantum-mechanical approach. Comparisons between theory and recent experiments are reported in terms of doubly and singly differential cross sections.

  20. Theoretical and experimental quantification of doubly and singly differential cross sections for electron-induced ionization of isolated tetrahydrofuran molecules

    SciTech Connect

    Champion, Christophe; Quinto, Michele A.; Bug, Marion U.; Baek, Woon Y.; Weck, Philippe F.

    2014-07-29

    Electron-induced ionization of the commonly used surrogate of the DNA sugar-phosphate backbone, namely, the tetrahydrofuran molecule, is here theoretically described within the 1st Born approximation by means of quantum-mechanical approach. Comparisons between theory and recent experiments are reported in terms of doubly and singly differential cross sections.

  1. Theoretical and experimental quantification of doubly and singly differential cross sections for electron-induced ionization of isolated tetrahydrofuran molecules

    NASA Astrophysics Data System (ADS)

    Champion, Christophe; Quinto, Michele A.; Bug, Marion U.; Baek, Woon Y.; Weck, Philippe F.

    2014-07-01

    Electron-induced ionization of the tetrahydrofuran molecule, the commonly used surrogate of the DNA sugar-phosphate backbone, is theoretically described in this study within the 1st Born approximation. Comparisons between theory and recent experiments are reported in terms of doubly and singly differential cross sections.

  2. Capsaicin Induces “Brite” Phenotype in Differentiating 3T3-L1 Preadipocytes

    PubMed Central

    Baboota, Ritesh K.; Singh, Dhirendra P.; Sarma, Siddhartha M.; Kaur, Jaspreet; Sandhir, Rajat; Boparai, Ravneet K.; Kondepudi, Kanthi K.; Bishnoi, Mahendra

    2014-01-01

    Objective Targeting the energy storing white adipose tissue (WAT) by pharmacological and dietary means in order to promote its conversion to energy expending “brite” cell type holds promise as an anti-obesity approach. Present study was designed to investigate/revisit the effect of capsaicin on adipogenic differentiation with special reference to induction of “brite” phenotype during differentiation of 3T3-L1 preadipocytes. Methods Multiple techniques such as Ca2+ influx assay, Oil Red-O staining, nutrigenomic analysis in preadipocytes and matured adipocytes have been employed to understand the effect of capsaicin at different doses. In addition to in-vitro experiments, in-vivo studies were carried out in high-fat diet (HFD) fed rats treated with resiniferatoxin (RTX) (a TRPV1 agonist) and in mice administered capsaicin. Results TRPV1 channels are expressed in preadipocytes but not in adipocytes. In preadipocytes, both capsaicin and RTX stimulate Ca2+ influx in dose-dependent manner. This stimulation may be prevented by capsazepine, a TRPV1 antagonist. At lower doses, capsaicin inhibits lipid accumulation and stimulates TRPV1 gene expression, while at higher doses it enhances accumulation of lipids and suppresses expression of its receptor. In doses of 0.1–100 µM, capsaicin promotes expression of major pro-adipogenic factor PPARγ and some of its downstream targets. In concentrations of 1 µM, capsaicin up-regulates anti-adipogenic genes. Low-dose capsaicin treatment of 3T3-L1 preadipocytes differentiating into adipocytes results in increased expression of brown fat cell marker genes. In white adipose of mice, capsaicin administration leads to increase in browning-specific genes. Global TRPV1 ablation (i.p. by RTX administration) leads to increase in locomotor activity with no change in body weight. Conclusion Our findings suggest the dual modulatory role of capsaicin in adipogenesis. Capsaicin inhibits adipogenesis in 3T3-L1 via TRPV1 activation and

  3. Effect of angiotensin II on proliferation and differentiation of mouse induced pluripotent stem cells into mesodermal progenitor cells

    SciTech Connect

    Ishizuka, Toshiaki; Goshima, Hazuki; Ozawa, Ayako; Watanabe, Yasuhiro

    2012-03-30

    Highlights: Black-Right-Pointing-Pointer Treatment with angiotensin II enhanced LIF-induced DNA synthesis of mouse iPS cells. Black-Right-Pointing-Pointer Angiotensin II may enhance the DNA synthesis via induction of superoxide. Black-Right-Pointing-Pointer Treatment with angiotensin II significantly increased JAK/STAT3 phosphorylation. Black-Right-Pointing-Pointer Angiotensin II enhanced differentiation into mesodermal progenitor cells. Black-Right-Pointing-Pointer Angiotensin II may enhance the differentiation via activation of p38 MAPK. -- Abstract: Previous studies suggest that angiotensin receptor stimulation may enhance not only proliferation but also differentiation of undifferentiated stem/progenitor cells. Therefore, in the present study, we determined the involvement of the angiotensin receptor in the proliferation and differentiation of mouse induced pluripotent stem (iPS) cells. Stimulation with angiotensin II (Ang II) significantly increased DNA synthesis in mouse iPS cells cultured in a medium with leukemia inhibitory factor (LIF). Pretreatment of the cells with either candesartan (a selective Ang II type 1 receptor [AT{sub 1}R] antagonist) or Tempol (a cell-permeable superoxide scavenger) significantly inhibited Ang II-induced DNA synthesis. Treatment with Ang II significantly increased JAK/STAT3 phosphorylation. Pretreatment with candesartan significantly inhibited Ang II- induced JAK/STAT3 phosphorylation. In contrast, induction of mouse iPS cell differentiation into Flk-1-positive mesodermal progenitor cells was performed in type IV collagen (Col IV)- coated dishes in a differentiation medium without LIF. When Col IV-exposed iPS cells were treated with Ang II for 5 days, the expression of Flk-1 was significantly increased compared with that in the cells treated with the vehicle alone. Pretreatment of the cells with both candesartan and SB203580 (a p38 MAPK inhibitor) significantly inhibited the Ang II- induced increase in Flk-1 expression

  4. Selection of the Inducer for the Differentiation of Chicken Embryonic Stem Cells into Male Germ Cells In Vitro

    PubMed Central

    Zhang, Yani; Wang, Yingjie; Zuo, Qisheng; Wang, Xiaoyan; Li, Dong; Tang, Beibei; Li, Bichun

    2016-01-01

    Several inducers have been used to differentiate embryonic stem cells (ESCs) into male germ cells but the induction process has been inefficient. To solve the problem of low efficiency of inducer for ESCs differentiation into male germ cells, all-trans retinoic acid (ATRA), Am80(the retinoic acid receptor agonist), and estradiol (E2) was used to induce ESCs to differentiate into male germ cells in vitro. ESCs were cultured in media containing ATRA, Am80, or E2 respectively which can differentiate ESCs into a germ cell lineage. In process of ATRA and Am80 induction Group, germ cell-like cells can be observed in 10 days; but have no in E2 induction Group. The marker genes of germ cell: Dazl, Stra8, C-kit, Cvh, integrinα6, and integrinβ1 all showed a significant up-regulation in the expression level. The ATRA-induction group showed high expression of C-kit and Cvh around 4 days, and integrinα6 and integrinβ1 were activated on day 10, respectively, while the E2-,Am80- induction group showed a high expression of C-kit as early as 4 days immunocytochemistry results shown that, integrinα6 and integrinβ1 could be detected in the ATRA-, Am80-, and E2-induction group, Positive clones in the ATRA group were greater in number than those in the other two groups. we conclued that ATRA, Am80, and E2 can promote the expression of the corresponding genes of germ cells, and had different effect on the differentiation of ESCs into male germ cells. ATRA was the most effective inducer of germ cell differentiation. PMID:27741318

  5. Shockwaves induce osteogenic differentiation of human mesenchymal stem cells through ATP release and activation of P2X7 receptors.

    PubMed

    Sun, Dahui; Junger, Wolfgang G; Yuan, Changji; Zhang, Wenyan; Bao, Yi; Qin, Daming; Wang, Chengxue; Tan, Lei; Qi, Baochang; Zhu, Dong; Zhang, Xizheng; Yu, Tiecheng

    2013-06-01

    Shockwave treatment promotes bone healing of nonunion fractures. In this study, we investigated whether this effect could be due to adenosine 5'-triphosphate (ATP) release-induced differentiation of human mesenchymal stem cells (hMSCs) into osteoprogenitor cells. Cultured bone marrow-derived hMSCs were subjected to shockwave treatment and ATP release was assessed. Osteogenic differentiation and mineralization of hMSCs were evaluated by examining alkaline phosphatase activity, osteocalcin production, and calcium nodule formation. Expression of P2X7 receptors and c-fos and c-jun mRNA was determined with real-time reverse transcription polymerase chain reaction and Western blotting. P2X7-siRNA, apyrase, P2 receptor antagonists, and p38 MAPK inhibitors were used to evaluate the roles of ATP release, P2X7 receptors, and p38 MAPK signaling in shockwave-induced osteogenic hMSCs differentiation. Shockwave treatment released significant amounts (≈ 7 μM) of ATP from hMSCs. Shockwaves and exogenous ATP induced c-fos and c-jun mRNA transcription, p38 MAPK activation, and hMSC differentiation. Removal of ATP with apyrase, targeting of P2X7 receptors with P2X7-siRNA or selective antagonists, or blockade of p38 MAPK with SB203580 prevented osteogenic differentiation of hMSCs. Our findings indicate that shockwaves release cellular ATP that activates P2X7 receptors and downstream signaling events that caused osteogenic differentiation of hMSCs. We conclude that shockwave therapy promotes bone healing through P2X7 receptor signaling, which contributes to hMSC differentiation.

