Note: This page contains sample records for the topic gene inhibiting oca2 from Science.gov.
While these samples are representative of the content of Science.gov,
they are not comprehensive nor are they the most current set.
We encourage you to perform a real-time search of Science.gov
to obtain the most current and comprehensive results.
Last update: August 15, 2014.
1

[Polymorphism of pigmentation genes (OCA2 and ASIP) in some populations of Russia].  

PubMed

In Russian populations, polymorphism of two pigmentation system genes, OCA2 (loci 305, 355, and 419, tested in Russians, Buryats, Chukchi, Koryaks, and Evens) and ASIP (locus 8818, tested in Russians and Buryats) was examined. Pairwise comparisons of the F(ST) distances between the populations showed that only the populations from Northeast Asia (Chukchi, Koryaks, and Evens) were statistically significantly different from all other populations, at least relative to one of the OCA2 locus. In Russians from Pskov oblast and Novgorod oblast, increased frequency (up to 6%) of the OCA2 allele 419A was revealed. In earlier studies, as association of this allele with green eye color was demonstrated. The data obtained in terms of their application for ethnic population genetics. PMID:19382693

Maliarchuk, B A; Perkova, M A; Derenko, M V

2009-03-01

2

Polymorphism of pigmentation genes ( OCA2 and ASIP ) in some populations of Russia  

Microsoft Academic Search

In Russian populations, polymorphism of two pigmentation system genes, OCA2 (loci 305, 355, and 419, tested in Russians, Buryats, Chukchi, Koryaks, and Evens) and ASIP (locus 8818, tested in Russians and Buryats) was examined. Pairwise comparisons of the F\\u000a ST distances between the populations showed that only the populations from Northeast Asia (Chukchi, Koryaks, and Evens) were\\u000a statistically significantly different

B. A. Malyarchuk; M. A. Perkova; M. V. Derenko

2009-01-01

3

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

SciTech Connect

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.

Spritz, R.; Fukai, K.; Holmes, S.A. [Univ. of Wisconsin, Madison, WI (United States)] [and others

1995-06-01

4

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

SciTech Connect

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.

Lee, S.T.; Nicholls, R.D.; Schnur, R. [Univ. of Wisconsin, Madison, WI (United States)]|[Case Western Reserve Univ., Cleveland, OH (United States)]|[Children`s Hospital of Philadelphia, PA (United States)] [and others

1994-09-01

5

Distribution of Two Asian-Related Coding SNPs in the MC1R and OCA2 Genes  

Microsoft Academic Search

Very little is known about the genes and mechanisms affecting skin lightening in Asian populations. In this study, two coding\\u000a SNPs, c.G1129A (R163Q) at the MC1R (melanocortin 1 receptor) gene and c.A1962G (H615R) at the OCA2 (oculocutaneous albinism type II) gene, were investigated in a total of 1,809 individuals in 16 populations from various\\u000a areas. The Q163 and R615 alleles

I. Yuasa; K. Umetsu; S. Harihara; A. Kido; A. Miyoshi; N. Saitou; B. Dashnyam; F. Jin; G. Lucotte; P. K. Chattopadhyay; L. Henke; J. Henke

2007-01-01

6

Inheritance of a novel mutated allele of the OCA2 gene associated with high incidence of oculocutaneous albinism in a Polynesian community  

Microsoft Academic Search

Oculocutaneous albinism type 2 (OCA2) is a human autosomal-recessive hypopigmentation disorder associated with pathological mutations of the OCA2 gene. In this study, we investigated a form of OCA in a Polynesian population with an observed phenotype characterized by fair skin, some brown nevi present in the sun-exposed areas and green or blue eyes. Hair presented with a unique red coloration

Helene C Johanson; Wei Chen; Carol Wicking; Richard A Sturm

2010-01-01

7

Inheritance of a novel mutated allele of the OCA2 gene associated with high incidence of oculocutaneous albinism in a Polynesian community.  

