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Sample records for gene inhibiting oca2

  1. Report of a Novel OCA2 Gene Mutation and an Investigation of Two OCA2 Variants on Melanoma Predisposition in a Familial Melanoma Pedigree

    PubMed Central

    Hawkes, Jason E.; Cassidy, Pamela B.; Manga, Prashiela; Boissy, Raymond E.; Goldgar, David; Cannon-Albright, Lisa; Florell, Scott R.; Leachman, Sancy A.

    2016-01-01

    Background Oculocutaneous albinism type 2 (OCA2) is caused by mutations of the OCA2 gene. Individuals affected by OCA2 as well as other types of albinism are at a significantly increased risk for sun-induced skin-cancers, including malignant melanoma (MM). Objective To identify the molecular etiology of oculocutaneous albinism in a previously uncharacterized melanoma pedigree and to investigate the relationship between two OCA2 variants and melanoma predisposition in this pedigree. Methods DNA and RNA were isolated from the peripheral blood of seven patients in a familial melanoma pedigree. Electron microscopy was performed on the individual with clinical oculocutaneous albinism. OCA2, TYRP1, MC1R, CDKN2A/p16, CDKN2A/p19ARF, and CDK4 genes were sequenced in affected individuals. The relationship between OCA2 variants and melanoma was assessed using a pedigree likelihood-based method. Results The proband was determined to be an OCA2 compound heterozygous mutation carrier with a previously reported conservative missense mutation (V443I) and a novel non-conservative missense mutation (L734R). The pedigree contained both cutaneous and iris melanoma. Based on co-segregation analysis, the odds of the V443I OCA2 variant being a high penetrance locus for melanoma was: 1.3-to-1 if we include the iris melanoma as affected and 6.6-to-1 if we only consider cutaneous melanoma as affected. Conclusion The discovery of this novel OCA2 variant adds to the body of evidence on the detrimental effects of OCA2 gene mutations on pigmentation, supports existing GWAS data on the relevance of the OCA2 gene in melanoma predisposition, and may ultimately result in the development of targeted molecular therapies in the treatment of OCA and melanoma. PMID:23103111

  2. Frequent intragenic deletion of the P gene in Tanzanian patients with Type II oculocutaneous albinism (OCA2)

    SciTech Connect

    Spritz, R.; Fukai, K.; Holmes, S.A.

    1995-06-01

    Type II oculocutaneous albinism (OCA2) is an autosomal recessive disorder in which the biosynthesis of melanin pigment is reduced in the skin, hair, and eyes. OCA2, which results from mutations of the P gene, is the most frequent type of albinism in African and African-American patients. OCA2 is especially frequent in Tanzania, where it occurs with an incidence of {approximately}1/1,400. We have identified abnormalities of the P gene in each of 13 unrelated patients with OCA2 from Tanzania. One of these, a deletion of exon 7, is strongly predominant, accounting for {approximately}77% of mutant alleles in this group of patients. 20 refs., 2 figs.

  3. Analysis of P gene mutations in patients with type II (tyrosinase-positive) oculocutaneous albinism (OCA2)

    SciTech Connect

    Lee, S.T.; Nicholls, R.D.; Schnur, R. ||

    1994-09-01

    OCA2 is an autosomal recessive disorder in which the biosynthesis of melanin pigment is greatly reduced in the skin, hair, and eyes. Recently, we showed that OCA2 results from mutations of the P gene, in chromosome segment 15q11-q13. In addition to OCA2, mutations of P account for OCA associated with the Prader-Willi syndrome and some cases of {open_quotes}autosomal recessive ocular albinism{close_quotes} (AROA). We have now studied 38 unrelated patients with various forms of OCA2 or AROA from a variety of different ethnic groups. None of these patients had detectable abnormalities of the tyrosinase (TYR) gene. Among 8 African-American patients with OCA2 we observed apparent locus homogeneity. We detected abnormalities of the P gene in all 8 patients, including 12 different mutations and deletions, most of which are unique to this group and none of which is predominant. In contrast, OCA2 in other populations appears to be genetically heterogeneous. Among 21 Caucasian patients we detected abnormalities of the P gene in only 8, comprising 9 different point mutations and deletions, some of which also occurred among the African-American patients. Among 3 Middle-Eastern, 3 Indo-Pakistani, and 3 Asian patients we detected mutations of the P gene in only one from each group. In a large Indo-Pakistani kindred with OCA2 we have excluded both the TYR and P genes on the basis of genetic linkage. The prevalence of mutations of the P gene thus appears to be much higher among African-Americans with OCA2 than among patients from other ethnic groups. The incidence of OCA2 in some parts of equatorial Africa is extremely high, as frequent as 1 per 1100, and the disease has been linked to P in South African Bantu. The eventual characterization of P gene mutations in Africans will be informative with regard to the origins of P gene mutations in African-American patients.

  4. A nonsense nucleotide substitution in the oculocutaneous albinism II gene underlies the original pink-eyed dilution allele (Oca2(p)) in mice.

    PubMed

    Shoji, Haruka; Kiniwa, Yukiko; Okuyama, Ryuhei; Yang, Mu; Higuchi, Keiichi; Mori, Masayuki

    2015-01-01

    The original pink-eyed dilution (p) on chromosome 7 is a very old spontaneous mutation in mice. The oculocutaneous albinism II (Oca2) gene has previously been identified as the p gene. Oca2 transcripts have been shown to be absent in the skin of SJL/J mice with the original p mutant allele (Oca2(p)); however, the molecular genetic lesion underlying the original Oca2(p) allele has never been reported. The NCT mouse (commonly known as Nakano cataract mouse) has a pink-eyed dilution phenotype, which prompted us to undertake a molecular genetic analysis of the Oca2 gene of this strain. Our genetic linkage analysis suggests that the locus for the pink-eyed dilution phenotype of NCT is tightly linked to the Oca2 locus. PCR cloning and nucleotide sequence analysis indicates that the NCT mouse has a nonsense nucleotide substitution at exon 7 of the Oca2 gene. Examination of three mouse strains (NZW/NSlc, SJL/J, and 129X1/SvJJmsSlc) with the original Oca2(p) allele revealed the presence of a nonsense nucleotide substitution identical to that in the NCT strain. RT-PCR analysis revealed that the Oca2 transcripts were absent in the skin of NCT mice, suggesting intervention of the nonsense-mediated mRNA decay pathway. Collectively, the data in this study indicate that the nonsense nucleotide substitution in the Oca2 gene underlies the Oca2(p) allele. Our data also indicate that the NCT mouse can be used not only as a cataract model, but also as a model for human type II oculocutaneous albinism. PMID:25736709

  5. Amelanism in the corn snake is associated with the insertion of an LTR-retrotransposon in the OCA2 gene.

    PubMed

    Saenko, Suzanne V; Lamichhaney, Sangeet; Martinez Barrio, Alvaro; Rafati, Nima; Andersson, Leif; Milinkovitch, Michel C

    2015-01-01

    The corn snake (Pantherophis guttatus) is a new model species particularly appropriate for investigating the processes generating colours in reptiles because numerous colour and pattern mutants have been isolated in the last five decades. Using our captive-bred colony of corn snakes, transcriptomic and genomic next-generation sequencing, exome assembly, and genotyping of SNPs in multiple families, we delimit the genomic interval bearing the causal mutation of amelanism, the oldest colour variant observed in that species. Proceeding with sequencing the candidate gene OCA2 in the uncovered genomic interval, we identify that the insertion of an LTR-retrotransposon in its 11(th) intron results in a considerable truncation of the p protein and likely constitutes the causal mutation of amelanism in corn snakes. As amelanistic snakes exhibit white, instead of black, borders around an otherwise normal pattern of dorsal orange saddles and lateral blotches, our results indicate that melanocytes lacking melanin are able to participate to the normal patterning of other colours in the skin. In combination with research in the zebrafish, this work opens the perspective of using corn snake colour and pattern variants to investigate the generative processes of skin colour patterning shared among major vertebrate lineages. PMID:26597053

  6. Amelanism in the corn snake is associated with the insertion of an LTR-retrotransposon in the OCA2 gene

    PubMed Central

    Saenko, Suzanne V.; Lamichhaney, Sangeet; Barrio, Alvaro Martinez; Rafati, Nima; Andersson, Leif; Milinkovitch, Michel C.

    2015-01-01

    The corn snake (Pantherophis guttatus) is a new model species particularly appropriate for investigating the processes generating colours in reptiles because numerous colour and pattern mutants have been isolated in the last five decades. Using our captive-bred colony of corn snakes, transcriptomic and genomic next-generation sequencing, exome assembly, and genotyping of SNPs in multiple families, we delimit the genomic interval bearing the causal mutation of amelanism, the oldest colour variant observed in that species. Proceeding with sequencing the candidate gene OCA2 in the uncovered genomic interval, we identify that the insertion of an LTR-retrotransposon in its 11th intron results in a considerable truncation of the p protein and likely constitutes the causal mutation of amelanism in corn snakes. As amelanistic snakes exhibit white, instead of black, borders around an otherwise normal pattern of dorsal orange saddles and lateral blotches, our results indicate that melanocytes lacking melanin are able to participate to the normal patterning of other colours in the skin. In combination with research in the zebrafish, this work opens the perspective of using corn snake colour and pattern variants to investigate the generative processes of skin colour patterning shared among major vertebrate lineages. PMID:26597053

  7. Genetic variation in regulatory DNA elements: the case of OCA2 transcriptional regulation.

    PubMed

    Visser, Mijke; Kayser, Manfred; Grosveld, Frank; Palstra, Robert-Jan

    2014-03-01

    Mutations within the OCA2 gene or the complete absence of the OCA2 protein leads to oculocutaneous albinism type 2. The OCA2 protein plays a central role in melanosome biogenesis, and it is a strong determinant of the eumelanin content in melanocytes. Transcript levels of the OCA2 gene are strongly correlated with pigmentation intensities. Recent studies demonstrated that the transcriptional level of OCA2 is to a large extent determined by the noncoding SNP rs12913832 located 21.5 kb upstream of the OCA2 gene promoter. In this review, we discuss current hypotheses and the available data on the mechanism of OCA2 transcriptional regulation and how this is influenced by genetic variation. Finally, we will explore how future epigenetic studies can be used to advance our insight into the functional biology that connects genetic variation to human pigmentation. PMID:24387780

  8. Localization to Mature Melanosomes by Virtue of Cytoplasmic Dileucine Motifs Is Required for Human OCA2 Function

    PubMed Central

    Sitaram, Anand; Piccirillo, Rosanna; Palmisano, Ilaria; Harper, Dawn C.; Dell'Angelica, Esteban C.; Schiaffino, M. Vittoria

    2009-01-01

    Oculocutaneous albinism type 2 is caused by defects in the gene OCA2, encoding a pigment cell-specific, 12-transmembrane domain protein with homology to ion permeases. The function of the OCA2 protein remains unknown, and its subcellular localization is under debate. Here, we show that endogenous OCA2 in melanocytic cells rapidly exits the endoplasmic reticulum (ER) and thus does not behave as a resident ER protein. Consistently, exogenously expressed OCA2 localizes within melanocytes to melanosomes, and, like other melanosomal proteins, localizes to lysosomes when expressed in nonpigment cells. Mutagenized OCA2 transgenes stimulate melanin synthesis in OCA2-deficient cells when localized to melanosomes but not when specifically retained in the ER, contradicting a proposed primary function for OCA2 in the ER. Steady-state melanosomal localization requires a conserved consensus acidic dileucine-based sorting motif within the cytoplasmic N-terminal region of OCA2. A second dileucine signal within this region confers steady-state lysosomal localization in melanocytes, suggesting that OCA2 might traverse multiple sequential or parallel trafficking routes. The two dileucine signals physically interact in a differential manner with cytoplasmic adaptors known to function in trafficking other proteins to melanosomes. We conclude that OCA2 is targeted to and functions within melanosomes but that residence within melanosomes may be regulated by secondary or alternative targeting to lysosomes. PMID:19116314

  9. The transcription factor TBX2 regulates melanogenesis in melanocytes by repressing Oca2.

    PubMed

    Chen, Yu; Pan, Li; Su, Zhongyuan; Wang, Jing; Li, Huirong; Ma, Xiaoyin; Liu, Yin; Lu, Fan; Qu, Jia; Hou, Ling

    2016-04-01

    The T-box transcription factor TBX2 is known for its role as a critical regulator of melanoma cell proliferation, but its role in regulating melanogenesis has not been widely studied. Here we use a series of experiments to show in primary and immortalized mouse melanocytes that TBX2 acts as regulator of melanogenesis by repressing the expression of the gene encoding the melanosomal protein OCA2. We find that α-MSH or forskolin, both of which stimulate melanogenesis, also reduce TBX2 expression, and that specific knockdown of TBX2 increases melanogenesis. This effect primarily involves an increase in Oca2 expression as the combined knockdown of both Tbx2 and Oca2 interferes with the Tbx2 knockdown-mediated increase in melanogenesis. Standard chromatin immunoprecipitation and reporter assays suggest that TBX2 represses Oca2 at least in part directly. Hence, the results suggest that TBX2 may act as a nexus linking cell proliferation and melanogenesis. PMID:26971330

  10. Analysis of cultured human melanocytes based on polymorphisms within the SLC45A2/MATP, SLC24A5/NCKX5, and OCA2/P loci.

    PubMed

    Cook, Anthony L; Chen, Wei; Thurber, Amy E; Smit, Darren J; Smith, Aaron G; Bladen, Timothy G; Brown, Darren L; Duffy, David L; Pastorino, Lorenza; Bianchi-Scarra, Giovanna; Leonard, J Helen; Stow, Jennifer L; Sturm, Richard A

    2009-02-01

    Single nucleotide polymorphisms (SNPs) within the SLC45A2/MATP, SLC24A5/NCKX5, and OCA2/P genes have been associated with natural variation of pigmentation traits in human populations. Here, we describe the characterization of human primary melanocytic cells genotyped for polymorphisms within the MATP, NCKX5, or OCA2 loci. On the basis of genotype, these cultured cells reflect the phenotypes observed by others in terms of both melanin content and tyrosinase (TYR) activity when comparing skin designated as either "White" or "Black". We found a statistically significant association of MATP-374L (darker skin) with higher TYR protein abundance that was not observed for any NCKX5-111 or OCA2 rs12913832 allele. MATP-374L/L homozygous strains displayed significantly lower MATP transcript levels compared to MATP-374F/F homozygous cells, but this did not reach statistical significance based on NCKX5 or OCA2 genotype. Similarly, we observed significantly increased levels of OCA2 mRNA in rs12913832-T (brown eye) homozygotes compared to rs12913832-C (blue eye) homozygous strains, which was not observed for MATP or NCKX5 gene transcripts. In genotype-phenotype associations performed on a collection of 226 southern European individuals using these same SNPs, we were able to show strong correlations in MATP-L374F, OCA2, and melanocortin-1 receptor with skin, eye, and hair color variation, respectively. PMID:18650849

  11. Loss of Oca2 disrupts the unfolded protein response and increases resistance to endoplasmic reticulum stress in melanocytes.

    PubMed

    Cheng, Tsing; Orlow, Seth J; Manga, Prashiela

    2013-11-01

    Accumulation of proteins in the endoplasmic reticulum (ER) typically induces stress and initiates the unfolded protein response (UPR) to facilitate recovery. If homeostasis is not restored, apoptosis is induced. However, adaptation to chronic UPR activation can increase resistance to subsequent acute ER stress. We therefore investigated adaptive mechanisms in Oculocutaneous albinism type 2 (Oca2)-null melanocytes where UPR signaling is arrested despite continued tyrosinase accumulation leading to resistance to the chemical ER stressor thapsigargin. Although thapsigargin triggers UPR activation, instead of Perk-mediated phosphorylation of eIF2α, in Oca2-null melanocytes, eIF2α was rapidly dephosphorylated upon treatment. Dephosphorylation was mediated by the Gadd34-PP1α phosphatase complex. Gadd34-complex inhibition blocked eIF2α dephosphorylation and significantly increased Oca2-null melanocyte sensitivity to thapsigargin. Thus, Oca2-null melanocytes adapt to acute ER stress by disruption of pro-apoptotic Perk signaling, which promotes cell survival. This is the first study to demonstrate rapid eIF2α dephosphorylation as an adaptive mechanism to ER stress. PMID:23962237

  12. Loss of Oca2 disrupts the unfolded protein response and increases resistance to endoplasmic reticulum stress in melanocytes

    PubMed Central

    Cheng, Tsing; Orlow, Seth J.; Manga, Prashiela

    2013-01-01

    Summary Accumulation of proteins in the endoplasmic reticulum (ER) typically induces stress and initiates the unfolded protein response (UPR) to facilitate recovery. If homeostasis is not restored, apoptosis is induced. However, adaptation to chronic UPR activation can increase resistance to subsequent acute ER stress. We therefore investigated adaptive mechanisms in Oculocutaneous albinism type 2 (Oca2)-null melanocytes where UPR signaling is arrested despite continued tyrosinase accumulation leading to resistance to the chemical ER stressor thapsigargin. Although thapsigargin triggers UPR activation, instead of Perk-mediated phosphorylation of eIF2α, in Oca2-null melanocytes, eIF2α was rapidly dephosphorylated upon treatment. Dephosphorylation was mediated by the Gadd34-PP1α phosphatase complex. Gadd34-complex inhibition blocked eIF2α dephosphorylation and significantly increased Oca2-null melanocyte sensitivity to thapsigargin. Thus, Oca2-null melanocytes adapt to acute ER stress by disruption of proapoptotic Perk signaling, which promotes cell survival. This is the first study to demonstrate rapid eIF2α dephosphorylation as an adaptive mechanism to ER stress. PMID:23962237

  13. Screening of TYR, OCA2, GPR143, and MC1R in patients with congenital nystagmus, macular hypoplasia, and fundus hypopigmentation indicating albinism

    PubMed Central

    Preising, Markus N.; Gonser, Miriam; Lorenz, Birgit

    2011-01-01

    Background A broad spectrum of pigmentation of the skin and hair is found among patients diagnosed with ocular albinism (OA) and oculocutaneous albinism (OCA). Even though complexion is variable, three ocular features, i.e., hypopigmentation of the fundus, hypoplasia of the macula, and nystagmus, are classical pathological findings in these patients. We screened 172 index patients with a clinical diagnosis of OA or OCA based on the classical findings, to evaluate the frequency of sequence variants in tyrosinase (TYR), P-gene, P-protein (OCA2), and the G-protein-coupled receptor 143 gene, OA1 (GPR143). In addition, we investigated the association of sequence variants in the melanocortin receptor 1 gene (MC1R) and OCA2. Methods Pigmentation of the hair, skin, iris, and fundus were included in the evaluation of OCA and OA. Male OA patients showing X-linked inheritance were screened for GPR143. Females showing OA without family history were regarded as representing autosomal recessive OA (OA3). Direct sequencing was applied to PCR products showing aberrant single-strand conformation polymorphism–banding patterns. Results Fifty-seven male index patients were screened for OA. We identified 16 potentially pathogenic sequence variations in GPR143 (10 novel) in 22 males. In TYR, we identified 23 (7 novel), and in OCA2 28 (11 novel) possibly pathogenic variants. Variants on both alleles were identified in TYR or OCA2 in 29/79 OCA patients and 14/71 OA patients. Sequence changes in TYR were identified almost exclusively in OCA patients, while sequence changes in OCA2 occurred in OCA and OA patients. MC1R sequencing was performed in 47 patients carrying mutations in OCA2 and revealed MC1R mutations in 42 of them. Conclusions TYR gene mutations have a more severe effect on pigmentation than mutations in OCA2 and the GPR143 gene. Nevertheless, mutations in these genes affect the development of visual function either directly or by interaction with other genes like MC1R, which can be deduced from a frequent association of MC1R variants with p.R305W or p.R419Q in OCA2. PMID:21541274

  14. HERC2 rs12913832 modulates human pigmentation by attenuating chromatin-loop formation between a long-range enhancer and the OCA2 promoter

    PubMed Central

    Visser, Mijke; Kayser, Manfred; Palstra, Robert-Jan

    2012-01-01

    Pigmentation of skin, eye, and hair reflects some of the most evident common phenotypes in humans. Several candidate genes for human pigmentation are identified. The SNP rs12913832 has strong statistical association with human pigmentation. It is located within an intron of the nonpigment gene HERC2, 21 kb upstream of the pigment gene OCA2, and the region surrounding rs12913832 is highly conserved among animal species. However, the exact functional role of HERC2 rs12913832 in human pigmentation is unknown. Here we demonstrate that the HERC2 rs12913832 region functions as an enhancer regulating OCA2 transcription. In darkly pigmented human melanocytes carrying the rs12913832 T-allele, we detected binding of the transcription factors HLTF, LEF1, and MITF to the HERC2 rs12913832 enhancer, and a long-range chromatin loop between this enhancer and the OCA2 promoter that leads to elevated OCA2 expression. In contrast, in lightly pigmented melanocytes carrying the rs12913832 C-allele, chromatin-loop formation, transcription factor recruitment, and OCA2 expression are all reduced. Hence, we demonstrate that allelic variation of a common noncoding SNP located in a distal regulatory element not only disrupts the regulatory potential of this element but also affects its interaction with the relevant promoter. We provide the key mechanistic insight that allele-dependent differences in chromatin-loop formation (i.e., structural differences in the folding of gene loci) result in differences in allelic gene expression that affects common phenotypic traits. This concept is highly relevant for future studies aiming to unveil the functional basis of genetically determined phenotypes, including diseases. PMID:22234890

  15. Functional interactions between OCA2 and the protein complexes BLOC-1, BLOC-2, and AP-3 inferred from epistatic analyses of mouse coat pigmentation.

    PubMed

    Hoyle, Diego J; Rodriguez-Fernandez, Imilce A; Dell'angelica, Esteban C

    2011-04-01

    The biogenesis of melanosomes is a multistage process that requires the function of cell-type-specific and ubiquitously expressed proteins. OCA2, the product of the gene defective in oculocutaneous albinism type 2, is a melanosomal membrane protein with restricted expression pattern and a potential role in the trafficking of other proteins to melanosomes. The ubiquitous protein complexes AP-3, BLOC-1, and BLOC-2, which contain as subunits the products of genes defective in various types of Hermansky-Pudlak syndrome, have been likewise implicated in trafficking to melanosomes. We have tested for genetic interactions between mutant alleles causing deficiency in OCA2 (pink-eyed dilution unstable), AP-3 (pearl), BLOC-1 (pallid), and BLOC-2 (cocoa) in C57BL/6J mice. The pallid allele was epistatic to pink-eyed dilution, and the latter behaved as a semi-dominant phenotypic enhancer of cocoa and, to a lesser extent, of pearl. These observations suggest functional links between OCA2 and these three protein complexes involved in melanosome biogenesis. PMID:21392365

  16. Distribution of two OCA2 polymorphisms associated with pigmentation in East-Asian populations

    PubMed Central

    Murray, Nicole; Norton, Heather L; Parra, Esteban J

    2015-01-01

    Two OCA2 polymorphisms (rs1800414 and rs74653330) have been associated with pigmentation in East Asians. We explored the distribution of these markers in a panel of samples from populations around the world. The derived allele of rs1800414 has high frequencies in a broad East-Asian region, whereas the derived allele of rs74653330 is primarily restricted to northern East Asia. Our data suggest that these polymorphisms may have been selected independently in different regions of East Asia. PMID:27081560

  17. Association Between a Germline OCA2 Polymorphism at Chromosome 15q13.1 and Estrogen ReceptorNegative Breast Cancer Survival

    PubMed Central

    Tyrer, Jonathan; Fasching, Peter A.; Beckmann, Matthias W.; Ekici, Arif B.; Schulz-Wendtland, Rdiger; Bojesen, Stig E.; Nordestgaard, Brge G.; Flyger, Henrik; Milne, Roger L.; Arias, Jos Ignacio; Menndez, Primitiva; Bentez, Javier; Chang-Claude, Jenny; Hein, Rebecca; Wang-Gohrke, Shan; Nevanlinna, Heli; Heikkinen, Tuomas; Aittomki, Kristiina; Blomqvist, Carl; Margolin, Sara; Mannermaa, Arto; Kosma, Veli-Matti; Kataja, Vesa; Beesley, Jonathan; Chen, Xiaoqing; Chenevix-Trench, Georgia; Couch, Fergus J.; Olson, Janet E.; Fredericksen, Zachary S.; Wang, Xianshu; Giles, Graham G.; Severi, Gianluca; Baglietto, Laura; Southey, Melissa C.; Devilee, Peter; Tollenaar, Rob A. E. M.; Seynaeve, Caroline; Garca-Closas, Montserrat; Lissowska, Jolanta; Sherman, Mark E.; Bolton, Kelly L.; Hall, Per; Czene, Kamila; Cox, Angela; Brock, Ian W.; Elliott, Graeme C.; Reed, Malcolm W. R.; Greenberg, David; Anton-Culver, Hoda; Ziogas, Argyrios; Humphreys, Manjeet; Easton, Douglas F.; Caporaso, Neil E.; Pharoah, Paul D. P.

    2010-01-01

    Background Traditional prognostic factors for survival and treatment response of patients with breast cancer do not fully account for observed survival variation. We used available genotype data from a previously conducted two-stage, breast cancer susceptibility genome-wide association study (ie, Studies of Epidemiology and Risk factors in Cancer Heredity [SEARCH]) to investigate associations between variation in germline DNA and overall survival. Methods We evaluated possible associations between overall survival after a breast cancer diagnosis and 10?621 germline single-nucleotide polymorphisms (SNPs) from up to 3761 patients with invasive breast cancer (including 647 deaths and 26?978 person-years at risk) that were genotyped previously in the SEARCH study with high-density oligonucleotide microarrays (ie, hypothesis-generating set). Associations with all-cause mortality were assessed for each SNP by use of Cox regression analysis, generating a per rare allele hazard ratio (HR). To validate putative associations, we used patient genotype information that had been obtained with 5? nuclease assay or mass spectrometry and overall survival information for up to 14?096 patients with invasive breast cancer (including 2303 deaths and 70?019 person-years at risk) from 15 international casecontrol studies (ie, validation set). Fixed-effects meta-analysis was used to generate an overall effect estimate in the validation dataset and in combined SEARCH and validation datasets. All statistical tests were two-sided. Results In the hypothesis-generating dataset, SNP rs4778137 (C>G) of the OCA2 gene at 15q13.1 was statistically significantly associated with overall survival among patients with estrogen receptornegative tumors, with the rare G allele being associated with increased overall survival (HR of death per rare allele carried = 0.56, 95% confidence interval [CI] = 0.41 to 0.75, P = 9.2 10?5). This association was also observed in the validation dataset (HR of death per rare allele carried = 0.88, 95% CI = 0.78 to 0.99, P = .03) and in the combined dataset (HR of death per rare allele carried = 0.82, 95% CI = 0.73 to 0.92, P = 5 10?4). Conclusion The rare G allele of the OCA2 polymorphism, rs4778137, may be associated with improved overall survival among patients with estrogen receptornegative breast cancer. PMID:20308648

  18. Albinism in phylogenetically and geographically distinct populations of Astyanax cavefish arises through the same loss-of-function Oca2 allele

    PubMed Central

    Gross, J B; Wilkens, H

    2013-01-01

    The Mexican tetra, Astyanax mexicanus, comprises 29 populations of cave-adapted fish distributed across a vast karst region in northeastern Mexico. These populations have a complex evolutionary history, having descended from ‘old' and ‘young' ancestral surface-dwelling stocks that invaded the region ∼6.7 and ∼2.8 MYa, respectively. This study investigates a set of captive, pigmented Astyanax cavefish collected from the Micos cave locality in 1970, in which albinism appeared over the past two decades. We combined novel coloration analyses, coding sequence comparisons and mRNA expression level studies to investigate the origin of albinism in captive-bred Micos cavefish. We discovered that albino Micos cavefish harbor two copies of a loss-of-function ocular and cutaneous albinism type II (Oca2) allele previously identified in the geographically distant Pachón cave population. This result suggests that phylogenetically young Micos cavefish and phylogenetically old Pachón cave fish inherited this Oca2 allele from the ancestral surface-dwelling taxon. This likely resulted from the presence of the loss-of-function Oca2 haplotype in the ‘young' ancestral surface-dwelling stock that colonized the Micos cave and also introgressed into the ancient Pachón cave population. The appearance of albinism in captive Micos cavefish, caused by the same loss-of-function allele present in Pachón cavefish, implies that geographically and phylogenetically distinct cave populations can evolve the same troglomorphic phenotype from standing genetic variation present in the ancestral taxon. PMID:23572122

  19. Tumor necrosis factor-alpha inhibits renin gene expression.

    PubMed

    Todorov, Vladimir; Müller, Markus; Schweda, Frank; Kurtz, Armin

    2002-11-01

    Renin, produced in renal juxtaglomerular (JG) cells, is a fundamental regulator of blood pressure. Accumulating evidence suggests that cytokines may directly influence renin production in the JG cells. TNF-alpha, which is one of the key mediators in immunity and inflammation, is known to participate in the control of vascular proliferation and contraction and hence in the pathogenesis of cardiovascular diseases. Thus TNF-alpha may exert its effects on the cardiovascular system through modulation of renal renin synthesis. Therefore we have tested the effect of TNF-alpha on renin transcription in As4.1 cells, which represent transformed mouse JG cells, and in native mouse JG cells in culture. Renin gene expression was also determined in mice lacking the gene for TNF-alpha (TNF-alpha knockout mice). TNF-alpha inhibited renin gene expression via an inhibition of the transcriptional activity, targeting the proximal 4.1 kb of the renin promoter in As4.1 cells. TNF-alpha also attenuated forskolin-stimulated renin gene expression in primary cultures of mouse JG cells. Mice lacking the TNF-alpha gene had almost threefold higher basal renal renin mRNA abundance relative to the control strain. The general physiological regulation of renin expression by salt was not disturbed in TNF-alpha knockout mice. Our data suggest that TNF-alpha inhibits renin gene transcription at the cellular level and thus may act as a modulator of renin synthesis in (physio)pathological situations. PMID:12376397

  20. Unrevealing the role of P-protein on melanosome biology and structure, using siRNA-mediated down regulation of OCA2.

    PubMed

    Park, Sangjoo; Morya, V K; Nguyen, Dong Hoang; Singh, Birendra K; Lee, Hyang-Bok; Kim, Eun-Ki

    2015-05-01

    The pink-eyed dilution protein (P-protein) plays a critical role in melanin synthesis in melanocytes and retinal pigment epithelium cells. Mutation in this protein may cause complete or partial albinism. Role of the P-protein ranges in melanin synthesis to maturation and trafficking of the melanosomes. The aim of the present study was to evaluate the effect of P-protein inhibition on melanosome biology by comparing the shape, size, count, and types of melanosomes in melan-a melanocytes. The cells were extensively examined by the transmission electron microscopy. The P-protein inhibition was carried by P-protein-siRNA transfection to melan-a melanocytes, B16F10 mouse melanoma, and melan-p1 cells. Measurement of melanin contents, cellular tyrosinase, and different tyrosinase related proteins were also determined to investigate the effect of P-protein siRNA transfection on melanocytes. Results suggested that the inhibition of P-protein can significantly change the melanosomal morphology, types and their respective numbers, and provided a novel strategy for the control of melanin synthesis. PMID:25656818

  1. Noggin Gene Delivery Inhibits Cementoblast-Induced Mineralization

    PubMed Central

    Jin, Q.-M.; Zhao, M.; Economides, A. N.; Somerman, M. J.; Giannobile, W.V.

    2008-01-01

    Bone morphogenetic proteins (BMPs) are known to promote periodontal tissue regeneration, while noggin inhibits the biological activities of BMP-2, -4, and -7. To investigate the effect of BMPs and noggin gene transfer on cementogenesis,we used cloned murine cementoblasts (OCCM). Cells were transduced using adenoviruses encoding BMP-7 (Ad-BMP-7), noggin devoid of the heparin binding site (Ad-NOGΔB2), or a control adenovirus encoding green fluorescent protein (Ad-GFP). Cells were seeded into 3D polymer scaffolds and implanted into SCID mice to determine the in vivo mineral-inducing ability of the cells. Cells transduced with Ad-NOG.B2 at 3 and 6 weeks postimplantation exhibited reduced mineral formation compared with all other groups. Although gene expression of osteocalcin and bone sialoprotein increased after Ad-BMP-7 transduction in vitro, following BMP-7 gene transfer in vivo, transcripts for OCN and BSP were not significantly different from controls, and mineral density was not significantly increased compared with Ad-GFP and NT groups. These results indicate that in mature cementoblast populations, gene transfer of noggin inhibits biomineralization induced by cementoblasts, whereas exogenous BMP has minimal effects on mineralization. PMID:15203940

  2. Inhibition of human immunodeficiency virus gene amplification by heparin.

    PubMed Central

    Holodniy, M; Kim, S; Katzenstein, D; Konrad, M; Groves, E; Merigan, T C

    1991-01-01

    Gene amplification of virus-specific sequences is widely used as a method to detect or confirm human immunodeficiency virus (HIV) infection. In this study we used an enzyme-linked affinity assay to quantify polymerase chain reaction products from whole blood, plasma, and separated mononuclear cells collected in the presence of four common anticoagulants: acid citrate dextrose, sodium EDTA, potassium oxalate, and sodium heparin. Attenuation of the product signal was observed after amplification of nucleic acid extraction from whole blood, washed mononuclear cells, and plasma from specimens collected in sodium heparin. These inhibitory effects on gene amplification could be reversed with heparinase. The addition of as little as 0.05 U of heparin completely inhibited amplification of an HLA-DQa sequence from placental DNA. We conclude that heparin can cause attenuation or inhibition of gene amplification. Acid citrate dextrose and EDTA, which lack inhibitory activity, are the most appropriate anticoagulants for clinical blood samples when polymerase chain reaction amplification is anticipated. Images PMID:1909709

  3. Protein inhibitor of activated STAT3 inhibits adipogenic gene expression

    SciTech Connect

    Deng Jianbei; Hua Kunjie; Caveney, Erica J.; Takahashi, Nobuyuki; Harp, Joyce B. . E-mail: jharp@unc.edu

    2006-01-20

    Protein inhibitor of activated STAT3 (PIAS3), a cytokine-induced repressor of signal transducer and activator of transcription 3 (STAT3) and a modulator of a broad array of nuclear proteins, is expressed in white adipose tissue, but its role in adipogenesis is not known. Here, we determined that PIAS3 was constitutively expressed in 3T3-L1 cells at all stages of adipogenesis. However, it translocated from the nucleus to the cytoplasm 4 days after induction of differentiation by isobutylmethylxanthine, dexamethasone, and insulin (MDI). In ob/ob mice, PIAS3 expression was increased in white adipose tissue depots compared to lean mice and was found in the cytoplasm of adipocytes. Overexpression of PIAS3 in differentiating preadipocytes, which localized primarily to the nucleus, inhibited mRNA level gene expression of adipogenic transcription factors C/EBP{alpha} and PPAR{gamma}, as well as their downstream target genes aP2 and adiponectin. PIAS3 also inhibited C/EBP{alpha} promoter activation mediated specifically by insulin, but not dexamethasone or isobutylmethylxanthine. Taken together, these data suggest that PIAS3 may play an inhibitory role in adipogenesis by modulating insulin-activated transcriptional activation events. Increased PIAS3 expression in adipose tissue may play a role in the metabolic disturbances of obesity.

  4. Gene therapeutic approaches to inhibit hepatitis B virus replication

    PubMed Central

    Gebbing, Maren; Bergmann, Thorsten; Schulz, Eric; Ehrhardt, Anja

    2015-01-01

    Acute and chronic hepatitis B virus (HBV) infections remain to present a major global health problem. The infection can be associated with acute symptomatic or asymptomatic hepatitis which can cause chronic inflammation of the liver and over years this can lead to cirrhosis and the development of hepatocellular carcinomas. Currently available therapeutics for chronically infected individuals aim at reducing viral replication and to slow down or stop the progression of the disease. Therefore, novel treatment options are needed to efficiently combat and eradicate this disease. Here we provide a state of the art overview of gene therapeutic approaches to inhibit HBV replication. We discuss non-viral and viral approaches which were explored to deliver therapeutic nucleic acids aiming at reducing HBV replication. Types of delivered therapeutic nucleic acids which were studied since many years include antisense oligodeoxynucleotides and antisense RNA, ribozymes and DNAzymes, RNA interference, and external guide sequences. More recently designer nucleases gained increased attention and were exploited to destroy the HBV genome. In addition we mention other strategies to reduce HBV replication based on delivery of DNA encoding dominant negative mutants and DNA vaccination. In combination with available cell culture and animal models for HBV infection, in vitro and in vivo studies can be performed to test efficacy of gene therapeutic approaches. Recent progress but also challenges will be specified and future perspectives will be discussed. This is an exciting time to explore such approaches because recent successes of gene therapeutic strategies in the clinic to treat genetic diseases raise hope to find alternative treatment options for patients chronically infected with HBV. PMID:25729471

  5. FAK and HAS inhibition synergistically decrease colon cancer cell viability and affect expression of critical genes.

    PubMed

    Heffler, Melissa; Golubovskaya, Vita M; Conroy, Jeffrey; Liu, Song; Wang, Dan; Cance, William G; Dunn, Kelli B

    2013-05-01

    Focal adhesion kinase (FAK), hyaluronan (HA), and hyaluronan synthase-3 (HAS3) have been implicated in cancer growth and progression. FAK inhibition with the small molecule inhibitor Y15 decreases colon cancer cell growth in vitro and in vivo. HAS3 inhibition in colon cancer cells decreases FAK expression and activation, and exogenous HA increases FAK activation. We sought to determine the genes affected by HAS and FAK inhibition and hypothesized that dual inhibition would synergistically inhibit viability. Y15 (FAK inhibitor) and the HAS inhibitor 4-methylumbelliferone (4-MU) decreased viability in a dose dependent manner; viability was further inhibited by treatment with Y15 and 4-MU in colon cancer cells. HAS inhibited cells treated with 2 μM of Y15 showed significantly decreased viability compared to HAS scrambled cells treated with the same dose (p < 0.05) demonstrating synergistic inhibition of viability with dual FAK/HAS inhibition. Microarray analysis showed more than 2-fold up- or down-regulation of 121 genes by HAS inhibition, and 696 genes by FAK inhibition (p < 0.05) and revealed 29 common genes affected by both signaling. Among the genes affected by FAK or HAS3 inhibition were genes, playing role in apoptosis, cell cycle regulation, adhesion, transcription, heatshock and WNT pathways. Thus, FAK or HAS inhibition decreases SW620 viability and affects several similar genes, which are involved in the regulation of tumor survival. Dual inhibition of FAK and HAS3 decreases viability to a greater degree than with either agent alone, and suggests that synergistic inhibition of colon cancer cell growth can result from affecting similar genetic pathways. PMID:22934709

  6. Gene therapy for dyslipidemia: a review of gene replacement and gene inhibition strategies

    PubMed Central

    Kassim, Sadik H; Wilson, James M; Rader, Daniel J

    2012-01-01

    Despite numerous technological and pharmacological advances and more detailed knowledge of molecular etiologies, cardiovascular diseases remain the leading cause of morbidity and mortality worldwide claiming over 17 million lives a year. Abnormalities in the synthesis, processing and catabolism of lipoprotein particles can result in severe hypercholesterolemia, hypertriglyceridemia or low HDL-C. Although a plethora of antidyslipidemic pharmacological agents are available, these drugs are relatively ineffective in many patients with Mendelian lipid disorders, indicating the need for new and more effective interventions. In vivo somatic gene therapy is one such intervention. This article summarizes current strategies being pursued for the development of clinical gene therapy for dyslipidemias that cannot effectively be treated with existing drugs. PMID:22505953

  7. Inhibition of p53-induced apoptosis without affecting expression of p53-regulated genes

    PubMed Central

    Lotem, Joseph; Gal, Hilah; Kama, Rachel; Amariglio, Ninette; Rechavi, Gideon; Domany, Eytan; Sachs, Leo; Givol, David

    2003-01-01

    Using DNA microarray and clustering of expressed genes we have analyzed the mechanism of inhibition of wild-type p53-induced apoptosis by the cytokine interleukin 6 (IL-6) and the calcium mobilizer thapsigargin (TG). Clustering analysis of 1,786 genes, the expression level of which changed after activation of wild-type p53 in the absence or presence of IL-6 or TG, showed that these compounds did not cause a general inhibition of the ability of p53 to up-regulate or down-regulate gene expression. Expression of various p53 targets implicated as mediators of p53-induced apoptosis was also not affected by IL-6 or TG. These compounds thus can bypass the effect of wild-type p53 on gene expression and inhibit apoptosis. IL-6 and TG activated different p53-independent pathways of gene expression that include up-regulation of antiapoptotic genes. IL-6 and TG also activated different differentiation-associated genes. The ability of compounds such as cytokines and calcium mobilizers to inhibit p53-mediated apoptosis without generally inhibiting gene expression regulated by p53 can facilitate tumor development and tumor resistance to radiation and chemotherapy in cells that retain wild-type p53. PMID:12743373

  8. Adherens Junction Formation Inhibits Lentivirus Entry and Gene Transfer

    PubMed Central

    Padmashali, Roshan; You, Hui; Karnik, Nikhila; Lei, Pedro; Andreadis, Stelios T.

    2013-01-01

    Although cellular signaling pathways that affect lentivirus infection have been investigated, the role of cell-cell interactions in lentiviral gene delivery remains elusive. In the course of our studies we observed that lentiviral gene transfer was a strong function of the position of epithelial cells within colonies. While peripheral cells were transduced efficiently, cells in the center of colonies were resistant to gene transfer. In addition, gene delivery was enhanced significantly under culture conditions that disrupted adherens junctions (AJ) but decreased upon AJ formation. In agreement, gene knockdown and gain-of-function approaches showed that α-catenin, a key component of the AJ complex prevented lentivirus gene transfer. Using a doxycycline regulatable system we showed that expression of dominant negative E-cadherin enhanced gene transfer in a dose-dependent manner. In addition, dissolution of AJ by doxycycline increased entry of lentiviral particles into the cell cytoplasm in a dose-dependent manner. Taken together our results demonstrate that AJ formation renders cells non-permissive to lentiviral gene transfer and may facilitate development of simple means to enhance gene delivery or combat virus infection. PMID:24236116

  9. Organization and sequence of the human P gene and identification of a new family of transport proteins

    SciTech Connect

    Lee, S.T.; Fukai, K.; Spritz, R.A.

    1995-03-20

    We have determined the structure, nucleotide sequence, and polymorphisms of the human P gene. Mutations of the P gene result in type H oculocutaneous albinism (OCA2) in humans and pink-eyed dilution (p) in mice. We find that the human P gene is quite large, consisting of 25 exons spanning 250 to 600 kb in chromosome segment 15q11-q13. The P polypeptide appears to define a novel family of small molecule transporters and may be involved in transport of tyrosine, the precursor to melanin synthesis, within the melanocyte. These results provide the basis for analyses of patients with OCA2 and may point toward eventual pharmacologic treatment of this and related disorders of pigmentation. 40 refs., 5 figs., 3 tabs.

  10. Adenoviral gene transfer is inhibited by soluble factors in malignant pleural effusions.

    PubMed

    Batra, R K; Dubinett, S M; Henkle, B W; Sharma, S; Gardner, B K

    2000-05-01

    Direct in vivo gene delivery is a prerequisite for many gene therapy strategies; however, efficacy has been limited by a lack of therapeutic gene transfer. In studying intrapleural malignancy as a model for the gene therapy of non-small cell lung cancer, we previously identified soluble chondroitin sulfate-proteoglycans/glycosaminoglycans (CS-PG/GAGs) in malignant pleural effusions (MPE) as factors that inhibit retroviral vector (RV) transduction. Similarly, we have observed inhibition to gene transfer in the fluid component of MPE using adenoviral (Ad) vectors. Analyses indicate that the factors responsible for the block are filterable, soluble, titrable, and heat stable (56 degrees C). Passage through microporous membranes fractionates the inhibitory factors into large (> 100 kD) components of the effusions. In contrast to RV transduction, hyaluronic acid or CS-PG/GAGs are not the inhibitors because the block is not reversed by pretreatment of the effusions with mammalian hyaluronidase, and exogenous addition of GAGs into the transduction media does not diminish Ad transduction. In considering the mechanism of action of the inhibitory factors, we observe that Ad entry, and specifically the binding of radiolabeled Ad to its target cell, is inhibited in the presence of MPE. Ad internalization may also be impaired; however, these studies exclude soluble fibronectin in MPE as a competitive inhibitor of Ad transduction. Lastly, sepharose A- mediated immunoglobulin depletion of MPE only partially reverses the block, and significant inhibition to Ad gene transfer persists at lower adenovirus:target cell ratios. Identifying the structural and functional basis for inhibition to Ad gene transfer may yield specific strategies to enable better in vivo translation of gene therapy approaches. PMID:10783134

  11. Effect of protein synthesis inhibition on gene expression during early development of Dictyostelium discoideum

    SciTech Connect

    Singleton, C.K.; Manning, S.S.; Feng, Y.

    1988-01-01

    Several genes which are deactivated on the initiation of development of Dictyostelium discoideum were identified by differential screening of various cDNA libraries. These genes have in common a decrease in the steady-state levels of their corresponding mRNAs on the onset of development and as development proceeds. When development was carried out in the absence of protein synthesis by inhibition with cycloheximide, the decrease in mRNA levels for most genes (V genes) was normal or slightly accelerated. For about 5% of the genes (H genes), however, cycloheximide caused an apparent induction of expression, as revealed by a slight or dramatic increase in mRNA levels, instead of the normal decrease. This effect was due to inhibition of protein synthesis and not to cycloheximide per se. The induction was found to be due to an enhancement of the transcription rate; normal rates of transcription for the H genes were dependent on continued protein synthesis during vegetative growth and development. Thus, two general regulatory classes exist for deactivation of gene expression on initiation of development, one of which is dependent on and one of which is independent of protein synthesis. Analysis of expression of these genes in mutant strains which are aggregation deficient allowed the classes to be subdivided further. Taken together, these characterizations allow several distinct regulatory mechanisms to be identified that are involved in the deactivation of gene expression on the onset of development in D. discoideum.

  12. Inhibition of HSV-1 Replication by Gene Editing Strategy

    PubMed Central

    Roehm, Pamela C.; Shekarabi, Masoud; Wollebo, Hassen S.; Bellizzi, Anna; He, Lifan; Salkind, Julian; Khalili, Kamel

    2016-01-01

    HSV-1 induced illness affects greater than 85% of adults worldwide with no permanent curative therapy. We used RNA-guided CRISPR/Cas9 gene editing to specifically target for deletion of DNA sequences of the HSV-1 genome that span the region directing expression of ICP0, a key viral protein that stimulates HSV-1 gene expression and replication. We found that CRISPR/Cas9 introduced InDel mutations into exon 2 of the ICP0 gene profoundly reduced HSV-1 infectivity in permissive human cell culture models and protected permissive cells against HSV-1 infection. CRISPR/Cas9 mediated targeting ICP0 prevented HSV-1-induced disintegration of promonocytic leukemia (PML) nuclear bodies, an intracellular event critical to productive HSV-1 infection that is initiated by interaction of the ICP0 N-terminus with PML. Combined treatment of cells with CRISPR targeting ICP0 plus the immediate early viral proteins, ICP4 or ICP27, completely abrogated HSV-1 infection. We conclude that RNA-guided CRISPR/Cas9 can be used to develop a novel, specific and efficacious therapeutic and prophylactic platform for targeted viral genomic ablation to treat HSV-1 diseases. PMID:27064617

  13. Inhibition of HSV-1 Replication by Gene Editing Strategy.

    PubMed

    Roehm, Pamela C; Shekarabi, Masoud; Wollebo, Hassen S; Bellizzi, Anna; He, Lifan; Salkind, Julian; Khalili, Kamel

    2016-01-01

    HSV-1 induced illness affects greater than 85% of adults worldwide with no permanent curative therapy. We used RNA-guided CRISPR/Cas9 gene editing to specifically target for deletion of DNA sequences of the HSV-1 genome that span the region directing expression of ICP0, a key viral protein that stimulates HSV-1 gene expression and replication. We found that CRISPR/Cas9 introduced InDel mutations into exon 2 of the ICP0 gene profoundly reduced HSV-1 infectivity in permissive human cell culture models and protected permissive cells against HSV-1 infection. CRISPR/Cas9 mediated targeting ICP0 prevented HSV-1-induced disintegration of promonocytic leukemia (PML) nuclear bodies, an intracellular event critical to productive HSV-1 infection that is initiated by interaction of the ICP0 N-terminus with PML. Combined treatment of cells with CRISPR targeting ICP0 plus the immediate early viral proteins, ICP4 or ICP27, completely abrogated HSV-1 infection. We conclude that RNA-guided CRISPR/Cas9 can be used to develop a novel, specific and efficacious therapeutic and prophylactic platform for targeted viral genomic ablation to treat HSV-1 diseases. PMID:27064617

  14. Inhibition of SIRT1 reactivates silenced cancer genes without loss of promoter DNA hypermethylation.

    PubMed

    Pruitt, Kevin; Zinn, Rebekah L; Ohm, Joyce E; McGarvey, Kelly M; Kang, Sung-Hae L; Watkins, D Neil; Herman, James G; Baylin, Stephen B

    2006-03-01

    The class III histone deactylase (HDAC), SIRT1, has cancer relevance because it regulates lifespan in multiple organisms, down-regulates p53 function through deacetylation, and is linked to polycomb gene silencing in Drosophila. However, it has not been reported to mediate heterochromatin formation or heritable silencing for endogenous mammalian genes. Herein, we show that SIRT1 localizes to promoters of several aberrantly silenced tumor suppressor genes (TSGs) in which 5' CpG islands are densely hypermethylated, but not to these same promoters in cell lines in which the promoters are not hypermethylated and the genes are expressed. Heretofore, only type I and II HDACs, through deactylation of lysines 9 and 14 of histone H3 (H3-K9 and H3-K14, respectively), had been tied to the above TSG silencing. However, inhibition of these enzymes alone fails to re-activate the genes unless DNA methylation is first inhibited. In contrast, inhibition of SIRT1 by pharmacologic, dominant negative, and siRNA (small interfering RNA)-mediated inhibition in breast and colon cancer cells causes increased H4-K16 and H3-K9 acetylation at endogenous promoters and gene re-expression despite full retention of promoter DNA hypermethylation. Furthermore, SIRT1 inhibition affects key phenotypic aspects of cancer cells. We thus have identified a new component of epigenetic TSG silencing that may potentially link some epigenetic changes associated with aging with those found in cancer, and provide new directions for therapeutically targeting these important genes for re-expression. PMID:16596166

  15. Dopamine inhibits somatolactin gene expression in tilapia pituitary cells through the dopamine D2 receptors.

    PubMed

    Jiang, Quan; Lian, Anji; He, Qi

    2016-07-01

    Dopamine (DA) is an important neurotransmitter in the central nervous system of vertebrates and possesses key hypophysiotropic functions. Early studies have shown that DA has a potent inhibitory effect on somatolactin (SL) release in fish. However, the mechanisms responsible for DA inhibition of SL gene expression are largely unknown. To this end, tilapia DA type-1 (D1) and type-2 (D2) receptor transcripts were examined in the neurointermediate lobe (NIL) of the tilapia pituitary by real-time PCR. In tilapia, DA not only was effective in inhibiting SL mRNA levels in vivo and in vitro, but also could abolish pituitary adenylate cyclase-activating polypeptide (PACAP)- and salmon gonadotropin-releasing hormone (sGnRH)-stimulated SL gene expression at the pituitary level. In parallel studies, the specific D2 receptor agonists quinpirole and bromocriptine could mimic the DA-inhibited SL gene expression. Furthermore, the D2 receptor antagonists domperidone and (-)-sulpiride could abolish the SL response to DA or the D2 agonist quinpirole, whereas D1 receptor antagonists SCH23390 and SKF83566 were not effective in this respect. In primary cultures of tilapia NIL cells, D2 agonist quinpirole-inhibited cAMP production could be blocked by co-treatment with the D2 antagonist domperidone and the ability of forskolin to increase cAMP production was also inhibited by quinpirole. Using a pharmacological approach, the AC/cAMP pathway was shown to be involved in quinpirole-inhibited SL mRNA expression. These results provide evidence that DA can directly inhibit SL gene expression at the tilapia pituitary level via D2 receptor through the AC/cAMP-dependent mechanism. PMID:26970582

  16. Genomic targets, and histone acetylation and gene expression profiling of neural HDAC inhibition

    PubMed Central

    Lopez-Atalaya, Jose P.; Ito, Satomi; Valor, Luis M.; Benito, Eva; Barco, Angel

    2013-01-01

    Histone deacetylase inhibitors (HDACis) have been shown to potentiate hippocampal-dependent memory and synaptic plasticity and to ameliorate cognitive deficits and degeneration in animal models for different neuropsychiatric conditions. However, the impact of these drugs on hippocampal histone acetylation and gene expression profiles at the genomic level, and the molecular mechanisms that underlie their specificity and beneficial effects in neural tissue, remains obscure. Here, we mapped four relevant histone marks (H3K4me3, AcH3K9,14, AcH4K12 and pan-AcH2B) in hippocampal chromatin and investigated at the whole-genome level the impact of HDAC inhibition on acetylation profiles and basal and activity-driven gene expression. HDAC inhibition caused a dramatic histone hyperacetylation that was largely restricted to active loci pre-marked with H3K4me3 and AcH3K9,14. In addition, the comparison of Chromatin immunoprecipitation sequencing and gene expression profiles indicated that Trichostatin A-induced histone hyperacetylation, like histone hypoacetylation induced by histone acetyltransferase deficiency, had a modest impact on hippocampal gene expression and did not affect the transient transcriptional response to novelty exposure. However, HDAC inhibition caused the rapid induction of a homeostatic gene program related to chromatin deacetylation. These results illuminate both the relationship between hippocampal gene expression and histone acetylation and the mechanism of action of these important neuropsychiatric drugs. PMID:23821663

  17. Genomic targets, and histone acetylation and gene expression profiling of neural HDAC inhibition.

    PubMed

    Lopez-Atalaya, Jose P; Ito, Satomi; Valor, Luis M; Benito, Eva; Barco, Angel

    2013-09-01

    Histone deacetylase inhibitors (HDACis) have been shown to potentiate hippocampal-dependent memory and synaptic plasticity and to ameliorate cognitive deficits and degeneration in animal models for different neuropsychiatric conditions. However, the impact of these drugs on hippocampal histone acetylation and gene expression profiles at the genomic level, and the molecular mechanisms that underlie their specificity and beneficial effects in neural tissue, remains obscure. Here, we mapped four relevant histone marks (H3K4me3, AcH3K9,14, AcH4K12 and pan-AcH2B) in hippocampal chromatin and investigated at the whole-genome level the impact of HDAC inhibition on acetylation profiles and basal and activity-driven gene expression. HDAC inhibition caused a dramatic histone hyperacetylation that was largely restricted to active loci pre-marked with H3K4me3 and AcH3K9,14. In addition, the comparison of Chromatin immunoprecipitation sequencing and gene expression profiles indicated that Trichostatin A-induced histone hyperacetylation, like histone hypoacetylation induced by histone acetyltransferase deficiency, had a modest impact on hippocampal gene expression and did not affect the transient transcriptional response to novelty exposure. However, HDAC inhibition caused the rapid induction of a homeostatic gene program related to chromatin deacetylation. These results illuminate both the relationship between hippocampal gene expression and histone acetylation and the mechanism of action of these important neuropsychiatric drugs. PMID:23821663

  18. Identification of Genes That Promote or Inhibit Olfactory Memory Formation in Drosophila

    PubMed Central

    Walkinshaw, Erica; Gai, Yunchao; Farkas, Caitlin; Richter, Daniel; Nicholas, Eric; Keleman, Krystyna; Davis, Ronald L.

    2015-01-01

    Genetic screens in Drosophila melanogaster and other organisms have been pursued to filter the genome for genetic functions important for memory formation. Such screens have employed primarily chemical or transposon-mediated mutagenesis and have identified numerous mutants including classical memory mutants, dunce and rutabaga. Here, we report the results of a large screen using panneuronal RNAi expression to identify additional genes critical for memory formation. We identified >500 genes that compromise memory when inhibited (low hits), either by disrupting the development and normal function of the adult animal or by participating in the neurophysiological mechanisms underlying memory formation. We also identified >40 genes that enhance memory when inhibited (high hits). The dunce gene was identified as one of the low hits and further experiments were performed to map the effects of the dunce RNAi to the α/β and γ mushroom body neurons. Additional behavioral experiments suggest that dunce knockdown in the mushroom body neurons impairs memory without significantly affecting acquisition. We also characterized one high hit, sickie, to show that RNAi knockdown of this gene enhances memory through effects in dopaminergic neurons without apparent effects on acquisition. These studies further our understanding of two genes involved in memory formation, provide a valuable list of genes that impair memory that may be important for understanding the neurophysiology of memory or neurodevelopmental disorders, and offer a new resource of memory suppressor genes that will aid in understanding restraint mechanisms employed by the brain to optimize resources. PMID:25644700

  19. Analysis of the mechanism by which glucose inhibits maltose induction of MAL gene expression in Saccharomyces.

    PubMed Central

    Hu, Z; Yue, Y; Jiang, H; Zhang, B; Sherwood, P W; Michels, C A

    2000-01-01

    Expression of the MAL genes required for maltose fermentation in Saccharomyces cerevisiae is induced by maltose and repressed by glucose. Maltose-inducible regulation requires maltose permease and the MAL-activator protein, a DNA-binding transcription factor encoded by MAL63 and its homologues at the other MAL loci. Previously, we showed that the Mig1 repressor mediates glucose repression of MAL gene expression. Glucose also blocks MAL-activator-mediated maltose induction through a Mig1p-independent mechanism that we refer to as glucose inhibition. Here we report the characterization of this process. Our results indicate that glucose inhibition is also Mig2p independent. Moreover, we show that neither overexpression of the MAL-activator nor elimination of inducer exclusion is sufficient to relieve glucose inhibition, suggesting that glucose acts to inhibit induction by affecting maltose sensing and/or signaling. The glucose inhibition pathway requires HXK2, REG1, and GSF1 and appears to overlap upstream with the glucose repression pathway. The likely target of glucose inhibition is Snf1 protein kinase. Evidence is presented indicating that, in addition to its role in the inactivation of Mig1p, Snf1p is required post-transcriptionally for the synthesis of maltose permease whose function is essential for maltose induction. PMID:10628974

  20. Ultrasound-mediated interferon {beta} gene transfection inhibits growth of malignant melanoma

    SciTech Connect

    Yamaguchi, Kazuki; Department of Anatomy, Fukuoka University School of Medicine, 7-45-1 Nanakuma, Jonan-ku, Fukuoka City 814-0180 ; Feril, Loreto B.; Tachibana, Katsuro; Takahashi, Akira; Matsuo, Miki; Endo, Hitomi; Harada, Yoshimi; Nakayama, Juichiro

    2011-07-22

    Highlights: {yields} Successful ultrasound-mediated transfection of melanoma (C32) cells with IFN-{beta} genes both in vitro and in vivo. {yields} Ultrasound-mediated IFN-{beta} transfection inhibited proliferation of melanoma cells in vitro. {yields} Ultrasound-mediated IFN-{beta} transfection inhibited melanoma tumor growth in vivo. -- Abstract: We investigated the effects of ultrasound-mediated transfection (sonotransfection) of interferon {beta} (IFN-{beta}) gene on melanoma (C32) both in vitro and in vivo. C32 cells were sonotransfected with IFN-{beta} in vitro. Subcutaneous C32 tumors in mice were sonicated weekly immediately after intra-tumor injection with IFN-{beta} genes mixed with microbubbles. Successful sonotransfection with IFN-{beta} gene in vitro was confirmed by ELISA, which resulted in C32 growth inhibition. In vivo, the growth ratio of tumors transfected with IFN-{beta} gene was significantly lower than the other experimental groups. These results may lead to a new method of treatment against melanoma and other hard-to-treat cancers.

  1. Resveratrol inhibits LXRα-dependent hepatic lipogenesis through novel antioxidant Sestrin2 gene induction

    SciTech Connect

    Jin, So Hee; Yang, Ji Hye; Shin, Bo Yeon; Seo, Kyuhwa; Shin, Sang Mi; Cho, Il Je; Ki, Sung Hwan

    2013-08-15

    Liver X receptor-α (LXRα), a member of the nuclear receptor superfamily of ligand-activated transcription factors, regulates de novo fatty acid synthesis that leads to stimulate hepatic steatosis. Although, resveratrol has beneficial effects on metabolic disease, it is not known whether resveratrol affects LXRα-dependent lipogenic gene expression. This study investigated the effect of resveratrol in LXRα-mediated lipogenesis and the underlying molecular mechanism. Resveratrol inhibited the ability of LXRα to activate sterol regulatory element binding protein-1c (SREBP-1c) and thereby inhibited target gene expression in hepatocytes. Moreover, resveratrol decreased LXRα–RXRα DNA binding activity and LXRE-luciferase transactivation. Resveratrol is known to activate Sirtuin 1 (Sirt1) and AMP-activated protein kinase (AMPK), although its precise mechanism of action remains controversial. We found that the ability of resveratrol to repress T0901317-induced SREBP-1c expression was not dependent on AMPK and Sirt1. It is well established that hepatic steatosis is associated with antioxidant and redox signaling. Our data showing that expression of Sestrin2 (Sesn2), which is a novel antioxidant gene, was significantly down-regulated in the livers of high-fat diet-fed mice. Moreover, resveratrol up-regulated Sesn2 expression, but not Sesn1 and Sesn3. Sesn2 overexpression repressed LXRα-activated SREBP-1c expression and LXRE-luciferase activity. Finally, Sesn2 knockdown using siRNA abolished the effect of resveratrol in LXRα-induced FAS luciferase gene transactivation. We conclude that resveratrol affects Sesn2 gene induction and contributes to the inhibition of LXRα-mediated hepatic lipogenesis. - Highlights: • We investigated the effect of resveratrol in LXRα-mediated lipogenesis. • Resveratrol attenuated the ability of the LXRα-mediated lipogenic gene expression. • Resveratrol’s effects on T090-induced lipogenesis is not dependent on Sirt1 or AMPK. • Sestrin2 induction by resveratrol contributes to the inhibition of the LXRα activity.

  2. Multiple pigmentation gene polymorphisms account for a substantial proportion of risk of cutaneous malignant melanoma

    PubMed Central

    Duffy, David L.; Zhao, Z. Z.; Sturm, Richard A.; Hayward, Nicholas K.; Martin, Nicholas G.; Montgomery, Grant W.

    2013-01-01

    We have previously described the role of red hair (Melanocortin 1 Receptor, MC1R) and blue eye (Oculocutaneous Albinism Type 2, OCA2) gene polymorphisms in modulating risk of cutaneous malignant melanoma (CMM) in a highly sun-exposed population of European descent. A number of recent studies, including genome-wide association studies (GWAS), have identified numerous polymorphisms controlling human hair, eye and skin colour. In this paper, we test a selected set of polymorphisms in pigmentation loci (ASIP, TYR, TYRP1, MC1R, OCA2, IRF4, SLC24A4, SLC45A2) for association with CMM risk in a large Australian population-based case control study. Variants in IRF4 and SLC24A4, despite being strongly associated with pigmentation in our sample, did not modify CMM risk, but the other six did. Three SNPs (rs28777, rs35391, rs16891982) in the MATP gene (SLC45A2) exhibited the strongest crude association with risk, but this was attenuated to approximately the same effect size as that of a MC1R red hair color allele by controlling for ancestry of cases and controls. We also detected significant epistatic interactions between SLC45A2 and OCA2 alleles, and MC1R and ASIP alleles. Overall, these measured variants account for 12% of the familial risk of CMM in our population. PMID:19710684

  3. Antisense oligodeoxynucleotide to the cystic fibrosis gene inhibits anion transport in normal cultured sweat duct cells

    SciTech Connect

    Sorscher, E.J.; Kirk, K.L.; Weaver, M.L.; Jilling, T.; Blalock, J.E.; LeBoeuf, R.D. )

    1991-09-01

    The authors have tested the hypothesis that the cystic fibrosis (CF) gene product, called the CF transmembrane conductance regulator (CFTR), mediates anion transport in normal human sweat duct cells. Sweat duct cells in primary culture were treated with oligodeoxynucleotides that were antisense to the CFTR gene transcript in order to block the expression of the wild-type CFTR. Anion transport in CFTR transcript antisense-treated cells was then assessed with a halide-specific dye, 6-methoxy-N-(3-sulfopropryl)quinolinium, and fluorescent digital imaging microscopy to monitor halide influx and efflux from single sweat duct cells. Antisense oligodeoxynucleotide treatment for 24 hr virtually abolished Cl{sup {minus}} transport in sweat duct cells compared with untreated cells or control cells treated with sense oligodeoxynucleotides. Br{sup {minus}} uptake into sweat duct cells was also blocked after a 24-hr CFTR transcript antisense treatments, but not after treatments for only 4 hr. Lower concentrations of antisense oligodeoxynucleotides were less effective at inhibiting Cl{sup {minus}} transport. These results indicate that oligodeoxynucleotides that are antisense to CFTR transcript inhibit sweat duct Cl{sup {minus}} permeability in both a time-dependent and dose-dependent manner. This approach provides evidence that inhibition of the expression of the wild-type CFTR gene in a normal, untransfected epithelial cell results in an inhibition of Cl{sup {minus}} permeability.

  4. Differential gene expression for investigation of Escherichia coli biofilm inhibition by plant extract ursolic acid.

    PubMed

    Ren, Dacheng; Zuo, Rongjun; González Barrios, Andrés F; Bedzyk, Laura A; Eldridge, Gary R; Pasmore, Mark E; Wood, Thomas K

    2005-07-01

    After 13,000 samples of compounds purified from plants were screened, a new biofilm inhibitor, ursolic acid, has been discovered and identified. Using both 96-well microtiter plates and a continuous flow chamber with COMSTAT analysis, 10 microg of ursolic acid/ml inhibited Escherichia coli biofilm formation 6- to 20-fold when added upon inoculation and when added to a 24-h biofilm; however, ursolic acid was not toxic to E. coli, Pseudomonas aeruginosa, Vibrio harveyi, and hepatocytes. Similarly, 10 microg of ursolic acid/ml inhibited biofilm formation by >87% for P. aeruginosa in both complex and minimal medium and by 57% for V. harveyi in minimal medium. To investigate the mechanism of this nontoxic inhibition on a global genetic basis, DNA microarrays were used to study the gene expression profiles of E. coli K-12 grown with or without ursolic acid. Ursolic acid at 10 and 30 microg/ml induced significantly (P < 0.05) 32 and 61 genes, respectively, and 19 genes were consistently induced. The consistently induced genes have functions for chemotaxis and mobility (cheA, tap, tar, and motAB), heat shock response (hslSTV and mopAB), and unknown functions (such as b1566 and yrfHI). There were 31 and 17 genes repressed by 10 and 30 microg of ursolic acid/ml, respectively, and 12 genes were consistently repressed that have functions in cysteine synthesis (cysK) and sulfur metabolism (cysD), as well as unknown functions (such as hdeAB and yhaDFG). Ursolic acid inhibited biofilms without interfering with quorum sensing, as shown with the V. harveyi AI-1 and AI-2 reporter systems. As predicted by the differential gene expression, deleting motAB counteracts ursolic acid inhibition (the paralyzed cells no longer become too motile). Based on the differential gene expression, it was also discovered that sulfur metabolism (through cysB) affects biofilm formation (in the absence of ursolic acid). PMID:16000817

  5. Differential Gene Expression for Investigation of Escherichia coli Biofilm Inhibition by Plant Extract Ursolic Acid

    PubMed Central

    Ren, Dacheng; Zuo, Rongjun; González Barrios, Andrés F.; Bedzyk, Laura A.; Eldridge, Gary R.; Pasmore, Mark E.; Wood, Thomas K.

    2005-01-01

    After 13,000 samples of compounds purified from plants were screened, a new biofilm inhibitor, ursolic acid, has been discovered and identified. Using both 96-well microtiter plates and a continuous flow chamber with COMSTAT analysis, 10 μg of ursolic acid/ml inhibited Escherichia coli biofilm formation 6- to 20-fold when added upon inoculation and when added to a 24-h biofilm; however, ursolic acid was not toxic to E. coli, Pseudomonas aeruginosa, Vibrio harveyi, and hepatocytes. Similarly, 10 μg of ursolic acid/ml inhibited biofilm formation by >87% for P. aeruginosa in both complex and minimal medium and by 57% for V. harveyi in minimal medium. To investigate the mechanism of this nontoxic inhibition on a global genetic basis, DNA microarrays were used to study the gene expression profiles of E. coli K-12 grown with or without ursolic acid. Ursolic acid at 10 and 30 μg/ml induced significantly (P < 0.05) 32 and 61 genes, respectively, and 19 genes were consistently induced. The consistently induced genes have functions for chemotaxis and mobility (cheA, tap, tar, and motAB), heat shock response (hslSTV and mopAB), and unknown functions (such as b1566 and yrfHI). There were 31 and 17 genes repressed by 10 and 30 μg of ursolic acid/ml, respectively, and 12 genes were consistently repressed that have functions in cysteine synthesis (cysK) and sulfur metabolism (cysD), as well as unknown functions (such as hdeAB and yhaDFG). Ursolic acid inhibited biofilms without interfering with quorum sensing, as shown with the V. harveyi AI-1 and AI-2 reporter systems. As predicted by the differential gene expression, deleting motAB counteracts ursolic acid inhibition (the paralyzed cells no longer become too motile). Based on the differential gene expression, it was also discovered that sulfur metabolism (through cysB) affects biofilm formation (in the absence of ursolic acid). PMID:16000817

  6. Strain-Specific Inhibition of nod Gene Induction in Bradyrhizobium japonicum by Flavonoid Compounds

    PubMed Central

    Kosslak, Renee M.; Joshi, Rita S.; Bowen, Benjamin A.; Paaren, Herbert E.; Appelbaum, Edward R.

    1990-01-01

    A broad-host-range plasmid, pEA2-21, containing a Bradyrhizobium japonicum nodABC'-'lacZ translational fusion was used to identify strain-specific inhibitors of the genes required for soybean nodulation, the common nod genes. The responses of type strains of B. japonicum serogroups USDA 110, USDA 123, USDA 127, USDA 129, USDA 122, and USDA 138 to nod gene inhibitors were compared. Few compounds inhibited nod gene expression in B. japonicum USDA 110. In contrast, nod gene expression in strains belonging to several other serogroups was inhibited by most of the flavonoids tested. However, the application of two of these strain-specific compounds, chrysin and naringenin, had little effect on the pattern of competition between indigenous and inoculum strains of B. japonicum in greenhouse and field trials. Preliminary studies with radiolabeled chrysin and naringenin suggest that the different responses to nod gene inhibitors may be partly due to the degree to which plant flavonoids can be metabolized by each strain. PMID:16348186

  7. Gene expression profile change and growth inhibition in Drosophila larvae treated with azadirachtin.

    PubMed

    Lai, Duo; Jin, Xiaoyong; Wang, Hao; Yuan, Mei; Xu, Hanhong

    2014-09-20

    Azadirachtin is a botanical insecticide that affects various biological processes. The effects of azadirachtin on the digital gene expression profile and growth inhibition in Drosophila larvae have not been investigated. In this study, we applied high-throughput sequencing technology to detect the differentially expressed genes of Drosophila larvae regulated by azadirachtin. A total of 15,322 genes were detected, and 28 genes were found to be significantly regulated by azadirachtin. Biological process and pathway analysis showed that azadirachtin affected starch and sucrose metabolism, defense response, signal transduction, instar larval or pupal development, and chemosensory behavior processes. The genes regulated by azadirachtin were mainly enriched in starch and sucrose metabolism. This study provided a general digital gene expression profile of dysregulated genes in response to azadirachtin and showed that azadirachtin provoked potent growth inhibitory effects in Drosophila larvae by regulating the genes of cuticular protein, amylase, and odorant-binding protein. Finally, we propose a potential mechanism underlying the dysregulation of the insulin/insulin-like growth factor signaling pathway by azadirachtin. PMID:24956222

  8. HDAC inhibition attenuates cardiac hypertrophy by acetylation and deacetylation of target genes

    PubMed Central

    Ooi, Jenny Y Y; Tuano, Natasha K; Rafehi, Haloom; Gao, Xiao-Ming; Ziemann, Mark; Du, Xiao-Jun; El-Osta, Assam

    2015-01-01

    Pharmacological histone deacetylase (HDAC) inhibitors attenuate pathological cardiac remodeling and hypertrophic gene expression; yet, the direct histone targets remain poorly characterized. Since the inhibition of HDAC activity is associated with suppressing hypertrophy, we hypothesized histone acetylation would target genes implicated in cardiac remodeling. Trichostatin A (TSA) regulates cardiac gene expression and attenuates transverse aortic constriction (TAC) induced hypertrophy. We used chromatin immunoprecipitation (ChIP) coupled with massive parallel sequencing (ChIP-seq) to map, for the first time, genome-wide histone acetylation changes in a preclinical model of pathological cardiac hypertrophy and attenuation of pathogenesis with TSA. Pressure overload-induced cardiac hypertrophy was associated with histone acetylation of genes implicated in cardiac contraction, collagen deposition, inflammation, and extracellular matrix identified by ChIP-seq. Gene set enrichment analysis identified NF-kappa B (NF-κB) transcription factor activation with load induced hypertrophy. Increased histone acetylation was observed on the promoters of NFκB target genes (Icam1, Vcam1, Il21r, Il6ra, Ticam2, Cxcl10) consistent with gene activation in the hypertrophied heart. Surprisingly, TSA attenuated pressure overload-induced cardiac hypertrophy and the suppression of NFκB target genes by broad histone deacetylation. Our results suggest a mechanism for cardioprotection subject to histone deacetylation as a previously unknown target, implicating the importance of inflammation by pharmacological HDAC inhibition. The results of this study provides a framework for HDAC inhibitor function in the heart and argues the long held views of acetylation is subject to more flexibility than previously thought. PMID:25941940

  9. Gene microarray analysis of human renal cell carcinoma: the effects of HDAC inhibition and retinoid treatment.

    PubMed

    Tavares, Trisha S; Nanus, David; Yang, Ximing J; Gudas, Lorraine J

    2008-10-01

    Histone deacetylase (HDAC) inhibitor treatments can augment the anti-tumor effects of retinoids in renal cancer cells. We studied the effects of the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) and 13-cis retinoic acid (cRA) on two human renal cell carcinoma (RCC) lines. Cells were cultured in the presence of each drug for six days to determine the responses to monotherapy and to combination therapy. The proliferation of SKRC06 was inhibited with cRA treatment; the proliferation of SKRC39 was not. However, both RCC lines were sensitive to growth inhibition by dibutyryl cyclic AMP, with or without 13-cis RA. SAHA alone also reduced cell proliferation in both cell lines. To identify the alterations in gene expression that correlate with the responsiveness to treatment, gene microarray analyses were performed. Several retinoid-regulated genes exhibited much higher mRNA levels in SKRC06 than in SKRC39, even in the absence of drugs; these included crabp2, rargamma and cyp26A1. Combination treatment of cells with both SAHA and cRA induced several transcripts with known anti-cancer/immunomodulatory effects, including dhrs9, gata3, il1beta, phlda1, txk and vhl. Immunostaining confirmed the decreased expression of gata3 in human RCC specimens compared to normal kidney. Together, our results show that treatment of RCC with cRA and/or SAHA increases the expression of several genes and gene families that result in reduced cell proliferation. PMID:18769122

  10. A novel gene, msa1, inhibits sexual differentiation in Schizosaccharomyces pombe.

    PubMed Central

    Jeong, Hee Tae; Ozoe, Fumiyo; Tanaka, Katsunori; Nakagawa, Tsuyoshi; Matsuda, Hideyuki; Kawamukai, Makoto

    2004-01-01

    Sexual differentiation in the fission yeast Schizosaccharomyces pombe is triggered by nutrient starvation or by the presence of mating pheromones. We identified a novel gene, msa1, which encodes a 533-aa putative RNA-binding protein that inhibits sexual differentiation. Disruption of the msa1 gene caused cells to hypersporulate. Intracellular levels of msa1 RNA and Msa1 protein diminished after several hours of nitrogen starvation. Genetic analysis suggested that the function of msa1 is independent of the cAMP pathway and stress-responsive pathway. Deletion of the ras1 gene in diploid cells inhibited sporulation and in haploid cells decreased expression of mating-pheromone-induced genes such as mei2, mam2, ste11, and rep1; simultaneous deletion of msa1 reversed both phenotypes. Overexpression of msa1 decreased activated Ras1(Val17)-induced expression of mam2. Phenotypic hypersporulation was similar between cells with deletion of only rad24 and both msa1 and rad24, but simultaneous deletion of msa1 and msa2/nrd1 additively increased hypersporulation. Therefore, we suggest that the primary function of Msa1 is to negatively regulate sexual differentiation by controlling the expression of Ste11-regulated genes, possibly through the pheromone-signaling pathway. PMID:15166138

  11. Lack of feedback inhibition of V kappa gene rearrangement by productively rearranged alleles.

    PubMed

    Harada, K; Yamagishi, H

    1991-02-01

    Circular DNAs excised by immunoglobulin kappa chain gene rearrangements were cloned and characterized. 16 of 17 clones examined were double recombination products containing a V kappa-J kappa rearrangement (coding joint) as well as the reciprocal element (signal joint) of another V kappa-J kappa rearrangement. These products suggested multiple recombination, primary inversion, and secondary excision. In primary events, 5 of 16 translational reading frames were in-phase. Thus, V kappa gene rearrangement may not be inhibited by the presence of a productively rearranged allele. An unusually large trinucleotide (P) insertion forming a palindrome of 12 nucleotides was also observed in one of the coding joints. PMID:1988542

  12. [Novel cationic liposome loading siRNA inhibits the expression of hepatitis B virus HBx gene].

    PubMed

    Wang, Ya-Juan; Gao, Yu-Qing; Ni, Bei-Bei; Li, Chan; Wen, Tian-Jiao; Wang, Yan-Ling; Wang, Jia; Li, Chun-Lei

    2014-09-01

    In order to solve the problem of selection and in vivo delivery problem in siRNA treatment, hepatitis B virus (HBV) HBx gene which could be targeted by siRNA was studied. The siRNA expression plasmid which specific inhibits HBx expression was obtained by in vitro selection via a dual-luciferase plasmid including HBx-Fluc fusion protein expression domain. The selected siRNA expression plasmid was then encapsulated in PEG-modified cationic liposome, which was devoted into pharmacodynamic studies at both cellular and animal level. The results illustrated that the cationic liposome which encapsulated siRNA expression plasmid could effectively inhibit HBx gene expression both in vitro and in vivo. PMID:25518334

  13. Polyamine metabolism-based dual functional gene delivery system to synergistically inhibit the proliferation of cancer.

    PubMed

    Cui, Peng-Fei; Xing, Lei; Qiao, Jian-Bin; Zhang, Jia-Liang; He, Yu-Jing; Zhang, Mei; Lyu, Jin-Yuan; Luo, Cheng-Qiong; Jin, Liang; Jiang, Hu-Lin

    2016-06-15

    Polyamine content, which is associated with tumor growth, can be regulated by ornithine decarboxylase (ODC) and S-adenosyl methionine decarboxylase (SAMDC), two key enzymes in polyamine biosynthesis. Here we aim to develop a pH-responsive cationic poly(agmatine) based on a polyamine analogue-agmatine that can dually function as a gene delivery vector as well as an anticancer agent by inhibiting ODC after intracellular degradation. The core-shell nanoparticles, formed by poly(agmatine)/SAMDC siRNA complex as a core, were coated with bovine serum albumin for better in vivo circulation stability and tumor targeting. When the nanoparticles were taken up by tumor cells via endocytosis and degraded in endosome, the released agmatine and SAMDC siRNA can synergistically inhibit polyamines biosynthesis, inducing inhibition of tumor proliferation. Our study offered a potential way in tumor therapy based on polyamine metabolism. PMID:27102990

  14. Biological effects of RNAi targeted inhibiting Tiam1 gene expression on cholangiocarcinoma cells

    PubMed Central

    Cheng, Wei; Liu, Yaling; Zuo, Zhi; Yin, Xinmin; Jiang, Bo; Chen, Daojin; Peng, Chuang; Yang, Jianhui

    2015-01-01

    Objective: To investigate the characteristics of Tiam1 gene expression in human cholangiocarcinoma tissues and benign bile duct tissues, and to analyze the correlations between Tiam1 gene expression and the degree of tumor differentiation, invasive and metastatic abilities. To explore the effect of targeted inhibiting Tiam1 gene expression on proliferation and migration activity of human cholangiocarcinoma cells. Methods: Expression of Tiam1 in 83 cases of cholangiocarcinoma tissues and 25 cases of benign bile tissues was detected using immunohistochemistry. The clinical data of patients with cholangiocarcinoma were collected. The correlations between Tiam1 gene expression and the clinicopathologic features in patients with cholangiocarcinoma were analyzed. The human cholangiocarcinoma RBE cells were divided into 3 groups. Cells in experimental group and control group were respectively transfected with Tiam1 shRNA lentiviral vectors and negative shRNA lentiviral control vectors. Cells in blank group received no treatment. Real-time PCR endogenesis was used to verify Tiam1 gene expression. Cell cycle experiments and MTT assay were used to measure cell proliferation activity. Transwell test was used to detect cell migration activity. Results: The negative rate Tiam1 protein expression in cholangiocarcinoma tissues was significantly higher than that in benign bile tissues (P<0.001). Tiam1 protein expression in cholangiocarcinoma tissues had correlations with cholangiocarcinoma differentiation degree, TNM stage and lymph node metastasis (P<0.05), and had no significant correlations with gender, age and distant metastasis (P>0.05). Real-time PCR detection indicated that Tiam1 expression of experimental group was significantly lower than that in control group and blank group (P<0.05), demonstrating that Tiam1 shRNA was effective on Tiam1 gene silencing in RBE cells. Cell cycle experiment showed that the percentage of S phase in cell cycle in experimental group was lower than that in control group and blank group (P<0.05), demonstrating that after the down-regulation of Tiam1 gene expression, the speed of cell proliferation was inhibited. MTT assay results showed that the total growth speed in experimental group was significantly lower than that in control group and blank group (P<0.05), indicating that the proliferation activity of cholangiocarcinoma cells was inhibited after targeted inhibition of Tiam1 gene expression. Transwell detection results showed that the metastasis rate in experimental group was significantly lower than that in control group and blank group (P<0.05), demonstrating that targeted inhibition of Tiam1 gene expression could significantly inhibit migration ability of RBE cells. Conclusion: Tiam1 expression significantly increased in cholangiocarcinoma tissues, and increased along with the degree of malignancy of cholangiocarcinoma. Targeted silencing Tiam1 expression could inhibit proliferation and migration activity of cholangiocarcinoma cells. PMID:26884821

  15. Selective Dysregulation of Hippocampal Inhibition in the Mouse Lacking Autism Candidate Gene CNTNAP2

    PubMed Central

    Jurgensen, Sofia

    2015-01-01

    Mutations in the human gene encoding contactin-associated protein-like 2 (CNTNAP2) have been strongly associated with autism spectrum disorders (ASDs). Cntnap2−/− mice recapitulate major features of ASD, including social impairment, reduced vocalizations, and repetitive behavior. In addition, Cntnap2−/− mice show reduced cortical neuronal synchrony and develop spontaneous seizures throughout adulthood. As suggested for other forms of ASDs, this phenotype could reflect some form of synaptic dysregulation. However, the impact of lifelong deletion of CNTNAP2 on synaptic function in the brain remains unknown. To address this issue, we have assessed excitatory and inhibitory synaptic transmission in acute hippocampal slices of Cntnap2−/− mice. We found that although excitatory transmission was mostly normal, inhibition onto CA1 pyramidal cells was altered in Cntnap2−/− mice. Specifically, putative perisomatic, but not dendritic, evoked IPSCs were significantly reduced in these mice. Whereas both inhibitory short-term plasticity and miniature IPSC frequency and amplitude were normal in Cntnap2−/− mice, we found an unexpected increase in the frequency of spontaneous, action potential-driven IPSCs. Altered hippocampal inhibition could account for the behavioral phenotype Cntnap2−/− mice present later in life. Overall, our findings that Cntnap2 deletion selectively impairs perisomatic hippocampal inhibition while sparing excitation provide additional support for synaptic dysfunction as a common mechanism underlying ASDs. SIGNIFICANCE STATEMENT The gene encoding contactin-associated protein-like 2 (CNTNAP2) stands out as one the first genes to have both rare and common mutations strongly associated with ASDs. Whereas Cntnap2−/− mice appear to recapitulate core behavioral endophenotypes (e.g., social impairment, language deficits, and repetitive behavior), the cellular and circuit bases of this phenotype remain poorly understood. Here, we report that synaptic inhibition specifically onto the perisomatic compartment of CA1 pyramidal cells is impaired, whereas dendritic inhibition and excitatory transmission remain basically unchanged in Cntnap2−/− mice. These findings provide additional support to the notion that disruption of inhibition, in particular perisomatic inhibition onto principal cells, could play a critical role in ASDs. PMID:26511255

  16. Caffeine inhibits gene conversion by displacing Rad51 from ssDNA

    PubMed Central

    Tsabar, Michael; Mason, Jennifer M.; Chan, Yuen-Ling; Bishop, Douglas K.; Haber, James E.