  6. Shockwaves Induce Osteogenic Differentiation of Human Mesenchymal Stem Cells Through ATP Release and Activation of P2X7 Receptors

    PubMed Central

    Sun, Dahui; Junger, Wolfgang G.; Yuan, Changji; Zhang, Wenyan; Bao, Yi; Qin, Daming; Wang, Chengxue; Tan, Lei; Qi, Baochang; Zhu, Dong; Zhang, Xizheng; Yu, Tiecheng

    2014-01-01

    Shockwave fractures treatment promotes bone healing of nonunion fractures. In this study, we investigated whether this effect could be due to adenosine 5’-triphosphate (ATP) release-induced differentiation of human mesenchymal stem cells (hMSCs) into osteoprogenitor cells. Cultured bone marrow-derived hMSCs were subjected to shockwave treatment and ATP release was assessed. Osteogenic differentiation and mineralization of hMSCs were evaluated by examining alkaline phosphatase activity, osteocalcin production, and calcium nodule formation. Expression of P2X7 receptors and c-fos and c-jun mRNA was determined with real-time reverse transcription polymerase chain reaction and Western blotting. P2X7-siRNA, apyrase, P2 receptor antagonists, and p38 MAPK inhibitors were used to evaluate the roles of ATP release, P2X7 receptors, and p38 MAPK sig naling in shockwave-induced osteogenic hMSCs differentiation. Shockwave treatment released significant amounts (~7 μM) of ATP from hMSCs. Shockwaves and exogenous ATP induced c-fos and c-jun mRNA transcription, p38 MAPK activation, and hMSC differentiation. Removal of ATP with apyrase, targeting of P2X7 receptors with P2X7-siRNA or selective antagonists, or blockade of p38 MAPK with SB203580 prevented osteogenic differentiation of hMSCs. Our findings indicate that shockwaves release cellular ATP that activates P2X7 receptors and downstream signaling events that caused osteogenic differentiation of hMSCs. We conclude that shockwave therapy promotes bone healing through P2X7 receptor signaling, which contributes to hMSC differentiation. PMID:23404811

  7. The Apoplastic Copper AMINE OXIDASE1 Mediates Jasmonic Acid-Induced Protoxylem Differentiation in Arabidopsis Roots1

    PubMed Central

    Ghuge, Sandip A.; Carucci, Andrea; Rodrigues-Pousada, Renato A.; Tisi, Alessandra; Franchi, Stefano; Tavladoraki, Paraskevi; Cona, Alessandra

    2015-01-01

    Polyamines are involved in key developmental processes and stress responses. Copper amine oxidases oxidize the polyamine putrescine (Put), producing an aldehyde, ammonia, and hydrogen peroxide (H2O2). The Arabidopsis (Arabidopsis thaliana) amine oxidase gene At4g14940 (AtAO1) encodes an apoplastic copper amine oxidase expressed at the early stages of vascular tissue differentiation in roots. Here, its role in root development and xylem differentiation was explored by pharmacological and forward/reverse genetic approaches. Analysis of the AtAO1 expression pattern in roots by a promoter::green fluorescent protein-β-glucuronidase fusion revealed strong gene expression in the protoxylem at the transition, elongation, and maturation zones. Methyl jasmonate (MeJA) induced AtAO1 gene expression in vascular tissues, especially at the transition and elongation zones. Early protoxylem differentiation was observed upon MeJA treatment along with Put level decrease and H2O2 accumulation in wild-type roots, whereas Atao1 loss-of-function mutants were unresponsive to the hormone. The H2O2 scavenger N,N1-dimethylthiourea reversed the MeJA-induced early protoxylem differentiation in wild-type seedlings. Likewise, Put, which had no effect on Atao1 mutants, induced early protoxylem differentiation in the wild type, this event being counteracted by N,N1-dimethylthiourea treatment. Consistently, AtAO1-overexpressing plants showed lower Put levels and early protoxylem differentiation concurrent with H2O2 accumulation in the root zone where the first protoxylem cells with fully developed secondary wall thickenings are found. These results show that the H2O2 produced via AtAO1-driven Put oxidation plays a role in MeJA signaling leading to early protoxylem differentiation in root. PMID:25883242

  8. Alterations in polyamine levels induced by phorbol diesters and other agents that promote differentiation in human promyelocytic leukemia cells

    SciTech Connect

    Huberman, E.; Weeks, C.; Herrmann, A.; Callaham, M.; Slaga, T.

    1981-02-01

    Polyamine levels were evaluated in human HL-60 promyelocytic leukemia cells after treatment with inducers of terminal differentiation. Differentiation in these cells was determined by increases in the percentage of morphologically mature cells and in lysozyme activity. Treatment of the HL-60 cells with phorbol 12-myristate-13-acetate (PMA), phorbol 12,13-didecanoate or other inducers of terminal differentiation such as dimethylsulfoxide and retinoic acid resulted in increased levels of putrescine. However, no increase in putrescine could be detected after PMA treatment of a HL-60 cell variant that exhibited a decreased susceptibility to PMA-induced terminal differentiation. Similarly, no increase in putrescine was observed with two nontumor-promoters (phorbol 12,13-diacetate and 4-O-methyl-PMA) or with anthralin, a non-phorbol tumor promoter. In addition to enhancing putrescine levels, PMA also increased the amount of spermidine and decreased the amount of spermine. The increase in putrescine and spermidine preceded the expression of the various differentiation markers. Unlike the changes observed in the polyamine levels after PMA treatment, the activities of ornithine and S-adenosylmethionine decarboxylases, which are polyamine biosynthetic enzymes, did not significantly change. ..cap alpha..-Methylornithine and ..cap alpha..-difluoromethylornithine and methylglyoxal bis(guanylhydrazone), which are inhibitors of the polyamine biosynthetic enzymes, did not affect differentiation in control or PMA-treated cells. Because of these observations, we suggest that the change in polyamine levels involve biochemical pathways other than the known biosynthetic ones. By-products of these pathways may perhaps be the controlling factors involved in the induction of terminal differentiation in the HL-60 and other cell types as well.

  9. 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

  10. microRNA-449a functions as a tumor suppressor in neuroblastoma through inducing cell differentiation and cell cycle arrest

    PubMed Central

    Zhao, Zhenze; Ma, Xiuye; Sung, Derek; Li, Monica; Kosti, Adam; Lin, Gregory; Chen, Yidong; Pertsemlidis, Alexander; Hsiao, Tzu-Hung; Du, Liqin

    2015-01-01

    microRNA-449a (miR-449a) has been identified to function as a tumor suppressor in several types of cancers. However, the role of miR-449a in neuroblastoma has not been intensively investigated. We recently found that the overexpression of miR-449a significantly induces neuroblastoma cell differentiation, suggesting its potential tumor suppressor function in neuroblastoma. In this study, we further investigated the mechanisms underlying the tumor suppressive function of miR-449a in neuroblastoma. We observed that miR-449a inhibits neuroblastoma cell survival and growth through 2 mechanisms—inducing cell differentiation and cell cycle arrest. Our comprehensive investigations on the dissection of the target genes of miR-449a revealed that 3 novel targets- MFAP4, PKP4 and TSEN15 -play important roles in mediating its differentiation-inducing function. In addition, we further found that its function in inducing cell cycle arrest involves down-regulating its direct targets CDK6 and LEF1. To determine the clinical significance of the miR-449a-mediated tumor suppressive mechanism, we examined the correlation between the expression of these 5 target genes in neuroblastoma tumor specimens and the survival of neuroblastoma patients. Remarkably, we noted that high tumor expression levels of all the 3 miR-449a target genes involved in regulating cell differentiation, but not the target genes involved in regulating cell cycle, are significantly correlated with poor survival of neuroblastoma patients. These results suggest the critical role of the differentiation-inducing function of miR-449a in determining neuroblastoma progression. Overall, our study provides the first comprehensive characterization of the tumor-suppressive function of miR-449a in neuroblastoma, and reveals the potential clinical significance of the miR-449a-mediated tumor suppressive pathway in neuroblastoma prognosis. PMID:25760387

  11. Critical role of AZI2 in GM-CSF-induced dendritic cell differentiation.

    PubMed

    Fukasaka, Masahiro; Ori, Daisuke; Kawagoe, Tatsukata; Uematsu, Satoshi; Maruyama, Kenta; Okazaki, Toshihiko; Kozaki, Tatsuya; Imamura, Tomoko; Tartey, Sarang; Mino, Takashi; Satoh, Takashi; Akira, Shizuo; Takeuchi, Osamu

    2013-06-01

    TNFR-associated factor family member-associated NF-κB activator (TANK)-binding kinase 1 (TBK1) is critical for the activation of IFN regulatory factor 3 and type I IFN production upon virus infection. A set of TBK1-binding proteins, 5-azacytidine-induced gene 2 (AZI2; also known as NAP1), TANK, and TBK1-binding protein 1 (TBKBP1), have also been implicated in the production of type I IFNs. Among them, TANK was found to be dispensable for the responses against virus infection. However, physiological roles of AZI2 and TBKBP1 have yet to be clarified. In this study, we found that none of these TBK1-binding proteins is critical for type I IFN production in mice. In contrast, AZI2, but not TBKBP1, is critical for the differentiation of conventional dendritic cells (cDCs) from bone marrow cells in response to GM-CSF. AZI2 controls GM-CSF-induced cell cycling of bone marrow cells via TBK1. GM-CSF-derived DCs from AZI2-deficient mice show severe defects in cytokine production and T cell activation both in vitro and in vivo. Reciprocally, overexpression of AZI2 results in efficient generation of cDCs, and the cells show enhanced T cell activation in response to Ag stimulation. Taken together, AZI2 expression is critical for the generation of cDCs by GM-CSF and can potentially be used to increase the efficiency of immunization by cDCs. PMID:23610142

  12. Differential subcellular distribution of rat brain dopamine receptors and subtype-specific redistribution induced by cocaine

    PubMed Central

    Voulalas, Pamela J.; Schetz, John; Undieh, Ashiwel S.