PubMed

Oculocutaneous albinism type 2 (OCA2) is a human autosomal-recessive hypopigmentation disorder associated with pathological mutations of the OCA2 gene. In this study, we investigated a form of OCA in a Polynesian population with an observed phenotype characterized by fair skin, some brown nevi present in the sun-exposed areas and green or blue eyes. Hair presented with a unique red coloration since birth, with tones ranging across individuals from Yellow-Red to Brown-Red, or Auburn. We genetically screened for mutations in the OCA2 and MC1R genes as their products have previously been shown to be associated with red hair/fair skin and OCA2. The SLC45A2 gene was also screened to identify any possible relation to skin color variation. We have identified a novel missense substitution in the OCA2 gene (Gly775Asp) responsible for OCA2 in individuals of Polynesian heritage from Tuvalu. The estimated incidence of this form of OCA2 in the primary study community is believed to occur at one of the highest recorded rates of albinism at approximately 1 per 669 individuals. In addition, we have analyzed four unrelated individuals with albinism who have Polynesian heritage from three other separate communities and found they carry the same OCA2 mutation. We also analyzed an out-group comprising three unrelated individuals with albinism of Melanesian ancestries from two separate communities, one Australian Aboriginal and three Australian Caucasians, and did not detect this mutation. We hypothesize that this mutation may be Polynesian specific and that it originated from a common founder. PMID:20019752

Johanson, Helene C; Chen, Wei; Wicking, Carol; Sturm, Richard A

2010-02-01

8

A Potential Benefit of Albinism in Astyanax Cavefish: Downregulation of the oca2 Gene Increases Tyrosine and Catecholamine Levels as an Alternative to Melanin Synthesis  

PubMed Central

Albinism, the loss of melanin pigmentation, has evolved in a diverse variety of cave animals but the responsible evolutionary mechanisms are unknown. In Astyanax mexicanus, which has a pigmented surface dwelling form (surface fish) and several albino cave-dwelling forms (cavefish), albinism is caused by loss of function mutations in the oca2 gene, which operates during the first step of the melanin synthesis pathway. In addition to albinism, cavefish have evolved differences in behavior, including feeding and sleep, which are under the control of the catecholamine system. The catecholamine and melanin synthesis pathways diverge after beginning with the same substrate, L-tyrosine. Here we describe a novel relationship between the catecholamine and melanin synthesis pathways in Astyanax. Our results show significant increases in L-tyrosine, dopamine, and norepinephrine in pre-feeding larvae and adult brains of Pachón cavefish relative to surface fish. In addition, norepinephrine is elevated in cavefish adult kidneys, which contain the teleost homologs of catecholamine synthesizing adrenal cells. We further show that the oca2 gene is expressed during surface fish development but is downregulated in cavefish embryos. A key finding is that knockdown of oca2 expression in surface fish embryos delays the development of pigmented melanophores and simultaneously increases L-tyrosine and dopamine. We conclude that a potential evolutionary benefit of albinism in Astyanax cavefish may be to provide surplus L-tyrosine as a precursor for the elevated catecholamine synthesis pathway, which could be important for adaptation to the challenging cave environment.

Parkhurst, Amy; Jeffery, William R.

2013-01-01

9

Distribution of OCA2?481Thr and OCA2?615Arg, associated with hypopigmentation, in several additional populations.  

PubMed

Two mutants, OCA2?481Thr (c.1441G>A, p.Ala481Thr) and OCA2?615Arg (c.1844A>G, p.His615Arg), in the OCA2 (oculocutaneous albinism type II) gene are associated with hypopigmentation in East Asians. Here, these two alleles were studied to assess the frequencies in five different populations. In addition, the allele frequency of OCA2?615Arg was investigated in seven populations. Among a total of 24 global populations investigated, Oroqens in Heihe showed the highest frequency for OCA2?481Thr (0.519), and among 26 populations, Han Chinese in Changsha showed the highest frequency for OCA2?615Arg (0.673). This study confirmed that these two East Asian-specific alleles are characteristic of northern and central-southern East Asian populations. PMID:21565543

Yuasa, Isao; Harihara, Shinji; Jin, Feng; Nishimukai, Hiroaki; Fujihara, Junko; Fukumori, Yasuo; Takeshita, Haruo; Umetsu, Kazuo; Saitou, Naruya

2011-07-01

10

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

Microsoft Academic Search

Oculocutaneous albinism type 2 is caused by defects in the gene OCA2, encoding a pigment cell-specific, 12-transmem- brane 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

Anand Sitaram; Rosanna Piccirillo; Ilaria Palmisano; Dawn C. Harper; Esteban C. Dell' Angelica; M. V. Schiaffino; Michael S. Marks

2009-01-01

11

Multilocus OCA2 genotypes specify human iris colors.  