    2015-01-01

    Efficient repair of chromosomal double-strand breaks (DSBs) by homologous recombination relies on the formation of a Rad51 recombinase filament that forms on single-stranded DNA (ssDNA) created at DSB ends. This filament facilitates the search for a homologous donor sequence and promotes strand invasion. Recently caffeine treatment has been shown to prevent gene targeting in mammalian cells by increasing non-productive Rad51 interactions between the DSB and random regions of the genome. Here we show that caffeine treatment prevents gene conversion in yeast, independently of its inhibition of the Mec1ATR/Tel1ATM-dependent DNA damage response or caffeine's inhibition of 5′ to 3′ resection of DSB ends. Caffeine treatment results in a dosage-dependent eviction of Rad51 from ssDNA. Gene conversion is impaired even at low concentrations of caffeine, where there is no discernible dismantling of the Rad51 filament. Loss of the Rad51 filament integrity is independent of Srs2's Rad51 filament dismantling activity or Rad51's ATPase activity and does not depend on non-specific Rad51 binding to undamaged double-stranded DNA. Caffeine treatment had similar effects on irradiated HeLa cells, promoting loss of previously assembled Rad51 foci. We conclude that caffeine treatment can disrupt gene conversion by disrupting Rad51 filaments. PMID:26019181

  17. Interleukin 1beta inhibits interleukin 6-mediated rat gamma fibrinogen gene expression.

    PubMed

    Zhang, Z; Fuller, G M

    2000-11-15

    Interleukin (IL)-1beta and IL-6 are the 2 major inducers of a group of hepatic genes during acute inflammation; however, each cytokine uses different intracellular signaling molecules. In most instances, the 2 cytokines interact positively to enhance hepatic gene expression, but in one class of acute-phase reactants, which includes fibrinogen, IL-1beta exerts a transient inhibitory effect over the IL-6 stimulatory signal. This study explored the effects of IL-1beta/nuclear factor kappaB (NF-kappaB) and IL-6/signal transducer and activator of transcription 3 (STAT3) combinatory signaling on the transcriptional regulation of the rat gamma fibrinogen gene. Northern blot and functional analyses employing luciferase reporter constructs driven by the rat gamma fibrinogen promoter demonstrated that IL-1beta inhibited the IL-6-mediated transcription of this gene. Exposing primary rat hepatocytes to IL-1beta had no effect on IL-6-mediated STAT3 activation; instead, IL-1beta-activated NF-kappaB associated with 2 IL-6 responsive elements (STAT3 binding site) on the rat gamma fibrinogen promoter and blocked STAT3 binding to these regions. The competitive binding of NF-kappaB and STAT3 on the overlapping binding site provides a mechanism for the inhibition by IL-1beta of the IL-6-mediated transactivation of rat gamma fibrinogen. PMID:11071643

  18. Efficient inhibition of ovarian cancer by degradable nanoparticle-delivered survivin T34A gene.

    PubMed

    Luo, Li; Du, Ting; Zhang, Jiumeng; Zhao, Wei; Cheng, Hao; Yang, Yuping; Wu, Yujiao; Wang, Chunmei; Men, Ke; Gou, Maling

    2016-01-01

    Gene therapy has promising applications in ovarian cancer therapy. Blocking the function of the survivin protein could lead to the growth inhibition of cancer cells. Herein, we used degradable heparin-polyethyleneimine (HPEI) nanoparticles to deliver a dominant-negative human survivin T34A (hs-T34A) gene to treat ovarian cancer. HPEI nanoparticles were characterized and were found to have a dynamic diameter of 66±4.5 nm and a zeta potential of 27.1±1.87 mV. The constructed hs-T34A gene expression plasmid could be effectively delivered into SKOV3 ovarian carcinoma cells by HPEI nanoparticles with low cytotoxicity. Intraperitoneal administration of HPEI/hs-T34A complexes could markedly inhibit tumor growth in a mouse xenograft model of SKOV3 human ovarian cancer. Moreover, according to our results, apparent apoptosis of cancer cells was observed both in vitro and in vivo. Taken together, the prepared HPEI/hs-T34A formulation showed potential applications in ovarian cancer gene therapy. PMID:26893558

  19. Efficient inhibition of ovarian cancer by degradable nanoparticle-delivered survivin T34A gene

    PubMed Central

    Luo, Li; Du, Ting; Zhang, Jiumeng; Zhao, Wei; Cheng, Hao; Yang, Yuping; Wu, Yujiao; Wang, Chunmei; Men, Ke; Gou, Maling

    2016-01-01

    Gene therapy has promising applications in ovarian cancer therapy. Blocking the function of the survivin protein could lead to the growth inhibition of cancer cells. Herein, we used degradable heparin–polyethyleneimine (HPEI) nanoparticles to deliver a dominant-negative human survivin T34A (hs-T34A) gene to treat ovarian cancer. HPEI nanoparticles were characterized and were found to have a dynamic diameter of 66±4.5 nm and a zeta potential of 27.1±1.87 mV. The constructed hs-T34A gene expression plasmid could be effectively delivered into SKOV3 ovarian carcinoma cells by HPEI nanoparticles with low cytotoxicity. Intraperitoneal administration of HPEI/hs-T34A complexes could markedly inhibit tumor growth in a mouse xenograft model of SKOV3 human ovarian cancer. Moreover, according to our results, apparent apoptosis of cancer cells was observed both in vitro and in vivo. Taken together, the prepared HPEI/hs-T34A formulation showed potential applications in ovarian cancer gene therapy. PMID:26893558

  20. Antidiabetic thiazolidinediones inhibit leptin (ob) gene expression in 3T3-L1 adipocytes.

    PubMed Central

    Kallen, C B; Lazar, M A

    1996-01-01

    Lack of leptin (ob) protein causes obesity in mice. The leptin gene product is important for normal regulation of appetite and metabolic rate and is produced exclusively by adipocytes. Leptin mRNA was induced during the adipose conversion of 3T3-L1 cells, which are useful for studying adipocyte differentiation and function under controlled conditions. We studied leptin regulation by antidiabetic thiazolidinedione compounds, which are ligands for the adipocyte-specific nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) that regulates the transcription of other adipocyte-specific genes. Remarkably, leptin gene expression was dramatically repressed within a few hours after thiazolidinedione treatment. The ED50 for inhibition of leptin expression by the thiazolidinedione BRL49653 was between 5 and 50 nM, similar to its Kd for binding to PPARgamma. The relatively weak, nonthiazolidinedione PPAR activator WY 14,643 also inhibited leptin expression, but was approximately 1000 times less potent than BRL49653. These results indicate that antidiabetic thiazolidinediones down-regulate leptin gene expression with potencies that correlate with their abilities to bind and activate PPARgamma. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8650171

  1. Therapeutic ultrasound facilitates antiangiogenic gene delivery and inhibits prostate tumor growth.

    PubMed

    Duvshani-Eshet, Maayan; Benny, Ofra; Morgenstern, Avigail; Machluf, Marcelle

    2007-08-01

    Gene therapy clinical trials are limited due to several hurdles concerning the type of vector used, particularly, the viral vectors, and transfection efficacy when non-viral vectors are used. Therapeutic ultrasound is a promising non-viral technology that can be used in the clinical setting. Here, for the first time, we show the efficacy of therapeutic ultrasound to deliver genes encoding for hemopexin-like domain fragment (PEX), an inhibitor of angiogenesis, to prostate tumors in vivo. Moreover, the addition of an ultrasound contrast agent (Optison) to the transfection process was evaluated. Prostate cancer cells and endothelial cells (EC) were transfected in vitro with cDNA-PEX using therapeutic ultrasound alone (TUS + pPEX) or with Optison (TUS + pPEX + Optison). The biological activity of the expressed PEX was assessed using proliferation, migration, and apoptosis assays done on EC and prostate cancer cells. TUS + pPEX + Optison led to the inhibition of EC and prostate cancer cell proliferation (<65%), migration (<50%), and an increase in apoptosis. In vivo, C57/black mice were inoculated s.c. with prostate cancer cells. The tumors were treated with TUS + pPEX and TUS + pPEX + Optison either once or repeatedly. Tumor growth was evaluated, after which histology and immunohistochemistry analyses were done. A single treatment of TUS + pPEX led to a 35% inhibition in tumor growth. Using TUS + PEX + Optison led to an inhibition of 50%. Repeated treatments of TUS + pPEX + Optison were found to significantly (P < 0.001) inhibit prostate tumor growth by 80%, along with the angiogenic indices, with no toxicity to the surrounding tissues. These results depict the efficacy of therapeutic ultrasound as a non-viral technology to efficiently deliver genes to tumors in general, and to deliver angiogenic inhibitors to prostate cancer in particular. PMID:17699732

  2. ?-D-glucan inhibits endocrine-resistant breast cancer cell proliferation and alters gene expression.

    PubMed

    Jafaar, Zainab M T; Litchfield, Lacey M; Ivanova, Margarita M; Radde, Brandie N; Al-Rayyan, Numan; Klinge, Carolyn M

    2014-04-01

    Endocrine therapies have been successfully used for breast cancer patients with estrogen receptor ? (ER?) positive tumors, but ~40% of patients relapse due to endocrine resistance. ?-glucans are components of plant cell walls that have immunomodulatory and anticancer activity. The objective of this study was to examine the activity of ?-D-glucan, purified from barley, in endocrine-sensitive MCF-7 versus endocrine-resistant LCC9 and LY2 breast cancer cells. ?-D-glucan dissolved in DMSO but not water inhibited MCF-7 cell proliferation in a concentration-dependent manner as measured by BrdU incorporation with an IC?? of ~164 12 g/ml. ?-D-glucan dissolved in DMSO inhibited tamoxifen/endocrine-resistant LCC9 and LY2 cell proliferation with IC?? values of 4.6 0.3 and 24.2 1.4 g/ml, respectively. MCF-10A normal breast epithelial cells showed a higher IC?? ~464 g/ml and the proliferation of MDA-MB-231 triple negative breast cancer cells was not inhibited by ?-D-glucan. Concentration-dependent increases in the BAX/BCL2 ratio and cell death with ?-D-glucan were observed in MCF-7 and LCC9 cells. PCR array analysis revealed changes in gene expression in response to 24-h treatment with 10 or 50 g/ml ?-D-glucan that were different between MCF-7 and LCC9 cells as well as differences in basal gene expression between the two cell lines. Select results were confirmed by quantitative real-time PCR demonstrating that ?-D-glucan increased RASSF1 expression in MCF-7 cells and IGFBP3, CTNNB1 and ER? transcript expression in LCC9 cells. Our data indicate that ?-D-glucan regulates breast cancer-relevant gene expression and may be useful for inhibiting endocrine-resistant breast cancer cell proliferation. PMID:24534923

  3. JAZF1 can regulate the expression of lipid metabolic genes and inhibit lipid accumulation in adipocytes

    SciTech Connect

    Ming, Guang-feng; Xiao, Di; Gong, Wei-jing; Liu, Hui-xia; Liu, Jun; Zhou, Hong-hao; Liu, Zhao-qian

    2014-03-14

    Highlights: • JAZF1 was significantly upregulated during the differentiation of 3T3-L1 preadipocytes. • JAZF1 overexpression inhibited lipid accumulation in differentiated mature 3T3-L1 adipocytes. • JAZF1 overexpression inhibited the expression of SREBP1, ACC, and FAS. • JAZF1 overexpression upregulated the expression of HSL and ATGL. • SREBP1 and JAZF1 could regulate each other in adipocytes. - Abstract: JAZF1 is a newly identified gene with unknown functions. A recent genome-wide association study showed that JAZF1 is associated with type 2 diabetes and is highly expressed in liver and adipose tissue. Studies have demonstrated that JAZF1 is the co-repressor for nuclear orphan receptor TAK1, whereas most nuclear orphan receptor family members are involved in the regulation of lipid metabolism. Therefore, JAZF1 could be closely related to glycolipid metabolism. In this study, JAZF1 was significantly upregulated during the induced differentiation process of 3T3-L1 preadipocytes. The overexpression of JAZF1 inhibited lipid accumulation in differentiated mature 3T3-L1 adipocytes and significantly inhibited the expression of SREBPl, ACC, and FAS, which were important in lipid synthesis, while upregulating the expression of key enzyme hormone-sensitive lipase in lipoclasis. Moreover, SREBPl exhibited an inhibitory function on the expression of JAZF1. SREBP1 reversed the inhibitory action on lipid accumulation of JAZF1. SREBP1 and JAZF1 were observed to regulate each other in adipocytes. Therefore, JAZF1 could regulate the expression of particular genes related to lipid metabolism and inhibit lipid accumulation in adipocytes. This result suggests that JAZF1 may be a potential target for the treatment of diseases, such as obesity and lipid metabolism disorders.

  4. Inhibition of Human Cytomegalovirus Immediate-Early Gene Expression by Cyclin A2-Dependent Kinase Activity

    PubMed Central

    Oduro, Jennifer D.; Uecker, Ralf

    2012-01-01

    Human cytomegalovirus (HCMV) starts its lytic replication cycle only in the G0/G1 phase of the cell division cycle. S/G2 cells can be infected but block the onset of immediate-early (IE) gene expression. This block can be overcome by inhibition of cyclin-dependent kinases (CDKs), suggesting that cyclin A2, the only cyclin with an S/G2-specific activity profile, may act as a negative regulator of viral gene expression. To directly test this hypothesis, we generated derivatives of an HCMV-permissive glioblastoma cell line that express cyclin A2 in a constitutive, cell cycle-independent manner. We demonstrate that even moderate cyclin A2 overexpression in G1 was sufficient to severely compromise the HCMV replicative cycle after high-multiplicity infection. This negative effect was composed of a strong but transient inhibition of IE gene transcription and a more sustained alteration of IE mRNA processing, resulting in reduced levels of UL37 and IE2, an essential transactivator of viral early gene expression. Consistently, cyclin A2-overexpressing cells showed a strong delay of viral early and late gene expression, as well as virus reproduction. All effects were dependent on CDK activity, as a cyclin A2 mutant deficient in CDK binding was unable to interfere with the HCMV infectious cycle. Interestingly, murine CMV, whose IE gene expression is known to be cell cycle independent, is not affected by cyclin A2. Instead, it upregulates cyclin A2-associated kinase activity upon infection. Understanding the mechanisms behind the HCMV-specific action of cyclin A2-CDK might reveal new targets for antiviral strategies. PMID:22718829

  5. BMP7 gene transfer via gold nanoparticles into stroma inhibits corneal fibrosis in vivo.

    PubMed

    Tandon, Ashish; Sharma, Ajay; Rodier, Jason T; Klibanov, Alexander M; Rieger, Frank G; Mohan, Rajiv R

    2013-01-01

    This study examined the effects of BMP7 gene transfer on corneal wound healing and fibrosis inhibition in vivo using a rabbit model. Corneal haze in rabbits was produced with the excimer laser performing -9 diopters photorefractive keratectomy. BMP7 gene was introduced into rabbit keratocytes by polyethylimine-conjugated gold nanoparticles (PEI2-GNPs) transfection solution single 5-minute topical application on the eye. Corneal haze and ocular health in live animals was gauged with stereo- and slit-lamp biomicroscopy. The levels of fibrosis [α-smooth muscle actin (αSMA), F-actin and fibronectin], immune reaction (CD11b and F4/80), keratocyte apoptosis (TUNEL), calcification (alizarin red, vonKossa and osteocalcin), and delivered-BMP7 gene expression in corneal tissues were quantified with immunofluorescence, western blotting and/or real-time PCR. Human corneal fibroblasts (HCF) and in vitro experiments were used to characterize the molecular mechanism mediating BMP7's anti-fibrosis effects. PEI2-GNPs showed substantial BMP7 gene delivery into rabbit keratocytes in vivo (2×10(4) gene copies/ug DNA). Localized BMP7 gene therapy showed a significant corneal haze decrease (1.68±0.31 compared to 3.2±0.43 in control corneas; p<0.05) in Fantes grading scale. Immunostaining and immunoblot analyses detected significantly reduced levels of αSMA (46±5% p<0.001) and fibronectin proteins (48±5% p<0.01). TUNEL, CD11b, and F4/80 assays revealed that BMP7 gene therapy is nonimmunogenic and nontoxic for the cornea. Furthermore, alizarin red, vonKossa and osteocalcin analyses revealed that localized PEI2-GNP-mediated BMP7 gene transfer in rabbit cornea does not cause calcification or osteoblast recruitment. Immunofluorescence of BMP7-transefected HCFs showed significantly increased pSmad-1/5/8 nuclear localization (>88%; p<0.0001), and immunoblotting of BMP7-transefected HCFs grown in the presence of TGFβ demonstrated significantly enhanced pSmad-1/5/8 (95%; p<0.001) and Smad6 (53%, p<0.001), and decreased αSMA (78%; p<0.001) protein levels. These results suggest that localized BMP7 gene delivery in rabbit cornea modulates wound healing and inhibits fibrosis in vivo by counter balancing TGFβ1-mediated profibrotic Smad signaling. PMID:23799103

  6. BMP7 Gene Transfer via Gold Nanoparticles into Stroma Inhibits Corneal Fibrosis In Vivo

    PubMed Central

    Tandon, Ashish; Sharma, Ajay; Rodier, Jason T.; Klibanov, Alexander M.; Rieger, Frank G.; Mohan, Rajiv R.

    2013-01-01

    This study examined the effects of BMP7 gene transfer on corneal wound healing and fibrosis inhibition in vivo using a rabbit model. Corneal haze in rabbits was produced with the excimer laser performing -9 diopters photorefractive keratectomy. BMP7 gene was introduced into rabbit keratocytes by polyethylimine-conjugated gold nanoparticles (PEI2-GNPs) transfection solution single 5-minute topical application on the eye. Corneal haze and ocular health in live animals was gauged with stereo- and slit-lamp biomicroscopy. The levels of fibrosis [α-smooth muscle actin (αSMA), F-actin and fibronectin], immune reaction (CD11b and F4/80), keratocyte apoptosis (TUNEL), calcification (alizarin red, vonKossa and osteocalcin), and delivered-BMP7 gene expression in corneal tissues were quantified with immunofluorescence, western blotting and/or real-time PCR. Human corneal fibroblasts (HCF) and in vitro experiments were used to characterize the molecular mechanism mediating BMP7’s anti-fibrosis effects. PEI2-GNPs showed substantial BMP7 gene delivery into rabbit keratocytes in vivo (2×104 gene copies/ug DNA). Localized BMP7 gene therapy showed a significant corneal haze decrease (1.68±0.31 compared to 3.2±0.43 in control corneas; p<0.05) in Fantes grading scale. Immunostaining and immunoblot analyses detected significantly reduced levels of αSMA (46±5% p<0.001) and fibronectin proteins (48±5% p<0.01). TUNEL, CD11b, and F4/80 assays revealed that BMP7 gene therapy is nonimmunogenic and nontoxic for the cornea. Furthermore, alizarin red, vonKossa and osteocalcin analyses revealed that localized PEI2-GNP-mediated BMP7 gene transfer in rabbit cornea does not cause calcification or osteoblast recruitment. Immunofluorescence of BMP7-transefected HCFs showed significantly increased pSmad-1/5/8 nuclear localization (>88%; p<0.0001), and immunoblotting of BMP7-transefected HCFs grown in the presence of TGFβ demonstrated significantly enhanced pSmad-1/5/8 (95%; p<0.001) and Smad6 (53%, p<0.001), and decreased αSMA (78%; p<0.001) protein levels. These results suggest that localized BMP7 gene delivery in rabbit cornea modulates wound healing and inhibits fibrosis in vivo by counter balancing TGFβ1-mediated profibrotic Smad signaling. PMID:23799103

  7. The RNAPII-CTD Maintains Genome Integrity through Inhibition of Retrotransposon Gene Expression and Transposition

    PubMed Central

    Aristizabal, Maria J.; Negri, Gian Luca; Kobor, Michael S.

    2015-01-01

    RNA polymerase II (RNAPII) contains a unique C-terminal domain that is composed of heptapeptide repeats and which plays important regulatory roles during gene expression. RNAPII is responsible for the transcription of most protein-coding genes, a subset of non-coding genes, and retrotransposons. Retrotransposon transcription is the first step in their multiplication cycle, given that the RNA intermediate is required for the synthesis of cDNA, the material that is ultimately incorporated into a new genomic location. Retrotransposition can have grave consequences to genome integrity, as integration events can change the gene expression landscape or lead to alteration or loss of genetic information. Given that RNAPII transcribes retrotransposons, we sought to investigate if the RNAPII-CTD played a role in the regulation of retrotransposon gene expression. Importantly, we found that the RNAPII-CTD functioned to maintaining genome integrity through inhibition of retrotransposon gene expression, as reducing CTD length significantly increased expression and transposition rates of Ty1 elements. Mechanistically, the increased Ty1 mRNA levels in the rpb1-CTD11 mutant were partly due to Cdk8-dependent alterations to the RNAPII-CTD phosphorylation status. In addition, Cdk8 alone contributed to Ty1 gene expression regulation by altering the occupancy of the gene-specific transcription factor Ste12. Loss of STE12 and TEC1 suppressed growth phenotypes of the RNAPII-CTD truncation mutant. Collectively, our results implicate Ste12 and Tec1 as general and important contributors to the Cdk8, RNAPII-CTD regulatory circuitry as it relates to the maintenance of genome integrity. PMID:26496706

  8. Mediator Kinase Inhibition Further Activates Super-Enhancer Associated Genes in AML

    PubMed Central

    Nitulescu, Ioana I.; Tangpeerachaikul, Anupong; Poss, Zachary C.; Da Silva, Diogo H.; Caruso, Brittany T.; Arefolov, Alexander; Fadeyi, Olugbeminiyi; Christie, Amanda L.; Du, Karrie; Banka, Deepti; Schneider, Elisabeth V.; Jestel, Anja; Zou, Ge; Si, Chong; Ebmeier, Christopher C.; Bronson, Roderick T.; Krivtsov, Andrei V.; Myers, Andrew G.; Kohl, Nancy E.; Kung, Andrew L.; Armstrong, Scott A.; Lemieux, Madeleine E.; Taatjes, Dylan J.; Shair, Matthew D.

    2015-01-01

    Super-enhancers (SEs), which are composed of large clusters of enhancers densely loaded with the Mediator complex, transcription factors (TFs), and chromatin regulators, drive high expression of genes implicated in cell identity and disease, such as lineage-controlling TFs and oncogenes 1, 2. BRD4 and CDK7 are positive regulators of SE-mediated transcription3,4,5. In contrast, negative regulators of SE-associated genes have not been well described. Here we report that Mediator-associated kinases cyclin-dependent kinase 8 (CDK8) and CDK19 restrain increased activation of key SE-associated genes in acute myeloid leukaemia (AML) cells. We determined that the natural product cortistatin A (CA) selectively inhibited Mediator kinases, had antileukaemic activity in vitro and in vivo, and disproportionately induced upregulation of SE-associated genes in CA-sensitive AML cell lines but not in CA-insensitive cell lines. In AML cells, CA upregulated SE-associated genes with tumour suppressor and lineage-controlling functions, including the TFs CEBPA, IRF8, IRF1 and ETV6 6, 7, 8. The BRD4 inhibitor I-BET151 downregulated these SE-associated genes, yet also has antileukaemic activity. Individually increasing or decreasing expression of these TFs suppressed AML cell growth, providing evidence that leukaemia cells are sensitive to dosage of SE-associated genes. Our results demonstrate that Mediator kinases can negatively regulate SE-associated gene expression in specific cell types and can be pharmacologically targeted as a therapeutic approach to AML. PMID:26416749

  9. The RNAPII-CTD Maintains Genome Integrity through Inhibition of Retrotransposon Gene Expression and Transposition.

    PubMed

    Aristizabal, Maria J; Negri, Gian Luca; Kobor, Michael S

    2015-10-01

    RNA polymerase II (RNAPII) contains a unique C-terminal domain that is composed of heptapeptide repeats and which plays important regulatory roles during gene expression. RNAPII is responsible for the transcription of most protein-coding genes, a subset of non-coding genes, and retrotransposons. Retrotransposon transcription is the first step in their multiplication cycle, given that the RNA intermediate is required for the synthesis of cDNA, the material that is ultimately incorporated into a new genomic location. Retrotransposition can have grave consequences to genome integrity, as integration events can change the gene expression landscape or lead to alteration or loss of genetic information. Given that RNAPII transcribes retrotransposons, we sought to investigate if the RNAPII-CTD played a role in the regulation of retrotransposon gene expression. Importantly, we found that the RNAPII-CTD functioned to maintaining genome integrity through inhibition of retrotransposon gene expression, as reducing CTD length significantly increased expression and transposition rates of Ty1 elements. Mechanistically, the increased Ty1 mRNA levels in the rpb1-CTD11 mutant were partly due to Cdk8-dependent alterations to the RNAPII-CTD phosphorylation status. In addition, Cdk8 alone contributed to Ty1 gene expression regulation by altering the occupancy of the gene-specific transcription factor Ste12. Loss of STE12 and TEC1 suppressed growth phenotypes of the RNAPII-CTD truncation mutant. Collectively, our results implicate Ste12 and Tec1 as general and important contributors to the Cdk8, RNAPII-CTD regulatory circuitry as it relates to the maintenance of genome integrity. PMID:26496706

  10. Gene expression profile of Xenopus A6 cells cultured under random positioning machine shows downregulation of ion transporter genes and inhibition of dome formation

    NASA Astrophysics Data System (ADS)

    Ikuzawa, Masayuki; Akiduki, Saori; Asashima, Makoto

    Random positioning machine (RPM) devices that generate a simulated microgravity environment of approximately 0 g prevent the formation of dome structures in Xenopus kidney-derived A6 cells. In the present study, the gene expression profile of A6 cells cultured under RPM was determined using the Xenopus 22K scale microarray, and those genes up- or downregulated twofold or more were investigated. We identified 29 genes (up, 25 genes; down, 4 genes) on day 5, 68 genes (up, 25 genes; down, 43 genes) on day 8, 111 genes (up, 69 genes; down, 42 genes) on day 10, and 283 genes (up, 153 genes; down, 130 genes) on day 15 of culture under RPM. These genes were classified according to categories described in the KOG database, such as "extracellular structure", "cytoskeleton", and "transcription". Almost all the genes involved in "inorganic ion transport and metabolism" were downregulated under RPM. Our study further investigated some of these including the epithelial Na + channel (ENaC) and Na +/K +-ATPase transporter genes. A specific inhibitor of Na +/K +-ATPases, ouabain, inhibited dome formation in the A6 cells, even under control culturing conditions of 1 g (the static condition). Together these data suggested that downregulation of sodium ion transporter gene expression plays a significant role in the RPM-dependent prevention of the dome formation in kidney epithelial cells.

  11. Antisense-mediated suppression of human heparanase gene expression inhibits pleural dissemination of human cancer cells.

    PubMed

    Uno, F; Fujiwara, T; Takata, Y; Ohtani, S; Katsuda, K; Takaoka, M; Ohkawa, T; Naomoto, Y; Nakajima, M; Tanaka, N

    2001-11-01

    Heparan sulfate proteoglycans is a major component of the cell surface and extracellular matrix and functions as a barrier against cationic molecules and macromolecules. Heparanase is an endoglucuronidase capable of specifically degrading heparan sulfate, and its activity is associated with the metastatic potential of tumor cells. To inhibit human heparanase expression in human cancer cells, we constructed an adenoviral vector carrying a full-length human heparanase cDNA in an antisense orientation (Ad-AS/hep). Increased heparanase expression in T.Tn human esophageal cancer cells and A549 human lung cancer cells after infection with an adenovirus vector expressing the human heparanase gene (Ad-S/hep) was specifically inhibited by simultaneous infection with Ad-AS/hep in a dose-dependent manner. A modified Boyden chamber assay demonstrated that infection with Ad-AS/hep significantly inhibited in vitro invasion of A549 cells after Ad-S/hep infection. Moreover, intrathoracic administration of Ad-AS/hep reduced the number and size of heparanase-expressing A549 tumors implanted intrathoracically into BALB/c-nu/nu mice. Our results suggest that heparanase contributes to the invasive phenotype of tumor cells, and that antisense-mediated inhibition of heparanase activity may be efficacious in the prevention of pleural dissemination. PMID:11691803

  12. Inhibition of Hepatitis B Virus Gene Expression and Replication by Ribonuclease P

    PubMed Central

    Xia, Chuan; Chen, Yuan-Chuan; Gong, Hao; Zeng, Wenbo; Vu, Gia-Phong; Trang, Phong; Lu, Sangwei; Wu, Jianguo; Liu, Fenyong

    2013-01-01

    Nucleic acid-based gene interfering approaches, such as those mediated by RNA interference and RNase P-associated external guide sequence (EGS), have emerged as promising antiviral strategies. The RNase P-based technology is unique, because a custom-designed EGS can bind to any complementary mRNA sequence and recruit intracellular RNase P for specific degradation of the target mRNA. In this study, a functional EGS was constructed to target hepatitis B virus (HBV) essential transcripts. Furthermore, an attenuated Salmonella strain was constructed and used for delivery of anti-HBV EGS in cells and in mice. Substantial reduction in the levels of HBV gene expression and viral DNA was detected in cells treated with the Salmonella vector carrying the functional EGS construct. Furthermore, oral inoculation of Salmonella carrying the EGS construct led to an inhibition of ~95% in the levels of HBV gene expression and a reduction of ~200,000-fold in viral DNA level in the livers and sera of the treated mice transfected with a HBV plasmid. Our results suggest that EGSs are effective in inhibiting HBV replication in cultured cells and mammalian livers, and demonstrate the use of Salmonella-mediated delivery of EGS as a promising therapeutic approach for human diseases including HBV infection. PMID:23481322

  13. The RECK gene and biological malignancy--its significance in angiogenesis and inhibition of matrix metalloproteinases.

    PubMed

    Alexius-Lindgren, Michelle; Andersson, Erika; Lindstedt, Ingrid; Engström, Wilhelm

    2014-08-01

    The RECK (reversion-inducing cysteine-rich protein with Kazal motifs) gene is a relatively newly discovered gene with important implications in cancer biology. RECK is normally expressed in all cells of the body and has an important role in the balance between destructive and constructive features of the extracellular matrix (ECM). The RECK protein is a membrane-bound glycoprotein that inhibits matrix metalloproteinases with the function of breaking-down the ECM. There is a significant correlation between RECK gene expression and the formation of new vessels, presumably via the mediation of vascular endothelial growth factor (VEGF), which is an important and powerful inducer of angiogenesis. Research has shown that down-regulation of RECK is caused by the rat sarcoma oncogene (RAS), which is also a common cause of tumor development in the early stages. For a tumor to progress and gain characteristics that classifies it as malignant, the degradation of the ECM and mobilization of new blood vessels are essential functions. If the tumor is inhibited with respect to these functions, it will cease to grow. RECK is, therefore, a potential tumor inhibitor but also a prognostic marker available at early clinical stages. PMID:25075007

  14. Regulation of neural gene transcription by optogenetic inhibition of the RE1-silencing transcription factor

    PubMed Central

    Paonessa, Francesco; Criscuolo, Stefania; Sacchetti, Silvio; Amoroso, Davide; Scarongella, Helena; Pecoraro Bisogni, Federico; Carminati, Emanuele; Pruzzo, Giacomo; Maragliano, Luca; Cesca, Fabrizia; Benfenati, Fabio

    2016-01-01

    Optogenetics provides new ways to activate gene transcription; however, no attempts have been made as yet to modulate mammalian transcription factors. We report the light-mediated regulation of the repressor element 1 (RE1)-silencing transcription factor (REST), a master regulator of neural genes. To tune REST activity, we selected two protein domains that impair REST-DNA binding or recruitment of the cofactor mSin3a. Computational modeling guided the fusion of the inhibitory domains to the light-sensitive Avena sativa light–oxygen–voltage-sensing (LOV) 2-phototrophin 1 (AsLOV2). By expressing AsLOV2 chimeras in Neuro2a cells, we achieved light-dependent modulation of REST target genes that was associated with an improved neural differentiation. In primary neurons, light-mediated REST inhibition increased Na+-channel 1.2 and brain-derived neurotrophic factor transcription and boosted Na+ currents and neuronal firing. This optogenetic approach allows the coordinated expression of a cluster of genes impinging on neuronal activity, providing a tool for studying neuronal physiology and correcting gene expression changes taking place in brain diseases. PMID:26699507

  15. Regulation of neural gene transcription by optogenetic inhibition of the RE1-silencing transcription factor.

    PubMed

    Paonessa, Francesco; Criscuolo, Stefania; Sacchetti, Silvio; Amoroso, Davide; Scarongella, Helena; Pecoraro Bisogni, Federico; Carminati, Emanuele; Pruzzo, Giacomo; Maragliano, Luca; Cesca, Fabrizia; Benfenati, Fabio

    2016-01-01

    Optogenetics provides new ways to activate gene transcription; however, no attempts have been made as yet to modulate mammalian transcription factors. We report the light-mediated regulation of the repressor element 1 (RE1)-silencing transcription factor (REST), a master regulator of neural genes. To tune REST activity, we selected two protein domains that impair REST-DNA binding or recruitment of the cofactor mSin3a. Computational modeling guided the fusion of the inhibitory domains to the light-sensitive Avena sativa light-oxygen-voltage-sensing (LOV) 2-phototrophin 1 (AsLOV2). By expressing AsLOV2 chimeras in Neuro2a cells, we achieved light-dependent modulation of REST target genes that was associated with an improved neural differentiation. In primary neurons, light-mediated REST inhibition increased Na(+)-channel 1.2 and brain-derived neurotrophic factor transcription and boosted Na(+) currents and neuronal firing. This optogenetic approach allows the coordinated expression of a cluster of genes impinging on neuronal activity, providing a tool for studying neuronal physiology and correcting gene expression changes taking place in brain diseases. PMID:26699507

  16. Cyclic strain inhibits acute pro-inflammatory gene expression in aortic valve interstitial cells.

    PubMed

    Smith, Kathryn E; Metzler, Scott A; Warnock, James N

    2010-02-01

    Mechanical in vitro preconditioning of tissue engineered heart valves is viewed as an essential process for tissue development prior to in vivo implantation. However, a number of pro-inflammatory genes are mechanosensitive and their elaboration could elicit an adverse response in the host. We hypothesized that the application of normal physiological levels of strain to isolated valve interstitial cells would inhibit the expression of pro-inflammatory genes. Cells were subjected to 0, 5, 10, 15 and 20% strain. Expression of VCAM-1, MCP-1, GM-CSF and OPN was then measured using qRT-PCR. With the exception of OPN, all genes were significantly up regulated when no strain was applied. MCP-1 expression was significantly lower in the presence of strain, although strain magnitude did not affect the expression level. VCAM-1 and GM-CSF had the lowest expression levels at 15% strain, which represent normal physiological conditions. These findings were confirmed using confocal microscopy. Additionally, pSMAD 2/3 and IkappaBalpha expression were imaged to elucidate potential mechanisms of gene expression. Data showed that 15% strain increased pSMAD 2/3 expression and prevented phosphorylation of IkappaBalpha. In conclusion, cyclic strain reduces expression of pro-inflammatory genes, which may be beneficial for the in vitro pre-conditioning of tissue engineered heart valves. PMID:19636599

  17. Prediction on the Inhibition Ratio of Pyrrolidine Derivatives on Matrix Metalloproteinase Based on Gene Expression Programming

    PubMed Central

    Li, Yuqin; You, Guirong; Jia, Baoxiu; Si, Hongzong; Yao, Xiaojun

    2014-01-01

    Quantitative structure-activity relationships (QSAR) were developed to predict the inhibition ratio of pyrrolidine derivatives on matrix metalloproteinase via heuristic method (HM) and gene expression programming (GEP). The descriptors of 33 pyrrolidine derivatives were calculated by the software CODESSA, which can calculate quantum chemical, topological, geometrical, constitutional, and electrostatic descriptors. HM was also used for the preselection of 5 appropriate molecular descriptors. Linear and nonlinear QSAR models were developed based on the HM and GEP separately and two prediction models lead to a good correlation coefficient (R2) of 0.93 and 0.94. The two QSAR models are useful in predicting the inhibition ratio of pyrrolidine derivatives on matrix metalloproteinase during the discovery of new anticancer drugs and providing theory information for studying the new drugs. PMID:24971318

  18. Inhibition of TC-1 tumor progression by cotransfection of Saxatilin and IL-12 genes mediated by lipofection or electroporation.

    PubMed

    Park, Y S; Kim, K S; Lee, Y K; Kim, J S; Baek, J Y; Huang, L

    2009-01-01

    Recently, a number of reports have demonstrated that coexpression of therapeutic genes having different anticancer mechanisms is a more effective strategy for anticancer gene therapy than single gene expression. Saxatilin, a novel disintegrin from snake venom, has recently been shown to have potent antiangiogenic functions, such as inhibition of platelet aggregation, bFGF-induced proliferation of HUVEC, and vitronectin-induced smooth muscle cell migration. IL-12 is a well-known immune modulator that promotes Thl-type antitumor immune responses and inhibits angiogenesis as well. The saxatilin and/or IL-12 genes were transfected intratumorally into C57BL/6 mice carrying TC-1 transformed mouse lung endothelial cells by either lipofection or electroporation. The plasmids encoding saxatilin and IL-12 were administered to tumor tissues via novel cationic liposomes consisting of dimyristyl-glutamyl-lysine (DMKE). On the other hand, expression of the genes was also induced by electroporation after naked pDNA injection to the tumor tissues. Lipofection of saxatilin and/or IL-12 genes appeared to be slightly more effective in inhibition of tumor growth than electroporation of the same genes. Cotransfection of saxatilin and IL-12 genes was clearly more effective than individual administration of either gene. This result implies that cotransfection of saxatilin and IL-12 genes represents an innovative modality for anticancer gene therapy. PMID:20225758

  19. In vitro expression of Escherichia coli ribosomal protein genes: autogenous inhibition of translation.

    PubMed Central

    Yates, J L; Arfsten, A E; Nomura, M

    1980-01-01

    Escherichia coli ribosomal protein L1 (0.5 micro M) was found to inhibit the synthesis of both proteins of the L11 operon, L11 and L1, but not the synthesis of other proteins directed by lambda rifd 18 DNA. Similarly, S4 (1 micro M) selectively inhibited the synthesis of three proteins of the alpha operon, S13, S11, and S4, directed by lambda spcI DNA or a restriction enzyme fragment obtained from this DNA. S8 (3.6 micro M) also showed preferential inhibitory effects on the synthesis of some proteins encoded in the spc operon, L24 and L5 (and probably S14 and S8), directed by lambda spcl DNA or a restriction enzyme fragment carrying the genes for these proteins. The inhibitory effect of L1 was observed only with L1 and not with other proteins examined, including S4 and S8. Similarly, the effect of S4 was not observed with L1 or S8, and that of S8 was not seen with L1 or S4. Inhibition was shown to take place at the level of translation rather than transcription. Thus, at least some ribosomal proteins (L1 S4, and S8) have the ability to cause selective translational inhibition of the synthesis of certain ribosomal proteins whose genes are in the same operon as their own. These results support the hypothesis that certain free ribosomal proteins not assembled into ribosomes act as "autogenous" feedback inhibitors to regulate the synthesis of ribosomal proteins. Images PMID:6445562

  20. Amino acids inhibit Agrp gene expression via an mTOR-dependent mechanism

    PubMed Central

    Morrison, Christopher D.; Xi, Xiaochun; White, Christy L.; Ye, Jianping; Martin, Roy J.

    2008-01-01

    Metabolic fuels act on hypothalamic neurons to regulate feeding behavior and energy homeostasis, but the signaling mechanisms mediating these effects are not fully clear. Rats placed on a low-protein diet (10% of calories) exhibited increased food intake (P < 0.05) and hypothalamic Agouti-related protein (Agrp) gene expression (P = 0.002). Direct intracerebroventricular injection of either an amino acid mixture (RPMI 1640) or leucine alone (1 μg) suppressed 24-h food intake (P < 0.05), indicating that increasing amino acid concentrations within the brain is sufficient to suppress food intake. To define a cellular mechanism for these direct effects, GT1–7 hypothalamic cells were exposed to low amino acids for 16 h. Decreasing amino acid availability increased Agrp mRNA levels in GT1–7 cells (P < 0.01), and this effect was attenuated by replacement of the amino acid leucine (P < 0.05). Acute exposure to elevated amino acid concentrations increased ribosomal protein S6 kinase phosphorylation via a rapamycin-sensitive mechanism, suggesting that amino acids directly stimulated mammalian target of rapamycin (mTOR) signaling. To test whether mTOR signaling contributes to amino acid inhibition of Agrp gene expression, GT1–7 cells cultured in either low or high amino acids for 16 h and were also treated with rapamcyin (50 nM). Rapamycin treatment increased Agrp mRNA levels in cells exposed to high amino acids (P = 0.01). Taken together, these observations indicate that amino acids can act within the brain to inhibit food intake and that a direct, mTOR-dependent inhibition of Agrp gene expression may contribute to this effect. PMID:17374702

  1. Orf virus inhibits interferon stimulated gene expression and modulates the JAK/STAT signalling pathway.

    PubMed

    Harvey, Ryan; McCaughan, Catherine; Wise, Lyn M; Mercer, Andrew A; Fleming, Stephen B

    2015-10-01

    Interferons (IFNs) play a critical role as a first line of defence against viral infection. Activation of the Janus kinase/signal transducer and activation of transcription (JAK/STAT) pathway by IFNs leads to the production of IFN stimulated genes (ISGs) that block viral replication. The Parapoxvirus, Orf virus (ORFV) induces acute pustular skin lesions of sheep and goats and is transmissible to man. The virus replicates in keratinocytes that are the immune sentinels of skin. We investigated whether or not ORFV could block the expression of ISGs. The human gene GBP1 is stimulated exclusively by type II IFN while MxA is stimulated exclusively in response to type I IFNs. We found that GBP1 and MxA were strongly inhibited in ORFV infected HeLa cells stimulated with IFN-? or IFN-? respectively. Furthermore we showed that ORFV inhibition of ISG expression was not affected by cells pretreated with adenosine N1-oxide (ANO), a molecule that inhibits poxvirus mRNA translation. This suggested that new viral gene synthesis was not required and that a virion structural protein was involved. We next investigated whether ORFV infection affected STAT1 phosphorylation in IFN-? or IFN-? treated HeLa cells. We found that ORFV reduced the levels of phosphorylated STAT1 in a dose-dependent manner and was specific for Tyr701 but not Ser727. Treatment of cells with sodium vanadate suggested that a tyrosine phosphatase was responsible for dephosphorylating STAT1-p. ORFV encodes a factor, ORFV057, with homology to the vaccinia virus structural protein VH1 that impairs the JAK/STAT pathway by dephosphorylating STAT1. Our findings show that ORFV has the capability to block ISG expression and modulate the JAK/STAT signalling pathway. PMID:26113305

  2. Cisplatin inhibits hippocampal cell proliferation and alters the expression of apoptotic genes.

    PubMed

    Manohar, Senthilvelan; Jamesdaniel, Samson; Salvi, Richard

    2014-05-01

    The hippocampus, which is critical for memory and spatial navigation, contains a proliferating stem cell niche that is especially vulnerable to antineoplastic drugs such as cisplatin. Although the damaging effects of cisplatin have recently been recognized, the molecular mechanisms underlying its toxic effects on this vital region are largely unknown. Using a focused apoptosis gene array, we analyzed the early cisplatin-induced changes in gene expression in the hippocampus of adult Sprague-Dawley rats and compared the results to those from the inferior colliculus, a non-mitotic auditory region resistant to cisplatin-induced cell death. Two days after a 12 mg/kg dose of cisplatin, significant increases were observed in five proapoptotic genes: Bik, Bid, Bok, Trp53p2, and Card6 and a significant decrease in one antiapoptotic gene Bcl2a1. In contrast, Nol3, an antiapoptotic gene, showed a significant increase in expression. The cisplatin-induced increase in Bid mRNA and decrease in Bcl2a1 mRNA were accompanied by a corresponding increase and decrease of their respective proteins in the hippocampus. In contrast, the cisplatin-induced changes in Bcl2a1, Bid, Bik, and Bok gene expression in the inferior colliculus were strikingly different from those in the hippocampus consistent with the greater susceptibility of the hippocampus to cisplatin toxicity. Cisplatin also significantly reduced immunolabeling of the cell proliferation marker Ki67 in the subgranular zone of the hippocampus 2 days post-treatment. These results indicate that cisplatin-induced hippocampal cell death is mediated by increased expression of proapoptotic and decreased antiapoptotic genes and proteins that likely inhibit hippocampal cell proliferation. PMID:24277158

  3. Cisplatin Inhibits Hippocampal Cell Proliferation and Alters the Expression of Apoptotic Genes

    PubMed Central

    Manohar, Senthilvelan; Jamesdaniel, Samson; Salvi, Richard

    2014-01-01

    The hippocampus, which is critical for memory and spatial navigation, contains a proliferating stem cell niche that is especially vulnerable to anti-neoplastic drugs such as cisplatin. Although the damaging effects of cisplatin have recently been recognized, the molecular mechanisms underlying its toxic effects on this vital region are largely unknown. Using a focused apoptosis gene array, we analyzed the early cisplatin-induced changes in gene expression in the hippocampus of adult Sprague-Dawley rats and compared the results to those from the inferior colliculus, a non-mitotic auditory region resistant to cisplatin-induced cell death. Two days after a 12 mg/kg dose of cisplatin, significant increases were observed in five proapoptotic genes Bik, Bid, Bok, Trp53p2 and Card6 and a significant decrease in one antiapoptotic gene Bcl2a1. In contrast, Nol3, an antiapoptotic gene showed a significant increase in expression. The cisplatin-induced increase in Bid mRNA and decrease in Bcl2a1 mRNA was accompanied by a corresponding increase and decrease of their respective proteins in the hippocampus. In contrast, the cisplatin-induced changes in Bcl2a1, Bid, Bik and Bok gene expression in the inferior colliculus were strikingly different from those in the hippocampus consistent with the greater susceptibility of the hippocampus to cisplatin toxicity. Cisplatin also significantly reduced immunolabeling of the cell proliferation marker Ki67 in the subgranular zone (SGZ) of the hippocampus two days post treatment. These results indicate that cisplatin-induced hippocampal cell death is mediated by increased expression of proapoptotic and antiapoptotic genes and proteins that likely inhibit hippocampal cell proliferation. PMID:24277158

  4. Dexamethasone inhibits human interleukin 2 but not interleukin 2 receptor gene expression in vitro at the level of nuclear transcription.

    PubMed Central

    Boumpas, D T; Anastassiou, E D; Older, S A; Tsokos, G C; Nelson, D L; Balow, J E

    1991-01-01

    Glucocorticosteroids have an inhibitory effect on the expression of interleukin 2 (IL-2) and interleukin 2 receptor (IL-2R) genes. To determine the mechanisms of this inhibition, human T lymphocytes were stimulated with mitogens in the presence of dexamethasone. Nuclear transcription run-off assays showed that high doses of dexamethasone inhibited the transcription of the IL-2 gene but not that of the IL-2R gene. Post-transcriptionally, high doses of dexamethasone (10(-4) M) were required to inhibit IL-2R mRNA levels by 50%, whereas lower doses (10(-6) M) inhibited by greater than 70% the accumulation of IL-2 mRNA. IL-2 mRNA half-life decreased in the presence of dexamethasone (10(-6) M) by approximately 50%. At the protein product level, dexamethasone inhibited both IL-2 production, as well as cell surface and soluble forms of IL-2R. IL-2R gene expression was inhibited for at least 72 h after exposure of cells to dexamethasone. In the presence of exogenous IL-2, dexamethasone failed to exert a significant effect on the production of IL-2R protein. These data indicate that dexamethasone has a greater effect on the expression of the IL-2 gene than on the IL-2R gene. Dexamethasone both inhibits transcription of the IL-2 gene and decreases the stability of IL-2 mRNA. The effect of dexamethasone on the IL-2R gene is post-transcriptional and may result indirectly from decreased IL-2 production. Images PMID:2022743

  5. Berberine inhibits arylamine N-acetyltransferase activity and gene expression in Salmonella typhi.

    PubMed

    Wu, Lii-Tzu; Tsou, Mei-Fen; Ho, Chin-Chin; Chuang, Jing-Yuan; Kuo, Hsiu-Maan; Chung, Jing-Gung

    2005-10-01

    The effects of berberine on growth, arylamine N-acetyltransferase (NAT) activity, and gene expression in Salmonella Typhi (Typhi) were described. The growth inhibition of Typhi was determined by measuring absorbance by optical density (OD at 650 nm). The NAT activity was determined by measuring the levels of 2-aminofluorene (AF) and N-acetyl-2-aminofluorene (AAF) by high-performance liquid chromatography. The results demonstrated that 24-h berberine treatment decreased bacteria growth and amounts of AAF in Typhi. Western blotting and flow cytometry were used for examining the levels of NAT after bacteria were cotreated with or without various concentrations of berberine, and results indicated that berberine decreased the levels of NAT in Typhi. Polymerase chain reaction was used for examining the gene expression of NAT (mRNA NAT), and results indicated that berberine affects mRNA NAT1 expression in Typhi. PMID:16086103

  6. Transcriptional inhibition of the Catalase gene in phosphine-induced oxidative stress in Drosophila melanogaster.

    PubMed

    Liu, Tao; Li, Li; Zhang, Fanhua; Wang, Yuejin

    2015-10-01

    Phosphine (PH3) is a toxic substance to pest insects and is therefore commonly used in pest control. The oxidative damage induced by PH3 is considered to be one of the primary mechanisms of its toxicity in pest insects; however, the precise mode of PH3 action in this process is still unclear. In this study, we evaluated the responses of several oxidative biomarkers and two of the main antioxidant enzymes, catalase (CAT) and superoxide dismutase (SOD), after fumigation treatment with PH3 in Drosophila melanogaster as a model system. The results showed that larvae exposed to sub-lethal levels of PH3 (0.028 mg/L) exhibited lower aerobic respiration rates and higher levels of hydrogen peroxide (H2O2) and lipid peroxidation (LPO). Furthermore, unlike SOD, the activity and expression of CAT and its encoding gene were downregulated by PH3 in a time- and dose-dependent manner. Finally, the responses of six potential transcription factors of PH3 were determined by real-time polymerase chain reaction to explore the regulation mechanism of DmCAT by PH3. There were no significant effects of PH3 on three nuclear factor-kappa B homologs (DORSAL, DIF, and RELISH) or two activator protein-1 genes (JUN and FOS), while dramatic inhibition of DNA replication-related element factor (DREF) expression was observed after fumigation with PH3, suggesting that PH3 could inhibit the expression of DmCAT via the DRE/DREF system. These results confirmed that PH3 induces oxidative stress and targets CAT by downregulating its encoding gene in Drosophila. Our results provide new insight into the signal transduction mechanism between PH3 and its target genes. PMID:26453223

  7. Systems Mechanobiology: Tension-Inhibited Protein Turnover Is Sufficient to Physically Control Gene Circuits

    PubMed Central

    Dingal, P.C. Dave P.; Discher, Dennis E.

    2014-01-01

    Mechanotransduction pathways convert forces that stress and strain structures within cells into gene expression levels that impact development, homeostasis, and disease. The levels of some key structural proteins in the nucleus, cytoskeleton, or extracellular matrix have been recently reported to scale with tissue- and cell-level forces or mechanical properties such as stiffness, and so the mathematics of mechanotransduction becomes important to understand. Here, we show that if a given structural protein positively regulates its own gene expression, then stresses need only inhibit degradation of that protein to achieve stable, mechanosensitive gene expression. This basic use-it-or-lose-it module is illustrated by application to meshworks of nuclear lamin A, minifilaments of myosin II, and extracellular matrix collagen fibers—all of which possess filamentous coiled-coil/supercoiled structures. Past experiments not only suggest that tension suppresses protein degradation mediated and/or initiated by various enzymes but also that transcript levels vary with protein levels because key transcription factors are regulated by these structural proteins. Coupling between modules occurs within single cells and between cells in tissue, as illustrated during embryonic heart development where cardiac fibroblasts make collagen that cardiomyocytes contract. With few additional assumptions, the basic module has sufficient physics to control key structural genes in both development and disease. PMID:25468352

  8. Use of Walnut Shell Powder to Inhibit Expression of Fe2+-Oxidizing Genes of Acidithiobacillus Ferrooxidans

    PubMed Central

    Li, Yuhui; Liu, Yehao; Tan, Huifang; Zhang, Yifeng; Yue, Mei

    2016-01-01

    Acidithiobacillus ferrooxidans is a Gram-negative bacterium that obtains energy by oxidizing Fe2+ or reduced sulfur compounds. This bacterium contributes to the formation of acid mine drainage (AMD). This study determined whether walnut shell powder inhibits the growth of A. ferrooxidans. First, the effects of walnut shell powder on Fe2+ oxidization and H+ production were evaluated. Second, the chemical constituents of walnut shell were isolated to determine the active ingredient(s). Third, the expression of Fe2+-oxidizing genes and rus operon genes was investigated using real-time polymerase chain reaction. Finally, growth curves were plotted, and a bioleaching experiment was performed to confirm the active ingredient(s) in walnut shells. The results indicated that both walnut shell powder and the phenolic fraction exert high inhibitory effects on Fe2+ oxidation and H+ production by A. ferrooxidans cultured in standard 9K medium. The phenolic components exert their inhibitory effects by down-regulating the expression of Fe2+-oxidizing genes and rus operon genes, which significantly decreased the growth of A. ferrooxidans. This study revealed walnut shell powder to be a promising substance for controlling AMD. PMID:27144574

  9. Use of Walnut Shell Powder to Inhibit Expression of Fe(2+)-Oxidizing Genes of Acidithiobacillus Ferrooxidans.

    PubMed

    Li, Yuhui; Liu, Yehao; Tan, Huifang; Zhang, Yifeng; Yue, Mei

    2016-01-01

    Acidithiobacillus ferrooxidans is a Gram-negative bacterium that obtains energy by oxidizing Fe(2+) or reduced sulfur compounds. This bacterium contributes to the formation of acid mine drainage (AMD). This study determined whether walnut shell powder inhibits the growth of A. ferrooxidans. First, the effects of walnut shell powder on Fe(2+) oxidization and H⁺ production were evaluated. Second, the chemical constituents of walnut shell were isolated to determine the active ingredient(s). Third, the expression of Fe(2+)-oxidizing genes and rus operon genes was investigated using real-time polymerase chain reaction. Finally, growth curves were plotted, and a bioleaching experiment was performed to confirm the active ingredient(s) in walnut shells. The results indicated that both walnut shell powder and the phenolic fraction exert high inhibitory effects on Fe(2+) oxidation and H⁺ production by A. ferrooxidans cultured in standard 9K medium. The phenolic components exert their inhibitory effects by down-regulating the expression of Fe(2+)-oxidizing genes and rus operon genes, which significantly decreased the growth of A. ferrooxidans. This study revealed walnut shell powder to be a promising substance for controlling AMD. PMID:27144574

  10. Butylated Hydroxyanisole Stimulates Heme Oxygenase-1 Gene Expression and Inhibits Neointima Formation in Rat Arteries

    PubMed Central

    Liu, Xiao-ming; Azam, Mohammed A.; Peyton, Kelly J.; Ensenat, Diana; Keswani, Amit N.; Wang, Hong; Durante, William

    2007-01-01

    Objective Butylated hydroxyanisole (BHA) is a synthetic phenolic compound that is a potent inducer of phase II genes. Since heme oxygenase-1 (HO-1) is a vasoprotective protein that is upregulated by phase II inducers, the present study examined the effects of BHA on HO-1 gene expression and vascular smooth muscle cell proliferation. Methods The regulation of HO-1 gene expression and vascular cell growth by BHA was studied in cultured rat aortic smooth muscle cells and in balloon injured rat carotid arteries. Results Treatment of cultured smooth muscle cells with BHA stimulated the expression of HO-1 protein, mRNA and promoter activity in a time- and concentration-dependent manner. BHA-mediated HO-1 expression was dependent on the activation of NF-E2-related factor-2 by p38 mitogen-activated protein kinase. BHA also inhibited cell cycle progression and DNA synthesis in a HO-1-dependent manner. In addition, the local perivascular delivery of BHA immediately after arterial injury of rat carotid arteries induced HO-1 protein expression and markedly attenuated neointima formation. Conclusions These studies demonstrate that BHA stimulates HO-1 gene expression in vascular smooth muscle cells, and that the induction of HO-1 contributes to the antiproliferative actions of this phenolic antioxidant. BHA represents a potentially novel therapeutic agent in treating or preventing vasculoproliferative disease. PMID:17320844

  11. Hypothalamic gene transfer of BDNF inhibits breast cancer progression and metastasis in middle age obese mice.

    PubMed

    Liu, Xianglan; McMurphy, Travis; Xiao, Run; Slater, Andrew; Huang, Wei; Cao, Lei

    2014-07-01

    Activation of the hypothalamus-adipocyte axis is associated with an antiobesity and anticancer phenotype in animal models of melanoma and colon cancer. Brain-derived neurotrophic factor (BDNF) is a key mediator in the hypothalamus leading to preferential sympathoneural activation of adipose tissue and the ensuing resistance to obesity and cancer. Here, we generated middle age obese mice by high fat diet feeding for a year and investigated the effects of hypothalamic gene transfer of BDNF on a hormone receptor-positive mammary tumor model. The recombinant adeno-associated viral vector-mediated overexpression of BDNF led to marked weight loss and decrease of adiposity without change of food intake. BDNF gene therapy improved glucose tolerance, alleviated steatosis, reduced leptin level, inhibited mouse breast cancer EO771 growth, and prevented the metastasis. The reduced tumor growth in BDNF-treated mice was associated with reduced angiogenesis, decreased proliferation, increased apoptosis, and reduced adipocyte recruitment and lipid accumulation. Moreover, BDNF gene therapy reduced inflammation markers in the hypothalamus, the mammary gland, the subcutaneous fat, and the mammary tumor. Our results suggest that manipulating a single gene in the brain may influence multiple mechanisms implicated in obesity-cancer association and provide a target for the prevention and treatment of both obesity and cancer. PMID:24637454

  12. Ammonium Inhibits Chromomethylase 3-Mediated Methylation of the Arabidopsis Nitrate Reductase Gene NIA2

    PubMed Central

    Kim, Joo Yong; Kwon, Ye Jin; Kim, Sung-Il; Kim, Do Youn; Song, Jong Tae; Seo, Hak Soo

    2016-01-01

    Gene methylation is an important mechanism regulating gene expression and genome stability. Our previous work showed that methylation of the nitrate reductase (NR) gene NIA2 was dependent on chromomethylase 3 (CMT3). Here, we show that CMT3-mediated NIA2 methylation is regulated by ammonium in Arabidopsis thaliana. CHG sequences (where H can be A, T, or C) were methylated in NIA2 but not in NIA1, and ammonium [(NH4)2SO4] treatment completely blocked CHG methylation in NIA2. By contrast, ammonium had no effect on CMT3 methylation, indicating that ammonium negatively regulates CMT3-mediated NIA2 methylation without affecting CMT3 methylation. Ammonium upregulated NIA2 mRNA expression, which was consistent with the repression of NIA2 methylation by ammonium. Ammonium treatment also reduced the overall genome methylation level of wild-type Arabidopsis. Moreover, CMT3 bound to specific promoter and intragenic regions of NIA2. These combined results indicate that ammonium inhibits CMT3-mediated methylation of NIA2 and that of other target genes, and CMT3 selectively binds to target DNA sequences for methylation. PMID:26834755

  13. Apigenin inhibits hepatoma cell growth through alteration of gene expression patterns.

    PubMed

    Cai, Jing; Zhao, Xiang-Li; Liu, An-Wen; Nian, Hua; Zhang, Shu-Hui

    2011-03-15

    Apigenin, a common plant flavonoid, has been shown to possess anti-tumor properties; however, the underlying molecular mechanisms are still not completely understood. In the present study, we investigated the effects of apigenin on human hepatoma Huh7 cell proliferation, cell cycle distribution, apoptosis, and colony formation in vitro, as well as on the tumorigenicity of Huh7 cells in vivo. To get more insight into the mechanism of apigenin action, we performed genome-wide expression profiling of apigenin-treated Huh7 cells using cDNA microarrays (Agilent Whole Human Genome Oligo Microarray) that contain 41,000 genes. Ten of the most differentially expressed genes (≧5-fold changes) were selected for further evaluation by quantitative RT-PCR (qPCR) and Western blot analyses. Notably, apigenin (5-20 μg/ml) remarkably inhibited Huh7 cell proliferation and colony formation as compared to the vehicle control, which was in a dose-dependent manner. Accompanying with the decreased growth, apigenin-treated cells showed a cell cycle arrest at G2/M phase and an increased rate of apoptosis. Moreover, the xenografts derived from Huh7 cells were significantly (p<0.05) retarded by the delivery of apigenin (50 μg/mouse/day) relative to the control counterparts. Gene expression profile analysis revealed that 1336 genes were up-regulated and 428 genes were down-regulated by apigenin. The down-regulation of interleukin-4 receptor and ubiquitin specific protease 18 and the up-regulation of SLC27A3 and chemokine (C-C motif) receptor 2 were further confirmed by the qPCR and Western blot results. In conclusion, apigenin exhibits inhibitory effects on hepatoma cell growth, which is likely mediated through alteration of gene expression profiles. PMID:20850954

  14. Silencing cathepsin S gene expression inhibits growth, invasion and angiogenesis of human hepatocellular carcinoma in vitro

    SciTech Connect

    Fan, Qi; Wang, Xuedi; Zhang, Hanguang; Li, Chuanwei; Fan, Junhua; Xu, Jing

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer Cat S is highly expressed in HCC cells with high metastatic potential. Black-Right-Pointing-Pointer Knockdown of Cat S inhibits growth and invasion of HCC cells. Black-Right-Pointing-Pointer Knockdown of Cat S inhibits HCC-associated angiogenesis. Black-Right-Pointing-Pointer Cat S might be a potential target for HCC therapy. -- Abstract: Cathepsin S (Cat S) plays an important role in tumor invasion and metastasis by its ability to degrade extracellular matrix (ECM). Our previous study suggested there could be a potential association between Cat S and hepatocellular carcinoma (HCC) metastasis. The present study was designed to determine the role of Cat S in HCC cell growth, invasion and angiogenesis, using RNA interference technology. Small interfering RNA (siRNA) sequences for the Cat S gene were synthesized and transfected into human HCC cell line MHCC97-H. The Cat S gene targeted siRNA-mediated knockdown of Cat S expression, leading to potent suppression of MHCC97-H cell proliferation, invasion and angiogenesis. These data suggest that Cat S might be a potential target for HCC therapy.

  15. Effects of redox modulation by inhibition of thioredoxin reductase on radiosensitivity and gene expression

    PubMed Central

    Selenius, Markus; Hedman, Mattias; Brodin, David; Gandin, Valentina; Rigobello, Maria Pia; Flygare, Jenny; Marzano, Christine; Bindoli, Alberto; Brodin, Ola; Bjrnstedt, Mikael; Fernandes, Aristi P

    2012-01-01

    Abstract The thioredoxin system is a promising target when aiming to overcome the problem of clinical radiation resistance. Altered cellular redox status and redox sensitive thiols contributing to induction of resistance strongly connect the ubiquitous redox enzyme thioredoxin reductase (TrxR) to the cellular response to ionizing radiation. To further investigate possible strategies in combating clinical radiation resistance, human radio-resistant lung cancer cells were subjected to a combination of single fractions of ?-radiation at clinically relevant doses and non-toxic levels of a well-characterized thioredoxin reductase inhibitor, the phosphine gold(I) compound [Au(SCN)(PEt3)]. The combination of the TrxR-inhibitor and ionizing radiation reduced the surviving fractions and impaired the ability of the U1810 cells to repopulate by approximately 50%. In addition, inhibition of thioredoxin reductase caused changes in the cell cycle distribution, suggesting a disturbance of the mitotic process. Global gene expression analysis also revealed clustered genetic expression changes connected to several major cellular pathways such as cell cycle, cellular response to stress and DNA damage. Specific TrxR-inhibition as a factor behind the achieved results was confirmed by correlation of gene expression patterns between gold and siRNA treatment. These results clearly demonstrate TrxR as an important factor conferring resistance to irradiation and the use of [Au(SCN)(PEt3)] as a promising radiosensitizing agent. PMID:22003958

  16. Auxin inhibits stomatal development through MONOPTEROS repression of a mobile peptide gene STOMAGEN in mesophyll

    PubMed Central

    Zhang, Jing-Yi; He, Sheng-Bo; Li, Ling; Yang, Hong-Quan

    2014-01-01

    Plants, as sessile organisms, must coordinate various physiological processes to adapt to ever-changing surrounding environments. Stomata, the epidermal pores facilitating gas and water exchange, play important roles in optimizing photosynthetic efficiency and adaptability. Stomatal development is under the control of an intrinsic program mediated by a secretory peptide gene family—namely, EPIDERMAL PATTERNING FACTOR, including positively acting STOMAGEN/EPFL9. The phytohormone brassinosteroids and environment factor light also control stomatal production. However, whether auxin regulates stomatal development and whether peptide signaling is coordinated with auxin signaling in the regulation of stomatal development remain largely unknown. Here we show that auxin negatively regulates stomatal development through MONOPTEROS (also known as ARF5) repression of the mobile peptide gene STOMAGEN in mesophyll. Through physiological, genetic, transgenic, biochemical, and molecular analyses, we demonstrate that auxin inhibits stomatal development through the nuclear receptor TIR1/AFB-mediated signaling, and that MONOPTEROS directly binds to the STOMAGEN promoter to suppress its expression in mesophyll and inhibit stomatal development. Our results provide a paradigm of cross-talk between phytohormone auxin and peptide signaling in the regulation of stomatal production. PMID:25002510

  17. β-D-glucan inhibits endocrine-resistant breast cancer cell proliferation and alters gene expression

    PubMed Central

    JAFAAR, ZAINAB M.T.; LITCHFIELD, LACEY M.; IVANOVA, MARGARITA M.; RADDE, BRANDIE N.; AL-RAYYAN, NUMAN; KLINGE, CAROLYN M.

    2014-01-01

    Endocrine therapies have been successfully used for breast cancer patients with estrogen receptor α (ERα) positive tumors, but ∼40% of patients relapse due to endocrine resistance. β-glucans are components of plant cell walls that have immunomodulatory and anticancer activity. The objective of this study was to examine the activity of β-D-glucan, purified from barley, in endocrine-sensitive MCF-7 versus endocrine-resistant LCC9 and LY2 breast cancer cells. β-D-glucan dissolved in DMSO but not water inhibited MCF-7 cell proliferation in a concentration-dependent manner as measured by BrdU incorporation with an IC50 of ∼164±12 μg/ml. β-D-glucan dissolved in DMSO inhibited tamoxifen/endocrine-resistant LCC9 and LY2 cell proliferation with IC50 values of 4.6±0.3 and 24.2±1.4 μg/ml, respectively. MCF-10A normal breast epithelial cells showed a higher IC50 ∼464 μg/ml and the proliferation of MDA-MB-231 triple negative breast cancer cells was not inhibited by β-D-glucan. Concentration-dependent increases in the BAX/BCL2 ratio and cell death with β-D-glucan were observed in MCF-7 and LCC9 cells. PCR array analysis revealed changes in gene expression in response to 24-h treatment with 10 or 50 μg/ml β-D-glucan that were different between MCF-7 and LCC9 cells as well as differences in basal gene expression between the two cell lines. Select results were confirmed by quantitative real-time PCR demonstrating that β-D-glucan increased RASSF1 expression in MCF-7 cells and IGFBP3, CTNNB1 and ERβ transcript expression in LCC9 cells. Our data indicate that β-D-glucan regulates breast cancer-relevant gene expression and may be useful for inhibiting endocrine-resistant breast cancer cell proliferation. PMID:24534923

  18. Borna disease virus P protein inhibits nitric oxide synthase gene expression in astrocytes

    SciTech Connect

    Peng Guiqing; Zhang Fengmin; Zhang Qi; Wu Kailang; Zhu Fan; Wu Jianguo

    2007-09-30

    Borna disease virus (BDV) is one of the potential infectious agents involved in the development of central nervous system (CNS) diseases. Neurons and astrocytes are the main targets of BDV infection, but little is known about the roles of BDV infection in the biological effects of astrocytes. Here we reported that BDV inhibits the activation of inducible nitric oxide synthase (iNOS) in murine astrocytes induced by bacterial LPS and PMA. To determine which protein of BDV is responsible for the regulation of iNOS expression, we co-transfected murine astrocytes with reporter plasmid iNOS-luciferase and plasmid expressing individual BDV proteins. Results from analyses of reporter activities revealed that only the phosphoprotein (P) of BDV had an inhibitory effect on the activation of iNOS. In addition, P protein inhibits nitric oxide production through regulating iNOS expression. We also reported that the nuclear factor kappa B (NF-{kappa}B) binding element, AP-1 recognition site, and interferon-stimulated response element (ISRE) on the iNOS promoter were involved in the repression of iNOS gene expression regulated by the P protein. Functional analysis indicated that sequences from amino acids 134 to 174 of the P protein are necessary for the regulation of iNOS. These data suggested that BDV may suppress signal transduction pathways, which resulted in the inhibition of iNOS activation in astrocytes.

  19. WIPI1 Coordinates Melanogenic Gene Transcription and Melanosome Formation via TORC1 Inhibition*

    PubMed Central

    Ho, Hsiang; Kapadia, Rubina; Al-Tahan, Sejad; Ahmad, Safoora; Ganesan, Anand K.

    2011-01-01

    Recent studies implicate a role for WD repeat domain, phosphoinositide-interacting 1 (WIPI1) in the biogenesis of melanosomes, cell type-specific lysosome-related organelles. In this study, we determined that WIPI1, an ATG18 homologue that is shown to localize to both autophagosomes and early endosomes, inhibited mammalian target of rapamycin (MTOR) signaling, leading to increased transcription of melanogenic enzymes and the formation of mature melanosomes. WIPI1 suppressed the target of rapamycin complex 1 (TORC1) activity, resulting in glycogen synthase kinase 3β inhibition, β-Catenin stabilization, and increased transcription of microphthalmia transcription factor and its target genes. WIPI1-depleted cells accumulated stage I melanosomes but lacked stage III-IV melanosomes. Inhibition of TORC1 by rapamycin treatment resulted in the accumulation of stage IV melanosomes but not autophagosomes, whereas starvation resulted in the formation of autophagosomes but not melanin accumulation. Taken together, our studies define a distinct role for WIPI1 and TORC1 signaling in controlling the transcription of melanogenic enzymes and melanosome maturation, a process that is distinct from starvation-induced autophagy. PMID:21317285

  20. Lactobacilli Reduce Helicobacter pylori Attachment to Host Gastric Epithelial Cells by Inhibiting Adhesion Gene Expression.

    PubMed

    de Klerk, Nele; Maudsdotter, Lisa; Gebreegziabher, Hanna; Saroj, Sunil D; Eriksson, Beatrice; Eriksson, Olaspers Sara; Roos, Stefan; Lindén, Sara; Sjölinder, Hong; Jonsson, Ann-Beth

    2016-05-01

    The human gastrointestinal tract, including the harsh environment of the stomach, harbors a large variety of bacteria, of which Lactobacillus species are prominent members. The molecular mechanisms by which species of lactobacilli interfere with pathogen colonization are not fully characterized. In this study, we aimed to study the effect of lactobacillus strains upon the initial attachment of Helicobacter pylori to host cells. Here we report a novel mechanism by which lactobacilli inhibit adherence of the gastric pathogen H. pylori In a screen with Lactobacillus isolates, we found that only a few could reduce adherence of H. pylori to gastric epithelial cells. Decreased attachment was not due to competition for space or to lactobacillus-mediated killing of the pathogen. Instead, we show that lactobacilli act on H. pylori directly by an effector molecule that is released into the medium. This effector molecule acts on H. pylori by inhibiting expression of the adhesin-encoding gene sabA Finally, we verified that inhibitory lactobacilli reduced H. pylori colonization in an in vivo model. In conclusion, certain Lactobacillus strains affect pathogen adherence by inhibiting sabA expression and thereby reducing H. pylori binding capacity. PMID:26930708

  1. Selective Dysregulation of Hippocampal Inhibition in the Mouse Lacking Autism Candidate Gene CNTNAP2.

    PubMed

    Jurgensen, Sofia; Castillo, Pablo E

    2015-10-28

    Mutations in the human gene encoding contactin-associated protein-like 2 (CNTNAP2) have been strongly associated with autism spectrum disorders (ASDs). Cntnap2(-/-) mice recapitulate major features of ASD, including social impairment, reduced vocalizations, and repetitive behavior. In addition, Cntnap2(-/-) mice show reduced cortical neuronal synchrony and develop spontaneous seizures throughout adulthood. As suggested for other forms of ASDs, this phenotype could reflect some form of synaptic dysregulation. However, the impact of lifelong deletion of CNTNAP2 on synaptic function in the brain remains unknown. To address this issue, we have assessed excitatory and inhibitory synaptic transmission in acute hippocampal slices of Cntnap2(-/-) mice. We found that although excitatory transmission was mostly normal, inhibition onto CA1 pyramidal cells was altered in Cntnap2(-/-) mice. Specifically, putative perisomatic, but not dendritic, evoked IPSCs were significantly reduced in these mice. Whereas both inhibitory short-term plasticity and miniature IPSC frequency and amplitude were normal in Cntnap2(-/-) mice, we found an unexpected increase in the frequency of spontaneous, action potential-driven IPSCs. Altered hippocampal inhibition could account for the behavioral phenotype Cntnap2(-/-) mice present later in life. Overall, our findings that Cntnap2 deletion selectively impairs perisomatic hippocampal inhibition while sparing excitation provide additional support for synaptic dysfunction as a common mechanism underlying ASDs. PMID:26511255

  2. Fibroblast growth factor 7 inhibits cholesterol 7{alpha}-hydroxylase gene expression in hepatocytes

    SciTech Connect

    Sun, Zhichao; Yu, Xuemei; Wu, Weibin; Jia, Dongwei; Chen, Yinle; Ji, Lingling; Liu, Xijun; Peng, Xiaomin; Li, Yintao; Yang, Lili; Ruan, Yuanyuan; Gu, Jianxin; Ren, Shifang; Zhang, Songwen

    2012-07-13

    Highlights: Black-Right-Pointing-Pointer FGF7 strongly and rapidly down-regulates the expression of CYP7A1 in hepatocytes. Black-Right-Pointing-Pointer FGF7 suppresses the expression of CYP7A1 via FGFR2 and downstream JNK activation. Black-Right-Pointing-Pointer Blocking FGF7 abrogates HSC-induced inhibition of CYP7A1 expression in hepatocytes. -- Abstract: Cholesterol 7{alpha}-hydroxylase (CYP7A1) is the initial and rate-limiting enzyme for bile acid synthesis. Transcription of the CYP7A1 gene is regulated by bile acids, nuclear receptors and cytokines. Fibroblast growth factor 7 (FGF7) secreted from activated hepatic stellate cells (HSC) during chronic liver fibrosis regulates hepatocyte survival and liver regeneration. In the carbon tetrachloride (CCl{sub 4})-induced fibrotic mouse liver, we demonstrated that the expression of CYP7A1 was largely decreased while the expression of FGF7 was significantly increased. We further demonstrated that FGF7 inhibited CYP7A1 gene expression in hepatocytes. Knockdown study by short interfering RNA, kinase inhibition and phosphorylation assays revealed that the suppression of CYP7A1 expression by FGF7 was mediated by FGFR2 and its downstream JNK signaling cascade. The FGF7 neutralizing antibody restored CYP7A1 expression in Hep3B cells treated with conditioned medium from HSC. In summary, the data suggest that FGF7 is a novel regulator of CYP7A1 expression in hepatocytes and may prevent hepatocytes from accumulating toxic bile acids during liver injury and fibrosis.

  3. Prostacyclin synthase gene transfer modulates cyclooxygenase-2-derived prostanoid synthesis and inhibits neointimal formation in rat balloon-injured arteries.

    PubMed

    Yamada, Michiharu; Numaguchi, Yasushi; Okumura, Kenji; Harada, Mitsunori; Naruse, Keiji; Matsui, Hideo; Ito, Takayuki; Hayakawa, Tetsuo

    2002-02-01

    Previous studies have shown that prostacyclin (PGI(2)) synthase (PCS) gene transfer inhibits neointimal formation in balloon-injured arteries. However, the role of each cyclooxygenase (COX) isoform in this healing mechanism remains unknown. We hypothesized that overexpression of PCS may modulate COX-2-mediated prostaglandin (PG) metabolism. That is to say, excessive PGH(2) derived from COX-2 after balloon injury may be converted into PGI(2) rather than PGE(2) or thromboxane (TX) A(2) by overexpressed PCS. We examined the expression of COX isoforms and evaluated the role of COX-2 with regard to the effects of PCS gene transfer by using 4-(4-cyclohexyl-2-methyloxazol-5-yl)-2-fluorobenzenesulfonamide (JTE-522), a selective COX-2 inhibitor. Rats were divided into 4 groups in conjunction with PCS gene transfer and JTE-522 treatment. The PCS gene (30 microg) was transfected into rat balloon-injured arteries by a lipotransfection method. JTE-522 (30 mg/kg per day) was administered for 14 days after balloon injury. Immunohistochemical analysis demonstrated marked COX-2 expression on the neointima. PCS gene transfer markedly inhibited neointimal formation, but JTE-522 reversed this beneficial effect. PCS gene transfer augmented PGI(2) production and decreased PGE(2) production without affecting TXA(2) production, but JTE-522 inhibited this increase in PGI(2) production. In conclusion, PCS gene transfer modulated COX-2-mediated prostanoid synthesis and inhibited neointimal formation after balloon injury. PMID:11834525

  4. Flavonoids inhibit cytokine-induced endothelial cell adhesion protein gene expression.

    PubMed Central

    Gerritsen, M. E.; Carley, W. W.; Ranges, G. E.; Shen, C. P.; Phan, S. A.; Ligon, G. F.; Perry, C. A.

    1995-01-01

    Treatment of human endothelial cells with cytokines such as interleukin-1, tumor necrosis factor-alpha (TNF-alpha) or interferon-gamma induces the expression of specific leukocyte adhesion molecules on the endothelial cell surface. Interfering with either leukocyte adhesion or adhesion protein upregulation is an important therapeutic target as evidenced by the potent anti-inflammatory actions of neutralizing antibodies to these ligands in various animal models and in patients. In the present study we report that cotreatment of human endothelial cells with certain hydroxyflavones and flavanols blocks cytokine-induced ICAM-1, VCAM-1, and E-selectin expression on human endothelial cells. One of the most potent flavones, apigenin, exhibited a dose- and time-dependent, reversible effect on adhesion protein expression as well as inhibiting adhesion protein upregulation at the transcriptional level. Apigenin also inhibited IL-1 alpha-induced prostaglandin synthesis and TNF-alpha-induced IL-6 and IL-8 production, suggesting that the hydroxyflavones may act as general inhibitors of cytokine-induced gene expression. Although apigenin did not inhibit TNF-alpha-induced nuclear translocation of NF-kappa B(p50(NFKB1)/p65(RelA)) we found this flavonoid did inhibit TNF-alpha induced beta-galactosidase activity in SW480 cells stably transfected with a beta-galactosidase reporter construct driven by four NF-kappa B elements, suggesting an action on NF-kappa B transcriptional activation. Adhesion of leukocytes to cytokine-treated endothelial cells was blocked in endothelial cells cotreated with apigenin. Finally, apigenin demonstrated potent anti-inflammatory activity in carrageenan induced rat paw edema and delayed type hypersensitivity in the mouse. We conclude that flavonoids offer important therapeutic potential for the treatment of a variety of inflammatory diseases involving an increase in leukocyte adhesion and trafficking. Images Figure 7 Figure 8 Figure 11 PMID:7543732

  5. IL-10 inhibits while calcitriol reestablishes placental antimicrobial peptides gene expression.

    PubMed

    Olmos-Ortiz, Andrea; Noyola-Martínez, Nancy; Barrera, David; Zaga-Clavellina, Verónica; Avila, Euclides; Halhali, Ali; Biruete, Benjamín; Larrea, Fernando; Díaz, Lorenza

    2015-04-01

    IL-10 and calcitriol help to achieve a successful pregnancy by suppressing active maternal immunity; however, these factors exert opposite effects upon microbial infections. In the skin and immune cells, IL-10 downregulates β-defensins while calcitriol induces cathelicidin gene expression in various tissues including placenta. Though, the regulation of human placental β-defensins by IL-10 and calcitriol has not been studied. Therefore, we explored the regulation of these antimicrobial peptides expression in cultured placental cells by calcitriol and IL-10 alone and combined. Real time PCR showed that calcitriol stimulated, while IL-10 inhibited, β-defensins and cathelicidin gene expression (P<0.05). In coincubations studies, calcitriol was able to maintain antimicrobial peptides gene expression above control values, overriding IL-10 inhibitory effects. Calcitriol downregulated endogenous IL-10 secretion. Interestingly, calcitriol and TNF-α cooperatively enhanced β-defensins, while TNF-α reduced basal and calcitriol-stimulated cathelicidin gene expression. In summary, calcitriol and IL-10 exerted opposite effects on antimicrobial peptides expression in the human placenta, suggesting that unbalanced production of IL-10 and calcitriol could be deleterious to innate immune responses during gestation. Our results suggest that calcitriol enhancement of placental defenses involves two mechanisms: (1) downregulation of IL-10 secretion and (2) direct upregulation of β-defensins and cathelicidin gene expression. Considering that IL-10 and calcitriol differentially regulate the innate immune response in the placenta, in the case of an infection, calcitriol might restrict IL-10 permissive actions towards microbial invasion while restrains inflammation, allowing for pregnancy to continue in quiescence. These results strongly advice maternal vitamin D sufficiency during pregnancy. PMID:25088189

  6. The tobacco smoke component acrolein induces glucocorticoid resistant gene expression via inhibition of histone deacetylase.

    PubMed

    Randall, Matthew J; Haenen, Guido R M M; Bouwman, Freek G; van der Vliet, Albert; Bast, Aalt

    2016-01-01

    Chronic obstructive pulmonary disease (COPD) is the leading cause of cigarette smoke-related death worldwide. Acrolein, a crucial reactive electrophile found in cigarette smoke mimics many of the toxic effects of cigarette smoke-exposure in the lung. In macrophages, cigarette smoke is known to hinder histone deacetylases (HDACs), glucocorticoid-regulated enzymes that play an important role in the pathogenesis of glucocorticoid resistant inflammation, a common feature of COPD. Thus, we hypothesize that acrolein plays a role in COPD-associated glucocorticoid resistance. To examine the role of acrolein on glucocorticoid resistance, U937 monocytes, differentiated with PMA to macrophage-like cells were treated with acrolein for 0.5h followed by stimulation with hydrocortisone for 8h, or treated simultaneously with LPS and hydrocortisone for 8h without acrolein. GSH and nuclear HDAC activity were measured, or gene expression was analyzed by qPCR. Acrolein-mediated TNFα gene expression was not suppressed by hydrocortisone whereas LPS-induced TNFα expression was suppressed. Acrolein also significantly inhibited nuclear HDAC activity in macrophage-like cells. Incubation of recombinant HDAC2 with acrolein led to the formation of an HDAC2-acrolein adduct identified by mass spectrometry. Therefore, these results suggest that acrolein-induced inflammatory gene expression is resistant to suppression by the endogenous glucocorticoid, hydrocortisone. PMID:26481333

  7. Inhibition of Interleukin-2 Gene Expression by Human Herpesvirus 6B U54 Tegument Protein

    PubMed Central

    Iampietro, Mathieu; Morissette, Guillaume; Gravel, Annie

    2014-01-01

    ABSTRACT Human herpesvirus 6B (HHV-6B) is a ubiquitous pathogen causing lifelong infections in approximately 95% of humans worldwide. To persist within its host, HHV-6B has developed several immune evasion mechanisms, such as latency, during which minimal proteins are expressed, and the ability to disturb innate and adaptive immune responses. The primary cellular targets of HHV-6B are CD4+ T cells. Previous studies by Flamand et al. (L. Flamand, J. Gosselin, I. Stefanescu, D. Ablashi, and J. Menezes, Blood 85:1263–1271, 1995) reported on the capacity of HHV-6A as well as UV-irradiated HHV-6A to inhibit interleukin-2 (IL-2) synthesis in CD4+ lymphocytes, suggesting that viral structural components could be responsible for this effect. In the present study, we identified the HHV-6B U54 tegument protein (U54) as being capable of inhibiting IL-2 expression. U54 binds the calcineurin (CaN) phosphatase enzyme, causing improper dephosphorylation and nuclear translocation of NFAT (nuclear factor of activated T cells) proteins, resulting in suboptimal IL-2 gene transcription. The U54 GISIT motif (amino acids 293 to 297), analogous to the NFAT PXIXIT motif, contributed to the inhibition of NFAT activation. IMPORTANCE Human herpesvirus 6A (HHV-6A) and HHV-6B are associated with an increasing number of pathologies. These viruses have developed strategies to avoid the immune response allowing them to persist in the host. Several studies have illustrated mechanisms by which HHV-6A and HHV-6B are able to disrupt host defenses (reviewed in L. Dagna, J. C. Pritchett, and P. Lusso, Future Virol. 8:273–287, 2013, doi:10.2217/fvl.13.7). Previous work informed us that HHV-6A is able to suppress synthesis of interleukin-2 (IL-2), a key immune growth factor essential for adequate T lymphocyte proliferation and expansion. We obtained evidence that HHV-6B also inhibits IL-2 gene expression and identified the mechanisms by which it does so. Our work led us to the identification of U54, a virion-associated tegument protein, as being responsible for suppression of IL-2. Consequently, we have identified HHV-6B U54 protein as playing a role in immune evasion. These results further contribute to our understanding of HHV-6 interactions with its human host and the efforts deployed to ensure its long-term persistence. PMID:25122797

  8. The Rel/NF-?B pathway and transcription of immediate early genes in T cell activation are inhibited by microgravity.

    PubMed

    Chang, Tammy T; Walther, Isabelle; Li, Chai-Fei; Boonyaratanakornkit, Jim; Galleri, Grazia; Meloni, Maria Antonia; Pippia, Proto; Cogoli, Augusto; Hughes-Fulford, Millie

    2012-12-01

    This study tested the hypothesis that transcription of immediate early genes is inhibited in T cells activated in ?g. Immunosuppression during spaceflight is a major barrier to safe, long-term human space habitation and travel. The goals of these experiments were to prove that ?g was the cause of impaired T cell activation during spaceflight, as well as understand the mechanisms controlling early T cell activation. T cells from four human donors were stimulated with Con A and anti-CD28 on board the ISS. An on-board centrifuge was used to generate a 1g simultaneous control to isolate the effects of ?g from other variables of spaceflight. Microarray expression analysis after 1.5 h of activation demonstrated that ?g- and 1g-activated T cells had distinct patterns of global gene expression and identified 47 genes that were significantly, differentially down-regulated in ?g. Importantly, several key immediate early genes were inhibited in ?g. In particular, transactivation of Rel/NF-?B, CREB, and SRF gene targets were down-regulated. Expression of cREL gene targets were significantly inhibited, and transcription of cREL itself was reduced significantly in ?g and upon anti-CD3/anti-CD28 stimulation in simulated ?g. Analysis of gene connectivity indicated that the TNF pathway is a major early downstream effector pathway inhibited in ?g and may lead to ineffective proinflammatory host defenses against infectious pathogens during spaceflight. Results from these experiments indicate that ?g was the causative factor for impaired T cell activation during spaceflight by inhibiting transactivation of key immediate early genes. PMID:22750545

  9. Antisense oligodeoxynucleotide inhibition as a potent diagnostic tool for gene function in plant biology

    SciTech Connect

    Jansson, Christer; Sun, Chuanxin; Ghebramedhin, Haile; Hoglund, Anna-Stina; Jansson, Christer

    2008-01-15

    Antisense oligodeoxynucleotide (ODN) inhibition emerges as an effective means for probing gene function in plant cells. Employing this method we have established the importance of the SUSIBA2 transcription factor for regulation of starch synthesis in barley endosperm, and arrived at a model for the role of the SUSIBAs in sugar signaling and source-sink commutation during cereal endosperm development. In this addendum we provide additional data demonstrating the suitability of the antisense ODN technology in studies on starch branching enzyme activities in barley leaves. We also comment on the mechanism for ODN uptake in plant cells. Antisense ODNs are short (12-25 nt-long) stretches of single-stranded ODNs that hybridize to the cognate mRNA in a sequence-specific manner, thereby inhibiting gene expression. They are naturally occurring in both prokaryotes and eukaryotes where they partake in gene regulation and defense against viral infection. The mechanisms for antisense ODN inhibition are not fully understood but it is generally considered that the ODN either sterically interferes with translation or promotes transcript degradation by RNase H activation. The earliest indication of the usefulness of antisense ODN technology for the purposes of molecular biology and medical therapy was the demonstration in 1978 that synthetic ODNs complementary to Raos sarcoma virus could inhibit virus replication in tissue cultures of chick embryo fibroblasts. Since then the antisense ODN technology has been widely used in animal sciences and as an important emerging therapeutic approach in clinical medicine. However, antisense ODN inhibition has been an under-exploited strategy for plant tissues, although the prospects for plant cells in suspension cultures to take up single-stranded ODNs was reported over a decade ago. In 2001, two reports from Malho and coworker demonstrated the use of cationic-complexed antisense ODNs to suppress expression of genes encoding pollen-signaling proteins in pollen tubes from the lilly Agapanthus umbellatus. For the uptake of DNA pollen tubes represent a unique system since the growing tip is surrounded by a loose matrix of hemicellulose and pectins, exposing the plasma membrane7 and the first uptake of ODNs by pollen tubes was reported as early as 1994. A breakthrough in the employment of antisense ODN inhibition as a powerful approach in plant biology was recently presented through our work on intact barley leaves. As was illustrated by confocal microscopy and fluorescently labeled ODNs, naked ODNs were taken up through the leaf petiole and efficiently imported into the plant cell and the nucleus. The work portrayed in that study demonstrate the applicability of antisense ODN inhibition in plant biology, e.g. as a rapid antecedent to time-consuming transgenic studies, and that it operates through RNase H degradation. We employed the antisense ODN strategy to demonstrate the importance of the SUSIBA2 transcription factor in regulation of starch synthesis, and to depict a possible mechanism for sugar signaling in plants and how it might confer endosperm-specific gene expression during seed development. We also described the employment of the antisense ODN strategy for studies on in vitro spike cultures of barley. Here we present further evidence as to the value of the antisense ODN approach in plant biology by following the effects on starch branching enzyme (SBE) accumulation in barley leaves after suppression of individual SBE genes. In agreement with transcript analyses of SBE expression in barley leaves, a zymogram assay (Fig. 1) revealed that sucrose treatment of barley leaves increased the number of SBE activity bands as compared to sorbitol treatment. In the presence of antisense SBEI or SBEIIA ODNs, zymograms of sucrose-treated leaves displayed only a subset of these activities with bands in the top portion of the zymogram gel missing or diminished. With antisense SBEIIB ODN, all activity bands in the top portion of the gel as well as the lowest band were absent. Based on these data we provide a tentative annotation for the various SBE activity bands. In animal experiments, naked ODNs are usually not taken up by the cells since both the ODNs and the outside of the plasma membrane carry a net negative charge. Thus the uptake of naked ODNs into barley leaf cells was surprising and called for an explanation. As demonstrated in our subsequent paper, the answer seems to be that the ODNs slip into the cells through sugar translocators as they are activated in the presence of the appropriate sugar (Fig. 2). Whether it is the structural resemblance between the sugar (deoxyribose) backbone of the ODNs and the transported sugars that allows for the ODNs to be transferred, or if other mechanisms are involved, remains to be elucidated.