    2011-01-01

    We investigated the subcellular distribution of dopamine D1, D2 and D5 receptor subtypes in rat frontal cortex, and examined whether psychostimulant-induced elevation of synaptic dopamine could alter the receptor distribution. Differential detergent solubilization and density gradient centrifugation were used to separate various subcellular fractions, followed by semi-quantitative determination of the relative abundance of specific receptor proteins in each fraction. D1 receptors were predominantly localized to detergent-resistant membranes, and a portion of these receptors also floated on sucrose gradients. These properties are characteristic of proteins found in lipid rafts and caveolae. D2 receptors exhibited variable distribution between cytoplasmic, detergent-soluble and detergent-resistant membrane fractions, yet were not present in buoyant membranes. Most D5 receptor immunoreactivity was distributed into the cytoplasmic fraction, failing to sediment at forces up to 300,000g, while the remainder was localized to detergent-soluble membranes in cortex. D5 receptors were undetectable in detergent-resistant fractions or raft-like subdomains. Following daily cocaine administration for seven days, a significant portion of D1 receptors translocated from detergent-resistant membranes to detergent-soluble membranes and the cytoplasmic fraction. The distributions of D5 and D2 receptor subtypes were not significantly altered by cocaine treatment. These data imply that D5 receptors are predominantly cytoplasmic, D2 receptors are diffusely distributed within the cell, whereas D1 receptors are mostly localized to lipid rafts within the rat frontal cortex. Dopamine receptor subtype localization is susceptible to modulation by pharmacological manipulations that elevate synaptic dopamine, however the functional implications of such drug-induced receptor warrant further investigation. PMID:21236347

  13. Salicylic acid induces differential antioxidant response in spring maize under high temperature stress.

    PubMed

    Khanna, Palak; Kaur, Kamaljit; Gupta, Anil K

    2016-06-01

    High temperature is one of the important stress factors that affect crops in tropical countries. Plants do evolve or adopt different mechanisms to overcome such stress for survival. It is an interesting subject and has attracted many researchers to work upon. Here, we studied the effect of salicylic acid (SA) on seedling growth and antioxidative defense system in two spring maize (Zea mays L.) genotypes viz., CML-32 (relatively heat tolerant) and LM-11 (relatively heat susceptible), under high temperature stress. High temperature induced greater reduction in dry biomass of LM-1 1 seedlings as compared to those of CML-32. There was a parallel increase in ascorbate peroxidase and glutathione reductase activities in the roots of CML-32 seedlings. However, the activities of catalase and superoxide dismutase decreased and the contents of H202, proline and malonaldialdehyde (MDA) increased in seedlings of both the genotypes. Application of SA (400 µM) led to increased dry biomass in heat stressed CML-32 seedlings. It improved the efficiency of Halliwell-Asada pathway in roots of CML-32 seedlings as was evidenced by the enhanced ascorbate peroxidase and glutathione reductase activities. The activities of catalase and superoxide dismutase increased in both the tissues of LM-11 seedlings, whereas in CML-32, it was only in shoots, after SA application. Peroxidase activity increased in SA treated seedlings of both the genotypes, though the increase was comparatively higher in CML-32. The contents of H₂O₂ and MDA decreased and that of proline increased in SA treated seedlings of both the genotypes, under stress conditions. It may be concluded that SA induced differential antioxidant response by upregulating Halliwell-Asada pathway in roots and attaining high POX activity in both the tissues of CML-32 seedlings, under high temperature stress.

  14. Effects of Inositol 1,4,5-triphosphate on Osteoclast Differentiation in RANKL-induced Osteoclastogenesis.

    PubMed

    Son, Aran; Kim, Min Seuk; Jo, Hae; Byun, Hae Mi; Shin, Dong Min

    2012-02-01

    The receptor activator of NF-κB ligand (RANKL) signal is an activator of tumor necrosis factor receptor-associated factor 6 (TRAF6), which leads to the activation of NF-κB and other signal transduction pathways essential for osteoclastogenesis, such as Ca(2+) signaling. However, the intracellular levels of inositol 1,4,5-trisphosphate (IP(3)) and IP(3)-mediated cellular function of RANKL during osteoclastogenesis are not known. In the present study, we determined the levels of IP(3) and evaluated IP(3)-mediated osteoclast differentiation and osteoclast activity by RANKL treatment of mouse leukemic macrophage cells (RAW 264.7) and mouse bone marrow-derived monocyte/macrophage precursor cells (BMMs). During osteoclastogenesis, the expression levels of Ca(2+) signaling proteins such as IP(3) receptors (IP(3)Rs), plasma membrane Ca(2+) ATPase, and sarco/endoplasmic reticulum Ca(2+) ATPase type2 did not change by RANKL treatment for up to 6 days in both cell types. At 24 h after RANKL treatment, a higher steady-state level of IP(3) was observed in RAW264.7 cells transfected with green fluorescent protein (GFP)-tagged pleckstrin homology (PH) domains of phospholipase C (PLC) δ, a probe specifically detecting intracellular IP(3) levels. In BMMs, the inhibition of PLC with U73122 [a specific inhibitor of phospholipase C (PLC)] and of IP(3)Rs with 2-aminoethoxydiphenyl borate (2APB; a non-specific inhibitor of IP(3)Rs) inhibited the generation of RANKL-induced multinucleated cells and decreased the bone-resorption rate in dentin slice, respectively. These results suggest that intracellular IP(3) levels and the IP(3)-mediated signaling pathway play an important role in RANKL-induced osteoclastogenesis.

  15. Proteomic Identification of Novel Differentiation Plasma Protein Markers in Hypobaric Hypoxia-Induced Rat Model

    PubMed Central

    Ahmad, Mohammad Faiz; Sharma, Manish; Garg, Iti; Bhargava, Kalpana

    2014-01-01

    Background Hypobaric hypoxia causes complex changes in the expression of genes, including stress related genes and corresponding proteins that are necessary to maintain homeostasis. Whereas most prior studies focused on single proteins, newer methods allowing the simultaneous study of many proteins could lead to a better understanding of complex and dynamic changes that occur during the hypobaric hypoxia. Methods In this study we investigated the temporal plasma protein alterations of rat induced by hypobaric hypoxia at a simulated altitude of 7620 m (25,000 ft, 282 mm Hg) in a hypobaric chamber. Total plasma proteins collected at different time points (0, 6, 12 and 24 h), separated by two-dimensional electrophoresis (2-DE) and identified using matrix assisted laser desorption ionization time of flight (MALDI-TOF/TOF). Biological processes that were enriched in the plasma proteins during hypobaric hypoxia were identified using Gene Ontology (GO) analysis. According to their properties and obvious alterations during hypobaric hypoxia, changes of plasma concentrations of Ttr, Prdx-2, Gpx -3, Apo A-I, Hp, Apo-E, Fetub and Nme were selected to be validated by Western blot analysis. Results Bioinformatics analysis of 25 differentially expressed proteins showed that 23 had corresponding candidates in the database. The expression patterns of the eight selected proteins observed by Western blot were in agreement with 2-DE results, thus confirming the reliability of the proteomic analysis. Most of the proteins identified are related to cellular defense mechanisms involving anti-inflammatory and antioxidant activity. Their presence reflects the consequence of serial cascades initiated by hypobaric hypoxia. Conclusion/Significance This study provides information about the plasma proteome changes induced in response to hypobaric hypoxia and thus identification of the candidate proteins which can act as novel biomarkers. PMID:24842778