PubMed

Human iris color is a quantitative, multifactorial phenotype that exhibits quasi-Mendelian inheritance. Recent studies have shown that OCA2 polymorphism underlies most of the natural variability in human iris pigmentation but to date, only a few associated polymorphisms in this gene have been described. Herein, we describe an iris color score (C) for quantifying iris melanin content in-silico and undertake a more detailed survey of the OCA2 locus (n = 271 SNPs). In 1,317 subjects, we confirmed six previously described associations and identified another 27 strongly associated with C that were not explained by continental population stratification (OR 1.5-17.9, P = 0.03 to <0.001). Haplotype analysis with respect to these 33 SNPs revealed six haplotype blocks and 11 hap-tags within these blocks. To identify genetic features for best-predicting iris color, we selected sets of SNPs by parsing P values among possible combinations and identified four discontinuous and non-overlapping sets across the LD blocks (p-Selected SNP sets). In a second, partially overlapping sample of 1,072, samples with matching diplotypes comprised of these p-Selected OCA2 SNPs exhibited a rate of C concordance of 96.3% (n = 82), which was significantly greater than that obtained from randomly selected samples (62.6%, n = 246, P<0.0001). In contrast, the rate of C concordance using diplotypes comprised of the 11 identified hap-tags was only 83.7%, and that obtained using diplotypes comprised of all 33 SNPs organized as contiguous sets along the locus (defined by the LD block structure) was only 93.3%. These results confirm that OCA2 is the major human iris color gene and suggest that using an empirical database-driven system, genotypes from a modest number of SNPs within this gene can be used to accurately predict iris melanin content from DNA. PMID:17619204

Frudakis, Tony; Terravainen, Timothy; Thomas, Matthew

2007-11-01

12

Interactive effects of MC1R and OCA2 on melanoma risk phenotypes.  

PubMed

The relationships between MC1R gene variants and red hair, skin reflectance, degree of freckling and nevus count were investigated in 2331 adolescent twins, their sibs and parents in 645 twin families. Penetrance of each MC1R variant allele was consistent with an allelic model where effects were multiplicative for red hair but additive for skin reflectance. Of nine MC1R variant alleles assayed, four common alleles were strongly associated with red hair and fair skin (Asp84Glu, Arg151Cys, Arg160Trp and Asp294His), with a further three alleles having low penetrance (Val60Leu, Val92Met and Arg163Gln). These variants were separately combined for the purposes of this analysis and designated as strong 'R' (OR=63.3; 95% CI 31.9-139.6) and weak 'r ' (OR=5.1; 95% CI 2.5-11.3) red hair alleles. Red-haired individuals are predominantly seen in the R/R and R/r groups with 67.1 and 10.8%, respectively. To assess the interaction of the brown eye color gene OCA2 on the phenotypic effects of variant MC1R alleles we included eye color as a covariate, and also genotyped two OCA2 SNPs (Arg305Trp and Arg419Gln), which were confirmed as modifying eye color. MC1R genotype effects on constitutive skin color, freckling and mole count were modified by eye color, but not genotype for these two OCA2 SNPs. This is probably due to the association of these OCA2 SNPs with brown/green not blue eye color. Amongst individuals with a R/R genotype (but not R/r), those who also had brown eyes had a mole count twice that of those with blue eyes. This suggests that other OCA2 polymorphisms influence mole count and remain to be described. PMID:14709592

Duffy, David L; Box, Neil F; Chen, Wei; Palmer, James S; Montgomery, Grant W; James, Michael R; Hayward, Nicholas K; Martin, Nicholas G; Sturm, Richard A

2004-02-15

13

Interactions between HERC2, OCA2 and MC1R may influence human pigmentation phenotype.  