  10. Exposure to synthetic gray water inhibits amoeba encystation and alters expression of Legionella pneumophila virulence genes.

    PubMed

    Buse, Helen Y; Lu, Jingrang; Ashbolt, Nicholas J

    2015-01-01

    Water conservation efforts have focused on gray water (GW) usage, especially for applications that do not require potable water quality. However, there is a need to better understand environmental pathogens and their free-living amoeba (FLA) hosts within GW, given their growth potential in stored gray water. Using synthetic gray water (sGW) we examined three strains of the water-based pathogen Legionella pneumophila and its FLA hosts Acanthamoeba polyphaga, A. castellanii, and Vermamoeba vermiformis. Exposure to sGW for 72 h resulted in significant inhibition (P < 0.0001) of amoebal encystation versus control-treated cells, with the following percentages of cysts in sGW versus controls: A. polyphaga (0.6 versus 6%), A. castellanii (2 versus 62%), and V. vermiformis (1 versus 92%), suggesting sGW induced maintenance of the actively feeding trophozoite form. During sGW exposure, L. pneumophila culturability decreased as early as 5 h (1.3 to 2.9 log10 CFU, P < 0.001) compared to controls (Δ0 to 0.1 log10 CFU) with flow cytometric analysis revealing immediate changes in membrane permeability. Furthermore, reverse transcription-quantitative PCR was performed on total RNA isolated from L. pneumophila cells at 0 to 48 h after sGW incubation, and genes associated with virulence (gacA, lirR, csrA, pla, and sidF), the type IV secretion system (lvrB and lvrE), and metabolism (ccmF and lolA) were all shown to be differentially expressed. These results suggest that conditions within GW may promote interactions between water-based pathogens and FLA hosts, through amoebal encystment inhibition and alteration of bacterial gene expression, thus warranting further exploration into FLA and L. pneumophila behavior in GW systems. PMID:25381242

  11. Exposure to Synthetic Gray Water Inhibits Amoeba Encystation and Alters Expression of Legionella pneumophila Virulence Genes

    PubMed Central

    Lu, Jingrang; Ashbolt, Nicholas J.

    2014-01-01

    Water conservation efforts have focused on gray water (GW) usage, especially for applications that do not require potable water quality. However, there is a need to better understand environmental pathogens and their free-living amoeba (FLA) hosts within GW, given their growth potential in stored gray water. Using synthetic gray water (sGW) we examined three strains of the water-based pathogen Legionella pneumophila and its FLA hosts Acanthamoeba polyphaga, A. castellanii, and Vermamoeba vermiformis. Exposure to sGW for 72 h resulted in significant inhibition (P < 0.0001) of amoebal encystation versus control-treated cells, with the following percentages of cysts in sGW versus controls: A. polyphaga (0.6 versus 6%), A. castellanii (2 versus 62%), and V. vermiformis (1 versus 92%), suggesting sGW induced maintenance of the actively feeding trophozoite form. During sGW exposure, L. pneumophila culturability decreased as early as 5 h (1.3 to 2.9 log10 CFU, P < 0.001) compared to controls (Δ0 to 0.1 log10 CFU) with flow cytometric analysis revealing immediate changes in membrane permeability. Furthermore, reverse transcription-quantitative PCR was performed on total RNA isolated from L. pneumophila cells at 0 to 48 h after sGW incubation, and genes associated with virulence (gacA, lirR, csrA, pla, and sidF), the type IV secretion system (lvrB and lvrE), and metabolism (ccmF and lolA) were all shown to be differentially expressed. These results suggest that conditions within GW may promote interactions between water-based pathogens and FLA hosts, through amoebal encystment inhibition and alteration of bacterial gene expression, thus warranting further exploration into FLA and L. pneumophila behavior in GW systems. PMID:25381242

  12. siRNA targeting YAP gene inhibits gastric carcinoma growth and tumor metastasis in SCID mice

    PubMed Central

    ZHOU, ZHOU; ZHU, JIN-SHUI; GAO, CAI-PING; LI, LIANG-PING; ZHOU, CHAO; WANG, HAN; LIU, XIAO-GANG

    2016-01-01

    Yes-associated protein (YAP) is constitutively activated in numerous types of cancer, including gastric carcinoma. The aim of the present study was to investigate the effects of YAP silencing on proliferation, apoptosis, metastasis and angiogenesis in a gastric orthotopic implantation cancer model of severe combined immunodeficiency mice. Small-hairpin RNA (shRNA) targeting the YAP gene was employed to inhibit YAP expression. SGC7901 cells transfected with YAP shRNA demonstrated significantly decreased gastric cancer growth and metastasis in the orthotopic implantation mouse model. Silencing of YAP additionally promoted tumor cell apoptosis, and inhibited tumor cell proliferation and angiogenesis. Notably, YAP shRNA also downregulated the expression of TEA domain family member 1, cyclinD1, vascular endothelial growth factor and fibroblast growth factor-2. The results of the present study suggested that YAP may have a significant role in the proliferation, metastasis and angiogenesis of gastric cancer. RNA interference-mediated silencing of YAP may provide an opportunity to develop a novel treatment strategy for gastric cancer. PMID:27073556

  13. Correlation between hammerhead ribozyme-mediated eggshell protein gene cleavage and reproduction inhibition of Schistosoma japonicum.

    PubMed

    Liang, Yu; Zhou, Yuelan; Yin, Weiguo; Li, Yingju; Yang, Qiulin; Gao, Yuan; Zhang, Yukuai; Yang, Yaofei; Peng, Li; Xiao, Jianhua

    2012-04-01

    Schistosoma japonicum (S. japonicum) is an extremely harmful pathogen, which infects humans and causes severe public health problems. To date, no effective therapeutic drugs for this pathogen are available. In this study, we designed and constructed three hammerhead ribozymes targeting the eggshell protein gene of S. japonicum (SjESG). The cleavage activities of these three ribozymes were determined using cleavage experiments. The in vitro cleavage results showed that among the three synthesized ribozymes (Rz1, Rz2 and Rz3), Rz1 and Rz3 cleaved their target RNAs effectively. However, Rz2 did not cleave its target RNA detectably. The putative therapeutic roles of these three ribozymes to inhibit the reproduction of S. japonicum in mice were studied in vivo. Compared with the negative controls, Rz1 and Rz3 treatments resulted in increased levels of IFN-γ but decreased levels of IL-4 in mice. Rz2 affected levels of IFN-γ and IL-4 to degrees similar with those caused by the vector controls. In addition, Rz1 and Rz3 reduced the amounts of adult worms and eggs in the livers of mice more extensively than Rz2 and the vector controls. Altogether, these results suggest a correlation between the in vitro cleavage abilities of Rz1 and Rz3 and their roles in reproduction inhibition of S. japonicum. PMID:22246067

  14. d-alpha-tocopherol inhibits collagen alpha 1(I) gene expression in cultured human fibroblasts. Modulation of constitutive collagen gene expression by lipid peroxidation.

    PubMed Central

    Houglum, K; Brenner, D A; Chojkier, M

    1991-01-01

    Ascorbic acid stimulates collagen gene transcription in cultured fibroblasts, and this effect is mediated through the induction of lipid peroxidation by ascorbic acid. Quiescent cultured fibroblasts in the absence of ascorbic acid have a high constitutive level of collagen production, but the mechanisms of collagen gene regulation in this unstimulated state are not known. Because lipid peroxidation also occurs in normal cells, we wondered if lipid peroxidation plays a role in the regulation of basal collagen gene expression. Inhibition of lipid peroxidation in cultured human fibroblasts with d-alpha-tocopherol or methylene blue decreased the synthesis of collagen, the steady-state levels of procollagen alpha 1(I) mRNA and the transcription of the procollagen alpha 1(I) gene. This effect on collagen gene expression was selective and not associated with cellular toxicity. Thus, these experiments suggest a role for lipid peroxidation in the modulation of constitutive collagen gene expression. Images PMID:2040703

  15. Human papillomavirus type 16 E7 protein inhibits DNA binding by the retinoblastoma gene product.

    PubMed Central

    Stirdivant, S M; Huber, H E; Patrick, D R; Defeo-Jones, D; McAvoy, E M; Garsky, V M; Oliff, A; Heimbrook, D C

    1992-01-01

    The human papillomavirus E7 gene can transform murine fibroblasts and cooperate with other viral oncogenes in transforming primary cell cultures. One biochemical property associated with the E7 protein is binding to the retinoblastoma tumor suppressor gene product (pRB). Biochemical properties associated with pRB include binding to viral transforming proteins (E1A, large T, and E7), binding to cellular proteins (E2F and Myc), and binding to DNA. The mechanism by which E7 stimulates cell growth is uncertain. However, E7 binding to pRB inhibits binding of cellular proteins to pRB and appears to block the growth-suppressive activity of pRB. We have found that E7 also inhibits binding of pRB to DNA. A 60-kDa version of pRB (pRB60) produced in reticulocyte translation reactions or in bacteria bound quantitatively to DNA-cellulose. Recombinant E7 protein used at a 1:1 or 10:1 molar ratio with pRB60 blocked 50 or greater than 95% of pRB60 DNA-binding activity, respectively. A mutant E7 protein (E7-Ala-24) with reduced pRB60-binding activity exhibited a parallel reduction in its blocking of pRB60 binding to DNA. An E7(20-29) peptide that blocks binding of E7 protein to pRB60 restored the DNA-binding activity of pRB60 in the presence of E7. Peptide E7(2-32) did not block pRB60 binding to DNA, while peptide E7(20-57) and an E7 fragment containing residues 1 to 60 partially blocked DNA binding. E7 species containing residues 3 to 75 were fully effective at blocking pRB60 binding to DNA. These studies indicate that E7 protein specifically blocks pRB60 binding to DNA and suggest that the E7 region responsible for this property lies between residues 32 and 75. The functional significance of these observations is unclear. However, we have found that a point mutation in pRB60 that impairs DNA-binding activity also blocks the ability of pRB60 to inhibit cell growth. This correlation suggests that the DNA-binding activity of retinoblastoma proteins contributes to their biological properties. Images PMID:1314947

  16. Algal sulfated carrageenan inhibits proliferation of MDA-MB-231 cells via apoptosis regulatory genes.

    PubMed

    Murad, Hossam; Ghannam, Ahmed; Al-Ktaifani, Mahmoud; Abbas, Assef; Hawat, Mohammad

    2015-03-01

    Marine algae are prolific sources of sulfated polysaccharides, which may explain the low incidence of certain cancers in countries that traditionally consume marine food. Breast cancer is one of the most common types of non‑skin cancer in females. In this study, extracted sulfated carrageenan (ESC), predominantly consisting of ι‑carrageenan extracted from the red alga Laurencia papillosa, was characterized using Fourier transform infrared spectrometry. The biological effects of the identified extract were investigated and its potential cytotoxic activity was tested against the MDA‑MB‑231 cancer cell line. The biological biometer of the inhibitory concentration of the polysaccharide‑treated MDA‑MB‑231 cells was determined as 50 µM. Treatment with 50 µM ESC inhibited cell proliferation and promptly induced cell death through nuclear condensation and DNA fragmentation. Characterization of polysaccharide‑treated MDA‑MB‑231 cell death revealed that induction of apoptosis occurred via the activation of the extrinsic apoptotic caspase‑8 gene. The apoptotic signaling pathway was regulated through caspase‑3, caspase‑9, p53, Bax and Bcl‑2 genes. These findings suggest that ESC may serve as a potential therapeutic agent to target breast cancer via prompting apoptosis. PMID:25384757

  17. NF-κB signaling inhibits ubiquitin carboxyl-terminal hydrolase L1 gene expression.

    PubMed

    Wang, Ruitao; Zhang, Mingming; Zhou, Weihui; Ly, Philip T T; Cai, Fang; Song, Weihong

    2011-03-01

    Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is a deubiquitinating enzyme that plays a regulatory role in targeting proteins for proteasomal degradation. UCH-L1 is highly expressed in neurons and has been demonstrated to promote cell viability and maintain neuronal integrity. Reduced UCH-L1 levels have been observed in various neurodegenerative diseases, and expression of UCH-L1 can rescue synaptic dysfunction and memory deficits in Alzheimer's Disease model mice. However, the mechanisms regulating UCH-L1 expression have not been determined. In this study, we cloned a 1782 bp of the 5' flanking region of the human UCH-L1 gene and identified a 43 bp fragment containing the transcription start site as the minimal region necessary for promoter activity. Sequence analysis revealed several putative regulatory elements including NF-κB, NFAT, CREB, NRSF, YY1, AP1, and STAT in the UCH-L1 promoter. A functional NF-κB response element was identified in the UCH-L1 promoter region. Expression of NF-κB suppressed UCH-L1 gene transcription. In the RelA knockout system where NF-κB activity is ablated, UCH-L1 expression was significantly increased. Furthermore, activation of NF-κB signaling by the inflammatory stimulator lipopolysaccharide and TNFα resulted in a decrease of UCH-L1 gene expression by inhibiting its transcription. As NF-κB is an important signaling module in inflammatory response, our study suggests a possibility that inflammation might compromise neuronal functions via the interaction of NF-κB and UCH-L1. A better understanding of the NF-κB-regulated UCH-L1 transcription will provide insights to the role of inflammatory responses in Alzheimer's disease and Parkinson's disease. PMID:21210816

  18. The murine Sim-2 gene product inhibits transcription by active repression and functional interference.

    PubMed Central

    Moffett, P; Reece, M; Pelletier, J

    1997-01-01

    The Drosophila single-minded (Dsim) gene encodes a master regulatory protein involved in cell fate determination during midline development. This protein is a member of a rapidly expanding family of gene products possessing basic helix-loop-helix (bHLH) and hydrophobic PAS (designated a conserved region among PER, ARNT [aryl hydrocarbon receptor nuclear translocator] and SIM) protein association domains. Members of this family function as central transcriptional regulators in cellular differentiation and in the response to environmental stimuli such as xenobiotics and hypoxia. We have previously identified a murine member of this family, called mSim-2, showing sequence homology to the bHLH and PAS domains of Dsim. Immunoprecipitation experiments with recombinant proteins indicate that mSIM-2 associates with the arnt gene product. In the present work, by using fine-structure mapping we found that the HLH and PAS motifs of both proteins are required for optimal association. Forced expression of GAL4/mSIM-2 fusion constructs in mammalian cells demonstrated the presence of two separable repression domains within the carboxy terminus of mSIM-2. We found that mSIM-2 is capable of repressing ARNT-mediated transcriptional activation in a mammalian two-hybrid system. This effect (i) is dependent on the ability of mSIM-2 and ARNT to heterodimerize, (ii) is dependent on the presence of the mSIM-2 carboxy-terminal repression domain, and (iii) is not specific to the ARNT activation domain. These results suggest that mSIM-2 repression activity can dominantly override the activation potential of adjacent transcription factors. We also demonstrated that mSIM-2 can functionally interfere with hypoxia-inducible factor 1alpha (HIF-1alpha)/ARNT transcription complexes, providing a second mechanism by which mSIM-2 may inhibit transcription. PMID:9271372

  19. Inhibition of Candida albicans biofilm formation and modulation of gene expression by probiotic cells and supernatant.

    PubMed

    James, K M; MacDonald, K W; Chanyi, R M; Cadieux, P A; Burton, J P

    2016-04-01

    Oral candidiasis is a disease caused by opportunistic species of Candida that normally reside on human mucosal surfaces. The transition of Candida from budding yeast to filamentous hyphae allows for covalent attachment to oral epithelial cells, followed by biofilm formation, invasion and tissue damage. In this study, combinations of Lactobacillus plantarum SD5870, Lactobacillus helveticus CBS N116411 and Streptococcus salivarius DSM 14685 were assessed for their ability to inhibit the formation of and disrupt Candida albicans biofilms. Co-incubation with probiotic supernatants under hyphae-inducing conditions reduced C. albicans biofilm formation by >75 % in all treatment groups. Likewise, combinations of live probiotics reduced biofilm formation of C. albicans by >67 %. When live probiotics or their supernatants were overlaid on preformed C. albicans biofilms, biofilm size was reduced by >63 and >65 % respectively. Quantitative real-time PCR results indicated that the combined supernatants of SD5870 and CBS N116411 significantly reduced the expression of several C. albicans genes involved in the yeast-hyphae transition: ALS3 (adhesin/invasin) by 70 % (P < 0.0001), EFG1 (hyphae-specific gene activator) by 47 % (P = 0.0061), SAP5 (secreted protease) by 49 % (P < 0.0001) and HWP1 (hyphal wall protein critical to biofilm formation) by >99 % (P < 0.0001). These findings suggest the combination of L. plantarum SD5870, L. helveticus CBS N116411 and S. salivarius DSM 14685 is effective at both preventing the formation of and removing preformed C. albicans biofilms. Our novel results point to the downregulation of several Candida genes critical to the yeast-hyphae transition, biofilm formation, tissue invasion and cellular damage. PMID:26847045

  20. Proliferation inhibition and apoptosis enhancement of human cervical cancer cells by ultrasound-targeted microbubble destruction delivered double suicide genes.

    PubMed

    Hao, Yi; Guo, Li; Abudula, Abulizi; Saidoula, Wuliyati; Guo, Xia

    2014-01-01

    Successful gene therapy requires safe and efficient gene vectors and gene delivery methods. This study is to investigate the effects of double suicide genes on the proliferation and apoptosis of HeLa cells by using the ultrasound-targeted microbubble destruction (UTMD). A lentiviral vector with the KDR promoter was constructed, packaged, and delivered into HeLa cells by UTMD. The results showed that the encapsulation efficiency was 90.6 ± 3.1% and the drug-loading efficiency was 29.2 ± 0.9% assessed by reversed-phase high performance liquid chromatography (RP-HPLC). Cell proliferation was determined by MTT assay and apoptosis was detected by flow cytometry. The proliferation rates of HeLa cells were significantly inhibited when treated with dual-gene lentiviral vectors or lentiviral vector-loaded microbubbles plus UTMD (P < 0.05). Moreover, the inhibiting effects were enhanced along with the increased ultrasonic intensities and declined at 24 h post-irradiation. Additionally, in comparison with the control group, the apoptotic rates of HeLa cells were significantly elevated in the lentiviral vector group and the lentiviral vector microbubble groups (P < 0.05). The apoptotic rates were also elevated as the ultrasonic irradiation intensities were increased (P < 0.05). The results suggest that dual-gene lentiviral vector-loaded microbubbles inhibit proliferation and enhance apoptosis of cervical cancer cells. PMID:25664039

  1. Chylomicron remnants and nonesterified fatty acids differ in their ability to inhibit genes involved in lipogenesis in rats.

    PubMed

    Kohan, Alison B; Qing, Yang; Cyphert, Holly A; Tso, Patrick; Salati, Lisa M

    2011-02-01

    Primary hepatocytes treated with nonesterified PUFA have been used as a model for analyzing the inhibitory effects of dietary polyunsaturated fats on lipogenic gene expression. Although nonesterified fatty acids play an important signaling role in starvation, they do not completely recapitulate the mechanism of dietary fat presentation to the liver, which is delivered via chylomicron remnants. To test the effect of remnant TG on lipogenic enzyme expression, chylomicron remnants were generated from the lymph of rats intubated with either safflower oil or lard. The remnants were added to the medium of primary rat hepatocytes in culture and the accumulation of mRNA for genes involved in carbohydrate and lipid metabolism was measured. Both PUFA-enriched remnants and nonesterified PUFA inhibited the expression and maturation of sterol response element binding protein-1c (SREBP-1c) and the expression of lipogenic genes regulated by this transcription factor. These remnants also inhibited the expression of glucose-6-phosphate dehydrogenase (G6PD), a gene regulated at post-transcriptional steps. In contrast, PUFA-enriched remnants did not inhibit the accumulation of mRNA for malic enzyme, glucokinase, and L-pyruvate kinase, whereas nonesterified fatty acids caused a decrease in these mRNA. These genes are regulated independently of SREBP-1c. SFA-enriched remnants did not inhibit lipogenic gene expression, which is consistent with a lack of inhibition of lipogenesis by dietary saturated fats. Thus, the inhibitory action of dietary polyunsaturated fats on lipogenesis involves a direct action of chylomicron remnants on the liver. PMID:21169224

  2. Neferine inhibits angiotensin II-induced rat aortic smooth muscle cell proliferation predominantly by downregulating fractalkine gene expression

    PubMed Central

    ZHENG, LULU; CAO, YONGWEN; LIU, SHAO; PENG, ZHENYU; ZHANG, SAIDAN

    2014-01-01

    Neferine inhibits the angiotensin II (AngII)-induced proliferation of vascular smooth muscle cells (SMCs), but the underlying mechanism is unclear. The aim of this study was to explore the mechanism underlying the effect of neferine on the proliferation of vascular SMCs. Rat aortic SMCs (RASMCs) were used and fractalkine (Fkn) gene expression was measured by quantitative polymerase chain reaction and western blot analysis. The proliferation of RASMCs was analyzed by MTT assay and flow cytometry. It was revealed that AngII induced Fkn expression in a dose- and time-dependent manner. Fkn-knockdown with small interfering RNA attenuated the AngII-induced RASMC proliferation. Furthermore, neferine inhibited Fkn expression and attenuated the AngII-induced RASMC proliferation. These findings suggest that the Fkn gene may play an important role in AngII-induced RASMC proliferation and that neferine acts to attenuate AngII-induced RASMC proliferation by inhibiting Fkn expression. PMID:25289057

  3. Growth arrest-specific gene 1 is downregulated and inhibits tumor growth in gastric cancer.

    PubMed

    Wang, Honghong; Zhou, Xiong; Zhang, Yongguo; Zhu, Hongwu; Zhao, Lina; Fan, Linni; Wang, Yingmei; Gang, Yi; Wu, Kaichun; Liu, Zhiguo; Fan, Daiming

    2012-10-01

    Gastric cancer is one of the leading causes of malignancy-related mortality in the world, and malignant growth is a crucial characteristic in gastric cancer. In our previous study, we found that growth arrest-specific gene 1 (GAS1) suppression was involved in making gastric cancer cells multidrug-resistant by protecting them from drug-induced apoptosis. In the present study, we investigated the potential role of GAS1 in the growth and proliferation of gastric cancer. We demonstrated that GAS1 expression was decreased in gastric cancer, and patients without GAS1 expression showed shorter survival times than those with GAS1 expression. Both gain-of-function (by overexpression of GAS1) and loss-of-function (by GAS1-specific small interfering RNA knockdown) studies showed that increased GAS1 expression significantly reduced the colony-forming ability of gastric cancer cells in vitro and reduced cell growth in vivo, whereas decreased GAS1 expression had the opposite effects. Moreover, upregulation of GAS1 induced cell apoptosis, and downregulation of GAS1 inhibited apoptosis. Furthermore, we demonstrated that GAS1 could induce gastric cancer cell apoptosis, at least in part through modulating the Bcl-2/Bax ratio and the activity of caspase-3. Taken together, our results strongly indicate that GAS1 expression was decreased in gastric cancer and was predictive of a poor prognosis. Restoration of GAS1 expression inhibited cell growth and promoted apoptosis of gastric cancer cells, at least in part through modulating the Bcl-2/Bax ratio and activating caspase-3, suggesting that GAS1 might be used as a novel therapeutic candidate for gastric cancer. PMID:22846196

  4. Inhibition of RecBCD in Klebsiella pneumoniae by Gam and its effect on the efficiency of gene replacement.

    PubMed

    Chen, Chuan; Wei, Dong; Liu, Pengfu; Wang, Min; Shi, Jiping; Jiang, Biao; Hao, Jian

    2016-02-01

    Gam protein is an inhibitor of the host RecBCD exonuclease, and this inhibition is essential to the proficiency of Red recombinase-mediated gene replacement. In Klebsiella pneumoniae, the efficiency of this gene replacement was lower than that in Escherichia coli, and the minimum length of homologous extensions required was longer. Thus, it was supposed that the inhibitory effect of Gam against RecBCD was weak in K. pneumoniae. To test this hypothesis, a Gam-deficient Red recombinase expression plasmid and a ΔrecB K. pneumoniae mutant were constructed. The Gam-deficient Red recombinase showed a reduced capacity for gene replacement compared with that of the complete Red recombinase. The efficiency of gene replacement in the ΔrecB mutant was 6-8 times higher than the wild-type strain, and the minimum length for the homologous extensions was reduced to 100 bp. These results indicate that Gam does inhibit the RecBCD exonuclease in K. pneumoniae, but that this inhibition is not stringent. Furthermore, mutation of recB presents a convenient and efficient method to enhance the Red recombinase assisted gene replacement in K. pneumoniae. PMID:26471352

  5. Adenovirus-mediated delivery of interferon-γ gene inhibits the growth of nasopharyngeal carcinoma

    PubMed Central

    2012-01-01

    Background Interferon-γ (IFN-γ) is regarded as a potent antitumor agent, but its clinical application is limited by its short half-life and significant side effects. In this paper, we tried to develop IFN-γ gene therapy by a replication defective adenovirus encoding the human IFN-γ (Ad-IFNγ), and evaluate the antitumoral effects of Ad-IFNγ on nasopharyngeal carcinoma (NPC) cell lines in vitro and in xenografts model. Methods The mRNA levels of human IFN-γ in Ad-IFNγ-infected NPC cells were detected by reverse transcription-polymerase chain reaction (RT-PCR), and IFN-γ protein concentrations were measured by enzyme-linked immunosorbent assay (ELISA) in the culture supernatants of NPC cells and tumor tissues and bloods of nude mice treated with Ad-IFNγ. The effects of Ad-IFNγ on NPC cell proliferation was determined using MTT assay, cell cycle distribution was determined by flow cytometry analysis for DNA content, and cells apoptosis were analyzed by Annexin V-FITC/7-AAD binding assay and hoechst 33342/PI double staining. The anti-tumor effects and toxicity of Ad-IFNγ were evaluated in BALB/c nude mice carrying NPC xenografts. Results The results demonstrated that Ad-IFNγ efficiently expressed human IFN-γ protein in NPC cell lines in vitro and in vivo. Ad-IFNγ infection resulted in antiproliferative effects on NPC cells by inducing G1 phase arrest and cell apoptosis. Intratumoral administration of Ad-IFNγ significantly inhibited the growth of CNE-2 and C666-1 cell xenografts in nude mice, while no significant toxicity was observed. Conclusions These findings indicate IFN-γ gene therapy mediated by replication defective adenoviral vector is likely a promising approach in the treatment of nasopharyngeal carcinoma. PMID:23272637

  6. Gene expression profiles in engineered cardiac tissues respond to mechanical loading and inhibition of tyrosine kinases.

    PubMed

    Ye, Fei; Yuan, Fangping; Li, Xiaohong; Cooper, Nigel; Tinney, Joseph P; Keller, Bradley B

    2013-10-01

    Engineered cardiac tissues (ECTs) are platforms to investigate cardiomyocyte maturation and functional integration, the feasibility of generating tissues for cardiac repair, and as models for pharmacology and toxicology bioassays. ECTs rapidly mature in vitro to acquire the features of functional cardiac muscle and respond to mechanical load with increased proliferation and maturation. ECTs are now being investigated as platforms for in vitro models for human diseases and for pharmacologic screening for drug toxicities. We tested the hypothesis that global ECT gene expression patterns are complex and sensitive to mechanical loading and tyrosine kinase inhibitors similar to the maturing myocardium. We generated ECTs from day 14.5 rat embryo ventricular cells, as previously published, and then conditioned constructs after 5 days in culture for 48 h with mechanical stretch (5%, 0.5 Hz) and/or the p38 MAPK (p38 mitogen-activated protein kinase) inhibitor BIRB796. RNA was isolated from individual ECTs and assayed using a standard Agilent rat 4 × 44k V3 microarray and Pathway Analysis software for transcript expression fold changes and changes in regulatory molecules and networks. Changes in expression were confirmed by quantitative-polymerase chain reaction (q-PCR) for selected regulatory molecules. At the threshold of a 1.5-fold change in expression, stretch altered 1559 transcripts, versus 1411 for BIRB796, and 1846 for stretch plus BIRB796. As anticipated, top pathways altered in response to these stimuli include cellular development, cellular growth and proliferation; tissue development; cell death, cell signaling, and small molecule biochemistry as well as numerous other pathways. Thus, ECTs display a broad spectrum of altered gene expression in response to mechanical load and/or tyrosine kinase inhibition, reflecting a complex regulation of proliferation, differentiation, and architectural alignment of cardiomyocytes and noncardiomyocytes within ECT. PMID:24303162

  7. Responses of growth inhibition and antioxidant gene expression in earthworms (Eisenia fetida) exposed to tetrabromobisphenol A, hexabromocyclododecane and decabromodiphenyl ether.

    PubMed

    Shi, Ya-juan; Xu, Xiang-bo; Zheng, Xiao-qi; Lu, Yong-long

    2015-01-01

    Tetrabromobisphenol A (TBBPA), hexabromocyclododecane (HBCD) and decabromodiphenyl ether (BDE 209), suspected ubiquitous contaminants, account for the largest volume of brominated flame retardants (BFRs) since penta-BDE and octa-BDE have been phased out globally. In this paper, the growth inhibition and gene transcript levels of antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT)) and the stress-response gene involved in the prevention of oxidative stress (Hsp70) of earthworms (Eisenia fetida) exposed to TBBPA, HBCD and BDE 209 were measured to identify the toxicity effects of selected BFRs on earthworms. The growth of earthworms treated by TBBPA at 200 and 400 mg/kg dw were inhibited at rate of 13.7% and 22.0% respectively, while there was no significant growth inhibition by HBCD and BDE 209. A significant (P<0.01) up-regulation of SOD expression level was observed in earthworms exposed to TBBPA at 50 mg/kg dw (1.77-fold) and to HBCD at 400 mg/kg dw (2.06-fold). The transcript level of Hsp70 gene was significantly up-regulated (P<0.01) when earthworms exposed to TBBPA at concentration of 50-200 mg/kg (2.16-2.19-fold) and HBCD at 400 mg/kg (2.61-fold). No significant variation of CAT gene expression in all the BFRs treatments was observed, neither does all the target gene expression level exposed to BDE 209. Assessed by growth inhibition and the changes at mRNA levels of encoding genes in earthworms, TBBPA showed the greatest toxicity, followed by HBCD and BDE 209, consistent with trends in molecular properties. The results help to understand the molecular mechanism of antioxidant defense. PMID:26117064

  8. Ajoene, a sulfur-rich molecule from garlic, inhibits genes controlled by quorum sensing.

    PubMed

    Jakobsen, Tim Holm; van Gennip, Maria; Phipps, Richard Kerry; Shanmugham, Meenakshi Sundaram; Christensen, Louise Dahl; Alhede, Morten; Skindersoe, Mette Eline; Rasmussen, Thomas Bovbjerg; Friedrich, Karlheinz; Uthe, Friedrich; Jensen, Peter Østrup; Moser, Claus; Nielsen, Kristian Fog; Eberl, Leo; Larsen, Thomas Ostenfeld; Tanner, David; Høiby, Niels; Bjarnsholt, Thomas; Givskov, Michael

    2012-05-01

    In relation to emerging multiresistant bacteria, development of antimicrobials and new treatment strategies of infections should be expected to become a high-priority research area. Quorum sensing (QS), a communication system used by pathogenic bacteria like Pseudomonas aeruginosa to synchronize the expression of specific genes involved in pathogenicity, is a possible drug target. Previous in vitro and in vivo studies revealed a significant inhibition of P. aeruginosa QS by crude garlic extract. By bioassay-guided fractionation of garlic extracts, we determined the primary QS inhibitor present in garlic to be ajoene, a sulfur-containing compound with potential as an antipathogenic drug. By comprehensive in vitro and in vivo studies, the effect of synthetic ajoene toward P. aeruginosa was elucidated. DNA microarray studies of ajoene-treated P. aeruginosa cultures revealed a concentration-dependent attenuation of a few but central QS-controlled virulence factors, including rhamnolipid. Furthermore, ajoene treatment of in vitro biofilms demonstrated a clear synergistic, antimicrobial effect with tobramycin on biofilm killing and a cease in lytic necrosis of polymorphonuclear leukocytes. Furthermore, in a mouse model of pulmonary infection, a significant clearing of infecting P. aeruginosa was detected in ajoene-treated mice compared to a nontreated control group. This study adds to the list of examples demonstrating the potential of QS-interfering compounds in the treatment of bacterial infections. PMID:22314537

  9. Ajoene, a Sulfur-Rich Molecule from Garlic, Inhibits Genes Controlled by Quorum Sensing

    PubMed Central

    Jakobsen, Tim Holm; van Gennip, Maria; Phipps, Richard Kerry; Shanmugham, Meenakshi Sundaram; Christensen, Louise Dahl; Alhede, Morten; Skindersoe, Mette Eline; Rasmussen, Thomas Bovbjerg; Friedrich, Karlheinz; Uthe, Friedrich; Jensen, Peter Østrup; Moser, Claus; Nielsen, Kristian Fog; Eberl, Leo; Larsen, Thomas Ostenfeld; Tanner, David; Høiby, Niels; Bjarnsholt, Thomas

    2012-01-01

    In relation to emerging multiresistant bacteria, development of antimicrobials and new treatment strategies of infections should be expected to become a high-priority research area. Quorum sensing (QS), a communication system used by pathogenic bacteria like Pseudomonas aeruginosa to synchronize the expression of specific genes involved in pathogenicity, is a possible drug target. Previous in vitro and in vivo studies revealed a significant inhibition of P. aeruginosa QS by crude garlic extract. By bioassay-guided fractionation of garlic extracts, we determined the primary QS inhibitor present in garlic to be ajoene, a sulfur-containing compound with potential as an antipathogenic drug. By comprehensive in vitro and in vivo studies, the effect of synthetic ajoene toward P. aeruginosa was elucidated. DNA microarray studies of ajoene-treated P. aeruginosa cultures revealed a concentration-dependent attenuation of a few but central QS-controlled virulence factors, including rhamnolipid. Furthermore, ajoene treatment of in vitro biofilms demonstrated a clear synergistic, antimicrobial effect with tobramycin on biofilm killing and a cease in lytic necrosis of polymorphonuclear leukocytes. Furthermore, in a mouse model of pulmonary infection, a significant clearing of infecting P. aeruginosa was detected in ajoene-treated mice compared to a nontreated control group. This study adds to the list of examples demonstrating the potential of QS-interfering compounds in the treatment of bacterial infections. PMID:22314537

  10. Inhibition of Competence Development, Horizontal Gene Transfer and Virulence in Streptococcus pneumoniae by a Modified Competence Stimulating Peptide

    PubMed Central

    Zhu, Luchang; Lau, Gee W.

    2011-01-01

    Competence stimulating peptide (CSP) is a 17-amino acid peptide pheromone secreted by Streptococcus pneumoniae. Upon binding of CSP to its membrane-associated receptor kinase ComD, a cascade of signaling events is initiated, leading to activation of the competence regulon by the response regulator ComE. Genes encoding proteins that are involved in DNA uptake and transformation, as well as virulence, are upregulated. Previous studies have shown that disruption of key components in the competence regulon inhibits DNA transformation and attenuates virulence. Thus, synthetic analogues that competitively inhibit CSPs may serve as attractive drugs to control pneumococcal infection and to reduce horizontal gene transfer during infection. We performed amino acid substitutions on conserved amino acid residues of CSP1 in an effort to disable DNA transformation and to attenuate the virulence of S. pneumoniae. One of the mutated peptides, CSP1-E1A, inhibited development of competence in DNA transformation by outcompeting CSP1 in time and concentration-dependent manners. CSP1-E1A reduced the expression of pneumococcal virulence factors choline binding protein D (CbpD) and autolysin A (LytA) in vitro, and significantly reduced mouse mortality after lung infection. Furthermore, CSP1-E1A attenuated the acquisition of an antibiotic resistance gene and a capsule gene in vivo. Finally, we demonstrated that the strategy of using a peptide inhibitor is applicable to other CSP subtype, including CSP2. CSP1-E1A and CSP2-E1A were able to cross inhibit the induction of competence and DNA transformation in pneumococcal strains with incompatible ComD subtypes. These results demonstrate the applicability of generating competitive analogues of CSPs as drugs to control horizontal transfer of antibiotic resistance and virulence genes, and to attenuate virulence during infection by S. pneumoniae. PMID:21909280

  11. Glaucine inhibits breast cancer cell migration and invasion by inhibiting MMP-9 gene expression through the suppression of NF-κB activation.

    PubMed

    Kang, Hyereen; Jang, Sung-Wuk; Pak, Jhang Ho; Shim, Sungbo

    2015-05-01

    Matrix metalloproteinase-9 (MMP-9) plays a central role in the invasion and metastasis of various types of cancer cells. Here, we demonstrate that glaucine, an alkaloid isolated from the plant Corydalis turtschaninovii tuber (Papaveraceae), can inhibit the migration and invasion of human breast cancer cells. We further show that glaucine significantly blocks phorbol 12-myristate 13-acetate (PMA)-induced MMP-9 expression and activity in a dose-dependent manner. Results from reporter gene and electrophoretic mobility shift assays revealed that glaucine inhibits MMP-9 expression by suppressing activation of the nuclear transcription factor nuclear factor-κB (NF-κB). Moreover, glaucine attenuates PMA-induced IκBα degradation and nuclear translocation of NF-κB. Finally, we also found that glaucine inhibits invasion and MMP-9 expression in the highly metastatic MDA-MB-231 breast cancer cell line. Taken together, our findings indicate that the MMP-9 inhibitory activity of glaucine and its abilities to attenuate IκBα and NF-κB activities may be therapeutically useful as a novel means of controlling breast cancer growth and invasiveness. PMID:25670016

  12. Indirubin derivatives alter DNA binding activity of the transcription factor NF-Y and inhibit MDR1 gene promoter.

    PubMed

    Tanaka, Toru; Ohashi, Sachiyo; Saito, Hiroaki; Higuchi, Takashi; Tabata, Keiichi; Kosuge, Yasuhiro; Suzuki, Takashi; Miyairi, Shinichi; Kobayashi, Shunsuke

    2014-10-15

    Indirubin derivatives exert antitumor activity. However, their effects on the expression of multidrug resistance gene 1 (MDR1) have not been investigated. Here we found three derivatives that inhibit the MDR1 gene promoter. To investigate the effects of indirubins on the DNA binding of NF-Y, a major MDR1 gene transcription factor that recognizes an inverted CCAAT element in the promoter, gel mobility shift assay was performed using the element as a probe with nuclear extracts from NG108-15, MCF7, HepG2, C2C12, and SK-N-SH cells. Among 17 compounds, 5-methoxyindirubin inhibited the DNA binding of NF-Y significantly, whereas indirubin-3'-oxime and 7-methoxyindirubin 3'-oxime increased the binding considerably. After evaluating a suitable concentration of each compound for transcription analysis using living tumor cells, we performed a reporter gene assay using a reporter DNA plasmid containing EGFP cDNA fused to the MDR1 gene promoter region. Indirubin-3'-oxime exerted a significant inhibitory effect on the MDR1 promoter activity in MCF7 and HepG2 cells, and 5-methoxyindirubin inhibited the activity only in MCF7 cells; 7-methoxyindirubin 3'-oxime suppressed the activity in all of the cell lines. We further confirmed that the compounds reduced endogenous MDR1 transcription without any inhibitory effect on NF-Y expression. Moreover, each compound increased the doxorubicin sensitivity of MCF7 cells. These results indicate that each indirubin derivative acts on the DNA binding of NF-Y and represses the MDR1 gene promoter with tumor cell-type specificity. PMID:25066113

  13. Silencing of Kv1.5 Gene Inhibits Proliferation and Induces Apoptosis of Osteosarcoma Cells

    PubMed Central

    Wu, Jin; Chen, Zhida; Liu, Qingjun; Zeng, Wenrong; Wu, Xinyu; Lin, Bin

    2015-01-01

    Kv1.5 (also known as KCNA5) is a protein encoded by the KCNA5 gene, which belongs to the voltage-gated potassium channel, shaker-related subfamily. Recently, a number of studies have suggested that Kv1.5 is overexpressed in numerous cancers and plays crucial roles in cancer development. However, until now, the expression and functions of Kv1.5 in osteosarcoma are still unclear. To characterize the potential biological functions of Kv1.5 in osteosarcoma, herein, we examined the expression levels of Kv1.5 in osteosarcoma cells and tissues using quantitative real-time polymerase chain reaction (qRT-PCR), western blot, and immunohistochemistry assays. Four short hairpin RNAs (shRNAs) targeting Kv1.5 were designed and homologous recombination technology was used to construct pGeneSil-Kv1.5 vectors. In addition, the vectors were transfected into osteosarcoma MG63 cells and Kv1.5 mRNA level was measured by qRT-PCR and the Kv1.5 protein level was examined by western blot. We also examined the effects of Kv1.5 silencing on proliferation, cell cycle and apoptosis of the osteosarcoma cells using CCK-8, colony formation, flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays. Our results showed that Kv1.5 was aberrantly expressed in osteosarcoma and that the synthesized shRNA targeting Kv1.5 reduced Kv1.5 mRNA and protein expression effectively. Silencing Kv1.5 expression in the osteosarcoma cells significantly inhibited the proliferation of osteosarcoma cells, induced cell cycle arrest at G0/G1 phase, and induced cell apoptosis through up-regulation of p21, p27, Bax, Bcl-XL and caspase-3 and down-regulation of cyclins A, cyclins D1, cyclins E, Bcl-2 and Bik. In summary, our results indicate that Kv1.5 silencing could suppress osteosarcoma progression through multiple signaling pathways and suggest that Kv1.5 may be a novel target for osteosarcoma therapeutics. PMID:26569226

  14. Gene 33/Mig6 inhibits hexavalent chromium-induced DNA damage and cell transformation in human lung epithelial cells.

    PubMed

    Park, Soyoung; Li, Cen; Zhao, Hong; Darzynkiewicz, Zbigniew; Xu, Dazhong

    2016-02-23

    Hexavalent Chromium [Cr(VI)] compounds are human lung carcinogens and environmental/occupational hazards. The molecular mechanisms of Cr(VI) carcinogenesis appear to be complex and are poorly defined. In this study, we investigated the potential role of Gene 33 (ERRFI1, Mig6), a multifunctional adaptor protein, in Cr(VI)-mediated lung carcinogenesis. We show that the level of Gene 33 protein is suppressed by both acute and chronic Cr(VI) treatments in a dose- and time-dependent fashion in BEAS-2B lung epithelial cells. The inhibition also occurs in A549 lung bronchial carcinoma cells. Cr(VI) suppresses Gene 33 expression mainly through post-transcriptional mechanisms, although the mRNA level of gene 33 also tends to be lower upon Cr(VI) treatments. Cr(VI)-induced DNA damage appears primarily in the S phases of the cell cycle despite the high basal DNA damage signals at the G2M phase. Knockdown of Gene 33 with siRNA significantly elevates Cr(VI)-induced DNA damage in both BEAS-2B and A549 cells. Depletion of Gene 33 also promotes Cr(VI)-induced micronucleus (MN) formation and cell transformation in BEAS-2B cells. Our results reveal a novel function of Gene 33 in Cr(VI)-induced DNA damage and lung epithelial cell transformation. We propose that in addition to its role in the canonical EGFR signaling pathway and other signaling pathways, Gene 33 may also inhibit Cr(VI)-induced lung carcinogenesis by reducing DNA damage triggered by Cr(VI). PMID:26760771

  15. Wnt/β-catenin pathway regulates MGMT gene expression in cancer and inhibition of Wnt signalling prevents chemoresistance

    PubMed Central

    Wickström, Malin; Dyberg, Cecilia; Milosevic, Jelena; Einvik, Christer; Calero, Raul; Sveinbjörnsson, Baldur; Sandén, Emma; Darabi, Anna; Siesjö, Peter; Kool, Marcel; Kogner, Per; Baryawno, Ninib; Johnsen, John Inge

    2015-01-01

    The DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) is commonly overexpressed in cancers and is implicated in the development of chemoresistance. The use of drugs inhibiting MGMT has been hindered by their haematologic toxicity and inefficiency. As a different strategy to inhibit MGMT we investigated cellular regulators of MGMT expression in multiple cancers. Here we show a significant correlation between Wnt signalling and MGMT expression in cancers with different origin and confirm the findings by bioinformatic analysis and immunofluorescence. We demonstrate Wnt-dependent MGMT gene expression and cellular co-localization between active β-catenin and MGMT. Pharmacological or genetic inhibition of Wnt activity downregulates MGMT expression and restores chemosensitivity of DNA-alkylating drugs in mouse models. These findings have potential therapeutic implications for chemoresistant cancers, especially of brain tumours where the use of temozolomide is frequently used in treatment. PMID:26603103

  16. Wnt/?-catenin pathway regulates MGMT gene expression in cancer and inhibition of Wnt signalling prevents chemoresistance.

    PubMed

    Wickstrm, Malin; Dyberg, Cecilia; Milosevic, Jelena; Einvik, Christer; Calero, Raul; Sveinbjrnsson, Baldur; Sandn, Emma; Darabi, Anna; Siesj, Peter; Kool, Marcel; Kogner, Per; Baryawno, Ninib; Johnsen, John Inge

    2015-01-01

    The DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) is commonly overexpressed in cancers and is implicated in the development of chemoresistance. The use of drugs inhibiting MGMT has been hindered by their haematologic toxicity and inefficiency. As a different strategy to inhibit MGMT we investigated cellular regulators of MGMT expression in multiple cancers. Here we show a significant correlation between Wnt signalling and MGMT expression in cancers with different origin and confirm the findings by bioinformatic analysis and immunofluorescence. We demonstrate Wnt-dependent MGMT gene expression and cellular co-localization between active ?-catenin and MGMT. Pharmacological or genetic inhibition of Wnt activity downregulates MGMT expression and restores chemosensitivity of DNA-alkylating drugs in mouse models. These findings have potential therapeutic implications for chemoresistant cancers, especially of brain tumours where the use of temozolomide is frequently used in treatment. PMID:26603103

  17. Constitutive expression of a grapevine polygalacturonase-inhibiting protein affects gene expression and cell wall properties in uninfected tobacco

    PubMed Central

    2011-01-01

    Background Polygalacturonase-inhibiting proteins (PGIPs) directly limit the effective ingress of fungal pathogens by inhibiting cell wall-degrading endopolygalacturonases (ePGs). Transgenic tobacco plants over-expressing grapevine (Vitis vinifera) Vvpgip1 have previously been shown to be resistant to Botrytis infection. In this study we characterized two of these PGIP over-expressing lines with known resistance phenotypes by gene expression and hormone profiling in the absence of pathogen infection. Results Global gene expression was performed by a cross-species microarray approach using a potato cDNA microarray. The degree of potential cross-hybridization between probes was modeled by a novel computational workflow designed in-house. Probe annotations were updated by predicting probe-to-transcript hybridizations and combining information derived from other plant species. Comparing uninfected Vvpgip1-overexpressing lines to wild-type (WT), 318 probes showed significant change in expression. Functional groups of genes involved in metabolism and associated to the cell wall were identified and consequent cell wall analysis revealed increased lignin-levels in the transgenic lines, but no major differences in cell wall-derived polysaccharides. GO enrichment analysis also identified genes responsive to auxin, which was supported by elevated indole-acetic acid (IAA) levels in the transgenic lines. Finally, a down-regulation of xyloglucan endotransglycosylase/hydrolases (XTHs), which are important in cell wall remodeling, was linked to a decrease in total XTH activity. Conclusions This evaluation of PGIP over-expressing plants performed under pathogen-free conditions to exclude the classical PGIP-ePG inhibition interaction indicates additional roles for PGIPs beyond the inhibition of ePGs. PMID:22078230

  18. Adeno-associated virus mediated gene transfer of Shepherdin inhibits gallbladder carcinoma growth in vitro and in vivo.

    PubMed

    Zhu, Aijun; Ren, Yu; Wang, Ning; Jin, Qiuyue; Zhang, Dongchang; Yang, Guangxiao; Wang, Quanying

    2015-11-01

    Gene therapy, a significantly crucial strategy for treatment of malignancies, has been gradually accepted in recent years. However, this therapeutic approach has being facing great challenges concerning problems which include complicated development of cancer with multiple gene control, effective target shortage, low efficiency of gene transferring and safety of the vector delivery system. Shepherdin, a novel peptidomimetic molecule designed from Lys-79 to Leu-87 of survivin, has been identified as a tumor suppressor with the function that can not only competitively interfere with the interaction between survivin and Hsp90 (heat shock protein-90) leading to the degradation of survivin to anti-tumor, but also competitively target the ATP-dependent binding pocket of Hsp90 resulting in the dysfunction of Hsp90 chaperone to cell apoptosis via a mitochondrial dependent or independent pathway. In the present study, we designed and constructed a recombinant Adeno-associated virus (rAAV) loading fusion gene NT4-TAT-6His-Shepherdin. The expression of Shepherdin in gallbladder carcinoma (GBC) cells was detected and its strong inhibitory effects against GBC growth were evaluated after AAV mediated gene transfer of Shepherdin into GBC cells and xenograft tumors. MTT assay and flow cytometric analysis demonstrated that rAAV containing Shepherdin gene could significantly inhibit the growth of GBC and this effect was closely associated with apoptosis. These results indicated that rAAV-NT4-TAT-6His-Shepherdin may be considered a novel therapeutic strategy in the gene therapy for gallbladder carcinoma. PMID:26143116

  19. A Gene Expression Profiling of Early Rice Stamen Development that Reveals Inhibition of Photosynthetic Genes by OsMADS58.

    PubMed

    Chen, Rui; Shen, Li-Ping; Wang, Dong-Hui; Wang, Fu-Gui; Zeng, Hong-Yun; Chen, Zhi-Shan; Peng, Yi-Ben; Lin, Ya-Nan; Tang, Xing; Deng, Ming-Hua; Yao, Nan; Luo, Jing-Chu; Xu, Zhi-Hong; Bai, Shu-Nong

    2015-07-01

    Stamen is a unique plant organ wherein germ cells or microsporocytes that commit to meiosis are initiated from somatic cells during its early developmental process. While genes determining stamen identity are known according to the ABC model of floral development, little information is available on how these genes affect germ cell initiation. By using the Affymetrix GeneChip Rice Genome Array to assess 51 279 transcripts, we established a dynamic gene expression profile (GEP) of the early developmental process of rice (Oryza sativa) stamen. Systematic analysis of the GEP data revealed novel expression patterns of some developmentally important genes including meiosis-, tapetum-, and phytohormone-related genes. Following the finding that a substantial amount of nuclear genes encoding photosynthetic proteins are expressed at the low levels in early rice stamen, through the ChIP-seq analysis we found that a C-class MADS box protein, OsMADS58, binds many nuclear-encoded genes participated in photosystem and light reactions and the expression levels of most of them are increased when expression of OsMADS58 is downregulated in the osmads58 mutant. Furthermore, more pro-chloroplasts are observed and increased signals of reactive oxygen species are detected in the osmads58 mutant anthers. These findings implicate a novel link between stamen identity determination and hypoxia status establishment. PMID:25684654

  20. Insulin inhibits transcription of IRS-2 gene in rat liver through an insulin response element (IRE) that resembles IREs of other insulin-repressed genes

    PubMed Central

    Zhang, Jiandi; Ou, Jiafu; Bashmakov, Yuriy; Horton, Jay D.; Brown, Michael S.; Goldstein, Joseph L.

    2001-01-01

    Recent data indicate that sustained elevations in plasma insulin suppress the mRNA for IRS-2, a component of the insulin signaling pathway in liver, and that this deficiency contributes to hepatic insulin resistance and inappropriate gluconeogenesis. Here, we use nuclear run-on assays to show that insulin inhibits transcription of the IRS-2 gene in the livers of intact rats. Insulin also inhibited transcription of a reporter gene driven by the human IRS-2 promoter that was transfected into freshly isolated rat hepatocytes. The human promoter contains a heptanucleotide sequence, TGTTTTG, that is identical to the insulin response element (IRE) identified previously in the promoters of insulin-repressed genes. Single base pair substitutions in this IRE decreased transcription of the IRS-2-driven reporter in the absence of insulin and abolished insulin-mediated repression. We conclude that insulin represses transcription of the IRS-2 gene by blocking the action of a positive factor that binds to the IRE. Sustained repression of IRS-2, as occurs in chronic hyperinsulinemia, contributes to hepatic insulin resistance and accelerates the development of the diabetic state. PMID:11259670

  1. Nitrification inhibition by hexavalent chromium Cr(VI) - Microbial ecology, gene expression and off-gas emissions.

    PubMed

    Kim, Young Mo; Park, Hongkeun; Chandran, Kartik

    2016-04-01

    The goal of this study was to investigate the responses in the physiology, microbial ecology and gene expression of nitrifying bacteria to imposition of and recovery from Cr(VI) loading in a lab-scale nitrification bioreactor. Exposure to Cr(VI) in the reactor strongly inhibited nitrification performance resulting in a parallel decrease in nitrate production and ammonia consumption. Cr(VI) exposure also led to an overall decrease in total bacterial concentrations in the reactor. However, the fraction of ammonia oxidizing bacteria (AOB) decreased to a greater extent than the fraction of nitrite oxidizing bacteria (NOB). In terms of functional gene expression, a rapid decrease in the transcript concentrations of amoA gene coding for ammonia oxidation in AOB was observed in response to the Cr(VI) shock. In contrast, transcript concentrations of the nxrA gene coding for nitrite oxidation in NOB were relatively unchanged compared to Cr(VI) pre-exposure levels. Therefore, Cr(VI) exposure selectively and directly inhibited activity of AOB, which indirectly resulted in substrate (nitrite) limitation to NOB. Significantly, trends in amoA expression preceded performance trends both during imposition of and recovery from inhibition. During recovery from the Cr(VI) shock, the high ammonia concentrations in the bioreactor resulted in an irreversible shift towards AOB populations, which are expected to be more competitive in high ammonia environments. An inadvertent impact during recovery was increased emission of nitrous oxide (N2O) and nitric oxide (NO), consistent with recent findings linking AOB activity and the production of these gases. Therefore, Cr(VI) exposure elicited multiple responses on the microbial ecology, gene expression and both aqueous and gaseous nitrogenous conversion in a nitrification process. A complementary interrogation of these multiple responses facilitated an understanding of both direct and indirect inhibitory impacts on nitrification. PMID:26874778

  2. [Pyramiding of senescence-inhibition IPT gene and Xa23 for resistance to bacterial blight in rice (Oryza sativa L.)].

    PubMed

    He, Guang-Ming; Sun, Chuan-Qing; Fu, Yong-Cai; Fu, Qiang; Zhao, Kai-Jun; Wang, Chun-Lian; Zhang, Qi; Ling, Zhong-Zhuan; Wang, Xiang-Kun

    2004-08-01

    Transgenic lines (GC-1) carrying a senescence-inhibition cheimeric gene, IPT (isopentenyl transferase) gene, CBB23, a isogenic lines carrying Xa23 gene for resistance to bacterial blight, and Hexi15, a commercial cultivar showing high resistance to blast disease, were used as donors to pyramid IPT gene and Xa23 by marker-assisted selection (MAS). Seventeen BC1F1 plants pyramiding Xa23 gene and IPT genes were obtained from three multi-cross combinations. Then, the plants carrying Xa23 and IPT genes were crossed with parental lines of two-line hybrid rice, such as 9311, E32, Pei' ai 64S and W9834S. The progenies were backcrossed the acceptor parents. A total of 17 plants carrying Xa23 and IPT genes were detected by PCR, disease resistance identification and analysis of CTK contents of in the four combinations of "(9311///Hexi15/CBB23// GC-1) x 9311", "(E32///Hexi15/CBB23//GC-1) x E32", "(Pei'ai 64S///Hexi15/CBB23//GC-1) x Pei' ai 64S" and "(GC-1/CBB23//W9834S/Hexi15) x W9834S". These plants showed resistance to blast disease by inoculating test using 21 the lines of Pyricularia grisea from Northern China. Six plants of BC2F1 pyramiding Xa23 and IPT genes were further obtained in the combinations of "[(9311///Hexi15/CBB23//GC-1) x 9311] x 9311", "[(E32///Hexi15/CBB23//GC-1) x E32] x E32". After backcrossed and self-crossed 1 approximately 2nd, the plants pyramiding Xa23 and IPT genes can be used in the program of hybrid rice breeding. PMID:15481540

  3. Characterization of a Beta Vulgaris polygalacturonase-inhibiting protein: a defense response gene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polygalacturonase-inhibiting proteins (PGIPs) are plant cell wall proteins that inhibit pathogen and pest polygalacturonases (PGs). PGIPs are members of the leucine-rich repeat (LRR) protein family that play crucial roles in development, pathogen defense and recognition of beneficial microbes in pl...

  4. Introduction of apple ANR genes into tobacco inhibits expression of both CHI and DFR genes in flowers, leading to loss of anthocyanin

    PubMed Central

    Han, Yuepeng; Vimolmangkang, Sornkanok; Soria-Guerra, Ruth Elena; Korban, Schuyler S.

    2012-01-01

    Three genes encoding anthocyanidin reductase (ANR) in apple (Malus×domestica Borkh.), designated MdANR1, MdANR2a, and MdANR2b, have been cloned and characterized. MdANR1 shows 91% identity in coding DNA sequences with MdANR2a and MdANR2b, while MdANR2a and MdANR2b are allelic and share 99% nucleotide sequence identity in the coding region. MdANR1 and MdANR2 genes are located on linkage groups 10 and 5, respectively. Expression levels of both MdANR1 and MdANR2 genes are generally higher in yellow-skinned cv. Golden Delicious than in red-skinned cv. Red Delicious. Transcript accumulation of MdANR1 and MdANR2 genes in fruits gradually decreased throughout fruit development. Ectopic expression of apple MdANR genes in tobacco positively and negatively regulates the biosynthesis of proanthocyanidins (PAs) and anthocyanin, respectively, resulting in white, pale pink-coloured, and white/red variegated flowers. The accumulation of anthocyanin is significantly reduced in all tobacco transgenic flowers, while catechin and epicatechin contents in transgenic flowers are significantly higher than those in flowers of wild-type plants. The inhibition of anthocyanin synthesis in tobacco transgenic flowers overexpressing MdANR genes is probably attributed to down-regulation of CHALCONE ISOMERASE (CHI) and DIHYDROFLAVONOL-4-REDUCTASE (DFR) genes involved in the anthocyanin pathway. Interestingly, several transgenic lines show no detectable transcripts of the gene encoding leucoanthocyanidin reductase (LAR) in flowers, but accumulate higher levels of catechin in flowers of transgenic plants than those of wild-type plants. This finding suggests that the ANR gene may be capable of generating catechin via an alternative route, although this mechanism is yet to be further elucidated. PMID:22238451

  5. Remote limb ischemic preconditioning (rIPC) activates antioxidant and antiapoptotic genes and inhibits proinflammatory cytokine genes in renal ischemia/reperfusion injury.

    PubMed

    Hussein, Abdelaziz M; Harraz, Ahmed M; Awadalla, Amira; Barakat, Nashwa; Khater, Shery; Shokeir, Ahmed A

    2016-01-01

    The mechanisms underlying the renoprotective effect for remote limb ischemic preconditioning (rIPC) against renal ischemia/reperfusion injury need further elucidation. In our work, one hundred and twenty male Sprague Dawley rats were randomized into 3 groups; sham, I/R group (left renal 45 min ischemia) and rIPC (as I/R group with 3 cycles of left femoral ischemic PC just before renal ischemia). Rats were sacrificed at 2 h, 24 h, 48 h and 7 days. Serum creatinine and urea were measured at the baseline and endpoints. Also, histopathological examination and assessment of the expression of inflammatory cytokines e.g. TNF-α, IL-1β and ICAM-1 and antioxidant genes: Nrf2, HO-1 and NQO-1 and anti-apoptotic gene Bcl-2 in left kidney were done by the end of experiment. The results of this study demonstrated that, rIPC caused significant improvement in serum creatinine and BUN levels and in the expression of antioxidant genes and Bcl-2 antiapoptotic gene with significant attenuation of pro-inflammatory cytokines and histopathological damage score at all-time points compared to I/R group (p ≤ 0.05). In conclusion, inhibition of inflammatory cytokine (TNF-α, IL-1β and ICAM-1) formation and activation of antioxidant genes: Nrf2, HO-1 and NQO-1 and anti-apoptotic gene Bcl-2 could be possible underlying mechanisms for the renoprotective effect of rIPC. PMID:26612920

  6. Reactivation of HIC-1 gene by saRNA inhibits clonogenicity and invasiveness in breast cancer cells.

    PubMed

    Zhao, Feng; Pan, Shengli; Gu, Yan; Guo, Shanyu; Dai, Qiancheng; Yu, Yingyan; Zhang, Wei

    2015-01-01

    Hypermethylated in cancer 1 (HIC-1) is a tumor suppressor gene, which is epigenetically silenced in breast cancer. It is known that the loss of HIC-1, caused by promoter hypermethylation, is associated with tumor aggression and poor survival in breast carcinoma. It has been shown that small activating RNA (saRNA) targeting promoter sequences may induce gene re-expression. In the current study, saRNA was used to restore HIC-1 expression, and the effects on colony formation, invasiveness and the cell cycle in breast cancer cells were explored. dsHIC1-2998, an saRNA, exhibited activating efficacy on MCF-7 and MDA-MB-231 cancer cell lines. A clonogenicity assay showed that evident colony inhibition was induced via saRNA-mediated re-expression of HIC-1 in the two cancer cell lines. Reactivation of HIC-1 significantly inhibited cell migration and invasion, resulting in G0/G1 cell cycle arrest in these cell lines. These findings suggest that HIC-1 may be a potential target in gene therapy for the treatment of breast cancer. saRNA may function as a therapeutic option for upregulating tumor suppressor genes in breast cancer. PMID:25435951

  7. Reactivation of HIC-1 gene by saRNA inhibits clonogenicity and invasiveness in breast cancer cells

    PubMed Central

    ZHAO, FENG; PAN, SHENGLI; GU, YAN; GUO, SHANYU; DAI, QIANCHENG; YU, YINGYAN; ZHANG, WEI

    2015-01-01

    Hypermethylated in cancer 1 (HIC-1) is a tumor suppressor gene, which is epigenetically silenced in breast cancer. It is known that the loss of HIC-1, caused by promoter hypermethylation, is associated with tumor aggression and poor survival in breast carcinoma. It has been shown that small activating RNA (saRNA) targeting promoter sequences may induce gene re-expression. In the current study, saRNA was used to restore HIC-1 expression, and the effects on colony formation, invasiveness and the cell cycle in breast cancer cells were explored. dsHIC1-2998, an saRNA, exhibited activating efficacy on MCF-7 and MDA-MB-231 cancer cell lines. A clonogenicity assay showed that evident colony inhibition was induced via saRNA-mediated re-expression of HIC-1 in the two cancer cell lines. Reactivation of HIC-1 significantly inhibited cell migration and invasion, resulting in G0/G1 cell cycle arrest in these cell lines. These findings suggest that HIC-1 may be a potential target in gene therapy for the treatment of breast cancer. saRNA may function as a therapeutic option for upregulating tumor suppressor genes in breast cancer. PMID:25435951

  8. 20(S)-protopanaxatriol inhibits liver X receptor α-mediated expression of lipogenic genes in hepatocytes.

    PubMed

    Oh, Gyun-Sik; Yoon, Jin; Lee, Gang Gu; Oh, Won Keun; Kim, Seung-Whan

    2015-06-01

    20(S)-protopanaxatriol (PPT) is an aglycone of ginsenosides isolated from Panax ginseng and has several interesting activities, including anti-inflammatory and anti-oxidative stress effects. Herein, PPT was identified as an inhibitor against the ligand-dependent transactivation of liver X receptor α (LXRα) using a Gal4-TK-luciferase reporter system. LXRα is a transcription factor of nuclear hormone receptor family and stimulates the transcription of many metabolic genes, such as lipogenesis- or reverse cholesterol transport (RCT)-related genes. Quantitative RT-PCR analysis showed that PPT inhibited the LXRα-dependent transcription of lipogenic genes, such as sterol regulatory element binding protein-1c (SREBP-1c), fatty acid synthase, and stearoyl CoA desaturase 1. These inhibitory effects of PPT are, at least in part, a consequence of the reduced recruitment of RNA polymerase II to the LXR response element (LXRE) of the SREBP-1c promoter. Furthermore, LXRα-dependent triglyceride accumulation in primary mouse hepatocytes was significantly reduced by PPT. Interestingly, PPT did not inhibit the LXRα-dependent transcription of ABCA1, a crucial LXRα target gene involved in RCT. Chromatin immunoprecipitation assays revealed that PPT repressed recruitment of the lipogenic coactivator TRAP80 to the SREBP-1c LXRE, but not the ABCA1 LXRE. Overall, these data suggest that PPT has selective inhibitory activity against LXRα-mediated lipogenesis, but not LXRα-stimulated RCT. PMID:26109499

  9. Mitogen-activated protein kinase kinase 1/2 inhibition and angiotensin II converting inhibition in mice with cardiomyopathy caused by lamin A/C gene mutation

    SciTech Connect

    Muchir, Antoine; Wu, Wei; Sera, Fusako; Homma, Shunichi; Worman, Howard J.

    2014-10-03

    Highlights: • Both ACE and MEK1/2 inhibition are beneficial on cardiac function in Lmna cardiomyopathy. • MEK1/2 inhibitor has beneficial effects beyond ACE inhibition for Lmna cardiomyopathy. • These results provide further preclinical rationale for a clinical trial of a MEK1/2 inhibitor. - Abstract: Background: Mutations in the LMNA gene encoding A-type nuclear lamins can cause dilated cardiomyopathy with or without skeletal muscular dystrophy. Previous studies have shown abnormally increased extracellular signal-regulated kinase 1/2 activity in hearts of Lmna{sup H222P/H222P} mice, a small animal model. Inhibition of this abnormal signaling activity with a mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitor has beneficial effects on heart function and survival in these mice. However, such treatment has not been examined relative to any standard of care intervention for dilated cardiomyopathy or heart failure. We therefore examined the effects of an angiotensin II converting enzyme (ACE) inhibitor on left ventricular function in Lmna{sup H222P/H222P} mice and assessed if adding a MEK1/2 inhibitor would provide added benefit. Methods: Male Lmna{sup H222P/H222P} mice were treated with the ACE inhibitor benazepril, the MEK1/2 inhibitor selumetinib or both. Transthoracic echocardiography was used to measure left ventricular diameters and fractional shortening was calculated. Results: Treatment of Lmna{sup H222P/H222P} mice with either benazepril or selumetinib started at 8 weeks of age, before the onset of detectable left ventricular dysfunction, lead to statistically significantly increased fractional shortening compared to placebo at 16 weeks of age. There was a trend towards a great value for fractional shortening in the selumetinib-treated mice. When treatment was started at 16 weeks of age, after the onset of left ventricular dysfunction, the addition of selumetinib treatment to benazepril lead to a statistically significant increase in left ventricular fractional shortening at 20 weeks of age. Conclusions: Both ACE inhibition and MEK1/2 inhibition have beneficial effects on left ventricular function in Lmna{sup H222P/H222P} mice and both drugs together have a synergistic benefit when initiated after the onset of left ventricular dysfunction. These results provide further preclinical rationale for a clinical trial of a MEK1/2 inhibitor in addition to standard of care in patients with dilated cardiomyopathy caused by LMNA mutations.

  10. Inhibition of protein translation by the DISC1-Boymaw fusion gene from a Scottish family with major psychiatric disorders.

    PubMed

    Ji, Baohu; Higa, Kerin K; Kim, Minjung; Zhou, Lynn; Young, Jared W; Geyer, Mark A; Zhou, Xianjin

    2014-11-01

    The t(1; 11) translocation appears to be the causal genetic lesion with 70% penetrance for schizophrenia, major depression and other psychiatric disorders in a Scottish family. Molecular studies identified the disruption of the disrupted-in-schizophrenia 1 (DISC1) gene by chromosome translocation at chromosome 1q42. Our previous studies, however, revealed that the translocation also disrupted another gene, Boymaw (also termed DISC1FP1), on chromosome 11. After translocation, two fusion genes [the DISC1-Boymaw (DB7) and the Boymaw-DISC1 (BD13)] are generated between the DISC1 and Boymaw genes. In the present study, we report that expression of the DB7 fusion gene inhibits both intracellular NADH oxidoreductase activities and protein translation. We generated humanized DISC1-Boymaw mice with gene targeting to examine the in vivo functions of the fusion genes. Consistent with the in vitro studies on the DB7 fusion gene, protein translation activity is decreased in the hippocampus and in cultured primary neurons from the brains of the humanized mice. Expression of Gad67, Nmdar1 and Psd95 proteins are also reduced. The humanized mice display prolonged and increased responses to the NMDA receptor antagonist, ketamine, on various mouse genetic backgrounds. Abnormal information processing of acoustic startle and depressive-like behaviors are also observed. In addition, the humanized mice display abnormal erythropoiesis, which was reported to associate with depression in humans. Expression of the DB7 fusion gene may reduce protein translation to impair brain functions and thereby contribute to the pathogenesis of major psychiatric disorders. PMID:24908665

  11. LY294002 inhibits glucocorticoid-induced COX-2 gene expression in cardiomyocytes through a phosphatidylinositol 3 kinase-independent mechanism

    SciTech Connect

    Sun Haipeng; Xu Beibei; Sheveleva, Elena; Chen, Qin M.

    2008-10-01

    Glucocorticoids induce COX-2 expression in rat cardiomyocytes. While investigating whether phosphatidylinositol 3 kinase (PI3K) plays a role in corticosterone (CT)-induced COX-2, we found that LY294002 (LY29) but not wortmannin (WM) attenuates CT from inducing COX-2 gene expression. Expression of a dominant-negative mutant of p85 subunit of PI3K failed to inhibit CT from inducing COX-2 expression. CT did not activate PI3K/AKT signaling pathway whereas LY29 and WM decreased the activity of PI3K. LY303511 (LY30), a structural analogue and a negative control for PI3K inhibitory activity of LY29, also suppressed COX-2 induction. These data suggest PI3K-independent mechanisms in regulating CT-induced COX-2 expression. LY29 and LY30 do not inhibit glucocorticoid receptor transactivity. Both compounds have been reported to inhibit Casein Kinase 2 activity and modulate potassium and calcium levels independent of PI3K, while LY29 has been reported to inhibit mammalian Target of Rapamycin (mTOR), and DNA-dependent Protein Kinase (DNA-PK). Inhibitor of Casein Kinase 2 (CK2), mTOR or DNA-PK failed to prevent CT from inducing COX-2 expression. Tetraethylammonium (TEA), a potassium channel blocker, and nimodipine, a calcium channel blocker, both attenuated CT from inducing COX-2 gene expression. CT was found to increase intracellular Ca{sup 2+} concentration, which can be inhibited by LY29, TEA or nimodipine. These data suggest a possible role of calcium instead of PI3K in CT-induced COX-2 expression in cardiomyocytes.

  12. LY294002 Inhibits Glucocorticoid Induced COX-2 Gene Expression in Cardiomyocytes through a Phosphatidylinositol 3 Kinase Independent Mechanism

    PubMed Central

    Sun, Haipeng; Xu, Beibei; Sheveleva, Elena; Chen, Qin M.

    2014-01-01

    Glucocorticoids induce COX-2 expression in rat cardiomyocytes. While investigating whether phosphatidylinositol 3 kinase (PI3K) plays a role in corticosterone (CT) induced COX-2, we found that LY294002 (LY29) but not wortmannin (WM) attenuates CT from inducing COX-2 gene expression. Expression of a dominant-negative mutant of p85 subunit of PI3K failed to inhibit CT from inducing COX-2 expression. CT did not activate PI3K/AKT signaling pathway whereas LY29 and WM decreased the activity of PI3K. LY303511 (LY30), a structural analogue and a negative control for PI3K inhibitory activity of LY29, also suppressed COX-2 induction. These data suggest PI3K independent mechanisms in regulating CT induced COX-2 expression. LY29 and LY30 do not inhibit glucocorticoid receptor transactivity. Both compounds have been reported to inhibit Casein Kinase 2 activity and modulate potassium and calcium levels independent of PI3K, while LY29 has been reported to inhibit mammalian Target of Rapamycin (mTOR), DNA-dependent Protein Kinase (DNA-PK). Inhibitor of Casein Kinase 2 (CK2), mTOR or DNA-PK failed to prevent CT from inducing COX-2 expression. Tetraethylammonium (TEA), a potassium channel blocker, and nimodipine, a calcium channel blocker, both attenuated CT from inducing COX-2 gene expression. CT was found to increase intracellular Ca2+ concentration, which can be inhibited by LY29, TEA or nimodipine. These data suggest a possible role of calcium instead of PI3K in CT induced COX-2 expression in cardiomyocytes. PMID:18657281

  13. Tumor-targeted inhibition by a novel strategy - mimoretrovirus expressing siRNA targeting the Pokemon gene.

    PubMed

    Tian, Zhiqiang; Wang, Huaizhi; Jia, Zhengcai; Shi, Jinglei; Tang, Jun; Mao, Liwei; Liu, Hongli; Deng, Yijing; He, Yangdong; Ruan, Zhihua; Li, Jintao; Wu, Yuzhang; Ni, Bing

    2010-12-01

    Pokemon gene has crucial but versatile functions in cell differentiation, proliferation and tumorigenesis. It is a master regulator of the ARF-HDM2-p53 and Rb-E2F pathways. The facts that the expression of Pokemon is essential for tumor formation and many kinds of tumors over-express the Pokemon gene make it an attractive target for therapeutic intervention for cancer treatment. In this study, we used an RNAi strategy to silence the Pokemon gene in a cervical cancer model. To address the issues involving tumor specific delivery and durable expression of siRNA, we applied the Arg-Gly-Asp (RGD) peptide ligand and polylysine (K(18)) fusion peptide to encapsulate a recombinant retrovirus plasmid expressing a siRNA targeting the Pokemon gene and produced the 'mimoretrovirus'. At charge ratio 2.0 of fusion peptide/plasmid, the mimoretrovirus formed stable and homogenous nanoparticles, and provided complete DNase I protection and complete gel retardation. This nanoparticle inhibited SiHa cell proliferation and invasion, while it promoted SiHa cell apoptosis. The binding of the nanoparticle to SiHa cells was mediated via the RGD-integrin α(v)β(3) interaction, as evidenced by the finding that unconjugated RGD peptide inhibited this binding significantly. This tumor-targeting mimoretrovirus exhibited excellent anti-tumor capacity in vivo in a nude mouse model. Moreover, the mimoretrovirus inhibited tumor growth with a much higher efficiency than recombinant retrovirus expressing siRNA or the K(18)/P4 nanoparticle lacking the RGD peptide. Results suggest that the RNAi/RGD-based mimoretrovirus developed in this study represents a novel anti-tumor strategy that may be applicable to most research involving cancer therapy and, thus, has promising potential as a cervical cancer treatment. PMID:20879980

  14. Monocular inhibition reveals temporal and spatial changes in gene expression in the primary visual cortex of marmoset

    PubMed Central

    Nakagami, Yuki; Watakabe, Akiya; Yamamori, Tetsuo

    2013-01-01

    We investigated the time course of the expression of several activity-dependent genes evoked by visual inputs in the primary visual cortex (V1) in adult marmosets. In order to examine the rapid time course of activity-dependent gene expression, marmosets were first monocularly inactivated by tetrodotoxin (TTX), kept in darkness for two days, and then exposed to various length of light stimulation. Activity-dependent genes including HTR1B, HTR2A, whose activity-dependency were previously reported by us, and well-known immediate early genes (IEGs), c-FOS, ZIF268, and ARC, were examined by in situ hybridization. Using this system, first, we demonstrated the ocular dominance type of gene expression pattern in V1 under this condition. IEGs were expressed in columnar patterns throughout layers II–VI of all the tested monocular marmosets. Second, we showed the regulation of HTR1B and HTR2A expressions by retinal spontaneous activity, because HTR1B and HTR2A mRNA expressions sustained a certain level regardless of visual stimulation and were inhibited by a blockade of the retinal activity with TTX. Third, IEGs dynamically changed its laminar distribution from half an hour to several hours upon a stimulus onset with the unique time course for each gene. The expression patterns of these genes were different in neurons of each layer as well. These results suggest that the regulation of each neuron in the primary visual cortex of marmosets is subjected to different regulation upon the change of activities from retina. It should be related to a highly differentiated laminar structure of marmoset visual systems, reflecting the functions of the activity-dependent gene expression in marmoset V1. PMID:23576954

  15. Sensitivity of Small Cell Lung Cancer to BET Inhibition Is Mediated by Regulation of ASCL1 Gene Expression.

    PubMed

    Lenhart, Ryan; Kirov, Stefan; Desilva, Heshani; Cao, Jian; Lei, Ming; Johnston, Kathy; Peterson, Russell; Schweizer, Liang; Purandare, Ashok; Ross-Macdonald, Petra; Fairchild, Craig; Wong, Tai; Wee, Susan

    2015-10-01

    The BET (bromodomain and extra-terminal) proteins bind acetylated histones and recruit protein complexes to promote transcription elongation. In hematologic cancers, BET proteins have been shown to regulate expression of MYC and other genes that are important to disease pathology. Pharmacologic inhibition of BET protein binding has been shown to inhibit tumor growth in MYC-dependent cancers, such as multiple myeloma. In this study, we demonstrate that small cell lung cancer (SCLC) cells are exquisitely sensitive to growth inhibition by the BET inhibitor JQ1. JQ1 treatment has no impact on MYC protein expression, but results in downregulation of the lineage-specific transcription factor ASCL1. SCLC cells that are sensitive to JQ1 are also sensitive to ASCL1 depletion by RNAi. Chromatin immunoprecipitation studies confirmed the binding of the BET protein BRD4 to the ASCL1 enhancer, and the ability of JQ1 to disrupt the interaction. The importance of ASCL1 as a potential driver oncogene in SCLC is further underscored by the observation that ASCL1 is overexpressed in >50% of SCLC specimens, an extent greater than that observed for other putative oncogenes (MYC, MYCN, and SOX2) previously implicated in SCLC. Our studies have provided a mechanistic basis for the sensitivity of SCLC to BET inhibition and a rationale for the clinical development of BET inhibitors in this disease with high unmet medical need. PMID:26253517

  16. Inhibition of Hedgehog-Signaling Driven Genes in Prostate Cancer Cells by Sutherlandia frutescens Extract

    PubMed Central

    Lu, Yuan; Starkey, Nicholas; Lei, Wei; Li, Jilong; Cheng, Jianlin; Folk, William R.; Lubahn, Dennis B.

    2015-01-01

    Sutherlandia frutescens (L) R. Br. (Sutherlandia) is a South African botanical that is traditionally used to treat a variety of health conditions, infections and diseases, including cancer. We hypothesized Sutherlandia might act through Gli/ Hedgehog (Hh)-signaling in prostate cancer cells and used RNA-Seq transcription profiling to profile gene expression in TRAMPC2 murine prostate cancer cells with or without Sutherlandia extracts. We found 50% of Hh-responsive genes can be repressed by Sutherlandia ethanol extract, including the canonical Hh-responsive genes Gli1 and Ptch1 as well as newly distinguished Hh-responsive genes Hsd11b1 and Penk. PMID:26710108

  17. Nanoceria inhibit expression of genes associated with inflammation and angiogenesis in the retina of Vldlr null mice

    PubMed Central

    Kyosseva, Svetlana V.; Chen, Lijuan; Seal, Sudipta; McGinnis, James J.

    2013-01-01

    Oxidative stress and inflammation are important pathological mechanisms in many neurodegenerative diseases, including age-related macular degeneration (AMD). The Very Low-Density Lipoprotein Receptor knockout mouse (Vldlr−/−) has been identified as a model for AMD and in particular for Retinal Angiomatous Proliferation (RAP). In this study we examined the effect of cerium oxide nanoparticles (nanoceria) that have been shown to have catalytic antioxidant activity, on expression of 88 major cytokines in the retinas of Vldlr−/− mice using a PCR array. A single intravitrial injection of nanoceria at P28 caused inhibition of pro-inflammatory cytokines and pro-angiogenic growth factors including Tslp, Lif, Il-3, Il-7, Vegfa, Fgf1, Fgf2, Fgf7, Egf, Efna 3, Lep, and up-regulation of several cytokines and anti-angiogenic genes in the Vldlr−/−retina within one week. We used the Ingenuity Pathway Analysis software to search for biological functions, pathways, and interrelationships between gene networks. Many of the genes whose activities were affected are involved in cell signaling, cellular development, growth and proliferation, and tissue development. Western blot analysis revealed that nanoceria inhibit the activation of ERK 1/2, JNK, p38 MAP kinase, and Akt. These data suggest that nanoceria may represent a novel therapeutic strategy to treat AMD, RAP, and other neurodegenerative diseases. PMID:23978600

  18. Theobroxide Treatment Inhibits Wild Fire Disease Occurrence in Nicotiana benthamiana by the Overexpression of Defense-related Genes

    PubMed Central

    Ahn, Soon Young; Baek, Kwang-Hyun; Moon, Yong Sun; Yun, Hae Keun

    2013-01-01

    Theobroxide, a novel compound isolated from a fungus Lasiodiplodia theobromae, stimulates potato tuber formation and induces flowering of morning glory by initiating the jasmonic acid synthesis pathway. To elucidate the effect of theobroxide on pathogen resistance in plants, Nicotiana benthamiana plants treated with theobroxide were immediately infiltrated with Pseudomonas syringae pv. tabaci. Exogenous application of theobroxide inhibited development of lesion symptoms, and growth of the bacterial cells was significantly retarded. Semi-quantitative RT-PCRs using the primers of 18 defense-related genes were performed to investigate the molecular mechanisms of resistance. Among the genes, the theobroxide treatment increased the expression of pathogenesis-related protein 1a (PR1a), pathogenesis-related protein 1b (PR1b), glutathione S-transferase (GST), allen oxide cyclase (AOC), and lipoxyganase (LOX). All these data strongly indicate that theobroxide treatment inhibits disease development by faster induction of defense responses, which can be possible by the induction of defense-related genes including PR1a, PR1b, and GST triggered by the elevated jasmonic acid. PMID:25288936

  19. Interleukin-6 inhibits human peroxisome proliferator activated receptor alpha gene expression via CCAAT/enhancer-binding proteins in hepatocytes.

    PubMed

    Chew, Choy-Hoong; Chew, Guat-Siew; Najimudin, Nazalan; Tengku-Muhammad, Tengku Sifzizul

    2007-01-01

    Peroxisome proliferator activated receptor alpha has been implicated as a regulator of acute phase response genes in hepatocytes. Interleukin-6 is widely known as a major cytokine responsible in the regulation of acute phase proteins and, therefore, acute phase response. Unfortunately, to date, very little is understood about the molecular mechanisms by which interleukin-6 regulates the gene expression of peroxisome proliferator activated receptor alpha. Here, we report the molecular mechanisms by which peroxisome proliferator activated receptor alpha was regulated by interleukin-6 in human HepG2 cells. Interleukin-6 was shown to down-regulate the peroxisome proliferator activated receptor alpha gene expression at the level of gene transcription. Functional dissection of human peroxisome proliferator activated receptor alpha promoter B revealed the role of predicted CCAAT/enhancer-binding protein binding site (-164/+34) in mediating the interleukin-6 inhibitory effects on peroxisome proliferator activated receptor alpha mRNA expression and electrophoretic mobility shift assay showed the binding of CCAAT/enhancer-binding protein isoforms to this cis-acting elements was increased in interleukin-6-treated HepG2 cells. Co-transfection experiments, then, demonstrated that CCAAT/enhancer-binding protein beta either in homodimer or heterodimer with CCAAT/enhancer-binding protein alpha and CCAAT/enhancer-binding protein delta plays a predominant role in inhibiting the transcriptional activity of peroxisome proliferator activated receptor alpha promoter B, thus, reducing the peroxisome proliferator activated receptor alpha mRNA expression. These studies, therefore, suggest a novel mechanism for interleukin-6-mediated inhibition of peroxisome proliferator activated receptor alpha gene expression that involves the activation of CCAAT/enhancer-binding protein isoforms with CCAAT/enhancer-binding protein beta may play a major role. PMID:17616429

  20. The mushroom Ganoderma lucidum suppresses breast-to-lung cancer metastasis through the inhibition of pro-invasive genes.

    PubMed

    Loganathan, Jagadish; Jiang, Jiahua; Smith, Amanda; Jedinak, Andrej; Thyagarajan-Sahu, Anita; Sandusky, George E; Nakshatri, Harikrishna; Sliva, Daniel

    2014-06-01

    Breast cancer metastasis is one of the major reasons for the high morbidity and mortality of breast cancer patients. In spite of surgical interventions, chemotherapy, radiation therapy and targeted therapy, some patients are considering alternative therapies with herbal/natural products. In the present study, we evaluated a well-characterized extract from the medicinal mushroom Ganoderma lucidum (GLE) for its affects on tumor growth and breast-to-lung cancer metastasis. MDA-MB-231 human breast cancer cells were implanted into the mammary fat pads of nude mice. GLE (100 mg/kg/every other day) was administered to the mice by an oral gavage for 4 weeks, and tumor size was measured using microcalipers. Lung metastases were evaluated by hematoxylin and eosin (H&E) staining. Gene expression in MDA-MB-231 cells was determined by DNA microarray analysis and confirmed by quantitative PCR. Identified genes were silenced by siRNA, and cell migration was determined in Boyden chambers and by wound-healing assay. Although an oral administration of GLE only slightly suppressed the growth of large tumors, the same treatment significantly inhibited the number of breast-to-lung cancer metastases. GLE also downregulated the expression of genes associated with invasive behavior (HRAS, VIL2, S100A4, MCAM, I2PP2A and FN1) in MDA-MB-231 cells. Gene silencing of HRAS, VIL2, S100A4, I2PP2A and FN1 by siRNA suppressed migration of MDA-MB‑231 cells. Our study suggests that an oral administration of GLE can inhibit breast-to-lung cancer metastases through the downregulation of genes responsible for cell invasiveness. The anti-metastatic benefits of GLE warrant further clinical studies. PMID:24718855

  1. The mushroom Ganoderma lucidum suppresses breast-to-lung cancer metastasis through the inhibition of pro-invasive genes

    PubMed Central

    LOGANATHAN, JAGADISH; JIANG, JIAHUA; SMITH, AMANDA; JEDINAK, ANDREJ; THYAGARAJAN-SAHU, ANITA; SANDUSKY, GEORGE E.; NAKSHATRI, HARIKRISHNA; SLIVA, DANIEL

    2014-01-01

    Breast cancer metastasis is one of the major reasons for the high morbidity and mortality of breast cancer patients. In spite of surgical interventions, chemotherapy, radiation therapy and targeted therapy, some patients are considering alternative therapies with herbal/natural products. In the present study, we evaluated a well-characterized extract from the medicinal mushroom Ganoderma lucidum (GLE) for its affects on tumor growth and breast-to-lung cancer metastasis. MDA-MB-231 human breast cancer cells were implanted into the mammary fat pads of nude mice. GLE (100 mg/kg/every other day) was administered to the mice by an oral gavage for 4 weeks, and tumor size was measured using microcalipers. Lung metastases were evaluated by hematoxylin and eosin (H&E) staining. Gene expression in MDA-MB-231 cells was determined by DNA microarray analysis and confirmed by quantitative PCR. Identified genes were silenced by siRNA, and cell migration was determined in Boyden chambers and by wound-healing assay. Although an oral administration of GLE only slightly suppressed the growth of large tumors, the same treatment significantly inhibited the number of breast-to-lung cancer metastases. GLE also downregulated the expression of genes associated with invasive behavior (HRAS, VIL2, S100A4, MCAM, I2PP2A and FN1) in MDA-MB-231 cells. Gene silencing of HRAS, VIL2, S100A4, I2PP2A and FN1 by siRNA suppressed migration of MDA-MB-231 cells. Our study suggests that an oral administration of GLE can inhibit breast-to-lung cancer metastases through the downregulation of genes responsible for cell invasiveness. The anti-metastatic benefits of GLE warrant further clinical studies. PMID:24718855

  2. [Effect of Chemical Prevention Drugs-based MicroRNAs and Their Target Genes on Tumor Inhibition].