  16. Salicylic acid induces differential antioxidant response in spring maize under high temperature stress.

    PubMed

    Khanna, Palak; Kaur, Kamaljit; Gupta, Anil K

    2016-06-01

    High temperature is one of the important stress factors that affect crops in tropical countries. Plants do evolve or adopt different mechanisms to overcome such stress for survival. It is an interesting subject and has attracted many researchers to work upon. Here, we studied the effect of salicylic acid (SA) on seedling growth and antioxidative defense system in two spring maize (Zea mays L.) genotypes viz., CML-32 (relatively heat tolerant) and LM-11 (relatively heat susceptible), under high temperature stress. High temperature induced greater reduction in dry biomass of LM-1 1 seedlings as compared to those of CML-32. There was a parallel increase in ascorbate peroxidase and glutathione reductase activities in the roots of CML-32 seedlings. However, the activities of catalase and superoxide dismutase decreased and the contents of H202, proline and malonaldialdehyde (MDA) increased in seedlings of both the genotypes. Application of SA (400 µM) led to increased dry biomass in heat stressed CML-32 seedlings. It improved the efficiency of Halliwell-Asada pathway in roots of CML-32 seedlings as was evidenced by the enhanced ascorbate peroxidase and glutathione reductase activities. The activities of catalase and superoxide dismutase increased in both the tissues of LM-11 seedlings, whereas in CML-32, it was only in shoots, after SA application. Peroxidase activity increased in SA treated seedlings of both the genotypes, though the increase was comparatively higher in CML-32. The contents of H₂O₂ and MDA decreased and that of proline increased in SA treated seedlings of both the genotypes, under stress conditions. It may be concluded that SA induced differential antioxidant response by upregulating Halliwell-Asada pathway in roots and attaining high POX activity in both the tissues of CML-32 seedlings, under high temperature stress. PMID:27468465

  17. Effects of Inositol 1,4,5-triphosphate on Osteoclast Differentiation in RANKL-induced Osteoclastogenesis

    PubMed Central

    Son, Aran; Kim, Min Seuk; Jo, Hae; Byun, Hae Mi

    2012-01-01

    The receptor activator of NF-κB ligand (RANKL) signal is an activator of tumor necrosis factor receptor-associated factor 6 (TRAF6), which leads to the activation of NF-κB and other signal transduction pathways essential for osteoclastogenesis, such as Ca2+ signaling. However, the intracellular levels of inositol 1,4,5-trisphosphate (IP3) and IP3-mediated cellular function of RANKL during osteoclastogenesis are not known. In the present study, we determined the levels of IP3 and evaluated IP3-mediated osteoclast differentiation and osteoclast activity by RANKL treatment of mouse leukemic macrophage cells (RAW 264.7) and mouse bone marrow-derived monocyte/macrophage precursor cells (BMMs). During osteoclastogenesis, the expression levels of Ca2+ signaling proteins such as IP3 receptors (IP3Rs), plasma membrane Ca2+ ATPase, and sarco/endoplasmic reticulum Ca2+ ATPase type2 did not change by RANKL treatment for up to 6 days in both cell types. At 24 h after RANKL treatment, a higher steady-state level of IP3 was observed in RAW264.7 cells transfected with green fluorescent protein (GFP)-tagged pleckstrin homology (PH) domains of phospholipase C (PLC) δ, a probe specifically detecting intracellular IP3 levels. In BMMs, the inhibition of PLC with U73122 [a specific inhibitor of phospholipase C (PLC)] and of IP3Rs with 2-aminoethoxydiphenyl borate (2APB; a non-specific inhibitor of IP3Rs) inhibited the generation of RANKL-induced multinucleated cells and decreased the bone-resorption rate in dentin slice, respectively. These results suggest that intracellular IP3 levels and the IP3-mediated signaling pathway play an important role in RANKL-induced osteoclastogenesis. PMID:22416217

  18. Bone morphogenetic protein 4 and retinoic acid trigger bovine VASA homolog expression in differentiating bovine induced pluripotent stem cells.

    PubMed

    Malaver-Ortega, Luis F; Sumer, Huseyin; Jain, Kanika; Verma, Paul J

    2016-02-01

    Primordial germ cells (PGCs) are the earliest identifiable and completely committed progenitors of female and male gametes. They are obvious targets for genome editing because they assure the transmission of desirable or introduced traits to future generations. PGCs are established at the earliest stages of embryo development and are difficult to propagate in vitro--two characteristics that pose a problem for their practical application. One alternative method to enrich for PGCs in vitro is to differentiate them from pluripotent stem cells derived from adult tissues. Here, we establish a reporter system for germ cell identification in bovine pluripotent stem cells based on green fluorescent protein expression driven by the minimal essential promoter of the bovine Vasa homolog (BVH) gene, whose regulatory elements were identified by orthologous modelling of regulatory units. We then evaluated the potential of bovine induced pluripotent stem cell (biPSC) lines carrying the reporter construct to differentiate toward the germ cell lineage. Our results showed that biPSCs undergo differentiation as embryoid bodies, and a fraction of the differentiating cells expressed BVH. The rate of differentiation towards BVH-positive cells increased up to tenfold in the presence of bone morphogenetic protein 4 or retinoic acid. Finally, we determined that the expression of key PGC genes, such as BVH or SOX2, can be modified by pre-differentiation cell culture conditions, although this increase is not necessarily mirrored by an increase in the rate of differentiation.

  19. Bile acids induce monocyte differentiation toward interleukin-12 hypo-producing dendritic cells via a TGR5-dependent pathway

    PubMed Central

    Ichikawa, Riko; Takayama, Tetsuro; Yoneno, Kazuaki; Kamada, Nobuhiko; Kitazume, Mina T; Higuchi, Hajime; Matsuoka, Katsuyoshi; Watanabe, Mitsuhiro; Itoh, Hiroshi; Kanai, Takanori; Hisamatsu, Tadakazu; Hibi, Toshifumi

    2012-01-01

    Dendritic cells (DCs) are known as antigen-presenting cells and play a central role in both innate and acquired immunity. Peripheral blood monocytes give rise to resident and recruited DCs in lymph nodes and non-lymphoid tissues. The ligands of nuclear hormone receptors can modulate DC differentiation and so influence various biological functions of DCs. The role of bile acids (BAs) as signalling molecules has recently become apparent, but the functional role of BAs in DC differentiation has not yet been elucidated. We show that DCs derived from human peripheral blood monocytes cultured with a BA produce lower levels of interleukin-12 (IL-12) and tumour necrosis factor-α in response to stimulation with commensal bacterial antigens. Stimulation through the nuclear receptor farnesoid X (FXR) did not affect the differentiation of DCs. However, DCs differentiated with the specific agonist for TGR5, a transmembrane BA receptor, showed an IL-12 hypo-producing phenotype. Expression of TGR5 could only be identified in monocytes and was rapidly down-regulated during monocyte differentiation to DCs. Stimulation with 8-bromoadenosine-cyclic AMP (8-Br-cAMP), which acts downstream of TGR5 signalling, also promoted differentiation into IL-12 hypo-producing DCs. These results indicate that BAs induce the differentiation of IL-12 hypo-producing DCs from monocytes via the TGR5-cAMP pathway. PMID:22236403

  20. Bone morphogenic protein-2 regulates the myogenic differentiation of PMVECs in CBDL rat serum-induced pulmonary microvascular remodeling

    SciTech Connect

    Liu, Chang; Chen, Lin; Zeng, Jing; Cui, Jian; Ning, Jiao-nin; Wang, Guan-song; Belguise, Karine; Wang, Xiaobo; Qian, Gui-sheng; Lu, Kai-zhi; Yi, Bin

    2015-08-01

    Hepatopulmonary syndrome (HPS) is characterized by an arterial oxygenation defect induced by intrapulmonary vasodilation (IPVD) that increases morbidity and mortality. In our previous study, it was determined that both the proliferation and the myogenic differentiation of pulmonary microvascular endothelial cells (PMVECs) play a key role in the development of IPVD. However, the molecular mechanism underlying the relationship between IPVD and the myogenic differentiation of PMVECs remains unknown. Additionally, it has been shown that bone morphogenic protein-2 (BMP2), via the control of protein expression, may regulate cell differentiation including cardiomyocyte differentiation, neuronal differentiation and odontoblastic differentiation. In this study, we observed that common bile duct ligation (CBDL)-rat serum induced the upregulation of the expression of several myogenic proteins (SM-α-actin, calponin, SM-MHC) and enhanced the expression levels of BMP2 mRNA and protein in PMVECs. We also observed that both the expression levels of Smad1/5 and the activation of phosphorylated Smad1/5 were significantly elevated in PMVECs following exposure to CBDL-rat serum, which was accompanied by the down-regulation of Smurf1. The blockage of the BMP2/Smad signaling pathway with Noggin inhibited the myogenic differentiation of PMVECs, a process that was associated with relatively low expression levels of both SM-α-actin and calponin in the setting of CBDL-rat serum exposure, although SM-MHC expression was not affected. These findings suggested that the BMP2/Smad signaling pathway is involved in the myogenic differentiation of the PMVECs. In conclusion, our data highlight the pivotal role of BMP2 in the CBDL-rat serum-induced myogenic differentiation of PMVECs via the activation of both Smad1 and Smad5 and the down-regulation of Smurf1, which may represent a potential therapy for HPS-induced pulmonary vascular remodeling. - Highlights: • CBDL-rat serum promotes the myogenic

  1. Curl flux induced drift in stochastic differential equations in the zero-mass limit

    NASA Astrophysics Data System (ADS)

    Wang, Jinhua; Yuan, Bo

    2016-11-01

    We consider the nonlinear stochastic dynamics of dissipative Hamiltonian systems with state-dependent friction and diffusion connected by the fluctuation-dissipation relation in high dimensions. The system under study has a close connection to Ao's framework in constructing a dynamical potential for non-equilibrium processes without detailed balance. We study the limiting case where the mass approaches zero and give a new and complete derivation of effective stochastic differential equations. Using the Ito stochastic integral convention, we show that the limiting effective Langevin equations have a new drift term. This extra term happens to be identical to the corresponding anti-Ito (or isothermal) integral (requiring constant temperature) in one dimension. We, however, cannot obtain this additional drift term using conventional stochastic integrals in high dimension. It is interesting to note that in a high-dimensional system, a curl flux induced drift may appear even if the diffusion matrix is constant. Our findings are supported by numerical simulations. We further analyze and discuss the role of this new drift term in calculating the classic escape time. For the first time, to our knowledge, the relation between the escape rate and the anti-Ito integral is presented. We also demonstrate that the derived diffusion equations give a new sampling algorithm which can increase convergence speed in a simple two-dimensional example.