PubMed

Human pigmentation is a polygenic trait which may be shaped by different kinds of gene-gene interactions. Recent studies have revealed that interactive effects between HERC2 and OCA2 may be responsible for blue eye colour determination in humans. Here we performed a population association study, examining important polymorphisms within the HERC2 and OCA2 genes. Furthermore, pooling these results with genotyping data for MC1R, ASIP and SLC45A2 obtained for the same population sample we also analysed potential genetic interactions affecting variation in eye, hair and skin colour. Our results confirmed the association of HERC2 rs12913832 with eye colour and showed that this SNP is also significantly associated with skin and hair colouration. It is also concluded that OCA2 rs1800407 is independently associated with eye colour. Finally, using various approaches we were able to show that there is an interaction between MC1R and HERC2 in determination of skin and hair colour in the studied population sample. PMID:19208107

Branicki, Wojciech; Brudnik, Urszula; Wojas-Pelc, Anna

2009-03-01

14

Analysis of Cultured Human Melanocytes Based on Polymorphisms within the SLC45A2\\/MATP, SLC24A5\\/NCKX5, and OCA2\\/P Loci  

Microsoft Academic Search

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

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

2009-01-01

15

A global view of the OCA2-HERC2 region and pigmentation.  

PubMed

Mutations in the gene OCA2 are responsible for oculocutaneous albinism type 2, but polymorphisms in and around OCA2 have also been associated with normal pigment variation. In Europeans, three haplotypes in the region have been shown to be associated with eye pigmentation and a missense SNP (rs1800407) has been associated with green/hazel eyes (Branicki et al. in Ann Hum Genet 73:160-170, 2009). In addition, a missense mutation (rs1800414) is a candidate for light skin pigmentation in East Asia (Yuasa et al. in Biochem Genet 45:535-542, 2007; Anno et al. in Int J Biol Sci 4, 2008). We have genotyped 3,432 individuals from 72 populations for 21 SNPs in the OCA2-HERC2 region including those previously associated with eye or skin pigmentation. We report that the blue-eye associated alleles at all three haplotypes were found at high frequencies in Europe; however, one is restricted to Europe and surrounding regions, while the other two are found at moderate to high frequencies throughout the world. We also observed that the derived allele of rs1800414 is essentially limited to East Asia where it is found at high frequencies. Long-range haplotype tests provide evidence of selection for the blue-eye allele at the three haplotyped systems but not for the green/hazel eye SNP allele. We also saw evidence of selection at the derived allele of rs1800414 in East Asia. Our data suggest that the haplotype restricted to Europe is the strongest marker for blue eyes globally and add further inferential evidence that the derived allele of rs1800414 is an East Asian skin pigmentation allele. PMID:22065085

Donnelly, Michael P; Paschou, Peristera; Grigorenko, Elena; Gurwitz, David; Barta, Csaba; Lu, Ru-Band; Zhukova, Olga V; Kim, Jong-Jin; Siniscalco, Marcello; New, Maria; Li, Hui; Kajuna, Sylvester L B; Manolopoulos, Vangelis G; Speed, William C; Pakstis, Andrew J; Kidd, Judith R; Kidd, Kenneth K

2012-05-01

16

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

PubMed

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

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

2012-03-01

17

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

PubMed Central

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.

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

2012-01-01

18

Association Between a Germline OCA2 Polymorphism at Chromosome 15q13.1 and Estrogen Receptor-Negative Breast Cancer Survival  

PubMed Central

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 case–control 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 receptor–negative 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 receptor–negative breast cancer.

Tyrer, Jonathan; Fasching, Peter A.; Beckmann, Matthias W.; Ekici, Arif B.; Schulz-Wendtland, Rudiger; Bojesen, Stig E.; Nordestgaard, B?rge G.; Flyger, Henrik; Milne, Roger L.; Arias, Jose Ignacio; Menendez, Primitiva; Benitez, Javier; Chang-Claude, Jenny; Hein, Rebecca; Wang-Gohrke, Shan; Nevanlinna, Heli; Heikkinen, Tuomas; Aittomaki, 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; Garcia-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

19

Differential recognition of a dileucine-based sorting signal by AP-1 and AP-3 reveals a requirement for both BLOC-1 and AP-3 in delivery of OCA2 to melanosomes.  