    PubMed

    Jiang, Yanhui; Song, Xin

    2015-04-01

    Chemopreventive drugs including natural chemopreventive drugs and synthetic chemopreventive drugs, it not only can prevent cancer, can also play a role in tumor treatment. MicroRNAs (miRNAs) is a kind of short chains of non-coding RNA, regulating the expression of many genes through the way of degradation of mRNA or inhibitting mRNA translation. In recent years, more and more studies have shown that chemopreventive drugs through influence the expression of miRNAs and their target genes play a role in the prevention and treatment in a variety of tumors, and chemopreventive drugs on the experimental study of miRNAs and their target genes in tumor have demonstrated a good safety and efficacy. Effect on chemopreventive drugs-based microRNAs and their target genes into cancer cells will be expected as a new starting point for cancer research. The thesis expounds and analyzes between the natural chemopreventive drugs and synthetic chemopreventive drugs and miRNAs and their target genes in tumor research progress. PMID:25936887

  3. Specific Inhibition of Histone Deacetylase 8 Reduces Gene Expression and Production of Proinflammatory Cytokines in Vitro and in Vivo*

    PubMed Central

    Li, Suzhao; Fossati, Gianluca; Marchetti, Carlo; Modena, Daniela; Pozzi, Pietro; Reznikov, Leonid L.; Moras, Maria Luisa; Azam, Tania; Abbate, Antonio; Mascagni, Paolo; Dinarello, Charles A.

    2015-01-01

    ITF2357 (generic givinostat) is an orally active, hydroxamic-containing histone deacetylase (HDAC) inhibitor with broad anti-inflammatory properties, which has been used to treat children with systemic juvenile idiopathic arthritis. ITF2357 inhibits both Class I and II HDACs and reduces caspase-1 activity in human peripheral blood mononuclear cells and the secretion of IL-1β and other cytokines at 25–100 nm; at concentrations >200 nm, ITF2357 is toxic in vitro. ITF3056, an analog of ITF2357, inhibits only HDAC8 (IC50 of 285 nm). Here we compared the production of IL-1β, IL-1α, TNFα, and IL-6 by ITF2357 with that of ITF3056 in peripheral blood mononuclear cells stimulated with lipopolysaccharide (LPS), heat-killed Candida albicans, or anti-CD3/anti-CD28 antibodies. ITF3056 reduced LPS-induced cytokines from 100 to 1000 nm; at 1000 nm, the secretion of IL-1β was reduced by 76%, secretion of TNFα was reduced by 88%, and secretion of IL-6 was reduced by 61%. The intracellular levels of IL-1α were 30% lower. There was no evidence of cell toxicity at ITF3056 concentrations of 100–1000 nm. Gene expression of TNFα was markedly reduced (80%), whereas IL-6 gene expression was 40% lower. Although anti-CD3/28 and Candida stimulation of IL-1β and TNFα was modestly reduced, IFNγ production was 75% lower. Mechanistically, ITF3056 reduced the secretion of processed IL-1β independent of inhibition of caspase-1 activity; however, synthesis of the IL-1β precursor was reduced by 40% without significant decrease in IL-1β mRNA levels. In mice, ITF3056 reduced LPS-induced serum TNFα by 85% and reduced IL-1β by 88%. These data suggest that specific inhibition of HDAC8 results in reduced inflammation without cell toxicity. PMID:25451941

  4. Antioxidative Dietary Compounds Modulate Gene Expression Associated with Apoptosis, DNA Repair, Inhibition of Cell Proliferation and Migration

    PubMed Central

    Wang, Likui; Gao, Shijuan; Jiang, Wei; Luo, Cheng; Xu, Maonian; Bohlin, Lars; Rosendahl, Markus; Huang, Wenlin

    2014-01-01

    Many dietary compounds are known to have health benefits owing to their antioxidative and anti-inflammatory properties. To determine the molecular mechanism of these food-derived compounds, we analyzed their effect on various genes related to cell apoptosis, DNA damage and repair, oxidation and inflammation using in vitro cell culture assays. This review further tests the hypothesis proposed previously that downstream products of COX-2 (cyclooxygenase-2) called electrophilic oxo-derivatives induce antioxidant responsive elements (ARE), which leads to cell proliferation under antioxidative conditions. Our findings support this hypothesis and show that cell proliferation was inhibited when COX-2 was down-regulated by polyphenols and polysaccharides. Flattened macrophage morphology was also observed following the induction of cytokine production by polysaccharides extracted from viili, a traditional Nordic fermented dairy product. Coix lacryma-jobi (coix) polysaccharides were found to reduce mitochondrial membrane potential and induce caspase-3- and 9-mediated apoptosis. In contrast, polyphenols from blueberries were involved in the ultraviolet-activated p53/Gadd45/MDM2 DNA repair system by restoring the cell membrane potential. Inhibition of hypoxia-inducible factor-1 by saponin extracts of ginsenoside (Ginsen) and Gynostemma and inhibition of S100A4 by coix polysaccharides inhibited cancer cell migration and invasion. These observations suggest that antioxidants and changes in cell membrane potential are the major driving forces that transfer signals through the cell membrane into the cytosol and nucleus, triggering gene expression, changes in cell proliferation and the induction of apoptosis or DNA repair. PMID:25226533

  5. Antioxidative dietary compounds modulate gene expression associated with apoptosis, DNA repair, inhibition of cell proliferation and migration.

    PubMed

    Wang, Likui; Gao, Shijuan; Jiang, Wei; Luo, Cheng; Xu, Maonian; Bohlin, Lars; Rosendahl, Markus; Huang, Wenlin

    2014-01-01

    Many dietary compounds are known to have health benefits owing to their antioxidative and anti-inflammatory properties. To determine the molecular mechanism of these food-derived compounds, we analyzed their effect on various genes related to cell apoptosis, DNA damage and repair, oxidation and inflammation using in vitro cell culture assays. This review further tests the hypothesis proposed previously that downstream products of COX-2 (cyclooxygenase-2) called electrophilic oxo-derivatives induce antioxidant responsive elements (ARE), which leads to cell proliferation under antioxidative conditions. Our findings support this hypothesis and show that cell proliferation was inhibited when COX-2 was down-regulated by polyphenols and polysaccharides. Flattened macrophage morphology was also observed following the induction of cytokine production by polysaccharides extracted from viili, a traditional Nordic fermented dairy product. Coix lacryma-jobi (coix) polysaccharides were found to reduce mitochondrial membrane potential and induce caspase-3- and 9-mediated apoptosis. In contrast, polyphenols from blueberries were involved in the ultraviolet-activated p53/Gadd45/MDM2 DNA repair system by restoring the cell membrane potential. Inhibition of hypoxia-inducible factor-1 by saponin extracts of ginsenoside (Ginsen) and Gynostemma and inhibition of S100A4 by coix polysaccharides inhibited cancer cell migration and invasion. These observations suggest that antioxidants and changes in cell membrane potential are the major driving forces that transfer signals through the cell membrane into the cytosol and nucleus, triggering gene expression, changes in cell proliferation and the induction of apoptosis or DNA repair. PMID:25226533

  6. Amygdalin inhibits genes related to cell cycle in SNU-C4 human colon cancer cells

    PubMed Central

    Park, Hae-Jeong; Yoon, Seo-Hyun; Han, Long-Shan; Zheng, Long-Tai; Jung, Kyung-Hee; Uhm, Yoon-Kyung; Lee, Je-Hyun; Jeong, Ji-Seon; Joo, Woo-Sang; Yim, Sung-Vin; Chung, Joo-Ho; Hong, Seon-Pyo

    2005-01-01

    AIM: The genes were divided into seven categories according to biological function; apoptosis-related, immune response-related, signal transduction-related, cell cycle-related, cell growth-related, stress response-related and transcription-related genes. METHODS: We compared the gene expression profiles of SNU-C4 cells between amygdalin-treated (5 mg/mL, 24 h) and non-treated groups using cDNA microarray analysis. We selected genes downregulated in cDNA microarray and investigated mRNA levels of the genes by RT-PCR. RESULTS: Microarray showed that amygdalin downregulated especially genes belonging to cell cycle category: exonuclease 1 (EXO1), ATP-binding cassette, sub-family F, member 2 (ABCF2), MRE11 meiotic recombination 11 homolog A (MRE11A), topoisomerase (DNA) I (TOP1), and FK506 binding protein 12-rapamycin-associated protein 1 (FRAP1). RT-PCR analysis revealed that mRNA levels of these genes were also decreased by amygdalin treatment in SNU-C4 human colon cancer cells. CONCLUSION: These results suggest that amygdalin have an anticancer effect via downregulation of cell cycle-related genes in SNU-C4 human colon cancer cells, and might be used for therapeutic anticancer drug. PMID:16127745

  7. Inhibiting expression of specific genes in mammalian cells with 5' end-mutated U1 small nuclear RNAs targeted to terminal exons of pre-mRNA.

    PubMed

    Fortes, Puri; Cuevas, Yolanda; Guan, Fei; Liu, Peng; Pentlicky, Sara; Jung, Stephen P; Martínez-Chantar, Maria L; Prieto, Jesús; Rowe, David; Gunderson, Samuel I

    2003-07-01

    Reducing or eliminating expression of a given gene is likely to require multiple methods to ensure coverage of all of the genes in a given mammalian cell. We and others [Furth, P. A., Choe, W. T., Rex, J. H., Byrne, J. C., and Baker, C. C. (1994) Mol. Cell. Biol. 14, 5278-5289] have previously shown that U1 small nuclear (sn) RNA, both natural or with 5' end mutations, can specifically inhibit reporter gene expression in mammalian cells. This inhibition occurs when the U1 snRNA 5' end base pairs near the polyadenylation signal of the reporter gene's pre-mRNA. This base pairing inhibits poly(A) tail addition, a key, nearly universal step in mRNA biosynthesis, resulting in degradation of the mRNA. Here we demonstrate that expression of endogenous mammalian genes can be efficiently inhibited by transiently or stably expressed 5' end-mutated U1 snRNA. Also, we determine the inhibitory mechanism and establish a set of rules to use this technique and to improve the efficiency of inhibition. Two U1 snRNAs base paired to a single pre-mRNA act synergistically, resulting in up to 700-fold inhibition of the expression of specific reporter genes and 25-fold inhibition of endogenous genes. Surprisingly, distance from the U1 snRNA binding site to the poly(A) signal is not critical for inhibition, instead the U1 snRNA must be targeted to the terminal exon of the pre-mRNA. This could reflect a disruption by the 5' end-mutated U1 snRNA of the definition of the terminal exon as described by the exon definition model. PMID:12826613

  8. Knockout Zbtb33 gene results in an increased locomotion, exploration and pre-pulse inhibition in mice.

    PubMed

    Kulikov, Alexander V; Korostina, Valeria S; Kulikova, Elizabeth A; Fursenko, Dariya V; Akulov, Andrey E; Moshkin, Mikhail P; Prokhortchouk, Egor B

    2016-01-15

    The Zbtb33 gene encodes the Kaiso protein-a bimodal transcriptional repressor. Here, the effects of Zbtb33 gene disruption on the brain and behaviour of the Kaiso-deficient (KO) and C57BL/6 (WT) male mice were investigated. Behaviour was studied using the open field, novel object, elevated plus maze and acoustic startle reflex tests. Brain morphology was investigated with magnetic resonance imaging. Biogenic amine levels and gene expression in the brain were measured with high-performance liquid chromatography and quantitative real-time RT-PCR, respectively. Zbtb33 gene mRNA was not detected in the brain of KO mice. KO mice exhibited increased locomotion, exploration in the open field, novel object and elevated plus-maze test. At the same time, Zbtb33 gene disruption did not alter anxiety-related behaviour in the elevated plus-maze test. KO mice showed elevated amplitudes and pre-pulse inhibitions of the acoustic startle reflex. These behavioural alterations were accompanied by significant reductions in the volumes of the lateral ventricles without significant alterations in the volumes of the hippocampus, striatum, thalamus and corpus callosum. Norepinephrine concentration was reduced in the hypothalami and hippocampi in KO mice, while the levels of serotonin, dopamine, their metabolites as well as mRNA of the gene coding brain-derived neurotrophic factor were not altered in the brain of KO mice compared to WT mice. This study is the first to reveal the involvement of the Zbtb33 gene in the regulation of behaviour and the central nervous system. PMID:26454239

  9. Specific inhibition of skeletal alpha-actin gene transcription by applied mechanical forces through integrins and actin.

    PubMed Central

    Lew, A M; Glogauer, M; Mculloch, C A

    1999-01-01

    Skeletal alpha-actin (skA), a prominent fetal actin isoform that is re-expressed by adult cardiac myocytes after chronic overload in vivo, provides a model for studying cytoskeletal gene regulation by mechanical forces in vitro. We have determined the mechanisms by which perpendicular applied forces acting through integrins and the actin cytoskeleton regulate the expression of skA. Rat-2 fibroblasts were transiently transfected with plasmids containing 5'-regulatory regions of the skA gene fused to luciferase coding sequences. A constant, perpendicular force (0.2 pN/micrometer(2)) was applied by using a collagen-magnetic bead model; a 25% deformation was obtained on the dorsal cell surface. In this system, force is applied through focal adhesion integrins and strongly induces actin assembly [Glogauer, Arora, Yao, Sokholov, Ferrier and McCulloch (1997) J. Cell Sci. 110, 11-21]. skA promoter activity was inhibited by 68% in cells subjected to 4 h of applied force, whereas Rous sarcoma virus promoter activity was unaffected. In cells transiently transfected with a skA expression vector there was also a parallel 40% decrease in skA protein levels by force, as shown by Western blotting. In L8 cells, constitutive skA expression was decreased by more than 50%. Analyses of specific motifs in the skA promoter revealed that transcriptional enhancer factor 1 and Yin and Yang 1 sites, but not serum response factor and Sp1 sites, mediated inhibitory responses to force. In cells treated with cycloheximide the force-induced inhibition was abrogated, indicating a dependence on new protein synthesis. Inhibition of actin filament assembly with either cytochalasin D or Ca(2+)-depleted medium blocked the inhibitory effect induced by the applied force, suggesting that actin filaments are required for the regulation of skA promoter activity. Western blot analysis showed that p38 kinase, but not Jun N-terminal kinase or extracellular signal-regulated protein kinase 1/2, was activated by force; indeed, the p38 kinase inhibitor SB203580 relieved the force-induced inhibition of skA. We conclude that the force-induced inhibition of skA promoter activity requires an intact actin cytoskeleton and can be mapped to two different response elements. This inhibition might be mediated through the p38 kinase. PMID:10417328

  10. Inhibition of FSS-induced actin cytoskeleton reorganization by silencing LIMK2 gene increases the mechanosensitivity of primary osteoblasts.

    PubMed

    Yang, Zhi; Tan, Shuyi; Shen, Yun; Chen, Rui; Wu, Changjing; Xu, Yajuan; Song, Zijun; Fu, Qiang

    2015-05-01

    Mechanical stimulation plays an important role in bone cell metabolic activity. However, bone cells lose their mechanosensitivity upon continuous mechanical stimulation (desensitization) and they can recover the sensitivity with insertion of appropriate rest period into the mechanical loading profiles. The concrete molecular mechanism behind the regulation of cell mechanosensitivity still remains unclear. As one kind of mechanosensitive cell to react to the mechanical stimulation, osteoblasts respond to fluid shear stress (FSS) with actin cytoskeleton reorganization, and the remodeling of actin cytoskeleton is closely associated with the alteration of cell mechanosensitivity. In order to find out whether inhibiting the actin cytoskeleton reorganization by silencing LIM-kinase 2 (LIMK2) gene would increase the mechanosensitivity of primary osteoblasts, we attenuated the formation of actin stress fiber under FSS in a more specific way: inhibiting the LIMK2 expression by RNA interference. We found that inhibition of LIMK2 expression by RNA interference attenuated the formation of FSS-induced actin stress fiber, and simultaneously maintained the integrity of actin cytoskeleton in primary osteoblasts. We confirmed that the decreased actin cytoskeleton reorganization in response to LIMK2 inhibition during FSS increased the mechanosensitivity of the osteoblasts, based on the increased c-Fos and COX-2 expression as well as the enhanced proliferative activity in response to FSS. These data suggest that osteoblasts can increase their mechanosensitivity under continuous mechanical stimulation by reducing the actin stress fiber formation through inhibiting the LIMK2 expression. This study provides us with a new and more specific method to regulate the osteoblast mechanosensitivity, and also a new therapeutic target to cure bone related diseases, which is of importance in maintaining bone mass and promoting osteogenesis. PMID:25549868

  11. fMRI Activation during Response Inhibition and Error Processing: The Role of the DAT1 Gene in Typically Developing Adolescents and Those Diagnosed with ADHD

    ERIC Educational Resources Information Center

    Braet, Wouter; Johnson, Katherine A.; Tobin, Claire T.; Acheson, Ruth; McDonnell, Caroline; Hawi, Ziarah; Barry, Edwina; Mulligan, Aisling; Gill, Michael; Bellgrove, Mark A.; Robertson, Ian H.; Garavan, Hugh

    2011-01-01

    The DAT1 gene codes for the dopamine transporter, which clears dopamine from the synaptic cleft, and a variant of this gene has previously been associated with compromised response inhibition in both healthy and clinical populations. This variant has also been associated with ADHD, a disorder that is characterised by disturbed dopamine function as

  12. fMRI Activation during Response Inhibition and Error Processing: The Role of the DAT1 Gene in Typically Developing Adolescents and Those Diagnosed with ADHD

    ERIC Educational Resources Information Center

    Braet, Wouter; Johnson, Katherine A.; Tobin, Claire T.; Acheson, Ruth; McDonnell, Caroline; Hawi, Ziarah; Barry, Edwina; Mulligan, Aisling; Gill, Michael; Bellgrove, Mark A.; Robertson, Ian H.; Garavan, Hugh

    2011-01-01

    The DAT1 gene codes for the dopamine transporter, which clears dopamine from the synaptic cleft, and a variant of this gene has previously been associated with compromised response inhibition in both healthy and clinical populations. This variant has also been associated with ADHD, a disorder that is characterised by disturbed dopamine function as…

  13. Mir-338-3p Inhibits Malignant Biological Behaviors of Glioma Cells by Targeting MACC1 Gene.

    PubMed

    Shang, Chao; Hong, Yang; Guo, Yan; Xue, Yixue

    2016-01-01

    BACKGROUND Human brain glioma is the most common endocranial tumor; its mortality and morbidity are very high. The objective of this study was to determine whether miR-338-3p can regulate malignant biological behaviors of glioma cells by targeted silencing of MACC1. MATERIAL AND METHODS The expression of miR-338-3p was detected by quantitative real-time PCR in brain glioma tissues and cell lines. Bioinformatics software was used to predict some potential target genes of miR-338-3p. Luciferase activities assay was used to verify the combination between target genes and miR-338-3p. And MACC1 protein expression was detected by Western blot. The apoptosis and proliferation ability were analyzed by MTT and flow cytometry assay. RESULTS Compared with normal brain tissues and cells, miR-338-3p in glioma tissues and cell lines was confirmed to be expressed at low levels, and down-regulation of miR-338-3p tended to be correlated with worse histological grade. Up-regulation of miR-338-3p promoted apoptosis and sharply inhibited cell proliferation ability of U251 and U87 cells. The luciferase activities assay, biotin-avidin pull-down assay, and western blot analysis verified that MACC1 was a specific target gene of miR-338-3p. Subsequent experiments found that up-regulation of MACC1 significantly inhibited the apoptosis and increased the cell proliferation ability of U251 and U87 cells. The regulation effects of miR-338-3p on malignant biological behaviors of glioma cells can be partly reversed by up-regulation of MACC1. CONCLUSIONS Down-regulation of miR-338-3p was an independent prognostic biomarker associated with poor prognosis in glioma patients; miR-338-3p acted as a tumor-suppressing gene whose silencing can inhibit malignant biological behaviors of glioma cells. MACC1 was a specific target gene of miR-338-3p, which regulates malignant biological behaviors of glioma cells partly through directly silencing MACC1 expression. PMID:26936749

  14. Mir-338-3p Inhibits Malignant Biological Behaviors of Glioma Cells by Targeting MACC1 Gene

    PubMed Central

    Shang, Chao; Hong, Yang; Guo, Yan; Xue, Yi-xue

    2016-01-01

    Background Human brain glioma is the most common endocranial tumor; its mortality and morbidity are very high. The objective of this study was to determine whether miR-338-3p can regulate malignant biological behaviors of glioma cells by targeted silencing of MACC1. Material/Methods The expression of miR-338-3p was detected by quantitative real-time PCR in brain glioma tissues and cell lines. Bioinformatics software was used to predict some potential target genes of miR-338-3p. Luciferase activities assay was used to verify the combination between target genes and miR-338-3p. And MACC1 protein expression was detected by Western blot. The apoptosis and proliferation ability were analyzed by MTT and flow cytometry assay. Results Compared with normal brain tissues and cells, miR-338-3p in glioma tissues and cell lines was confirmed to be expressed at low levels, and down-regulation of miR-338-3p tended to be correlated with worse histological grade. Up-regulation of miR-338-3p promoted apoptosis and sharply inhibited cell proliferation ability of U251 and U87 cells. The luciferase activities assay, biotin-avidin pull-down assay, and western blot analysis verified that MACC1 was a specific target gene of miR-338-3p. Subsequent experiments found that up-regulation of MACC1 significantly inhibited the apoptosis and increased the cell proliferation ability of U251 and U87 cells. The regulation effects of miR-338-3p on malignant biological behaviors of glioma cells can be partly reversed by up-regulation of MACC1. Conclusions Down-regulation of miR-338-3p was an independent prognostic biomarker associated with poor prognosis in glioma patients; miR-338-3p acted as a tumor-suppressing gene whose silencing can inhibit malignant biological behaviors of glioma cells. MACC1 was a specific target gene of miR-338-3p, which regulates malignant biological behaviors of glioma cells partly through directly silencing MACC1 expression. PMID:26936749

  15. Identification of zebrafish LPTS: a gene with similarities to human LPTS/PinX1 that inhibits telomerase activity.

    PubMed

    Sun, Chengfu; Wu, Zhili; Jia, Fangjun; Wang, Yifei; Li, Tsaiping; Zhao, Mujun

    2008-08-15

    Human LPTS/PinX1 is a newly identified telomerase inhibitory protein. Overexpression of the LPTS/PinX1 gene suppresses telomerase activity, results in shortened telomeres. To investigate the role of the LPTS gene in zebrafish, we cloned the homologous gene, zLPTS, which encodes a protein of 355 amino acids. Sequence analysis revealed that, like human LPTS/PinX1, the zLPTS protein has a conserved G-patch domain at its N-terminus and a lysine-rich domain at its C-terminus. Bioinformatics analysis showed the evolutionary conservation of zLPTS. Using RT-PCR and northern blot, we found that zLPTS was expressed in all zebrafish tissues with higher level in ovary, and in all embryonic developmental stages examined. Whole mount in situ hybridization revealed that zLPTS was expressed in all regions of early developmental embryos. The subcellular localization of zLPTS protein was showed in the nucleolus and telomeres. We also cloned the gene for zebrafish Telomerase Reverse Transcriptase (zTERT), a catalytic subunit of telomerase, and demonstrated that zLPTS protein can interact with zTERT through the TR-binding domain of zTERT. Further, we verified that zLPTS could inhibit telomerase activities in zebrafish embryos and human cancer cell line by TRAP assay. Our results clearly demonstrate that zLPTS is ubiquitously expressed in tissues and embryos and plays a function of inhibiting telomerase activity. This study may provide a useful system for further investigating the mechanism of telomere length regulation. PMID:18583067

  16. Prenylation differentially inhibits insulin-dependent immediate early gene mRNA expression.

    PubMed

    Franklin, J Lee; Amsler, Maggie O; Messina, Joseph L

    2016-06-01

    Increased activity of prenyl transferases is observed in pathological states of insulin resistance, diabetes, and obesity. Thus, functional inhibitors of farnesyl transferase (FTase) and geranylgeranyl transferase (GGTase) may be promising therapeutic treatments. We previously identified insulin responsive genes from a rat H4IIE hepatoma cell cDNA library, including β-actin, EGR1, Pip92, c-fos, and Hsp60. In the present study, we investigated whether acute treatment with FTase and GGTase inhibitors would alter insulin responsive gene initiation and/or elongation rates. We observed differential regulation of insulin responsive gene expression, suggesting a differential sensitivity of these genes to one or both of the specific protein prenylation inhibitors. PMID:27086854

  17. Lysimachia foenum-graecum Herba Extract, a Novel Biopesticide, Inhibits ABC Transporter Genes and Mycelial Growth of Magnaporthe oryzae

    PubMed Central

    Lee, Youngjin

    2016-01-01

    To identify a novel biopesticide controlling rice blast disease caused by Magnaporthe oryzae, 700 plant extracts were evaluated for their inhibitory effects on mycelial growth of M. oryzae. The L. foenum-graecum Herba extract showed the lowest inhibition concentration (IC50) of 39.28 μg/ml, which is lower than the IC50 of blasticidin S (63.06 μg/ml), a conventional fungicide for rice blast disease. When treatments were combined, the IC50 of blasticidin S was dramatically reduced to 10.67 μg/ml. Since ABC transporter genes are involved in fungicide resistance of many organisms, we performed RT-PCR to investigate the transcriptional changes of 40 ABC transporter family genes of M. oryzae treated with the plant extract, blasticidin S, and tetrandrine, a recognized ABC transporter inhibitor. Four ABC transporter genes were prominently activated by blasticidin S treatment, but were suppressed by combinational treatment of blasticidin S with the plant extract, or with tetrandrine that didn’t show cellular toxicity by itself in this study. Mycelial death was detected via confocal microscopy at 24 h after plant extract treatment. Finally, subsequent rice field study revealed that the plant extract had high control efficacy of 63.3% and should be considered a biopesticide for rice blast disease. These results showed that extract of L. foenum graecum Herba suppresses M. oryzae ABC transporter genes inducing mycelial death and therefore may be a potent novel biopesticide. PMID:26889110

  18. Dopaminergic Control of Attentional Flexibility: Inhibition of Return is Associated with the Dopamine Transporter Gene (DAT1)

    PubMed Central

    Colzato, Lorenza S.; Pratt, Jay; Hommel, Bernhard

    2010-01-01

    Genetic variability related to the dopamine (DA) transporter gene (DAT1) has received increasing attention as a possible modulator of human cognition. The 9-repeat allele of the DAT1 gene is presumably associated with higher striatal DA levels than the 10-repeat allele, which might support inhibitory control functions. We investigated the impact of the DAT1 gene on the inhibition of return (IOR) effect, which refers to the fact that people are slower to detect a target if it appears in a previously attended location. 140 healthy adults, genotyped for the DAT1 gene, performed an IOR task with stimulus-onset asynchronies (SOAs) between attention cue and target of 150–1200 ms. Nine-repeat carriers showed more pronounced IOR effect than 10/10 homozygous at short SOAs but both groups of subjects eventually reached the same magnitude of IOR. Our findings support the idea that striatal DA levels promote IOR, presumably by biasing the interplay between prefrontal and striatal networks towards greater cognitive flexibility. PMID:20661460

  19. Lysimachia foenum-graecum Herba Extract, a Novel Biopesticide, Inhibits ABC Transporter Genes and Mycelial Growth of Magnaporthe oryzae.

    PubMed

    Lee, Youngjin

    2016-02-01

    To identify a novel biopesticide controlling rice blast disease caused by Magnaporthe oryzae, 700 plant extracts were evaluated for their inhibitory effects on mycelial growth of M. oryzae. The L. foenum-graecum Herba extract showed the lowest inhibition concentration (IC50) of 39.28 μg/ml, which is lower than the IC50 of blasticidin S (63.06 μg/ml), a conventional fungicide for rice blast disease. When treatments were combined, the IC50 of blasticidin S was dramatically reduced to 10.67 μg/ml. Since ABC transporter genes are involved in fungicide resistance of many organisms, we performed RT-PCR to investigate the transcriptional changes of 40 ABC transporter family genes of M. oryzae treated with the plant extract, blasticidin S, and tetrandrine, a recognized ABC transporter inhibitor. Four ABC transporter genes were prominently activated by blasticidin S treatment, but were suppressed by combinational treatment of blasticidin S with the plant extract, or with tetrandrine that didn't show cellular toxicity by itself in this study. Mycelial death was detected via confocal microscopy at 24 h after plant extract treatment. Finally, subsequent rice field study revealed that the plant extract had high control efficacy of 63.3% and should be considered a biopesticide for rice blast disease. These results showed that extract of L. foenum graecum Herba suppresses M. oryzae ABC transporter genes inducing mycelial death and therefore may be a potent novel biopesticide. PMID:26889110

  20. Short-chain fatty acids inhibit growth hormone and prolactin gene transcription via cAMP/PKA/CREB signaling pathway in dairy cow anterior pituitary cells.

    PubMed

    Wang, Jian-Fa; Fu, Shou-Peng; Li, Su-Nan; Hu, Zhong-Ming; Xue, Wen-Jing; Li, Zhi-Qiang; Huang, Bing-Xu; Lv, Qing-Kang; Liu, Ju-Xiong; Wang, Wei

    2013-01-01

    Short-chain fatty acids (SCFAs) play a key role in altering carbohydrate and lipid metabolism, influence endocrine pancreas activity, and as a precursor of ruminant milk fat. However, the effect and detailed mechanisms by which SCFAs mediate bovine growth hormone (GH) and prolactin (PRL) gene transcription remain unclear. In this study, we detected the effects of SCFAs (acetate, propionate, and butyrate) on the activity of the cAMP/PKA/CREB signaling pathway, GH, PRL, and Pit-1 gene transcription in dairy cow anterior pituitary cells (DCAPCs). The results showed that SCFAs decreased intracellular cAMP levels and a subsequent reduction in PKA activity. Inhibition of PKA activity decreased CREB phosphorylation, thereby inhibiting GH and PRL gene transcription. Furthermore, PTX blocked SCFAs- inhibited cAMP/PKA/CREB signaling pathway. These data showed that the inhibition of GH and PRL gene transcription induced by SCFAs is mediated by Gi activation and that propionate is more potent than acetate and butyrate in inhibiting GH and PRL gene transcription. In conclusion, this study identifies a biochemical mechanism for the regulation of SCFAs on bovine GH and PRL gene transcription in DCAPCs, which may serve as one of the factors that regulate pituitary function in accordance with dietary intake. PMID:24177567

  1. Androgen Inhibits Abdominal Fat Accumulation and Negatively Regulates the PCK1 Gene in Male Chickens

    PubMed Central

    Shao, Yonggang; Li, Junying; Ling, Yao; Teng, Kedao; Li, Hongwei; Wu, Changxin

    2013-01-01

    Capons are male chickens whose testes have been surgically incised. Capons show a significant increase in fat accumulation compared to intact male chickens. However, while caponization leads to a significant reduction in androgen levels in roosters, little is known about the molecular mechanisms through which androgen status affects lipogenesis in avian species. Therefore, investigation of the influence of androgens on fat accumulation in the chicken will provide insights into this process. In this study, Affymetrix microarray technology was used to analyze the gene expression profiles of livers from capons and intact male chickens because the liver is the major site of lipogenesis in avian species. Through gene ontology, we found that genes involved in hepatic lipogenic biosynthesis were the most highly enriched. Interestingly, among the upregulated genes, the cytosolic form of the phosphoenolpyruvate carboxykinase (PCK1) gene showed the greatest fold change. Additionally, in conjunction with quantitative real-time PCR data, our results suggested that androgen status negatively regulated the PCK1 gene in male chickens. PMID:23544081

  2. CD26/DPPIV inhibition alters the expression of immune response-related genes in the thymi of NOD mice.

    PubMed

    Julián, María Teresa; Alonso, Núria; Colobran, Roger; Sánchez, Alex; Miñarro, Antoni; Pujol-Autonell, Irma; Carrascal, Jorge; Rodríguez-Fernández, Silvia; Ampudia, Rosa María; Vives-Pi, Marta; Puig-Domingo, Manel

    2016-05-01

    The transmembrane glycoprotein CD26 or dipeptidyl peptidase IV (DPPIV) is a multifunctional protein. In immune system, CD26 plays a role in T-cell function and is also involved in thymic maturation and emigration patterns. In preclinical studies, treatment with DPPIV inhibitors reduces insulitis and delays or even reverses the new -onset of type 1 diabetes (T1D) in non-obese diabetic (NOD) mice. However, the specific mechanisms involved in these effects remain unknown. The aim of the present study was to investigate how DPPIV inhibition modifies the expression of genes in the thymus of NOD mice by microarray analysis. Changes in the gene expression of β-cell autoantigens and Aire in thymic epithelial cells (TECs) were also evaluated by using qRT-PCR. A DPPIV inhibitor, MK626, was orally administered in the diet for 4 and 6 weeks starting at 6-8 weeks of age. Thymic glands from treated and control mice were obtained for each study checkpoint. Thymus transcriptome analysis revealed that 58 genes were significantly over-expressed in MK626-treated mice after 6 weeks of treatment. Changes in gene expression in the thymus were confined mainly to the immune system, including innate immunity, chemotaxis, antigen presentation and immunoregulation. Most of the genes are implicated in central tolerance mechanisms through several pathways. No differences were observed in the expression of Aire and β-cell autoantigens in TECs. In the current study, we demonstrate that treatment with the DPPIV inhibitor MK626 in NOD mice alters the expression of the immune response-related genes in the thymus, especially those related to immunological central tolerance, and may contribute to the prevention of T1D. PMID:26911933

  3. Hydroxytyrosol Inhibits Cannabinoid CB1 Receptor Gene Expression in 3T3-L1 Preadipocyte Cell Line.

    PubMed

    Tutino, Valeria; Orlando, Antonella; Russo, Francesco; Notarnicola, Maria

    2016-02-01

    The 3T3-L1 preadipocyte cell line is a well characterized cell model for studying the adipocyte status and the molecular mechanisms involved in differentiation of these cells. 3T3-L1 preadipocytes have the ability to synthesize and degrade endocannabinoid anandamide (AEA) and their differentiation into adipocytes increases the expression of cannabinoid (CB1) and PPAR-γ receptors. Clinically, the blocking stimulation of the endocannabinoid pathway has been one of the first approaches proposed to counteract the obesity and obesity-associated diseases (such as diabetes, metabolic syndrome and cancer). In this connection, here we studied in cultured 3T3-L1 pre-adipocytes the effects of n-3-PUFA, α-Linolenic acid (OM-3), n-6-PUFA, Linoleic acid (OM-6), and hydroxytyrosol (HT) on the expression of CB1 receptor gene and the adipogenesis-related genes PPAR-γ, Fatty Acid Synthase (FAS) and Lipoprotein Lipase (LPL). HT was able to inhibit 3T3-L1 cell differentiation by down-regulating cell proliferation and CB1 receptor gene expression. HT exhibited anti-adipogenic effects, whereas OM-3 and OM-6 exerted an inhibitory action on cell proliferation associated with an induction of the preadipocytes differentiation and CB1 receptor gene expression. Moreover, the expression of FAS and LPL genes resulted increased after treatment with both HT and OM-3 and OM-6. The present study points out that the intake of molecules such as HT, contained in extra virgin olive oil, may be considered also in view of antiobesity and antineoplastic properties by acting directly on the adipose tissue and modulating CB1 receptor gene transcription. PMID:26189725

  4. The rolB-like part of the Agrobacterium rhizogenes orf8 gene inhibits sucrose export in tobacco.

    PubMed

    Umber, Marie; Voll, Lars; Weber, Andreas; Michler, Pierre; Otten, Léon

    2002-09-01

    Many Agrobacterium T-DNA genes belong to the highly diverse rolB family. The mode of action of most of these genes is still unknown. rolB-like sequences also are present at the 5' ends of the T-DNA-located iaaM genes and the iaaM homolog orf8, whereas iaaM genes from Pseudomonas and Erwinia spp. lack such sequences. iaaM genes encode tryptophan monooxygenases; these enzymes convert tryptophan into indole-3-acetamide, a precursor of indole-3-acetic acid. Tobacco plants expressing the rolB-like part of the A4 orf8 gene (2x35S-A4-Norf8 plants) accumulate glucose, fructose, sucrose, and starch and resemble sucrose transporter (NtSUT1) antisense plants. Different lines of evidence indicate that 2x35S-A4-Norf8 plants export less sucrose from source leaves. Glucose, fructose, sucrose, and starch accumulate in source leaves during sink-source transition, whereas sink tissues like petioles and midveins contain lower levels than normal. Petiole exudation experiments demonstrate a significant decrease in export of label after 14C-sucrose infiltration and after 14CO2 labeling. Grafting of stunted homozygous 2x35S-A4-Norf8 plants onto wild-type rootstocks restores growth, indicating that unloading is not affected. Growth of 2x35S-A4-Norf8 seedlings is inhibited on naphthalene acetic acid-containing media, suggesting a link between sucrose transport and auxin sensitivity. PMID:12236602

  5. Advanced In vivo Use of CRISPR/Cas9 and Anti-sense DNA Inhibition for Gene Manipulation in the Brain

    PubMed Central

    Walters, Brandon J.; Azam, Amber B.; Gillon, Colleen J.; Josselyn, Sheena A.; Zovkic, Iva B.

    2016-01-01

    Gene editing tools are essential for uncovering how genes mediate normal brain–behavior relationships and contribute to neurodegenerative and neuropsychiatric disorders. Recent progress in gene editing technology now allows neuroscientists unprecedented access to edit the genome efficiently. Although many important tools have been developed, here we focus on approaches that allow for rapid gene editing in the adult nervous system, particularly CRISPR/Cas9 and anti-sense nucleotide-based techniques. CRISPR/Cas9 is a flexible gene editing tool, allowing the genome to be manipulated in diverse ways. For instance, CRISPR/Cas9 has been successfully used to knockout genes, knock-in mutations, overexpress or inhibit gene activity, and provide scaffolding for recruiting specific epigenetic regulators to individual genes and gene regions. Moreover, the CRISPR/Cas9 system may be modified to target multiple genes at one time, affording simultaneous inhibition and overexpression of distinct genetic targets. Although many of the more advanced applications of CRISPR/Cas9 have not been applied to the nervous system, the toolbox is widely accessible, such that it is poised to help advance neuroscience. Anti-sense nucleotide-based technologies can be used to rapidly knockdown genes in the brain. The main advantage of anti-sense based tools is their simplicity, allowing for rapid gene delivery with minimal technical expertise. Here, we describe the main applications and functions of each of these systems with an emphasis on their many potential applications in neuroscience laboratories. PMID:26793235

  6. Inhibition of nonviral cationic liposome-mediated gene transfer into primary human respiratory cells by interferon-gamma.

    PubMed

    Sersale, Giovanna; Carpani, Daniela; Casotti, Valeria; Livraghi, Alessandra; Carrabino, Salvatore; Di Cicco, Maurizio; Assael, Baroukh M; Giunta, Annamaria; Conese, Massimo

    2002-08-01

    The effect of interferon (IFN) gamma on cationic liposome-mediated gene transfer into primary respiratory epithelial cells was investigated. Treatment of primary respiratory epithelial cells with IFN-gamma resulted in a dose-dependent increase in the intermediate filament cytokeratin 13 and a decrease in cellular proliferation, indicating that respiratory cells underwent squamous differentiation. IFN-gamma pretreatment resulted in a dramatic inhibition of transfection efficiency mediated by a cationic liposome (DOTAP). Incubation of squamous nasal cells with DOTAP/DNA complexes for various periods at 4 degrees C and evaluation of luciferase levels suggested that IFN-gamma pretreatment inhibits complex binding to the cells. In primary nasal and bronchial cells cytofluorimetric analysis demonstrated that IFN-gamma reduces binding of FITC-labeled complexes. The data indicate that differentiation of respiratory epithelial cells to a squamous phenotype, which may occur in chronic respiratory diseases such as cystic fibrosis, induces a refractory condition to gene transfer by nonviral cationic liposomes. PMID:12185450

  7. In vitro RNA interference targeting the DNA polymerase gene inhibits orf virus replication in primary ovine fetal turbinate cells.

    PubMed

    Wang, Gaili; He, Wenqi; Song, Deguang; Li, Jida; Bao, Yingfu; Lu, Rongguang; Bi, Jingying; Zhao, Kui; Gao, Feng

    2014-05-01

    Orf, which is caused by orf virus (ORFV), is distributed worldwide and is endemic in most sheep- and/or goat-raising countries. RNA interference (RNAi) pathways have emerged as important regulators of virus-host cell interactions. In this study, the specific effect of RNAi on the replication of ORFV was explored. The application of RNA interference (RNAi) inhibited the replication of ORFV in cell culture by targeting the ORF025 gene of ORFV, which encodes the viral polymerase. Three small interfering RNA (siRNA) (named siRNA704, siRNA1017 and siRNA1388) were prepared by in vitro transcription. The siRNAs were evaluated for antiviral activity against the ORFV Jilin isolate by the observation of cytopathic effects (CPE), virus titration, and real-time PCR. After 48 h of infection, siRNA704, siRNA1017 and siRNA1388 reduced virus titers by 59- to 199-fold and reduced the level of viral replication by 73-89 %. These results suggest that these three siRNAs can efficiently inhibit ORFV genome replication and infectious virus production. RNAi targeting of the DNA polymerase gene is therefore potentially useful for studying the replication of ORFV and may have potential therapeutic applications. PMID:24178308

  8. siRNA associated with immunonanoparticles directed against cd99 antigen improves gene expression inhibition in vivo in Ewing's sarcoma.

    PubMed

    Ramon, A L; Bertrand, J R; de Martimprey, H; Bernard, G; Ponchel, G; Malvy, C; Vauthier, C

    2013-07-01

    Ewing's sarcoma is a rare, mostly pediatric bone cancer that presents a chromosome abnormality called EWS/Fli-1, responsible for the development of the tumor. In vivo, tumor growth can be inhibited specifically by delivering small interfering RNA (siRNA) associated with nanoparticles. The aim of the work was to design targeted nanoparticles against the cell membrane glycoprotein cd99, which is overexpressed in Ewing's sarcoma cells to improve siRNA delivery to tumor cells. Biotinylated poly(isobutylcyanoacrylate) nanoparticles were conceived as a platform to design targeted nanoparticles with biotinylated ligands and using the biotin-streptavidin coupling method. The targeted nanoparticles were validated in vivo for the targeted delivery of siRNA after systemic administration to mice bearing a tumor model of the Ewing's sarcoma. The expression of the gene responsible of Ewing's sarcoma was inhibited at 78% ± 6% by associating the siRNA with the cd99-targeted nanoparticles compared with an inhibition of only 41% ± 9% achieved with the nontargeted nanoparticles. PMID:23657987

  9. Kamebakaurin inhibits the expression of hypoxia-inducible factor-1α and its target genes to confer antitumor activity.

    PubMed

    Wang, Ke Si; Ma, Juan; Mi, Chunliu; Li, Jing; Lee, Jung Joon; Jin, Xuejun

    2016-04-01

    Hypoxia-inducible factor 1 (HIF-1), a heterodimeric transcription factor that mediates the adaptation of tumor cells and tissues to the hypoxic microenvironment, has attracted considerable interest as a potential therapeutic target. Kamebakaurin is a diterpenoid compound isolated from Isodon excia (Maxin.) Hara, which has been used for anti-inflammatory activities. However, its antitumor activity along with molecular mechanism has not been reported. Kamebakaurin showed potent inhibitory activity against HIF-1 activation induced by hypoxia or CoCl2 in various human cancer cell lines. This compound significantly decreased the hypoxia-induced accumulation of HIF-1α protein, whereas it did not affect the expression of topoisomerase-I (Topo-I). Further analysis revealed that kamebakaurin inhibited HIF-1α protein synthesis, without affecting the expression level of HIF-1α mRNA or degradation of HIF-1α protein. Furthermore, kamebakaurin prevented hypoxia-induced expression of HIF-1 target genes for vascular endothelial growth factor (VEGF) and erythropoietin (EPO). However, kamebakaurin caused cell growth inhibition via cell cycle arrest at G1 phase in tumor cells. In vivo studies, we further confirmed the inhibitory effect of kamebakaurin on the expression of HIF-1α proteins, leading to growth inhibition of HCT116 cells in a xenograft tumor model. These results show that kamebakaurin is an effective inhibitor of HIF-1 and provide new perspectives into its anticancer activity. PMID:26781327

  10. Bresol inhibits phosphodiesterase 4 gene expression and modulates the levels of select mediators of inflammation in human monocytic cells.

    PubMed

    Sandeep Varma, R; Ashok, G; Vidyashankar, S; Nandakumar, K S; Patki, P S

    2011-01-01

    Bresol-a poly-herbal formulation, has been reported to be effective against bronchial asthma and allergic rhinitis in children. In vivo studies have supported the anti-histaminic and anti-anaphylactic action of bresol. However, the mechanism of action of bresol in modulation of inflammation has not been studied at the cellular and molecular level. The present study was aimed to elucidate the mechanism(s) of action of bresol at the cellular and molecular levels, using human monocyte leukemia cells. The effects of bresol on phosphodiesterase 4B (PDE4B) gene expression were analyzed using human monocytic U937 leukemia cells. The ability of bresol to stimulate cAMP formation in these cells, as well as its effects on mediators of inflammation like tumor necrosis factor-α (TNFα), nitric oxide (NO), and cycloxygenase-2 (COX-2) in lipopolysaccharide (LPS)-stimulated U937 cells, were also studied. The results here indicated that bresol exhibited potential anti-inflammatory properties by inhibiting LPS-induced PDE4B gene expression in the cells. Bresol also dose dependently activated cAMP formation, and inhibited TNFα, NO, as well as COX-2 formation in the LPS-stimulated cells. Based upon the results, we concluded that the reported anti-inflammatory activity of bresol might be attributed to its abilities to inhibit PDE4B and thus elevate cAMP levels in human monocytes. The anti-inflammatory effects of bresol might also be a result of the capacity of bresol to modulate the formation of TNFα, NO, and COX-2 in monocytes. PMID:21854221

  11. HBx inhibits CYP2E1 gene expression via downregulating HNF4α in human hepatoma cells.

    PubMed

    Liu, Hongming; Lou, Guiyu; Li, Chongyi; Wang, Xiaodong; Cederbaum, Arthur I; Gan, Lixia; Xie, Bin

    2014-01-01

    CYP2E1, one of the cytochrome P450 mixed-function oxidases located predominantly in liver, plays a key role in metabolism of xenobiotics including ethanol and procarcinogens. Recently, down-expression of CYP2E1 was found in hepatocellular carcinoma (HCC) with the majority to be chronic hepatitis B virus (HBV) carriers. In this study, we tested a hypothesis that HBx may inhibit CYP2E1 gene expression via hepatocyte nuclear factor 4α (HNF4α). By enforced HBx gene expression in cultured HepG2 cells, we determined the effect of HBx on CYP2E1 mRNA and protein expression. With a bioinformatics analysis, we found a consensus HNF-4α binding sequence located on -318 to -294 bp upstream of human CYP2E1 promoter. Using reporter gene assay and site-directed mutagenesis, we have shown that mutation of this site dramatically decreased CYP2E1 promoter activity. By silencing endogenous HNF-4α, we have further validated knockdown of HNF-4α significantly decreased CYP2E1 expression. Ectopic overexpression of HBx in HepG2 cells inhibits HNF-4α expression, and HNF-4α levels were inversely correlated with viral proteins both in HBV-infected HepG2215 cells and as well as HBV positive HCC liver tissues. Moreover, the HBx-induced CYP2E1 reduction could be rescued by ectopic supplement of HNF4α protein expression. Furthermore, human hepatoma cells C34, which do not express CYP2E1, shows enhanced cell growth rate compared to E47, which constitutively expresses CYP2E1. In addition, the significantly altered liver proteins in CYP2E1 knockout mice were detected with proteomics analysis. Together, HBx inhibits human CYP2E1 gene expression via downregulating HNF4α which contributes to promotion of human hepatoma cell growth. The elucidation of a HBx-HNF4α-CYP2E1 pathway provides novel insight into the molecular mechanism underlining chronic HBV infection associated hepatocarcinogenesis. PMID:25238230

  12. PTPRG inhibition By DNA methylation and cooperation with RAS gene activation in childhood acute lymphoblastic leukemia

    PubMed Central

    Xiao, Jianqiao; Lee, Seung-Tae; Xiao, Yuanyuan; Ma, Xiaomei; Houseman, E. Andres; Hsu, Ling-I; Roy, Ritu; Wrensch, Margaret; de Smith, Adam J.; Chokkalingam, Anand; Buffler, Patricia; Wiencke, John K.; Wiemels, Joseph L.

    2014-01-01

    While the cytogenetic and genetic characteristics of childhood acute lymphoblastic leukemias (ALL) are well studied, less clearly understood are the contributing epigenetic mechanisms that influence the leukemia phenotype. Our previous studies and others identified gene mutation (RAS) and DNA methylation (FHIT) to be associated with the most common cytogenetic subgroup of childhood ALL, high hyperdiploidy (having 5 more chromosomes). We screened DNA methylation profiles, using a genome-wide high dimension platform, of 166 childhood ALLs and 6 normal pre-B cell samples and observed a strong association of DNA methylation status at the PTPRG locus in human samples with levels of PTPRG gene expression as well as with RAS gene mutation status. In the 293 cell line, we found that PTPRG expression induces de-phosphorylation of ERK, a downstream RAS target which may be critical for mutant RAS-induced cell growth. In addition, PTPRG expression is up-regulated by RAS activation under DNA hypomethylating conditions. An element within the PTPRG promoter is bound by the RAS-responsive transcription factor RREB1, also under hypomethylating conditions. In conclusion, we provide evidence that DNA methylation of the PTPRG gene is a complementary event in oncogenesis induced by RAS mutations. Evidence for additional roles for PTPR family member genes is also suggested. This provides a potential therapeutic target for RAS-related leukemias as well as insight into childhood ALL etiology and pathophysiology. PMID:24496747

  13. The Drosophila Over Compensating Males Gene Genetically Inhibits Dosage Compensation in Males

    PubMed Central

    Lim, Chiat Koo; Kelley, Richard L.

    2013-01-01

    Male Drosophila are monosomic for the X chromosome, but survive due to dosage compensation. They use the Male Specific Lethal (MSL) complex composed of noncoding roX RNA and histone modifying enzymes to hypertranscribe most genes along the X ?1.61.8 fold relative to each female allele. It is not known how the MSL complex achieves this precise adjustment to a large and diverse set of target genes. We carried out a genetic screen searching for novel factors that regulate dosage compensation in flies. This strategy generated thirty alleles in a previously uncharacterized gene, over compensating males (ocm) that antagonizes some aspect of MSL activity. The mutations were initially recovered because they derepressed an MSL-dependent eye color reporter. Null ocm mutations are lethal to both sexes early in development revealing an essential function. Combinations of hypomorphic ocm alleles display a male specific lethality similar to mutations in the classic msl genes, but ocm males die due to excessive, rather than lack of dosage compensation. Males that die due to very low MSL activity can be partially rescued by ocm mutations. Likewise, males that would die from ocm mutations can be rescued by reducing the dose of various msl and roX genes. ocm encodes a large nuclear protein that shares a novel cysteine rich motif with known transcription factors. PMID:23565249

  14. DNA-Dependent Protein Kinase Inhibits AID-Induced Antibody Gene Conversion

    PubMed Central

    Cook, Adam J. L; Raftery, Joanna M; Lau, K. K. Edwin; Jessup, Andrew; Harris, Reuben S; Takeda, Shunichi; Jolly, Christopher J

    2007-01-01

    Affinity maturation and class switching of antibodies requires activation-induced cytidine deaminase (AID)-dependent hypermutation of Ig V(D)J rearrangements and Ig S regions, respectively, in activated B cells. AID deaminates deoxycytidine bases in Ig genes, converting them into deoxyuridines. In V(D)J regions, subsequent excision of the deaminated bases by uracil-DNA glycosylase, or by mismatch repair, leads to further point mutation or gene conversion, depending on the species. In Ig S regions, nicking at the abasic sites produced by AID and uracil-DNA glycosylases results in staggered double-strand breaks, whose repair by nonhomologous end joining mediates Ig class switching. We have tested whether nonhomologous end joining also plays a role in V(D)J hypermutation using chicken DT40 cells deficient for Ku70 or the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Inactivation of the Ku70 or DNA-PKcs genes in DT40 cells elevated the rate of AID-induced gene conversion as much as 5-fold. Furthermore, DNA-PKcs-deficiency appeared to reduce point mutation. The data provide strong evidence that double-strand DNA ends capable of recruiting the DNA-dependent protein kinase complex are important intermediates in Ig V gene conversion. PMID:17355182

  15. Downregulation of N1 gene expression inhibits the initial heartbeating and heart development in axolotls.

    PubMed

    Zhang, C; Meng, F; Huang, X P; Zajdel, R; Lemanski, S L; Foster, D; Erginel-Unaltuna, N; Dube, D K; Lemanski, L F

    2004-02-01

    Recessive mutant gene c in the axolotl results in a failure of affected embryos to develop contracting hearts. This abnormality can be corrected by treating the mutant heart with RNA isolated from normal anterior endoderm or from endoderm conditioned medium. A cDNA library was constructed from the total conditioned medium RNA using a random priming technique in a pcDNAII vector. We have previously identified a clone (designated as N1) from the constructed axolotl cDNA library, which has a unique nucleotide sequence. We have also discovered that the N1 gene product is related to heart development in the Mexican axolotl [Cell Mol. Biol. Res. 41 (1995) 117]. In the present studies, we further investigate the role of N1 on heartbeating and heart development in axolotls. N1 mRNA expression has been determined by using semi-quantitative RT-PCR with specifically designed primers. Normal embryonic hearts (at stages 30-31) have been transfected with anti-sense oligonucleotides against N1 to determine if downregulation of N1 gene expression has any effect on normal heart development. Our results show that cardiac N1 mRNA expression is partially blocked in the hearts transfected with anti-sense nucleotides and the downregulation of N1 gene expression results in a decrease of heartbeating in normal embryos, although the hearts remain alive as indicated by calcium spike movement throughout the hearts. Confocal microscopy data indicate some myofibril disorganization in the hearts transfected with the anti-sense N1 oligonucleotides. Interestingly, we also find that N1 gene expression is significantly decreased in the mutant axolotl hearts. Our results suggest that N1 is a novel gene in Mexican axolotls and it probably plays an important role in myofibrillogenesis and in the initiation of heartbeating during heart development. PMID:14729455

  16. 4'-Acetoamido-4-hydroxychalcone, a chalcone derivative, inhibits glioma growth and invasion through regulation of the tropomyosin 1 gene

    SciTech Connect

    Ku, Bo Mi; Ryu, Hyung Won; Lee, Yeon Kyung; Ryu, Jinhyun; Jeong, Joo Yeon; Choi, Jungil; Cho, Hee Jun; Park, Ki Hun; Kang, Sang Soo

    2010-11-19

    Research highlights: {yields} 4'-Acetoamido-4-hydroxychalcone (AHC) has anti-cancer property for glioma. {yields} 4'-Acetoamido-4-hydroxychalcone (AHC) increased tropomyosin expreesion through activattion of PKA signaling. {yields} 4'-Acetoamido-4-hydroxychalcone (AHC) inhibits glioma cell migration and invasion. {yields} In vivo administration of 4'-acetoamido-4-hydroxychalcone (AHC) reduced tumor growth. -- Abstract: Chalcones are precursors of flavonoids and have been shown to have anti-cancer activity. Here, we identify the synthetic chalcone derivative 4'-acetoamido-4-hydroxychalcone (AHC) as a potential therapeutic agent for the treatment of glioma. Treatment with AHC reduced glioma cell invasion, migration, and colony formation in a concentration-dependent manner. In addition, AHC inhibited vascular endothelial growth factor-induced migration, invasion, and tube formation in HUVECs. To determine the mechanism underlying the inhibitory effect of AHC on glioma cell invasion and migration, we investigated the effect of AHC on the gene expression change and found that AHC affects actin dynamics in U87MG glioma cells. In actin cytoskeleton regulating system, AHC increased tropomyosin expression and stress fiber formation, probably through activation of PKA. Suppression of tropomyosin expression by siRNA or treatment with the PKA inhibitor H89 reduced the inhibitory effects of AHC on glioma cell invasion and migration. In vivo experiments also showed that AHC inhibited tumor growth in a xenograft mouse tumor model. Together, these data suggest that the synthetic chalcone derivative AHC has potent anti-cancer activity through inhibition of glioma proliferation, invasion, and angiogenesis and is therefore a potential chemotherapeutic candidate for the treatment of glioma.

  17. Soluble Epoxide Hydrolase Gene Deficiency or Inhibition Attenuates Chronic Active Inflammatory bowel disease in IL-10(−/−) Mice

    PubMed Central

    Zhang, Wanying; Yang, Allison L.; Liao, Jie; Li, Haonan; Dong, Hua; Chung, Yeon Tae; Bai, Han; Matkowskyj, Kristina A.; Hammock, Bruce D.; Yang, Guang-Yu

    2013-01-01

    Background Soluble epoxide hydrolase (sEH) metabolizes anti-inflammatory epoxyeicosatrienoic acids (EETs) into their much less active dihydroxy derivatives dihydroxyeicosatrienoic acids (DHETs). Thus, targeting sEH would be important for inflammation. Aims To determine whether knockout or inhibition of sEH would attenuate the development of inflammatory bowel disease (IBD) in a mouse model of IBD in IL-10(−/−) mice. Methods Either the small molecule sEH inhibitor trans/-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB) or sEH knockout mice were used in combination with IL-10(−/−) mice. t-AUCB was administered to mice in drinking fluid. Extensive histopathologic, immunochemical and biochemical analyses were performed to evaluate effect of sEH inhibition or deficiency on chronic active inflammation and related mechanism in the bowel. Results Compared to IL-10 (−/−) mice, sEH inhibition or sEH deficiency in IL-10(−/−) mice resulted in significantly lower incidence of active ulcer formation and transmural inflammation, along with a significant decrease in myeloperoxidase-labeled neutrophil infiltration in the inflamed bowel. The levels of IFN-γ, TNF-α, and MCP-1, as well VCAM-1 and NF-kB/IKK-α signals were significantly decreased as compared to control animals. Moreover, an eicosanoid profile analysis revealed a significant increase in the ratio of EETs/DHET and EpOME/DiOME, and a slightly down-regulation of inflammatory mediators LTB4 and 5-HETE. Conclusion These results indicate that sEH gene deficiency or inhibition reduces inflammatory activities in the IL-10 (−/−) mouse model of IBD, and that sEH inhibitor could be a highly potential in the treatment of IBD. PMID:22588244

  18. Astragaloside IV Inhibits NF-κB Activation and Inflammatory Gene Expression in LPS-Treated Mice

    PubMed Central

    Zhang, Wei-Jian; Frei, Balz

    2015-01-01

    In this study we investigated the role of astragaloside IV (AS-IV), one of the major active constituents purified from the Chinese medicinal herb Astragalus membranaceus, in LPS-induced acute inflammatory responses in mice in vivo and examined possible underlying mechanisms. Mice were assigned to four groups: vehicle-treated control animals; AS-IV-treated animals (10 mg/kg b.w. AS-IV daily i.p. injection for 6 days); LPS-treated animals; and AS-IV plus LPS-treated animals. We found that AS-IV treatment significantly inhibited LPS-induced increases in serum levels of MCP-1 and TNF by 82% and 49%, respectively. AS-IV also inhibited LPS-induced upregulation of inflammatory gene expression in different organs. Lung mRNA levels of cellular adhesion molecules, MCP-1, TNFα, IL-6, and TLR4 were significantly attenuated, and lung neutrophil infiltration and activation were strongly inhibited, as reflected by decreased myeloperoxidase content, when the mice were pretreated with AS-IV. Similar results were observed in heart, aorta, kidney, and liver. Furthermore, AS-IV significantly suppressed LPS-induced NF-κB and AP-1 DNA-binding activities in lung and heart. In conclusion, our data provide new in vivo evidence that AS-IV effectively inhibits LPS-induced acute inflammatory responses by modulating NF-κB and AP-1 signaling pathways. Our results suggest that AS-IV may be useful for the prevention or treatment of inflammatory diseases. PMID:25960613

  19. Inhibition of autophagy in EBV-positive Burkitt's lymphoma cells enhances EBV lytic genes expression and replication

    PubMed Central

    De Leo, A; Colavita, F; Ciccosanti, F; Fimia, G M; Lieberman, P M; Mattia, E

    2015-01-01

    Autophagy, an important degradation system involved in maintaining cellular homeostasis, serves also to eliminate pathogens and process their fragments for presentation to the immune system. Several viruses have been shown to interact with the host autophagic machinery to suppress or make use of this cellular catabolic pathway to enhance their survival and replication. Epstein Barr virus (EBV) is a γ-herpes virus associated with a number of malignancies of epithelial and lymphoid origin in which establishes a predominantly latent infection. Latent EBV can periodically reactivate to produce infectious particles that allow the virus to spread and can lead to the death of the infected cell. In this study, we analyzed the relationship between autophagy and EBV reactivation in Burkitt's lymphoma cells. By monitoring autophagy markers and EBV lytic genes expression, we demonstrate that autophagy is enhanced in the early phases of EBV lytic activation but decreases thereafter concomitantly with increased levels of EBV lytic proteins. In a cell line defective for late antigens expression, we found an inverse correlation between EBV early antigens expression and autophagosomes formation, suggesting that early after activation, the virus is able to suppress autophagy. We report here for the first time that inhibition of autophagy by Bafilomycin A1 or shRNA knockdown of Beclin1 gene, highly incremented EBV lytic genes expression as well as intracellular viral DNA and viral progeny yield. Taken together, these findings indicate that EBV activation induces the autophagic response, which is soon inhibited by the expression of EBV early lytic products. Moreover, our findings open the possibility that pharmacological inhibitors of autophagy may be used to enhance oncolytic viral therapy of EBV-related lymphomas. PMID:26335716

  20. The histone acetyltransferase p300 inhibitor C646 reduces pro-inflammatory gene expression and inhibits histone deacetylases.

    PubMed

    van den Bosch, Thea; Boichenko, Alexander; Leus, Niek G J; Ourailidou, Maria E; Wapenaar, Hannah; Rotili, Dante; Mai, Antonello; Imhof, Axel; Bischoff, Rainer; Haisma, Hidde J; Dekker, Frank J

    2016-02-15

    Lysine acetylations are reversible posttranslational modifications of histone and non-histone proteins that play important regulatory roles in signal transduction cascades and gene expression. Lysine acetylations are regulated by histone acetyltransferases as writers and histone deacetylases as erasers. Because of their role in signal transduction cascades, these enzymes are important players in inflammation. Therefore, histone acetyltransferase inhibitors could reduce inflammatory responses. Among the few histone acetyltransferase inhibitors described, C646 is one of the most potent (Ki of 0.4μM for histone acetyltransferase p300). C646 was described to affect the NF-κB pathway; an important pathway in inflammatory responses, which is regulated by acetylation. This pathway has been implicated in asthma and COPD. Therefore, we hypothesized that via regulation of the NF-κB signaling pathway, C646 can inhibit pro-inflammatory gene expression, and have potential for the treatment of inflammatory lung diseases. In line with this, we demonstrate here that C646 reduces pro-inflammatory gene expression in RAW264.7 murine macrophages and murine precision-cut lung slices. To unravel its effects on cellular substrates we applied mass spectrometry and found, counterintuitively, a slight increase in acetylation of histone H3. Based on this finding, and structural features of C646, we presumed inhibitory activity of C646 on histone deacetylases, and indeed found inhibition of histone deacetylases from 7μM and higher concentrations. This indicates that C646 has potential for further development towards applications in the treatment of inflammation, however, its newly discovered lack of selectivity at higher concentrations needs to be taken into account. PMID:26718586

  1. Inhibition of autophagy in EBV-positive Burkitt's lymphoma cells enhances EBV lytic genes expression and replication.

    PubMed

    De Leo, A; Colavita, F; Ciccosanti, F; Fimia, G M; Lieberman, P M; Mattia, E

    2015-01-01

    Autophagy, an important degradation system involved in maintaining cellular homeostasis, serves also to eliminate pathogens and process their fragments for presentation to the immune system. Several viruses have been shown to interact with the host autophagic machinery to suppress or make use of this cellular catabolic pathway to enhance their survival and replication. Epstein Barr virus (EBV) is a γ-herpes virus associated with a number of malignancies of epithelial and lymphoid origin in which establishes a predominantly latent infection. Latent EBV can periodically reactivate to produce infectious particles that allow the virus to spread and can lead to the death of the infected cell. In this study, we analyzed the relationship between autophagy and EBV reactivation in Burkitt's lymphoma cells. By monitoring autophagy markers and EBV lytic genes expression, we demonstrate that autophagy is enhanced in the early phases of EBV lytic activation but decreases thereafter concomitantly with increased levels of EBV lytic proteins. In a cell line defective for late antigens expression, we found an inverse correlation between EBV early antigens expression and autophagosomes formation, suggesting that early after activation, the virus is able to suppress autophagy. We report here for the first time that inhibition of autophagy by Bafilomycin A1 or shRNA knockdown of Beclin1 gene, highly incremented EBV lytic genes expression as well as intracellular viral DNA and viral progeny yield. Taken together, these findings indicate that EBV activation induces the autophagic response, which is soon inhibited by the expression of EBV early lytic products. Moreover, our findings open the possibility that pharmacological inhibitors of autophagy may be used to enhance oncolytic viral therapy of EBV-related lymphomas. PMID:26335716

  2. Inhibition of bacterial cell wall-induced leukocyte recruitment and hepatic granuloma formation by TGF-beta gene transfer.

    PubMed

    Song, X; Zeng, L; Pilo, C M; Zagorski, J; Wahl, S M

    1999-10-01

    Intraperitoneal injection of streptococcal cell walls (SCW) into Lewis rats results in dissemination of SCW to the liver, spleen, bone marrow, and peripheral joints. The uptake of SCW by Kupffer cells in the liver initiates a chain of events largely mediated by T lymphocytes and macrophages. Local synthesis and secretion of cytokines and growth factors in response to the persistent SCW lead to the evolution and maintenance of a chronic T cell-dependent granulomatous response and result in granuloma formation and irreversible hepatic fibrosis. In an attempt to impede the development of the chronic granulomatous lesions in the liver, we injected a plasmid DNA encoding TGF-beta 1 i.m. to the SCW animals to determine the effect of TGF-beta 1 gene transfer on the course of liver inflammation and fibrosis. A single injection of plasmid DNA encoding TGF-beta 1 resulted in virtual abolition of the development of the SCW-induced hepatic granuloma formation and matrix expansion. TGF-beta 1 DNA not only reduced key proinflammatory cytokines including TNF-alpha, IL-1 beta, IFN-gamma, and IL-18, but also inhibited both CXC and CC chemokine production, thereby blocking inflammatory cell recruitment and accumulation in the liver. Moreover, TGF-beta 1 gene delivery inhibited its own expression in the liver tissue, which is otherwise up-regulated in SCW-injected animals. Our study suggests that TGF-beta 1 gene transfer suppresses hepatic granuloma formation by blocking the recruitment of inflammatory cells to the liver, and thus may provide a new approach to the control of hepatic granulomatous and fibrotic diseases. PMID:10491005

  3. Caffeine Induces High Expression of cyp-35A Family Genes and Inhibits the Early Larval Development in Caenorhabditis elegans

    PubMed Central

    Min, Hyemin; Kawasaki, Ichiro; Gong, Joomi; Shim, Yhong-Hee

    2015-01-01

    Intake of caffeine during pregnancy can cause retardation of fetal development. Although the significant influence of caffeine on animal development is widely recognized, much remains unknown about its mode of action because of its pleiotropic effects on living organisms. In the present study, by using Caenorhabditis elegans as a model organism, the effects of caffeine on development were examined. Brood size, embryonic lethality, and percent larval development were investigated, and caffeine was found to inhibit the development of C. elegans at most of the stages in a dosage-dependent fashion. Upon treatment with 30 mM caffeine, the majority (86.1 ± 3.4%) of the L1 larvae were irreversibly arrested without further development. In contrast, many of the late-stage larvae survived and grew to adults when exposed to the same 30 mM caffeine. These results suggest that early-stage larvae are more susceptible to caffeine than later-stage larvae. To understand the metabolic responses to caffeine treatment, the levels of expression of cytochrome P450 (cyp) genes were examined with or without caffeine treatment using comparative micro-array, and it was found that the expression of 24 cyp genes was increased by more than 2-fold (p < 0.05). Among them, induction of the cyp-35A gene family was the most prominent. Interestingly, depletion of the cyp-35A family genes one-by-one or in combination through RNA interference resulted in partial rescue from early larval developmental arrest caused by caffeine treatment, suggesting that the high-level induction of cyp-35A family genes can be fatal to the development of early-stage larvae. PMID:25591395

  4. Phosphodiesterase-4 Inhibition Alters Gene Expression and Improves Isoniazid – Mediated Clearance of Mycobacterium tuberculosis in Rabbit Lungs

    PubMed Central

    Subbian, Selvakumar; Tsenova, Liana; O'Brien, Paul; Yang, Guibin; Koo, Mi-Sun; Peixoto, Blas; Fallows, Dorothy; Dartois, Veronique; Muller, George; Kaplan, Gilla

    2011-01-01

    Tuberculosis (TB) treatment is hampered by the long duration of antibiotic therapy required to achieve cure. This indolent response has been partly attributed to the ability of subpopulations of less metabolically active Mycobacterium tuberculosis (Mtb) to withstand killing by current anti-TB drugs. We have used immune modulation with a phosphodiesterase-4 (PDE4) inhibitor, CC-3052, that reduces tumor necrosis factor alpha (TNF-α) production by increasing intracellular cAMP in macrophages, to examine the crosstalk between host and pathogen in rabbits with pulmonary TB during treatment with isoniazid (INH). Based on DNA microarray, changes in host gene expression during CC-3052 treatment of Mtb infected rabbits support a link between PDE4 inhibition and specific down-regulation of the innate immune response. The overall pattern of host gene expression in the lungs of infected rabbits treated with CC-3052, compared to untreated rabbits, was similar to that described in vitro in resting Mtb infected macrophages, suggesting suboptimal macrophage activation. These alterations in host immunity were associated with corresponding down-regulation of a number of Mtb genes that have been associated with a metabolic shift towards dormancy. Moreover, treatment with CC-3052 and INH resulted in reduced expression of those genes associated with the bacterial response to INH. Importantly, CC-3052 treatment of infected rabbits was associated with reduced ability of Mtb to withstand INH killing, shown by improved bacillary clearance, from the lungs of co-treated animals compared to rabbits treated with INH alone. The results of our study suggest that changes in Mtb gene expression, in response to changes in the host immune response, can alter the responsiveness of the bacteria to antimicrobial agents. These findings provide a basis for exploring the potential use of adjunctive immune modulation with PDE4 inhibitors to enhance the efficacy of existing anti-TB treatment. PMID:21949656

  5. Activation of PPARgamma is required for curcumin to induce apoptosis and to inhibit the expression of extracellular matrix genes in hepatic stellate cells in vitro.

    PubMed

    Zheng, Shizhong; Chen, Anping

    2004-11-15

    During liver fibrogenesis, quiescent HSC (hepatic stellate cells) become active, a transformation that is associated with enhanced cell proliferation and overproduction of ECM (extracellular matrix). Inhibition of cell proliferation and induction of apoptosis are potential strategies to block the activation of HSC for the prevention and treatment of liver fibrosis. Levels of PPARgamma (peroxisome proliferator-activated receptor gamma) are dramatically diminished in parallel with HSC activation. Stimulation of PPARgamma by its agonists inhibits HSC activation in vitro and in vivo. We demonstrated recently that curcumin, the yellow pigment in curry, inhibited HSC activation in vitro, reducing cell proliferation, inducing apoptosis and inhibiting ECM gene expression. Further studies indicated that curcumin induced the gene expression of PPARgamma and stimulated its activity in activated HSC in vitro, which was required for curcumin to inhibit HSC proliferation. The aims of the present study were to evaluate the roles of PPARgamma activation in the induction of apoptosis and suppression of ECM gene expression by curcumin in activated HSC, and to elucidate the underlying mechanisms. Our results demonstrated that blocking PPARgamma activation abrogated the effects of curcumin on the induction of apoptosis and inhibition of the expression of ECM genes in activated HSC in vitro. Further experiments demonstrated that curcumin suppressed the gene expression of TGF-beta (transforming growth factor-beta) receptors and interrupted the TGF-beta signalling pathway in activated HSC, which was mediated by PPARgamma activation. Taken together, our results demonstrate that curcumin stimulated PPARgamma activity in activated HSC in vitro, which was required for curcumin to reduce cell proliferation, induce apoptosis and suppress ECM gene expression. These results provide novel insight into the mechanisms responsible for the inhibition of HSC activation by curcumin. The characteristics of curcumin, which has no adverse health effects, make it a potential candidate for prevention and treatment of hepatic fibrosis. PMID:15320868

  6. Transfer gene expression during fertility inhibition of the Escherichia coli K12 sex factor F by the I-like plasmid R62.

    PubMed

    Gasson, M; Willetts, N

    1976-12-22

    Further understanding of how the FinQ fertility inhibition system of the I-like plasmid R62 inhibits transfer of the sex factor F has been gained by the use of a genetic assay for individual transfer gene products. The technique involved construction of a series of Flac plasmids carrying suppressible mutations in individual transfer genes together with a FinQ inhibitor-insensitive traQ mutation. The transfer of the Flac double mutants from a strain carrying wild-type Fhis and R62 then indicated the effect of R62-encoded transfer inhibition on the expression of individual F transfer genes. During such inhibition the products of genes traJ, traA, traE, traB and traC were present in quantities sufficient to permit efficient F transfer, whereas the levels of the traF, traH, traG and traD gene products were so reduced as to limit F transfer. These findings and a failure to obtain recombination between traC and traQ mutations suggest that the R62 fertility inhibition system terminates transcription of the transfer operon between traC and traF. PMID:799252

  7. A NATURALLY OCCURRING EPIGENETIC MUTATION IN AN SBP-BOX GENE INHIBITS TOMATO FRUIT RIPENING

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A major player in the regulatory network controlling fruit ripening is likely to be the gene at the tomato Colorless non-ripening (Cnr) locus 1,2. The Cnr mutation results in colorless fruits with a significant loss of cell to cell adhesion. The nature of the mutation and the identity of the Cnr g...

  8. Knockdown of Five Genes Encoding Uncharacterized Proteins Inhibits Entamoeba histolytica Phagocytosis of Dead Host Cells.

    PubMed

    Sateriale, Adam; Miller, Peter; Huston, Christopher D

    2016-04-01

    Entamoeba histolyticais the protozoan parasite that causes invasive amebiasis, which is endemic to many developing countries and characterized by dysentery and liver abscesses. The virulence ofE. histolyticacorrelates with the degree of host cell engulfment, or phagocytosis, andE. histolyticaphagocytosis alters amebic gene expression in a feed-forward manner that results in an increased phagocytic ability. Here, we used a streamlined RNA interference screen to silence the expression of 15 genes whose expression was upregulated in phagocyticE. histolyticatrophozoites to determine whether these genes actually function in the phagocytic process. When five of these genes were silenced, amebic strains with significant decreases in the ability to phagocytose apoptotic host cells were produced. Phagocytosis of live host cells, however, was largely unchanged, and the defects were surprisingly specific for phagocytosis. Two of the five encoded proteins, which we namedE. histolyticaILWEQ (EhILWEQ) andE. histolyticaBAR (EhBAR), were chosen for localization via SNAP tag labeling and localized to the site of partially formed phagosomes. Therefore, both EhILWEQ and EhBAR appear to contribute toE. histolyticavirulence through their function in phagocytosis, and the large proportion (5/15 [33%]) of gene-silenced strains with a reduced ability to phagocytose host cells validates the previously published microarray data set demonstrating feed-forward control ofE. histolyticaphagocytosis. Finally, although only limited conclusions can be drawn from studies using the virulence-deficient G3Entamoebastrain, the relative specificity of the defects induced for phagocytosis of apoptotic cells but not healthy cells suggests that cell killing may play a rate-limiting role in the process ofEntamoeba histolyticahost cell engulfment. PMID:26810036

  9. A possible mechanism for the inhibition of ribosomal RNA gene transcription during mitosis.

    PubMed

    Weisenberger, D; Scheer, U

    1995-05-01

    When cells enter mitosis, RNA synthesis ceases. Yet the RNA polymerase I (pol I) transcription machinery involved in the production of pre-rRNA remains bound to the nucleolus organizing region (NOR), the chromosome site harboring the tandemly repeated rRNA genes. Here we examine whether rDNA transcription units are transiently blocked or "frozen" during mitosis. By using fluorescent in situ hybridization we were unable to detect nascent pre-rRNA chains on the NORs of mouse 3T3 and rat kangaroo PtK2 cells. Appropriate controls showed that our approach was sensitive enough to visualize, at the light microscopic level, individual transcriptionally active rRNA genes both in situ after experimental unfolding of nucleoli and in chromatin spreads ("Miller spreads"). Analysis of the cell cycle-dependent redistribution of transcript-associated components also revealed that most transcripts are released from the rDNA at mitosis. Upon disintegration of the nucleolus during mitosis, U3 small nucleolar RNA (snoRNA) and the nucleolar proteins fibrillarin and nucleolin became dispersed throughout the cytoplasm and were excluded from the NORs. Together, our data rule out the presence of "frozen Christmas-trees" at the mitotic NORs but are compatible with the view that inactive pol I remains on the rDNA. We propose that expression of the rRNA genes is regulated during mitosis at the level of transcription elongation, similarly to what is known for a number of genes transcribed by pol II. Such a mechanism may explain the decondensed state of the NOR chromatin and the immediate transcriptional reactivation of the rRNA genes following mitosis. PMID:7730396

  10. Prenatal genotyping of four common oculocutaneous albinism genes in 51 Chinese families.

    PubMed

    Wei, Ai-Hua; Zang, Dong-Jie; Zhang, Zhao; Yang, Xiu-Min; Li, Wei

    2015-06-20

    Oculocutaneous albinism (OCA) is an autosomal recessive disorder characterized by hypopigmentation in eyes, hair and skin, accompanied with vision loss. Currently, six genes have been identified as causative genes for non-syndromic OCA (OCA-1∼4, 6, 7), and ten genes for syndromic OCA (HPS-1-9, CHS-1). Genetic counseling of 51 Chinese OCA families (39 OCA-1 with mutations in the TYR gene, 6 OCA-2 with mutations in the OCA2 gene, 4 OCA-4 with mutations in the SLC45A2 gene, 1 HPS-1 (Hermansky-Pudlak syndrome-1) with mutation in the HPS1 gene, and 1 mixed OCA-1 and OCA-4) led us to perform the prenatal genetic testing of OCA using amniotic fluid cells through the implementation of our optimized strategy. In our cohort, eleven previously unidentified alleles (PUAs) (5 in TYR, 2 in OCA2, and 4 in SLC45A2) were found. Three missense PUAs (p.C112R, p.H363R and p.G379V of TYR) and one in-frame deletional PUA (p.S222del of SLC24A5) led to fetuses with OCA when co-inherited with other disease causative alleles. Three PUAs (p.P152H and p.W272X of TYR, p.A486T of SLC24A5) identified in the OCA probands did not co-transmit with known pathological alleles and thus gave rise to unaffected fetuses. Four PUAs (p.Q83X and p.A658T of TYR, p.G161R and p.G366R of SLC24A5) did not transmit to the unaffected fetuses. In addition, the in vitro transfection assays showed that the p.S192Y variant of TYR produced less pigment compared to the wild-type allele. A fetus with a digenic carrier of OCA-1 and OCA-4 was unaffected. In combination with functional assays, the family inheritance pattern is useful for the evaluation of pathogenicity of PUAs and genetic counseling of OCA. PMID:26165494

  11. Inhibition of Chlamydiae by Primary Alcohols Correlates with the Strain-Specific Complement of Plasticity Zone Phospholipase D Genes

    PubMed Central

    Nelson, David E.; Crane, Deborah D.; Taylor, Lacey D.; Dorward, David W.; Goheen, Morgan M.; Caldwell, Harlan D.

    2006-01-01

    Members of the genus Chlamydia are obligate intracellular pathogens that have a unique biphasic developmental cycle and interactions with host cells. Many genes that dictate host infection tropism and, putatively, pathogenic manifestations of disease are clustered in a hypervariable region of the genome termed the plasticity zone (PZ). Comparative genomics studies have determined that an uncharacterized family of PZ genes encoding orthologs of eukaryotic and prokaryotic members of the phospholipase D (PLD) enzyme family varies among chlamydiae. Here, we show that the PZ PLD (pzPLD) of Chlamydia trachomatis are transcribed during both normal and persistent infection and that the corresponding PLD proteins are predominately localized in reticulate bodies on the inner leaflet of the inclusion membrane. Further, we show that strains of chlamydiae encoding the pzPLD, but not a strain lacking these genes, are inhibited by primary alcohols, potent PLD inhibitors, during growth in HeLa 229 cells. This inhibitory effect is amplified approximately 5,000-fold during recovery from persistent infection. These findings suggest that the chlamydial pzPLD may be important, strain-specific, pathogenesis factors in vivo. PMID:16368959

  12. Inhibition of Intracellular Antiviral Defense Mechanisms Augments Lentiviral Transduction of Human Natural Killer Cells: Implications for Gene Therapy

    PubMed Central

    Sutlu, Tolga; Nyström, Sanna; Gilljam, Mari; Stellan, Birgitta; Applequist, Steven E.

    2012-01-01

    Abstract Adoptive immunotherapy with genetically modified natural killer (NK) cells is a promising approach for cancer treatment. Yet, optimization of highly efficient and clinically applicable gene transfer protocols for NK cells still presents a challenge. In this study, we aimed at identifying conditions under which optimum lentiviral gene transfer to NK cells can be achieved. Our results demonstrate that stimulation of NK cells with interleukin (IL)-2 and IL-21 supports efficient transduction using a VSV-G pseudotyped lentiviral vector. Moreover, we have identified that inhibition of innate immune receptor signaling greatly enhances transduction efficiency. We were able to boost the efficiency of lentiviral genetic modification on average 3.8-fold using BX795, an inhibitor of the TBK1/IKKɛ complex acting downstream of RIG-I, MDA-5, and TLR3. We have also observed that the use of BX795 enhances lentiviral transduction efficiency in a number of human and mouse cell lines, indicating a broadly applicable, practical, and safe approach that has the potential of being applicable to various gene therapy protocols. PMID:22779406

  13. Lactobacillus zeae Protects Caenorhabditis elegans from Enterotoxigenic Escherichia coli-Caused Death by Inhibiting Enterotoxin Gene Expression of the Pathogen

    PubMed Central

    Zhou, Mengzhou; Yu, Hai; Yin, Xianhua; Sabour, Parviz M.; Chen, Wei; Gong, Joshua

    2014-01-01

    Background The nematode Caenorhabditis elegans has become increasingly used for screening antimicrobials and probiotics for pathogen control. It also provides a useful tool for studying microbe-host interactions. This study has established a C. elegans life-span assay to preselect probiotic bacteria for controlling K88+ enterotoxigenic Escherichia coli (ETEC), a pathogen causing pig diarrhea, and has determined a potential mechanism underlying the protection provided by Lactobacillus. Methodology/Principal Findings Life-span of C. elegans was used to measure the response of worms to ETEC infection and protection provided by lactic acid-producing bacteria (LAB). Among 13 LAB isolates that varied in their ability to protect C. elegans from death induced by ETEC strain JG280, Lactobacillus zeae LB1 offered the highest level of protection (86%). The treatment with Lactobacillus did not reduce ETEC JG280 colonization in the nematode intestine. Feeding E. coli strain JFF4 (K88+ but lacking enterotoxin genes of estA, estB, and elt) did not cause death of worms. There was a significant increase in gene expression of estA, estB, and elt during ETEC JG280 infection, which was remarkably inhibited by isolate LB1. The clone with either estA or estB expressed in E. coli DH5α was as effective as ETEC JG280 in killing the nematode. However, the elt clone killed only approximately 40% of worms. The killing by the clones could also be prevented by isolate LB1. The same isolate only partially inhibited the gene expression of enterotoxins in both ETEC JG280 and E. coli DH5α in-vitro. Conclusions/Significance The established life-span assay can be used for studies of probiotics to control ETEC (for effective selection and mechanistic studies). Heat-stable enterotoxins appeared to be the main factors responsible for the death of C. elegans. Inhibition of ETEC enterotoxin production, rather than interference of its intestinal colonization, appears to be the mechanism of protection offered by Lactobacillus. PMID:24558463

  14. Lentiviral vector PLV-PI3KCG gene transfer inhibits hypoxic cardiomyocytes apoptosis

    PubMed Central

    Li, Yan-Yan; Zhang, Hui; Lu, Xin-Zheng

    2015-01-01

    The PI3K/Akt signal pathway was suggested to be associated with apoptosis. However, it was still unclear whether activated PI3K/Akt signaling pathway could inhibit hypoxic cardiomyocytes apoptosis. In this research, the recombinant PI3KCG lentiviral vector plasmid (PLV-PI3KCG) was constructed and transfected into neonatal rat hypoxia/reoxygenation (H/R) injury cardiomyocytes models which were randomly divided into five groups as the normal control group, H/R group, HR empty plasmid group (HRE group), HR PLV-PI3KCG transfection preconditioning group (HRP group), and HR PLV-PI3KCG transfection + LY294002 group (HRPL group). Compared with the H/R, HRE and HRPL groups, the cardiomyocytes beat frequency and survival rate in the HRP group were significantly increased (P<0.05) and the released LDH were significantly decreased (P<0.05). The Bcl-2/Bax ratio was significantly lower in H/R, HRE and HRPL groups than that in HRP group (P<0.05). Activated PI3K/Akt signaling pathway could play a protection role in the cardiomyocytes H/R injury process which could be inhibited by LY294002. PMID:26884933

  15. Inhibition of P-glycoprotein Gene Expression and Function Enhances Triptolide-induced Hepatotoxicity in Mice

    PubMed Central

    Kong, Ling-Lei; zhuang, Xiao-Mei; Yang, Hai-Ying; Yuan, Mei; Xu, Liang; Li, Hua

    2015-01-01

    Triptolide (TP) is the major active principle of Tripterygium wilfordii Hook f. and very effective in treatment of autoimmune diseases. However, TP induced hepatotoxicity limited its clinical applications. Our previous study found that TP was a substrate of P-glycoprotein and its hepatobiliary clearance was markedly affected by P-gp modulation in sandwich-cultured rat hepatocytes. In this study, small interfering RNA (siRNA) and specific inhibitor tariquidar were used to investigate the impact of P-gp down regulation on TP-induced hepatotoxicity. The results showed that when the function of P-gp was inhibited by mdr1a-1 siRNA or tariquidar, the systemic and hepatic exposures of TP were significantly increased. The aggravated hepatotoxicity was evidenced with the remarkably lifted levels of serum biomarkers (ALT and AST) and pathological changes in liver. The other toxicological indicators (MDA, SOD and Bcl-2/Bax) were also significantly changed by P-gp inhibition. The data analysis showed that the increase of TP exposure in mice was quantitatively correlated to the enhanced hepatotoxicity, and the hepatic exposure was more relevant to the toxicity. P-gp mediated clearance played a significant role in TP detoxification. The risk of herb-drug interaction likely occurs when TP is concomitant with P-gp inhibitors or substrates in clinic. PMID:26134275

  16. N-Nicotinoyl tyramine, a novel niacinamide derivative, inhibits melanogenesis by suppressing MITF gene expression.

    PubMed

    Kim, Bora; Lee, Soung-Hoon; Choi, Kang-Yell; Kim, Hyun-Soo

    2015-10-01

    We synthesized and investigated the inhibitory effects of a novel niacinamide derivative, N-nicotinoyltyramine (NNT) on melanogenesis. NNT inhibited melanin production in B16F10 murine melanoma cells stimulated with α-melanocyte stimulating hormone (α-MSH), in human melanocyte and in three-dimensional cultured human skin model. NNT did not affect the catalytic activity of tyrosinase, but acted as an inhibitor of microphthalmia-associated transcription factor (MITF) and tyrosinase expressions in B16F10 cells. These findings suggest that the hypopigmentary effect of NNT results from the down-regulation of MITF and subsequently of tyrosinase, although NNT did not directly inhibit tyrosinase activity. In addition, safety of NNT was verified through performing neural stem cell morphology assay and Human repeated insult patch test as whitening agent. Our findings indicate that NNT may be a potential and non-skin irritant whitening agent for use in cosmetics and in the medical treatment of pigmentary disorders. PMID:26118836

  17. Genome wide transcriptional profiling in breast cancer cells reveals distinct changes in hormone receptor target genes and chromatin modifying enzymes after proteasome inhibition

    PubMed Central

    Kinyamu, H. Karimi; Collins, Jennifer B.; Grissom, Sherry F.; Hebbar, Pratibha B.; Archer, Trevor K.