  2. Human germ cell differentiation from fetal- and adult-derived induced pluripotent stem cells

    PubMed Central

    Panula, Sarita; Medrano, Jose V.; Kee, Kehkooi; Bergström, Rosita; Nguyen, Ha Nam; Byers, Blake; Wilson, Kitchener D.; Wu, Joseph C.; Simon, Carlos; Hovatta, Outi; Reijo Pera, Renee A.

    2011-01-01

    Historically, our understanding of molecular genetic aspects of human germ cell development has been limited, at least in part due to inaccessibility of early stages of human development to experimentation. However, the derivation of pluripotent stem cells may provide the necessary human genetic system to study germ cell development. In this study, we compared the potential of human induced pluripotent stem cells (iPSCs), derived from adult and fetal somatic cells to form primordial and meiotic germ cells, relative to human embryonic stem cells. We found that ∼5% of human iPSCs differentiated to primordial germ cells (PGCs) following induction with bone morphogenetic proteins. Furthermore, we observed that PGCs expressed green fluorescent protein from a germ cell-specific reporter and were enriched for the expression of endogenous germ cell-specific proteins and mRNAs. In response to the overexpression of intrinsic regulators, we also observed that iPSCs formed meiotic cells with extensive synaptonemal complexes and post-meiotic haploid cells with a similar pattern of ACROSIN staining as observed in human spermatids. These results indicate that human iPSCs derived from reprogramming of adult somatic cells can form germline cells. This system may provide a useful model for molecular genetic studies of human germline formation and pathology and a novel platform for clinical studies and potential therapeutical applications. PMID:21131292

  3. Chrysanthemum zawadskii extract induces hair growth by stimulating the proliferation and differentiation of hair matrix.

    PubMed

    Li, Zheng; Li, Jingjie; Gu, Lijuan; Begum, Shahnaz; Wang, Yunbo; Sun, Baishen; Lee, Mira; Sung, Changkeun

    2014-07-01

    Chrysanthemum zawadskii has been proven to possess hair growth activity and has been used as treatment for hair loss. The aim of this study was to provide a novel explanation of the mechanism by which Chrysanthemum zawadskii extracts (CZe) promote hair growth and to characterize the affected hair follicle (HF) regions and the progression of growth. The n-butanol and water fractions of CZe were used for hair growth induction by topical application to the backs of C57BL/6 mice for up to 30 days. To investigate cell development during HF morphogenesis, bromodeoxyuridine-labeled skin sections were detected using immunohistochemistry. The results showed that the water fraction of CZe promoted hair shaft production and induced premature entry of telogen HFs into the anagen. Subsequently, immunohistochemical studies indicated that the water fraction of CZe stimulated the differentiation and proliferation of pluripotent epidermal matrix cells in the matrix region and epithelial stem cells in the basal layer of the epidermis. Additionally, flavonoids were identified as effective constituents. Therefore, the findings of this study suggested that the water fraction of CZe may be developed as a therapeutic agent for the prevention of hair loss. PMID:24807783

  4. Light-induced negative differential resistance in graphene/Si-quantum-dot tunneling diodes.

    PubMed

    Lee, Kyeong Won; Jang, Chan Wook; Shin, Dong Hee; Kim, Jong Min; Kang, Soo Seok; Lee, Dae Hun; Kim, Sung; Choi, Suk-Ho; Hwang, Euyheon

    2016-01-01

    One of the interesing tunneling phenomena is negative differential resistance (NDR), the basic principle of resonant-tunneling diodes. NDR has been utilized in various semiconductor devices such as frequency multipliers, oscillators, relfection amplifiers, logic switches, and memories. The NDR in graphene has been also reported theoretically as well as experimentally, but should be further studied to fully understand its mechanism, useful for practical device applications. Especially, there has been no observation about light-induced NDR (LNDR) in graphene-related structures despite very few reports on the LNDR in GaAs-based heterostructures. Here, we report first observation of LNDR in graphene/Si quantum dots-embedded SiO2 (SQDs:SiO2) multilayers (MLs) tunneling diodes. The LNDR strongly depends on temperature (T) as well as on SQD size, and the T dependence is consistent with photocurrent (PC)-decay behaviors. With increasing light power, the PC-voltage curves are more structured with peak-to-valley ratios over 2 at room temperature. The physical mechanism of the LNDR, governed by resonant tunneling of charge carriers through the minibands formed across the graphene/SQDs:SiO2 MLs and by their nonresonant phonon-assisted tunneling, is discussed based on theoretical considerations.

  5. Effects of methylazoxymethanol-induced micrencephaly on temporal response differentiation and progressive ratio responding in rats.

    PubMed

    Ferguson, S A; Holson, R R; Paule, M G

    1994-07-01

    Micrencephalic Sprague-Dawley rats were produced by an injection of 20 mg/kg methylazoxymethanol acetate on gestational Day 14. Brain weights of the offspring were 70% of controls while weights of frontal cortex and hippocampus were approximately 58% (Ferguson, Racey, Paule, & Holson, 1993). Operant performance was measured with particular emphasis on assessment of time estimation. The temporal response differentiation (TRD) and the progressive ratio (PR) tasks, previously used in the NCTR operant test battery for monkeys, were chosen for evaluation. The TRD schedule is notably different from other temporal tasks in that it requires subjects to initiate and maintain a lever press for 10-14 s. The PR task was included as a measure of motivation to work for food reinforces. Micrencephalics acquired and performed both tasks comparably to controls. During extinction, however, micrencephalics exhibited an increased TRD lever hold duration. This suggests an atypical response perservation, that is, perseverating the previously correct response. Previously, frontal cortical alterations were suggested to contribute heavily to micrencephalic-induced behavioral alterations (Ferguson et al., 1993). This study provides further evidence that response perseveration, a hallmark of frontal cortical lesions, is expressed in micrencephalic rats.

  6. Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

    PubMed Central

    Di Pasquale, Elisa; Song, Belle; Condorelli, Gianluigi

    2013-01-01

    In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells. PMID:23851455

  7. Gap state charge induced spin-dependent negative differential resistance in tunnel junctions

    NASA Astrophysics Data System (ADS)

    Jiang, Jun; Zhang, X.-G.; Han, X. F.

    2016-04-01

    We propose and demonstrate through first-principles calculation a new spin-dependent negative differential resistance (NDR) mechanism in magnetic tunnel junctions (MTJ) with cubic cation disordered crystals (CCDC) AlO x or Mg1-x Al x O as barrier materials. The CCDC is a class of insulators whose band gap can be changed by cation doping. The gap becomes arched in an ultrathin layer due to the space charge formed from metal-induced gap states. With an appropriate combination of an arched gap and a bias voltage, NDR can be produced in either spin channel. This mechanism is applicable to 2D and 3D ultrathin junctions with a sufficiently small band gap that forms a large space charge. It provides a new way of controlling the spin-dependent transport in spintronic devices by an electric field. A generalized Simmons formula for tunneling current through junction with an arched gap is derived to show the general conditions under which ultrathin junctions may exhibit NDR.

  8. Light-induced negative differential resistance in graphene/Si-quantum-dot tunneling diodes.

    PubMed

    Lee, Kyeong Won; Jang, Chan Wook; Shin, Dong Hee; Kim, Jong Min; Kang, Soo Seok; Lee, Dae Hun; Kim, Sung; Choi, Suk-Ho; Hwang, Euyheon

    2016-01-01

    One of the interesing tunneling phenomena is negative differential resistance (NDR), the basic principle of resonant-tunneling diodes. NDR has been utilized in various semiconductor devices such as frequency multipliers, oscillators, relfection amplifiers, logic switches, and memories. The NDR in graphene has been also reported theoretically as well as experimentally, but should be further studied to fully understand its mechanism, useful for practical device applications. Especially, there has been no observation about light-induced NDR (LNDR) in graphene-related structures despite very few reports on the LNDR in GaAs-based heterostructures. Here, we report first observation of LNDR in graphene/Si quantum dots-embedded SiO2 (SQDs:SiO2) multilayers (MLs) tunneling diodes. The LNDR strongly depends on temperature (T) as well as on SQD size, and the T dependence is consistent with photocurrent (PC)-decay behaviors. With increasing light power, the PC-voltage curves are more structured with peak-to-valley ratios over 2 at room temperature. The physical mechanism of the LNDR, governed by resonant tunneling of charge carriers through the minibands formed across the graphene/SQDs:SiO2 MLs and by their nonresonant phonon-assisted tunneling, is discussed based on theoretical considerations. PMID:27465107

  9. Gravimetric analysis and differential scanning calorimetric studies on glycerin-induced skin hydration.

    PubMed

    Lee, Ae-Ri Cho; Moon, Hee Kyung

    2007-11-01

    A thermal gravimetric analysis (TGA) and a differential scanning calorimetry (DSC) were carried out to characterize the water property and an alteration of lipid phase transition of stratum corneum (SC) by glycerin. In addition, the relationship between steady state skin permeation rate and skin hydration in various concentrations of glycerin was investigated. Water vapor absorption-desorption was studied in the hairless mouse stratum corneum. Dry SC samples were exposed to different conc. of glycerin (0-50%) followed by exposure to dry air and the change in weight property was monitored over time by use of TGA. In DSC study, significant decrease in DeltaH of the lipid transition in 10% glycerin and water treated sample: the heat of lipid transition of normal, water, 10% glycerin treated SC were 6.058, 4.412 and 4.316 mJ/mg, respectively. In 10% glycerin treated SCs, the Tc of water shifts around 129 degrees C, corresponding to the weakly bound secondary water. In 40% glycerin treated SC, the Tc of water shifts to 144 degrees C corresponding to strongly bound primary water. There was a good correlation between the hydration property of the skin and the steady state skin flux with the correlation coefficient (r2=0.94). As the hydration increased, the steady state flux increased. As glycerin concentration increased, hydration property decreased. High diffusivity induced by the hydration effect of glycerin and water could be the major contributing factor for the enhanced skin permeation of nicotinic acid (NA).

  10. Electrospun tilapia collagen nanofibers accelerating wound healing via inducing keratinocytes proliferation and differentiation.

    PubMed

    Zhou, Tian; Wang, Nanping; Xue, Yang; Ding, Tingting; Liu, Xin; Mo, Xiumei; Sun, Jiao

    2016-07-01

    The development of biomaterials with the ability to induce skin wound healing is a great challenge in biomedicine. In this study, tilapia skin collagen sponge and electrospun nanofibers were developed for wound dressing. The collagen sponge was composed of at least two α-peptides. It did not change the number of spleen-derived lymphocytes in BALB/c mice, the ratio of CD4(+)/CD8(+) lymphocytes, and the level of IgG or IgM in Sprague-Dawley rats. The tensile strength and contact angle of collagen nanofibers were 6.72±0.44MPa and 26.71±4.88°, respectively. They also had good thermal stability and swelling property. Furthermore, the nanofibers could significantly promote the proliferation of human keratinocytes (HaCaTs) and stimulate epidermal differentiation through the up-regulated gene expression of involucrin, filaggrin, and type I transglutaminase in HaCaTs. The collagen nanofibers could also facilitate rat skin regeneration. In the present study, electrospun biomimetic tilapia skin collagen nanofibers were succesfully prepared, were proved to have good bioactivity and could accelerate rat wound healing rapidly and effectively. These biological effects might be attributed to the biomimic extracellular matrix structure and the multiple amino acids of the collagen nanofibers. Therefore, the cost-efficient tilapia collagen nanofibers could be used as novel wound dressing, meanwhile effectively avoiding the risk of transmitting animal disease in the future clinical apllication. PMID:27037778

  11. Light-induced negative differential resistance in graphene/Si-quantum-dot tunneling diodes

    PubMed Central

    Lee, Kyeong Won; Jang, Chan Wook; Shin, Dong Hee; Kim, Jong Min; Kang, Soo Seok; Lee, Dae Hun; Kim, Sung; Choi, Suk-Ho; Hwang, Euyheon

    2016-01-01

    One of the interesing tunneling phenomena is negative differential resistance (NDR), the basic principle of resonant-tunneling diodes. NDR has been utilized in various semiconductor devices such as frequency multipliers, oscillators, relfection amplifiers, logic switches, and memories. The NDR in graphene has been also reported theoretically as well as experimentally, but should be further studied to fully understand its mechanism, useful for practical device applications. Especially, there has been no observation about light-induced NDR (LNDR) in graphene-related structures despite very few reports on the LNDR in GaAs-based heterostructures. Here, we report first observation of LNDR in graphene/Si quantum dots-embedded SiO2 (SQDs:SiO2) multilayers (MLs) tunneling diodes. The LNDR strongly depends on temperature (T) as well as on SQD size, and the T dependence is consistent with photocurrent (PC)-decay behaviors. With increasing light power, the PC-voltage curves are more structured with peak-to-valley ratios over 2 at room temperature. The physical mechanism of the LNDR, governed by resonant tunneling of charge carriers through the minibands formed across the graphene/SQDs:SiO2 MLs and by their nonresonant phonon-assisted tunneling, is discussed based on theoretical considerations. PMID:27465107

  12. Gap state charge induced spin-dependent negative differential resistance in tunnel junctions

    NASA Astrophysics Data System (ADS)

    Jiang, Jun; Zhang, X.-G.; Han, X. F.

    2016-04-01

    We propose and demonstrate through first-principles calculation a new spin-dependent negative differential resistance (NDR) mechanism in magnetic tunnel junctions (MTJ) with cubic cation disordered crystals (CCDC) AlO x or Mg1‑x Al x O as barrier materials. The CCDC is a class of insulators whose band gap can be changed by cation doping. The gap becomes arched in an ultrathin layer due to the space charge formed from metal-induced gap states. With an appropriate combination of an arched gap and a bias voltage, NDR can be produced in either spin channel. This mechanism is applicable to 2D and 3D ultrathin junctions with a sufficiently small band gap that forms a large space charge. It provides a new way of controlling the spin-dependent transport in spintronic devices by an electric field. A generalized Simmons formula for tunneling current through junction with an arched gap is derived to show the general conditions under which ultrathin junctions may exhibit NDR.

  13. Differential effects of aging and sex on stroke induced inflammation across the lifespan.

    PubMed

    Manwani, Bharti; Liu, Fudong; Scranton, Victoria; Hammond, Matthew D; Sansing, Lauren H; McCullough, Louise D

    2013-11-01

    Aging and biological sex are critical determinants of stroke outcome. Post-ischemic inflammatory response strongly contributes to the extent of ischemic brain injury, but how this response changes with age and sex is unknown. We subjected young (5-6 months), middle aged (14-15 months) and aged (20-22 months), C57BL/6 male and female mice to transient middle cerebral artery occlusion (MCAO) and found that a significant age by sex interaction influenced histological stroke outcomes. Acute functional outcomes were worse with aging. Neutrophils, inflammatory macrophages, macrophages, dendritic cells (DCs) and microglia significantly increased in the brain post MCAO. Cycling females had higher Gr1(-) non-inflammatory macrophages and lower T cells in the brain after stroke and these correlated with serum estradiol levels. Estrogen loss in acyclic aged female mice exacerbated stroke induced splenic contraction. Advanced age increased T cells, DCs and microglia at the site of injury, which may be responsible for the exacerbated behavioral deficits in the aged. We conclude that aging and sex have differential effects on the post stroke inflammatory milieu. Putative immunomodulatory therapies need to account for this heterogeneity.

  14. Berberine Sulfate Attenuates Osteoclast Differentiation through RANKL Induced NF-κB and NFAT Pathways.

    PubMed

    Zhou, Lin; Song, Fangming; Liu, Qian; Yang, Mingli; Zhao, Jinmin; Tan, Renxiang; Xu, Jun; Zhang, Ge; Quinn, Julian M W; Tickner, Jennifer; Xu, Jiake

    2015-11-13

    Osteoporosis, a metabolic bone disease, is characterized by an excessive formation and activation of osteoclasts. Anti-catabolic treatment using natural compounds has been proposed as a potential therapeutic strategy against the osteoclast related osteolytic diseases. In this study, the activity of berberine sulfate (an orally available form of berberine) on osteoclast differentiation and its underlying molecular mechanisms of action were investigated. Using bone marrow macrophages (BMMs) derived osteoclast culture system, we showed that berberine sulfate at the dose of 0.25, 0.5 and 1 μM significantly inhibited the formation of osteoclasts. Notably, berberine sulfate at these doses did not affect the BMM viability. In addition, we observed that berberine sulfate inhibited the expression of osteoclast marker genes, including cathepsin K (Ctsk), nuclear factor of activated T cells cytoplasmic 1 (NFATc1), tartrate resistant acid phosphatase (TRAcP, Acp5) and Vacuolar-type H+-ATPase V0 subunit D2 (V-ATPase d2). Luciferase reporter gene assay and Western blot analysis further revealed that berberine sulfate inhibits receptor for activation of nuclear factor ligand (RANKL)-induced NF-κB and NFAT activity. Taken together, our results suggest that berberine sulfate is a natural compound potentially useful for the treatment of osteoporosis.