    2010-01-01

    Steroid hormone receptors, like glucocorticoid (GR) and estrogen receptors (ER), are master regulators of genes that control many biological processes implicated in health and disease. Gene expression is dependent on receptor levels which are tightly regulated by the ubiquitin-proteasome system. Previous studies have shown that proteasome inhibition increases GR, but decreases ER-mediated gene expression. At the gene expression level this divergent role of the proteasome in receptor-dependent transcriptional regulation is not well understood. We have used a genomic approach to examine the impact of proteasome activity on GR and ER-mediated gene expression in MCF-7 breast cancer cells treated with dexamethasone (DEX) or 17β-estradiol (E2), the proteasome inhibitor MG132 (MG) or MG132 and either hormone (MD or ME2) for 24h. Transcript profiling reveals that inhibiting proteasome activity modulates gene expression by GR and ER in a similar manner in that several GR and ER target genes are up-regulated and down-regulated after proteasome inhibition. In addition, proteasome inhibition modulates receptor-dependent genes involved in the etiology of a number of human pathological states, including multiple myeloma, leukemia, breast/prostate cancer, HIV/AIDS and neurodegenerative disorders. Importantly, our analysis reveals that a number of transcripts encoding histone and DNA modifying enzymes, prominently histone/DNA methyltransferases and demethylases, are altered after proteasome inhibition. As proteasome inhibitors are currently in clinical trials as therapy for multiple myeloma, HIV/AIDs and leukemia, the possibility that some of the target molecules are hormone regulated and by chromatin modifying enzymes is intriguing in this era of epigenetic therapy. PMID:18381591

  18. The Rel/NF-κB pathway and transcription of immediate early genes in T cell activation are inhibited by microgravity

    PubMed Central

    Chang, Tammy T.; Walther, Isabelle; Li, Chai-Fei; Boonyaratanakornkit, Jim; Galleri, Grazia; Meloni, Maria Antonia; Pippia, Proto; Cogoli, Augusto; Hughes-Fulford, Millie

    2012-01-01

    This study tested the hypothesis that transcription of immediate early genes is inhibited in T cells activated in μg. Immunosuppression during spaceflight is a major barrier to safe, long-term human space habitation and travel. The goals of these experiments were to prove that μg was the cause of impaired T cell activation during spaceflight, as well as understand the mechanisms controlling early T cell activation. T cells from four human donors were stimulated with Con A and anti-CD28 on board the ISS. An on-board centrifuge was used to generate a 1g simultaneous control to isolate the effects of μg from other variables of spaceflight. Microarray expression analysis after 1.5 h of activation demonstrated that μg- and 1g-activated T cells had distinct patterns of global gene expression and identified 47 genes that were significantly, differentially down-regulated in μg. Importantly, several key immediate early genes were inhibited in μg. In particular, transactivation of Rel/NF-κB, CREB, and SRF gene targets were down-regulated. Expression of cREL gene targets were significantly inhibited, and transcription of cREL itself was reduced significantly in μg and upon anti-CD3/anti-CD28 stimulation in simulated μg. Analysis of gene connectivity indicated that the TNF pathway is a major early downstream effector pathway inhibited in μg and may lead to ineffective proinflammatory host defenses against infectious pathogens during spaceflight. Results from these experiments indicate that μg was the causative factor for impaired T cell activation during spaceflight by inhibiting transactivation of key immediate early genes. PMID:22750545

  19. Complete inhibition of tobamovirus multiplication by simultaneous mutations in two homologous host genes.

    PubMed

    Yamanaka, Takuya; Imai, Takahiro; Satoh, Rena; Kawashima, Arata; Takahashi, Miki; Tomita, Kayo; Kubota, Kenji; Meshi, Tetsuo; Naito, Satoshi; Ishikawa, Masayuki

    2002-03-01

    The TOM1 gene of Arabidopsis thaliana encodes a putative multipass transmembrane protein which is necessary for the efficient multiplication of tobamoviruses. We have previously shown that mutations severely destructive to the TOM1 gene reduce tobamovirus multiplication to low levels but do not impair it completely. In this report, we subjected one of the tom1 mutants (tom1-1) to another round of mutagenesis and isolated a new mutant which did not permit a detectable level of tobamovirus multiplication. In addition to tom1-1, this mutant carried a mutation referred to as tom3-1. Positional cloning showed that TOM3 was one of two TOM1-like genes in Arabidopsis. Based on the similarity between the amino acid sequences of TOM1 and TOM3, together with the results of a Sos recruitment assay suggesting that both TOM1 and TOM3 bind tobamovirus-encoded replication proteins, we propose that TOM1 and TOM3 play parallel and essential roles in the replication of tobamoviruses. PMID:11836427

  20. Engineered External Guide Sequences Are Highly Effective in Inhibiting Gene Expression and Replication of Hepatitis B Virus in Cultured Cells

    PubMed Central

    Xia, Chuan; Chen, Yuan-Chuan; Liu, Fenyong; Wu, Jianguo; Lu, Sangwei

    2013-01-01

    External guide sequences (EGSs) are RNA molecules that consist of a sequence complementary to a target mRNA and recruit intracellular ribonuclease P (RNase P), a tRNA processing enzyme, for specific degradation of the target mRNA. We have previously used an in vitro selection procedure to generate EGS variants that efficiently induce human RNase P to cleave a target mRNA in vitro. In this study, we constructed EGSs from a variant to target the overlapping region of the S mRNA, pre-S/L mRNA, and pregenomic RNA (pgRNA) of hepatitis B virus (HBV), which are essential for viral replication and infection. The EGS variant was about 50-fold more efficient in inducing human RNase P to cleave the mRNA in vitro than the EGS derived from a natural tRNA. Following Salmonella-mediated gene delivery, the EGSs were expressed in cultured HBV-carrying cells. A reduction of about 97% and 75% in the level of HBV RNAs and proteins and an inhibition of about 6,000- and 130-fold in the levels of capsid-associated HBV DNA were observed in cells treated with Salmonella vectors carrying the expression cassette for the variant and the tRNA-derived EGS, respectively. Our study provides direct evidence that the EGS variant is more effective in blocking HBV gene expression and DNA replication than the tRNA-derived EGS. Furthermore, these results demonstrate the feasibility of developing Salmonella-mediated gene delivery of highly active EGS RNA variants as a novel approach for gene-targeting applications such as anti-HBV therapy. PMID:23776459

  1. N-acetylcysteine inhibits the upregulation of mitochondrial biogenesis genes in livers from rats fed ethanol chronically

    PubMed Central

    Caro, Andres A.; Bell, Matthew; Ejiofor, Shannon; Zurcher, Grant; Petersen, Dennis R.; Ronis, Martin J. J.

    2014-01-01

    Background Chronic ethanol administration to experimental animals induces hepatic oxidative stress and upregulates mitochondrial biogenesis. The mechanisms by which chronic ethanol upregulates mitochondrial biogenesis have not been fully explored. In this work, we hypothesized that oxidative stress is a factor that triggers mitochondrial biogenesis after chronic ethanol feeding. If our hypothesis is correct, co-administration of antioxidants should prevent upregulation of mitochondrial biogenesis genes. Methods Rats were fed an ethanol-containing diet intragastrically by total enteral nutrition for 150 days, in the absence or presence of the antioxidant N-acetylcysteine (NAC) at 1.7 g/kg/day; control rats were administered isocaloric diets where carbohydrates substituted for ethanol calories. Results Ethanol administration significantly increased hepatic oxidative stress, evidenced as decreased liver total glutathione and GSH/GSSG ratio. These effects were inhibited by co-administration of ethanol and NAC. Chronic ethanol increased the expression of mitochondrial biogenesis genes including peroxisome proliferator activated receptor gamma-coactivator-1 alpha and mitochondrial transcription factor A, and mitochondrial DNA; co-administration of ethanol and NAC prevented these effects. Chronic ethanol administration was associated with decreased mitochondrial mass, inactivation and depletion of mitochondrial complex I and complex IV, and increased hepatic mitochondrial oxidative damage, effects that were not prevented by NAC. Conclusions These results suggest that oxidative stress caused by chronic ethanol triggered the upregulation of mitochondrial biogenesis genes in rat liver, because an antioxidant such as NAC prevented both effects. Because NAC did not prevent liver mitochondrial oxidative damage, extra-mitochondrial effects of reactive oxygen species may regulate mitochondrial biogenesis. In spite of the induction of hepatic mitochondrial biogenesis genes by chronic ethanol, mitochondrial mass and function decreased probably in association with mitochondrial oxidative damage. These results also predict that the effectiveness of NAC as an antioxidant therapy for chronic alcoholism will be limited by its limited antioxidant effects in mitochondria, and its inhibitory effect on mitochondrial biogenesis. PMID:25581647

  2. Targeted transcriptional activation of silent oct4 pluripotency gene by combining designer TALEs and inhibition of epigenetic modifiers

    PubMed Central

    Bultmann, Sebastian; Morbitzer, Robert; Schmidt, Christine S.; Thanisch, Katharina; Spada, Fabio; Elsaesser, Janett; Lahaye, Thomas; Leonhardt, Heinrich

    2012-01-01

    Specific control of gene activity is a valuable tool to study and engineer cellular functions. Recent studies uncovered the potential of transcription activator-like effector (TALE) proteins that can be tailored to activate user-defined target genes. It remains however unclear whether and how epigenetic modifications interfere with TALE-mediated transcriptional activation. We studied the activity of five designer TALEs (dTALEs) targeting the oct4 pluripotency gene. In vitro assays showed that the five dTALEs that target distinct sites in the oct4 promoter had the expected DNA specificity and comparable affinities to their corresponding DNA targets. In contrast to their similar in vitro properties, transcriptional activation of oct4 by these distinct dTALEs varied up to 25-fold. While dTALEs efficiently upregulated transcription of the active oct4 promoter in embryonic stem cells (ESCs) they failed to activate the silenced oct4 promoter in ESC-derived neural stem cells (NSCs), indicating that as for endogenous transcription factors also dTALE activity is limited by repressive epigenetic mechanisms. We therefore targeted the activity of epigenetic modulators and found that chemical inhibition of histone deacetylases by valproic acid or DNA methyltransferases by 5-aza-2′-deoxycytidine facilitated dTALE-mediated activation of the epigenetically silenced oct4 promoter in NSCs. Notably, demethylation of the oct4 promoter occurred only if chemical inhibitors and dTALEs were applied together but not upon treatment with inhibitors or dTALEs only. These results show that dTALEs in combination with chemical manipulation of epigenetic modifiers facilitate targeted transcriptional activation of epigenetically silenced target genes. PMID:22387464

  3. Myc Inhibits p27-Induced Erythroid Differentiation of Leukemia Cells by Repressing Erythroid Master Genes without Reversing p27-Mediated Cell Cycle Arrest▿ ‡

    PubMed Central

    Acosta, Juan C.; Ferrándiz, Nuria; Bretones, Gabriel; Torrano, Verónica; Blanco, Rosa; Richard, Carlos; O'Connell, Brenda; Sedivy, John; Delgado, M. Dolores; León, Javier

    2008-01-01

    Inhibition of differentiation has been proposed as an important mechanism for Myc-induced tumorigenesis, but the mechanisms involved are unclear. We have established a genetically defined differentiation model in human leukemia K562 cells by conditional expression of the cyclin-dependent kinase (Cdk) inhibitor p27 (inducible by Zn2+) and Myc (activatable by 4-hydroxy-tamoxifen). Induction of p27 resulted in erythroid differentiation, accompanied by Cdk inhibition and G1 arrest. Interestingly, activation of Myc inhibited p27-mediated erythroid differentiation without affecting p27-mediated proliferation arrest. Microarray-based gene expression indicated that, in the presence of p27, Myc blocked the upregulation of several erythroid-cell-specific genes, including NFE2, JUNB, and GATA1 (transcription factors with a pivotal role in erythropoiesis). Moreover, Myc also blocked the upregulation of Mad1, a transcriptional antagonist of Myc that is able to induce erythroid differentiation. Cotransfection experiments demonstrated that Myc-mediated inhibition of differentiation is partly dependent on the repression of Mad1 and GATA1. In conclusion, this model demonstrates that Myc-mediated inhibition of differentiation depends on the regulation of a specific gene program, whereas it is independent of p27-mediated cell cycle arrest. Our results support the hypothesis that differentiation inhibition is an important Myc tumorigenic mechanism that is independent of cell proliferation. PMID:18838534

  4. Myc inhibits p27-induced erythroid differentiation of leukemia cells by repressing erythroid master genes without reversing p27-mediated cell cycle arrest.

    PubMed

    Acosta, Juan C; Ferrándiz, Nuria; Bretones, Gabriel; Torrano, Verónica; Blanco, Rosa; Richard, Carlos; O'Connell, Brenda; Sedivy, John; Delgado, M Dolores; León, Javier

    2008-12-01

    Inhibition of differentiation has been proposed as an important mechanism for Myc-induced tumorigenesis, but the mechanisms involved are unclear. We have established a genetically defined differentiation model in human leukemia K562 cells by conditional expression of the cyclin-dependent kinase (Cdk) inhibitor p27 (inducible by Zn(2+)) and Myc (activatable by 4-hydroxy-tamoxifen). Induction of p27 resulted in erythroid differentiation, accompanied by Cdk inhibition and G(1) arrest. Interestingly, activation of Myc inhibited p27-mediated erythroid differentiation without affecting p27-mediated proliferation arrest. Microarray-based gene expression indicated that, in the presence of p27, Myc blocked the upregulation of several erythroid-cell-specific genes, including NFE2, JUNB, and GATA1 (transcription factors with a pivotal role in erythropoiesis). Moreover, Myc also blocked the upregulation of Mad1, a transcriptional antagonist of Myc that is able to induce erythroid differentiation. Cotransfection experiments demonstrated that Myc-mediated inhibition of differentiation is partly dependent on the repression of Mad1 and GATA1. In conclusion, this model demonstrates that Myc-mediated inhibition of differentiation depends on the regulation of a specific gene program, whereas it is independent of p27-mediated cell cycle arrest. Our results support the hypothesis that differentiation inhibition is an important Myc tumorigenic mechanism that is independent of cell proliferation. PMID:18838534

  5. Inhibition of the IKK/NF-κB pathway by AAV gene transfer improves muscle regeneration in older mdx mice.

    PubMed

    Tang, Y; Reay, D P; Salay, M N; Mi, M Y; Clemens, P R; Guttridge, D C; Robbins, P D; Huard, J; Wang, B

    2010-12-01

    The IκB kinase (IKKα, β and the regulatory subunit IKKγ) complex regulates nuclear factor of κB (NF-κB) transcriptional activity, which is upregulated in many chronic inflammatory diseases. NF-κB signaling promotes inflammation and limits muscle regeneration in Duchenne muscular dystrophy (DMD), resulting in fibrotic and fatty tissue replacement of muscle that exacerbates the wasting process in dystrophic muscles. Here, we examined whether dominant-negative forms of IKKα (IKKα-dn) and IKKβ (IKKβ-dn) delivered by adeno-associated viral (AAV) vectors to the gastrocnemius (GAS) and tibialis anterior (TA) muscles of 1, 2 and 11-month-old mdx mice, a murine DMD model, block NF-κB activation and increase muscle regeneration. At 1 month post-treatment, the levels of nuclear NF-κB in locally treated muscle were decreased by gene transfer with either AAV-CMV-IKKα-dn or AAV-CMV-IKKβ-dn, but not by IKK wild-type controls (IKKα and β) or phosphate-buffered saline (PBS). Although treatment with AAV-IKKα-dn or AAV-IKKβ-dn vectors had no significant effect on muscle regeneration in young mdx mice treated at 1 and 2 months of age and collected 1 month later, treatment of old (11 months) mdx with AAV-CMV-IKKα-dn or AAV-CMV-IKKβ-dn significantly increased levels of muscle regeneration. In addition, there was a significant decrease in myofiber necrosis in the AAV-IKKα-dn- and AAV-IKKβ-dn-treated mdx muscle in both young and old mice. These results demonstrate that inhibition of IKKα or IKKβ in dystrophic muscle reduces the adverse effects of NF-κB signaling, resulting in a therapeutic effect. Moreover, these results clearly demonstrate the therapeutic benefits of inhibiting NF-κB activation by AAV gene transfer in dystrophic muscle to promote regeneration, particularly in older mdx mice, and block necrosis. PMID:20720575

  6. Heme oxygenase-1 gene delivery by Sleeping Beauty inhibits vascular stasis in a murine model of sickle cell disease.

    PubMed

    Belcher, John D; Vineyard, Julie V; Bruzzone, Carol M; Chen, Chunsheng; Beckman, Joan D; Nguyen, Julia; Steer, Clifford J; Vercellotti, Gregory M

    2010-07-01

    Increases in heme oxygenase-1 (HO-1) and administration of heme degradation products CO and biliverdin inhibit vascular inflammation and vasoocclusion in mouse models of sickle cell disease (SCD). In this study, an albumin (alb) promoter-driven Sleeping Beauty (SB) transposase plasmid with a wild-type rat hmox-1 (wt-HO-1) transposable element was delivered by hydrodynamic tail vein injections to SCD mice. Eight weeks after injection, SCD mice had three- to five-fold increases in HO-1 activity and protein expression in liver, similar to hemin-treated mice. Immunohistochemistry demonstrated increased perinuclear HO-1 staining in hepatocytes. Messenger RNA transcription of the hmox-1 transgene in liver was confirmed by quantitative real-time polymerase chain reaction restriction fragment length polymorphism (qRT-PCR RFLP) with no detectible transgene expression in other organs. The livers of all HO-1 overexpressing mice had activation of nuclear phospho-p38 mitogen-activated protein kinase (MAPK) and phospho-Akt, decreased nuclear expression of nuclear factor-kappa B (NF-kappaB) p65, and decreased soluble vascular cell adhesion molecule-1 (sVCAM-1) in serum. Hypoxia-induced stasis, a characteristic of SCD, but not normal mice, was inhibited in dorsal skin fold chambers in wt-HO-1 SCD mice despite the absence of hmox-1 transgene expression in the skin suggesting distal effects of HO activity on the vasculature. No protective effects were seen in SCD mice injected with nonsense (ns-) rat hmox-1 that encodes carboxy-truncated HO-1 with little or no enzyme activity. We speculate that HO-1 gene delivery to the liver is beneficial in SCD mice by degrading pro-oxidative heme, releasing anti-inflammatory heme degradation products CO and biliverdin/bilirubin into circulation, activating cytoprotective pathways and inhibiting vascular stasis at sites distal to transgene expression. PMID:20306336

  7. Anesthetic drug midazolam inhibits cardiac human ether-à-go-go-related gene channels: mode of action

    PubMed Central

    Vonderlin, Nadine; Fischer, Fathima; Zitron, Edgar; Seyler, Claudia; Scherer, Daniel; Thomas, Dierk; Katus, Hugo A; Scholz, Eberhard P

    2015-01-01

    Midazolam is a short-acting benzodiazepine that is in wide clinical use as an anxiolytic, sedative, hypnotic, and anticonvulsant. Midazolam has been shown to inhibit ion channels, including calcium and potassium channels. So far, the effects of midazolam on cardiac human ether-à-go-go-related gene (hERG) channels have not been analyzed. The inhibitory effects of midazolam on heterologously expressed hERG channels were analyzed in Xenopus oocytes using the double-electrode voltage clamp technique. We found that midazolam inhibits hERG channels in a concentration-dependent manner, yielding an IC50 of 170 μM in Xenopus oocytes. When analyzed in a HEK 293 cell line using the patch-clamp technique, the IC50 was 13.6 μM. Midazolam resulted in a small negative shift of the activation curve of hERG channels. However, steady-state inactivation was not significantly affected. We further show that inhibition is state-dependent, occurring within the open and inactivated but not in the closed state. There was no frequency dependence of block. Using the hERG pore mutants F656A and Y652A we provide evidence that midazolam uses a classical binding site within the channel pore. Analyzing the subacute effects of midazolam on hERG channel trafficking, we further found that midazolam does not affect channel surface expression. Taken together, we show that the anesthetic midazolam is a low-affinity inhibitor of cardiac hERG channels without additional effects on channel surface expression. These data add to the current understanding of the pharmacological profile of the anesthetic midazolam. PMID:25733807

  8. The Flavones Apigenin and Luteolin Induce FOXO1 Translocation but Inhibit Gluconeogenic and Lipogenic Gene Expression in Human Cells

    PubMed Central

    Bumke-Vogt, Christiane; Osterhoff, Martin A.; Borchert, Andrea; Guzman-Perez, Valentina; Sarem, Zeinab; Birkenfeld, Andreas L.; Bähr, Volker; Pfeiffer, Andreas F. H.

    2014-01-01

    The flavones apigenin (4′,5,7,-trihydroxyflavone) and luteolin (3′,4′,5,7,-tetrahydroxyflavone) are plant secondary metabolites with antioxidant, antiinflammatory, and anticancer activities. We evaluated their impact on cell signaling pathways related to insulin-resistance and type 2 diabetes. Apigenin and luteolin were identified in our U-2 OS (human osteosarcoma) cell screening assay for micronutrients triggering rapid intracellular translocation of the forkhead box transcription factor O1 (FOXO1), an important mediator of insulin signal transduction. Insulin reversed the translocation of FOXO1 as shown by live cell imaging. The impact on the expression of target genes was evaluated in HepG2 (human hepatoma) cells. The mRNA-expression of the gluconeogenic enzymes phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pc), the lipogenic enzymes fatty-acid synthase (FASN) and acetyl-CoA-carboxylase (ACC) were down-regulated by both flavones with smaller effective dosages of apigenin than for luteolin. PKB/AKT-, PRAS40-, p70S6K-, and S6-phosphorylation was reduced by apigenin and luteolin but not that of the insulin-like growth factor receptor IGF-1R by apigenin indicating a direct inhibition of the PKB/AKT-signaling pathway distal to the IGF-1 receptor. N-acetyl-L-cysteine did not prevent FOXO1 nuclear translocation induced by apigenin and luteolin, suggesting that these flavones do not act via oxidative stress. The roles of FOXO1, FOXO3a, AKT, sirtuin1 (SIRT1), and nuclear factor (erythroid-derived2)-like2 (NRF2), investigated by siRNA knockdown, showed differential patterns of signal pathways involved and a role of NRF2 in the inhibition of gluconeogenic enzyme expression. We conclude that these flavones show an antidiabetic potential due to reduction of gluconeogenic and lipogenic capacity despite inhibition of the PKB/AKT pathway which justifies detailed investigation in vivo. PMID:25136826

  9. The flavones apigenin and luteolin induce FOXO1 translocation but inhibit gluconeogenic and lipogenic gene expression in human cells.

    PubMed

    Bumke-Vogt, Christiane; Osterhoff, Martin A; Borchert, Andrea; Guzman-Perez, Valentina; Sarem, Zeinab; Birkenfeld, Andreas L; Bähr, Volker; Pfeiffer, Andreas F H

    2014-01-01

    The flavones apigenin (4',5,7,-trihydroxyflavone) and luteolin (3',4',5,7,-tetrahydroxyflavone) are plant secondary metabolites with antioxidant, antiinflammatory, and anticancer activities. We evaluated their impact on cell signaling pathways related to insulin-resistance and type 2 diabetes. Apigenin and luteolin were identified in our U-2 OS (human osteosarcoma) cell screening assay for micronutrients triggering rapid intracellular translocation of the forkhead box transcription factor O1 (FOXO1), an important mediator of insulin signal transduction. Insulin reversed the translocation of FOXO1 as shown by live cell imaging. The impact on the expression of target genes was evaluated in HepG2 (human hepatoma) cells. The mRNA-expression of the gluconeogenic enzymes phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pc), the lipogenic enzymes fatty-acid synthase (FASN) and acetyl-CoA-carboxylase (ACC) were down-regulated by both flavones with smaller effective dosages of apigenin than for luteolin. PKB/AKT-, PRAS40-, p70S6K-, and S6-phosphorylation was reduced by apigenin and luteolin but not that of the insulin-like growth factor receptor IGF-1R by apigenin indicating a direct inhibition of the PKB/AKT-signaling pathway distal to the IGF-1 receptor. N-acetyl-L-cysteine did not prevent FOXO1 nuclear translocation induced by apigenin and luteolin, suggesting that these flavones do not act via oxidative stress. The roles of FOXO1, FOXO3a, AKT, sirtuin1 (SIRT1), and nuclear factor (erythroid-derived2)-like2 (NRF2), investigated by siRNA knockdown, showed differential patterns of signal pathways involved and a role of NRF2 in the inhibition of gluconeogenic enzyme expression. We conclude that these flavones show an antidiabetic potential due to reduction of gluconeogenic and lipogenic capacity despite inhibition of the PKB/AKT pathway which justifies detailed investigation in vivo. PMID:25136826

  10. Anesthetic drug midazolam inhibits cardiac human ether-à-go-go-related gene channels: mode of action.

    PubMed

    Vonderlin, Nadine; Fischer, Fathima; Zitron, Edgar; Seyler, Claudia; Scherer, Daniel; Thomas, Dierk; Katus, Hugo A; Scholz, Eberhard P

    2015-01-01

    Midazolam is a short-acting benzodiazepine that is in wide clinical use as an anxiolytic, sedative, hypnotic, and anticonvulsant. Midazolam has been shown to inhibit ion channels, including calcium and potassium channels. So far, the effects of midazolam on cardiac human ether-à-go-go-related gene (hERG) channels have not been analyzed. The inhibitory effects of midazolam on heterologously expressed hERG channels were analyzed in Xenopus oocytes using the double-electrode voltage clamp technique. We found that midazolam inhibits hERG channels in a concentration-dependent manner, yielding an IC50 of 170 μM in Xenopus oocytes. When analyzed in a HEK 293 cell line using the patch-clamp technique, the IC50 was 13.6 μM. Midazolam resulted in a small negative shift of the activation curve of hERG channels. However, steady-state inactivation was not significantly affected. We further show that inhibition is state-dependent, occurring within the open and inactivated but not in the closed state. There was no frequency dependence of block. Using the hERG pore mutants F656A and Y652A we provide evidence that midazolam uses a classical binding site within the channel pore. Analyzing the subacute effects of midazolam on hERG channel trafficking, we further found that midazolam does not affect channel surface expression. Taken together, we show that the anesthetic midazolam is a low-affinity inhibitor of cardiac hERG channels without additional effects on channel surface expression. These data add to the current understanding of the pharmacological profile of the anesthetic midazolam. PMID:25733807

  11. Mxi1 inhibits the proliferation of U87 glioma cells through down-regulation of cyclin B1 gene expression

    PubMed Central

    Manni, I; Tunici, P; Cirenei, N; Albarosa, R; Colombo, B M; Roz, L; Sacchi, A; Piaggio, G; Finocchiaro, G

    2002-01-01

    Mxi1 is a Mad family member that plays a role in cell proliferation and differentiation. To test the role of Mxi1 on tumorigenesis of glioma cells we transfected a CMV-driven MXI1 cDNA in U87 human glioblastoma cells. Two clones were isolated expressing MXI1 levels 18- and 3.5-fold higher than wild-type U87 cells (clone U87.Mxi1.14 and U87.Mxi1.22, respectively). In vivo, U87.Mxi1.14 cells were not tumorigenic in nude mice and delayed development of tumours was observed with U87.Mxi1.22 cells. In vitro, the proliferation rate was partially and strongly inhibited in U87.Mxi1.22 and U87.Mxi1.14 cells respectively. The cell cycle analysis revealed a relevant accumulation of U87.Mxi1.14 cells in the G2/M phase. Interestingly, the expression of cyclin B1 was inhibited to about 60% in U87.Mxi1.14 cells. This inhibition occurs at the transcriptional level and depends, at least in part, on the E-box present on the cyclin B1 promoter. Consistent with this, the endogenous Mxi1 binds this E-box in vitro. Thus, our findings indicate that Mxi1 can act as a tumour suppressor in human glioblastomas through a molecular mechanism involving the transcriptional down-regulation of cyclin B1 gene expression. British Journal of Cancer (2002) 86, 477–484. DOI: 10.1038/sj/bjc/6600065 www.bjcancer.com © 2002 The Cancer Research Campaign PMID:11875718

  12. Vasopressin inhibits type-I collagen and albumin gene expression in primary cultures of adult rat hepatocytes

    SciTech Connect

    Chojkier, M.; Brenner, D.A.; Leffert, H.L.

    1989-06-05

    The mechanisms that regulate collagen gene expression in hepatic cells are poorly understood. Accelerated Ca2+ fluxes are associated with inhibiting collagen synthesis selectively in human fibroblasts. In suspension cultures of isolated hepatocytes, the Ca2+ agonist vasopressin increases cytosolic levels of free Ca2+. However, whether vasopressin's interactions with plasma membrane V1 receptors attenuate hepatic collagen production is unknown. We investigated this problem by studying vasopressin's effects on collagen synthesis and Ca2+ efflux in long-term primary cultures of differentiated and proliferation-competent adult rat hepatocytes. Twelve-day-old quiescent cultures were exposed to test substances and labeled with (5-3H)proline. Determinations of radioactivity in collagenase-sensitive and collagenase-resistant proteins were used to calculate the relative levels of collagen production. Synthetic (8-arg)vasopressin stimulated 45Ca2+ efflux within 1 min and inhibited hepatocyte collagen production within 3 h by 50%; overall rates of protein synthesis were not affected significantly. In cultures labeled with (35S)methionine, vasopressin also decreased the levels of newly synthesized and secreted albumin, but not fibrinogen, detected in specific immunoprecipitates analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Northern blot analyses using specific (32P)cDNA probes revealed 70% decreases in hybridizable levels of collagen alpha 1(I) mRNA in hepatocyte cultures treated with either vasopressin or Ca2+ ionophore A23187; hybridizable levels of albumin mRNA also fell approximately 50% following vasopressin treatment.

  13. A Chemical Probe Targets DNA 5-Formylcytosine Sites and Inhibits TDG Excision, Polymerases Bypass, and Gene Expression.

    PubMed

    Xu, Liang; Chen, Ying-Chu; Nakajima, Satoshi; Chong, Jenny; Wang, Lanfeng; Lan, Li; Zhang, Chao; Wang, Dong

    2014-02-01

    Dynamic regulation and faithful maintenance of proper DNA methylation patterns are essential for many cellular functions. 5-Formylcytosine (5fC), a newly discovered oxidized form of methylcytosine (mC) is involved in active DNA demethylation process. The latest progresses suggest exciting novel functional roles of this residue. Chemical tools are desired to further elucidate the functional roles of 5fC and to modulate dynamics of DNA demethylation and downstream biological processes. Here we designed and constructed a chemical probe, consisting of an aldehyde targeting group and an intercalation group. This molecule can selectively react with 5fC and subsequently inhibit base excision by thymine DNA glycosylase (TDG) and cause significant pausing for both DNA and RNA polymerase elongation. Further investigation using a GFP reporter system in living cells revealed that the ligand modification in 5fC sites at 5'-UTR of the GFP gene greatly inhibited the GFP expression level. These results altogether confirmed our successful design and established a new approach for generating functional ligands that target the formylcytosine sites and modulate 5fC-related biological processes. PMID:24883182

  14. Hair cell stereociliary bundle regeneration by espin gene transduction after aminoglycoside damage and hair cell induction by Notch inhibition.

    PubMed

    Taura, A; Taura, K; Koyama, Y; Yamamoto, N; Nakagawa, T; Ito, J; Ryan, A F

    2016-05-01

    Once inner ear hair cells (HCs) are damaged by drugs, noise or aging, their apical structures including the stereociliary arrays are frequently the first cellular feature to be lost. Although this can be followed by progressive loss of HC somata, a significant number of HC bodies often remain even after stereociliary loss. However, in the absence of stereocilia they are nonfunctional. HCs can sometimes be regenerated by Atoh1 transduction or Notch inhibition, but they also may lack stereociliary bundles. It is therefore important to develop methods for the regeneration of stereocilia, in order to achieve HC functional recovery. Espin is an actin-bundling protein known to participate in sterociliary elongation during development. We evaluated stereociliary array regeneration in damaged vestibular sensory epithelia in tissue culture, using viral vector transduction of two espin isoforms. Utricular HCs were damaged with aminoglycosides. The utricles were then treated with a γ-secretase inhibitor, followed by espin or control transduction and histochemistry. Although γ-secretase inhibition increased the number of HCs, few had stereociliary arrays. In contrast, 46 h after espin1 transduction, a significant increase in hair-bundle-like structures was observed. These were confirmed to be immature stereociliary arrays by scanning electron microscopy. Increased uptake of FM1-43 uptake provided evidence of stereociliary function. Espin4 transduction had no effect. The results demonstrate that espin1 gene therapy can restore stereocilia on damaged or regenerated HCs. PMID:26886463

  15. Inhibition of proprotein convertase SKI-1 blocks transcription of key extracellular matrix genes regulating osteoblastic mineralization.

    PubMed

    Gorski, Jeff P; Huffman, Nichole T; Chittur, Sridar; Midura, Ronald J; Black, Claudine; Oxford, Julie; Seidah, Nabil G

    2011-01-21

    Mineralization, a characteristic phenotypic property of osteoblastic lineage cells, was blocked by 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) and decanoyl-Arg-Arg-Leu-Leu-chloromethyl ketone (dec-RRLL-cmk), inhibitors of SKI-1 (site 1; subtilisin kexin like-1) protease. Because SKI-1 is required for activation of SREBP and CREB (cAMP-response element-binding protein)/ATF family transcription factors, we tested the effect of these inhibitors on gene expression. AEBSF decreased expression of 140 genes by 1.5-3.0-fold including Phex, Dmp1, COL1A1, COL11A1, and fibronectin. Direct comparison of AEBSF and dec-RRLL-cmk, a more specific SKI-1 inhibitor, demonstrated that expression of Phex, Dmp1, COL11A1, and fibronectin was reduced by both, whereas COL1A2 and HMGCS1 were reduced only by AEBSF. AEBSF and dec-RRLL-cmk decreased the nuclear content of SKI-1-activated forms of transcription factors SREBP-1, SREBP-2, and OASIS. In contrast to AEBSF, the actions of dec-RRLL-cmk represent the sum of its direct actions on SKI-1 and indirect actions on caspase-3. Specifically, dec-RRLL-cmk reduced intracellular caspase-3 activity by blocking the formation of activated 19-kDa caspase-3. Conversely, overexpression of SKI-1-activated SREBP-1a and CREB-H in UMR106-01 osteoblastic cells increased the number of mineralized foci and altered their morphology to yield mineralization nodules, respectively. In summary, SKI-1 regulates the activation of transmembrane transcription factor precursors required for expression of key genes required for mineralization of osteoblastic cultures in vitro and bone formation in vivo. Our results indicate that the differentiated phenotype of osteoblastic cells and possibly osteocytes depends upon the non-apoptotic actions of SKI-1. PMID:21075843

  16. Liver kinase B1 inhibits the expression of inflammation-related genes postcontraction in skeletal muscle.

    PubMed

    Chen, Ting; Moore, Timothy M; Ebbert, Mark T W; McVey, Natalie L; Madsen, Steven R; Hallowell, David M; Harris, Alexander M; Char, Robin E; Mackay, Ryan P; Hancock, Chad R; Hansen, Jason M; Kauwe, John S; Thomson, David M

    2016-04-15

    Skeletal muscle-specific liver kinase B1 (LKB1) knockout mice (skmLKB1-KO) exhibit elevated mitogen-activated protein kinase (MAPK) signaling after treadmill running. MAPK activation is also associated with inflammation-related signaling in skeletal muscle. Since exercise can induce muscle damage, and inflammation is a response triggered by damaged tissue, we therefore hypothesized that LKB1 plays an important role in dampening the inflammatory response to muscle contraction, and that this may be due in part to increased susceptibility to muscle damage with contractions in LKB1-deficient muscle. Here we studied the inflammatory response and muscle damage with in situ muscle contraction or downhill running. After in situ muscle contractions, the phosphorylation of both NF-κB and STAT3 was increased more in skmLKB1-KO vs. wild-type (WT) muscles. Analysis of gene expression via microarray and RT-PCR shows that expression of many inflammation-related genes increased after contraction only in skmLKB1-KO muscles. This was associated with mild skeletal muscle fiber membrane damage in skmLKB1-KO muscles. Gene markers of oxidative stress were also elevated in skmLKB1-KO muscles after contraction. Using the downhill running model, we observed significantly more muscle damage after running in skmLKB1-KO mice, and this was associated with greater phosphorylation of both Jnk and STAT3 and increased expression of SOCS3 and Fos. In conclusion, we have shown that the lack of LKB1 in skeletal muscle leads to an increased inflammatory state in skeletal muscle that is exacerbated by muscle contraction. Increased susceptibility of the muscle to damage may underlie part of this response. PMID:26796753

  17. A Threshold Neurotoxic Amphetamine Exposure Inhibits Parietal Cortex Expression of Synaptic Plasticity-Related Genes

    PubMed Central

    Bowyer, John F.; Pogge, Amy R.; Delongchamp, Robert R.; O'Callaghan, James P.; Patel, Kruti M.; Vrana, Kent E.; Freeman, Willard M.

    2007-01-01

    Compulsive drug abuse has been conceptualized as a behavioral state where behavioral stimuli override normal decision making. Clinical studies of methamphetamine users have detailed decision making changes and imaging studies have found altered metabolism and activation in the parietal cortex. To examine the molecular effects of amphetamine on the parietal cortex, gene expression responses to amphetamine challenge (7.5mg/kg) were examined in the parietal cortex of rats pretreated for nine days with either saline, non-neurotoxic AMPH, or neurotoxic AMPH dosing regimens. The neurotoxic AMPH exposure [3 doses of 7.5 mg/kg/day AMPH (6 hr between doses), for nine days] produced histological signs of neurotoxicity in the parietal cortex while a non-neurotoxic dosing regimen (2.0 mg/kg/day × 3) did not. Neurotoxic AMPH pretreatment resulted in significantly diminished AMPH challenge-induced mRNA increases of activity-regulated cytoskeletal protein (ARC), nerve growth-factor inducible protein A (NGFI-A), and nerve growth-factor inducible protein B (NGFI-B) in the parietal cortex while neither saline pretreatment nor non-neurotoxic AMPH pretreatment did. This effect was specific to these genes as tissue plasminogen activator (t-PA), neuropeptide Y (NPY) and c-jun expression in response to AMPH challenge was unaltered or enhanced by amphetamine pretreatements. In the striatum, there were no differences between saline, neurotoxic AMPH, and non-neurotoxic AMPH pretreatments on ARC, NGFI-A or NGFI-B expression elicited by the AMPH challenge. These data indicate that the responsiveness of synaptic plasticity related genes are sensitive to disruption specifically in the parietal cortex by threshold neurotoxic AMPH exposures. PMID:17049170

  18. Significant Inhibition of Corneal Scarring In Vivo with Tissue-Selective, Targeted AAV5 Decorin Gene Therapy

    PubMed Central

    Tandon, Ashish; Sharma, Ajay; Cowden, John W.; Tovey, Jonathan C. K.

    2011-01-01

    Purpose. This study tested a hypothesis that tissue-selective targeted decorin gene therapy delivered to the stroma with adeno-associated virus serotype 5 (AAV5) inhibits corneal fibrosis in vivo without significant side effects. Methods. An in vivo rabbit model of corneal fibrosis was used. Targeted decorin gene therapy was delivered to the rabbit cornea by a single topical application of AAV5 (100 μL; 6.5 × 1012 μg/mL) onto the bare stroma for 2 minutes. The levels of corneal fibrosis were determined with stereomicroscopy, slit lamp biomicroscopy, α-smooth muscle actin (αSMA), fibronectin, and F-actin immunocytochemistry, and/or immunoblotting. CD11b, F4/80 immunocytochemistry, and TUNEL assay were used to examine immunogenicity and cytotoxicity of AAV5 to the cornea. Transmission electron microscopy (TEM) was used to investigate ultrastructural features. Slot-blot–quantified the copy number of AAV5-delivered decorin genes. Results. Selective decorin delivery into the stroma showed a significant (P < 0.01) decrease in corneal haze (1.3 ± 0.3) compared with the no-decorin-delivered control rabbit corneas (3 ± 0.4) quantified using slit lamp biomicroscopy. Immunostaining and immunoblot analyses detected significantly reduced levels of αSMA, F-actin, and fibronectin proteins (59%–73%; P < 0.001 or <0.01) in decorin-delivered rabbit corneas compared with the no-decorin-delivered controls. The visual clinical eye examination, slit lamp clinical studies, TUNEL, CD11b, and F4/80 assays revealed that AAV5-mediated decorin gene therapy is nonimmunogenic and nontoxic for the cornea. TEM studies suggested that decorin gene delivery does not jeopardize collagen fibrillogenesis as no significant differences in collagen fibril diameter and arrangement were observed in decorin-delivered and no-decorin-delivered control corneas. Conclusions. Tissue-targeted AAV5-mediated decorin gene therapy is effective and safe for treating corneal fibrosis in vivo. PMID:21551414

  19. No evidence of inhibition of horizontal gene transfer by CRISPR-Cas on evolutionary timescales.

    PubMed

    Gophna, Uri; Kristensen, David M; Wolf, Yuri I; Popa, Ovidiu; Drevet, Christine; Koonin, Eugene V

    2015-09-01

    The CRISPR (clustered, regularly, interspaced, short, palindromic repeats)-Cas (CRISPR-associated genes) systems of archaea and bacteria provide adaptive immunity against viruses and other selfish elements and are believed to curtail horizontal gene transfer (HGT). Limiting acquisition of new genetic material could be one of the sources of the fitness cost of CRISPR-Cas maintenance and one of the causes of the patchy distribution of CRISPR-Cas among bacteria, and across environments. We sought to test the hypothesis that the activity of CRISPR-Cas in microbes is negatively correlated with the extent of recent HGT. Using three independent measures of HGT, we found no significant dependence between the length of CRISPR arrays, which reflects the activity of the immune system, and the estimated number of recent HGT events. In contrast, we observed a significant negative dependence between the estimated extent of HGT and growth temperature of microbes, which could be explained by the lower genetic diversity in hotter environments. We hypothesize that the relevant events in the evolution of resistance to mobile elements and proclivity for HGT, to which CRISPR-Cas systems seem to substantially contribute, occur on the population scale rather than on the timescale of species evolution. PMID:25710183

  20. Silencing of Paternally Expressed Gene 10 Inhibits Trophoblast Proliferation and Invasion

    PubMed Central

    Chen, Haiying; Sun, Manni; Liu, Jing; Tong, Chunxiao; Meng, Tao

    2015-01-01

    Paternally expressed gene 10 (PEG10) is an imprinted and monoallelic expressed gene. Previous study using a knockout mouse model revealed a crucial role of PEG10 in placental development, yet the exact function of PEG10 during placentation remains to be elucidated. In this study, denuded chorionic villi were prepared from first trimester human placentas, and transduced with PEG10 small interference RNA (siRNA) or non-targeting control sequence by lentiviral infection. Immunohistochemical staining revealed that silencing of PEG10 in the chorionic villous explants resulted in reduced immune-reactivity to CK7, Ki67 and integrin α5, implying that silencing of PEG10 impaired the proliferation of villous trophoblasts and may interfere with the activity of extravillous trophoblasts. We further investigated the role of PEG10 in the proliferation, migration and invasion of JEG-3 trophoblast cell line and the primary chorionic villous cells. PEG10-silenced JEG-3 cells and primary chorionic villous cells displayed a reduced proliferation rate and impaired invasiveness in vitro. Silencing of PEG10 in trophoblast cells led to upregulated expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) as well as downregulated expression of matrix metalloproteinase (MMP)-2 and MMP-9. Furthermore, knockdown of TIMP-1 reversed the suppressed invasiveness of PEG10 siRNA-transduced JEG-3 cells. In conclusion, our study demonstrates that PEG10 plays an important role in trophoblast proliferation and promotes trophoblast invasion through TIMP-1. PMID:26680220

  1. Small molecule mediated inhibition of RORγ-dependent gene expression and autoimmune disease pathology in vivo.

    PubMed

    Banerjee, Daliya; Zhao, Linlin; Wu, Lan; Palanichamy, Arumugam; Ergun, Ayla; Peng, Liaomin; Quigley, Catherine; Hamann, Stefan; Dunstan, Robert; Cullen, Patrick; Allaire, Norm; Guertin, Kevin; Wang, Tao; Chao, Jianhua; Loh, Christine; Fontenot, Jason D

    2016-04-01

    Retinoic acid receptor-related orphan nuclear receptor γ (RORγ) orchestrates a pro-inflammatory gene expression programme in multiple lymphocyte lineages including T helper type 17 (Th17) cells, γδ T cells, innate lymphoid cells and lymphoid tissue inducer cells. There is compelling evidence that RORγ-expressing cells are relevant targets for therapeutic intervention in the treatment of autoimmune and inflammatory diseases. Unlike Th17 cells, where RORγ expression is induced under specific pro-inflammatory conditions, γδ T cells and other innate-like immune cells express RORγ in the steady state. Small molecule mediated disruption of RORγ function in cells with pre-existing RORγ transcriptional complexes represents a significant and challenging pharmacological hurdle. We present data demonstrating that a novel, selective and potent small molecule RORγ inhibitor can block the RORγ-dependent gene expression programme in both Th17 cells and RORγ-expressing γδ T cells as well as a disease-relevant subset of human RORγ-expressing memory T cells. Importantly, systemic administration of this inhibitor in vivo limits pathology in an innate lymphocyte-driven mouse model of psoriasis. PMID:26694902

  2. BDE-209 inhibits pluripotent genes expression and induces apoptosis in human embryonic stem cells.

    PubMed

    Du, Lili; Sun, Wen; Zhang, Huili; Chen, Dunjin

    2016-05-01

    Decabromodiphenyl ether (BDE-209) has been detected in human serum, semen, placenta, cord blood and milk worldwide. However, little is known regarding the potential effects on the early human embryonic development of BDE-209. In this study, human embryonic stem cell lines FY-hES-10 and FY-hES-26 were used to evaluate the potential effects and explore the toxification mechanisms using low-level BDE-209 exposure. Our data showed that BDE-209 exposure (1, 10 and 100 nM) reduced the expression of pluripotent genes such as OCT4, SOX2 and NANOG and induced human embryonic stem cells (hESCs) apoptosis. The downregulation of BIRC5/BCL2 and upregulation of BAX were related to apoptosis of hESCs induced by BDE-209 exposure. A mechanism study showed that OCT4 down-regulation accompanied by OCT4 promoter hypermethylation and increasing miR-145/miR-335 levels, OCT4 inhibitors. Moreover, BDE-209 could increase the generation of intracellular reactive oxygen species (ROS) and decrease SOD2 expression. The ROS increase and OCT4 downregulation after BDE-209 exposure could be reversed partly by antioxidant N-acetylcysteine supplement. These findings showed that BDE-209 exposure could decrease pluripotent genes expression via epigenetic regulation and induce apoptosis through ROS generation in human embryonic stem cells in vitro. Copyright © 2015 John Wiley & Sons, Ltd. PMID:26206603

  3. NF-kappaB inhibits transcription of the H(+)-K(+)-ATPase alpha(2)-subunit gene: role of histone deacetylases.

    PubMed

    Zhang, Wenzheng; Kone, Bruce C

    2002-11-01

    The H(+)-K(+)-ATPase alpha(2) (HKalpha(2)) gene plays a central role in potassium homeostasis, yet little is known about its transcriptional control. We recently demonstrated that the proximal promoter confers basal transcriptional activity in mouse inner medullary collecting duct 3 cells. We sought to determine whether the kappaB DNA binding element at -104 to -94 influences basal HKalpha(2) gene transcription in these cells. Recombinant NF-kappaB p50 footprinted the region -116/-94 in vitro. Gel shift and supershift analysis revealed NF-kappaB p50- and p65-containing DNA-protein complexes in nuclear extracts of mouse inner medullary collecting duct 3 cells. A promoter-luciferase construct with a mutated -104/-94 NF-kappaB element exhibited higher activity than the wild-type promoter in transfection assays. Overexpression of NF-kappaB p50, p65, or their combination trans-repressed the HKalpha(2) promoter. The histone deacetylase (HDAC) inhibitor trichostatin A partially reversed NF-kappaB-mediated trans-repression of the HKalpha(2) promoter. HDAC6 overexpression inhibited HKalpha(2) promoter activity, and HDAC6 coimmunoprecipitated with NF-kappaB p50 and p65. These results suggest that HDAC6, recruited to the DNA protein complex, acts with NF-kappaB to suppress HKalpha(2) transcription and identify NF-kappaB p50 and p65 as novel binding partners for HDAC6. PMID:12372765

  4. Systematic screen for mutants resistant to TORC1 inhibition in fission yeast reveals genes involved in cellular ageing and growth

    PubMed Central

    Rallis, Charalampos; López-Maury, Luis; Georgescu, Teodora; Pancaldi, Vera; Bähler, Jürg

    2014-01-01

    Summary Target of rapamycin complex 1 (TORC1), which controls growth in response to nutrients, promotes ageing in multiple organisms. The fission yeast Schizosaccharomyces pombe emerges as a valuable genetic model system to study TORC1 function and cellular ageing. Here we exploited the combinatorial action of rapamycin and caffeine, which inhibit fission yeast growth in a TORC1-dependent manner. We screened a deletion library, comprising ∼84% of all non-essential fission yeast genes, for drug-resistant mutants. This screen identified 33 genes encoding functions such as transcription, kinases, mitochondrial respiration, biosynthesis, intra-cellular trafficking, and stress response. Among the corresponding mutants, 5 showed shortened and 21 showed increased maximal chronological lifespans; 15 of the latter mutants showed no further lifespan increase with rapamycin and might thus represent key targets downstream of TORC1. We pursued the long-lived sck2 mutant with additional functional analyses, revealing that the Sck2p kinase functions within the TORC1 network and is required for normal cell growth, global protein translation, and ribosomal S6 protein phosphorylation in a nutrient-dependent manner. Notably, slow cell growth was associated with all long-lived mutants while oxidative-stress resistance was not. PMID:24463365

  5. Influence of lipid components on gene delivery by polycation liposomes: Transfection efficiency, intracellular kinetics and in vivo tumor inhibition.

    PubMed

    Chen, Jinliang; Sun, Xiaoyi; Yu, Zhenwei; Gao, Jianqing; Liang, Wenquan

    2012-01-17

    Transfection efficiency of non-viral gene vectors is influenced by many factors, including chemical makeup, cellular uptake pathway and intracellular delivery. To investigate the effect of lipid saturation on transfection efficiency of polycation liposomes (PCLs), a soybean phospholipids (SPL), egg phospholipids (EPL) and hydrogenated soybean phosphatidylcholine (HSPC) series was used to prepare PCLs. Testing these PCLs in a luciferase assay indicated that with increasing saturation (SPLgene expression decreased. The effect of protamine combined with these PCLs was also studied in different cell lines. Improved transfection because of protamine incorporation was dependent on lipid saturation and on the cell line tested. The kinetics of cellular uptake and intracellular distribution was studied using flow cytometry and laser scanning confocal microscope, which showed that naked oligonucleotide (ODN) and PCLs/ODN complexes became equilibrium after 4h incubation. PCLs containing SPL (PCLs-S) and 1,2-dieleoyl-sn-glycero-3-phosphoethanolamine (PCLs-D) increased uptake rates by 2.20- and 5.45-fold, respectively. Furthermore, pCMV-IL-12 transfection mediated by PCLs-D showed excellent tumor inhibition efficiency compared with control and naked pCMV-IL-12 treatments in vivo. PMID:22119962

  6. Suppressing N-Myc downstream regulated gene 1 reactivates senescence signaling and inhibits tumor growth in hepatocellular carcinoma

    PubMed Central

    Lu, Wen-Jing; Chua, Mei-Sze; So, Samuel K.

    2014-01-01

    Hepatocellular carcinoma (HCC) is the fifth most lethal malignancy worldwide with no curative therapies. To discover potentially novel therapeutic targets for HCC, we previously studied the gene expression profiles of HCC patients and identified that significant upregulation of N-Myc downstream regulated gene 1 (NDRG1) is associated with more aggressive phenotypes and poorer overall survival of HCC patients. In this study, we further used a loss-of-function approach (RNA interference) to understand the role of NDRG1 in hepatocarcinogenesis. We found that suppression of NDRG1 significantly impaired HCC cell growth through inducing extensive cellular senescence of HCC cells both in vitro and in vivo, accompanied by cell cycle arrest at the G1 phase. The observed antitumor effects of NDRG1 suppression were correlated with activation of major senescence-associated signaling pathways, such as upregulation of tumor suppressors p53, p21 and p16, and decreased phosphorylated Rb. To obtain further insights into the clinical significance of NDRG1-modulated senescence in HCC patients, immunohistochemistry staining of 92 cases of HCC patients was done. We found that high NDRG1 expression (n = 66) is associated with low p21 (n = 82; P < 0.001) and low p16 (n = 86; P < 0.001) levels. In conclusion, this study demonstrated that NDRG1 is a potential therapeutic target for HCC because its suppression triggers senescence of HCC cells through activating glycogen synthase kinase-3β–p53 pathway, thereby inhibiting tumor progression. PMID:24302615

  7. Stem cell gene therapy for HIV: strategies to inhibit viral entry and replication.

    PubMed

    DiGiusto, David L

    2015-03-01

    Since the demonstration of a cure of an HIV+ patient with an allogeneic stem cell transplant using naturally HIV-resistant cells, significant interest in creating similar autologous products has fueled the development of a variety of "cell engineering" approaches to stem cell therapy for HIV. Among the more well-studied strategies is the inhibition of viral entry through disruption of expression of viral co-receptors or through competitive inhibitors of viral fusion with the cell membrane. Preclinical evaluation of these approaches often starts in vitro but ultimately is tested in animal models prior to clinical implementation. In this review, we trace the development of several key approaches (meganucleases, short hairpin RNA (shRNA), and fusion inhibitors) to modification of hematopoietic stem cells designed to impart resistance to HIV to their T-cell and monocytic progeny. The basic evolution of technologies through in vitro and in vivo testing is discussed as well as the pros and cons of each approach and how the addition of postentry inhibitors may enhance the overall antiviral efficacy of these approaches. PMID:25578054

  8. Inhibition of Experimental Liver Cirrhosis in Mice by Telomerase Gene Delivery

    NASA Astrophysics Data System (ADS)

    Rudolph, Karl Lenhard; Chang, Sandy; Millard, Melissa; Schreiber-Agus, Nicole; DePinho, Ronald A.

    2000-02-01

    Accelerated telomere loss has been proposed to be a factor leading to end-stage organ failure in chronic diseases of high cellular turnover such as liver cirrhosis. To test this hypothesis directly, telomerase-deficient mice, null for the essential telomerase RNA (mTR) gene, were subjected to genetic, surgical, and chemical ablation of the liver. Telomere dysfunction was associated with defects in liver regeneration and accelerated the development of liver cirrhosis in response to chronic liver injury. Adenoviral delivery of mTR into the livers of mTR-/- mice with short dysfunctional telomeres restored telomerase activity and telomere function, alleviated cirrhotic pathology, and improved liver function. These studies indicate that telomere dysfunction contributes to chronic diseases of continual cellular loss-replacement and encourage the evaluation of ``telomerase therapy'' for such diseases.

  9. Inhibition of Hyperhomocysteinemia-Induced Inflammasome Activation and Glomerular Sclerosis by NLRP3 Gene Deletion

    PubMed Central

    Xia, Min; Conley, Sabena M.; Li, Guangbi; Li, Pin-Lan; Boini, Krishna M.

    2016-01-01

    Background/Aims Hyperhomocysteinemia (hHcys) has been reported to initiate Nod-like receptor protein 3 (NLRP3) inflammasome formation and activation in podocytes, leading to glomerular dysfunction and sclerosis. However, it remains unknown whether Nlrp3 gene is critical for the formation and activation of inflammasomes in glomeruli of hHcys mice. Methods Plasma homocysteine concentration was estimated utilizing HPLC, inflammasome formation and immunofluorescence expression from confocal microscopy, IL-1β production from ELISA. Results Uninephrectomized Nlrp3 knockout (Nlrp3-/-) and wild type (Nlrp3+/+) and intra renal Nlrp3 shRNA–transfected wild type mice (Nlrp3 shRNA) were fed a folate free (FF) diet or normal chow (ND) for 4 weeks to produce hHcys. The plasma Hcys levels were significantly elevated in both Nlrp3-/- and Nlrp3+/+ mice fed a FF diet compared to ND fed mice. The FF diet significantly increased the colocalization of Nlrp3 with apoptosis-associated speck-like protein (ASC) or caspase-1, caspase-1 activity and IL-1β production in glomeruli of Nlrp3+/+, but not in Nlrp3-/- mice and local Nlrp3 shRNA transfected mice. Correspondingly, the glomerular damage index (GDI) and urinary protein excretion were significantly higher in Nlrp3+/+ mice compared to ND fed mice. However, the hHcys-induced increase in GDI and proteinuria were significantly lower in Nlrp3-/- and local Nlrp3 shRNA transfected mice than in Nlrp3+/+ mice. Immunocytochemical analysis showed that hHcys decreased expression of podocin and nephrin, but increased desmin expression in glomeruli of Nlrp3+/+ mice compared to Nlrp3-/- mice. Conclusion Nlrp3 gene is an essential component of Nlrp3 inflammasomes and that targeting Nlrp3 may be important therapeutic strategy to prevent inflammasome activation and thereby protect podocytes and glomeruli from hHcys-induced injury PMID:25171193

  10. Clove bud oil reduces kynurenine and inhibits pqs A gene expression in P. aeruginosa.

    PubMed

    H, Jayalekshmi; Omanakuttan, Athira; Pandurangan, N; S Vargis, Vidhu; Maneesh, M; G Nair, Bipin; B Kumar, Geetha

    2016-04-01

    Quorum sensing (QS), a communication system involved in virulence of pathogenic bacteria like Pseudomonas aeruginosa is a promising target to combat multiple drug resistance. In vitro studies using clove bud oil (CBO) in P. aeruginosa revealed a concentration dependent attenuation of a variety of virulence factors including motility, extracellular DNA, exopolysaccharides and pigment production. Furthermore, treatment with CBO demonstrated a distinct dose-dependent reduction in biofilm formation as well as promoting dispersion of already formed biofilm, observations that were also supported by porcine skin ex vivo studies. Expression studies of genes involved in signalling systems of P. aeruginosa indicated a specific decrease in transcription of pqsA, but not in the lasI or rhlI levels. Additionally, the expression of vfr and gacA genes, involved in regulation, was also not affected by CBO treatment. CBO also influenced the PQS signalling pathway by decreasing the levels of kynurenine, an effect which was reversed by the addition of exogenous kynurenine. Though the synthesis of the signalling molecules of the Las and Rhl pathways was not affected by CBO, their activity was significantly affected, as observed by decrease in levels of their various effectors. Molecular modelling studies demonstrated that eugenol, the major component of CBO, favourably binds to the QS receptor by hydrophobic interactions as well as by hydrogen bonding with Arg61 and Tyr41 which are key amino acid residues of the LasR receptor. These results thus elucidate the molecular mechanism underlying the action of CBO and provide the basis for the identification of an attractive QS inhibitor. PMID:26821927

  11. Fenugreek extract diosgenin and pure diosgenin inhibit the hTERT gene expression in A549 lung cancer cell line.

    PubMed

    Rahmati-Yamchi, Mohammad; Ghareghomi, Somayyeh; Haddadchi, Gholamreza; Milani, Morteza; Aghazadeh, Mohammad; Daroushnejad, Hasan

    2014-09-01

    Trigonella foenum-graecum generally known as fenugreek, has been normally cultivated in Asia and Africa for the edible and medicinal values of its seeds. Fenugreek leaves and seeds have been used widely for therapeutic purposes. Fenugreek seed is recognized to show anti-diabetic and anti-nociceptive properties and other things such as hypocholesterolaemic, and anti-cancer. Diosgenin is a steroidal saponin from therapeutic herbs, fenugreek (T. foenum-graceum L.), has been well-known to have anticancer properties. Telomerase activity is not identified in usual healthy cells, while in carcinogenic cell telomerase expression is reactivated. Therefore telomerase illustrates a promising cancer therapeutic target. We deliberate the inhibitory effect of pure diosgenin and fenugreek extract diosgenin on human telomerase reverse transcriptase gene (hTERT) expression which is critical for telomerase activity. MTT-assay and qRT-PCR analysis were achieved to discover cytotoxicity effects and hTERT gene expression inhibition properties, separately. MTT results exhibited that IC50 for pure diosgenin were 47, 44 and 43 M and for fenugreek extract diosgenin were 49, 48 and 47 M for 24, 48 and 72 h after treatment. Culturing cells with pure diosgenin and fenugreek extract diosgenin treatment caused in down regulation of hTERT expression. These results indication that pure and impure diosgenin prevents telomerase activity by down regulation of the hTERT gene expression in A549 lung cancer cell line, with the difference that pure compound is more effective than another. PMID:24973886

  12. The mood stabilizer valproate activates human FGF1 gene promoter through inhibiting HDAC and GSK-3 activities.

    PubMed

    Kao, Chien-Yu; Hsu, Yi-Chao; Liu, Jen-Wei; Lee, Don-Ching; Chung, Yu-Fen; Chiu, Ing-Ming

    2013-07-01

    Valproic acid (VPA) is the primary mood-stabilizing drug to exert neuroprotective effects and to treat bipolar disorder in clinic. Fibroblast growth factor 1 (FGF1) has been shown to regulate cell proliferation, cell division, and neurogenesis. Human FGF1 gene 1B promoter (-540 to +31)-driven green fluorescence (F1BGFP) has been shown to recapitulate endogenous FGF1 gene expression and facilitates the isolation of neural stem/progenitor cells (NSPCs) from developing and adult mouse brains. In this study, we provide several lines of evidence to demonstrate the underlying mechanisms of VPA in activating FGF-1B promoter activity: (i) VPA significantly increased the FGF-1B mRNA expression and the percentage of F1BGFP(+) cells; (ii) the increase of F1BGFP expression by VPA involves changes of regulatory factor X (RFX) 1-3 transcriptional complexes and the increase of histone H3 acetylation on the 18-bp cis-element of FGF-1B promoter; (iii) treatments of other histone deacetylases (HDAC) inhibitors, sodium butyrate and trichostatin A, significantly increased the expression levels of FGF-1B, RFX2, and RFX3 transcripts; (iv) treatments of glycogen synthase kinase 3 (GSK-3) inhibitor, lithium, or GSK-3 siRNAs also significantly activated FGF-1B promoter; (v) VPA specifically enhanced neuronal differentiation in F1BGFP(+) embryonic stem cells and NSPCs rather than GFP(-) cells. This study suggested, for the first time, that VPA activates human FGF1 gene promoter through inhibiting HDAC and GSK-3 activities. PMID:23647222

  13. Nitric Oxide Synthase Gene Transfer Overcomes the Inhibition of Wound Healing by Sulfur Mustard in a Human Keratinocyte In Vitro Model

    PubMed Central

    Ishida, Hiroshi; Ray, Radharaman; Amnuaysirikul, Jack; Ishida, Keiko; Ray, Prabhati

    2012-01-01

    Sulfur mustard (SM) is a chemical warfare agent that causes extensive skin injury. Previously we reported that SM exposure resulted in suppression of inducible nitric oxide synthase (iNOS) expression to inhibit the healing of scratch wounds in a cultured normal human epidermal keratinocyte (NHEK) model. Based on this finding, the present study was to use adenovirus-mediated gene transfer of iNOS to restore the nitric oxide (NO) supply depleted by exposure to SM and to evaluate the effect of NO on wound healing inhibited by SM in NHEKs. The effect of the iNOS gene transfer on iNOS protein expression and NO generation were monitored by Western blot and flow cytometry, respectively. Wound healing with or without the iNOS gene transfer after SM exposure was assessed by light and confocal microscopy. The iNOS gene transfer via adenovirus resulted in overexpression of the iNOS and an increase in NO production regardless of SM exposure in the NHEK model. The gene transfer was also effective in overcoming the inhibition of wound healing due to SM exposure leading to the promotion of wound closure. The findings in this study suggest that the iNOS gene transfer is a promising therapeutic strategy for SM-induced skin injury. PMID:23762631

  14. A gene therapy strategy using a transcription factor decoy of the E2F binding site inhibits smooth muscle proliferation in vivo.

    PubMed Central

    Morishita, R; Gibbons, G H; Horiuchi, M; Ellison, K E; Nakama, M; Zhang, L; Kaneda, Y; Ogihara, T; Dzau, V J

    1995-01-01

    The application of DNA technology to regulate the transcription of disease-related genes in vivo has important therapeutic potentials. The transcription factor E2F plays a pivotal role in the coordinated transactivation of cell cycle-regulatory genes such as c-myc, cdc2, and the gene encoding proliferating-cell nuclear antigen (PCNA) that are involved in lesion formation after vascular injury. We hypothesized that double-stranded DNA with high affinity for E2F may be introduced in vivo as a decoy to bind E2F and block the activation of genes mediating cell cycle progression and intimal hyperplasia after vascular injury. Gel mobility-shift assays showed complete competition for E2F binding protein by the E2F decoy. Transfection with E2F decoy inhibited expression of c-myc, cdc2, and the PCNA gene as well as vascular smooth muscle cell proliferation both in vitro and in the in vivo model of rat carotid injury. Furthermore, 2 weeks after in vivo transfection, neointimal formation was significantly prevented by the E2F decoy, and this inhibition continued up to 8 weeks after a single transfection in a dose-dependent manner. Transfer of an E2F decoy can therefore modulate gene expression and inhibit smooth muscle proliferation and vascular lesion formation in vivo. Images Fig. 1 Fig. 3 Fig. 4 PMID:7597041

  15. Inhibition of de novo Palmitate Synthesis by Fatty Acid Synthase Induces Apoptosis in Tumor Cells by Remodeling Cell Membranes, Inhibiting Signaling Pathways, and Reprogramming Gene Expression

    PubMed Central

    Ventura, Richard; Mordec, Kasia; Waszczuk, Joanna; Wang, Zhaoti; Lai, Julie; Fridlib, Marina; Buckley, Douglas; Kemble, George; Heuer, Timothy S.

    2015-01-01

    Inhibition of de novo palmitate synthesis via fatty acid synthase (FASN) inhibition provides an unproven approach to cancer therapy with a strong biological rationale. FASN expression increases with tumor progression and associates with chemoresistance, tumor metastasis, and diminished patient survival in numerous tumor types. TVB-3166, an orally-available, reversible, potent, and selective FASN inhibitor induces apoptosis, inhibits anchorage-independent cell growth under lipid-rich conditions, and inhibits in-vivo xenograft tumor growth. Dose-dependent effects are observed between 20–200 nM TVB-3166, which agrees with the IC50 in biochemical FASN and cellular palmitate synthesis assays. Mechanistic studies show that FASN inhibition disrupts lipid raft architecture, inhibits biological pathways such as lipid biosynthesis, PI3K–AKT–mTOR and β-catenin signal transduction, and inhibits expression of oncogenic effectors such as c-Myc; effects that are tumor-cell specific. Our results demonstrate that FASN inhibition has anti-tumor activities in biologically diverse preclinical tumor models and provide mechanistic and pharmacologic evidence that FASN inhibition presents a promising therapeutic strategy for treating a variety of cancers, including those expressing mutant K-Ras, ErbB2, c-Met, and PTEN. The reported findings inform ongoing studies to link mechanisms of action with defined tumor types and advance the discovery of biomarkers supporting development of FASN inhibitors as cancer therapeutics. Research in context Fatty acid synthase (FASN) is a vital enzyme in tumor cell biology; the over-expression of FASN is associated with diminished patient prognosis and resistance to many cancer therapies. Our data demonstrate that selective and potent FASN inhibition with TVB-3166 leads to selective death of tumor cells, without significant effect on normal cells, and inhibits in vivo xenograft tumor growth at well-tolerated doses. Candidate biomarkers for selecting tumors highly sensitive to FASN inhibition are identified. These preclinical data provide mechanistic and pharmacologic evidence that FASN inhibition presents a promising therapeutic strategy for treating a variety of cancers. PMID:26425687

  16. Exogenous C2 Ceramide Suppresses Matrix Metalloproteinase Gene Expression by Inhibiting ROS Production and MAPK Signaling Pathways in PMA-Stimulated Human Astroglioma Cells

    PubMed Central

    Jung, Ji-Sun; Ahn, Young-Ho; Moon, Byung-In; Kim, Hee-Sun

    2016-01-01

    Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases, which play a pivotal role in invasion, migration, and angiogenesis of glioma. Therefore, controlling MMPs is potentially an important therapeutic strategy for glioma. In the present study, we found that exogenous cell-permeable short-chain C2 ceramide inhibits phorbol myristate acetate (PMA)-induced MMP-1, -3, and -9 gene expressions in U87MG and U373MG human astroglioma cells. In addition, C2 ceramide inhibited the protein secretion and enzymatic activities of MMP-1, -3, and -9. The Matrigel invasion assay and wound healing assay showed that C2 ceramide suppresses the in vitro invasion and migration of glioma cells, which appears to be involved in strong inhibition of MMPs by C2 ceramide. Subsequent mechanistic studies revealed that C2 ceramide inhibits PMA-induced mitogen-activated protein kinase (MAPK) phosphorylation and nuclear factor (NF)-κB/activator protein (AP)-1 DNA binding activities. Furthermore, C2 ceramide significantly inhibited PMA-induced reactive oxygen species (ROS) production and NADPH oxidase 4 (NOX4) expression, and inhibition of ROS by diphenylene iodonium (DPI, NADPH oxidase inhibitor) mimicked the effects of C2 ceramide on MMP expression and NF-κB/AP-1 via inhibition of p38 MAPK. The results suggest C2 ceramide inhibits MMP expression and glioma invasion, at least partly, by modulating ROS-p38 MAPK signaling axis and other MAPK signaling pathways. PMID:27043542

  17. Novel mechanism of gene regulation: the protein Rv1222 of Mycobacterium tuberculosis inhibits transcription by anchoring the RNA polymerase onto DNA

    PubMed Central

    Rudra, Paulami; Prajapati, Ranjit Kumar; Banerjee, Rajdeep; Sengupta, Shreya; Mukhopadhyay, Jayanta

    2015-01-01

    We propose a novel mechanism of gene regulation in Mycobacterium tuberculosis where the protein Rv1222 inhibits transcription by anchoring RNA polymerase (RNAP) onto DNA. In contrast to our existing knowledge that transcriptional repressors function either by binding to DNA at specific sequences or by binding to RNAP, we show that Rv1222-mediated transcription inhibition requires simultaneous binding of the protein to both RNAP and DNA. We demonstrate that the positively charged C-terminus tail of Rv1222 is responsible for anchoring RNAP on DNA, hence the protein slows down the movement of RNAP along the DNA during transcription elongation. The interaction between Rv1222 and DNA is electrostatic, thus the protein could inhibit transcription from any gene. As Rv1222 slows down the RNA synthesis, upon expression of the protein in Mycobacterium smegmatis or Escherichia coli, the growth rate of the bacteria is severely impaired. The protein does not possess any significant affinity for DNA polymerase, thus, is unable to inhibit DNA synthesis. The proposed mechanism by which Rv1222 inhibits transcription reveals a new repertoire of prokaryotic gene regulation. PMID:25999340

  18. Acetylbritannilatone suppresses NO and PGE2 synthesis in RAW 264.7 macrophages through the inhibition of iNOS and COX-2 gene expression.

    PubMed

    Han, Mei; Wen, Jin-Kun; Zheng, Bin; Zhang, Di-Qun

    2004-06-25

    In order to elucidate the mechanism of anti-inflammatory effect of 1-o-acetylbritannilatone (ABL) isolated from Inula Britannica-F, we investigated ABL for its ability to inhibit the inflammatory factor production in RAW 264.7 macrophages. The studies showed that ABL not only inhibited LPS/IFN-gamma-mediated nitric oxide (NO) production and inducible nitric synthase (iNOS) expression, but also decreased LPS/IFN-gamma-induced prostaglandin E2 (PGE2) production and cyclo-oxygenase-2 (COX-2) expression in a concentration-dependent manner. EMSA demonstrated that ABL inhibited effectively the association of NF-kappaB, which is necessary for the expression of iNOS and COX-2, with its binding motif in the promoter of target genes. These data suggest that ABL suppress NO and PGE2 synthesis in RAW 264.7 macrophages through the inhibition of iNOS and COX-2 gene expression, respectively. The anti-inflammatory effect of ABL involves blocking the binding of NF-kappaB to the promoter in the target genes and inhibiting the expression of iNOS and COX-2. PMID:15172177

  19. Momordica charantia (bitter melon) inhibits primary human adipocyte differentiation by modulating adipogenic genes

    PubMed Central

    2010-01-01

    Background Escalating trends of obesity and associated type 2 diabetes (T2D) has prompted an increase in the use of alternative and complementary functional foods. Momordica charantia or bitter melon (BM) that is traditionally used to treat diabetes and complications has been demonstrated to alleviate hyperglycemia as well as reduce adiposity in rodents. However, its effects on human adipocytes remain unknown. The objective of our study was to investigate the effects of BM juice (BMJ) on lipid accumulation and adipocyte differentiation transcription factors in primary human differentiating preadipocytes and adipocytes. Methods Commercially available cryopreserved primary human preadipocytes were treated with and without BMJ during and after differentiation. Cytotoxicity, lipid accumulation, and adipogenic genes mRNA expression was measured by commercial enzymatic assay kits and semi-quantitative RT-PCR (RT-PCR). Results Preadipocytes treated with varying concentrations of BMJ during differentiation demonstrated significant reduction in lipid content with a concomitant reduction in mRNA expression of adipocyte transcription factors such as, peroxisome proliferator-associated receptor γ (PPARγ) and sterol regulatory element-binding protein 1c (SREBP-1c) and adipocytokine, resistin. Similarly, adipocytes treated with BMJ for 48 h demonstrated reduced lipid content, perilipin mRNA expression, and increased lipolysis as measured by the release of glycerol. Conclusion Our data suggests that BMJ is a potent inhibitor of lipogenesis and stimulator of lipolysis activity in human adipocytes. BMJ may therefore prove to be an effective complementary or alternative therapy to reduce adipogenesis in humans. PMID:20587058

  20. Mutations in the Gabrb1 gene promote alcohol consumption through increased tonic inhibition.

    PubMed

    Anstee, Quentin M; Knapp, Susanne; Maguire, Edward P; Hosie, Alastair M; Thomas, Philip; Mortensen, Martin; Bhome, Rohan; Martinez, Alonso; Walker, Sophie E; Dixon, Claire I; Ruparelia, Kush; Montagnese, Sara; Kuo, Yu-Ting; Herlihy, Amy; Bell, Jimmy D; Robinson, Iain; Guerrini, Irene; McQuillin, Andrew; Fisher, Elizabeth M C; Ungless, Mark A; Gurling, Hugh M D; Morgan, Marsha Y; Brown, Steve D M; Stephens, David N; Belelli, Delia; Lambert, Jeremy J; Smart, Trevor G; Thomas, Howard C

    2013-01-01

    Alcohol dependence is a common, complex and debilitating disorder with genetic and environmental influences. Here we show that alcohol consumption increases following mutations to the γ-aminobutyric acidA receptor (GABAAR) β1 subunit gene (Gabrb1). Using N-ethyl-N-nitrosourea mutagenesis on an alcohol-averse background (F1 BALB/cAnN x C3H/HeH), we develop a mouse model exhibiting strong heritable preference for ethanol resulting from a dominant mutation (L285R) in Gabrb1. The mutation causes spontaneous GABA ion channel opening and increases GABA sensitivity of recombinant GABAARs, coupled to increased tonic currents in the nucleus accumbens, a region long-associated with alcohol reward. Mutant mice work harder to obtain ethanol, and are more sensitive to alcohol intoxication. Another spontaneous mutation (P228H) in Gabrb1 also causes high ethanol consumption accompanied by spontaneous GABA ion channel opening and increased accumbal tonic current. Our results provide a new and important link between GABAAR function and increased alcohol consumption that could underlie some forms of alcohol abuse. PMID:24281383

  1. Resveratrol Inhibits Sodium/Iodide Symporter Gene Expression and Function in Rat Thyroid Cells

    PubMed Central

    Giuliani, Cesidio; Bucci, Ines; Di Santo, Serena; Rossi, Cosmo; Grassadonia, Antonino; Mariotti, Marianna; Piantelli, Mauro; Monaco, Fabrizio; Napolitano, Giorgio

    2014-01-01

    Resveratrol is a polyphenol found in grapes and berries that has antioxidant, antiproliferative and anti-inflammatory properties. For these reasons, it is available as a dietary supplement, and it is under investigation in several clinical trials. Few data are available regarding the effects of resveratrol on thyroid function. A previous study showed that resveratrol transiently increases iodide influx in FRTL-5 rat thyroid cells. Indeed, this increase arises after short treatment times (6–12 h), and no further effects are seen after 24 h. The aim of the present study was to investigate the effects of resveratrol on iodide uptake and sodium/iodide symporter expression in thyroid cells after longer times of treatment. For this purpose, the effects of resveratrol were evaluated both in vitro and in vivo using the rat thyroid FRTL-5 cell line and Sprague-Dawley rats, respectively. In FRTL-5 cells, resveratrol decreased the sodium/iodide symporter RNA and protein expression as a function of time. Furthermore, resveratrol decreased cellular iodide uptake after 48 h of treatment. The inhibitory effect of resveratrol on iodide uptake was confirmed in vivo in Sprague-Dawley rats. This study demonstrates that with longer-term treatment, resveratrol is an inhibitor of sodium/iodide symporter gene expression and function in the thyroid. These data suggest that resveratrol can act as a thyroid disruptor, which indicates the need for caution as a supplement and in therapeutic use. PMID:25251397

  2. Luteolin inhibited the gene expression, production and secretion of MUC5AC mucin via regulation of nuclear factor kappa B signaling pathway in human airway epithelial cells.

    PubMed

    Lee, Hyun Jae; Seo, Hyo-Seok; Ryu, Jiho; Yoon, Yong Pill; Park, Su Hyun; Lee, Choong Jae

    2015-04-01

    Luteolin, a flavonoidal compound derived from Lonicera japonica Thunb. and Chrysanthemum indicum L., has been reported to show anti-inflammatory, anti-oxidative and anti-carcinogenic effects. In this study, we investigated whether luteolin significantly affects the secretion, production and gene expression of airway mucin. Confluent NCI-H292 cells were pretreated with luteolin for 30 min and then stimulated with EGF (epidermal growth factor) or PMA (phorbol 12-myristate 13-acetate) for 24 h or the indicated periods. The MUC5AC mucin gene expression was measured by RT-PCR. Production and secretion of MUC5AC mucin protein were measured by ELISA. To elucidate the action mechanism of luteolin, effect of luteolin on PMA-induced NF-κB signaling pathway was investigated by western blot analysis. The results were as follows: (1) Luteolin inhibited the secretion of MUC5AC mucin protein induced by EGF or PMA; (2) Luteolin inhibited the production of MUC5AC mucin protein and the expression of MUC5AC mucin gene induced by EGF or PMA; (3) Luteolin inhibited PMA-induced phosphorylation and degradation of inhibitory kappa Bα (IκBα); (4) Luteolin inhibited PMA-induced phosphorylation and nuclear translocation of nuclear factor kappa B (NF-κB) p65. This result suggests that luteolin can regulate the secretion, production and gene expression of mucin by acting on airway epithelial cells via regulation of NF-kB signaling pathway. PMID:25285988

  3. Growth Inhibition of Head and Neck Squamous Cell Carcinoma Cells by sgRNA Targeting the Cyclin D1 mRNA Based on TRUE Gene Silencing

    PubMed Central

    Iizuka, Satoshi; Oridate, Nobuhiko; Nashimoto, Masayuki; Fukuda, Satoshi; Tamura, Masato

    2014-01-01

    Head and neck squamous cell carcinoma (HNSCC) exhibits increased expression of cyclin D1 (CCND1). Previous studies have shown a correlation between poor prognosis of HNSCC and cyclin D1 overexpression. tRNase ZL-utilizing efficacious gene silencing (TRUE gene silencing) is one of the RNA-mediated gene expression control technologies that have therapeutic potential. This technology is based on a unique enzymatic property of mammalian tRNase ZL, which is that it can cleave any target RNA at any desired site by recognizing a pre-tRNA-like complex formed between the target RNA and an artificial small guide RNA (sgRNA). In this study, we designed several sgRNAs targeting human cyclin D1 mRNA to examine growth inhibition of HNSCC cells. Transfection of certain sgRNAs decreased levels of cyclin D1 mRNA and protein in HSC-2 and HSC-3 cells, and also inhibited their proliferation. The combination of these sgRNAs and cisplatin showed more than additive inhibition of cancer cell growth. These findings demonstrate that TRUE gene silencing of cyclin D1 leads to inhibition of the growth of HNSCC cells and suggest that these sgRNAs alone or combined with cisplatin may be a useful new therapy for HNSCCs. PMID:25437003

  4. POLY I:C INHIBITS THE EXPRESSION OF CHANNEL CATFISH VIRUS IMMEDIATE-EARLY GENE ORF 1 AT EARLY TIMES AFTER INFECTION

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Channel catfish virus (CCV) is a herpes virus that infects channel catfish fry and fingerlings. Previous research has demonstrated that Type I interferons inhibit the expression of immediate-early (IE) genes of some mammalian herpesviruses. However, CCV is distantly related to the mammalian herpesvi...

  5. Alteration of splice site selection in the LMNA gene and inhibition of progerin production via AMPK activation.

    PubMed

    Finley, Jahahreeh

    2014-11-01

    Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare genetic condition characterized by an accelerated aging phenotype and an average life span of 13years. Patients typically exhibit extensive pathophysiological vascular alterations, eventually resulting in death from stroke or myocardial infarction. A silent point mutation at position 1824 (C1824T) of the LMNA gene, generating a truncated form of lamin A (progerin), has been shown to be the cause of most cases of HGPS. Interestingly, this mutation induces the use of an internal 5' cryptic splice site within exon 11 of the LMNA pre-mRNA, leading to the generation of progerin via aberrant alternative splicing. The serine-arginine rich splicing factor 1 (SRSF1 or ASF/SF2) has been shown to function as an oncoprotein and is upregulated in many cancers and other age-related disorders. Indeed, SRSF1 inhibition results in a splicing ratio in the LMNA pre-mRNA favoring lamin A production over that of progerin. It is our hypothesis that activation of AMP-activated protein kinase (AMPK), a master regulator of cellular metabolism, may lead to a reduction in SRSF1 and thus a decrease in the use of the LMNA 5' cryptic splice site in exon 11 through upregulation of p32, a splicing factor-associated protein and putative mitochondrial chaperone that has been shown to inhibit SRSF1 and enhance mitochondrial DNA (mtDNA) replication and oxidative phosphorylation. AMPK activation by currently available compounds such as metformin, resveratrol, and berberine may thus have wide-ranging implications for disorders associated with increased production and accumulation of progerin. PMID:25216752

  6. Inhibition of stimulated meningeal blood flow by a calcitonin gene-related peptide binding mirror-image RNA oligonucleotide

    PubMed Central

    Denekas, Thomas; Tröltzsch, Markus; Vater, Axel; Klussmann, Sven; Messlinger, Karl

    2006-01-01

    Calcitonin gene-related peptide (CGRP) released from trigeminal afferents is known to play an important role in the control of intracranial blood flow. In a rat preparation with exposed cranial dura mater, periods of electrical stimulation induce increases in meningeal blood flow. These responses are due to arterial vasodilatation mediated in part by the release of CGRP. In this preparation, the effect of a CGRP-binding mirror-image oligonucleotide (Spiegelmer NOX-C89) was examined. Spiegelmer NOX-C89 applied topically at concentrations between 10−7and 10−5 M to the exposed dura mater led to a dose-dependent inhibition of the electrically evoked blood flow increases. The highest dose reduced the mean increases in flow to 56% of the respective control levels. A nonfunctional control Spiegelmer (not binding to CGRP) was ineffective in changing blood flow increases. Intravenous injection of NOX-C89 (5 mg kg−1) reduced the evoked blood flow increases to an average of 65.5% of the control. The basal blood flow was not changed by any of the applied substances. In addition, an ex vivo preparation of the hemisected rat skull was used to determine CGRP release from the cranial dura mater caused by antidromic activation of meningeal afferents. In this model, 10−6 M of NOX-C89 reduced the evoked CGRP release by about 50%. We conclude that increases in meningeal blood flow due to afferent activation can be reduced by sequestering the released CGRP and thus preventing it from activating vascular CGRP receptors. Moreover, the Spiegelmer NOX-C89 may inhibit CGRP release from meningeal afferents. Therefore, the approach to interfere with the CGRP/CGRP receptor system by binding the CGRP may open a new opportunity for the therapy of diseases that are linked to excessive CGRP release such as some forms of primary headaches. PMID:16633354

  7. Calcitonin gene-related peptide inhibits autophagic-lysosomal proteolysis through cAMP/PKA signaling in rat skeletal muscles.

    PubMed

    Machado, Juliano; Manfredi, Leandro H; Silveira, Wilian A; Gonçalves, Dawit A P; Lustrino, Danilo; Zanon, Neusa M; Kettelhut, Isis C; Navegantes, Luiz C

    2016-03-01

    Calcitonin gene-related peptide (CGRP) is a neuropeptide released by motor neuron in skeletal muscle and modulates the neuromuscular transmission by induction of synthesis and insertion of acetylcholine receptor on postsynaptic muscle membrane; however, its role in skeletal muscle protein metabolism remains unclear. We examined the in vitro and in vivo effects of CGRP on protein breakdown and signaling pathways in control skeletal muscles and muscles following denervation (DEN) in rats. In isolated muscles, CGRP (10(-10) to 10(-6)M) reduced basal and DEN-induced activation of overall proteolysis in a concentration-dependent manner. The in vitro anti-proteolytic effect of CGRP was completely abolished by CGRP8-37, a CGRP receptor antagonist. CGRP down-regulated the lysosomal proteolysis, the mRNA levels of LC3b, Gabarapl1 and cathepsin L and the protein content of LC3-II in control and denervated muscles. In parallel, CGRP elevated cAMP levels, stimulated PKA/CREB signaling and increased Foxo1 phosphorylation in both conditions. In denervated muscles and starved C2C12 cells, Rp-8-Br-cAMPs or PKI, two PKA inhibitors, completely abolished the inhibitory effect of CGRP on Foxo1, 3 and 4 and LC3 lipidation. A single injection of CGRP (100μgkg(-1)) in denervated rats increased the phosphorylation levels of CREB and Akt, inhibited Foxo transcriptional activity, the LC3 lipidation as well as the mRNA levels of LC3b and cathepsin L, two bona fide targets of Foxo. This study shows for the first time that CGRP exerts a direct inhibitory action on autophagic-lysosomal proteolysis in control and denervated skeletal muscle by recruiting cAMP/PKA signaling, effects that are related to inhibition of Foxo activity and LC3 lipidation. PMID:26718975

  8. Cyclophilin A and calcineurin functions investigated by gene inactivation, cyclosporin A inhibition and cDNA arrays approaches in the phytopathogenic fungus Botrytis cinerea.

    PubMed

    Viaud, Muriel; Brunet-Simon, Adeline; Brygoo, Yves; Pradier, Jean-Marc; Levis, Caroline

    2003-12-01

    Calcineurin phosphatase and cyclophilin A are cellular components involved in fungal morphogenesis and virulence. Their roles were investigated in the phytopathogenic fungus Botrytis cinerea using gene inactivation, drug inhibition and cDNA macroarrays approaches. First, the BCP1 gene coding for cyclophilin A was identified and inactivated by homologous recombination. The bcp1Delta null mutant obtained was still able to develop infection structures but was altered in symptom development on bean and tomato leaves. Opposite to this, calcineurin inhibition using cyclosporin A (CsA) modified hyphal morphology and prevented infection structure formation. CsA drug pattern signature on macroarrays allowed the identification of 18 calcineurin-dependent (CND) genes among 2839 B. cinerea genes. Among the co-regulated CND genes, three were shown to be organized as a physical cluster that could be involved in secondary metabolism. The signature of BCP1 inactivation on macroarrays allowed the identification of only three BCP1 cyclophilin-dependent (CPD) genes that were different from CND genes. Finally, no CsA drug pattern signature was observed in the bcp1Delta null mutant which provided a molecular target validation of the drug. PMID:14651630

  9. Prolonged Proteasome Inhibition Cyclically Upregulates Oct3/4 and Nanog Gene Expression, but Reduces Induced Pluripotent Stem Cell Colony Formation

    PubMed Central

    Floyd, Elizabeth Z.; Staszkiewicz, Jaroslaw; Power, Rachel A.; Kilroy, Gail; Kirk-Ballard, Heather; Barnes, Christian W.; Strickler, Karen L.; Rim, Jong S.; Harkins, Lettie L.; Gao, Ru; Kim, Jeong

    2015-01-01

    Abstract There is ample evidence that the ubiquitin–proteasome system is an important regulator of transcription and its activity is necessary for maintaining pluripotency and promoting cellular reprogramming. Moreover, proteasome activity contributes to maintaining the open chromatin structure found in pluripotent stem cells, acting as a transcriptional inhibitor at specific gene loci generally associated with differentiation. The current study was designed to understand further the role of proteasome inhibition in reprogramming and its ability to modulate endogenous expression of pluripotency-related genes and induced pluripotent stem cells (iPSCs) colony formation. Herein, we demonstrate that acute combinatorial treatment with the proteasome inhibitors MG101 or MG132 and the histone deacetylase (HDAC) inhibitor valproic acid (VPA) increases gene expression of the pluripotency marker Oct3/4, and that MG101 alone is as effective as VPA in the induction of Oct3/4 mRNA expression in fibroblasts. Prolonged proteasome inhibition cyclically upregulates gene expression of Oct3/4 and Nanog, but reduces colony formation in the presence of the iPSC induction cocktail. In conclusion, our results demonstrate that the 26S proteasome is an essential modulator in the reprogramming process. Its inhibition enhances expression of pluripotency-related genes; however, efficient colony formation requires proteasome activity. Therefore, discovery of small molecules that increase proteasome activity might lead to more efficient cell reprogramming and generation of pluripotent cells. PMID:25826722

  10. Mucin 1 gene silencing inhibits the growth of SMMC-7721 human hepatoma cells through Bax-mediated mitochondrial and caspase-8-mediated death receptor apoptotic pathways

    PubMed Central

    YUAN, HONGYAN; WANG, JUAN; WANG, FENGLI; ZHANG, NANNAN; LI, QIONGSHU; XIE, FEI; CHEN, TANXIU; ZHAI, RUIPING; WANG, FANG; GUO, YINGYING; NI, WEIHUA; TAI, GUIXIANG

    2015-01-01

    Mucin 1 (MUC1) is an oncogene that has a crucial role in the pathogenesis and progression of the majority of epithelial malignant tumors. Our previous study demonstrated that MUC1 gene silencing inhibited the growth of SMMC-7721 cells in vitro and in vivo, however, whether this growth inhibition is associated with apoptotic cell death remains to be elucidated. In the present study, it was found that MUC1 gene silencing not only resulted in the inhibition of SMMC-7721 cell growth, determined using a clone formation assay in vitro and a tumor xenograft mouse model with an in vivo imaging system, but also induced apoptotic alterations in SMMC-7721 cells, determined using Hoechst 33342 staining, flow cytometry with an Annexin V-PE staining and a DNA ladder assay. Further investigation using western blotting revealed that cytochrome c was released from the mitochondria into the cytoplasm, and caspase-8 and caspase-9 were activated in MUC1 gene-silenced SMMC-7721 cells. The pro-apoptotic protein Bcl-2-associated X protein (Bax) and the tumor suppressor p53 were increased, while the anti-apoptotic protein B-cell lymphoma 2 was decreased in MUC1 gene-silenced cells. In addition, results from the co-immunoprecipitation experiments demonstrated that the MUC1 cytoplasmic tail can bind directly to Bax or caspase-8 and these interactions were reduced upon MUC1 gene silencing in SMMC-7721 cells. The above results indicate that MUC1 gene silencing induces growth inhibition in SMMC-7721 cells through Bax-mediated mitochondrial and caspase-8-mediated death receptor apoptotic pathways. PMID:26398332

  11. The Procambium Specification Gene Oshox1 Promotes Polar Auxin Transport Capacity and Reduces Its Sensitivity toward Inhibition1

    PubMed Central

    Scarpella, Enrico; Boot, Kees J.M.; Rueb, Saskia; Meijer, Annemarie H.

    2002-01-01

    The auxin-inducible homeobox gene Oshox1 of rice (Oryza sativa) is a positive regulator of procambial cell fate commitment, and its overexpression reduces the sensitivity of polar auxin transport (PAT) to the PAT inhibitor 1-N-naphthylphthalamic acid (NPA). Here, we show that wild-type rice leaves formed under conditions of PAT inhibition display vein hypertrophy, reduced distance between longitudinal veins, and increased distance between transverse veins, providing experimental evidence for a role of PAT in vascular patterning in a monocot species. Furthermore, we show that Oshox1 overexpression confers insensitivity to these PAT inhibitor-induced vascular-patterning defects. Finally, we show that in the absence of any overt phenotypical change, Oshox1 overexpression specifically reduces the affinity of the NPA-binding protein toward NPA and enhances PAT and its sensitivity toward auxin. These results are consistent with the hypothesis that Oshox1 promotes fate commitment of procambial cells by increasing their auxin conductivity properties and stabilizing this state against modulations of PAT by an endogenous NPA-like molecule. PMID:12428000

  12. Antisense inhibition of a pectate lyase gene supports a role for pectin depolymerization in strawberry fruit softening.

    PubMed

    Santiago-Doménech, Nieves; Jiménez-Bemúdez, Silvia; Matas, Antonio J; Rose, Jocelyn K C; Muñoz-Blanco, Juan; Mercado, José A; Quesada, Miguel A

    2008-01-01

    Cell wall disassembly in softening fruits is a complex process involving the cumulative action of many families of wall-modifying proteins on interconnected polysaccharide matrices. One strategy to elucidate the in vivo substrates of specific enzymes and their relative importance and contribution to wall modification is to suppress their expression in transgenic fruit. It has been reported previously that inhibiting the expression of pectate lyase genes by antisense technology in strawberry (Fragaria x ananassa Duch.) fruit resulted in prolonged fruit firmness. This suggested that pectin depolymerization might make a more important contribution to strawberry fruit softening than is often stated. In this present study, three independent transgenic lines were identified exhibiting a greater than 90% reduction in pectate lyase transcript abundance. Analyses of sequential cell wall extracts from the transgenic and control fruit collectively showed clear quantitative and qualitative differences in the extractability and molecular masses of populations of pectin polymers. Wall extracts from transgenic fruits showed a reduction in pectin solubility and decreased depolymerization of more tightly bound polyuronides. Additional patterns of differential extraction of other wall-associated pectin subclasses were apparent, particularly in the sodium carbonate- and chelator-soluble polymers. In addition, microscopic studies revealed that the typical ripening-associated loss of cell-cell adhesion was substantially reduced in the transgenic fruits. These results indicate that pectate lyase plays an important degradative role in the primary wall and middle lamella in ripening strawberry fruit, and should be included in synergistic models of cell wall disassembly. PMID:18522930

  13. Pentacyclic triterpene derivatives possessing polyhydroxyl ring A inhibit gram-positive bacteria growth by regulating metabolism and virulence genes expression.

    PubMed

    Huang, Lirong; Luo, Heng; Li, Qiji; Wang, Daoping; Zhang, Jianxin; Hao, Xiaojiang; Yang, Xiaosheng

    2015-05-01

    The hydroxyl group in ring A of pentacyclic triterpene is essential for antibacterial activity. Pentacyclic triterpenes bearing three hydroxyl groups in ring A were mainly found in plants and displayed significant antibacterial activity. However, no study reported how to obtain this type of compounds by chemical modification. In this study, twenty-five new pentacyclic triterpenes bearing polyhydroxyl ring A were synthesized from parental compounds ursolic acid (UA) and oleanolic acid (OA). Here, we showed that most of these derivatives displayed a significantly increased activity against Gram-positive bacteria compared to parental compounds in vitro. Some of these compounds exhibited minimum inhibitory concentration values of 1-3-fold more potent than the positive controls. The structure-activity relationship studies demonstrated that introducing two hydroxyl groups at positions C-1 and C-2 together with a small alkyl ester group at C-17 of UA and OA strongly enhanced growth-inhibiting activity against Gram-positive bacteria. The antibacterial mechanism of the active derivatives was shown to be involved in regulating the expression of genes associated with peptidoglycan and respiratory metabolisms, as well as virulence factor in bacteria. The enhanced potency and unique mechanism of action of these new pentacyclic triterpenes make them a promising antibacterial agent for further studies. PMID:25794790

  14. Antisense inhibition of a pectate lyase gene supports a role for pectin depolymerization in strawberry fruit softening

    PubMed Central

    Santiago-Doménech, Nieves; Jiménez-Bemúdez, Silvia; Matas, Antonio J.; Rose, Jocelyn K. C.; Muñoz-Blanco, Juan; Mercado, José A.; Quesada, Miguel A.

    2008-01-01

    Cell wall disassembly in softening fruits is a complex process involving the cumulative action of many families of wall-modifying proteins on interconnected polysaccharide matrices. One strategy to elucidate the in vivo substrates of specific enzymes and their relative importance and contribution to wall modification is to suppress their expression in transgenic fruit. It has been reported previously that inhibiting the expression of pectate lyase genes by antisense technology in strawberry (Fragaria×ananassa Duch.) fruit resulted in prolonged fruit firmness. This suggested that pectin depolymerization might make a more important contribution to strawberry fruit softening than is often stated. In this present study, three independent transgenic lines were identified exhibiting a greater than 90% reduction in pectate lyase transcript abundance. Analyses of sequential cell wall extracts from the transgenic and control fruit collectively showed clear quantitative and qualitative differences in the extractability and molecular masses of populations of pectin polymers. Wall extracts from transgenic fruits showed a reduction in pectin solubility and decreased depolymerization of more tightly bound polyuronides. Additional patterns of differential extraction of other wall-associated pectin subclasses were apparent, particularly in the sodium carbonate- and chelator-soluble polymers. In addition, microscopic studies revealed that the typical ripening-associated loss of cell–cell adhesion was substantially reduced in the transgenic fruits. These results indicate that pectate lyase plays an important degradative role in the primary wall and middle lamella in ripening strawberry fruit, and should be included in synergistic models of cell wall disassembly. PMID:18522930

  15. Orally delivered thioketal nanoparticles loaded with TNF-α-siRNA target inflammation and inhibit gene expression in the intestines

    NASA Astrophysics Data System (ADS)

    Wilson, D. Scott; Dalmasso, Guillaume; Wang, Lixin; Sitaraman, Shanthi V.; Merlin, Didier; Murthy, Niren

    2010-11-01

    Small interfering RNAs (siRNAs) directed against proinflammatory cytokines have the potential to treat numerous diseases associated with intestinal inflammation; however, the side-effects caused by the systemic depletion of cytokines demands that the delivery of cytokine-targeted siRNAs be localized to diseased intestinal tissues. Although various delivery vehicles have been developed to orally deliver therapeutics to intestinal tissue, none of these strategies has demonstrated the ability to protect siRNA from the harsh environment of the gastrointestinal tract and target its delivery to inflamed intestinal tissue. Here, we present a delivery vehicle for siRNA, termed thioketal nanoparticles (TKNs), that can localize orally delivered siRNA to sites of intestinal inflammation, and thus inhibit gene expression in inflamed intestinal tissue. TKNs are formulated from a polymer, poly-(1,4-phenyleneacetone dimethylene thioketal), that degrades selectively in response to reactive oxygen species (ROS). Therefore, when delivered orally, TKNs release siRNA in response to the abnormally high levels of ROS specific to sites of intestinal inflammation. Using a murine model of ulcerative colitis, we demonstrate that orally administered TKNs loaded with siRNA against the proinflammatory cytokine tumour necrosis factor-alpha (TNF-α) diminish TNF-α messenger RNA levels in the colon and protect mice from ulcerative colitis.

  16. DLC1 tumor suppressor gene inhibits migration and invasion of multiple myeloma cells through RhoA GTPase pathway

    PubMed Central

    Ullmannova-Benson, Veronika; Guan, Ming; Zhou, Xiaoling; Tripathi, Veenu; Yang, Xu-Yu; Zimonjic, Drazen B.; Popescu, Nicholas C

    2009-01-01

    DLC1, a tumor suppressor gene that encodes a RhoGTPase-activating protein, is recurrently downregulated or silenced in various solid tumors and hematological malignancies due to epigenetic modifications or genomic deletion. Here, we identified DLC-1 promoter hypermethylation in 43 out of 44 multiple myeloma (MM) cell lines, which resulted in downregulation or silencing of DLC1 in 41 samples. High frequency of tumor-specific methylation and attenuation or silencing of DLC1 expression could serve as an independent diagnostic marker for MM. Combined treatment with demethylating and acetylating agents significantly elevated the expression of DLC1 and suppressed MM cell proliferation. Two cell lines exhibiting complete promoter methylation and absence of DLC1 expression were transduced by an adenoviral vector containing DLC1 cDNA. In both cell lines reexpression of DLC1 inhibited myeloma cell invasion and migration, reduced RhoA activity and resulted in reorganization of actin cytoskeleton. These results provide the first evidence for antiproliferative effect of DLC1 in a hematological cancer and implicate RhoA pathway in suppression of MM migration and invasion. Given the myeloma cells sensitivity to reactivation of DLC-1 function, the potential for molecular targeted therapy of DLC-1 mediated pathways as well as epigenetic therapies hold prospects. PMID:18923442

  17. HoxD10 gene delivery using adenovirus/adeno-associate hybrid virus inhibits the proliferation and tumorigenicity of GH4 pituitary lactotrope tumor cells

    SciTech Connect

    Cho, Mi Ae; Yashar, Parham; Kim, Suk Kyoung; Noh, Taewoong; Gillam, Mary P.; Lee, Eun Jig Jameson, J. Larry

    2008-07-04

    Prolactinoma is one of the most common types of pituitary adenoma. It has been reported that a variety of growth factors and cytokines regulating cell growth and angiogenesis play an important role in the growth of prolactinoma. HoxD10 has been shown to impair endothelial cell migration, block angiogenesis, and maintain a differentiated phenotype of cells. We investigated whether HoxD10 gene delivery could inhibit the growth of prolactinoma. Rat GH4 lactotrope tumor cells were infected with adenovirus/adeno-associated virus (Ad/AAV) hybrid vectors carrying the mouse HoxD10 gene (Hyb-HoxD10) or the {beta}-galactosidase gene (Hyb-Gal). Hyb-HoxD10 expression inhibited GH4 cell proliferation in vitro. The expression of FGF-2 and cyclin D2 was inhibited in GH4 cells infected with Hyb-HoxD10. GH4 cells transduced with Hyb-HoxD10 did not form tumors in nude mice. These results indicate that the delivery of HoxD10 could potentially inhibit the growth of PRL-secreting tumors. This approach may be a useful tool for targeted therapy of prolactinoma and other neoplasms.

  18. Cross-resistance to herbicides of five ALS-inhibiting groups and sequencing of the ALS gene in Cyperus difformis L.

    PubMed

    Merotto, Aldo; Jasieniuk, Marie; Osuna, Maria D; Vidotto, Francesco; Ferrero, Aldo; Fischer, Albert J

    2009-02-25

    Resistance to ALS-inhibiting herbicides in Cyperus difformis has evolved rapidly in many rice areas worldwide. This study identified the mechanism of resistance, assessed cross-resistance patterns to all five chemical groups of ALS-inhibiting herbicides in four C. difformis biotypes, and attempted to sequence the ALS gene. Whole-plant and ALS enzyme activity dose-response assays indicated that the WA biotype was resistant to all ALS-inhibiting herbicides evaluated. The IR biotype was resistant to bensulfuron-methyl, orthosulfamuron, imazethapyr, and propoxycarbazone-sodium and less resistant to bispyribac-sodium and halosulfuron-methyl, and susceptible to penoxsulam. ALS enzyme activity assays indicated that resistance is due to an altered target site yet mutations previously found to endow target-site resistance in weeds were not detected in the sequences obtained. The inability to detect resistance mutations in C. difformis may result from the presence of additional ALS genes, which were not amplified by the primers used. This study reports the first ALS gene sequence from Cyperus difformis. Certain ALS-inhibiting herbicides can still be used to control some resistant C. difformis biotypes. However, because cross-resistance to all five classes of ALS-inhibitors was detected in other resistant biotypes, these herbicides should only be used within an integrated weed management program designed to delay the evolution of herbicide resistance. PMID:19191488

  19. Sulindac, a nonsteroidal anti-inflammatory drug, selectively inhibits interferon-{gamma}-induced expression of the chemokine CXCL9 gene in mouse macrophages

    SciTech Connect

    Sakaeda, Yoshiichi; Hiroi, Miki; Shimojima, Takahiro; Iguchi, Mayumi; Kanegae, Haruhide; Ohmori, Yoshihiro . E-mail: ohmori@dent.meikai.ac.jp

    2006-11-17

    Sulindac, a non-steroidal anti-inflammatory drug, has been shown to exert an anti-tumor effect on several types of cancer. To determine the effect of sulindac on intracellular signaling pathways in host immune cells such as macrophages, we investigated the effect of the drug on interferon gamma (IFN{gamma})-induced expression of signal transducer and activator of transcription 1 (STAT1) and other genes in mouse macrophage-like cell line RAW264.7 cells. Sulindac, but not aspirin or sodium salicylate, inhibited IFN{gamma}-induced expression of the CXC ligand 9 (CXCL9) mRNA, a chemokine for activated T cells, whereas the interferon-induced expression of CXCL10 or IFN regulatory factor-1 was not affected by sulindac. Luciferase reporter assay demonstrated that sulindac inhibited IFN{gamma}-induced promoter activity of the CXCL9 gene. Surprisingly, sulindac had no inhibitory effect on IFN{gamma}-induced STAT1 activation; however, constitutive nuclear factor {kappa}B activity was suppressed by the drug. These results indicate that sulindac selectively inhibited IFN{gamma}-inducible gene expression without inhibiting STAT1 activation.

  20. Differences in Expression of Key DNA Damage Repair Genes after Epigenetic-Induced BRCAness Dictate Synthetic Lethality with PARP1 Inhibition.

    PubMed

    Wiegmans, Adrian P; Yap, Pei-Yi; Ward, Ambber; Lim, Yi Chieh; Khanna, Kum Kum

    2015-10-01

    The triple-negative breast cancer (TNBC) subtype represents a cancer that is highly aggressive with poor patient outcome. Current preclinical success has been gained through synthetic lethality, targeting genome instability with PARP inhibition in breast cancer cells that harbor silencing of the homologous recombination (HR) pathway. Histone deacetylase inhibitors (HDACi) are a class of drugs that mediate epigenetic changes in expression of HR pathway genes. Here, we compare the activity of the pan-HDAC inhibitor suberoylanilide hydroxamic acid (SAHA), the class I/IIa HDAC inhibitor valproic acid (VPA), and the HDAC1/2-specific inhibitor romidepsin (ROMI) for their capability to regulate DNA damage repair gene expression and in sensitizing TNBC to PARPi. We found that two of the HDACis tested, SAHA and ROMI, but not VPA, indeed inhibit HR repair and that RAD51, BARD1, and FANCD2 represent key proteins whose inhibition is required for HDACi-mediated therapy with PARP inhibition in TNBC. We also observed that restoration of BRCA1 function stabilizes the genome compared with mutant BRCA1 that results in enhanced polyploid population after combination treatment with HDACi and PARPi. Furthermore, we found that overexpression of the key HR protein RAD51 represents a mechanism for this resistance, promoting aberrant repair and the enhanced polyploidy observed. These findings highlight the key components of HR in guiding synthetic lethality with PARP inhibition and support the rationale for utilizing the novel combination of HDACi and PARPi against TNBC in the clinical setting. PMID:26294743

  1. GATA-1 Inhibits PU.1 Gene via DNA and Histone H3K9 Methylation of Its Distal Enhancer in Erythroleukemia

    PubMed Central

    Burda, Pavel; Vargova, Jarmila; Curik, Nikola; Salek, Cyril; Papadopoulos, Giorgio Lucio; Strouboulis, John; Stopka, Tomas

    2016-01-01

    GATA-1 and PU.1 are two important hematopoietic transcription factors that mutually inhibit each other in progenitor cells to guide entrance into the erythroid or myeloid lineage, respectively. PU.1 controls its own expression during myelopoiesis by binding to the distal URE enhancer, whose deletion leads to acute myeloid leukemia (AML). We herein present evidence that GATA-1 binds to the PU.1 gene and inhibits its expression in human AML-erythroleukemias (EL). Furthermore, GATA-1 together with DNA methyl Transferase I (DNMT1) mediate repression of the PU.1 gene through the URE. Repression of the PU.1 gene involves both DNA methylation at the URE and its histone H3 lysine-K9 methylation and deacetylation as well as the H3K27 methylation at additional DNA elements and the promoter. The GATA-1-mediated inhibition of PU.1 gene transcription in human AML-EL mediated through the URE represents important mechanism that contributes to PU.1 downregulation and leukemogenesis that is sensitive to DNA demethylation therapy. PMID:27010793

  2. Antisense inhibition of gene expression in cells by oligonucleotides incorporating locked nucleic acids: effect of mRNA target sequence and chimera design

    PubMed Central

    Braasch, Dwaine A.; Liu, Yinghui; Corey, David R.

    2002-01-01

    Use of antisense oligonucleotides is a versatile strategy for achieving control of gene expression. Unfortunately, the interpretation of antisense-induced phenotypes is sometimes difficult, and chemical modifications that improve the potency and specificity of antisense action would be useful. The introduction of locked nucleic acid (LNA) bases into oligonucleotides confers exceptional improvement in binding affinity, up to 10°C per substitution, making LNAs an exciting option for the optimization of antisense efficacy. Here we examine the rules governing antisense gene inhibition within cells by oligonucleotides that contain LNA bases. LNA- containing oligomers were transfected into cells using cationic lipid and accumulated in the nucleus. We tested antisense gene inhibition by LNAs and LNA–DNA chimeras complementary to the 5′-untranslated region, the region surrounding the start codon and the coding region of mRNA, and identified effective antisense agents targeted to each of these locations. Our data suggest that LNA bases can be used to develop antisense oligonucleotides and that their use is a versatile approach for efficiently inhibiting gene expression inside cells. PMID:12466540

  3. Design, Synthesis, and Evaluation of Triazole Derivatives That Induce Nrf2 Dependent Gene Products and Inhibit the Keap1-Nrf2 Protein-Protein Interaction.

    PubMed

    Bertrand, Hélène C; Schaap, Marjolein; Baird, Liam; Georgakopoulos, Nikolaos D; Fowkes, Adrian; Thiollier, Clarisse; Kachi, Hiroko; Dinkova-Kostova, Albena T; Wells, Geoff

    2015-09-24

    The transcription factor Nrf2 regulates the expression of a large network of cytoprotective and metabolic enzymes and proteins. Compounds that directly and reversibly inhibit the interaction between Nrf2 and its main negative regulator Keap1 are potential pharmacological agents for a range of disease types including neurodegenerative conditions and cancer. We describe the development of a series of 1,4-diphenyl-1,2,3-triazole compounds that inhibit the Nrf2-Keap1 protein-protein interaction (PPI) in vitro and in live cells and up-regulate the expression of Nrf2-dependent gene products. PMID:26348784

  4. Coptisine inhibits RANKL-induced NF-?B phosphorylation in osteoclast precursors and suppresses function through the regulation of RANKL and OPG gene expression in osteoblastic cells.

    PubMed

    Lee, Ji-Won; Iwahashi, Ayumi; Hasegawa, Shin-ichi; Yonezawa, Takayuki; Jeon, Won Bae; Cha, Byung-Yoon; Nagai, Kazuo; Woo, Je-Tae

    2012-01-01

    Excessive receptor activator of NF-?B ligand (RANKL) signaling causes enhanced osteoclast formation and bone resorption. The downregulation of RANKL expression and its downstream signals may be an effective therapeutic approach to the treatment of bone loss diseases such as osteoporosis. Here, we found that coptisine, one of the isoquinoline alkaloids from Coptidis Rhizoma, exhibited inhibitory effects on osteoclastogenesis in vitro. Although coptisine has been studied for its antipyretic, antiphotooxidative, dampness dispelling, antidote, antinociceptive, and anti-inflammatory activities in vitro and in vivo, its effects on osteoclastogenesis have not been investigated. Therefore, we evaluated the effects of coptisine on osteoblastic cells as well as osteoclast precursors for osteoclastogenesis in vitro. The addition of coptisine to cocultures of mouse bone marrow cells and primary osteoblastic cells with 10(-8) M 1?,25(OH)(2)D(3) caused significant inhibition of osteoclast formation in a dose-dependent manner. Reverse transcriptase polymerase chain reaction (RT-PCR) analyses revealed that coptisine inhibited RANKL gene expression and stimulated the osteoprotegerin gene expression induced by 1?,25(OH)(2)D(3) in osteoblastic cells. Coptisine strongly inhibited RANKL-induced osteoclast formation when added during the early stage of bone marrow macrophage (BMM) cultures, suggesting that it acts on osteoclast precursors to inhibit RANKL/RANK signaling. Among the RANK signaling pathways, coptisine inhibited NF-?B p65 phosphorylations, which are regulated in response to RANKL in BMMs. Coptisine also inhibited the RANKL-induced expression of NFATc1, which is a key transcription factor. In addition, 10 ?M coptisine significantly inhibited both the survival of mature osteoclasts and their pit-forming activity in cocultures. Thus, coptisine has potential for the treatment or prevention of several bone diseases characterized by excessive bone destruction. PMID:21656335

  5. Conjugated Linoleic Acid (CLA) inhibits expression of the Spot 14 (THRSP) and fatty acid synthase genes and impairs the growth of human breast cancer and liposarcoma cells

    PubMed Central

    Donnelly, Christina; Olsen, Arne M.; Lewis, Lionel D.; Eisenberg, Burton L.; Eastman, Alan; Kinlaw, William B.

    2010-01-01

    Spot 14 (THRSP, S14) is a nuclear protein involved in the regulation of genes required for fatty acid synthesis in normal and malignant mammary epithelial and adipose cells. Havartine and Bauman reported that conjugated linoleic acid (CLA) inhibits S14 gene expression in bovine mammary and mouse adipose tissues, and reduces milk fat production in cows. We hypothesized that CLA inhibits S14 gene expression in human breast cancer and liposarcoma cells, and that this will retard their growth. Exposure of T47D breast cancer cells to a mixture of CLA isomers reduced the expression of the S14 and fatty acid synthase (FAS) genes. The mixture caused a dose-related inhibition of T47D cell growth, as did pure c9, t11- and t10, c12-CLA, but not linoleic acid. Similar effects were observed in MDA-MB-231 breast cancer cells. Provision of 8 μM palmitate fully (CLA mix, t10, c12-CLA) or partially (c9, t11-CLA) reversed the antiproliferative effect in T47D cells. CLA likewise suppressed levels of S14 and FAS mRNAs in liposarcoma cells, and caused growth inhibition that was prevented by palmitic acid. CLA did not affect the growth of nonlipogenic HeLa cells or human fibroblasts. We conclude that, as in bovine mammary and mouse adipose cells, CLA suppresses S14 and FAS gene expression in human breast cancer and liposarcoma cells. Rescue from the antiproliferative effect of CLA by palmitic acid indicates that reduced tumor lipogenesis is a major mechanism for the anticancer effects of CLA. PMID:19116881

  6. Rhesus macaque θ-defensin RTD-1 inhibits proinflammatory cytokine secretion and gene expression by inhibiting the activation of NF-κB and MAPK pathways.

    PubMed

    Tongaonkar, Prasad; Trinh, Katie K; Schaal, Justin B; Tran, Dat; Gulko, Percio S; Ouellette, André J; Selsted, Michael E

    2015-12-01

    θ-Defensins are pleiotropic, macrocyclic peptides that are expressed uniquely in Old World monkeys. The peptides are potent, broad-spectrum microbicides that also modulate inflammatory responses in vitro and in animal models of viral infection and polymicrobial sepsis. θ-Defensins suppress proinflammatory cytokine secretion by leukocytes stimulated with diverse Toll-like receptor (TLR) ligands. Studies were performed to delineate anti-inflammatory mechanisms of rhesus θ-defensin 1 (RTD-1), the most abundant θ-defensin isoform in macaque granulocytes. RTD-1 reduced the secretion of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-8 in lipopolysaccharide (LPS)-stimulated human blood monocytes and THP-1 macrophages, and this was accompanied by inhibition of nuclear factor κB (NF-κB) activation and mitogen-activated protein kinase (MAPK) pathways. Peptide inhibition of NF-κB activation occurred following stimulation of extracellular (TLRs 1/2 and 4) and intracellular (TLR9) receptors. Although RTD-1 did not inhibit MAPK in unstimulated cells, it induced phosphorylation of Akt in otherwise untreated monocytes and THP-1 cells. In the latter, this occurred within 10 min of RTD-1 treatment and produced a sustained elevation of phosphorylated Akt (pAkt) for at least 4 h. pAkt is a negative regulator of MAPK and NF-κB activation. RTD-1 inhibited IκBα degradation and p38 MAPK phosphorylation, and stimulated Akt phosphorylation in LPS-treated human primary monocytes and THP-1 macrophages. Specific inhibition of phosphatidylinositol 3-kinase (PI3K) blocked RTD-1-stimulated Akt phosphorylation and reversed the suppression of NF-κB activation by the peptide. These studies indicate that the anti-inflammatory properties of θ-defensins are mediated by activation of the PI3K/Akt pathway and suppression of proinflammatory signals in immune-stimulated cells. PMID:26269197

  7. Both the fipA gene of pKM101 and the pifC gene of F inhibit conjugal transfer of RP1 by an effect on traG.

    PubMed

    Santini, J M; Stanisich, V A

    1998-08-01

    The mechanisms by which gene products inhibit the conjugal transfer of IncP plasmids (e.g., RP1) have been little studied. We have isolated and characterized one such gene, fipA (624 nucleotides), from the SmaI (14.8 kb)-AatII (15.6 kb) region of pKM101(IncN). This gene, which is also conserved in other IncN plasmids, is transcribed in an anticlockwise direction, probably as part of a transfer operon that includes traHI. The FipA protein (24 kDa) appears to be cytoplasmic and, when expressed from a multicopy plasmid, retards the growth of Escherichia coli WP2. The mode of action of fipA was compared with that of the apparently unrelated pifC gene from F(IncFI). Both genes inhibit the transfer of IncPalpha and IncPbeta plasmids but to different degrees. They also inhibit the mobilization of RSF1010 (which requires the RP1 pilus genes and traG) but not of CloDF13 (which encodes a traG homolog). Evidence that traG was the specific target of inhibition was obtained in an artificial system in which cloned traG was used to enhance RSF1010 mobilization via the N pilus system. Such enhancement did not occur in the presence of fipA or pifC. The availability of an in vivo assay of PifC enabled us to show that F pif operon expression increased in cells carrying F'lac and traG, but only if the traG coding sequence was intact. This finding suggested that conjugal inhibition of RP1 was most likely due to a PifC-TraG protein interaction. On phenotypic grounds inhibition of traG by fipA is also likely to occur posttranscriptionally. Whether or not the selection of traG as the inhibition target is an evolutionary tactic to limit the spread of P plasmids, we anticipate that fipA and pifC will prove useful in further investigation of the conjugal roles of traG and its homologs. PMID:9696755

  8. Superiority of combined phosphodiesterase PDE3/PDE4 inhibition over PDE4 inhibition alone on glucocorticoid- and long-acting β2-adrenoceptor agonist-induced gene expression in human airway epithelial cells.

    PubMed

    BinMahfouz, Hawazen; Borthakur, Bibhusana; Yan, Dong; George, Tresa; Giembycz, Mark A; Newton, Robert

    2015-01-01

    Glucocorticoids, also known as corticosteroids, induce effector gene transcription as a part of their anti-inflammatory mechanisms of action. Such genomic effects can be significantly enhanced by long-acting β2-adrenoceptor agonists (LABAs) and may contribute to the clinical superiority of inhaled corticosteroid (ICS)/LABA combinations in asthma and chronic obstructive pulmonary disease (COPD) over ICSs alone. Using models of cAMP- and glucocorticoid-induced transcription in human bronchial epithelial BEAS-2B cells, we show that combining inhibitors of phosphodiesterase (PDE) 3 and PDE4 provides greater benefits compared with inhibiting either PDE alone. In respect to cAMP-dependent transcription, inhibitors of PDE3 (siguazodan, cilostazol) and PDE4 (rolipram, GSK256066, roflumilast N-oxide) each sensitized to the LABA, formoterol. This effect was magnified by dual PDE3 and PDE4 inhibition. Siguazodan plus rolipram was also more effective at inducing cAMP-dependent transcription than either inhibitor alone. Conversely, the concentration-response curve describing the enhancement of dexamethasone-induced, glucocorticoid response element-dependent transcription by formoterol was displaced to the left by PDE4, but not PDE3, inhibition. Overall, similar effects were described for bona fide genes, including RGS2, CD200, and CRISPLD2. Importantly, the combination of siguazodan plus rolipram prolonged the duration of gene expression induced by formoterol, dexamethasone, or dexamethasone plus formoterol. This was most apparent for RGS2, a bronchoprotective gene that may also reduce the proinflammatory effects of constrictor mediators. Collectively, these data provide a rationale for the use of PDE3 and PDE4 inhibitors in the treatment of COPD and asthma where they may enhance, sensitize, and prolong the effects of LABA/ICS combination therapies. PMID:25324049

  9. Targeting activating mutations of EZH2 leads to potent cell growth inhibition in human melanoma by derepression of tumor suppressor genes.

    PubMed

    Tiffen, Jessamy C; Gunatilake, Dilini; Gallagher, Stuart J; Gowrishankar, Kavitha; Heinemann, Anja; Cullinane, Carleen; Dutton-Regester, Ken; Pupo, Gulietta M; Strbenac, Dario; Yang, Jean Y; Madore, Jason; Mann, Graham J; Hayward, Nicholas K; McArthur, Grant A; Filipp, Fabian V; Hersey, Peter

    2015-09-29

    The epigenetic modifier EZH2 is part of the polycomb repressive complex that suppresses gene expression via histone methylation. Activating mutations in EZH2 are found in a subset of melanoma that contributes to disease progression by inactivating tumor suppressor genes. In this study we have targeted EZH2 with a specific inhibitor (GSK126) or depleted EZH2 protein by stable shRNA knockdown. We show that inhibition of EZH2 has potent effects on the growth of both wild-type and EZH2 mutant human melanoma in vitro particularly in cell lines harboring the EZH2Y646 activating mutation. This was associated with cell cycle arrest, reduced proliferative capacity in both 2D and 3D culture systems, and induction of apoptosis. The latter was caspase independent and mediated by the release of apoptosis inducing factor (AIFM1) from mitochondria. Gene expression arrays showed that several well characterized tumor suppressor genes were reactivated by EZH2 inhibition. This included activating transcription factor 3 (ATF3) that was validated as an EZH2 target gene by ChIP-qPCR. These results emphasize a critical role for EZH2 in the proliferation and viability of melanoma and highlight the potential for targeted therapy against EZH2 in treatment of patients with melanoma. PMID:26304929

  10. Silencing SlELP2L, a tomato Elongator complex protein 2-like gene, inhibits leaf growth, accelerates leaf, sepal senescence, and produces dark-green fruit

    PubMed Central

    Zhu, Mingku; Li, Yali; Chen, Guoping; Ren, Lijun; Xie, Qiaoli; Zhao, Zhiping; Hu, Zongli

    2015-01-01

    The multi-subunit complex Elongator interacts with elongating RNA polymerase II (RNAPII) and is thought to facilitate transcription through histone acetylation. Elongator is highly conserved in eukaryotes, yet has multiple kingdom-specific functions in diverse organisms. Recent genetic studies performed in Arabidopsis have demonstrated that Elongator functions in plant growth and development, and in response to biotic and abiotic stress. However, little is known about its roles in other plant species. Here, we study the function of an Elongator complex protein 2-like gene in tomato, here designated as SlELP2L, through RNAi-mediated gene silencing. Silencing SlELP2L in tomato inhibits leaf growth, accelerates leaf and sepal senescence, and produces dark-green fruit with reduced GA and IAA contents in leaves, and increased chlorophyll accumulation in pericarps. Gene expression analysis indicated that SlELP2L-silenced plants had reduced transcript levels of ethylene- and ripening-related genes during fruit ripening with slightly decreased carotenoid content in fruits, while the expression of DNA methyltransferase genes was up-regulated, indicating that SlELP2L may modulate DNA methylation in tomato. Besides, silencing SlELP2L increases ABA sensitivity in inhibiting seedling growth. These results suggest that SlELP2L plays important roles in regulating plant growth and development, as well as in response to ABA in tomato. PMID:25573793

  11. Targeting activating mutations of EZH2 leads to potent cell growth inhibition in human melanoma by derepression of tumor suppressor genes

    PubMed Central

    Tiffen, Jessamy C.; Gunatilake, Dilini; Gallagher, Stuart J.; Gowrishankar, Kavitha; Heinemann, Anja; Cullinane, Carleen; Dutton-Regester, Ken; Pupo, Gulietta M.; Strbenac, Dario; Yang, Jean Y.; Madore, Jason; Mann, Graham J.; Hayward, Nicholas K.; McArthur, Grant A.; Filipp, Fabian V.; Hersey, Peter

    2015-01-01

    The epigenetic modifier EZH2 is part of the polycomb repressive complex that suppresses gene expression via histone methylation. Activating mutations in EZH2 are found in a subset of melanoma that contributes to disease progression by inactivating tumor suppressor genes. In this study we have targeted EZH2 with a specific inhibitor (GSK126) or depleted EZH2 protein by stable shRNA knockdown. We show that inhibition of EZH2 has potent effects on the growth of both wild-type and EZH2 mutant human melanoma in vitro particularly in cell lines harboring the EZH2Y646 activating mutation. This was associated with cell cycle arrest, reduced proliferative capacity in both 2D and 3D culture systems, and induction of apoptosis. The latter was caspase independent and mediated by the release of apoptosis inducing factor (AIFM1) from mitochondria. Gene expression arrays showed that several well characterized tumor suppressor genes were reactivated by EZH2 inhibition. This included activating transcription factor 3 (ATF3) that was validated as an EZH2 target gene by ChIP-qPCR. These results emphasize a critical role for EZH2 in the proliferation and viability of melanoma and highlight the potential for targeted therapy against EZH2 in treatment of patients with melanoma. PMID:26304929

  12. Lipopolysaccharide derived from the digestive tract activates inflammatory gene expression and inhibits casein synthesis in the mammary glands of lactating dairy cows.

    PubMed

    Zhang, Kai; Chang, Guangjun; Xu, Tianle; Xu, Lei; Guo, Junfei; Jin, Di; Shen, Xiangzhen

    2016-03-01

    To meet the nutrition requirements of lactation, dairy cows are usually fed a high concentrate diet (HC). However, high-grain feeding causes subacute ruminal acidosis (SARA), a metabolic disorder that causes milk protein depression. This study aimed to investigate the effect of lipopolysaccharide (LPS) released in the rumen on inflammatory gene expression and casein synthesis in mammary glands of lactating dairy cows fed a HC diet. We found that milk protein was significantly decreased in the HC group after 15 weeks of feeding. Overall, LPS concentrations in the rumen fluid, lacteal artery and vein were increased in the HC group. Transcriptome microarray was used to evaluate alterations in the signaling pathway in mammary glands. Signaling pathways involved in inflammatory responses were activated, whereas those involved in protein synthesis were inhibited in the HC group. mRNA expression involved in inflammatory responses, including that of TLR4, NF-кB and pro-inflammatory genes, was increased in the HC group, while αs1-casein (CSN1S1), β-casein (CSN2), mTOR and S6K gene expression were decreased. Moreover, protein expression was consistent with the corresponding gene expression. After feeding with an HC diet, LPS derived from the rumen increased inflammatory gene expression and inhibited casein synthesis in the mammary glands of lactating dairy cows fed a HC diet. PMID:26893357

  13. Molecular mechanism of inhibition of estrogen-induced cathepsin D gene expression by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in MCF-7 cells

    SciTech Connect

    Krishnan, V.; Porter, W.; Santostefano, M.; Wang, Xiahong

    1995-12-01

    This report describes how 17{beta}-estradiol (E2) induces cathepsin D gene expression, but is inhibited by the aryl hydrocarbon receptor by disruption of the estrogen receptor/pBC12/S1/pac plasmid complex by interaction with an overlapping xenobiotic responsive element. It was also determined that 2,3,7,8-tetrachlorobenzo-p-dioxin (TCDD) alone does not affect cathepsin D gene expression but can together with E2 to affect the rate of transcription and levels of immunoreactive protein. 85 refs., 6 figs., 2 tabs.

  14. Efficient Inhibition of Ovarian Cancer by Gelonin Toxin Gene Delivered by Biodegradable Cationic Heparin-polyethyleneimine Nanogels.

    PubMed

    Bai, Yu; Gou, Maling; Yi, Tao; Yang, Li; Liu, Lili; Lin, Xiaojuan; Su, Dan; Wei, Yuquan; Zhao, Xia

    2015-01-01

    The use of toxins for cancer therapy has great promise. Gelonin, a potent plant toxin, causes cell death by inactivating the 60S ribosomal subunit. Recently, we developed a novel gene delivery system using biodegradable cationic heparin-polyethyleneimine (HPEI) nanogels. In the current study, the antitumor activity of a recombinant plasmid expressing gelonin (pGelonin) on human ovarian cancer was assessed. The application of HPEI nanogels, was also evaluated. Gelonin-cDNA was cloned into the pVAX1 plasmid vector and transfected into SKOV3 human ovarian cancer cells using biodegradable cationic HPEI nanogels. The expression of gelonin in vitro and in vivo was confirmed using RT-PCR and western blot analysis. Cell viability and apoptosis were examined using an MTT assay and flow cytometric analysis. For the in vivo study, an SKOV3 intraperitoneal ovarian carcinomatosis model was established, and nude mice were randomly assigned into four groups receiving i.p. administration of pGelonin/HPEI complexes, pVAX/HPEI complexes, HPEI alone and 5% glucose solution. The tumor weight was monitored, and a TUNEL assay and Ki-67 immunohistochemistry were performed to evaluate apoptosis and cell proliferation in the tumor tissue sections, respectively. Gelonin was efficiently expressed in SKOV3 cancer cells in vitro and in vivo using pGelonin incorporated with HPEI nanogels. The pGelonin/HPEI complexes inhibited cell viability and induced apoptosis in the cell culture. Treatment for intraperitoneal carcinomatosis with pGelonin/HPEI complexes reduced the tumor weight by ~58.55% compared to the control groups (P<0.05). The antitumor effect was accompanied by increased apoptosis and reduced cell proliferation (P<0.05). No significant side effects were observed with i.p. administration of the pGelonin/HPEI complexes. Our data indicate that HPEI nanogel-delivered pGelonin may have promising applications against human ovarian cancer. PMID:26005374

  15. Dynamin inhibition interferes with inflammasome activation and cytokine gene expression in Streptococcus pyogenes-infected human macrophages.

    PubMed

    Latvala, S; Mäkelä, S M; Miettinen, M; Charpentier, E; Julkunen, I

    2014-11-01

    In the present study, we have analysed the ability of Streptococcus pyogenes [Group A streptococcus (GAS)] to activate the NACHT-domain-, leucine-rich repeat- and PYD-containing protein 3 (NALP3) inflammasome complex in human monocyte-derived macrophages and the molecules and signalling pathways involved in GAS-induced inflammatory responses. We focused upon analysing the impact of dynamin-dependent endocytosis and the role of major streptococcal virulence factors streptolysin O (SLO) and streptolysin S (SLS) in the immune responses induced by GAS. These virulence factors are involved in immune evasion by forming pores in host cell membranes, and aid the bacteria to escape from the endosome-lysosome pathway. We analysed cytokine gene expression in human primary macrophages after stimulation with live or inactivated wild-type GAS as well as with live SLO and SLS defective bacteria. Interleukin (IL)-1β, IL-10, tumour necrosis factor (TNF)-α and chemokine (C-X-C motif) ligand (CXCL)-10 cytokines were produced after bacterial stimulation in a dose-dependent manner and no differences in cytokine levels were seen between live, inactivated or mutant bacteria. These data suggest that streptolysins or other secreted bacterial products are not required for the inflammatory responses induced by GAS. Our data indicate that inhibition of dynamin-dependent endocytosis in macrophages attenuates the induction of IL-1β, TNF-α, interferon (IFN)-β and CXCL-10 mRNAs. We also observed that pro-IL-1β protein was expressed and efficiently cleaved into mature-IL-1β via inflammasome activation after bacterial stimulation. Furthermore, we demonstrate that multiple signalling pathways are involved in GAS-stimulated inflammatory responses in human macrophages. PMID:25079511

  16. Histone Deacetylase Inhibition Rescues Gene Knockout Levels Achieved with Integrase-Defective Lentiviral Vectors Encoding Zinc-Finger Nucleases

    PubMed Central

    Pelascini, Laetitia P.L.; Maggio, Ignazio; Liu, Jin; Holkers, Maarten; Cathomen, Toni

    2013-01-01

    Abstract Zinc-finger nucleases (ZFNs) work as dimers to induce double-stranded DNA breaks (DSBs) at predefined chromosomal positions. In doing so, they constitute powerful triggers to edit and to interrogate the function of genomic sequences in higher eukaryotes. A preferred route to introduce ZFNs into somatic cells relies on their cotransduction with two integrase-defective lentiviral vectors (IDLVs) each encoding a monomer of a functional heterodimeric pair. The episomal nature of IDLVs diminishes the risk of genotoxicity and ensures the strict transient expression profile necessary to minimize deleterious effects associated with long-term ZFN activity. However, by deploying IDLVs and conventional lentiviral vectors encoding HPRT1- or eGFP-specific ZFNs, we report that DSB formation at target alleles is limited after IDLV-mediated ZFN transfer. This IDLV-specific underperformance stems, to a great extent, from the activity of chromatin-remodeling histone deacetylases (HDACs). Importantly, the prototypic and U.S. Food and Drug Administration–approved inhibitors of metal-dependent HDACs, trichostatin A and vorinostat, respectively, did not hinder illegitimate recombination-mediated repair of targeted chromosomal DSBs. This allowed rescuing IDLV-mediated site-directed mutagenesis to levels approaching those achieved by using their isogenic chromosomally integrating counterparts. Hence, HDAC inhibition constitutes an efficacious expedient to incorporate in genome-editing strategies based on transient IDLV-mediated ZFN expression. Finally, we compared two of the most commonly used readout systems to measure targeted gene knockout activities based on restriction and mismatch-sensitive endonucleases. These experiments indicate that these enzymatic assays display a similar performance. PMID:24059449

  17. Mucin 1 gene silencing inhibits the growth of SMMC-7721 human hepatoma cells through Bax-mediated mitochondrial and caspase-8-mediated death receptor apoptotic pathways.

    PubMed

    Yuan, Hongyan; Wang, Juan; Wang, Fengli; Zhang, Nannan; Li, Qiongshu; Xie, Fei; Chen, Tanxiu; Zhai, Ruiping; Wang, Fang; Guo, Yingying; Ni, Weihua; Tai, Guixiang

    2015-11-01

    Mucin 1 (MUC1) is an oncogene that has a crucial role in the pathogenesis and progression of the majority of epithelial malignant tumors. Our previous study demonstrated that MUC1 gene silencing inhibited the growth of SMMC‑7721 cells in vitro and in vivo, however, whether this growth inhibition is associated with apoptotic cell death remains to be elucidated. In the present study, it was found that MUC1 gene silencing not only resulted in the inhibition of SMMC‑7721 cell growth, determined using a clone formation assay in vitro and a tumor xenograft mouse model with an in vivo imaging system, but also induced apoptotic alterations in SMMC‑7721 cells, determined using Hoechst 33342 staining, flow cytometry with an Annexin V-PE staining and a DNA ladder assay. Further investigation using western blotting revealed that cytochrome c was released from the mitochondria into the cytoplasm, and caspase‑8 and caspase‑9 were activated in MUC1 gene‑silenced SMMC‑7721 cells. The pro‑apoptotic protein Bcl‑2‑associated X protein (Bax) and the tumor suppressor p53 were increased, while the anti‑apoptotic protein B‑cell lymphoma 2 was decreased in MUC1 gene‑silenced cells. In addition, results from the co‑immunoprecipitation experiments demonstrated that the MUC1 cytoplasmic tail can bind directly to Bax or caspase‑8 and these interactions were reduced upon MUC1 gene silencing in SMMC‑7721 cells. The above results indicate that MUC1 gene silencing induces growth inhibition in SMMC‑7721 cells through Bax‑mediated mitochondrial and caspase-8-mediated death receptor apoptotic pathways. PMID:26398332

  18. The Flavonoid 7,4'-Dihydroxyflavone Inhibits MUC5AC Gene Expression, Production, and Secretion via Regulation of NF-κB, STAT6, and HDAC2.

    PubMed

    Liu, Changda; Weir, David; Busse, Paula; Yang, Nan; Zhou, Zhenwen; Emala, Charles; Li, Xiu-Min

    2015-06-01

    Mucus overproduction is a significant component of the pathophysiology of obstructive lung diseases. Currently, there are only a few medications available that inhibit mucus production. Previous studies showed that glycyrrhizin, a triterpenoid in Glycyrrhiza uralensis inhibits mucin 5AC (MUC5AC) mRNA and protein expression. Other potential mucus production inhibitory compounds contained within in G. uralensis have not been fully investigated. The aim of the present study was to determine if the G. uralensis flavonoid 7,4'-dihydroxyflavone (7,4'-DHF) inhibits MUC5AC gene expression, mucus production, and secretion, and if so, to elucidate the mechanism of this inhibition. 7,4'-Dihydroxyflavone significantly decreased phorbol 12-myristate 13-acetate-stimulated NCI-H292 human airway epithelial cell MUC5AC gene expression and mucus production, at a 28-fold lower concentration than glycyrrhizin (The half maximal inhibitory concentration IC50 value of 1.4 μM vs 38 μM, respectively); 7,4'-DHF also inhibited MUC5AC mucus secretion. Inhibition was associated with the suppression of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), signal transducer and activator of transcription 6 (STAT6) activation, and enhanced histone deacetylase 2 (HDAC2) expression. In a murine model of asthma, 7,4'-DHF-treated mice exhibited a marked reduction in MUC5AC secretion in the bronchoalveolar lavage fluid compared with control mice. These findings, together with previous findings linking NF-κB, STAT6, and HDAC2 modulation to the control of MUC5AC expression, demonstrate that 7,4'-DHF is a newly identified component of G. uralensis that regulates MUC5AC expression and secretion via regulation of NF-κB, STAT6, and HDAC2. PMID:25809288

  19. Inhibition of the ADP-glucose pyrophosphorylase in transgenic potatoes leads to sugar-storing tubers and influences tuber formation and expression of tuber storage protein genes.

    PubMed Central

    Müller-Röber, B; Sonnewald, U; Willmitzer, L

    1992-01-01

    Transgenic potato plants were created in which the expression of ADP-glucose pyrophosphorylase (AGPase) was inhibited by introducing a chimeric gene containing the coding region of one of the subunits of the AGPase linked in an antisense orientation to the CaMV 35S promoter. Partial inhibition of the AGPase enzyme was achieved in leaves and almost complete inhibition in tubers. This resulted in the abolition of starch formation in tubers, thus proving that AGPase has a unique role in starch biosynthesis in plants. Instead up to 30% of the dry weight of the transgenic potato tubers was represented by sucrose and up to 8% by glucose. The process of tuber formation also changed, resulting in significantly more tubers both per plant and per stolon. The accumulation of soluble sugars in tubers of antisense plants resulted in a significant increase of the total tuber fresh weight, but a decrease in dry weight of tubers. There was no significant change in the RNA levels of several other starch biosynthetic enzymes, but there was a great increase in the RNA level of the major sucrose synthesizing enzyme sucrose phosphate synthase. In addition, the inhibition of starch biosynthesis was accompanied by a massive reduction in the expression of the major storage protein species of potato tubers, supporting the idea that the expression of storage protein genes is in some way connected to carbohydrate formation in sink storage tissues. Images PMID:1373373

  20. Salt Stress Inhibits the Repair of Photodamaged Photosystem II by Suppressing the Transcription and Translation of psbA Genes in Synechocystis1

    PubMed Central

    Allakhverdiev, Suleyman I.; Nishiyama, Yoshitaka; Miyairi, Sachio; Yamamoto, Hiroshi; Inagaki, Noritoshi; Kanesaki, Yu; Murata, Norio

    2002-01-01

    Light stress and salt stress are major environmental factors that limit the efficiency of photosynthesis. However, we have found that the effects of light and salt stress on photosystem II (PSII) in the cyanobacterium Synechocystis sp. PCC 6803 are completely different. Strong light induced photodamage to PSII, whereas salt stress inhibited the repair of the photodamaged PSII and did not accelerate damage to PSII directly. The combination of light and salt stress appeared to inactivate PSII very rapidly as a consequence of their synergistic effects. Radioactive labeling of cells revealed that salt stress inhibited the synthesis of proteins de novo and, in particular, the synthesis of the D1 protein. Northern- and western-blotting analyses demonstrated that salt stress inhibited the transcription and the translation of psbA genes, which encode D1 protein. DNA microarray analysis indicated that the light-induced expression of various genes was suppressed by salt stress. Thus, our results suggest that salt stress inhibits the repair of PSII via suppression of the activities of the transcriptional and translational machinery. PMID:12428009

  1. Inhibition of Gene Expression of Organic Cation/Carnitine Transporter and Antioxidant Enzymes in Oxazaphosphorines-Induced Acute Cardiomyopathic Rat Models

    PubMed Central

    Sayed-Ahmed, Mohamed M.; Aldelemy, Meshan Lafi; Hafez, Mohamed M.; Al-Shabanah, Othman A.

    2012-01-01

    It is well documented that high therapeutic doses of oxazaphosphorines, cyclophosphamide (CP) and ifosfamide (IFO), are associated with cardiomyopathy. This study investigated whether oxazaphosphorines alter the expression of organic cation/carnitine transporter (OCTN2) and antioxidant genes and if so, whether these alterations contribute to CP and IFO-induced cardiotoxicity. Adult male Wistar albino rats were assigned to one of six treatment groups namely, control, L carnitine, CP, IFO, CP plus L carnitine and IFO plus L carnitine. In cardiac and kidney tissues, CP and IFO significantly decreased mRNA and protein expression of OCTN2. Oxazaphosphorines significantly increased serum acyl-carnitine/free carnitine ratio and urinary carnitine excretion and significantly decreased total carnitine in cardiac tissues. Interestingly, carnitine supplementation completely reversed the biochemical and gene expression changes-induced by oxazaphosphorines to the control values, except OCTN2 expression remained inhibited by IFO. Data from this study suggest that: (1) Oxazaphosphorines decreased myocardial carnitine content following the inhibition of OCTN2 mRNA and protein expression in cardiac tissues. (2) Oxazaphosphorine therapy increased urinary loss of carnitine secondary to the inhibition of OCTN2 mRNA and protein expression in proximal tubules of the kidney. (3) Carnitine supplementation attenuates CP but not IFO-induced inhibition of OCTN2 mRNA and protein expression in heart and kidney tissues. PMID:22701146

  2. The miR-193a-3p-regulated ING5 gene activates the DNA damage response pathway and inhibits multi-chemoresistance in bladder cancer

    PubMed Central

    Qian, Liting; Xiao, Jun; Zhao, Weidong; Liu, Qi; Zhang, Daming; Wang, Yingwei; Yan, Jun; Zhang, Hongyu; He, Yinghua; Zhu, Jingde

    2015-01-01

    As the major barrier to curative cancer chemotherapy, chemoresistance presents a formidable challenge to both cancer researchers and clinicians. We have previously shown that the bladder cancer (BCa) cell line 5637 is significantly more sensitive to the cytoxicity of five chemotherapeutic agents than H-bc cells. Using an RNA-seq-based omic analysis and validation at both the mRNA and protein levels, we found that the inhibitor of growth 5 (ING5) gene was upregulated in 5637 cells compared with H-bc cells, indicating that it has an inhibitory role in BCa chemoresistance. siRNA-mediated inhibition of ING5 increased the chemoresistance and inhibited the DNA damage response pathway in 5637 cells. Conversely, forced expression of EGFP-ING5 decreased the chemoresistance of and activated the DNA damage response pathway in H-bc cells. We also showed that ING5 gene expression is inhibited by miR-193a-3p and is instrumental in miR-193a-3p's role in activating BCa chemoresistance. Our results demonstrate both the role and mechanism of inhibition of BCa chemoresistance by ING5. PMID:25991669

  3. Mycobacterium tuberculosis EIS gene inhibits macrophage autophagy through up-regulation of IL-10 by increasing the acetylation of histone H3.

    PubMed

    Duan, Liang; Yi, Min; Chen, Juan; Li, Shengjin; Chen, Weixian

    2016-05-13

    Autophagy plays a crucial role in the progress of Mycobacterium tuberculosis (MTB) infection. Recently, MTB enhanced intracellular survival (EIS) protein was reported to be secreted from MTB cells and linked to the inhibition of autophagy and the intracellular persistence of the pathogen. Here, we investigated the mechanism of EIS-mediated inhibition of autophagy in a human phorbol myristate acetate (PMA)-treated THP-1 cell line as well as in murine macrophages. We confirmed that the presence of EIS led to the inhibition of rapamycin (Rapa)-induced autophagy, while IL-10 gene expression was increased and Akt/mTOR/p70S6K pathway was activated during the process. IL-10 gene silencing led to a significant recovery of EIS-mediated autophagy suppression and decreased activity of the Akt/mTOR/p70S6K pathway. IL-10 promoter activity was unaffected by EIS. Remarkably, EIS increased the acetylation level of histone H3 (Ac-H3), which binds to the SP1 and STAT3 region of the human IL-10 gene promoter sequence. Thus, EIS protein possibly increased IL-10 expression through the regulation of Ac-H3 of its promoter. Our data demonstrated that one possible mechanism of the MTB evasion of autophagy is that the EIS protein up-regulates IL-10 via Ac-H3 and thus activates Akt/mTOR/p70S6K pathway. PMID:27079235

  4. GATA4 represses an ileal program of gene expression in the proximal small intestine by inhibiting the acetylation of histone H3, lysine 27.

    PubMed

    Aronson, B E; Rabello Aronson, S; Berkhout, R P; Chavoushi, S F; He, A; Pu, W T; Verzi, M P; Krasinski, S D

    2014-11-01

    GATA4 is expressed in the proximal 85% of small intestine where it promotes a proximal intestinal ('jejunal') identity while repressing a distal intestinal ('ileal') identity, but its molecular mechanisms are unclear. Here, we tested the hypothesis that GATA4 promotes a jejunal versus ileal identity in mouse intestine by directly activating and repressing specific subsets of absorptive enterocyte genes by modulating the acetylation of histone H3, lysine 27 (H3K27), a mark of active chromatin, at sites of GATA4 occupancy. Global analysis of mouse jejunal epithelium showed a statistically significant association of GATA4 occupancy with GATA4-regulated genes. Occupancy was equally distributed between down- and up-regulated targets, and occupancy sites showed a dichotomy of unique motif over-representation at down- versus up-regulated genes. H3K27ac enrichment at GATA4-binding loci that mapped to down-regulated genes (activation targets) was elevated, changed little upon conditional Gata4 deletion, and was similar to control ileum, whereas H3K27ac enrichment at GATA4-binding loci that mapped to up-regulated genes (repression targets) was depleted, increased upon conditional Gata4 deletion, and approached H3K27ac enrichment in wild-type control ileum. These data support the hypothesis that GATA4 both activates and represses intestinal genes, and show that GATA4 represses an ileal program of gene expression in the proximal small intestine by inhibiting the acetylation of H3K27. PMID:24878542

  5. Guava leaf extract inhibits quorum-sensing and Chromobacterium violaceum induced lysis of human hepatoma cells: whole transcriptome analysis reveals differential gene expression.

    PubMed

    Ghosh, Runu; Tiwary, Bipransh Kumar; Kumar, Anoop; Chakraborty, Ranadhir

    2014-01-01

    Quorum sensing (QS) is a process mediated via small molecules termed autoinducers (AI) that allow bacteria to respond and adjust according to the cell population density by altering the expression of multitudinous genes. Since QS governs numerous bioprocesses in bacteria, including virulence, its inhibition promises to be an ideal target for the development of novel therapeutics. We found that the aqueous leaf extract of Psidium guajava (GLE) exhibited anti-QS properties as evidenced by inhibition of violacein production in Chromobacterium violaceum and swarming motility of Pseudomonas aeruginosa. The gram-negative bacterium, C. violaceum is a rare pathogen with high mortality rate. In this study, perhaps for the first time, we identified the target genes of GLE in C. violaceum MTCC 2656 by whole transcriptome analysis on Ion Torrent. Our data revealed that GLE significantly down-regulated 816 genes at least three fold, with p value ≤ 0.01, which comprises 19% of the C. violaceum MTCC 2656 genome. These genes were distributed throughout the genome and were associated with virulence, motility and other cellular processes, many of which have been described as quorum regulated in C. violaceum and other gram negative bacteria. Interestingly, GLE did not affect the growth of the bacteria. However, consistent with the gene expression pattern, GLE treated C. violaceum cells were restrained from causing lysis of human hepatoma cell line, HepG2, indicating a positive relationship between the QS-regulated genes and pathogenicity. Overall, our study proposes GLE as a QS inhibitor (QSI) with the ability to attenuate virulence without affecting growth. To the best of our knowledge, this is the first report which provides with a plausible set of candidate genes regulated by the QS system in the neglected pathogen C. violaceum. PMID:25229331

  6. Guava Leaf Extract Inhibits Quorum-Sensing and Chromobacterium violaceum Induced Lysis of Human Hepatoma Cells: Whole Transcriptome Analysis Reveals Differential Gene Expression

    PubMed Central

    Tiwary, Bipransh Kumar; Kumar, Anoop

    2014-01-01

    Quorum sensing (QS) is a process mediated via small molecules termed autoinducers (AI) that allow bacteria to respond and adjust according to the cell population density by altering the expression of multitudinous genes. Since QS governs numerous bioprocesses in bacteria, including virulence, its inhibition promises to be an ideal target for the development of novel therapeutics. We found that the aqueous leaf extract of Psidium guajava (GLE) exhibited anti-QS properties as evidenced by inhibition of violacein production in Chromobacterium violaceum and swarming motility of Pseudomonas aeruginosa. The gram-negative bacterium, C. violaceum is a rare pathogen with high mortality rate. In this study, perhaps for the first time, we identified the target genes of GLE in C. violaceum MTCC 2656 by whole transcriptome analysis on Ion Torrent. Our data revealed that GLE significantly down-regulated 816 genes at least three fold, with p value?0.01, which comprises 19% of the C. violaceum MTCC 2656 genome. These genes were distributed throughout the genome and were associated with virulence, motility and other cellular processes, many of which have been described as quorum regulated in C. violaceum and other gram negative bacteria. Interestingly, GLE did not affect the growth of the bacteria. However, consistent with the gene expression pattern, GLE treated C. violaceum cells were restrained from causing lysis of human hepatoma cell line, HepG2, indicating a positive relationship between the QS-regulated genes and pathogenicity. Overall, our study proposes GLE as a QS inhibitor (QSI) with the ability to attenuate virulence without affecting growth. To the best of our knowledge, this is the first report which provides with a plausible set of candidate genes regulated by the QS system in the neglected pathogen C. violaceum. PMID:25229331

  7. A Novel Neurotoxin Gene ar1b Recombination Enhances the Efficiency of Helicoverpa armigera Nucleopolyhedrovirus as a Pesticide by Inhibiting the Host Larvae Ability to Feed and Grow

    PubMed Central

    Yu, Huan; Meng, Jiao; Xu, Jian; Liu, Tong-xian; Wang, Dun

    2015-01-01

    A recombinant Helicoverpa armigera nucleopolyhedrovirus (HearNPV), Ar1b-HearNPV, was constructed and identified as an improved bio-control agent of Helicoverpa armigera larvae. The HearNPV polyhedrin promoter was used to express the insect-specific neurotoxin gene, ar1b, which was originally isolated from the Australian funnel-web spider (Atrax robustus). RT-PCR and Western blotting analysis showed that both the ar1b transcript and protein were produced successfully in Ar1b-HearNPV-infected HzAM1 cells. In order to investigate the influence of foreign gene insertion in HearNPV, including the ar1b gene, chloramphenicol resistance gene, lacZ, kanamycin resistance gene, and the gentamicin resistance gene, two virus strains (HZ8-HearNPV and wt-HearNPV) were used as controls in the cell transfection analysis. As expected, foreign gene insertion had no impact on budded virus production and viral DNA replication. Both optical microscopy and electron microscopy observations indicated that the formation of the occlusion bodies of recombinant virus was similar to wild type virus. The Ar1b-HearNPV-infected H. armigera larvae exhibited paralysis and weight loss before dying. This recombinant virus also showed a 32.87% decrease in LT50 assays compared with the wild type virus. Besides, Ar1b-HearNPV also inhibited host larval growth and diet consumption. This inhibition was still significant in the older instar larvae treated with the recombinant virus. All of these positive properties of this novel recombinant HearNPV provide a further opportunity to develop this virus strain into a commercial product to control the cotton bollworm. PMID:26296090

  8. Transcriptional up-regulation of antioxidant genes by PPAR{delta} inhibits angiotensin II-induced premature senescence in vascular smooth muscle cells

    SciTech Connect

    Kim, Hyo Jung; Ham, Sun Ah; Paek, Kyung Shin; Hwang, Jung Seok; Jung, Si Young; Kim, Min Young; Jin, Hanna; Kang, Eun Sil; Woo, Im Sun; Kim, Hye Jung; Lee, Jae Heun; Chang, Ki Churl; Han, Chang Woo; Seo, Han Geuk

    2011-03-25

    Research highlights: {yields} Activation of PPAR{delta} by GW501516 significantly inhibited Ang II-induced premature senescence in hVSMCs. {yields} Agonist-activated PPAR{delta} suppressed generation of Ang II-triggered ROS with a concomitant reduction in DNA damage. {yields} GW501516 up-regulated expression of antioxidant genes, such as GPx1, Trx1, Mn-SOD and HO-1. {yields} Knock-down of these antioxidant genes abolished the effects of GW501516 on ROS production and premature senescence. -- Abstract: This study evaluated peroxisome proliferator-activated receptor (PPAR) {delta} as a potential target for therapeutic intervention in Ang II-induced senescence in human vascular smooth muscle cells (hVSMCs). Activation of PPAR{delta} by GW501516, a specific agonist of PPAR{delta}, significantly inhibited the Ang II-induced premature senescence of hVSMCs. Agonist-activated PPAR{delta} suppressed the generation of Ang II-triggered reactive oxygen species (ROS) with a concomitant reduction in DNA damage. Notably, GW501516 up-regulated the expression of antioxidant genes, such as glutathione peroxidase 1, thioredoxin 1, manganese superoxide dismutase and heme oxygenase 1. siRNA-mediated down-regulation of these antioxidant genes almost completely abolished the effects of GW501516 on ROS production and premature senescence in hVSMCs treated with Ang II. Taken together, the enhanced transcription of antioxidant genes is responsible for the PPAR{delta}-mediated inhibition of premature senescence through sequestration of ROS in hVSMCs treated with Ang II.

  9. ATF4-dependent Regulation of the JMJD3 Gene during Amino Acid Deprivation Can Be Rescued in Atf4-deficient Cells by Inhibition of Deacetylation*

    PubMed Central

    Shan, Jixiu; Fu, Lingchen; Balasubramanian, Mukundh N.; Anthony, Tracy; Kilberg, Michael S.

    2012-01-01

    Following amino acid deprivation, the amino acid response (AAR) induces transcription from specific genes through a collection of signaling mechanisms, including the GCN2-eIF2-ATF4 pathway. The present report documents that the histone demethylase JMJD3 is an activating transcription factor 4 (ATF4)-dependent target gene. The JMJD3 gene contains two AAR-induced promoter activities and chromatin immunoprecipitation (ChIP) analysis showed that the AAR leads to enhanced ATF4 recruitment to the C/EBP-ATF response element (CARE) upstream of Promoter-1. AAR-induced histone modifications across the JMJD3 gene locus occur upon ATF4 binding. Jmjd3 transcription is not induced in Atf4-knock-out cells, but the AAR-dependent activation was rescued by inhibition of histone deacetylation with trichostatin A (TSA). The TSA rescue of AAR activation in the absence of Atf4 also occurred for the Atf3 and C/EBP homology protein (Chop) genes, but not for the asparagine synthetase gene. ChIP analysis of the Jmjd3, Atf3, and Chop genes in Atf4 knock-out cells documented that activation of the AAR in the presence of TSA led to specific changes in acetylation of histone H4. The results suggest that a primary function of ATF4 is to recruit histone acetyltransferase activity to a sub-set of AAR target genes. Thus, absolute binding of ATF4 to these particular genes is not required and no ATF4 interaction with the general transcription machinery is necessary. The data are consistent with the hypothesis that ATF4 functions as a pioneer factor to alter chromatin structure and thus, enhance transcription in a gene-specific manner. PMID:22955275

  10. Identification of functional domains of the IR2 protein of equine herpesvirus 1 required for inhibition of viral gene expression and replication

    SciTech Connect

    Kim, Seong K. Kim, Seongman; Dai Gan; Zhang Yunfei; Ahn, Byung C.; O'Callaghan, Dennis J.

    2011-09-01

    The equine herpesvirus 1 (EHV-1) negative regulatory IR2 protein (IR2P), an early 1,165-amino acid (aa) truncated form of the 1487-aa immediate-early protein (IEP), lacks the trans-activation domain essential for IEP activation functions but retains domains for binding DNA, TFIIB, and TBP and the nuclear localization signal. IR2P mutants of the N-terminal region which lack either DNA-binding activity or TFIIB-binding activity were unable to down-regulate EHV-1 promoters. In EHV-1-infected cells expressing full-length IR2P, transcription and protein expression of viral regulatory IE, early EICP0, IR4, and UL5, and late ETIF genes were dramatically inhibited. Viral DNA levels were reduced to 2.1% of control infected cells, but were vey weakly affected in cells that express the N-terminal 706 residues of IR2P. These results suggest that IR2P function requires the two N-terminal domains for binding DNA and TFIIB as well as the C-terminal residues 707 to 1116 containing the TBP-binding domain. - Highlights: > We examine the functional domains of IR2P that mediates negative regulation. > IR2P inhibits at the transcriptional level. > DNA-binding mutant or TFIIB-binding mutant fails to inhibit. > C-terminal aa 707 to 1116 are required for full inhibition. > Inhibition requires the DNA-binding domain, TFIIB-binding domain, and C-terminus.

  11. IL-10 gene-deficient mice lack TGF-beta/Smad signaling and fail to inhibit proinflammatory gene expression in intestinal epithelial cells after the colonization with colitogenic Enterococcus faecalis.

    PubMed

    Ruiz, Pedro A; Shkoda, Anna; Kim, Sandra C; Sartor, R Balfour; Haller, Dirk

    2005-03-01

    Nonpathogenic enteric bacterial species initiate and perpetuate experimental colitis in IL-10 gene-deficient mice (IL-10(-/-)). Bacteria-specific effects on the epithelium are difficult to dissect due to the complex nature of the gut microflora. We showed that IL-10(-/-) mice compared with wild-type mice fail to inhibit proinflammatory gene expression in native intestinal epithelial cells (IEC) after the colonization with colitogenic Gram-positive Enterococcus faecalis. Interestingly, proinflammatory gene expression was transient after 1 wk of E. faecalis monoassociation in IEC from wild-type mice, but persisted after 14 wk of bacterial colonization in IL-10(-/-) mice. Accordingly, wild-type IEC expressed phosphorylated NF-kappaB subunit RelA (p65) and phosphorylated Smad2 only at day 7 after bacterial colonization, whereas E. faecalis-monoassociated IL-10(-/-) mice triggered persistent RelA, but no Smad2 phosphorylation in IEC at days 3, 7, 14, and 28. Consistent with the induction of TLR2-mediated RelA phosphorylation and proinflammatory gene expression in E. faecalis-stimulated cell lines, TLR2 protein expression was absent after day 7 from E. faecalis-monoassociated wild-type mice, but persisted in IL-10(-/-) IEC. Of note, TGF-beta1-activated Smad signaling was associated with the loss of TLR2 protein expression and the inhibition of NF-kappaB-dependent gene expression in IEC lines. In conclusion, E. faecalis-monoassociated IL-10(-/-), but not wild-type mice lack protective TGF-beta/Smad signaling and fail to inhibit TLR2-mediated proinflammatory gene expression in the intestinal epithelium, suggesting a critical role for IL-10 and TGF-beta in maintaining normal epithelial cell homeostasis in the interplay with commensal enteric bacteria. PMID:15728512

  12. Grb7 Upregulation Is a Molecular Adaptation to HER2 Signaling Inhibition Due to Removal of Akt-Mediated Gene Repression

    PubMed Central

    Nencioni, Alessio; Cea, Michele; Garuti, Anna; Passalacqua, Mario; Raffaghello, Lizzia; Soncini, Debora; Moran, Eva; Zoppoli, Gabriele; Pistoia, Vito; Patrone, Franco; Ballestrero, Alberto

    2010-01-01

    The efficacy of anti-HER2 therapeutics, such as lapatinib and trastuzumab, is limited by primary and acquired resistance. Cellular adaptations that allow breast cancer cell to survive prolonged HER2 inhibition include de-repression of the transcription factor FOXO3A with consequent estrogen receptor activation, and/or increased HER3 signaling. Here, we used low-density arrays, quantitative PCR, and western blotting to determine how HER2 signaling inhibition with lapatinib or PI3K inhibitors affects the expression of genes involved in breast cancer metastatic spread and overall prognosis. Retroviral transgenesis was used to express constitutively active forms of Akt in the HER2+ breast cancer cell line SKBR3, and Grb7 in MCF7 cells. Specific gene silencing was obtained by siRNAs transfection. A murine BT474 xenograft cancer model was used to assess the effect of lapatinib on gene expression in vivo. We found that lapatinib induces upregulation of Grb7, an adaptor protein involved in receptor tyrosine kinase signaling and promoting cell survival and cell migration. Grb7 upregulation induced by lapatinib was found to occur in cancer cells in vitro and in vivo. We demonstrate that Grb7 upregulation is recreated by PI3K inhibitors while being prevented by constitutively active Akt. Thus, Grb7 is repressed by PI3K signaling and lapatinib-mediated Akt inhibition is responsible for Grb7 de-repression. Finally, we show that Grb7 removal by RNA-interference reduces breast cancer cell viability and increases the activity of lapatinib. In conclusion, Grb7 upregulation is a potentially adverse consequence of HER2 signaling inhibition. Preventing Grb7 accumulation and/or its interaction with receptor tyrosine kinases may increase the benefit of HER2-targeting drugs. PMID:20126311

  13. Honokiol reverses alcoholic fatty liver by inhibiting the maturation of sterol regulatory element binding protein-1c and the expression of its downstream lipogenesis genes

    SciTech Connect

    Yin Huquan; Kim, Youn-Chul; Chung, Young-Suk; Kim, Young-Chul; Shin, Young-Kee; Lee, Byung-Hoon

    2009-04-01

    Ethanol induces hepatic steatosis via a complex mechanism that is not well understood. Among the variety of molecules that have been proposed to participate in this mechanism, the sterol regulatory element (SRE)-binding proteins (SREBPs) have been identified as attractive targets for therapeutic intervention. In the present study, we evaluated the effects of honokiol on alcoholic steatosis and investigated its possible effect on the inhibition of SREBP-1c maturation. In in vitro studies, H4IIEC3 rat hepatoma cells developed increased lipid droplets when exposed to ethanol, but co-treatment with honokiol reversed this effect. Honokiol inhibited the maturation of SREBP-1c and its translocation to the nucleus, the binding of nSREBP-1c to SRE or SRE-related sequences of its lipogenic target genes, and the expression of genes for fatty acid synthesis. In contrast, magnolol, a structural isomer of honokiol, had no effect on nSREBP-1c levels. Male Wistar rats fed with a standard Lieber-DeCarli ethanol diet for 4 weeks exhibited increased hepatic triglyceride and decreased hepatic glutathione levels, with concomitantly increased serum alanine aminotransferase and TNF-{alpha} levels. Daily administration of honokiol (10 mg/kg body weight) by gavage during the final 2 weeks of ethanol treatment completely reversed these effects on hepatotoxicity markers, including hepatic triglyceride, hepatic glutathione, and serum TNF-{alpha}, with efficacious abrogation of fat accumulation in the liver. Inhibition of SREBP-1c protein maturation and of the expression of Srebf1c and its target genes for hepatic lipogenesis were also observed in vivo. A chromatin immunoprecipitation assay demonstrated inhibition of specific binding of SREBP-1c to the Fas promoter by honokiol in vivo. These results demonstrate that honokiol has the potential to ameliorate alcoholic steatosis by blocking fatty acid synthesis regulated by SREBP-1c.

  14. Structure–activity relationship studies of naphthol AS-E and its derivatives as anticancer agents by inhibiting CREB-mediated gene transcription

    PubMed Central

    Li, Bingbing X.; Yamanaka, Kinrin; Xiao, Xiangshu

    2012-01-01

    CREB (cyclic AMP-response element binding protein) is a downstream transcription factor of a multitude of signaling pathways emanating from receptor tyrosine kinases or G-protein coupled receptors. CREB is not activated until it is phosphorylated at Ser133 and its subsequent binding to CREB-binding protein (CBP) through kinase-inducible domain (KID) in CREB and KID-interacting (KIX) domain in CBP. Tumor tissues from various organs present higher level of expression and activation of CREB. Thus CREB has been proposed as a promising cancer drug target. We previously described naphthol AS-E (1a) as a small molecule inhibitor of CREB-mediated gene transcription in living cells. Here we report the structure–activity relationship (SAR) studies of 1a by modifying the appendant phenyl ring. All the compounds were evaluated for in vitro inhibition of KIX–KID interaction, cellular inhibition of CREB-mediated gene transcription and inhibition of proliferation of four cancer cell lines (A549, MCF-7, MDA-MB-231 and MDA-MB-468). SAR indicated that a small and electron-withdrawing group was preferred at the para-position for KIX–KID interaction inhibition. Compound 1a was selected for further biological characterization and it was found that 1a down-regulated the expression of endogenous CREB target genes. Expression of a constitutively active CREB mutant, VP16-CREB in MCF-7 cells rendered the cells resistant to 1a, suggesting that CREB was critical in mediating its anticancer activity. Furthermore, 1a was not toxic to normal human cells. Collectively, these data support that 1a represents a structural template for further development into potential cancer therapeutics with a novel mechanism of action. PMID:23102993

  15. Canopy architectural and physiological characterization of near-isogenic wheat lines differing in the tiller inhibition gene tin.

    PubMed

    Moeller, Carina; Evers, Jochem B; Rebetzke, Greg

    2014-01-01

    Tillering is a core constituent of plant architecture, and influences light interception to affect plant and crop performance. Near-isogenic lines (NILs) varying for a tiller inhibition (tin) gene and representing two genetic backgrounds were investigated for tillering dynamics, organ size distribution, leaf area, light interception, red: far-red ratio, and chlorophyll content. Tillering ceased earlier in the tin lines to reduce the frequencies of later primary and secondary tillers compared to the free-tillering NILs, and demonstrated the genetically lower tillering plasticity of tin-containing lines. The distribution of organ sizes along shoots varied between NILs contrasting for tin. Internode elongation commenced at a lower phytomer, and the peduncle was shorter in the tin lines. The flag leaves of tin lines were larger, and the longest leaf blades were observed at higher phytomers in the tin than in free-tillering lines. Total leaf area was reduced in tin lines, and non-tin lines invested more leaf area at mid-canopy height. The tiller economy (ratio of seed-bearing shoots to numbers of shoots produced) was 10% greater in the tin lines (0.73-0.76) compared to the free-tillering sisters (0.62-0.63). At maximum tiller number, the red: far-red ratio (light quality stimulus that is thought to induce the cessation of tillering) at the plant-base was 0.18-0.22 in tin lines and 0.09-0.11 in free-tillering lines at levels of photosynthetic active radiation of 49-53% and 30-33%, respectively. The tin lines intercepted less radiation compared to their free-tillering sisters once genotypic differences in tiller numbers had established, and maintained green leaf area in the lower canopy later into the season. Greater light extinction coefficients (k) in tin lines prior to, but reduced k after, spike emergence indicated that differences in light interception between NILs contrasting in tin cannot be explained by leaf area alone but that geometric and optical canopy properties contributed. The careful characterization of specifically-developed NILs is refining the development of a physiology-based model for tillering to improve understanding of the value of architectural traits for use in cereal improvement. PMID:25520724

  16. Canopy architectural and physiological characterization of near-isogenic wheat lines differing in the tiller inhibition gene tin

    PubMed Central

    Moeller, Carina; Evers, Jochem B.; Rebetzke, Greg

    2014-01-01

    Tillering is a core constituent of plant architecture, and influences light interception to affect plant and crop performance. Near-isogenic lines (NILs) varying for a tiller inhibition (tin) gene and representing two genetic backgrounds were investigated for tillering dynamics, organ size distribution, leaf area, light interception, red: far-red ratio, and chlorophyll content. Tillering ceased earlier in the tin lines to reduce the frequencies of later primary and secondary tillers compared to the free-tillering NILs, and demonstrated the genetically lower tillering plasticity of tin-containing lines. The distribution of organ sizes along shoots varied between NILs contrasting for tin. Internode elongation commenced at a lower phytomer, and the peduncle was shorter in the tin lines. The flag leaves of tin lines were larger, and the longest leaf blades were observed at higher phytomers in the tin than in free-tillering lines. Total leaf area was reduced in tin lines, and non-tin lines invested more leaf area at mid-canopy height. The tiller economy (ratio of seed-bearing shoots to numbers of shoots produced) was 10% greater in the tin lines (0.73–0.76) compared to the free-tillering sisters (0.62–0.63). At maximum tiller number, the red: far-red ratio (light quality stimulus that is thought to induce the cessation of tillering) at the plant-base was 0.18–0.22 in tin lines and 0.09–0.11 in free-tillering lines at levels of photosynthetic active radiation of 49–53% and 30–33%, respectively. The tin lines intercepted less radiation compared to their free-tillering sisters once genotypic differences in tiller numbers had established, and maintained green leaf area in the lower canopy later into the season. Greater light extinction coefficients (k) in tin lines prior to, but reduced k after, spike emergence indicated that differences in light interception between NILs contrasting in tin cannot be explained by leaf area alone but that geometric and optical canopy properties contributed. The careful characterization of specifically-developed NILs is refining the development of a physiology-based model for tillering to improve understanding of the value of architectural traits for use in cereal improvement. PMID:25520724

  17. AAV9 delivering a modified human Mullerian inhibiting substance as a gene therapy in patient-derived xenografts of ovarian cancer

    PubMed Central

    Pépin, David; Sosulski, Amanda; Zhang, Lihua; Wang, Dan; Vathipadiekal, Vinod; Hendren, Katherine; Coletti, Caroline M.; Yu, Aaron; Castro, Cesar M.; Birrer, Michael J.; Gao, Guangping; Donahoe, Patricia K.

    2015-01-01

    To improve ovarian cancer patient survival, effective treatments addressing chemoresistant recurrences are particularly needed. Mullerian inhibiting substance (MIS) has been shown to inhibit the growth of a stem-like population of ovarian cancer cells. We have recently engineered peptide modifications to human MIS [albumin leader Q425R MIS (LRMIS)] that increase production and potency in vitro and in vivo. To test this novel therapeutic peptide, serous malignant ascites from highly resistant recurrent ovarian cancer patients were isolated and amplified to create low-passage primary cell lines. Purified recombinant LRMIS protein successfully inhibited the growth of cancer spheroids in vitro in a panel of primary cell lines in four of six patients tested. Adeno-associated virus (AAV) -delivered gene therapy has undergone a clinical resurgence with a good safety profile and sustained gene expression. Therefore, AAV9 was used as a single i.p. injection to deliver LRMIS to test its efficacy in inhibiting growth of palpable tumors in patient-derived ovarian cancer xenografts from ascites (PDXa). AAV9-LRMIS monotherapy resulted in elevated and sustained blood concentrations of MIS, which significantly inhibited the growth of three of five lethal chemoresistant serous adenocarcinoma PDXa models without signs of measurable or overt toxicity. Finally, we tested the frequency of MIS type II receptor expression in a tissue microarray of serous ovarian tumors by immunohistochemistry and found that 88% of patients bear tumors that express the receptor. Taken together, these preclinical data suggest that AAV9-LRMIS provides a potentially well-tolerated and effective treatment strategy poised for testing in patients with chemoresistant serous ovarian cancer. PMID:26216943

  18. Inhibition of β-Defensin Gene Expression in Airway Epithelial Cells by Low Doses of Residual Oil Fly Ash is Mediated by Vanadium

    PubMed Central

    Klein-Patel, Marcia E.; Diamond, Gill; Boniotto, Michele; Saad, Sherif; Ryan, Lisa K.

    2007-01-01

    Poor ambient air quality is associated with increased morbidity and mortality, including respiratory infections. However, its effects on various host-defense mechanisms are poorly understood. This study utilized an in vitro model to study the effect of particulate matter (PM2.5) on one antimicrobial mechanism of host defense in the airway, β-defensin-2 and its bovine homologue, tracheal antimicrobial peptide (TAP) induction in response to lipopolysaccharide (LPS) and IL-1β. Our model utilized cultured primary bovine tracheal epithelial (BTE) cells and the human alveolar type II epithelial cell line, A549, treated with 0–20 μg/cm2 residual oil fly ash (ROFA) for 6 h. The cells were then washed and stimulated for 18 h with 100 ng/ml LPS or for 6 h with 100 ng/ml IL-1β. ROFA inhibited the LPS-induced increase in TAP mRNA and protein without inducing significant cytotoxicity. As little as 2.5 μg/cm2 of ROFA inhibited LPS-induced TAP gene expression by 30%. The inhibitory activity was associated with the soluble fraction and not the washed particle. The activity in the leachate was attributed to vanadium, but not nickel or iron. SiO2 and TiO2 were utilized as controls and did not inhibit LPS induction of TAP gene expression in BTE. ROFA also inhibited the increase of IL-1β–induced human β-defensin-2, a homologue of TAP, in A549 cells. The results show that ROFA, V2O5, and VOSO4 inhibit the ability of airway epithelial cells to respond to inflammatory stimuli at low, physiologically relevant doses and suggest that exposure to these agents could result in an impairment of defense against airborne pathogens. PMID:16641320

  19. Inhibition of beta-defensin gene expression in airway epithelial cells by low doses of residual oil fly ash is mediated by vanadium.

    PubMed

    Klein-Patel, Marcia E; Diamond, Gill; Boniotto, Michele; Saad, Sherif; Ryan, Lisa K

    2006-07-01

    Poor ambient air quality is associated with increased morbidity and mortality, including respiratory infections. However, its effects on various host-defense mechanisms are poorly understood. This study utilized an in vitro model to study the effect of particulate matter (PM(2.5)) on one antimicrobial mechanism of host defense in the airway, beta-defensin-2 and its bovine homologue, tracheal antimicrobial peptide (TAP) induction in response to lipopolysaccharide (LPS) and IL-1beta. Our model utilized cultured primary bovine tracheal epithelial (BTE) cells and the human alveolar type II epithelial cell line, A549, treated with 0-20 microg/cm(2) residual oil fly ash (ROFA) for 6 h. The cells were then washed and stimulated for 18 h with 100 ng/ml LPS or for 6 h with 100 ng/ml IL-1beta. ROFA inhibited the LPS-induced increase in TAP mRNA and protein without inducing significant cytotoxicity. As little as 2.5 microg/cm(2) of ROFA inhibited LPS-induced TAP gene expression by 30%. The inhibitory activity was associated with the soluble fraction and not the washed particle. The activity in the leachate was attributed to vanadium, but not nickel or iron. SiO(2) and TiO(2) were utilized as controls and did not inhibit LPS induction of TAP gene expression in BTE. ROFA also inhibited the increase of IL-1beta-induced human beta-defensin-2, a homologue of TAP, in A549 cells. The results show that ROFA, V(2)O(5), and VOSO(4) inhibit the ability of airway epithelial cells to respond to inflammatory stimuli at low, physiologically relevant doses and suggest that exposure to these agents could result in an impairment of defense against airborne pathogens. PMID:16641320

  20. AAV9 delivering a modified human Mullerian inhibiting substance as a gene therapy in patient-derived xenografts of ovarian cancer.