  15. Light-induced negative differential resistance in graphene/Si-quantum-dot tunneling diodes

    NASA Astrophysics Data System (ADS)

    Lee, Kyeong Won; Jang, Chan Wook; Shin, Dong Hee; Kim, Jong Min; Kang, Soo Seok; Lee, Dae Hun; Kim, Sung; Choi, Suk-Ho; Hwang, Euyheon

    2016-07-01

    One of the interesing tunneling phenomena is negative differential resistance (NDR), the basic principle of resonant-tunneling diodes. NDR has been utilized in various semiconductor devices such as frequency multipliers, oscillators, relfection amplifiers, logic switches, and memories. The NDR in graphene has been also reported theoretically as well as experimentally, but should be further studied to fully understand its mechanism, useful for practical device applications. Especially, there has been no observation about light-induced NDR (LNDR) in graphene-related structures despite very few reports on the LNDR in GaAs-based heterostructures. Here, we report first observation of LNDR in graphene/Si quantum dots-embedded SiO2 (SQDs:SiO2) multilayers (MLs) tunneling diodes. The LNDR strongly depends on temperature (T) as well as on SQD size, and the T dependence is consistent with photocurrent (PC)-decay behaviors. With increasing light power, the PC-voltage curves are more structured with peak-to-valley ratios over 2 at room temperature. The physical mechanism of the LNDR, governed by resonant tunneling of charge carriers through the minibands formed across the graphene/SQDs:SiO2 MLs and by their nonresonant phonon-assisted tunneling, is discussed based on theoretical considerations.

  16. Resveratrol sensitizes glioblastoma-initiating cells to temozolomide by inducing cell apoptosis and promoting differentiation.

    PubMed

    Li, Hao; Liu, Yaodong; Jiao, Yumin; Guo, Anchen; Xu, Xiaoxue; Qu, Xianjun; Wang, Shuo; Zhao, Jizong; Li, Ye; Cao, Yong

    2016-01-01

    Glioblastoma-initiating cells play crucial roles in the origin, growth, and recurrence of glioblastoma multiforme. The elimination of glioblastoma-initiating cells is believed to be a key strategy for achieving long-term survival of glioblastoma patients due to the highly resistant property of glioblastoma-initiating cells to temozolomide. Resveratrol, a naturally occurring polyphenol, has been widely studied as a promising candidate for cancer prevention and treatment. Whether resveratrol could enhance the sensitivity of glioblastoma-initiating cells to temozolomide therapy has not yet been reported. Here, using patient-derived glioblastoma-initiating cell lines, we found that resveratrol sensitized glioblastoma-initiating cells to temozolomide both in vitro and in vivo. Furthermore, we showed that resveratrol enhanced glioblastoma-initiating cells to temozolomide-induced apoptosis through DNA double-stranded breaks/pATM/pATR/p53 pathway activation, and promoted glioblastoma-initiating cell differentiation involving p-STAT3 inactivation. Our results propose that temozolomide and resveratrol combination strategy may be effective in the management of glioblastoma patients, particularly for those patients who have been present with a high abundance of glioblastoma-initiating cells in their tumors and show slight responsiveness to temozolomide.

  17. Differential effects of aging and sex on stroke induced inflammation across the lifespan

    PubMed Central

    Manwani, Bharti; Liu, Fudong; Scranton, Victoria; Hammond, Matthew D.; Sansing, Lauren H.; McCullough, Louise D.

    2013-01-01

    Aging and biological sex are critical determinants of stroke outcome. Post-ischemic inflammatory response strongly contributes to the extent of ischemic brain injury, but how this response changes with age and sex is unknown. We subjected young (5–6 months), middle aged (14–15 months) and aged (20–22 months), C57BL/6 male and female mice to transient middle cerebral artery occlusion (MCAO) and found that a significant age by sex interaction influenced histological stroke outcomes. Acute functional outcomes were worse with aging. Neutrophils, inflammatory macrophages, macrophages, dendritic cells (DCs) and microglia significantly increased in the brain post MCAO. Cycling females had higher Gr1− non-inflammatory macrophages and lower T cells in the brain after stroke and these correlated with serum estradiol levels. Estrogen loss in acyclic aged female mice exacerbated stroke induced splenic contraction. Advanced age increased T cells, DCs and microglia at the site of injury, which may be responsible for the exacerbated behavioral deficits in the aged. We conclude that aging and sex have differential effects on the post stroke inflammatory milieu. Putative immunomodulatory therapies need to account for this heterogeneity. PMID:23994069

  18. Differentiation of silicates from H2O ice in an icy body induced by ripening

    NASA Astrophysics Data System (ADS)

    Sirono, Sin-iti

    2013-12-01

    One of the probable scenarios of differentiation between silicate-ice in an icy object is the settling of a silicate particle in water after the melting of the object. In order for settling to proceed or occur, the size of the particle should be sufficiently large such that the settling velocity of the particle exceeds the background flow velocity induced by thermal convection. The sizes of the particles change because of dissolution and precipitation. This process is called ripening. In this study, the critical particle sizes required for settling, and the timescales for the growth of the particles to these sizes through ripening, are analytically derived. It is observed that settling is possible if the silicate particles coagulate with each other to form a network in water. If the particles do not coagulate, the probability of the occurrence of settling is low, because the time duration required for the particle growth to the critical size is large. The coagulation of silicate particles strongly depends on the pH of the water.

  19. High mobility group box 1 induced human lung myofibroblasts differentiation and enhanced migration by activation of MMP-9.

    PubMed

    Lee, Chen-Chen; Wang, Chien-Neng; Lee, Yueh-Lun; Tsai, Yi-Ru; Liu, Jau-Jin

    2015-01-01

    High mobility group box 1 (HMGB1) is a nuclear protein that involves the binding with DNA and influences chromatin regulation and transcription. HMGB1 is also a cytokine that can activate monocytes and neutrophils involved in inflammation. In this study, we investigated the role of HMGB1 on cellular activation using human fibroblast cell line WI-38. After treatment with 1, 10, and 100 ng/mL of HMGB1 for 24 h, we did not find obviously cytotoxicity and cellular proliferation of WI-38 cells by MTT and BrdU incorporation assay, respectively. However, we found that treatment with 10 and 100 ng/mL of HMGB1 induced the differentiation of lung fibroblasts into myofibroblasts and myofibroblasts showed higher migration ability through activation of matrix metalloproteinase (MMP)-9 activation. To delineate the mechanism underlying HMGB1-induced cellular migration, we examined HMGB1-induced mitogen activated protein kinases (MAPKs), including extracellular signal related kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen activated protein kinase (p38) phosphorylation, as well as nuclear factor (NF)-κB nuclear translocation. Using specific inhibitors and shRNAs of protein kinases, we observed that repression of ERK, JNK, p38, and NF-κB all inhibited HMGB1-induced cellular differentiation, migration and MMP-9 activation in WI-38 cells. In addition, knocking down of RAGE but not TLR2 and TLR4 by shRNAs attenuated HMGB1-induced myofibroblast differentiation and migration. In conclusion, our study demonstrated that HMGB1 induced lung fibroblasts' differentiation into myofibroblasts and enhanced cell migration through induction of MMP-9 activation and the RAGE-MAPK and NF-κB interaction signaling pathways. Targeting HMGB1 might be a potential therapeutic approach for alleviation of airway remodeling seen in chronic airway inflammatory diseases.

  20. Differential responses in central dopaminergic activity induced by apomorphine in IPL nude rat.

    PubMed

    Estrella, Cecilia Ruth; Bregonzio, Claudia; Cabrera, Ricardo Jorge

    2002-07-18

    increase in the DA synthesis rate in nucleus accumbens, while 5 mg/kg decrease it in both strains. The DA turnover rate in the same area was lower in IPL nude than in SD rats after saline injection. Apomorphine enhances the DA turnover rate in both strains. We conclude that the modifications of the oral spontaneous and induced stereotypical patterns observed in the IPL nude rats could be related to the differential responses in dopaminergic activity in the two brain areas examined. PMID:12110447

  1. Saponin-induced release of single cells from filaments and rhizoid differentiation in Spirogyra.

    PubMed

    Ikegaya, Hisato; Yamada, Shin-Ya; Sonobe, Seiji; Shimmen, Teruo

    2004-12-01

    Some species of Spirogyra living in streams can anchor to the substratum by differentiating a rhizoid from a terminal cell of a filament. Rhizoid differentiation occurs in the light but not in the dark. When a filament of Spirogyra sp. competent for rhizoid differentiation was incubated in a medium containing 0.1% saponin, terminal cells were released one by one, forming single cells. Single cells effectively differentiated to be rhizoids when saponin in the incubation medium was removed. The single-cell system developed in the present study seems suitable for analysis of gene expression during rhizoid differentiation of Spirogyra.