    PubMed

    Pépin, David; Sosulski, Amanda; Zhang, Lihua; Wang, Dan; Vathipadiekal, Vinod; Hendren, Katherine; Coletti, Caroline M; Yu, Aaron; Castro, Cesar M; Birrer, Michael J; Gao, Guangping; Donahoe, Patricia K

    2015-08-11

    To improve ovarian cancer patient survival, effective treatments addressing chemoresistant recurrences are particularly needed. Mullerian inhibiting substance (MIS) has been shown to inhibit the growth of a stem-like population of ovarian cancer cells. We have recently engineered peptide modifications to human MIS [albumin leader Q425R MIS (LRMIS)] that increase production and potency in vitro and in vivo. To test this novel therapeutic peptide, serous malignant ascites from highly resistant recurrent ovarian cancer patients were isolated and amplified to create low-passage primary cell lines. Purified recombinant LRMIS protein successfully inhibited the growth of cancer spheroids in vitro in a panel of primary cell lines in four of six patients tested. Adeno-associated virus (AAV) -delivered gene therapy has undergone a clinical resurgence with a good safety profile and sustained gene expression. Therefore, AAV9 was used as a single i.p. injection to deliver LRMIS to test its efficacy in inhibiting growth of palpable tumors in patient-derived ovarian cancer xenografts from ascites (PDXa). AAV9-LRMIS monotherapy resulted in elevated and sustained blood concentrations of MIS, which significantly inhibited the growth of three of five lethal chemoresistant serous adenocarcinoma PDXa models without signs of measurable or overt toxicity. Finally, we tested the frequency of MIS type II receptor expression in a tissue microarray of serous ovarian tumors by immunohistochemistry and found that 88% of patients bear tumors that express the receptor. Taken together, these preclinical data suggest that AAV9-LRMIS provides a potentially well-tolerated and effective treatment strategy poised for testing in patients with chemoresistant serous ovarian cancer. PMID:26216943

  1. Activity of the Agrobacterium Ti plasmid conjugal transfer regulator TraR is inhibited by the product of the traM gene.

    PubMed Central

    Fuqua, C; Burbea, M; Winans, S C

    1995-01-01

    The Agrobacterium Ti plasmid tra regulon was previously found to be positively regulated by the TraR protein in the presence of a diffusible N-acyl homoserine lactone designated Agrobacterium autoinducer (AAI). TraR and AAI are similar to LuxR from Vibrio fischeri and the Vibrio autoinducer (VAI), which regulate target bioluminescence (lux) genes in a cell density-dependent manner. We now show that tra genes are also regulated by a second protein, designated TraM, which acts to antagonize TraR-dependent activation. The traM gene is closely linked to traR, and the two genes are transcribed convergently. The predicted TraM proteins of two different Ti plasmids are 77% identical but are not significantly similar to other protein sequences in the database, and thus TraM may represent a novel regulatory protein. Null mutations in traM cause strongly increased conjugation, tra gene transcription, and AAI production. A functional copy of traM introduced into traM mutants decreased conjugation, tra gene transcription, and AAI synthesis. TraM inhibits transcription of traA, traI, and traM. Although traM was first identified by its octopine-inducible promoter, we now show that induction by octopine requires traR, strongly suggesting that TraR is the direct traM activator. PMID:7868612

  2. NCYM, a Cis-Antisense Gene of MYCN, Encodes a De Novo Evolved Protein That Inhibits GSK3β Resulting in the Stabilization of MYCN in Human Neuroblastomas

    PubMed Central

    Suenaga, Yusuke; Islam, S. M. Rafiqul; Alagu, Jennifer; Kaneko, Yoshiki; Kato, Mamoru; Tanaka, Yukichi; Kawana, Hidetada; Hossain, Shamim; Matsumoto, Daisuke; Yamamoto, Mami; Shoji, Wataru; Itami, Makiko; Shibata, Tatsuhiro; Nakamura, Yohko; Ohira, Miki; Haraguchi, Seiki; Takatori, Atsushi; Nakagawara, Akira

    2014-01-01

    The rearrangement of pre-existing genes has long been thought of as the major mode of new gene generation. Recently, de novo gene birth from non-genic DNA was found to be an alternative mechanism to generate novel protein-coding genes. However, its functional role in human disease remains largely unknown. Here we show that NCYM, a cis-antisense gene of the MYCN oncogene, initially thought to be a large non-coding RNA, encodes a de novo evolved protein regulating the pathogenesis of human cancers, particularly neuroblastoma. The NCYM gene is evolutionally conserved only in the taxonomic group containing humans and chimpanzees. In primary human neuroblastomas, NCYM is 100% co-amplified and co-expressed with MYCN, and NCYM mRNA expression is associated with poor clinical outcome. MYCN directly transactivates both NCYM and MYCN mRNA, whereas NCYM stabilizes MYCN protein by inhibiting the activity of GSK3β, a kinase that promotes MYCN degradation. In contrast to MYCN transgenic mice, neuroblastomas in MYCN/NCYM double transgenic mice were frequently accompanied by distant metastases, behavior reminiscent of human neuroblastomas with MYCN amplification. The NCYM protein also interacts with GSK3β, thereby stabilizing the MYCN protein in the tumors of the MYCN/NCYM double transgenic mice. Thus, these results suggest that GSK3β inhibition by NCYM stabilizes the MYCN protein both in vitro and in vivo. Furthermore, the survival of MYCN transgenic mice bearing neuroblastoma was improved by treatment with NVP-BEZ235, a dual PI3K/mTOR inhibitor shown to destabilize MYCN via GSK3β activation. In contrast, tumors caused in MYCN/NCYM double transgenic mice showed chemo-resistance to the drug. Collectively, our results show that NCYM is the first de novo evolved protein known to act as an oncopromoting factor in human cancer, and suggest that de novo evolved proteins may functionally characterize human disease. PMID:24391509

  3. Pattern Triggered Immunity (PTI) in Tobacco: Isolation of Activated Genes Suggests Role of the Phenylpropanoid Pathway in Inhibition of Bacterial Pathogens

    PubMed Central

    Szatmári, Ágnes; Zvara, Ágnes; Móricz, Ágnes M.; Besenyei, Eszter; Szabó, Erika; Ott, Péter G.; Puskás, László G.; Bozsó, Zoltán

    2014-01-01

    Background Pattern Triggered Immunity (PTI) or Basal Resistance (BR) is a potent, symptomless form of plant resistance. Upon inoculation of a plant with non-pathogens or pathogenicity-mutant bacteria, the induced PTI will prevent bacterial proliferation. Developed PTI is also able to protect the plant from disease or HR (Hypersensitive Response) after a challenging infection with pathogenic bacteria. Our aim was to reveal those PTI-related genes of tobacco (Nicotiana tabacum) that could possibly play a role in the protection of the plant from disease. Methodology/Principal Findings Leaves were infiltrated with Pseudomonas syringae pv. syringae hrcC- mutant bacteria to induce PTI, and samples were taken 6 and 48 hours later. Subtraction Suppressive Hybridization (SSH) resulted in 156 PTI-activated genes. A cDNA microarray was generated from the SSH clone library. Analysis of hybridization data showed that in the early (6 hpi) phase of PTI, among others, genes of peroxidases, signalling elements, heat shock proteins and secondary metabolites were upregulated, while at the late phase (48 hpi) the group of proteolysis genes was newly activated. Microarray data were verified by real time RT-PCR analysis. Almost all members of the phenyl-propanoid pathway (PPP) possibly leading to lignin biosynthesis were activated. Specific inhibition of cinnamic-acid-4-hydroxylase (C4H), rate limiting enzyme of the PPP, decreased the strength of PTI - as shown by the HR-inhibition and electrolyte leakage tests. Quantification of cinnamate and p-coumarate by thin-layer chromatography (TLC)-densitometry supported specific changes in the levels of these metabolites upon elicitation of PTI. Conclusions/Significance We believe to provide first report on PTI-related changes in the levels of these PPP metabolites. Results implicated an actual role of the upregulation of the phenylpropanoid pathway in the inhibition of bacterial pathogenic activity during PTI. PMID:25101956

  4. N-Octanoyl Dopamine Inhibits the Expression of a Subset of ?B Regulated Genes: Potential Role of p65 Ser276 Phosphorylation

    PubMed Central

    Gaertner, Sophie; Stamellou, Eleni; Kraaij, Tineke; Mandel, Linda; Loesel, Ralf; Sticht, Carsten; Hoeger, Simone; Ait-Hsiko, Lamia; Schedel, Angelika; Hafner, Mathias; Yard, Benito; Tsagogiorgas, Charalambos

    2013-01-01

    Background and Purpose Catechol containing compounds have anti-inflammatory properties, yet for catecholamines these properties are modest. Since we have previously demonstrated that the synthetic dopamine derivative N-octanoyl dopamine (NOD) has superior anti-inflammatory properties compared to dopamine, we tested NOD in more detail and sought to elucidate the molecular entities and underlying mechanism by which NOD down-regulates inflammation. Experimental Approach Genome wide gene expression profiling of human umbilical vein endothelial cells (HUVECs) was performed after stimulation with TNF-? or in the combination with NOD. Confirmation of these differences, NF?B activation and the molecular entities that were required for the anti-inflammatory properties were assessed in subsequent experiments. Key Results Down regulation of inflammatory genes by NOD occurred predominantly for ?B regulated genes, however not all ?B regulated genes were affected. These findings were explained by inhibition of RelA phosphorylation at Ser276. Leukocyte adherence to TNF-? stimulated HUVECs was inhibited by NOD and was reflected by a diminished expression of adhesion molecules on HUVECs. NOD induced HO-1 expression, but this was not required for inhibition of NF?B. The anti-inflammatory effect of NOD seems to involve the redox active catechol structure, although the redox active para-dihydroxy benzene containing compounds also displayed anti-inflammatory effects, provided that they were sufficiently hydrophobic. Conclusions and Implications The present study highlighted important mechanisms and molecular entities by which dihydroxy benzene compounds exert their potential anti-inflammatory action. Since NOD does not have hemodynamic properties, NOD seems to be a promising candidate drug for the treatment of inflammatory diseases. PMID:24023820

  5. Linking the response of endocrine regulated genes to adverse effects on sex differentiation improves comprehension of aromatase inhibition in a Fish Sexual Development Test.

    PubMed

    Muth-Köhne, Elke; Westphal-Settele, Kathi; Brückner, Jasmin; Konradi, Sabine; Schiller, Viktoria; Schäfers, Christoph; Teigeler, Matthias; Fenske, Martina

    2016-07-01

    The Fish Sexual Development Test (FSDT) is a non-reproductive test to assess adverse effects of endocrine disrupting chemicals. With the present study it was intended to evaluate whether gene expression endpoints would serve as predictive markers of endocrine disruption in a FSDT. For proof-of-concept, a FSDT according to the OECD TG 234 was conducted with the non-steroidal aromatase inhibitor fadrozole (test concentrations: 10μg/L, 32μg/L, 100μg/L) using zebrafish (Danio rerio). Gene expression analyses using quantitative RT-PCR were included at 48h, 96h, 28days and 63days post fertilization (hpf, dpf). The selection of genes aimed at finding molecular endpoints which could be directly linked to the adverse apical effects of aromatase inhibition. The most prominent effects of fadrozole exposure on the sexual development of zebrafish were a complete sex ratio shift towards males and an acceleration of gonad maturation already at low fadrozole concentrations (10μg/L). Due to the specific inhibition of the aromatase enzyme (Cyp19) by fadrozole and thus, the conversion of C19-androgens to C18-estrogens, the steroid hormone balance controlling the sex ratio of zebrafish was altered. The resulting key event is the regulation of directly estrogen-responsive genes. Subsequently, gene expression of vitellogenin 1 (vtg1) and of the aromatase cyp19a1b isoform (cyp19a1b), were down-regulated upon fadrozole treatment compared to controls. For example, mRNA levels of vtg1 were down-regulated compared to the controls as early as 48 hpf and 96 hpf. Further regulated genes cumulated in pathways suggested to be controlled by endocrine mechanisms, like the steroid and terpenoid synthesis pathway (e.g. mevalonate (diphospho) decarboxylase (mvd), lanosterol synthase (2,3-oxidosqualene-lanosterol cyclase; lss), methylsterol monooxygenase 1 (sc4mol)) and in lipid transport/metabolic processes (steroidogenic acute regulatory protein (star), apolipoprotein Eb (apoEb)). Taken together, this study demonstrated that the existing Adverse Outcome Pathway (AOP) for aromatase inhibition in fish can be translated to the life-stage of sexual differentiation. We were further able to identify MoA-specific marker gene expression which can be instrumental in defining new measurable key events (KE) of existing or new AOPs related to endocrine disruption. PMID:27130971

  6. IL-10 gene-deficient mice lack TGF-beta/Smad-mediated TLR2 degradation and fail to inhibit proinflammatory gene expression in intestinal epithelial cells under conditions of chronic inflammation.

    PubMed

    Ruiz, Pedro A; Shkoda, Anna; Kim, Sandra C; Sartor, R Balfour; Haller, Dirk

    2006-08-01

    Nonpathogenic enteric bacterial species initiate and perpetuate experimental colitis in interleukin-10 geneeficient mice (IL-10(-/-)). Bacteria-specific effects on the epithelium are difficult to distinguish because of the complex nature of the gut microflora. We showed that IL-10(-/-) mice compared to wild-type mice fail to inhibit pro-inflammatory gene expression in native intestinal epithelial cells after the colonization with colitogenic Gram-positive Enterococcus faecalis. Of interest, pro-inflammatory gene expression was transient after 1 week of E. faecalis monoassociation in IECs from wild-type mice but persisted after 14 weeks of bacterial colonization in IL-10(-/-) mice. Accordingly, wild-type IECs expressed phosphorylated NF-kappaB subunit RelA (p65) and phosphorylated Smad2 only at day 7 after bacterial colonization, whereas E. faecalis-monoassociated IL-10(-/-) mice triggered persistent RelA but no Smad2 phosphorylation in IECs at days 3, 7, 14, and 28. Consistent with the induction of TLR2-mediated RelA phosphorylation and pro-inflammatory gene expression in E. faecalis-stimulated cell lines, TLR2 protein expression was absent after day 7 from E. faecalis-monoassociated wild-type mice but persisted in IL-10(-/-) IECs. Of note, TGF-beta-activated Smad signaling was associated with the loss of TLR2 protein expression and the inhibition of NF-kappa Bependent gene expression in E. faecalis-stimulated IEC lines. In conclusion, E. faecalis-monoassociated IL-10(-/-) but not wild-type mice lack protective TGF-beta/Smad signaling and fail to inhibit TLR2-mediated pro-inflammatory gene expression in the intestinal epithelium, suggesting a critical role for IL-10 and TGF-beta in maintaining normal epithelial cell homeostasis in the interplay with commensal enteric bacteria. PMID:17057220

  7. Heat shock selectively inhibits ribosomal RNA gene transcription and down-regulates E1BF/Ku in mouse lymphosarcoma cells.

    PubMed Central

    Ghoshal, K; Jacob, S T

    1996-01-01

    The effect of heat shock on RNA polymerase I (pol I)-directed transcription of the rRNA gene was studied in S-100 extract derived from mouse lymphosarcoma cells, and by in vivo labelling of rRNA. Exposure of cells to 42 degrees C for 2 h resulted in complete inhibition of rRNA synthesis in vivo. Pol I transcription was inhibited by 50% within 2 h of heat shock and was abolished after 3 h exposure at 42 degrees C. Under this condition, the core-promoter-binding activity of the factor (CPBF) that modulates pol I transcription was unaffected. In contrast, the promoter-binding activity of enhancer-1-binding factor, a protein related to the Ku autoantigen, which is involved in pol I transcription initiation, was reduced by 50 and 90% after 2 and 3 h of heat shock respectively. Western-blot analysis with antibodies specific for the two subunits of Ku protein showed the absence of p72 subunit after 3 h of heat shock. Under this condition, pol II transcription from the adenovirus major late promoter and pol III transcription of 5 S RNA gene remained unaffected. Mixing experiments ruled out the possibility that the inhibition of transcription was due to activation of nucleases or other inhibitors. This is the first report to show selective down-regulation of pol I transcription in vitro by heat shock and of the potential involvement of a pol I transcription factor in this process. PMID:8760351

  8. Overexpression of G0/G1 Switch Gene 2 in Adipose Tissue of Transgenic Quail Inhibits Lipolysis Associated with Egg Laying.

    PubMed

    Chen, Paula Renee; Shin, Sangsu; Choi, Young Min; Kim, Elizabeth; Han, Jae Yong; Lee, Kichoon

    2016-01-01

    In avians, yolk synthesis is regulated by incorporation of portomicrons from the diet, transport of lipoproteins from the liver, and release of lipids from adipose tissue; however, the extent to which lipolysis in adipose tissue contributes to yolk synthesis and egg production has yet to be elucidated. G0/G1 switch gene 2 (G0S2) is known to bind and inhibit adipose triglyceride lipase (ATGL), the rate-limiting enzyme in lipolysis. The objective of this study was to determine whether overexpression of the G0S2 gene in adipose tissue could successfully inhibit endogenous ATGL activity associated with egg laying. Two independent lines of transgenic quail overexpressing G0S2 had delayed onset of egg production and reduced number of eggs over a six-week period compared to non-transgenic quail. Although no differences in measured parameters were observed at the pre-laying stage (5 weeks of age), G0S2 transgenic quail had significantly larger interclavicular fat pad weights and adipocyte sizes and lower NEFA concentrations in the serum at early (1 week after laying first egg) and active laying (5 weeks after laying first egg) stages. Overexpression of G0S2 inhibited lipolysis during early and active laying, which drastically shifted the balance towards a net accumulation of triacylglycerols and increased adipose tissue mass. Thereby, egg production was negatively affected as less triacylglycerols were catabolized to produce lipids for the yolk. PMID:26999108

  9. Hydrogen sulfide inhibits opioid withdrawal-induced pain sensitization in rats by down-regulation of spinal calcitonin gene-related peptide expression in the spine.

    PubMed

    Yang, Hai-Yu; Wu, Zhi-Yuan; Bian, Jin-Song

    2014-09-01

    Hyperalgesia often occurs in opioid-induced withdrawal syndrome. In the present study, we found that three hourly injections of DAMGO (a μ-opioid receptor agonist) followed by naloxone administration at the fourth hour significantly decreased rat paw nociceptive threshold, indicating the induction of withdrawal hyperalgesia. Application of NaHS (a hydrogen sulfide donor) together with each injection of DAMGO attenuated naloxone-precipitated withdrawal hyperalgesia. RT-PCR and Western blot analysis showed that NaHS significantly reversed the gene and protein expression of up-regulated spinal calcitonin gene-related peptide (CGRP) in naloxone-treated animals. NaHS also inhibited naloxone-induced cAMP rebound and cAMP response element-binding protein (CREB) phosphorylation in rat spinal cord. In SH-SY5Y neuronal cells, NaHS inhibited forskolin-stimulated cAMP production and adenylate cyclase (AC) activity. Moreover, NaHS pre-treatment suppressed naloxone-stimulated activation of protein kinase C (PKC) α, Raf-1, and extracellular signal-regulated kinase (ERK) 1/2 in rat spinal cord. Our data suggest that H2S prevents the development of opioid withdrawal-induced hyperalgesia via suppression of synthesis of CGRP in spine through inhibition of AC/cAMP and PKC/Raf-1/ERK pathways. PMID:24824948

  10. Overexpression of G0/G1 Switch Gene 2 in Adipose Tissue of Transgenic Quail Inhibits Lipolysis Associated with Egg Laying

    PubMed Central

    Chen, Paula Renee; Shin, Sangsu; Choi, Young Min; Kim, Elizabeth; Han, Jae Yong; Lee, Kichoon

    2016-01-01

    In avians, yolk synthesis is regulated by incorporation of portomicrons from the diet, transport of lipoproteins from the liver, and release of lipids from adipose tissue; however, the extent to which lipolysis in adipose tissue contributes to yolk synthesis and egg production has yet to be elucidated. G0/G1 switch gene 2 (G0S2) is known to bind and inhibit adipose triglyceride lipase (ATGL), the rate-limiting enzyme in lipolysis. The objective of this study was to determine whether overexpression of the G0S2 gene in adipose tissue could successfully inhibit endogenous ATGL activity associated with egg laying. Two independent lines of transgenic quail overexpressing G0S2 had delayed onset of egg production and reduced number of eggs over a six-week period compared to non-transgenic quail. Although no differences in measured parameters were observed at the pre-laying stage (5 weeks of age), G0S2 transgenic quail had significantly larger interclavicular fat pad weights and adipocyte sizes and lower NEFA concentrations in the serum at early (1 week after laying first egg) and active laying (5 weeks after laying first egg) stages. Overexpression of G0S2 inhibited lipolysis during early and active laying, which drastically shifted the balance towards a net accumulation of triacylglycerols and increased adipose tissue mass. Thereby, egg production was negatively affected as less triacylglycerols were catabolized to produce lipids for the yolk. PMID:26999108

  11. Sodium houttuyfonate inhibits biofilm formation and alginate biosynthesis-associated gene expression in a clinical strain of Pseudomonas aeruginosa in vitro

    PubMed Central

    WU, DA-QIANG; CHENG, HUIJUAN; DUAN, QIANGJUN; HUANG, WEIFENG

    2015-01-01

    The increasing multidrug resistance of Pseudomonas aeruginosa has become a serious public-health problem. In the present study, the inhibitory activities of sodium houttuyfonate (SH) against biofilm formation and alginate production in a clinical strain of P. aeruginosa (AH16) were investigated in vitro using crystal violet dying and standard curve methods, respectively. The cellular morphology of P. aeruginosa treated with SH was observed using a scanning electron microscope. Furthermore, reverse transcription-quantitative polymerase chain reaction was used to identify differences in the expression levels of genes associated with alginate biosynthesis as a result of the SH treatment. The results indicated that SH significantly inhibited biofilm formation, and decreased the levels of the primary biofilm constituent, alginate, in P. aeruginosa AH16 at various stages of biofilm development. In addition, scanning electron microscopy observations demonstrated that SH markedly altered the cellular morphology and biofilm structure of P. aeruginosa. Furthermore, the results from the reverse transcription-quantitative polymerase chain reaction analysis indicated that SH inhibited biofilm formation by mitigating the expression of the algD and algR genes, which are associated with alginate biosynthesis. Therefore, the present study has provided novel insights into the potent effects and underlying mechanisms of SH-induced inhibition of biofilm formation in a clinical strain of P. aeruginosa. PMID:26622388

  12. Synergistic inhibition of PARP-1 and NF-κB signaling downregulates immune response against recombinant AAV2 vectors during hepatic gene therapy.

    PubMed

    Hareendran, Sangeetha; Ramakrishna, Banumathi; Jayandharan, Giridhara R

    2016-01-01

    Host immune response remains a key obstacle to widespread application of adeno-associated virus (AAV) based gene therapy. Thus, targeted inhibition of the signaling pathways that trigger such immune responses will be beneficial. Previous studies have reported that DNA damage response proteins such as poly(ADP-ribose) polymerase-1 (PARP-1) negatively affect the integration of AAV in the host genome. However, the role of PARP-1 in regulating AAV transduction and the immune response against these vectors has not been elucidated. In this study, we demonstrate that repression of PARP-1 improves the transduction of single-stranded AAV vectors both in vitro (∼174%) and in vivo (two- to 3.4-fold). Inhibition of PARP-1, also significantly downregulated the expression of several proinflammatory and cytokine markers such as TLRs, ILs, NF-κB subunit proteins associated with the host innate response against self-complementary AAV2 vectors. The suppression of the inflammatory response targeted against these vectors was more effective upon combined inhibition of PARP-1 and NF-κB signaling. This strategy also effectively attenuated the AAV capsid-specific cytotoxic T-cell response, with minimal effect on vector transduction, as demonstrated in normal C57BL/6 and hemophilia B mice. These data suggest that targeting specific host cellular proteins could be useful to attenuate the immune barriers to AAV-mediated gene therapy. PMID:26443873

  13. Orostachys japonicus Inhibits Expression of the TLR4, NOD2, iNOS, and COX-2 Genes in LPS-Stimulated Human PMA-Differentiated THP-1 Cells by Inhibiting NF-κB and MAPK Activation

    PubMed Central

    Woo, Hong-Jung; Kim, Youngchul

    2015-01-01

    Orostachys japonicus is traditionally used as an inflammatory agent. In this report, we investigated the effects of O. japonicus extract on the expression of genes encoding pathogen-recognition receptors (TLR2, TLR4, NOD1, and NOD2) and proinflammatory factors (iNOS, COX-2, and cytokines) in LPS-stimulated PMA-differentiated THP-1 cells and the NF-κB and MAPK pathways. O. japonicus induced toxicity at high concentrations but had no effect at concentrations lower than 25 μg/mL. O. japonicus inhibited LPS-induced TLR4 and NOD2 mRNA levels, suppressed LPS-induced iNOS and COX-2 transcription and translocation, and downregulated LPS-induced proinflammatory cytokine (IL-1β, IL-6, IL-8, and TNF-α) mRNA levels. In addition, O. japonicus inhibited LPS-induced NF-κB activation and IκBα degradation and suppressed LPS-induced JNK, p38 MAPK, and ERK phosphorylation. Overall, our results demonstrate that the anti-inflammatory effects of O. japonicus are mediated by suppression of NF-κB and MAPK signaling, resulting in reduced TLR4, NOD2, iNOS, and COX-2 expression and inhibition of inflammatory cytokine expression. PMID:25810745

  14. p53 inhibits the expression of p125 and the methylation of POLD1 gene promoter by downregulating the Sp1-induced DNMT1 activities in breast cancer

    PubMed Central

    Zhang, Liang; Yang, Weiping; Zhu, Xiao; Wei, Changyuan

    2016-01-01

    p125 is one of four subunits of human DNA polymerases – DNA Pol δ as well as one of p53 target protein encoded by POLD1. However, the function and significance of p125 and the role that p53 plays in regulating p125 expression are not fully understood in breast cancer. Tissue sections of human breast cancer obtained from 70 patients whose median age was 47.6 years (range: 38–69 years) with stage II–III breast cancer were studied with normal breast tissue from the same patients and two human breast cell lines (MCF-7 and MCF-10A). p53 expression levels were reduced, while p125 protein expression was increased in human breast cancer tissues and cell line detected by Western blot and quantitative reverse transcriptase-polymerase chain reaction. The methylation level of the POLD1 gene promoter was greater in breast cancer tissues and cells when compared with normal tissues and cells. In MCF-7 cell model, p53 overexpression caused a decrease in the level of p125 protein, while the methylation level of the p125 gene promoter was also inhibited by p53 overexpression. To further investigate the regulating mechanism of p53 on p125 expression, our study focused on DNA methyltransferase 1 (DNMT1) and transcription factor Sp1. Both DNMT1 and Sp1 protein expression were reduced when p53 was overexpressed in MCF-7 cells. The Sp1 binding site appears to be important for DNMT1 gene transcription; Sp1 and p53 can bind together, which means that DNMT1 gene expression may be downregulated by p53 through binding to Sp1. Because DNMT1 methylation level of the p125 gene promoter can affect p125 gene transcription, we propose that p53 may indirectly regulate p125 gene promoter expression through the control of DNMT1 gene transcription. In conclusion, the data from this preliminary study have shown that p53 inhibits the methylation of p125 gene promoter by downregulating the activities of Sp1 and DNMT1 in breast cancer. PMID:27022290

  15. Hexokinase-mediated sugar signaling controls expression of the calcineurin B-like interacting protein kinase 15 gene and is perturbed by oxidative phosphorylation inhibition.

    PubMed

    Yim, Hui-Kyeong; Lim, Mi-Na; Lee, Sung-Eun; Lim, Jun; Lee, Yew; Hwang, Yong-Sic

    2012-10-15

    Calcineurin B-like (CBL) interacting protein kinase 15 (CIPK15) is a newly identified positive regulator which is critical to directing the O(2) deficiency signal to the sugar signaling cascade as part of Amy3D (representative Amy3 gene) regulation in rice. It is located upstream and probably contributes to reserve mobilization under anoxia. In isolated starving embryos, the temporal pattern of accumulation of CIPK15 transcripts and leaky suppression of this gene suggests that factors other than CIPK15 may also be involved in the regulation of Amy3D expression. Probing of a variety of sugars and sugar analogs has shown that hexokinase mediates the sugar regulation of CIPK15. For example, hexokinase substrates, such as mannose, 2-deoxyglucose, and other metabolizable sugars, repressed CIPK15 expression, whereas 3-O-methylglucose and 6-deoxyglucose did not. By using glucosamine, a hexokinase inhibitor, to release glucose-dependent CIPK15 suppression, we confirmed that hexokinase mediates regulation of this gene. Chemical inhibitors of mitochondrial electron transfer, proton separation or ATP synthase also effectively abolished sugar-induced repression of CIPK15. This type of interference, the release from glucose-induced repression of gene expression by inhibition of oxidative phosphorylation, was previously identified for the Amy3D gene, which suggests that hexokinase-mediated sugar signaling may be coordinated with the cellular energy status. Analysis of a transgenic rice cell line harboring the GUS reporter gene under the control of the CIPK15 promoter, and transient expression assay for 3' UTR of the CIPK15 gene indicate that sugar regulation of the rice CIPK15 gene is likely mediated by 2548-bp 5'-flanking region, with no additional post-transcriptional control. PMID:22796010

  16. Metformin inhibits epithelial–mesenchymal transition in prostate cancer cells: Involvement of the tumor suppressor miR30a and its target gene SOX4

    SciTech Connect

    Zhang, Jing; Shen, Chengwu; Wang, Lin; Ma, Quanping; Xia, Pingtian; Qi, Mei; Yang, Muyi; Han, Bo

    2014-09-26

    Highlights: • Metformin inhibits TGF-β-induced EMT in prostate cancer (PCa) cells. • Metformin upregulates tumor suppressor miR30a and downregulates SOX4 in PCa cells. • SOX4 is a target gene of miR30a. - Abstract: Tumor metastasis is the leading cause of mortality and morbidity of prostate cancer (PCa) patients. Epithelial–mesenchymal transition (EMT) plays a critical role in cancer progression and metastasis. Recent evidence suggested that diabetic patients treated with metformin have lower PCa risk and better prognosis. This study was aimed to investigate the effects of metformin on EMT in PCa cells and the possible microRNA (miRNA)-based mechanisms. MiRNAs have been shown to regulate various processes of cancer metastasis. We herein showed that metformin significantly inhibits proliferation of Vcap and PC-3 cells, induces G0/G1 cell cycle arrest and inhibits invasiveness and motility capacity of Vcap cells. Metformin could inhibit TGF-β-induced EMT in Vcap cells, as manifested by inhibition of the increase of N-cadherin (p = 0.013), Vimentin (p = 0.002) and the decrease of E-cadherin (p = 0.0023) and β-catenin (p = 0.034) at mRNA and protein levels. Notably, we demonstrated significant upregulation of miR30a levels by metformin (P < 0.05) and further experiments indicated that miR30a significantly inhibits proliferation and EMT process of Vcap cells. Interestingly, we identified that SOX4, a previously reported oncogenic transcriptional factor and modulator of EMT, is a direct target gene of miR30a. Finally, we screened the expression of miR30a and SOX4 in 84 PCa cases with radical prostatectomy. Of note, SOX4 overexpression is significantly associated with decreased levels of miR30a in PCa cases. In all, our study suggested that inhibition of EMT by metformin in PCa cells may involve upregulation of miR30a and downregulation of SOX4.

  17. The Mg-chelatase H subunit of Arabidopsis antagonizes a group of WRKY transcription repressors to relieve ABA-responsive genes of inhibition.

    PubMed

    Shang, Yi; Yan, Lu; Liu, Zhi-Qiang; Cao, Zheng; Mei, Chao; Xin, Qi; Wu, Fu-Qing; Wang, Xiao-Fang; Du, Shu-Yuan; Jiang, Tao; Zhang, Xiao-Feng; Zhao, Rui; Sun, Hai-Li; Liu, Rui; Yu, Yong-Tao; Zhang, Da-Peng

    2010-06-01

    The phytohormone abscisic acid (ABA) plays a vital role in plant development and response to environmental challenges, but the complex networks of ABA signaling pathways are poorly understood. We previously reported that a chloroplast protein, the magnesium-protoporphyrin IX chelatase H subunit (CHLH/ABAR), functions as a receptor for ABA in Arabidopsis thaliana. Here, we report that ABAR spans the chloroplast envelope and that the cytosolic C terminus of ABAR interacts with a group of WRKY transcription factors (WRKY40, WRKY18, and WRKY60) that function as negative regulators of ABA signaling in seed germination and postgermination growth. WRKY40, a central negative regulator, inhibits expression of ABA-responsive genes, such as ABI5. In response to a high level of ABA signal that recruits WRKY40 from the nucleus to the cytosol and promotes ABAR-WRKY40 interaction, ABAR relieves the ABI5 gene of inhibition by repressing WRKY40 expression. These findings describe a unique ABA signaling pathway from the early signaling events to downstream gene expression. PMID:20543028

  18. A dominant negative mutant of an IFN regulatory factor family protein inhibits both type I and type II IFN-stimulated gene expression and antiproliferative activity of IFNs.

    PubMed

    Thornton, A M; Ogryzko, V V; Dent, A; Sharf, R; Levi, B Z; Kanno, Y; Staudt, L M; Howard, B H; Ozato, K

    1996-12-01

    Type I (alpha,beta) and type II (gamma) IFNs elicit antiproliferative and antiviral activities through two distinct transcription pathways involving 1) IRF family proteins and ISGF3, and 2) STAT1. We have employed a dominant negative strategy to study the role of IRF family proteins in eliciting the biologic activities of IFN. A truncated IRF protein retaining the DNA-binding domain (DBD) of ICSBP (a member of the IRF family) was stably transfected into U937 monocytic cells. Clones expressing DBD had markedly reduced ISRE-binding activity and were defective in expressing several type I IFN-inducible genes. STAT1 was one such type I IFN-inducible gene whose expression was also inhibited in DBD clones. As a result, the expression of several IFN-gamma-inducible genes was also inhibited in these clones, indicating functional coupling of the type I and type II IFN transcription pathways. Furthermore, DBD clones grew more slowly than control clones and were refractory to antiproliferative effects of both types of IFNs. We found that IFN treatment of U937 cells leads to a G1 arrest and an increase in underphosphorylated retinoblastoma gene product. However, IFN treatment did not change the cell cycle profile, nor retinoblastoma gene product phosphorylation state in DBD clones. These data indicate that expression of DBD disrupts cell cycle regulatory mechanisms. Combined with the previously noted failure of DBD clones to elicit antiviral activity, the present work shows that IRF family proteins play an integral part in growth control activities of IFNs. PMID:8943426

  19. Transgenic Cotton Plants Expressing Double-stranded RNAs Target HMG-CoA Reductase (HMGR) Gene Inhibits the Growth, Development and Survival of Cotton Bollworms

    PubMed Central

    Tian, Geng; Cheng, Linlin; Qi, Xuewei; Ge, Zonghe; Niu, Changying; Zhang, Xianlong; Jin, Shuangxia

    2015-01-01

    RNA interference (RNAi) has been developed as a powerful technique in the research of functional genomics as well as plant pest control. In this report, double-stranded RNAs (dsRNA) targeting 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene, which catalyze a rate-limiting enzymatic reaction in the mevalonate pathway of juvenile hormone (JH) synthesis in cotton bollworm, was expressed in cotton plants via Agrobacterium tumefaciens-mediated transformation. PCR and Sothern analysis revealed the integration of HMGR gene into cotton genome. RT-PCR and qRT-PCR confirmed the high transcription level of dsHMGR in transgenic cotton lines. The HMGR expression both in transcription and translation level was significantly downregulated in cotton bollworms (helicoverpa armigera) larvae after feeding on the leaves of HMGR transgenic plants. The transcription level of HMGR gene in larvae reared on transgenic cotton leaves was as much as 80.68% lower than that of wild type. In addition, the relative expression level of vitellogenin (Vg, crucial source of nourishment for offspring embryo development) gene was also reduced by 76.86% when the insect larvae were fed with transgenic leaves. The result of insect bioassays showed that the transgenic plant harboring dsHMGR not only inhibited net weight gain but also delayed the growth of cotton bollworm larvae. Taken together, transgenic cotton plant expressing dsRNAs successfully downregulated HMGR gene and impaired the development and survival of target insect, which provided more option for plant pest control. PMID:26435695

  20. Transgenic Cotton Plants Expressing Double-stranded RNAs Target HMG-CoA Reductase (HMGR) Gene Inhibits the Growth, Development and Survival of Cotton Bollworms.

    PubMed

    Tian, Geng; Cheng, Linlin; Qi, Xuewei; Ge, Zonghe; Niu, Changying; Zhang, Xianlong; Jin, Shuangxia

    2015-01-01

    RNA interference (RNAi) has been developed as a powerful technique in the research of functional genomics as well as plant pest control. In this report, double-stranded RNAs (dsRNA) targeting 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene, which catalyze a rate-limiting enzymatic reaction in the mevalonate pathway of juvenile hormone (JH) synthesis in cotton bollworm, was expressed in cotton plants via Agrobacterium tumefaciens-mediated transformation. PCR and Sothern analysis revealed the integration of HMGR gene into cotton genome. RT-PCR and qRT-PCR confirmed the high transcription level of dsHMGR in transgenic cotton lines. The HMGR expression both in transcription and translation level was significantly downregulated in cotton bollworms (helicoverpa armigera) larvae after feeding on the leaves of HMGR transgenic plants. The transcription level of HMGR gene in larvae reared on transgenic cotton leaves was as much as 80.68% lower than that of wild type. In addition, the relative expression level of vitellogenin (Vg, crucial source of nourishment for offspring embryo development) gene was also reduced by 76.86% when the insect larvae were fed with transgenic leaves. The result of insect bioassays showed that the transgenic plant harboring dsHMGR not only inhibited net weight gain but also delayed the growth of cotton bollworm larvae. Taken together, transgenic cotton plant expressing dsRNAs successfully downregulated HMGR gene and impaired the development and survival of target insect, which provided more option for plant pest control. PMID:26435695

  1. Interleukin-4 inhibits prostaglandin E2 production by freshly prepared adherent rheumatoid synovial cells via inhibition of biosynthesis and gene expression of cyclo-oxygenase II but not of cyclo-oxygenase I.

    PubMed Central

    Sugiyama, E; Taki, H; Kuroda, A; Mino, T; Yamashita, N; Kobayashi, M

    1996-01-01

    OBJECTIVE: To characterise the effect of interleukin-4 (IL-4) on the biosynthesis of cyclo-oxygenases I (COX I) and II (COX II), the rate limiting enzymes of the synthesis of prostaglandin E2 (PGE2), in freshly prepared rheumatoid synovial cells. METHODS: Adherent synovial cells were obtained from rheumatoid synovium by collagenase digestion. The concentrations of PGE2 in culture supernatants were determined by enzyme linked immunosorbent assay. The protein and mRNA concentrations of COX I and COX II were determined by Western blotting and reverse transcription polymerase chain reaction, respectively. RESULTS: Freshly prepared synovial cells produced large amounts of PGE2. They also showed increased gene expression of COX I and COX II, and synthesised these proteins. IL-4 had suppressive effects on the production of PGE2 by untreated or lipopolysaccharide (LPS) stimulated synovial cells. In addition, IL-4 inhibited the biosynthesis of COX II at the mRNA level. In contrast, it did not modify the protein concentration of COX I. In tests of cell specificity, IL-4 did not reduce the mRNA concentration of COX II in interleukin-1 alpha (IL-1 alpha) stimulated cultured synovial fibroblasts at passages 3-6, but it reduced considerably the mRNA concentrations of COX II in an LPS or IL-1 alpha stimulated U937 monocyte/macrophage cell line. CONCLUSIONS: These results suggest that IL-4 might inhibit overproduction of PGE2 in rheumatoid synovia via selective inhibition of the biosynthesis of COX II, and that this inhibition might be specific to macrophage-like synovial cells. Images PMID:8694577

  2. Arvelexin from Brassica rapa suppresses NF-κB-regulated pro-inflammatory gene expression by inhibiting activation of IκB kinase

    PubMed Central

    Shin, Ji-Sun; Noh, Young-Su; Lee, Yong Sup; Cho, Young-Wuk; Baek, Nam-In; Choi, Myung-Sook; Jeong, Tae-Sook; Kang, Eunkyung; Chung, Hae-Gon; Lee, Kyung-Tae

    2011-01-01

    BACKGROUND AND PURPOSE Brassica rapa species constitute one of the major sources of food. In the present study, we investigated the anti-inflammatory effects and the underlying molecular mechanism of arvelexin, isolated from B. rapa, on lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages and on a model of septic shock induced by LPS. EXPERIMENTAL APPROACH The expression of Inducible nitric oxide synthase (iNOS) and COX-2, TNF-α, IL-6 and IL-1β were determined by Western blot and/or RT-PCR respectively. To elucidate the underlying mechanism(s), activation of NF-κB activation and its pathways were investigated by electrophoretic mobility shift assay, reporter gene and Western blot assays. In addition, the in vivo anti-inflammatory effects of arvelexin were evaluated in endotoxaemia induced with LPS. KEY RESULTS Promoter assays for iNOS and COX-2 revealed that arvelexin inhibited LPS-induced NO and prostaglandin E2 production through the suppression of iNOS and COX-2 at the level of gene transcription. In addition, arvelexin inhibited NF-κB-dependent inflammatory responses by modulating a series of intracellular events of IκB kinase (IKK)-inhibitor κBα (IκBα)-NF-κB signalling. Moreover, arvelexin inhibited IKKβ-elicited NF-κB activation as well as iNOS and COX-2 expression. Serum levels of NO and inflammatory cytokines and mortality in mice challenged injected with LPS were significantly reduced by arvelexin. CONCLUSION AND IMPLICATIONS Arvelexin down-regulated inflammatory iNOS, COX-2, TNF-α, IL-6 and IL-1β gene expression in macrophages interfering with the activation of IKKβ and p38 mitogen-activated protein kinase, and thus, preventing NF-κB activation. PMID:21434881

  3. GATA4 represses an ileal program of gene expression in the proximal small intestine by inhibiting the acetylation of histone H3, lysine 27

    PubMed Central

    Aronson, B. E.; Aronson, S. Rabello; Berkhout, R. P.; Chavoushi, S. F.; He, A.; Pu, W. T.; Verzi, M. P.; Krasinski, S. D.

    2015-01-01

    GATA4 is expressed in the proximal 85% of small intestine where it promotes a proximal intestinal (‘jejunal’) identity while repressing a distal intestinal (‘ileal’) identity, but its molecular mechanisms are unclear. Here, we tested the hypothesis that GATA4 promotes a jejunal vs. ileal identity in mouse intestine by directly activating and repressing specific subsets of absorptive enterocyte genes by modulating the acetylation of histone H3, lysine 27 (H3K27), a mark of active chromatin, at sites of GATA4 occupancy. Global analysis of mouse jejunal epithelium showed a statistically significant association of GATA4 occupancy with GATA4-regulated genes. Occupancy was equally distributed between down- and up-regulated targets, and occupancy sites showed a dichotomy of unique motif over-representation at down- vs. up-regulated genes. H3K27ac enrichment at GATA4-binding loci that mapped to down-regulated genes (activation targets) was elevated, changed little upon conditional Gata4 deletion, and was similar to control ileum, whereas H3K27ac enrichment at GATA4-binding loci that mapped to up-regulated genes (repression targets) was depleted, increased upon conditional Gata4 deletion, and approached H3K27ac enrichment in wildtype control ileum. These data support the hypothesis that GATA4 both activates and represses intestinal genes, and show that GATA4 represses an ileal program of gene expression in the proximal small intestine by inhibiting the acetylation of H3K27. PMID:24878542

  4. Fat-1 gene inhibits human oral squamous carcinoma cell proliferation through downregulation of β-catenin signaling pathways

    PubMed Central

    NIE, DAIBANG; WANG, ZUOZHAO; ZHANG, YING; PANG, DAXIN; OUYANG, HONGSHENG; LI, LI

    2016-01-01

    The ω-3 fatty acid desaturase (fat-1) gene encodes the enzyme that converts ω-6 polyunsaturated fatty acids (PUFAs) to ω-3 PUFAs. Numerous studies have suggested that the ratio of ω-6/ω-3 PUFAs has an impact on tumorigenesis. To investigate the biological function of the fat-1 gene in human oral squamous cell carcinoma (OSCC), the fat-1 gene was introduced into OSCC cells by transfection. The uptake of the gene was confirmed by reverse transcription-polymerase chain reaction and analyzed using gas chromatography. The antitumor effects and mechanisms of the fat-1 gene were evaluated by studying cell survival and tumor growth in vitro and in vivo. Gas chromatography results revealed that the cells transfected with the fat-1 gene had a higher ω-3/ω-6 PUFA ratio than cells transfected with the control vector. An MTT and DNA fragmentation assay indicated that the presence of the fat-1 gene in vitro significantly decreased OSCC cell proliferation and significantly increased the rate of apoptosis. Similar antitumor effects of the fat-1 gene were also observed in vivo. Immunohistochemistry analysis confirmed that Tca8113 cell tumors displayed a significant reduction in cell growth and cell survival following the introduction of the fat-1 gene. The current study suggests that the inhibitory effect of the fat-1 gene on tumor growth may be a result of a reduction in the expression of the tumor survival protein β-catenin. The results also support the theory that the ratio of ω-3/ω-6 PUFAs has an impact on OSCC tumor growth. The findings of the study provide notable molecular insight into the theory suggesting that ω-3 PUFAs are an intermediate for the chemoprevention and treatment of human OSCC. PMID:26889238

  5. Targeted gene delivery in tumor xenografts by the combination of ultrasound-targeted microbubble destruction and polyethylenimine to inhibit survivin gene expression and induce apoptosis

    PubMed Central

    2010-01-01

    Background Noninvasive and tissue-specific technologies of gene transfection would be valuable in clinical gene therapy. This present study was designed to determine whether it could enhance gene transfection in vivo by the combination of ultrasound-targeted microbubble destruction (UTMD) with polyethylenimine (PEI) in tumor xenografts, and illuminate the effects of gene silencing and apoptosis induction with short hairpin RNA (shRNA) interference therapy targeting human survivin by this novel technique. Methods Two different expression vectors (pCMV-LUC and pSIREN) were incubated with PEI to prepare cationic complexes (PEI/DNA) and confirmed by the gel retardation assay. Human cervical carcinoma (Hela) tumors were planted subcutaneously in both flanks of nude mice. Tumor-bearing mice were administered by tail vein with PBS, plasmid, plasmid and SonoVue microbubble, PEI/DNA and SonoVue microbubble. One tumor was exposed to ultrasound irradiation, while the other served as control. The feasibility of targeted delivery and tissue specificity facilitated by UTMD and PEI were investigated. Moreover, immunohistochemistry analyses about gene silencing and apoptosis induction were detected. Results Electrophoresis experiment revealed that PEI could condense DNA efficiently. The application of UTMD significantly increases the tissue transfection. Both expression vectors showed that gene expressions were present in all sections of tumors that received ultrasound exposure but not in control tumors. More importantly, the increases in transgene expression were related to UTMD with the presence of PEI significantly. Silencing of the survivin gene could induce apoptosis effectively by downregulating survivin and bcl-2 expression, also cause up-regulation of bax and caspase-3 expression. Conclusions This noninvasive, novel combination of UTMD with PEI could enhance targeted gene delivery and gene expression in tumor xenografts at intravenous administration effectively without causing any apparently adverse effect, and might be a promising candidate for gene therapy. Silencing of survivin gene expression with shRNA could be facilitated by this non-viral technique, and lead to significant cell apoptosis. PMID:21092274

  6. Acetylaminofluorene and aminofluorene adducts inhibit in vitro transcription of a Xenopus 5S RNA gene only when located on the coding strand

    SciTech Connect

    Yenhui Chen; Matsumoto, Yoshihiro; Shibutani, Shinya; Bogenhagen, D.F. )

    1991-11-01

    Unique N-acetyl-2-aminofluorene (AAF) or 2-aminofluorene (AF) adducts were introduced into the Xenopus borealis somatic 5S RNA gene between the intragenic control region and the transcription termination site. The effects of these bulky adducts on transcription were studied in a cell-free extract derived from Xenopus laevis oocytes. AAF and AF adducts inhibit transcription only when they are on the template strand, whereas transcription passes through these adducts when they are played on the nontemplate strand. In the presence of the AAF or AF adduct on the template strand, transcription usually terminates one nucleotide before the altered guanine residue. Premature termination at these bulky adducts does not block reinitiation of transcription, since several transcripts are produced per gene per hour on these damaged templates.

  7. Novel Dominant-Negative Mutation Within the Six Domain of the Conserved Eye Specification Gene sine oculis Inhibits Eye Development in Drosophila

    PubMed Central

    Roederer, Kristin; Cozy, Loralyn; Anderson, Jason; Kumar, Justin P.

    2009-01-01

    The development of the compound eye of Drosophila is controlled, in part, by the concerted actions of several nuclear proteins that form an intricate regulatory system. One member of this network is sine oculis (so), the founding member of the Six gene family. Mutations within so affect the entire visual system, including the compound eye. The vertebrate homologs Six3 and Six6 also appear to play crucial roles in retinal formation. Mutations in Six3 inhibit retinal formation in chickens and fish, whereas those in Six6 are the underlying cause of bilateral anophthalmia in humans. Together, these phenotypes suggest a conserved role for the Six genes in eye development. In this report, we describe the effects of a dominant-negative mutation of sine oculis on the development of the compound eye of Drosophila. The mutation resides within the Six domain and may have implications for eye development and disease. PMID:15704100

  8. Low Concentrations of o,p’-DDT Inhibit Gene Expression and Prostaglandin Synthesis by Estrogen Receptor-Independent Mechanism in Rat Ovarian Cells

    PubMed Central

    Liu, Jing; Zhao, Meirong; Zhuang, Shulin; Yang, Yan; Yang, Ye; Liu, Weiping

    2012-01-01

    o,p’-DDT is an infamous xenoestrogen as well as a ubiquitous and persistent pollutant. Biomonitoring studies show that women have been internally exposed to o,p’-DDT at range of 0.3–500 ng/g (8.46×10−10 M−1.41×10−6 M) in blood and other tissues. However, very limited studies have investigated the biological effects and mechanism(s) of o,p’-DDT at levels equal to or lower than current exposure levels in human. In this study, using primary cultures of rat ovarian granulosa cells, we determined that very low doses of o,p’-DDT (10−12−10−8 M) suppressed the expression of ovarian genes and production of prostaglandin E2 (PGE2). In vivo experiments consistently demonstrated that o,p’-DDT at 0.5–1 mg/kg inhibited the gene expression and PGE2 levels in rat ovary. The surprising results from the receptor inhibitors studies showed that these inhibitory effects were exerted independently of either classical estrogen receptors (ERs) or G protein-coupled receptor 30 (GPR30). Instead, o,p’-DDT altered gene expression or hormone action via inhibiting the activation of protein kinase A (PKA), rather than protein kinase C (PKC). We further revealed that o,p’-DDT directly interfered with the PKA catalytic subunit. Our novel findings support the hypothesis that exposure to low concentrations of o,p’-DDT alters gene expression and hormone synthesis through signaling mediators beyond receptor binding, and imply that the current exposure levels of o,p’-DDT observed in the population likely poses a health risk to female reproduction. PMID:23209616

  9. Inhibition of DNA-repair genes Ercc1 and Mgmt enhances temozolomide efficacy in gliomas treatment: a pre-clinical study

    PubMed Central

    Boccard, Sandra G.; Marand, Sandie V.; Geraci, Sandra; Pycroft, Laurie; Berger, François R.; Pelletier, Laurent A.

    2015-01-01

    Gliomas are the most common primary brain tumors. To date, therapies do not allow curing patients, and glioblastomas (GBMs) are associated with remarkably poor prognosis. This situation is at least partly due to intrinsic or acquired resistance to treatment, especially to chemotherapy. In 2005, temozolomide (TMZ) has become the first chemotherapeutic drug validated for GBM. Nevertheless TMZ efficacy depends on Mgmt status. While the methylation of Mgmt promoter was considered so far as a prognostic marker, its targeting is becoming an effective therapeutic opportunity. Thus, arrival of both TMZ and Mgmt illustrated that considerable progress can still be realized by optimizing adjuvant chemotherapy. A part of this progress could be accomplished in the future by overcoming residual resistance. The aim of the present study was to investigate the involvement of a set of other DNA-repair genes in glioma resistance to temozolomide. We focused on DNA-repair genes located in the commonly deleted chromosomal region in oligodendroglioma (1p/19q) highly correlated with patient response to chemotherapy. We measured effects of inhibition of ten DNA-repair genes expression using siRNAs on astrocytoma cell response to cisplatin (CDDP) and TMZ. SiRNAs targeting ercc1, ercc2, mutyh, and pnkp significantly sensitized cells to chemotherapy, increasing cell death by up to 25%. In vivo we observed a decrease of subcutaneous glioma tumor growth after injection of siRNA in conjunction with absorption of TMZ. We demonstrated in this pre-clinical study that targeting of DNA-repair genes such as Ercc1 could be used as an adjuvant chemosensitization treatment, similarly to Mgmt inhibition. PMID:26336131

  10. PRMT5 Is Upregulated in HTLV-1-Mediated T-Cell Transformation and Selective Inhibition Alters Viral Gene Expression and Infected Cell Survival

    PubMed Central

    Panfil, Amanda R.; Al-Saleem, Jacob; Howard, Cory M.; Mates, Jessica M.; Kwiek, Jesse J.; Baiocchi, Robert A.; Green, Patrick L.

    2015-01-01

    Human T-cell leukemia virus type-1 (HTLV-1) is a tumorigenic retrovirus responsible for development of adult T-cell leukemia/lymphoma (ATLL). This disease manifests after a long clinical latency period of up to 2–3 decades. Two viral gene products, Tax and HBZ, have transforming properties and play a role in the pathogenic process. Genetic and epigenetic cellular changes also occur in HTLV-1-infected cells, which contribute to transformation and disease development. However, the role of cellular factors in transformation is not completely understood. Herein, we examined the role of protein arginine methyltransferase 5 (PRMT5) on HTLV-1-mediated cellular transformation and viral gene expression. We found PRMT5 expression was upregulated during HTLV-1-mediated T-cell transformation, as well as in established lymphocytic leukemia/lymphoma cell lines and ATLL patient PBMCs. shRNA-mediated reduction in PRMT5 protein levels or its inhibition by a small molecule inhibitor (PRMT5i) in HTLV-1-infected lymphocytes resulted in increased viral gene expression and decreased cellular proliferation. PRMT5i also had selective toxicity in HTLV-1-transformed T-cells. Finally, we demonstrated that PRMT5 and the HTLV-1 p30 protein had an additive inhibitory effect on HTLV-1 gene expression. Our study provides evidence for PRMT5 as a host cell factor important in HTLV-1-mediated T-cell transformation, and a potential target for ATLL treatment. PMID:26729154

  11. Reprogrammed CRISPR-Cas9 targeting the conserved regions of HPV6/11 E7 genes inhibits proliferation and induces apoptosis in E7-transformed keratinocytes.

    PubMed

    Liu, Yu-Chen; Cai, Zhi-Ming; Zhang, Xue-Jun

    2016-01-01

    The persistence infection of low-risk type (type 6 or type 11) of human papillomavirus (HPV) is the main cause of genital warts. Given the high rate of recurrence after treatment, the use of a new molecular agent is certain to be of value. The aim of this study was to achieve targeted inactivation of viral E 7 gene in keratinocytes using the reprogrammed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system. To accomplish this, a universal CRISPR-Cas9 system for targeting both HPV6/11 E 7 genes was constructed by using a dual guide RNA vector. After transfection of the vector into E 7-transfromed keratinocytes, the expression level of E 7 protein was measured using western-blot analysis and the sequence of the E 7 gene was determined using Sanger sequencing. Cell proliferation was analyzed by CCK-8 assay, and cell apoptosis was evaluated by Hoechst 33258 staining, flow cytometry analysis and ELISA assay. The results indicated that both HPV6/11 E 7 genes can be inactivated by the single CRISPR-Cas9 system. Furthermore, silencing of E 7 led to inhibition of cell proliferation and induction of apoptosis in E 7-transfromed keratinocytes but not in normal keratinocytes. Our data suggested that the reprogrammed CRISPR-Cas9 system has the potential for the development of an adjuvant therapy for genital warts. PMID:26228041

  12. Cloning, characterization, expression analysis and inhibition studies of a novel gene encoding Bowman-Birk type protease inhibitor from rice bean.

    PubMed

    Katoch, Rajan; Singh, Sunil Kumar; Thakur, Neelam; Dutt, Som; Yadav, Sudesh Kumar; Shukle, Rich

    2014-08-10

    This paper presents the first study describing the isolation, cloning and characterization of a full length gene encoding Bowman-Birk protease inhibitor (RbTI) from rice bean (Vigna umbellata). A full-length protease inhibitor gene with complete open reading frame of 327 bp encoding 109 amino acids was cloned from rice bean seeds using degenerate primer set. BlastP search revealed that the RbTI encoded amino acid of approx 13.0 kDa and shared 99% homology each with BBI from Phaseolus parvulus, Vigna trilobata and Vigna vexilata. Phylogenetic tree also showed close relationship of RbTI with BBI from other members of Leguminaceae family. RbTI gene was further confirmed as intronless (GenBank accession no. KJ159908). The secondary and 3D-structural models for the RbTI were predicted with homology modeling. qRT-PCR studies revealed the highest RbTI expression in the seeds nearing maturity, whereas the low expression of the gene was noticed in young leaves. The isolated RbTI was successfully expressed in Escherichiacoli and the highest expression was recorded after 5.5h of induction. Study on the inhibitory activity of expressed protein against the gut proteases of Hessian fly larvae revealed 87% inhibition. The novel RbTI gene will further broaden the pool of plant defense genes and could be an ideal choice for developing transgenic crops resistant to insect pests with high economic value. In addition, it has the potential to be used as a probe for selection of insect- and pathogen-resistant genotypes. PMID:24905651

  13. Inhibition of the NF-?B pathway by R65 ribozyme gene via adeno-associatedvirus serotype 9 ameliorated oxidized LDL induced human umbilical vein endothelial cell injury

    PubMed Central

    Zhai, Hui; Chen, Qing-Jie; Gao, Xiao-Ming; Ma, Yi-Tong; Chen, Bang-Dang; Yu, Zi-Xiang; Li, Xiao-Mei; Liu, Fen; Xiang, Yang; Xie, Jia; Yang, Yi-Ning

    2015-01-01

    Objective: NF-?B signaling plays a central role in the regulation of inflammatory responses in atherosclerosis. R65 ribozyme gene suppresses activation of NF-?B pathway, therefore we studied whether R65 gene therapy can ameliorate oxidized low-density lipoprotein (ox-LDL) induced human umbilical vein endothelial cells (HUVECs) injury. Methods and results: Recombinant adeno-associated virus serotype 9 (rAVV9) vector was used to transfect the R65 ribozyme gene (rAVV9-R65) into HUVECs then following ox-LDL stimulation, expression of NF-?B p65 and p50 subunits, inflammatory mediators and cell apoptosis were examined. First, rAVV9-enhanced green fluorescent protein (eGFP)-R65 at 1107 v.g./cell multiplicity of infection reached a long-lasting and significant increase in R65 gene expression. Second, ox-LDL treatment led to time- and dose-dependent activation of NF-?B pathway, and enhanced inflammatory response and cell death evidenced by increased expression of nuclear NF-?B p65 and p50 subunits, greater production of tumor necrosis factor ?, interleukin-6 and von willebrand factor and 20.57% increasedapoptotic HUVECs. Third, over-expression ofR65 gene was 2-fold increased in HUVECs attenuated ox-LDL induced unclear accumulation and expression of p65 subunit and ameliorated inflammation and cell death (all P < 0.05). Conclusion: rAAV9-mediated R65 ribozyme gene transfection in cultured HUVECs effectively inhibits ox-LDL induced activation of NF-?B and production of inflammatory cytokines and prevents cell apoptosis. PMID:26617700

  14. A Luciferase Reporter Gene Assay to Measure Ebola Virus Viral Protein 35-Associated Inhibition of Double-Stranded RNA-Stimulated, Retinoic Acid-Inducible Gene 1-Mediated Induction of Interferon β.

    PubMed

    Cannas, Valeria; Daino, Gian Luca; Corona, Angela; Esposito, Francesca; Tramontano, Enzo

    2015-10-01

    During Ebola virus (EBOV) infection, the type I interferon α/β (IFN-α/β) innate immune response is suppressed by EBOV viral protein 35 (VP35), a validated drug target. Identification of EBOV VP35 inhibitors requires a cellular system able to assess the VP35-based inhibitory functions of viral double-stranded RNA (dsRNA) IFN-β induction. We established a miniaturized luciferase gene reporter assay in A549 cells that measures IFN-β induction by viral dsRNA and is dose-dependently inhibited by VP35 expression. When compared to influenza A virus NS1 protein, EBOV VP35 showed improved inhibition of viral dsRNA-based IFN-β induction. This assay can be used to screen for EBOV VP35 inhibitors. PMID:25926684

  15. An Essential Role of cAMP Response Element Binding Protein in Ginsenoside Rg1-Mediated Inhibition of Na+/Glucose Cotransporter 1 Gene Expression.

    PubMed

    Wang, Chun-Wen; Su, Shih-Chieh; Huang, Shu-Fen; Huang, Yu-Chuan; Chan, Fang-Na; Kuo, Yu-Han; Hung, Mei-Whey; Lin, Hang-Chin; Chang, Wen-Liang; Chang, Tsu-Chung

    2015-12-01

    The Na(+)/glucose cotransporter 1 (SGLT1) is responsible for glucose uptake in intestinal epithelial cells. It has been shown that the intestinal SGLT1 level is significantly increased in diabetic individuals and positively correlated with the pathogenesis of diabetes. The development of targeted therapeutics that can reduce the intestinal SGLT1 expression level is, therefore, important. In this study, we showed that ginsenoside Rg1 effectively decreased intestinal glucose uptake through inhibition of SGLT1 gene expression in vivo and in vitro. Transient transfection analysis of the SGLT1 promoter revealed an essential cAMP response element (CRE) that confers the Rg1-mediated inhibition of SGLT1 gene expression. Chromatin immunoprecipitation assay and targeted CRE-binding protein (CREB) silencing demonstrated that Rg1 reduced the promoter binding of CREB and CREB binding protein associated with an inactivated chromatin status. In addition, further studies showed that the epidermal growth factor receptor (EGFR) signaling pathway also plays an essential role in the inhibitory effect of Rg1; taken together, our study demonstrates the involvement of the EGFR-CREB signaling pathway in the Rg1-mediated downregulation of SGLT1 expression, which offers a potential strategy in the development of antihyperglycemic and antidiabetic treatments. PMID:26429938

  16. The von Hippel-Lindau gene product inhibits vascular permeability factor/vascular endothelial growth factor expression in renal cell carcinoma by blocking protein kinase C pathways.

    PubMed

    Pal, S; Claffey, K P; Dvorak, H F; Mukhopadhyay, D

    1997-10-31

    Mutation or loss of function of the von Hippel-Lindau (VHL) tumor suppressor gene is regularly found in sporadic renal cell carcinomas (RCC), well vascularized malignant tumors that characteristically overexpress vascular permeability factor/vascular endothelial growth factor (VPF/VEGF). The wild-type VHL (wt-VHL) gene product acts to suppress VPF/VEGF expression, which is overexpressed when wt-VHL is inactive. The present study investigated the pathways by which VHL regulates VPF/VEGF expression. We found that inhibition of protein kinase C (PKC) represses VPF/VEGF expression in RCC cells that regularly overexpress VPF/VEGF. The wt-VHL expressed by stably transfected RCC cells forms cytoplasmic complexes with two specific PKC isoforms, zeta and delta, and prevents their translocation to the cell membrane where they otherwise would engage in signaling steps that lead to VPF/VEGF overexpression. Other experiments implicated mitogen-activated protein kinase (MAPK) phosphorylation as a downstream step in PKC regulation of VPF/VEGF expression. Taken together, these data demonstrate that wt-VHL, by neutralizing PKC isoforms zeta and delta and thereby inhibiting MAPK activation, plays an important role in preventing aberrant VPF/VEGF overexpression and the angiogenesis that results from such overexpression. PMID:9346879

  17. Delivery of inhibitor of growth 4 (ING4) gene significantly inhibits proliferation and invasion and promotes apoptosis of human osteosarcoma cells

    PubMed Central

    Li, Mei; Zhu, Ye; Zhang, Hongbin; Li, Lihua; He, Peng; Xia, Hong; Zhang, Yu; Mao, Chuanbin

    2014-01-01

    Growing evidence has suggested that inhibitor of growth 4 (ING4), a novel member of ING family proteins, plays a critical role in the development and progression of different tumors via multiple pathways. However, the function of ING4 in human osteosarcoma remains unclear. To understand its potential roles and mechanisms in inhibiting osteosarcoma, we constructed an expression vector pEGFP-ING4 and transfected the human osteosarcoma cells using this vector. We then studied the effects of over-expressed ING4 in the transfected cells on the proliferation, apoptosis and invasion of the osteosarcoma cells. The up-regulation of ING4 in the osteosarcoma cells, arising from the stable pEGFP-ING4 gene transfection, was found to significantly inhibit the cell proliferation by the cell cycle alteration with S phase reduction and G0/G1 phase arrest, induce cell apoptosis via the activation of the mitochondria pathway, and suppress cell invasion through the down-regulation of the matrix metalloproteinase 2 (MMP-2) and MMP-9 expression. In addition, increased ING4 level evoked the blockade of NF-κB signaling pathway and down-regulation of its target proteins. Our work suggests that ING4 can suppress osteosarcoma progression through signaling pathways such as mitochondria pathway and NF-κB signaling pathway and ING4 gene therapy is a promising approach to treating osteosarcoma. PMID:25490312

  18. Auxin inhibition of decapitation-induced branching is dependent on graft-transmissible signals regulated by genes Rms1 and Rms2.

    PubMed

    Beveridge, C A; Symons, G M; Turnbull, C G

    2000-06-01

    Decapitation-induced axillary bud outgrowth is a vital mechanism whereby shoots are able to continue normal growth and development. In many plants, including wild-type garden pea (Pisum sativum L.), this process can be inhibited by exogenous auxin. Using the ramosus (rms) increased branching mutants of pea, we present evidence that this response to auxin is dependent on graft-transmissible substance(s) regulated by the genes Rms1 and Rms2. The response to exogenous auxin is massively diminished in decapitated rms1 and rms2 mutant plants. However, basipetal auxin transport is not reduced in intact or decapitated mutants. Grafting rms1 or rms2 shoots onto wild-type rootstocks restored the auxin response, indicating that Rms1 and Rms2 gene action in the rootstock is sufficient to enable an auxin response in mutant shoots. We conclude that Rms1 and Rms2 act in the rootstock and shoot to control levels of mobile substance(s) that interact with exogenous auxin in the inhibition of bud outgrowth after decapitation. At least for rms1, the reduced auxin response is unlikely to be due to an inability of auxin to decrease xylem sap cytokinin content, as this is already low in intact rms1 plants. Consequently, we have genetic evidence that auxin action in decapitated plants depends on at least one novel long-distance signal. PMID:10859199

  19. Auxin Inhibition of Decapitation-Induced Branching Is Dependent on Graft-Transmissible Signals Regulated by Genes Rms1 and Rms21

    PubMed Central

    Beveridge, Christine A.; Symons, Gregory M.; Turnbull, Colin G.N.

    2000-01-01

    Decapitation-induced axillary bud outgrowth is a vital mechanism whereby shoots are able to continue normal growth and development. In many plants, including wild-type garden pea (Pisum sativum L.), this process can be inhibited by exogenous auxin. Using the ramosus (rms) increased branching mutants of pea, we present evidence that this response to auxin is dependent on graft-transmissible substance(s) regulated by the genes Rms1 and Rms2. The response to exogenous auxin is massively diminished in decapitated rms1 and rms2 mutant plants. However, basipetal auxin transport is not reduced in intact or decapitated mutants. Grafting rms1 or rms2 shoots onto wild-type rootstocks restored the auxin response, indicating that Rms1 and Rms2 gene action in the rootstock is sufficient to enable an auxin response in mutant shoots. We conclude that Rms1 and Rms2 act in the rootstock and shoot to control levels of mobile substance(s) that interact with exogenous auxin in the inhibition of bud outgrowth after decapitation. At least for rms1, the reduced auxin response is unlikely to be due to an inability of auxin to decrease xylem sap cytokinin content, as this is already low in intact rms1 plants. Consequently, we have genetic evidence that auxin action in decapitated plants depends on at least one novel long-distance signal. PMID:10859199

  20. Short-hairpin RNA-mediated Heat shock protein 90 gene silencing inhibits human breast cancer cell growth in vitro and in vivo

    SciTech Connect

    Zuo, Keqiang; Li, Dan; Pulli, Benjamin; Yu, Fei; Cai, Haidong; Yuan, Xueyu; Zhang, Xiaoping; Lv, Zhongwei

    2012-05-04

    Highlights: Black-Right-Pointing-Pointer Hsp90 is over-expressed in human breast cancer. Black-Right-Pointing-Pointer The shRNA-mediated gene silencing of Hsp90 resulted in inhibition of cell growth. Black-Right-Pointing-Pointer Akt and NF-kB were down-regulation after transfection due to Hsp90 silencing. Black-Right-Pointing-Pointer The tumor growth ratio was decline due to Hsp90 silencing. Black-Right-Pointing-Pointer The PCNA expression was down-regulation due to Hsp90 silencing. -- Abstract: Hsp90 interacts with proteins that mediate signaling pathways involved in the regulation of essential processes such as proliferation, cell cycle control, angiogenesis and apoptosis. Hsp90 inhibition is therefore an attractive strategy for blocking abnormal pathways that are crucial for cancer cell growth. In the present study, the role of Hsp90 in human breast cancer MCF-7 cells was examined by stably silencing Hsp90 gene expression with an Hsp90-silencing vector (Hsp90-shRNA). RT-PCR and Western blot analyses showed that Hsp90-shRNA specifically and markedly down-regulated Hsp90 mRNA and protein expression. NF-kB and Akt protein levels were down-regulated in Hsp90-shRNA transfected cells, indicating that Hsp90 knockout caused a reduction of survival factors and induced apoptosis. Treatment with Hsp90-shRNA significantly increased apoptotic cell death and caused cell cycle arrest in the G1/S phase in MCF-7 cells, as shown by flow cytometry. Silencing of Hsp90 also reduced cell viability, as determined by MTT assay. In vivo experiments showed that MCF-7 cells stably transfected with Hsp90-shRNA grew slowly in nude mice as compared with control groups. In summary, the Hsp90-shRNA specifically silenced the Hsp90 gene, and inhibited MCF-7 cell growth in vitro and in vivo. Possible molecular mechanisms underlying the effects of Hsp90-shRNA include the degradation of Hsp90 breast cancer-related client proteins, the inhibition of survival signals and the upregulation of apoptotic pathways. shRNA-mediated interference may have potential therapeutic utility in human breast cancer.

  1. MiR224-3p inhibits hypoxia-induced autophagy by targeting autophagy-related genes in human glioblastoma cells

    PubMed Central

    Guo, Xing; Xue, Hao; Guo, Xiaofan; Gao, Xiao; Xu, Shugang; Yan, Shaofeng; Han, Xiao; Li, Tong; Shen, Jie; Li, Gang

    2015-01-01

    Human glioblastoma multiforme (GBM) is a malignant solid tumor characterized by severe hypoxia. Autophagy plays a protective role in cancer cells under hypoxia. However, the microRNA (miRNA)-related molecular mechanisms underlying hypoxia-reduced autophagy remain poorly understood in GBM. In this study, we performed a miRNA microarray analysis on GBM cells and found that numerous miRNAs were differentially expressed under hypoxic conditions. Further research showed that miR224-3p, one of the significantly down-regulated miRNAs, was involved in regulating hypoxia-induced autophagy in GBM cells. Overexpression of miR224-3p abolished hypoxia-induced autophagy, whereas knocking down endogenous miR224-3p increased autophagic activity under normoxia. In addition, we demonstrated that miR224-3p inhibited autophagy by directly suppressing the expression of two autophagy-related genes (ATGs), ATG5 and FAK family-interacting protein of 200 kDa (FIP200). Furthermore, in vitro, miR224-3p attenuated cell proliferation and promoted hypoxia-induced apoptosis, and in vivo, overexpression of miR224-3p inhibited tumorigenesis of GBM cells. Collectively, our study identified a novel hypoxia-down-regulated miRNA, miR224-3p, as a key modulator of autophagy by inhibiting ATGs in GBM cells. PMID:26536662

  2. (Z)-1,1-Dichloro-2-(4-methoxyphenyl)-3-phenylcyclopropane induces concentration-dependent growth inhibition, apoptosis, and coordinates regulation of apoptotic genes in TRAMP cells

    PubMed Central

    Thomas, Catherine A.; Grant, Stephen G.; Pflug, Beth R.; Getzenberg, Robert H.; Day, Billy W.

    2016-01-01

    (Z)-1-1-Dichloro-2,3-diphenylcyclopropane (AII) and (Z)-1,1-dichloro-2-(4-methoxyphenyl)-3-phenylcyclopropane [2–(4-methoxyphenyl)-AII] inhibit tubulin polymerization, PSA production, and the proliferation of human prostate cancer cells. The actions of the agents were studied in three transgenic adenocarcinomas of the mouse prostate (TRAMP) cell lines. Antiproliferative potencies were determined and cells treated with the more potent 2-(4-methoxyphenyl)-AII were examined for induction of apoptosis. Microarray analyses were conducted to determine the apoptosis-related genes up- and down-regulated by the agent. 2-(4-Methoxyphenyl)-AII concentration-dependently inhibited growth of all three cell lines. Fifty percent and 100% growth inhibitory and 50% lethal concentrations were determined to be 0.3, 1.5, and 5 µM, respectively. Minimum detectable apoptosis-inducing concentrations by ELISA were 0.10 to 0.14 µM. PARP cleavage and two-color flow cytometry assays verified apoptosis induction. Microarray analyses showed Bok and Siva-pending to be up-regulated and that Birc, Dad1, and Atf5 were down-regulated. 2-(4-methoxyphenyl)-AII inhibits proliferation and induces apoptosis in the in vivo-adaptable TRAMP cells, suggesting the compound should be further examined in preclinical models. PMID:18367102

  3. Transcriptional and Posttranscriptional Regulation of Cytokine Gene Expression in HIV-1 Antigen-Specific CD8+ T Cells That Mediate Virus Inhibition

    PubMed Central

    Payne, Tamika L.; Blackinton, Jeff; Frisbee, Alyse; Pickeral, Joy; Sawant, Sheetal; Vandergrift, Nathan A.; Freel, Stephanie A.; Ferrari, Guido; Keene, Jack D.

    2014-01-01

    ABSTRACT The ability of CD8+ T cells to effectively limit HIV-1 replication and block HIV-1 acquisition is determined by the capacity to rapidly respond to HIV-1 antigens. Understanding both the functional properties and regulation of an effective CD8+ response would enable better evaluation of T cell-directed vaccine strategies and may inform the design of new therapies. We assessed the antigen specificity, cytokine signature, and mechanisms that regulate antiviral gene expression in CD8+ T cells from a cohort of HIV-1-infected virus controllers (VCs) (<5,000 HIV-1 RNA copies/ml and CD4+ lymphocyte counts of >400 cells/μl) capable of soluble inhibition of HIV-1. Gag p24 and Nef CD8+ T cell-specific soluble virus inhibition was common among the VCs and correlated with substantial increases in the abundance of mRNAs encoding the antiviral cytokines macrophage inflammatory proteins MIP-1α, MIP-1αP (CCL3L1), and MIP-1β; granulocyte-macrophage colony-stimulating factor (GM-CSF); lymphotactin (XCL1); tumor necrosis factor receptor superfamily member 9 (TNFRSF9); and gamma interferon (IFN-γ). The induction of several of these mRNAs was driven through a coordinated response of both increased transcription and stabilization of mRNA, which together accounted for the observed increase in mRNA abundance. This coordinated response allows rapid and robust induction of mRNA messages that can enhance the CD8+ T cells' ability to inhibit virus upon antigen encounter. IMPORTANCE We show that mRNA stability, in addition to transcription, is key in regulating the direct anti-HIV-1 function of antigen-specific memory CD8+ T cells. Regulation at the level of RNA helps enable rapid recall of memory CD8+ T cell effector functions for HIV-1 inhibition. By uncovering and understanding the mechanisms employed by CD8+ T cell subsets with antigen-specific anti-HIV-1 activity, we can identify new strategies for comprehensive identification of other important antiviral genes. This will, in turn, enhance our ability to inhibit virus replication by informing both cure strategies and HIV-1 vaccine designs that aim to reduce transmission and can aid in blocking HIV-1 acquisition. PMID:24899193

  4. Inhibition of the binding of MSG-intermolt-specific complex, MIC, to the sericin-1 gene promoter and sericin-1 gene expression by POU-M1/SGF-3.

    PubMed

    Kimoto, Mai; Kitagawa, Tsuyuki; Kobayashi, Isao; Nakata, Tomohiro; Kuroiwa, Asato; Takiya, Shigeharu

    2012-11-01

    The sericin-1 gene encoding a glue protein is expressed in the middle silk gland (MSG) of the silkworm, Bombyx mori. A member of the class III POU domain transcription factors, POU-M1, was cloned as the factor bound to the SC site of the sericin-1 promoter and has been proposed to be a positive transcription factor. In this study, we analyzed the expression pattern of the POU-M1 gene in fourth and fifth instars in comparison with the pattern of the sericin-1 gene. The POU-M1 gene was expressed strongly in the region anterior to the sericin-1-expressing portion of the silk gland at both feeding stages. As the sericin-1-expressing region expands from the posterior to middle portions of the MSG in the fifth instar, the POU-M1-expressing region retreated from the middle to anterior portion. Introduction of the expression vector of POU-M1 into the silk glands by gene gun technology repressed promoter activity of the sericin-1 gene, suggesting that POU-M1 regulates the sericin-1 gene negatively. An in vitro binding assay showed that POU-M1 bound not only to the SC site but also to other promoter elements newly detected in vivo. Another spatiotemporal specific factor MIC binds to these elements, and POU-M1 competed with MIC to bind at the -70 site essential for promoter activity. These results suggest that POU-M1 is involved in restricting the anterior boundary of the sericin-1-expressing region in the silk gland by inhibiting the binding of the transcriptional activator to the promoter elements. PMID:23070540

  5. Cardiac CaM Kinase II Genes δ and γ Contribute to Adverse Remodeling but Redundantly Inhibit Calcineurin-Induced Myocardial Hypertrophy

    PubMed Central

    Kreusser, Michael M.; Lehmann, Lorenz H.; Keranov, Stanislav; Hoting, Marc-Oscar; Oehl, Ulrike; Kohlhaas, Michael; Reil, Jan-Christian; Neumann, Kay; Schneider, Michael D.; Hill, Joseph A.; Dobrev, Dobromir; Maack, Christoph; Maier, Lars S.; Gröne, Hermann-Josef; Katus, Hugo A.; Olson, Eric N.; Backs, Johannes

    2014-01-01

    Background Ca2+-dependent signaling through CaM Kinase II (CaMKII) and calcineurin was suggested to contribute to adverse cardiac remodeling. However, the relative importance of CaMKII versus calcineurin for adverse cardiac remodeling remained unclear. Methods and Results We generated double-knockout mice (DKO) lacking the 2 cardiac CaMKII genes δ and γ specifically in cardiomyocytes. We show that both CaMKII isoforms contribute redundantly to phosphorylation not only of phospholamban, ryanodine receptor 2, and histone deacetylase 4, but also calcineurin. Under baseline conditions, DKO mice are viable and display neither abnormal Ca2+ handling nor functional and structural changes. On pathological pressure overload and β-adrenergic stimulation, DKO mice are protected against cardiac dysfunction and interstitial fibrosis. But surprisingly and paradoxically, DKO mice develop cardiac hypertrophy driven by excessive activation of endogenous calcineurin, which is associated with a lack of phosphorylation at the auto-inhibitory calcineurin A site Ser411. Likewise, calcineurin inhibition prevents cardiac hypertrophy in DKO. On exercise performance, DKO mice show an exaggeration of cardiac hypertrophy with increased expression of the calcineurin target gene RCAN1-4 but no signs of adverse cardiac remodeling. Conclusions We established a mouse model in which CaMKII’s activity is specifically and completely abolished. By the use of this model we show that CaMKII induces maladaptive cardiac remodeling while it inhibits calcineurin-dependent hypertrophy. These data suggest inhibition of CaMKII but not calcineurin as a promising approach to attenuate the progression of heart failure. PMID:25124496

  6. HOS3, an ELO-Like Gene, Inhibits Effects of ABA and Implicates a S-1-P/Ceramide Control System for Abiotic Stress Responses in Arabidopsis thaliana

    PubMed Central

    Quist, Tanya M.; Sokolchik, Irina; Shi, Huazhong; Joly, Robert J.; Bressan, Ray A.; Maggio, Albino; Narsimhan, Meena; Li, Xia

    2009-01-01

    A hyper-osmotically sensitive mutant of Arabidopsis thaliana, designated hos3-1 (high expression of osmotically responsive genes), was identified based on its hyper-luminescence of RD29A:LUC promoter fusion plants upon treatment with NaCl and ABA. These responses implicate the disrupted gene as a direct or indirect negative regulator of the RD29A stress-responsive pathway. By sequencing the flanking regions of the T-DNA borders, it was determined that the disrupted gene is at locus At4g36830, annotated as encoding a putative protein with high homology to CIG30 (ELO2/FEN1). CIG30 has been implicated in synthesis of very long chain fatty acids (VLCFA), which are essential precursors for sphingolipids and ceramides. Altered stress responses characteristic of ABA-hypersensitivity, including reduced root growth inhibition and reduced germination with ABA treatment and reduced water loss from leaves, were exhibited by allelic hos3-1 and hos3-2 mutants. The hos3-2 mutant is partially suppressed in its transcript abundance and is inherited as a recessive trait. Further, the HOS3 ORF under the control of the 35SCaMV promoter restored wild-type NaCl- and ABA-root growth sensitivity as well as RD29A:LUC luminescence in mutant plants. We also show here that the HOS3 wild-type gene functionally complements the sensitivity of elo2 and elo3 yeast mutants to monensin. Furthermore, both hos3-1 and hos3-2 alleles shared increased sensitivity to the herbicide Metolachlor, which inhibits acyl chain elongation in synthesis of VLCFA, and HOS3 functionally complemented both elo2 and elo3 and restored levels of VLCFA. Together, these data establish that HOS3 inhibits ABA-mediated stress responses and implicate the VLCFA pathway and products as control points for several aspects of abiotic stress signaling and responses. The results also provide support for a role of ceramide in the control of stomatal behavior. PMID:19529829

  7. Promoter specific sensitivity to inhibition of histone deacetylases: implications for hormonal gene control, cellular differentiation and cancer.

    PubMed

    Dressel, U; Renkawitz, R; Baniahmad, A

    2000-01-01

    Alterations in histone acetylation status appear to play a central role in the regulation of neoplasia, tumor suppression, cell cycle control, hormone responsiveness and senescence. These alterations of chromatin control gene transcription. The histone acetylation status is regulated by the equilibrium of histone acetyl-transferase activity (HAT) and the histone deacetylase activity (HDAC). Commonly, DNA-transfection assays are used to measure the effect of histone acetylation and deacetylation on gene transcription. Here we have analyzed the response of various viral long terminal repeats and vertebrate promoters to the specific histone deacetylase inhibitor trichostatin A (TSA). We show that the activity of many, but not all, promoters is increased upon TSA treatment. Interestingly, the lysozyme promoter exhibited TSA resistance, while the activity of metallothionine, the human growth hormone, and the thymidine kinase promoters was increased. Furthermore, we found that all tested viral promoters are induced by TSA. Analysis of the transcriptional behaviour of the thyroid hormone receptor (TR), the cellular homologue of the v-erbA oncogene, revealed that TSA reduced the gene silencing function but had no influence on the hormone-induced gene activation function of the receptor. These results on gene specific effects, together with the HDAC structural data (1), may be a basis for the development of HDAC inhibitors as antitumor agents. PMID:10810390

  8. Agrobacterium-Mediated Root Transformation Is Inhibited by Mutation of an Arabidopsis Cellulose Synthase-Like Gene1

    PubMed Central

    Zhu, Yanmin; Nam, Jaesung; Carpita, Nicholas C.; Matthysse, Ann G.; Gelvin, Stanton B.

    2003-01-01

    Agrobacterium-mediated plant genetic transformation involves a complex interaction between the bacterium and the host plant. Relatively little is known about the role plant genes and proteins play in this process. We previously identified an Arabidopsis mutant, rat4, that is resistant to Agrobacterium transformation. We show here that rat4 contains a T-DNA insertion into the 3′-untranslated region of the cellulose synthase-like gene CSLA9. CSLA9 transcripts are greatly reduced in the rat4 mutant. Genetic complementation of rat4 with wild-type genomic copies of the CSLA9 gene restores both transformation competence and the wild-type level of CSLA9 transcripts. The CSLA9 promoter shows a distinct pattern of expression in Arabidopsis plants. In particular, the promoter is active in the elongation zone of roots, the root tissue that we previously showed is most susceptible to Agrobacterium-mediated transformation. Disruption of the CSLA9 gene in the rat4 mutant results in reduced numbers and rate of growth of lateral roots and reduced ability of the roots to bind A. tumefaciens cells under certain conditions. No major differences in the linkage structure of the non-cellulosic polysaccharides could be traced to the defective CSLA9 gene. PMID:14612582

  9. Histone Deacetylase Inhibition Induces Long-Lasting Changes in Maternal Behavior and Gene Expression in Female Mice

    PubMed Central

    Stevens, Jacqueline S.; Rissman, Emilie F.

    2014-01-01

    In many species, including mice, maternal responsiveness is experience-dependent and permanent, lasting for long periods (months to years). We have shown that after brief exposures to pups, virgin female mice continue to respond maternally toward pups for at least one month. Administration of a histone deacetylase inhibitor (HDACi) reduces the amount of maternal experience required to affect maternal behavior and gene expression. In this set of studies, we examined the epigenetic mechanisms that underlie these motivated behaviors. We assessed whether the effects of HDACi persisted 1 month after the initial experience (in the absence of continued pup experience or HDACi treatment) and whether the maintenance of maternal memory was associated with stable changes in gene expression. Using chromatin immunoprecipitation, we examined whether Esr2 and Oxt gene expression might be mediated by recruitment of the histone acetyltransferase cAMP response element binding protein (CBP) to their promoter regions after maternal memory consolidation. We report that HDACi treatment induced long-lasting changes in maternal responsiveness. Maternal learning was associated with increased recruitment of CBP to the Esr2 and Oxt gene promoters during the consolidation of maternal memory as well as a persistent increase in estrogen receptor-β (Esr2) mRNA and decreased expression of the de novo DNA methyltransferase Dnmt3a within the medial preoptic area. The consolidation of the maternal experience may involve the CBP recruitment and stable changes in gene expression, which maintain increased maternal responsiveness for long periods of time. PMID:24932804

  10. Knockdown of astrocyte elevated gene-1 inhibits tumor growth and modifies microRNAs expression profiles in human colorectal cancer cells

    SciTech Connect

    Huang, Sujun; Wu, Binwen; Li, Dongfeng; Zhou, Weihong; Deng, Gang; Zhang, Kaijun; Li, Youjia

    2014-02-14

    Highlights: • AEG-1 expression in CRC cell lines and down-regulation or upregulation of AEG-1 in vitro. • Knockdown of AEG-1 inhibits cell proliferation, colony formation and invasion. • Upregulation of AEG-1 enhances proliferation, invasion and colony formation. • Knockdown of AEG-1 accumulates G0/G1-phase cells and promotes apoptosis in CRC cells. • AEG-1 knockdown increases 5-FU cytotoxicity. - Abstract: Astrocyte elevated gene-1 (AEG-1), upregulated in various types of malignancies including colorectal cancer (CRC), has been reported to be associated with the carcinogenesis. MicroRNAs (miRNAs) are widely involved in the initiation and progression of cancer. However, the functional significance of AEG-1 and the relationship between AEG-1 and microRNAs in human CRC remains unclear. The aim of this study was to investigate whether AEG-1 could serve as a potential therapeutic target of human CRC and its possible mechanism. We adopted a strategy of ectopic overexpression or RNA interference to upregulate or downregulate expression of AEG-1 in CRC models. Their phenotypic changes were analyzed by Western blot, MTT and transwell matrix penetration assays. MicroRNAs expression profiles were performed using microarray analysis followed by validation using qRT-PCR. Knockdown of AEG-1 could significantly inhibit colon cancer cell proliferation, colony formation, invasion and promotes apoptosis. Conversely, upregulation of AEG-1 could significantly enhance cell proliferation, invasion and reduced apoptisis. AEG-1 directly contributes to resistance to chemotherapeutic drug. Targeted downregulation of AEG-1 might improve the expression of miR-181a-2{sup ∗}, -193b and -193a, and inversely inhibit miR-31 and -9{sup ∗}. Targeted inhibition of AEG-1 can lead to modification of key elemental characteristics, such as miRNAs, which may become a potential effective therapeutic strategy for CRC.

  11. The TMPRSS2-ERG Gene Fusion Blocks XRCC4-Mediated Nonhomologous End-Joining Repair and Radiosensitizes Prostate Cancer Cells to PARP Inhibition.

    PubMed

    Chatterjee, Payel; Choudhary, Gaurav S; Alswillah, Turkeyah; Xiong, Xiahui; Heston, Warren D; Magi-Galluzzi, Cristina; Zhang, Junran; Klein, Eric A; Almasan, Alexandru

    2015-08-01

    Exposure to genotoxic agents, such as ionizing radiation (IR), produces DNA damage, leading to DNA double-strand breaks (DSB); IR toxicity is augmented when the DNA repair is impaired. We reported that radiosensitization by a PARP inhibitor (PARPi) was highly prominent in prostate cancer cells expressing the TMPRSS2-ERG gene fusion protein. Here, we show that TMPRSS2-ERG blocks nonhomologous end-joining (NHEJ) DNA repair by inhibiting DNA-PKcs. VCaP cells, which harbor TMPRSS2-ERG and PC3 cells that stably express it, displayed γH2AX and 53BP1 foci constitutively, indicating persistent DNA damage that was absent if TMPRSS2-ERG was depleted by siRNA in VCaP cells. The extent of DNA damage was enhanced and associated with TMPRSS2-ERG's ability to inhibit DNA-PKcs function, as indicated by its own phosphorylation (Thr2609, Ser2056) and that of its substrate, Ser1778-53BP1. DNA-PKcs deficiency caused by TMPRSS2-ERG destabilized critical NHEJ components on chromatin. Thus, XRCC4 was not recruited to chromatin, with retention of other NHEJ core factors being reduced. DNA-PKcs autophosphorylation was restored to the level of parental cells when TMPRSS2-ERG was depleted by siRNA. Following IR, TMPRSS2-ERG-expressing PC3 cells had elevated Rad51 foci and homologous recombination (HR) activity, indicating that HR compensated for defective NHEJ in these cells, hence addressing why TMPRSS2-ERG alone did not lead to radiosensitization. However, the presence of TMPRSS2-ERG, by inhibiting NHEJ DNA repair, enhanced PARPi-mediated radiosensitization. IR in combination with PARPi resulted in enhanced DNA damage in TMPRSS2-ERG-expressing cells. Therefore, by inhibiting NHEJ, TMPRSS2-ERG provides a synthetic lethal interaction with PARPi in prostate cancer patients expressing TMPRSS2-ERG. PMID:26026052

  12. Molecular characterization of an Enterobacter cloacae gene (romA) which pleiotropically inhibits the expression of Escherichia coli outer membrane proteins.

    PubMed Central

    Komatsu, T; Ohta, M; Kido, N; Arakawa, Y; Ito, H; Mizuno, T; Kato, N

    1990-01-01

    The introduction of a newly cloned Enterobacter cloacae chromosomal gene romA, into Escherichia coli and E. cloacae resulted in enhancement of resistance to quinolones, beta-lactams, chloramphenicol, and tetracycline. The primary effect of romA on a multicopy vector in E. coli was almost complete inhibition of OmpF expression in the outer membrane. From the experiments with ompR and envZ mutants or with ompF-lacZ and ompC-lacZ fusion plasmids, it was concluded that this inhibition is posttranscriptional. The introduction of romA on a multicopy vector into strains with micF deletion elicited only a moderate decrease in OmpF protein expression. This indicates that reduction of OmpF expression by romA is partly mediated posttranscriptionally by the activation of micF. Moreover, the overexpression of RomA protein from an isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible promoter resulted in nearly complete inhibition of expression of OmpC and OmpA, as well as OmpF. Taken together with an observation in a recent study that overexpressed OmpC inhibited the synthesis of OmpA and LamB, a possible inhibitory mechanism at the translational stage of the synthesis of outer membrane proteins should also be considered. By Southern hybridization, romA was generally detected in the chromosomes of all E. cloacae strains tested but not in the E. coli K-12 chromosome. Sequence data show that there is an open reading frame specifying 368 amino acids residues including a putative signal peptide. RomA appears to belong to the outer membrane protein family since it was extractable from an outer membrane preparation, but no sequence homology to other outer membrane proteins was detected. Images PMID:2193928

  13. Inhibition of Both HIV-1 Reverse Transcription and Gene Expression by a Cyclic Peptide that Binds the Tat-Transactivating Response Element (TAR) RNA

    PubMed Central

    Lalonde, Matthew S.; Lobritz, Michael A.; Ratcliff, Annette; Chamanian, Mastooreh; Athanassiou, Zafiria; Tyagi, Mudit; Wong, Julian; Robinson, John A.; Karn, Jonathan; Varani, Gabriele; Arts, Eric J.

    2011-01-01

    The RNA response element TAR plays a critical role in HIV replication by providing a binding site for the recruitment of the viral transactivator protein Tat. Using a structure-guided approach, we have developed a series of conformationally-constrained cyclic peptides that act as structural mimics of the Tat RNA binding region and block Tat-TAR interactions at nanomolar concentrations in vitro. Here we show that these compounds block Tat-dependent transcription in cell-free systems and in cell-based reporter assays. The compounds are also cell permeable, have low toxicity, and inhibit replication of diverse HIV-1 strains, including both CXCR4-tropic and CCR5-tropic primary HIV-1 isolates of the divergent subtypes A, B, C, D and CRF01_AE. In human peripheral blood mononuclear cells, the cyclic peptidomimetic L50 exhibited an IC50 ∼250 nM. Surprisingly, inhibition of LTR-driven HIV-1 transcription could not account for the full antiviral activity. Timed drug-addition experiments revealed that L-50 has a bi-phasic inhibition curve with the first phase occurring after HIV-1 entry into the host cell and during the initiation of HIV-1 reverse transcription. The second phase coincides with inhibition of HIV-1 transcription. Reconstituted reverse transcription assays confirm that HIV-1 (−) strand strong stop DNA synthesis is blocked by L50-TAR RNA interactions in-vitro. These findings are consistent with genetic evidence that TAR plays critical roles both during reverse transcription and during HIV gene expression. Our results suggest that antiviral drugs targeting TAR RNA might be highly effective due to a dual inhibitory mechanism. PMID:21625572

  14. Inhibition of both HIV-1 reverse transcription and gene expression by a cyclic peptide that binds the Tat-transactivating response element (TAR) RNA.

    PubMed

    Lalonde, Matthew S; Lobritz, Michael A; Ratcliff, Annette; Chamanian, Mastooreh; Athanassiou, Zafiria; Tyagi, Mudit; Wong, Julian; Robinson, John A; Karn, Jonathan; Varani, Gabriele; Arts, Eric J

    2011-05-01

    The RNA response element TAR plays a critical role in HIV replication by providing a binding site for the recruitment of the viral transactivator protein Tat. Using a structure-guided approach, we have developed a series of conformationally-constrained cyclic peptides that act as structural mimics of the Tat RNA binding region and block Tat-TAR interactions at nanomolar concentrations in vitro. Here we show that these compounds block Tat-dependent transcription in cell-free systems and in cell-based reporter assays. The compounds are also cell permeable, have low toxicity, and inhibit replication of diverse HIV-1 strains, including both CXCR4-tropic and CCR5-tropic primary HIV-1 isolates of the divergent subtypes A, B, C, D and CRF01_AE. In human peripheral blood mononuclear cells, the cyclic peptidomimetic L50 exhibited an IC(50) ∼250 nM. Surprisingly, inhibition of LTR-driven HIV-1 transcription could not account for the full antiviral activity. Timed drug-addition experiments revealed that L-50 has a bi-phasic inhibition curve with the first phase occurring after HIV-1 entry into the host cell and during the initiation of HIV-1 reverse transcription. The second phase coincides with inhibition of HIV-1 transcription. Reconstituted reverse transcription assays confirm that HIV-1 (-) strand strong stop DNA synthesis is blocked by L50-TAR RNA interactions in-vitro. These findings are consistent with genetic evidence that TAR plays critical roles both during reverse transcription and during HIV gene expression. Our results suggest that antiviral drugs targeting TAR RNA might be highly effective due to a dual inhibitory mechanism. PMID:21625572

  15. Rho-kinase inhibition alleviates pulmonary hypertension in transgenic mice expressing a dominant-negative type II bone morphogenetic protein receptor gene.

    PubMed

    Yasuda, Tadashi; Tada, Yuji; Tanabe, Nobuhiro; Tatsumi, Koichiro; West, James

    2011-11-01

    Pulmonary arterial hypertension (PAH) is a life-threatening disease characterized by a sustained elevation in the pulmonary artery pressure and subsequent right heart failure. The activation of Rho/Rho-kinase activity and the beneficial effect of Rho-kinase inhibition have been demonstrated in several experimental models of pulmonary hypertension. However, it remains unclear whether Rho-kinase inhibitors can also be used against pulmonary hypertension associated with mutations in the type II bone morphogenetic protein receptor (BMPRII) gene. Transgenic mice expressing a dominant-negative BMPRII gene (with an arginine to termination mutation at amino acid 899) in smooth muscle by a tetracycline-gene switch system (SM22-tet-BMPR2(R899X) mice) were examined. They developed an elevated right ventricular systolic pressure (RVSP), right ventricular (RV) hypertrophy, muscularization of small pulmonary arteries, and an associated disturbed blood flow in their lungs. The Rho/Rho-kinase activity and Smad activity were determined by a Western blot analysis by detecting GTP-RhoA and the phosphorylation of myosin phosphatase target subunit 1, Smad1, and Smad2. In the lungs of SM22-tet-BMPR2(R899X) mice, the Rho/Rho-kinase activity was elevated significantly, whereas the Smad activity was almost unchanged. Fasudil, a Rho-kinase inhibitor, significantly decreased RVSP, alleviated RV hypertrophy and muscularization of small pulmonary arteries, and improved blood flow in SM22-tet-BMPR2(R899X) mice, although it did not alter Smad signaling. Our study demonstrates that Rho/Rho-kinase signaling is activated via a Smad-independent pathway in an animal model of pulmonary hypertension with a BMPRII mutation in the cytoplasmic tail domain. Rho-kinase inhibition is therefore a possible therapeutic approach for the treatment of PAH associated with genetic mutation. PMID:21856816

  16. Stimulation of Neutrophil Granulocytes with Mycobacterium bovis Bacillus Calmette-Guérin Induces Changes in Phenotype and Gene Expression and Inhibits Spontaneous Apoptosis

    PubMed Central

    Suttmann, Henrik; Lehan, Nadine; Böhle, Andreas; Brandau, Sven

    2003-01-01

    Polymorphonuclear neutrophil granulocytes (PMN) have been implicated in the early inflammatory response against mycobacteria besides monocytes/macrophages. Yet, little is known about the interaction of mycobacteria with PMN. We investigated the potential of Mycobacterium bovis bacillus Calmette-Guérin (BCG) to stimulate and influence PMN phenotype, gene expression profile and spontaneous apoptosis. Flow cytometric analyses revealed an upregulation of the function-associated molecules Fcγ receptor III (FcγR III) and II (CD16 and CD32) as well as MAC-1 (CD11b and CD18) on BCG-stimulated PMN. As determined by cDNA microarrays and multiplex reverse transcriptase PCR, stimulation with BCG alters the expression of various genes for proinflammatory cytokines/chemokines or receptors in PMN. We detected an upregulation or de novo synthesis of interleukin 1α (IL-1α), IL-1β, IL-8, macrophage inflammatory protein 1α (MIP-1α), MIP-1β, GRO-α, transforming growth factor β, MCP-1, IL-2 receptor γ (IL-2Rγ), IL-10Rα, and IL-6R. Genes for IL-9, IL-12α, IL-15, IL-5Rα, and IL-13Rα1 were found to be downregulated or switched off. Furthermore, Giemsa and annexin V-propidium iodide double staining demonstrated an inhibition of spontaneous PMN apoptosis following BCG stimulation. Changes in phenotype and inhibition of apoptosis did not depend on direct mycobacterial stimulation alone, but were a result of an autocrine-paracrine stimulation mechanism. Our findings support the hypothesis that PMN become activated at the site of mycobacterial infections and that this activation might set the stage for a subsequent antimycobacterial immune response. PMID:12874345

  17. Imatinib inhibits the expression of SCO2 and FRATAXIN genes that encode mitochondrial proteins in human Bcr-Abl⁺ leukemia cells.

    PubMed

    Papadopoulou, Lefkothea C; Kyriazou, Angeliki V; Bonovolias, Ioannis D; Tsiftsoglou, Asterios S

    2014-01-01

    Imatinib mesylate (IM/Gleevec®), a selective inhibitor of chimeric Bcr-Abl tyrosine kinase, was developed as a first line drug to treat CML and ALL Ph(+) patients. Earlier studies have shown that hemin counteracts the IM-induced cell killing in human K-562 CML cells. In this study, we investigated whether IM disrupts the heme-dependent Cytochrome c Oxidase (COX) Biosynthesis and Assembly Pathway (HDCBAP) in Bcr-Abl(+) and Bcr-Abl(-) cells by affecting the expression of key-genes. Cells were exposed to IM and evaluated at time intervals for cell growth, cell death, expression of various genes by RT-PCR analysis as well as Sco2 mature protein levels by western blot analysis and COX enzymatic activity. IM at 1 μM induced extensive cell growth inhibition and cell death as well as marked suppression of the expression of SCO2 and FRATAXIN (FXN) genes in human K-562 and KU-812 Bcr-Abl(+) CML cells. IM also reduced the protein level of mature Sco2 mitochondrial protein as well as COX activity in these cell lines. However, treatment of human MOLT-4 Bcr-Abl(-) cells with 1μM and even with higher concentrations (4×10(-5)M) of IM neither reduced the expression of SCO2 and FXN genes nor suppressed the protein level of mature Sco2 protein and COX activity. Our findings indicate that SCO2 and FXN genes, involved in HDCBAP, are repressed by IM in human Bcr-Abl(+) CML cells and may represent novel target sites in leukemia therapy. PMID:24726617

  18. Inhibition by recombinant human interleukin-6 of the glucagon-dependent induction of phosphoenolpyruvate carboxykinase and of the insulin-dependent induction of glucokinase gene expression in cultured rat hepatocytes: regulation of gene transcription and messenger RNA degradation.

    PubMed

    Christ, B; Nath, A; Heinrich, P C; Jungermann, K

    1994-12-01

    The influence of recombinant human interleukin-6, the major mediator of the inflammatory response in liver, on the glucagon- and insulin-dependent induction of the phosphoenolpyruvate carboxykinase and glucokinase gene, respectively, was monitored on the level of gene transcription, mRNA abundance and enzyme activity in cultured rat hepatocytes. As control markers of the interleukin-6-induced acute-phase response the mRNA levels of the acute phase proteins alpha 2-macroglobulin and beta-fibrinogen were determined. In cultured rat hepatocytes, recombinant human interleukin-6, added simultaneously with glucagon and insulin, lowered the maximal increase in glucagon-induced phosphoenolpyruvate carboxykinase mRNA levels after 2 hr and the maximal increase in glucokinase mRNA levels after 3 hr to about 30%, respectively. It inhibited the glucagon-induced increase in phosphoenolpyruvate carboxykinase gene transcription and phosphoenolpyruvate carboxykinase enzyme activity, as well as the insulin-induced increases in glucokinase gene transcription and glucokinase enzyme activity. Recombinant human interleukin-6 increased the mRNA levels of the acute-phase proteins alpha 2-macroglobulin and beta-fibrinogen gradually over 4 to 6 hr. Recombinant human interleukin-6, added 2 hr after glucagon or 3 hr after insulin at the maximum of the hormone-induced enzyme mRNA levels, almost doubled the decay rate of phosphoenolpyruvate carboxykinase mRNA and glucokinase mRNA. The results show that interleukin-6 induced the expression of inflammatory proteins and simultaneously inhibited the hormone-induced expression of enzymes of intermediary metabolism.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7527006

  19. TET2 Inhibits Differentiation of Embryonic Stem Cells but Does Not Overcome Methylation-Induced Gene Silencing

    PubMed Central

    2014-01-01

    TET2 is a methylcytosine dioxygenase that is frequently mutated in myeloid malignancies, notably myelodysplasia and acute myeloid leukemia. TET2 catalyses the conversion of 5′-methylcytosine to 5′-hydroxymethylcytosine within DNA and has been implicated in the process of genomic demethylation. However, the mechanism by which TET2 loss of function results in hematopoietic dysplasia and leukemogenesis is poorly understood. Here, we show that TET2 is expressed in undifferentiated embryonic stem cells and that its knockdown results in reduction of 5′-hydroxymethylcytosine in genomic DNA. We also present DNA methylation data from bone marrow samples obtained from patients with TET2-mutated myelodysplasia. Based on these findings, we sought to identify the role of TET2 in regulating pluripotency and differentiation. We show that overexpression of TET2 in a stably integrated transgene leads to increased alkaline phosphatase expression in differentiating ES cells and impaired differentiation in methylcellulose culture. We speculate that this effect is due to TET2-mediated expression of stem cell genes in ES cells via hydroxylation of 5′-methylcytosines at key promoter sequences within genomic DNA. This leads to relative hypomethylation of gene promoters as 5′-hydroxymethylcytosine is not a substrate for DNMT1-mediated maintenance methylation. We sought to test this hypothesis by cotransfecting the TET2 gene with methylated reporter genes. The results of these experiments are presented. PMID:25276435

  20. TET2 Inhibits Differentiation of Embryonic Stem Cells but Does Not Overcome Methylation-Induced Gene Silencing.

    PubMed

    Norman, Louis; Tarrant, Paul; Chevassut, Timothy

    2014-01-01

    TET2 is a methylcytosine dioxygenase that is frequently mutated in myeloid malignancies, notably myelodysplasia and acute myeloid leukemia. TET2 catalyses the conversion of 5'-methylcytosine to 5'-hydroxymethylcytosine within DNA and has been implicated in the process of genomic demethylation. However, the mechanism by which TET2 loss of function results in hematopoietic dysplasia and leukemogenesis is poorly understood. Here, we show that TET2 is expressed in undifferentiated embryonic stem cells and that its knockdown results in reduction of 5'-hydroxymethylcytosine in genomic DNA. We also present DNA methylation data from bone marrow samples obtained from patients with TET2-mutated myelodysplasia. Based on these findings, we sought to identify the role of TET2 in regulating pluripotency and differentiation. We show that overexpression of TET2 in a stably integrated transgene leads to increased alkaline phosphatase expression in differentiating ES cells and impaired differentiation in methylcellulose culture. We speculate that this effect is due to TET2-mediated expression of stem cell genes in ES cells via hydroxylation of 5'-methylcytosines at key promoter sequences within genomic DNA. This leads to relative hypomethylation of gene promoters as 5'-hydroxymethylcytosine is not a substrate for DNMT1-mediated maintenance methylation. We sought to test this hypothesis by cotransfecting the TET2 gene with methylated reporter genes. The results of these experiments are presented. PMID:25276435

  1. Differential regulation of adipose tissue and vascular inflammatory gene expression by chronic systemic inhibition of NOS in lean and obese rats

    PubMed Central

    Padilla, Jaume; Jenkins, Nathan T.; Thorne, Pamela K.; Lansford, Kasey A.; Fleming, Nicholas J.; Bayless, David S.; Sheldon, Ryan D.; Rector, R. Scott; Laughlin, M. Harold

    2014-01-01

    Abstract We tested the hypothesis that a decrease in bioavailability of nitric oxide (NO) would result in increased adipose tissue (AT) inflammation. In particular, we utilized the obese Otsuka Long Evans Tokushima Fatty rat model (n = 20) and lean Long Evans Tokushima Otsuka counterparts (n = 20) to determine the extent to which chronic inhibition of NO synthase (NOS) with Nω‐nitro‐l‐arginine methyl ester (L‐NAME) treatment (for 4 weeks) upregulates expression of inflammatory genes and markers of immune cell infiltration in retroperitoneal white AT, subscapular brown AT, periaortic AT as well as in its contiguous aorta free of perivascular AT. As expected, relative to lean rats (% body fat = 13.5 ± 0.7), obese rats (% body fat = 27.2 ± 0.8) were hyperlipidemic (total cholesterol 77.0 ± 2.1 vs. 101.0 ± 3.3 mg/dL), hyperleptinemic (5.3 ± 0.9 vs. 191.9 ± 59.9 pg/mL), and insulin‐resistant (higher HOMA IR index [3.9 ± 0.8 vs. 25.2 ± 4.1]). Obese rats also exhibited increased expression of proinflammatory genes in perivascular, visceral, and brown ATs. L‐NAME treatment produced a small but statistically significant decrease in percent body fat (24.6 ± 0.9 vs. 27.2 ± 0.8%) and HOMA IR index (16.9 ± 2.3 vs. 25.2 ± 4.1) in obese rats. Further, contrary to our hypothesis, we found that expression of inflammatory genes in all AT depots examined were generally unaltered with L‐NAME treatment in both lean and obese rats. This was in contrast with the observation that L‐NAME produced a significant upregulation of inflammatory and proatherogenic genes in the aorta. Collectively, these findings suggest that chronic NOS inhibition alters transcriptional regulation of proinflammatory genes to a greater extent in the aortic wall compared to its adjacent perivascular AT, or visceral white and subscapular brown AT depots. PMID:24744894

  2. 6-Methoxy Podophyllotoxin Induces Apoptosis via Inhibition of TUBB3 and TOPIIA Gene Expressions in 5637 and K562 Cancer Cell Lines

    PubMed Central

    Sadeghi, Iman; Behmanesh, Mehrdad; Ahmadian Chashmi, Najmeh; Sharifi, Mohsen; Soltani, Bahram Mohammad

    2015-01-01

    Objective Podophyllotoxin (PTOX), a natural compound in numerous plants, contains remarkable biological properties that include anti-tumor, anti-viral such as anti-human im- munodeficiency virus (HIV) activities. In order to avoid its adverse effects, various com- pounds have been derived from PTOX. 6-methoxy PTOX (MPTOX) is one of the natural PTOX derivatives with an extra methoxy group. MPTOX is mostly isolated from the Linum species. This study has sought to determine the biological effects of MPTOX on cancer cell lines, 5637 and K562. Materials and Methods In this experimental study, we treated the 5637 and K562 cancer cell lines with MPTOX in a doseand time-dependent manner. Apoptosis was examined by flow cytometry and viability rate was analyzed by the MTT assay. Expressions of the tubulin (TUBB3) and topoisomerase II (TOPIIA) genes were determined by real-time poly- merase chain reaction (PCR). Results Treatment with MPTOX led to significant induction of apoptosis in cancer cells compared to control cells. Gene expression analysis showed reduced levels of TUBB3 and TOPIIA mRNA following MPTOX treatment. Conclusion MPTOX inhibited TUBB3 and TOPIIA gene expression and subsequently induced cell death through apoptosis. These results suggested that MPTOX could be considered a potential anti-tumor agent. PMID:26464822

  3. Cold exposure inhibits hypothalamic Kiss-1 gene expression, serum leptin concentration, and delays reproductive development in male Brandt's vole ( Lasiopodomys brandtii)

    NASA Astrophysics Data System (ADS)

    Zhang, Qiang; Lin, Yi; Zhang, Xue-Ying; Wang, De-Hua

    2015-06-01

    Cold commonly affects growth and reproductive development in small mammals. Here, we test the hypothesis that low ambient temperature will affect growth and puberty onset, associated with altered hypothalamic Kiss-1 gene expression and serum leptin concentration in wild rodents. Male Brandt's voles ( Lasiopodomys brandtii) were exposed to cold (4 ± 1 °C) and warm (23 ± 1 °C) conditions from the birth and sacrificed on different developmental stages (day 26, day 40, day 60, and day 90, respectively). Brandt's voles increased the thermogenic capacity of brown adipose tissue, mobilized body fat, decreased serum leptin levels, and delayed the reproductive development especially on day 40 in the cold condition. They increased food intake to compensate for the high energy demands in the cold. The hypothalamic Kiss-1 gene expression on day 26 was decreased, associated with lower wet testis mass and testis testosterone concentration on day 40, in the cold-exposed voles compared to that in the warm. Serum leptin was positively correlated with body fat, testis mass, and testosterone concentration. These data suggested that cold exposure inhibited hypothalamic Kiss-1 gene expression during the early stage of development, decreased serum leptin concentration, and delayed reproductive development in male Brandt's voles.

  4. The effects of drugs inhibiting protein secretion in the filamentous fungus Trichoderma reesei. Evidence for down-regulation of genes that encode secreted proteins in the stressed cells.

    PubMed

    Pakula, Tiina M; Laxell, Marjukka; Huuskonen, Anne; Uusitalo, Jaana; Saloheimo, Markku; Penttilä, Merja

    2003-11-01

    To study the mechanisms of protein secretion as well as the cellular responses to impaired protein folding and transport in filamentous fungi, we have analyzed Trichoderma reesei cultures treated with chemical agents that interfere with these processes, dithiothreitol, brefeldin A, and the Ca(2+)-ionophore A23187. The effects of the drugs on the kinetics of protein synthesis and transport were characterized using metabolic labeling of synthesized proteins. Cellobiohydrolase I (CBHI, Cel7A), the major secreted cellulase, was analyzed as a model protein. Northern analysis showed that under conditions where protein transport was inhibited (treatments with dithiothreitol or brefeldin A) the unfolded protein response pathway was activated. The active form of the hac1 mRNA that mediates unfolded protein response signaling was induced, followed by induction of the foldase and chaperone genes pdi1 and bip1. Concomitant with the activation of the unfolded protein response pathway, the transcript levels of genes encoding secreted proteins, like cellulases and xylanases, were drastically decreased, suggesting a novel type of feedback mechanism activated in response to impairment in protein folding or transport (repression under secretion stress (RESS)). By studying expression of the reporter gene lacZ under cbh1 promoters of different length, it was shown that the feedback response was mediated through the cellulase promoter. PMID:12941955

  5. Selection on Glycine β-1,3-Endoglucanase Genes Differentially Inhibited by a Phytophthora Glucanase Inhibitor Protein

    PubMed Central

    Bishop, J. G.; Ripoll, D. R.; Bashir, S.; Damasceno, C. M. B.; Seeds, J. D.; Rose, J. K. C.

    2005-01-01

    Plant endo-β-1,3-glucanases (EGases) degrade the cell wall polysaccharides of attacking pathogens and release elicitors of additional plant defenses. Isozymes EGaseA and EGaseB of soybean differ in susceptibility to a glucanase inhibitor protein (GIP1) produced by Phytophthora sojae, a major soybean pathogen. EGaseA, the major elicitor-releasing isozyme, is a high-affinity ligand for GIP1, which completely inhibits it, whereas EGaseB is unaffected by GIP1. We tested for departures from neutral evolution on the basis of partial sequences of EGaseA and EGaseB from 20 widespread accessions of Glycine soja (the wild progenitor of soybean), from 4 other Glycine species, and across dicotyledonous plants. G. soja exhibited little intraspecific variation at either locus. Phylogeny-based codon evolution models detected strong evidence of positive selection on Glycine EGaseA and weaker evidence for selection on dicot EGases and Glycine EGaseB. Positively selected peptide sites were identified and located on a structural model of EGase bound to GIP1. Positively selected sites and highly variable sites were found disproportionately within 4.5 Å of bound GIP1. Low variation within G. soja EGases, coupled with positive selection in both Glycine and dicot lineages and the proximity of rapidly evolving sites to GIP1, suggests an arms race involving repeated adaptation to pathogen attack and inhibition. PMID:15545660

  6. Leptin, BMI, and a Metabolic Gene Expression Signature Associated with Clinical Outcome to VEGF Inhibition in Colorectal Cancer.

    PubMed

    Pommier, Aurélien J C; Farren, Matthew; Patel, Bhavika; Wappett, Mark; Michopoulos, Filippos; Smith, Neil R; Kendrew, Jane; Frith, Jeremy; Huby, Russell; Eberlein, Catherine; Campbell, Hayley; Womack, Christopher; Smith, Paul D; Robertson, Jane; Morgan, Shethah; Critchlow, Susan E; Barry, Simon T

    2016-01-12

    VEGF (vascular endothelial growth factor) signaling inhibitors are widely used in different cancer types; however, patient selection remains a challenge. Analyses of samples from a phase III clinical trial in metastatic colorectal cancer testing chemotherapy versus chemotherapy with the small molecule VEGF receptors inhibitor cediranib identified circulating leptin levels, BMI, and a tumor metabolic and angiogenic gene expression signature associated with improved clinical outcome in patients treated with cediranib. Patients with a glycolytic and hypoxic/angiogenic profile were associated with increased benefit from cediranib, whereas patients with a high lipogenic, oxidative phosphorylation and serine biosynthesis signature did not gain benefit. These findings translated to pre-clinical tumor xenograft models where the same metabolic gene expression profiles were associated with in vivo sensitivity to cediranib as monotherapy. These findings suggest a link between patient physiology, tumor biology, and response to antiangiogenics, which may guide patient selection for VEGF therapy in the future. PMID:26626460

  7. High Temperature Inhibits Ascorbate Recycling and Light Stimulation of the Ascorbate Pool in Tomato despite Increased Expression of Biosynthesis Genes

    PubMed Central

    Massot, Capucine; Bancel, Doriane; Lopez Lauri, Félicie; Truffault, Vincent; Baldet, Pierre; Stevens, Rebecca; Gautier, Hélène

    2013-01-01

    Understanding how the fruit microclimate affects ascorbate (AsA) biosynthesis, oxidation and recycling is a great challenge in improving fruit nutritional quality. For this purpose, tomatoes at breaker stage were harvested and placed in controlled environment conditions at different temperatures (12, 17, 23, 27 and 31°C) and irradiance regimes (darkness or 150 µmol m-2 s-1). Fruit pericarp tissue was used to assay ascorbate, glutathione, enzymes related to oxidative stress and the AsA/glutathione cycle and follow the expression of genes coding for 5 enzymes of the AsA biosynthesis pathway (GME, VTC2, GPP, L-GalDH, GLDH). The AsA pool size in pericarp tissue was significantly higher under light at temperatures below 27°C. In addition, light promoted glutathione accumulation at low and high temperatures. At 12°C, increased AsA content was correlated with the enhanced expression of all genes of the biosynthesis pathway studied, combined with higher DHAR and MDHAR activities and increased enzymatic activities related to oxidative stress (CAT and APX). In contrast, at 31°C, MDHAR and GR activities were significantly reduced under light indicating that enzymes of the AsA/glutathione cycle may limit AsA recycling and pool size in fruit pericarp, despite enhanced expression of genes coding for AsA biosynthesis enzymes. In conclusion, this study confirms the important role of fruit microclimate in the regulation of fruit pericarp AsA content, as under oxidative conditions (12°C, light) total fruit pericarp AsA content increased up to 71%. Moreover, it reveals that light and temperature interact to regulate both AsA biosynthesis gene expression in tomato fruits and AsA oxidation and recycling. PMID:24367665

  8. A long-acting suicide gene toxin, 6-methylpurine, inhibits slow growing tumors after a single administration.

    PubMed

    Gadi, Vijayakrishna K; Alexander, Sherrie D; Waud, William R; Allan, Paula W; Parker, William B; Sorscher, Eric J

    2003-03-01

    We have demonstrated antitumor activity against refractory human glioma and pancreatic tumors with 6-methylpurine (MeP) using either a suicide gene therapy strategy to selectively release 6-methylpurine in tumor cells or direct intratumoral injection of 6-methylpurine itself. A single i.p. injection in mice of the prodrug 9-beta-D-[2-deoxyribofuranosyl]-6-methylpurine (MeP-dR; 134 mg/kg) caused sustained regression lasting over 70 days of D54 (human glioma) tumors transduced with the Escherichia coli purine nucleoside phosphorylase (PNP), and a single intratumoral injection of 6-methylpurine (5-10 mg/kg) elicited prolonged delays of the growth of D54 tumors and CFPAC human pancreatic carcinoma. Because the D54 tumor doubling time is >15 days, the experiments indicate that prodrug activation by E. coli PNP engenders destruction of both dividing and nondividing tumor compartments in vivo and, therefore, address a fundamental barrier that has limited the development of suicide gene strategies in the past. A prolonged retention time of 6-methylpurine metabolites in tumors was noted in vivo (T(1/2) >24 h compared with a serum half-life of <1 h). By high-pressure liquid chromatography, metabolites of [(3)H]MeP-dR were 5- to 6-fold higher in tumors expressing E. coli PNP. These experiments point to new endpoints for monitoring E. coli PNP suicide gene therapy, including intratumoral enzymatic activity, in situ (intratumoral) prodrug conversion, and tumor regressions after direct injection of a suicide gene toxin. The findings also help explain the strong in vivo bystander killing mechanism ascribed by several laboratories to E. coli PNP in the past. PMID:12604707

  9. Inhibition of interleukin-1 responsiveness by type II receptor gene transfer: a surface "receptor" with anti-interleukin-1 function.

    PubMed

    Re, F; Sironi, M; Muzio, M; Matteucci, C; Introna, M; Orlando, S; Penton-Rol, G; Dower, S K; Sims, J E; Colotta, F; Mantovani, A

    1996-04-01

    The hypothesis that the type II receptor (RII) acts as a decoy for interleukin-1 (IL-1) was tested by gene transfer in cells expressing only the type I receptor (8387 fibroblasts). RII-transfected cells showed defective responsiveness to IL-1 in terms of NFkappaB activation, cytokine gene expression and production. Blocking monoclonal antibodies against RII restored the capacity of RII-transfected cells to respond to IL-1 beta. Hence defective IL-1 responsiveness of RII-transfected cells requires surface expression of the molecule. RII-transfected cells showed normal responsiveness to TNF, which shares functional properties and elements in the signal transduction pathway with IL-1. Cells transfected with a deletion mutant of RII missing 26 of 29 amino acids of the cytoplasmic portion of the molecule showed impaired responsiveness to IL-2. Cells transfected with full-length or the cytoplasmic deletion mutant of RII released copious amounts of RII in the supernatant. However, transfected cells showed defective responsiveness to brief exposure to IL-1, in the absence of measurable released RII. These results indicate that impairment of the responsiveness to IL-1 following RII gene transfer was dependent upon surface expression of the molecule, specific for IL-1 and unaffected by truncation of the cytoplasmic portion. Thus, the type II "receptor" is a decoy surface molecule, regulated by antiinflammatory signals, whose only known function is to capture and block IL-1. PMID:8666940

  10. A whole-genome RNAi screen identifies an 8q22 gene cluster that inhibits death receptor-mediated apoptosis.

    PubMed

    Dompe, Nicholas; Rivers, Celina Sanchez; Li, Li; Cordes, Shaun; Schwickart, Martin; Punnoose, Elizabeth A; Amler, Lukas; Seshagiri, Somasekar; Tang, Jerry; Modrusan, Zora; Davis, David P

    2011-10-25

    Deregulation of apoptosis is a common occurrence in cancer, for which emerging oncology therapeutic agents designed to engage this pathway are undergoing clinical trials. With the aim of uncovering strategies to activate apoptosis in cancer cells, we used a pooled shRNA screen to interrogate death receptor signaling. This screening approach identified 16 genes that modulate the sensitivity to ligand induced apoptosis, with several genes exhibiting frequent overexpression and/or copy number gain in cancer. Interestingly, two of the top hits, EDD1 and GRHL2, are found 50 kb apart on chromosome 8q22, a region that is frequently amplified in many cancers. By using a series of silencing and overexpression studies, we show that EDD1 and GRHL2 suppress death-receptor expression, and that EDD1 expression is elevated in breast, pancreas, and lung cancer cell lines resistant to death receptor-mediated apoptosis. Supporting the relevance of EDD1 and GRHL2 as therapeutic candidates to engage apoptosis in cancer cells, silencing the expression of either gene sensitizes 8q22-amplified breast cancer cell lines to death receptor induced apoptosis. Our findings highlight a mechanism by which cancer cells may evade apoptosis, and therefore provide insight in the search for new targets and functional biomarkers for this pathway. PMID:21949371

  11. Genome-wide RNAi analysis reveals that simultaneous inhibition of specific mevalonate pathway genes potentiates tumor cell death.

    PubMed

    Pandyra, Aleksandra A; Mullen, Peter J; Goard, Carolyn A; Ericson, Elke; Sharma, Piyush; Kalkat, Manpreet; Yu, Rosemary; Pong, Janice T; Brown, Kevin R; Hart, Traver; Gebbia, Marinella; Lang, Karl S; Giaever, Guri; Nislow, Corey; Moffat, Jason; Penn, Linda Z

    2015-09-29

    The mevalonate (MVA) pathway is often dysregulated or overexpressed in many cancers suggesting tumor dependency on this classic metabolic pathway. Statins, which target the rate-limiting enzyme of this pathway, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), are promising agents currently being evaluated in clinical trials for anti-cancer efficacy. To uncover novel targets that potentiate statin-induced apoptosis when knocked down, we carried out a pooled genome-wide short hairpin RNA (shRNA) screen. Genes of the MVA pathway were amongst the top-scoring targets, including sterol regulatory element binding transcription factor 2 (SREBP2), 3-hydroxy-3-methylglutaryl-coenzyme A synthase 1 (HMGCS1) and geranylgeranyl diphosphate synthase 1 (GGPS1). Each gene was independently validated and shown to significantly sensitize A549 cells to statin-induced apoptosis when knocked down. SREBP2 knockdown in lung and breast cancer cells completely abrogated the fluvastatin-induced upregulation of sterol-responsive genes HMGCR and HMGCS1. Knockdown of SREBP2 alone did not affect three-dimensional growth of lung and breast cancer cells, yet in combination with fluvastatin cell growth was disrupted. Taken together, these results show that directly targeting multiple levels of the MVA pathway, including blocking the sterol-feedback loop initiated by statin treatment, is an effective and targetable anti-tumor strategy. PMID:26353928

  12. Garlic inhibits microsomal triglyceride transfer protein gene expression in human liver and intestinal cell lines and in rat intestine.

    PubMed

    Lin, Marie C; Wang, Er-Jia; Lee, Catherine; Chin, K T; Liu, Depei; Chiu, Jen-Fu; Kung, Hsiang-Fu

    2002-06-01

    Epidemiologic studies have suggested that fresh garlic has lipid-lowering activity. Because the microsomal triglyceride transfer protein (MTP) plays a pivotal role in the assembly and secretion of apolipoprotein B (apoB)-containing lipoproteins, we evaluated the effect of garlic on the expression of the MTP gene in vitro in cell lines and in vivo in rats. Fresh garlic extract (FGE) reduced MTP mRNA levels in both the human hepatoma HepG2 and intestinal carcinoma Caco-2 cells in dose-dependent fashion; significant reductions were detected with 3 g/L FGE. Maximal 72 and 59% reductions, respectively, were observed with 6 g/L FGE. To evaluate the in vivo effect of garlic on MTP gene expression, rats were given a single oral dose of fresh garlic homogenate (FGH), with hepatic and intestinal MTP mRNA measured 3 h after dosing. Rats fed FGH had significantly (46% of the control) lower intestinal MTP mRNA levels compared with the control rats, whereas hepatic MTP mRNA levels were not affected. These results suggest a new mechanism for the hypolipidemic effect of fresh garlic. Long-term dietary supplementation of fresh garlic may exert a lipid-lowering effect partly through reducing intestinal MTP gene expression, thus suppressing the assembly and secretion of chylomicrons from intestine to the blood circulation. PMID:12042427

  13. Krüppel Homolog 1 Inhibits Insect Metamorphosis via Direct Transcriptional Repression of Broad-Complex, a Pupal Specifier Gene.

    PubMed

    Kayukawa, Takumi; Nagamine, Keisuke; Ito, Yuka; Nishita, Yoshinori; Ishikawa, Yukio; Shinoda, Tetsuro

    2016-01-22

    The Broad-Complex gene (BR-C) encodes transcription factors that dictate larval-pupal metamorphosis in insects. The expression of BR-C is induced by molting hormone (20-hydroxyecdysone (20E)), and this induction is repressed by juvenile hormone (JH), which exists during the premature larval stage. Krüppel homolog 1 gene (Kr-h1) has been known as a JH-early inducible gene responsible for repression of metamorphosis; however, the functional relationship between Kr-h1 and repression of BR-C has remained unclear. To elucidate this relationship, we analyzed cis- and trans elements involved in the repression of BR-C using a Bombyx mori cell line. In the cells, as observed in larvae, JH induced the expression of Kr-h1 and concurrently suppressed 20E-induced expression of BR-C. Forced expression of Kr-h1 repressed the 20E-dependent activation of the BR-C promoter in the absence of JH, and Kr-h1 RNAi inhibited the JH-mediated repression, suggesting that Kr-h1 controlled the repression of BR-C. A survey of the upstream sequence of BR-C gene revealed a Kr-h1 binding site (KBS) in the BR-C promoter. When KBS was deleted from the promoter, the repression of BR-C was abolished. Electrophoresis mobility shift demonstrated that two Kr-h1 molecules bound to KBS in the BR-C promoter. Based on these results, we conclude that Kr-h1 protein molecules directly bind to the KBS sequence in the BR-C promoter and thereby repress 20E-dependent activation of the pupal specifier, BR-C. This study has revealed a considerable portion of the picture of JH signaling pathways from the reception of JH to the repression of metamorphosis. PMID:26518872

  14. Inhibition of hTERT Gene Expression by Silibinin-Loaded PLGA-PEG-Fe3O4 in T47D Breast Cancer Cell Line

    PubMed Central

    Ebrahimnezhad, Zohreh; Zarghami, Nosratollah; Keyhani, Manoutchehr; Amirsaadat, Soumaye; Akbarzadeh, Abolfazl; Rahmati, Mohammad; Mohammad Taheri, Zohreh; Nejati-Koshki, Kazem

    2013-01-01

    Introduction: Nowadays, using drug delivery is an essential method to improve cancer therapy through decreasing drug toxicity and increasing efficiency of treatment. Silibinin (C25H22O10), a polyphenolic flavonoid which is isolated from the milk thistle plant, has various applications in cancer therapy but it has hydrophobic structure with low water solubility and bioavailability. To increase the effect of silibinin, silibinin-loaded PLGA-PEG-Fe3O4 was prepared to determine the inhibitory effect of this nanodrug on Telomerase gene expression. Methods: The rate of silibinin loaded into PLGA-PEG-Fe3O4 was measured. Then, the cytotoxic effect of silibinin-loaded PLGA-PEG-Fe3O4 was determined by Methyl Thiazol Tetrazolium (MTT) assay. After that, inhibition of Telomerase gene expression was indicated through Real-time PCR. Results: Data analysis from MTT assay showed that silibinin-loaded PLGA-PEG-Fe3O4 had dose dependent cytotoxic effect on T47D cell line. MTT assay showed no cytotoxic effect of free PLGA-PEG-Fe3O4 on T47D breast cancer cell line. Real Time PCR analysis showed that the level of telomerase gene expression more efficiently decreased with silibinin-loaded PLGA-PEG-Fe3O4 than with free silibinin alone. Conclusion: The present study indicates that this nanodrug causes down-regulation of Telomerase gene expression in cancer cells. Therefore, PLGA-PEG-Fe3O4 could be an appropriate carrier for hydrophobic agents such as silibinin to improve their action in cancer therapy. PMID:23878789

  15. Selective expression of constitutively active pro-apoptotic protein BikDD gene in primary mammary tumors inhibits tumor growth and reduces tumor initiating cells.

    PubMed

    Rahal, Omar M; Nie, Lei; Chan, Li-Chuan; Li, Chia-Wei; Hsu, Yi-Hsin; Hsu, Jennifer; Yu, Dihua; Hung, Mien-Chie

    2015-01-01

    Our previous study showed that specifically delivering BikDD, a constitutive active mutant of pro-apoptotic protein Bik, to breast cancer cell xenografts in immunocompromised mice has a potent activity against tumor initiating cells (TICs), and that the combination between tyrosine kinase inhibitors (TKI) and BikDD gene therapy yielded synergistic effect on EGFR and HER2 positive breast cancer cells in immunodeficient nude mice. Those encouraging results have allowed us to propose a clinical trial using the liposome-complexing plasmid DNA expressing BikDD gene which has been approved by the NIH RAC Advisory committee. However, it is imperative to test whether systemic delivery of BikDD-expressing plasmid DNAs with liposomes into immunocompetent mice has therapeutic efficacy and tolerable side effects as what we observed in the nude mice model. In this study, we investigated the effects of BikDD gene-therapy on the primary mammary tumors, especially on tumor initiating cells (TICs), of a genetically engineered immunocompetent mouse harboring normal microenvironment and immune response. The effects on TIC population in tumors were determined by FACS analysis with different sets of murine specific TIC markers, CD49f(high)CD61(high) and CD24(+)Jagged1(-). First we showed in vitro that ectopic expression of BikDD in murine N202 cells derived from MMTV-HER2/Neu transgenic mouse tumors induced apoptosis and decreased the number of TICs. Consistently, systemic delivery of VISA-Claudin4-BikDD by liposome complexes significantly inhibited mammary tumor growth and slowed down residual tumor growth post cessation of therapy in MMTV-HER2/Neu transgenic mice compared to the controls. In addition, the anti-tumor effects of BikDD in vivo were consistent with decreased TIC population assessed by FACS analysis and in vitro tumorsphere formation assay of freshly isolated tumor cells. Importantly, systemic administration of BikDD did not cause significant cytotoxic response in standard toxicity assays or body weight changes. Taken together, our findings validated that selective expression of BikDD in the primary mammary tumors in immunocompetent hosts significantly reduced tumor burden and inhibited the residual tumor growth at off-therapy stage by eliminating TICs. Hence, the VISA-Claudin4-BikDD-mediated gene therapy is worthy of further investigation in breast cancer clinical trials. PMID:26885451

  16. Selective expression of constitutively active pro-apoptotic protein BikDD gene in primary mammary tumors inhibits tumor growth and reduces tumor initiating cells

    PubMed Central

    Rahal, Omar M; Nie, Lei; Chan, Li-Chuan; Li, Chia-Wei; Hsu, Yi-Hsin; Hsu, Jennifer; Yu, Dihua; Hung, Mien-Chie

    2015-01-01

    Our previous study showed that specifically delivering BikDD, a constitutive active mutant of pro-apoptotic protein Bik, to breast cancer cell xenografts in immunocompromised mice has a potent activity against tumor initiating cells (TICs), and that the combination between tyrosine kinase inhibitors (TKI) and BikDD gene therapy yielded synergistic effect on EGFR and HER2 positive breast cancer cells in immunodeficient nude mice. Those encouraging results have allowed us to propose a clinical trial using the liposome-complexing plasmid DNA expressing BikDD gene which has been approved by the NIH RAC Advisory committee. However, it is imperative to test whether systemic delivery of BikDD-expressing plasmid DNAs with liposomes into immunocompetent mice has therapeutic efficacy and tolerable side effects as what we observed in the nude mice model. In this study, we investigated the effects of BikDD gene-therapy on the primary mammary tumors, especially on tumor initiating cells (TICs), of a genetically engineered immunocompetent mouse harboring normal microenvironment and immune response. The effects on TIC population in tumors were determined by FACS analysis with different sets of murine specific TIC markers, CD49fhighCD61high and CD24+Jagged1-. First we showed in vitro that ectopic expression of BikDD in murine N202 cells derived from MMTV-HER2/Neu transgenic mouse tumors induced apoptosis and decreased the number of TICs. Consistently, systemic delivery of VISA-Claudin4-BikDD by liposome complexes significantly inhibited mammary tumor growth and slowed down residual tumor growth post cessation of therapy in MMTV-HER2/Neu transgenic mice compared to the controls. In addition, the anti-tumor effects of BikDD in vivo were consistent with decreased TIC population assessed by FACS analysis and in vitro tumorsphere formation assay of freshly isolated tumor cells. Importantly, systemic administration of BikDD did not cause significant cytotoxic response in standard toxicity assays or body weight changes. Taken together, our findings validated that selective expression of BikDD in the primary mammary tumors in immunocompetent hosts significantly reduced tumor burden and inhibited the residual tumor growth at off-therapy stage by eliminating TICs. Hence, the VISA-Claudin4-BikDD-mediated gene therapy is worthy of further investigation in breast cancer clinical trials. PMID:26885451

  17. Silencing gene expression of the ethylene-forming enzyme results in a reversible inhibition of ovule development in transgenic tobacco plants

    PubMed Central

    De Martinis D; Mariani, C

    1999-01-01

    To study the role of ethylene in plant reproduction, we constructed transgenic tobacco plants in which the expression of a pistil-specific gene coding for the ethylene-forming enzyme 1-aminocyclopropane-1-carboxylate oxidase was inhibited. Flowers from transgenic plants showed female sterility due to an arrest in ovule development. Megasporogenesis did not occur, and ovules did not reach maturity. When pollinated, pollen tubes were able to reach the ovary but did not penetrate into the immature ovule in transgenic plants. Flower treatment with an ethylene source resulted in a functional recovery of ovule development and restored guidance of the pollen tube tip into the ovule micropyle that resulted in seed set. The recovery was abolished if inhibitors of ethylene action were present. These results demonstrate that the plant hormone ethylene is required during the very early stages of female sporogenesis and ultimately to enable fertilization. PMID:10368177

  18. Inhibition of Schistosoma mansoni ether-a-go-go related gene-encoded potassium channels leads to hypermotility and impaired egg production.

    PubMed

    Parker-Manuel, S J; Hahnel, S; Grevelding, C G

    2015-11-01

    The purpose of this work was to investigate the effect of ether-a-go-go related gene (ERG) potassium channel inhibition on Schistosoma mansoni. Use of dofetilide to block the schistosome ERGs resulted in a striking 'corkscrew' effect. The worms were unable to control their motility; they were hypermotile. The treated worms produced abnormal eggs, some of which consisted of little more than a spine. One of the S. mansoni ERGs (SmERGs), Smp_161140, was chosen for further study by RNAi. The transcript was knocked down to 50% compared to the controls. These RNAi-treated worms demonstrated seizure-like movements. In S. mansoni, as in other organisms, ERG channels seem to play a role in regulating muscle excitability. This work shows that egg production can be greatly reduced by effectively targeting muscle coordination in these important parasites. PMID:26188142

  19. 2',5'-linked oligo-3'-deoxyribonucleoside phosphorothioate chimeras: thermal stability and antisense inhibition of gene expression.

    PubMed Central

    Bhan, P; Bhan, A; Hong, M; Hartwell, J G; Saunders, J M; Hoke, G D

    1997-01-01

    2',5'-Linked oligo-3'-deoxyribonucleotides bind selectively to complementary RNA but not to DNA. These oligonucleotides (ODNs) do not recognize double-stranded DNA by Hoogsteen triplex formation and the complexes formed by these ODNs with RNA are not substrates for Escherichia coli RNase H. Substitution of the 2',5'-phosphodiester backbone by phosphorothioate linkages gives 2',5'-linked oligo-3'-deoxynucleoside phosphorothioate ODNs that exhibit significantly less non-specific binding to cellular proteins or thrombin. Incorporation of a stretch of seven contiguous 3',5'-linked oligo-2'-deoxynucleoside phosphorothioate linkages in the center of 2',5'-linked ODNs (as a putative RNase H recognition site) afford chimeric antisense ODNs that retain the ability to inhibit steroid 5alpha-reductase (5alphaR) expression in cell culture. PMID:9241246

  20. Inhibition of cell proliferation and induction of apoptosis by oleanane triterpenoid (CDDO-Me) in pancreatic cancer cells is associated with the suppression of hTERT gene expression and its telomerase activity

    SciTech Connect

    Deeb, Dorrah; Gao, Xiaohua; Liu, Yongbo; Kim, Sahn-Ho; Pindolia, Kirit R.; Arbab, Ali S.; Gautam, Subhash C.

    2012-06-15

    Highlights: Black-Right-Pointing-Pointer CDDO-Me inhibits hTERT gene expression. Black-Right-Pointing-Pointer CDDO-Me inhibits hTERT protein expression. Black-Right-Pointing-Pointer CDDO-Me inhibits hTERT telomerase activity. Black-Right-Pointing-Pointer CDDO-Me inhibits hTERT regulatory proteins. -- Abstract: Methyl-2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oate (CDDO-Me) is a multifunctional oleanane synthetic triterpenoid with potent anti-inflammatory and antitumorigenic properties. The mechanisms of the antisurvival and apoptosis-inducing activities of CDDO-Me and related derivatives of oleanolic acid have been defined; however, to date, no study has been carried out on the effect of CDDOs on human telomerase reverse transcriptase (hTERT) gene or telomerase activity. Here we report for the first time that inhibition of cell proliferation and induction of apoptosis by CDDO-Me in pancreatic cancer cell lines is associated with the inhibition of hTERT gene expression, hTERT telomerase activity and a number of proteins that regulate hTERT expression and activity. Furthermore, abrogation or overexpression of hTERT protein altered the susceptibility of tumor cells to CDDO-Me. These findings suggest that telomerase (hTERT) is a relevant target of CDDO-Me in pancreatic cancer cells.

  1. Specific NF-kappaB blockade selectively inhibits tumour necrosis factor-alpha-induced COX-2 but not constitutive COX-1 gene expression in HT-29 cells.

    PubMed Central

    Jobin, C; Morteau, O; Han, D S; Balfour Sartor, R

    1998-01-01

    Cyclo-oxygenase (COX) is the key regulatory enzyme of the prostaglandin/eicosanoid pathway. While COX-1 is mostly constitutively expressed, the COX-2 isoform is inducible by proinflammatory cytokines. We used an adenoviral vector containing an NF-kappaB super-repressor (Ad5IkappaB) to investigate the role of NF-kappaB in tumour necrosis factor-alpha (TNF-alpha)-mediated COX-2 gene expression in a colonic epithelial cell line. COX-1 mRNA and protein were constitutively expressed in uninfected, control Ad5LacZ- or Ad5IkappaB-infected HT-29 cells with no apparent change following TNF-alpha exposure. COX-2 mRNA and protein expression was undetectable in unstimulated cells but was strongly up-regulated after TNF-alpha stimulation in uninfected and Ad5LacZ-infected HT-29 cells. This induction was prevented in Ad5IkappaB cells. TNF-alpha increased prostaglandin E2 production by 20-fold in Ad5LacZ-infected HT-29 cells compared with uninfected cells and was significantly inhibited in Ad5IkappaB-infected cells in agreement with the COX-2 mRNA findings. We conclude that NF-kappaB activation is critical in mediating COX-2, but not COX-1 gene expression in HT-29 cells. Selective inhibition of COX-2 expression with the NF-kappaB super-repressor may be useful in distinguishing the role of inducible versus constitutive prostaglandins in intestinal function and provides greater specificity than pharmacological inhibitors. Images Figure 1 Figure 2 Figure 3 PMID:9893042

  2. Inhibition of Lipolysis in the Novel Transgenic Quail Model Overexpressing G0/G1 Switch Gene 2 in the Adipose Tissue during Feed Restriction

    PubMed Central

    Shin, Sangsu; Choi, Young Min; Han, Jae Yong; Lee, Kichoon

    2014-01-01

    In addition to the issue of obesity in humans, the production of low-fat meat from domestic animals is important in the agricultural industry to satisfy consumer demand. Understanding the regulation of lipolysis in adipose tissue could advance our knowledge to potentially solve both issues. Although the G0/G1 switch gene 2 (G0S2) was recently identified as an inhibitor of adipose triglyceride lipase (ATGL) in vitro, its role in vivo has not been fully clarified. This study was conducted to investigate the role of G0S2 gene in vivo by using two independent transgenic quail lines during different energy conditions. Unexpectedly, G0S2 overexpression had a negligible effect on plasma NEFA concentration, fat cell size and fat pad weight under ad libitum feeding condition when adipose lipolytic activity is minimal. A two-week feed restriction in non-transgenic quail expectedly caused increased plasma NEFA concentration and dramatically reduced fat cell size and fat pad weight. Contrary, G0S2 overexpression under a feed restriction resulted in a significantly less elevation of plasma NEFA concentration and smaller reductions in fat pad weights and fat cell size compared to non-transgenic quail, demonstrating inhibition of lipolysis and resistance to loss of fat by G0S2. Excessive G0S2 inhibits lipolysis in vivo during active lipolytic conditions, such as food restriction and fasting, suggesting G0S2 as a potential target for treatment of obesity. In addition, transgenic quail are novel models for studying lipid metabolism and mechanisms of obesity. PMID:24964090

  3. Tissue inhibitor of metalloproteinases-1 stimulates gene expression in MDA-MB-435 human breast cancer cells by means of its ability to inhibit metalloproteinases.

    PubMed

    Porter, Joseph F; Sharma, Shashi; Wilson, Donna L; Kappil, Maya A; Hart, Ronald P; Denhardt, David T

    2005-11-01

    Tissue inhibitor of metalloproteinases-1 (TIMP-1) is a widely expressed, secreted protein that functions primarily to inhibit members of a large family of metalloproteinases (MPs). Because of the ability of TIMP-1 to inhibit MPs, it functions in many of the same pathophysiological processes as these enzymes, e.g. wound healing, ovulation, angiogenesis, and cancer cell metastasis. TIMP-1 can also stimulate proliferation ([3H]thymidine incorporation) and cellular anabolic processes (Alamar Blue reduction). This stimulation has been shown to be dependent on the MP-inhibitory ability of TIMP-1 in the human breast cancer cell line MDA-MB-435 (Porter et al., Br J Cancer 90: 463, 2004). To shed light on the mechanism by which TIMP-1 stimulates cellular anabolic processes, an oligonucleotide microarray analysis was performed over a time course of TIMP-1 treatment of MDA-MB-435 cells. Fifteen genes whose mRNAs were differentially regulated were identified. Six (Importin-7, MGC10471, FOXC1, subunit p20 of Arp2/3 complex, mitochondrial ribosomal protein L32, and the serine/threonine kinase-4 (MST1)) of these genes were confirmed by quantitative real time PCR. These same mRNAs were shown to be regulated by the synthetic hydroxamate MP-inhibitor GM6001 but not by its inactive derivative GM6001*, suggesting that the differential regulation occurs through the MP-inhibitory ability of TIMP-1. These results suggest a complex action of TIMP-1 on cancer cells mediated by constitutively active cell surface metalloproteinases that release factors regulating cell signaling pathways; they may account for the paradoxical observation that elevated levels of TIMP-1 in tumors can correlate with an adverse prognosis. PMID:16142437

  4. A Pit-1 threonine 220 phosphomimic reduces binding to monomeric DNA sites to inhibit Ras and estrogen stimulation of the prolactin gene promoter.

    PubMed

    Jean, Annie; Gutierrez-Hartmann, Arthur; Duval, Dawn L

    2010-01-01

    Pit-1 is a POU-homeodomain transcription factor that dictates the ontogeny of pituitary somatotrophs, lactotrophs, and thyrotrophs through regulation of their respective protein hormone genes: GH, prolactin (PRL), and TSHbeta. Although Pit-1 threonine 220 (T220) and serine 115 are protein kinase phospho-acceptor sites, the transcriptional role of Pit-1 phosphorylation remains unclear. In the rat PRL promoter (rPRL), Ras-stimulated transcription is mediated by binding of Ets-1 and Pit-1 at a composite site (FPIV). Ets-1 and Pit-1 physically interact, and Pit-1 T220 is a major Ets-1 contact point. T220 was mutated to aspartic acid (D, to mimic phosphorylation) or a neutral alanine (A), and DNA binding and transcriptional activity were tested. The Pit-1 T220D mutation reduced binding at monomeric Pit-1 sites (FPIV, PRL-1d), but not dimeric Pit-1 sites (FPI). Pit-1 T220A bound all sites with wild-type (WT) affinity. In transfections of HeLa cells, each Pit-1 mutant transcriptionally activated the -425rPRL promoter and cooperated with Ets-1 to WT levels. In contrast, Pit-1-mediated Ras activation of the -425 rPRL promoter was significantly inhibited by T220D. Finally, Pit-1 synergistic activation of the 2500-bp rPRL promoter with estrogen receptor was reduced by T220D compared with T220A and WT Pit-1. Thus, phosphorylation of Pit-1 T220 reduces binding to monomeric sites blunting Ras and estrogen/estrogen receptor stimulation of the rPRL gene promoter. Consequently, T220 phosphorylation of Pit-1 by protein kinase A, protein kinase C, or cell cycle-dependent kinases appears to serve as a regulatory switch, inhibiting Ras and estrogen/estrogen receptor regulatory pathways, while enhancing the cAMP/protein kinase A response, thus allowing a more precise integration of pituitary responses to distinct signaling stimuli. PMID:19887646

  5. Salinosporamide A (NPI-0052) potentiates apoptosis, suppresses osteoclastogenesis, and inhibits invasion through down-modulation of NF-kappaB regulated gene products.

    PubMed

    Ahn, Kwang Seok; Sethi, Gautam; Chao, Ta-Hsiang; Neuteboom, Saskia T C; Chaturvedi, Madan M; Palladino, Michael A; Younes, Anas; Aggarwal, Bharat B

    2007-10-01

    Salinosporamide A (also called NPI-0052), recently identified from the marine bacterium Salinispora tropica, is a potent inhibitor of 20S proteasome and exhibits therapeutic potential against a wide variety of tumors through a poorly understood mechanism. Here we demonstrate that salinosporamide A potentiated the apoptosis induced by tumor necrosis factor alpha (TNF), bortezomib, and thalidomide, and this correlated with down-regulation of gene products that mediate cell proliferation (cyclin D1, cyclooxygenase-2 [COX-2], and c-Myc), cell survival (Bcl-2, Bcl-xL, cFLIP, TRAF1, IAP1, IAP2, and survivin), invasion (matrix metallopro-teinase-9 [MMP-9] and ICAM-1), and angiogenesis (vascular endothelial growth factor [VEGF]). Salinosporamide A also suppressed TNF-induced tumor cell invasion and receptor activator of nuclear factor kappaB ligand (RANKL)-induced osteoclastogenesis. We also found that it suppressed both constitutive and inducible NF-kappaB activation. Compared with bortezomib, MG-132, N-acetyl-leucyl-leucyl-norleucinal (ALLN), and lactacystin, salinosporamide A was found to be the most potent suppressor of NF-kappaB activation. Further studies showed that salinosporamide A inhibited TNF-induced inhibitory subunit of NF-kappaB alpha (IkappaBalpha) degradation, nuclear translocation of p65, and NF-kappaB-dependent reporter gene expression but had no effect on IkappaBalpha kinase activation, IkappaBalpha phosphorylation, or IkappaBalpha ubiquitination. Thus, overall, our results indicate that salinosporamide A enhances apoptosis, suppresses osteoclastogenesis, and inhibits invasion through suppression of the NF-kappaB pathway. PMID:17609425

  6. Co-Inoculation with Rhizobia and AMF Inhibited Soybean Red Crown Rot: From Field Study to Plant Defense-Related Gene Expression Analysis

    PubMed Central

    Gao, Xiang; Lu, Xing; Wu, Man; Zhang, Haiyan; Pan, Ruqian; Tian, Jiang; Li, Shuxian; Liao, Hong

    2012-01-01

    Background Soybean red crown rot is a major soil-borne disease all over the world, which severely affects soybean production. Efficient and sustainable methods are strongly desired to control the soil-borne diseases. Principal Findings We firstly investigated the disease incidence and index of soybean red crown rot under different phosphorus (P) additions in field and found that the natural inoculation of rhizobia and arbuscular mycorrhizal fungi (AMF) could affect soybean red crown rot, particularly without P addition. Further studies in sand culture experiments showed that inoculation with rhizobia or AMF significantly decreased severity and incidence of soybean red crown rot, especially for co-inoculation with rhizobia and AMF at low P. The root colony forming unit (CFU) decreased over 50% when inoculated by rhizobia and/or AMF at low P. However, P addition only enhanced CFU when inoculated with AMF. Furthermore, root exudates of soybean inoculated with rhizobia and/or AMF significantly inhibited pathogen growth and reproduction. Quantitative RT-PCR results indicated that the transcripts of the most tested pathogen defense-related (PR) genes in roots were significantly increased by rhizobium and/or AMF inoculation. Among them, PR2, PR3, PR4 and PR10 reached the highest level with co-inoculation of rhizobium and AMF. Conclusions Our results indicated that inoculation with rhizobia and AMF could directly inhibit pathogen growth and reproduction, and activate the plant overall defense system through increasing PR gene expressions. Combined with optimal P fertilization, inoculation with rhizobia and AMF could be considered as an efficient method to control soybean red crown rot in acid soils. PMID:22442737

  7. Sequence-specific inhibition of gene expression by a novel antisense oligodeoxynucleotide phosphorothioate directed against a nonregulatory region of the human immunodeficiency virus type 1 genome.

    PubMed Central

    Anazodo, M I; Wainberg, M A; Friesen, A D; Wright, J A

    1995-01-01

    Previous studies have demonstrated that oligodeoxynucleotide phosphorothioates complementary to human immunodeficiency virus type 1 (HIV-1) RNA are more nuclease resistant and are effective inhibitors of HIV-1 replication than their unmodified counterpart. In this study, antisense oligodeoxynucleotide sequences were evaluated for therapeutic potential in the treatment of HIV infections. The use of HIV-infected lymphocytes to test the efficacy of a drug is very complex, and therefore it is difficult to draw conclusions about the mechanism. We used a COS-like Monkey kidney cell line (CMT3) stably transfected with plasmids pCMVgagpol-rre-r (containing gag and pol genes) and pCMVrev (containing the rev gene of HIV-1), derived from cDNA clone BH10, as a model. A biologically active provirus that transcribes and translates their nucleotide sequences into viral proteins p24, p39/41, p55, and p160 was generated. Sequence-specific and dose-dependent inhibition of HIV-1 viral protein synthesis and significant inhibition at the mRNA level were demonstrated by antisense construct GPI2A, directed against a nonregulatory region of the HIV-1 genome. Also, our studies demonstrated enhancement of the antisense effect through encapsulation in a cationic lipid preparation. The observed attenuation of HIV-1 mRNA levels suggests that, at least in part, the mechanism of action of GPI2A was at the transcript level. Further studies have also shown antiviral activity of this construct as determined by the reverse transcriptase assay using acutely and chronically infected cells of lymphoid origin (H9 cells). Toxicological studies involving cell growth characteristics, colony-forming ability, effects on cellular proteins, specific activities of labeled proteins, and DNA synthesis in cell culture showed no cytotoxic effects of GPI2A. PMID:7853519

  8. A Nongenomic Mechanism for Progesterone-mediated Immunosuppression: Inhibition of K+ Channels, Ca2+ Signaling, and Gene Expression in T Lymphocytes

    PubMed Central

    Ehring, George R.; Kerschbaum, Hubert H.; Eder, Claudia; Neben, Amber L.; Fanger, Christopher M.; Khoury, Rosana M.; Negulescu, Paul A.; Cahalan, Michael D.

    1998-01-01

    The mechanism by which progesterone causes localized suppression of the immune response during pregnancy has remained elusive. Using human T lymphocytes and T cell lines, we show that progesterone, at concentrations found in the placenta, rapidly and reversibly blocks voltage-gated and calcium-activated K+ channels (KV and KCa, respectively), resulting in depolarization of the membrane potential. As a result, Ca2+ signaling and nuclear factor of activated T cells (NF-AT)-driven gene expression are inhibited. Progesterone acts distally to the initial steps of T cell receptor (TCR)-mediated signal transduction, since it blocks sustained Ca2+ signals after thapsigargin stimulation, as well as oscillatory Ca2+ signals, but not the Ca2+ transient after TCR stimulation. K+ channel blockade by progesterone is specific; other steroid hormones had little or no effect, although the progesterone antagonist RU 486 also blocked KV and KCa channels. Progesterone effectively blocked a broad spectrum of K+ channels, reducing both Kv1.3 and charybdotoxin–resistant components of KV current and KCa current in T cells, as well as blocking several cloned KV channels expressed in cell lines. Progesterone had little or no effect on a cloned voltage-gated Na+ channel, an inward rectifier K+ channel, or on lymphocyte Ca2+ and Cl− channels. We propose that direct inhibition of K+ channels in T cells by progesterone contributes to progesterone-induced immunosuppression. PMID:9802971

  9. Molecular cloning and functional analysis of three genes encoding polygalacturonase-inhibiting proteins from Capsicum annuum, and their relation to increased resistance to two fungal pathogens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polygalacturonase-inhibiting proteins (PGIPs) are plant cell wall glycoproteins that can inhibit fungal endopolygalacturonases (PGs). Inhibiting by PGIPs directly reduces potential PG activity in specific plant pathogenic fungi, reducing their aggressiveness. Here, we isolated and functionally chara...

  10. Suppression of invasion and metastasis in aggressive salivary cancer cells through targeted inhibition of ID1 gene expression.

    PubMed

    Murase, Ryuichi; Sumida, Tomoki; Kawamura, Rumi; Onishi-Ishikawa, Akiko; Hamakawa, Hiroyuki; McAllister, Sean D; Desprez, Pierre-Yves

    2016-07-10

    Salivary gland cancer (SGC) represents the most common malignancy in the head and neck region, and often metastasizes to the lungs. The helix-loop-helix ID1 protein has been shown to control metastatic progression in many types of cancers. Using two different approaches to target the expression of ID1 (genetic knockdown and progesterone receptor introduction combined with progesterone treatment), we previously determined that the aggressiveness of salivary gland tumor ACCM cells in culture was suppressed. Here, using the same approaches to target ID1 expression, we investigated the ability of ACCM cells to generate lung metastatic foci in nude mice. Moreover, since both approaches would be challenging for applications in humans, we added a third approach, i.e., treatment of mice with a non-toxic cannabinoid compound known to down-regulate ID1 gene expression. All approaches aimed at targeting the pro-metastatic ID1 gene led to a significant reduction in the formation of lung metastatic foci. Therefore, targeting a key transcriptional regulator using different means results in the same reduction of the metastatic spread of SGC cells in animal models, suggesting a novel approach for the treatment of patients with aggressive SGC. PMID:27087608

  11. Sodium butyrate inhibits Staphylococcus aureus internalization in bovine mammary epithelial cells and induces the expression of antimicrobial peptide genes.

    PubMed

    Ochoa-Zarzosa, Alejandra; Villarreal-Fernández, Edith; Cano-Camacho, Horacio; López-Meza, Joel E

    2009-07-01

    A distinctive feature of bovine milk fat is the presence of butyrate, molecule with recognized antimicrobial and antiinflammatory properties. Bovine mastitis is a pathology characterized by inflammatory and infectious processes; however, the role of sodium butyrate on Staphylococcus aureus infection in mammary epithelium has not been studied. In this work we assess the role of sodium butyrate on the invasion of bovine mammary epithelial cells (bMEC) by S. aureus responsible of mastitis and on the expression of antimicrobial peptide genes. Our data show that sodium butyrate (0.25-0.5mM) reduces approximately 50% the internalization of S. aureus (ATCC 27543) into bMEC. By RT-PCR analysis, we showed that sodium butyrate is able to up-regulate the expression of tracheal antimicrobial peptide (TAP), beta-defensin and inducible nitric oxide synthase (iNOS) mRNAs, as well as nitric oxide production. Also, sodium butyrate and infection increased acetylation of histone H3 in bMEC. These results indicate that sodium butyrate could be effective to modulate innate immune gene expression in mammary gland that leads to a better defense against bacterial infection. To our knowledge, this is the first report that shows a role of sodium butyrate during the internalization of S. aureus into bMEC. PMID:19393738

  12. Ephrin-A1 inhibits NSCLC tumor growth via induction of Cdx-2 a tumor suppressor gene

    PubMed Central

    2012-01-01

    Background Tumor formation is a complex process which involves constitutive activation of oncogenes and suppression of tumor suppressor genes. Receptor EphA2 and its ligand ephrin-A1 form an important cell communication system with its functional role in cell-cell interaction and tumor growth. Loss of cell-cell adhesion is central to the cellular transformation and acquisition of metastatic potential. Claudins, the integrated tight junction (TJ) cell-cell adhesion proteins located on the apico-lateral portion of epithelial cells, functions in maintaining cell polarity. There is extensive evidence implicating Eph receptors and ephrins in malignancy, but the mechanisms how these molecular players affect TJ proteins and regulate tumor growth are not clear. In the present study we hypothesized that EphA2 signaling modulates claudin-2 gene expression via induction of cdx-2, a tumor suppressor gene in NSCLC cells. Methods The expression of EphA2, claudin-2 was determined in various NSCLC cell lines by using real-time quantitative polymerase chain reaction and Western blot analysis. The claudin-2 expression was also analyzed by immunofluorescence analysis. EphA2 and erk1/erk2 phosphorylation in ephrin-A1 activated cells was evaluated by Western blot analysis. The cell proliferation and tumor colony formation were determined by WST-1 and 3-D matrigel assays respectively. Results NSCLC cells over expressed receptor EphA2 and claudin-2. Ephrin-A1 treatment significantly down regulated the claudin-2 and EphA2 expression in NSCLC cells. The transient transfection of cells with vector containing ephrin-A1 construct (pcDNA-EFNA1) decreased the expression of claudin-2, EphA2 when compared to empty vector. In addition ephrin-A1 activation increased cdx-2 expression in A549 cells. In contrast over-expression of EphA2 with plasmid pcDNA-EphA2 up regulated claudin-2 mRNA expression and decreased cdx-2 expression. The transient transfection of cells with vector containing cdx-2 construct (pcMV-cdx-2) decreased the expression of claudin-2 in A549 cells. Moreover, silencing the expression of receptor EphA2 by siRNA significantly reduced claudin-2 expression and decreased cell proliferation and tumor formation. Furthermore, silencing cdx-2 gene expression before ephrin-A1 treatment increased claudin-2 expression along with increased cell proliferation and tumor growth in A549 cells. Conclusions Our study suggests that EphA2 signaling up-regulates the expression of the TJ-protein claudin-2 that plays an important role in promoting cell proliferation and tumor growth in NSCLC cells. We conclude that receptor EphA2 activation by ephrin-A1 induces tumor suppressor gene cdx-2 expression which attenuates cell proliferation, tumor growth and thus may be a promising therapeutic target against NSCLC. PMID:22824143

  13. TBX-3, the gene mutated in Ulnar-Mammary Syndrome, is a negative regulator of p19ARF and inhibits senescence.

    PubMed

    Brummelkamp, Thijn R; Kortlever, Roderik M; Lingbeek, Merel; Trettel, Flavia; MacDonald, Marcy E; van Lohuizen, Maarten; Bernards, René

    2002-02-22

    Prolonged culturing of rodent cells in vitro activates p19(ARF) (named p14(ARF) in man), resulting in a p53-dependent proliferation arrest known as senescence. The p19(ARF)-Mdm2-p53 pathway also serves to protect primary cells against oncogenic transformation. We have used a genetic screen in mouse neuronal cells, conditionally immortalized by a temperature-sensitive mutant of SV40 large T antigen, to identify genes that allow bypass of senescence. Using retroviral cDNA expression libraries, we have identified TBX-3 as a potent inhibitor of senescence. TBX-3 is a T-box gene, which is found mutated in the human developmental disorder Ulnar-Mammary Syndrome. We have shown that TBX-3 potently represses expression of both mouse p19(ARF) and human p14(ARF). We have also shown here that point mutants of TBX-3, which are found in Ulnar-Mammary Syndrome, have lost the ability to inhibit senescence and fail to repress mouse p19(ARF) and human p14(ARF) expression. These data suggest that the hypoproliferative features of this genetic disorder may be caused, at least in part, by deregulated expression of p14(ARF). PMID:11748239

  14. Adenovirus-mediated gene transfer of endostatin in vivo results in high level of transgene expression and inhibition of tumor growth and metastases

    NASA Astrophysics Data System (ADS)

    Sauter, Bernhard V.; Martinet, Olivier; Zhang, Wei-Jian; Mandeli, John; Woo, Savio L. C.

    2000-04-01

    Inhibition of angiogenesis has been shown to be an effective strategy in cancer therapy in mice. However, its widespread application has been hampered by difficulties in the large-scale production of the antiangiogenic proteins. This limitation may be resolved by in vivo delivery and expression of the antiangiogenic genes. We have constructed a recombinant adenovirus that expresses murine endostatin that is biologically active both in vitro, as determined in endothelial cell proliferation assays, and in vivo, by suppression of angiogenesis induced by vascular endothelial growth factor 165. Persistent high serum levels of endostatin (605-1740 ng/ml; mean, 936 ng/ml) were achieved after systemic administration of the vector to nude mice, which resulted in significant reduction of the growth rates and the volumes of JC breast carcinoma and Lewis lung carcinoma (P < 0.001 and P < 0.05, respectively). In addition, the endostatin vector treatment completely prevented the formation of pulmonary micrometastases in Lewis lung carcinoma (P = 0.0001). Immunohistochemical staining of the tumors demonstrated a decreased number of blood vessels in the treatment group versus the controls. In conclusion, the present study clearly demonstrates the potential of vector-mediated antiangiogenic gene therapy as a component in cancer therapy.

  15. Stabilization of SIRT7 deacetylase by viral oncoprotein HBx leads to inhibition of growth restrictive RPS7 gene and facilitates cellular transformation.

    PubMed

    Pandey, Vijaya; Kumar, Vijay

    2015-01-01

    Sirtuin-7 (SIRT7) deacetylase exhibits a high selectivity for acetylated H3K18 and has been implicated in the maintenance of malignant phenotype. However, it remains unclear if SIRT7 and H3K18ac play a role in the tumorigenic program driven by oncogenic viruses. We show that ectopically expressed HBx oncoprotein of hepatitis B virus promoted intracellular stability of SIRT7 by salvaging it from ubiquitin-mediated proteasomal degradation. HBx-dependent accumulation of SIRT7 favored H3K18 deacetylation and down-regulated the small ribosomal protein gene, RPS7, involved in cell death and DNA damage response. HBx facilitated the recruitment of SIRT7 to RPS7 promoter thus impeding H3K18ac occupancy and hindering RPS7 transcription. The antagonistic relationship between SIRT7 and RPS7 was also observed in the HBx transgenic mice, where elevated levels of SIRT7 protein were coincident with low levels of H3K18ac and RPS7. Strikingly, inhibition of cellular deubiquitinase activity restored RPS7 gene transcription. Further, depletion of endogenous SIRT7 led to decreased cell viability and transformation. The biological relevance of RPS7 suppression by HBx-SIRT7 axis was evident from ectopic expression of RPS7 which attenuated clonogenicity of cells. Thus, our findings suggest that SIRT7 is a critical regulator of HBx-driven oncogenic program, through its antagonistic impact on growth restrictive ribosomal protein RPS7. PMID:26442981

  16. MIF inhibition reverts the gene expression profile of human melanoma cell line-induced MDSCs to normal monocytes

    PubMed Central

    Waigel, Sabine; Rendon, Beatriz E.; Lamont, Gwyneth; Richie, Jamaal; Mitchell, Robert A.; Yaddanapudi, Kavitha

    2016-01-01

    Myeloid-derived suppressor cells (MDSCs) are potently immunosuppressive innate immune cells that accumulate in advanced cancer patients and actively inhibit anti-tumor T lymphocyte responses [1]. Increased numbers of circulating MDSCs directly correlate with melanoma patient morbidity and reduced anti-tumor immune responses [2], [3]. Previous studies have revealed that monocyte-derived macrophage migration inhibitory factor (MIF) is necessary for the immune suppressive function of MDSCs in mouse models of melanoma [4], [5]. To investigate whether MIF participates in human melanoma-induced MDSC differentiation and/or suppressive function, we have established an in vitro MDSC induction model using primary, normal human monocytes co-cultured with human melanoma cell lines in the presence or absence of the MIF antagonist—4-IPP [4], [6], [7], [8], [9]. To identify potential mechanistic effectors, we have performed transcriptome analyses on cultured monocytes and on melanoma-induced MDSCs obtained from either untreated or 4-IPP-treated A375:monocyte co-cultures. Here, we present a detailed protocol, which can facilitate easy reproduction of the microarray results (NCBI GEO accession number GSE73333) published by Yaddanapudi et al. (2015) in Cancer Immunology Research [10].

  17. MIF inhibition reverts the gene expression profile of human melanoma cell line-induced MDSCs to normal monocytes.

    PubMed

    Waigel, Sabine; Rendon, Beatriz E; Lamont, Gwyneth; Richie, Jamaal; Mitchell, Robert A; Yaddanapudi, Kavitha

    2016-03-01

    Myeloid-derived suppressor cells (MDSCs) are potently immunosuppressive innate immune cells that accumulate in advanced cancer patients and actively inhibit anti-tumor T lymphocyte responses [1]. Increased numbers of circulating MDSCs directly correlate with melanoma patient morbidity and reduced anti-tumor immune responses [2], [3]. Previous studies have revealed that monocyte-derived macrophage migration inhibitory factor (MIF) is necessary for the immune suppressive function of MDSCs in mouse models of melanoma [4], [5]. To investigate whether MIF participates in human melanoma-induced MDSC differentiation and/or suppressive function, we have established an in vitro MDSC induction model using primary, normal human monocytes co-cultured with human melanoma cell lines in the presence or absence of the MIF antagonist-4-IPP [4], [6], [7], [8], [9]. To identify potential mechanistic effectors, we have performed transcriptome analyses on cultured monocytes and on melanoma-induced MDSCs obtained from either untreated or 4-IPP-treated A375:monocyte co-cultures. Here, we present a detailed protocol, which can facilitate easy reproduction of the microarray results (NCBI GEO accession number GSE73333) published by Yaddanapudi et al. (2015) in Cancer Immunology Research [10]. PMID:26981417

  18. The mouse pink-eyed dilution allele of the P-gene greatly inhibits eumelanin but not pheomelanin synthesis.

    PubMed

    Hirobe, Tomohisa; Ito, Shosuke; Wakamatsu, Kazumasa

    2011-02-01

    The mouse pink-eyed dilution (p) locus is known to control eumelanin synthesis, melanosome morphology, and tyrosinase activity in melanocytes. However, it has not been fully determined whether the mutant allele, p affects pheomelanin synthesis. Effects of the p allele on eumelanin and phemelanin synthesis were investigated by chemical analysis of dorsal hairs of 5-week-old mice obtained from the F(2) generations (black, pink-eyed black, recessive yellow, pink-eyed recessive yellow, agouti, and pink-eyed agouti) between C57BL/10JHir (B10)-congenic pink-eyed black mice (B10-p/p) and recessive yellow (B10-Mc1r(e)/Mc1r(e)) or agouti (B10-A/A) mice. The eumelanin content was dramatically (>20-fold) decreased in pink-eyed black and pink-eyed agouti mice, whereas the pheomelanin content did not decrease in pink-eyed black, pink-eyed recessive yellow, or pink-eyed agouti mice compared to the corresponding P/- mice. These results suggest that the pink-eyed dilution allele greatly inhibits eumelanin synthesis, but not pheomelanin synthesis. PMID:21232027

  19. Antisense RNA inhibits endogenous gene expression in mouse preimplantation embryos: lack of double-stranded RNA "melting" activity.

    PubMed Central

    Bevilacqua, A; Erickson, R P; Hieber, V

    1988-01-01

    beta-Glucuronidase activity increases 60-fold from the 4-cell to the blastocyst stage during in vitro development of mouse preimplantation embryos, secondary to a 13-fold increase in beta-glucuronidase mRNA. Injections of antisense RNA from a beta-glucuronidase cDNA clone lacking the 5'-untranslated region and the coding sequences for approximately equal to 150 N-terminal amino acids were effective in partially blocking the appearance of beta-glucuronidase activity. Injection of the same RNA, capped with guanosine(5')triphospho(5')guanosine (GpppG), into each blastomere at the 4-cell stage yielded 75% inhibition of enzyme activity at the blastocyst stage. Injections of the sense strand or of an unrelated RNA did not alter the normal increase in activity of the enzyme. These results are in accord with our inability to detect RNA-duplex "melting" activity in 1-cell mouse embryos. We suggest that it may be possible to analyze genetics of mammalian development by antisense techniques. Images PMID:2448782

  20. Overexpression of cellular repressor of E1A-stimulated genes inhibits TNF-{alpha}-induced apoptosis via NF-{kappa}B in mesenchymal stem cells

    SciTech Connect

    Peng, Cheng-Fei; Cardiovascular Research Institute and Department of Cardiology, Shenyang Northern Hospital, Shenyang ; Han, Ya-Ling; Jie-Deng,; Yan, Cheng-Hui; Jian-Kang,; Bo-Luan,; Jie-Li

    2011-03-25

    Research highlights: {yields} CREG protected MSCs from tumor necrosis factor-{alpha} (TNF-{alpha}) induced apoptosis. {yields} CREG inhibits the phosphorylation of I{kappa}B{alpha} and prevents the activation of NF-{kappa}B. {yields} CREG inhibits NF-{kappa}B nuclear translocation and pro-apoptosis protein transcription. {yields} CREG anti-apoptotic effect involves inhibition of the death receptor pathway. {yields} p53 is downregulated by CREG via NF-{kappa}B pathway under TNF-{alpha} stimulation. -- Abstract: Bone marrow-derived mesenchymal stem cells (MSCs) show great potential for therapeutic repair after myocardial infarction. However, poor viability of transplanted MSCs in the ischemic heart has limited their use. Cellular repressor of E1A-stimulated genes (CREG) has been identified as a potent inhibitor of apoptosis. This study therefore aimed to determine if rat bone marrow MSCs transfected with CREG-were able to effectively resist apoptosis induced by inflammatory mediators, and to demonstrate the mechanism of CREG action. Apoptosis was determined by flow cytometric and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling assays. The pathways mediating these apoptotic effects were investigated by Western blotting. Overexpression of CREG markedly protected MSCs from tumor necrosis factor-{alpha} (TNF-{alpha}) induced apoptosis by 50% after 10 h, through inhibition of the death-receptor-mediated apoptotic pathway, leading to attenuation of caspase-8 and caspase-3. Moreover, CREG resisted the serine phosphorylation of I{kappa}B{alpha} and prevented the nuclear translocation of the transcription factor nuclear factor-{kappa}B (NF-{kappa}B) under TNF-{alpha} stimulation. Treatment of cells with the NF-{kappa}B inhibitor pyrrolidine dithiocarbamate (PDTC) significantly increased the transcription of pro-apoptosis proteins (p53 and Fas) by NF-{kappa}B, and attenuated the anti-apoptotic effects of CREG on MSCs. The results of this study indicate that CREG acts as a novel and potent survival factor in MSCs, and may therefore be a useful therapeutic adjunct for transplanting MSCs into the damaged heart after myocardial infarction.

  1. Long-Term Gene Therapy with Thrombospondin 2 Inhibits TGF-β Activation, Inflammation and Angiogenesis in Chronic Allograft Nephropathy

    PubMed Central

    Daniel, Christoph; Vogelbacher, Regina; Stief, Andrea; Grigo, Christina; Hugo, Christian

    2013-01-01

    We recently identified Thrombospondin-2 (TSP-2) as a regulator of matrix remodelling and inflammation in experimental kidney disease by using TSP-2 null mice and successfully proved TSP-2 overexpression as a therapeutic concept in a short term glomerulonephritis model in the rat. In this current study, we investigated if long-term TSP-2 overexpression is also capable to ameliorate the progression of chronic kidney disease in the setting of the chronic allograft nephropathy F344-Lewis model in the rat. Two weeks after renal transplantation, two rat thigh muscles were transfected once only with either a TSP-2 overexpressing plasmid (n = 8) or a luciferase-expressing plasmid as control (n = 8). Rats were monitored for renal function, histological changes and gene expression in the graft for up to 30 weeks after transplantation. Unexpectedly, only in the TSP-2 treated group 2 rats died before the end of the experiment and renal function tended to be worsened in the TSP-2 group compared to the luciferase-treated controls. In addition, glomerular sclerosis and tubular interstitial injury as well as cortical fibronectin deposition was significantly increased in the TSP-2 treated kidneys despite reduced TGF-β activation and marked anti-inflammatory (macrophages, T-cells and B-cells) effects in this group. Long-term TSP-2 therapy impaired repair of renal endothelium, as demonstrated by significant higher glomerular and peritubular endothelial rarefaction and reduced endothelial cell proliferation in the transplanted kidneys from TSP-2 treated rats compared to controls. This TSP-2 effect was associated with decreased levels of renal VEGF but not VEGF1 receptor. In conclusion, despite its anti-inflammatory and TGF-β activation blocking effects, TSP-2 gene therapy did not ameliorate but rather worsened experimental chronic allograft nephropathy most likely via its anti-angiogenic properties on the renal microvasculature. PMID:24376766

  2. Carfilzomib-induced cardiotoxicity mitigated by dexrazoxane through inhibition of hypertrophic gene expression and oxidative stress in rats.

    PubMe