  2. Nitric oxide synthase mediates PC12 differentiation induced by the surface topography of nanostructured TiO2

    PubMed Central

    2013-01-01

    Background Substrate nanoscale topography influences cell proliferation and differentiation through mechanisms that are at present poorly understood. In particular the molecular mechanism through which cells 'sense’ and adapt to the substrate and activate specific intracellular signals, influencing cells survival and behavior, remains to be clarified. Results To characterize these processes at the molecular level we studied the differentiation of PC12 cells on nanostructured TiO2 films obtained by supersonic cluster beam deposition. Our findings indicate that, in PC12 cells grown without Nerve Growth Factor (NGF), the roughness of nanostructured TiO2 triggers neuritogenesis by activating the expression of nitric oxide synthase (NOS) and the phospho-extracellular signal-regulated kinase 1/2 (pERK1/2) signaling. Differentiation is associated with an increase in protein nitration as observed in PC12 cells grown on flat surfaces in the presence of NGF. We demonstrate that cell differentiation and protein nitration induced by topography are not specific for PC12 cells but can be regarded as generalized effects produced by the substrate on different neuronal-like cell types, as shown by growing the human neuroblastoma SH-SY5Y cell line on nanostructured TiO2. Conclusion Our data provide the evidence that the nitric oxide (NO) signal cascade is involved in the differentiation process induced by nanotopography, adding new information on the mechanism and proteins involved in the neuritogenesis triggered by the surface properties. PMID:24119372

  3. Mutant GDF5 enhances ameloblast differentiation via accelerated BMP2-induced Smad1/5/8 phosphorylation.

    PubMed

    Liu, Jia; Saito, Kan; Maruya, Yuriko; Nakamura, Takashi; Yamada, Aya; Fukumoto, Emiko; Ishikawa, Momoko; Iwamoto, Tsutomu; Miyazaki, Kanako; Yoshizaki, Keigo; Ge, Lihong; Fukumoto, Satoshi

    2016-01-01

    Bone morphogenetic proteins (BMPs) regulate hard tissue formation, including bone and tooth. Growth differentiation factor 5 (GDF5), a known BMP, is expressed in cartilage and regulates chondrogenesis, and mutations have been shown to cause osteoarthritis. Notably, GDF5 is also expressed in periodontal ligament tissue; however, its role during tooth development is unclear. Here, we used cell culture and in vivo analyses to determine the role of GDF5 during tooth development. GDF5 and its associated BMP receptors are expressed at the protein and mRNA levels during postnatal tooth development, particularly at a stage associated with enamel formation. Furthermore, whereas BMP2 was observed to induce evidently the differentiation of enamel-forming ameloblasts, excess GDF5 induce mildly this differentiation. A mouse model harbouring a mutation in GDF5 (W408R) showed enhanced enamel formation in both the incisors and molars, but not in the tooth roots. Overexpression of the W408R GDF5 mutant protein was shown to induce BMP2-mediated mRNA expression of enamel matrix proteins and downstream phosphorylation of Smad1/5/8. These results suggest that mutant GDF5 enhances ameloblast differentiation via accelerated BMP2-signalling. PMID:27030100

  4. Interleukin-17A Differentially Induces Inflammatory and Metabolic Gene Expression in the Adipose Tissues of Lean and Obese Mice

    PubMed Central

    Qu, Yine; Zhang, Qiuyang; Ma, Siqi; Liu, Sen; Chen, Zhiquan; Mo, Zhongfu; You, Zongbing

    2016-01-01

    The functions of interleukin-17A (IL-17A) in adipose tissues and adipocytes have not been well understood. In the present study, male mice were fed with a regular diet (n = 6, lean mice) or a high-fat diet (n = 6, obese mice) for 30 weeks. Subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) were analyzed for IL-17A levels. SAT and VAT were treated with IL-17A and analyzed for inflammatory and metabolic gene expression. Mouse 3T3-L1 pre-adipocytes were differentiated into adipocytes, followed with IL-17A treatment and analysis for inflammatory and metabolic gene expression. We found that IL-17A levels were higher in obese SAT than lean SAT; the basal expression of inflammatory and metabolic genes was different between SAT and VAT and between lean and obese adipose tissues. IL-17A differentially induced expression of inflammatory and metabolic genes, such as tumor necrosis factor α, Il-6, Il-1β, leptin, and glucose transporter 4, in adipose tissues of lean and obese mice. IL-17A also differentially induced expression of inflammatory and metabolic genes in pre-adipocytes and adipocytes, and IL-17A selectively activated signaling pathways in adipose tissues and adipocytes. These findings suggest that IL-17A differentially induces inflammatory and metabolic gene expression in the adipose tissues of lean and obese mice. PMID:27070576

  5. Interleukin-17A Differentially Induces Inflammatory and Metabolic Gene Expression in the Adipose Tissues of Lean and Obese Mice.

    PubMed

    Qu, Yine; Zhang, Qiuyang; Ma, Siqi; Liu, Sen; Chen, Zhiquan; Mo, Zhongfu; You, Zongbing

    2016-04-07

    The functions of interleukin-17A (IL-17A) in adipose tissues and adipocytes have not been well understood. In the present study, male mice were fed with a regular diet (n = 6, lean mice) or a high-fat diet (n = 6, obese mice) for 30 weeks. Subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) were analyzed for IL-17A levels. SAT and VAT were treated with IL-17A and analyzed for inflammatory and metabolic gene expression. Mouse 3T3-L1 pre-adipocytes were differentiated into adipocytes, followed with IL-17A treatment and analysis for inflammatory and metabolic gene expression. We found that IL-17A levels were higher in obese SAT than lean SAT; the basal expression of inflammatory and metabolic genes was different between SAT and VAT and between lean and obese adipose tissues. IL-17A differentially induced expression of inflammatory and metabolic genes, such as tumor necrosis factor α, Il-6, Il-1β, leptin, and glucose transporter 4, in adipose tissues of lean and obese mice. IL-17A also differentially induced expression of inflammatory and metabolic genes in pre-adipocytes and adipocytes, and IL-17A selectively activated signaling pathways in adipose tissues and adipocytes. These findings suggest that IL-17A differentially induces inflammatory and metabolic gene expression in the adipose tissues of lean and obese mice.

  6. Interleukin-17A Differentially Induces Inflammatory and Metabolic Gene Expression in the Adipose Tissues of Lean and Obese Mice.

    PubMed

    Qu, Yine; Zhang, Qiuyang; Ma, Siqi; Liu, Sen; Chen, Zhiquan; Mo, Zhongfu; You, Zongbing

    2016-01-01

    The functions of interleukin-17A (IL-17A) in adipose tissues and adipocytes have not been well understood. In the present study, male mice were fed with a regular diet (n = 6, lean mice) or a high-fat diet (n = 6, obese mice) for 30 weeks. Subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) were analyzed for IL-17A levels. SAT and VAT were treated with IL-17A and analyzed for inflammatory and metabolic gene expression. Mouse 3T3-L1 pre-adipocytes were differentiated into adipocytes, followed with IL-17A treatment and analysis for inflammatory and metabolic gene expression. We found that IL-17A levels were higher in obese SAT than lean SAT; the basal expression of inflammatory and metabolic genes was different between SAT and VAT and between lean and obese adipose tissues. IL-17A differentially induced expression of inflammatory and metabolic genes, such as tumor necrosis factor α, Il-6, Il-1β, leptin, and glucose transporter 4, in adipose tissues of lean and obese mice. IL-17A also differentially induced expression of inflammatory and metabolic genes in pre-adipocytes and adipocytes, and IL-17A selectively activated signaling pathways in adipose tissues and adipocytes. These findings suggest that IL-17A differentially induces inflammatory and metabolic gene expression in the adipose tissues of lean and obese mice. PMID:27070576

  7. Mutant GDF5 enhances ameloblast differentiation via accelerated BMP2-induced Smad1/5/8 phosphorylation

    PubMed Central

    Liu, Jia; Saito, Kan; Maruya, Yuriko; Nakamura, Takashi; Yamada, Aya; Fukumoto, Emiko; Ishikawa, Momoko; Iwamoto, Tsutomu; Miyazaki, Kanako; Yoshizaki, Keigo; Ge, Lihong; Fukumoto, Satoshi

    2016-01-01

    Bone morphogenetic proteins (BMPs) regulate hard tissue formation, including bone and tooth. Growth differentiation factor 5 (GDF5), a known BMP, is expressed in cartilage and regulates chondrogenesis, and mutations have been shown to cause osteoarthritis. Notably, GDF5 is also expressed in periodontal ligament tissue; however, its role during tooth development is unclear. Here, we used cell culture and in vivo analyses to determine the role of GDF5 during tooth development. GDF5 and its associated BMP receptors are expressed at the protein and mRNA levels during postnatal tooth development, particularly at a stage associated with enamel formation. Furthermore, whereas BMP2 was observed to induce evidently the differentiation of enamel-forming ameloblasts, excess GDF5 induce mildly this differentiation. A mouse model harbouring a mutation in GDF5 (W408R) showed enhanced enamel formation in both the incisors and molars, but not in the tooth roots. Overexpression of the W408R GDF5 mutant protein was shown to induce BMP2-mediated mRNA expression of enamel matrix proteins and downstream phosphorylation of Smad1/5/8. These results suggest that mutant GDF5 enhances ameloblast differentiation via accelerated BMP2-signalling. PMID:27030100

  8. Bone Morphogenetic Protein-9 Effectively Induces Osteo/Odontoblastic Differentiation of the Reversibly Immortalized Stem Cells of Dental Apical Papilla

    PubMed Central

    Wang, Jinhua; Zhang, Hongmei; Zhang, Wenwen; Huang, Enyi; Wang, Ning; Wu, Ningning; Wen, Sheng; Chen, Xian; Liao, Zhan; Deng, Fang; Yin, Liangjun; Zhang, Junhui; Zhang, Qian; Yan, Zhengjian; Liu, Wei; Zhang, Zhonglin; Ye, Jixing; Deng, Youlin; Luu, Hue H.; Haydon, Rex C.

    2014-01-01

    Dental pulp/dentin regeneration using dental stem cells combined with odontogenic factors may offer great promise to treat and/or prevent premature tooth loss. We previously demonstrated that bone morphogenetic protein 9 (BMP9) is one of the most potent factors in inducing bone formation. Here, we investigate whether BMP9 can effectively induce odontogenic differentiation of the stem cells from m