PubMed

Cell types that generate unique lysosome-related organelles (LROs), such as melanosomes in melanocytes, populate nascent LROs with cargoes that are diverted from endosomes. Cargo sorting toward melanosomes correlates with binding via cytoplasmically exposed sorting signals to either heterotetrameric adaptor AP-1 or AP-3. Some cargoes bind both adaptors, but the relative contribution of each adaptor to cargo recognition and their functional interactions with other effectors during transport to melanosomes are not clear. Here we exploit targeted mutagenesis of the acidic dileucine-based sorting signal in the pigment cell-specific protein OCA2 to dissect the relative roles of AP-1 and AP-3 in transport to melanosomes. We show that binding to AP-1 or AP-3 depends on the primary sequence of the signal and not its position within the cytoplasmic domain. Mutants that preferentially bound either AP-1 or AP-3 each trafficked toward melanosomes and functionally complemented OCA2 deficiency, but AP-3 binding was necessary for steady-state melanosome localization. Unlike tyrosinase, which also engages AP-3 for optimal melanosomal delivery, both AP-1- and AP-3-favoring OCA2 variants required BLOC-1 for melanosomal transport. These data provide evidence for distinct roles of AP-1 and AP-3 in OCA2 transport to melanosomes and indicate that BLOC-1 can cooperate with either adaptor during cargo sorting to LROs. PMID:22718909

Sitaram, Anand; Dennis, Megan K; Chaudhuri, Rittik; De Jesus-Rojas, Wilfredo; Tenza, Danièle; Setty, Subba Rao Gangi; Wood, Christopher S; Sviderskaya, Elena V; Bennett, Dorothy C; Raposo, Graça; Bonifacino, Juan S; Marks, Michael S

2012-08-01

20

Linkage and Association Analysis of Spectrophotometrically Quantified Hair Color in Australian Adolescents: the Effect of OCA2 and HERC2  

Microsoft Academic Search

Genetic studies of pigmentation have benefited from spectrophotometric measures of light–dark hair color. Here we use one of those measures, absorbance at 650 nm, to look for chromosomal regions that harbor genes affecting hair pigmentation. At 7p15.1, marker D7S1808 was suggestive of linkage to light–dark hair color (LOD?2.99). Marker D1S235 at 1q42.3 was suggestive of linkage to hair color (light–dark

Sri N. Shekar; David L. Duffy; Tony Frudakis; Richard A. Sturm; Zhen Z. Zhao; Grant W. Montgomery; Nicholas G. Martin

2008-01-01

21

A single SNP in an evolutionary conserved region within intron 86 of the HERC2 gene determines human blue-brown eye color.  

PubMed

We have previously demonstrated that haplotypes of three single nucleotide polymorphisms (SNPs) within the first intron of the OCA2 gene are extremely strongly associated with variation in human eye color. In the present work, we describe additional fine association mapping of eye color SNPs in the intergenic region upstream of OCA2 and within the neighboring HERC2 (hect domain and RLD2) gene. We screened an additional 92 SNPs in 300-3000 European individuals and found that a single SNP in intron 86 of HERC2, rs12913832, predicted eye color significantly better (ordinal logistic regression R(2) = 0.68, association LOD = 444) than our previous best OCA2 haplotype. Comparison of sequence alignments of multiple species showed that this SNP lies in the center of a short highly conserved sequence and that the blue-eye-associated allele (frequency 78%) breaks up this conserved sequence, part of which forms a consensus binding site for the helicase-like transcription factor (HLTF). We were also able to demonstrate the OCA2 R419Q, rs1800407, coding SNP acts as a penetrance modifier of this new HERC2 SNP for eye color, and somewhat independently, of melanoma risk. We conclude that the conserved region around rs12913832 represents a regulatory region controlling constitutive expression of OCA2 and that the C allele at rs12913832 leads to decreased expression of OCA2, particularly within iris melanocytes, which we postulate to be the ultimate cause of blue eye color. PMID:18252222

Sturm, Richard A; Duffy, David L; Zhao, Zhen Zhen; Leite, Fabio P N; Stark, Mitchell S; Hayward, Nicholas K; Martin, Nicholas G; Montgomery, Grant W

2008-02-01

22

A Single SNP in an Evolutionary Conserved Region within Intron 86 of the HERC2 Gene Determines Human Blue-Brown Eye Color  

PubMed Central

We have previously demonstrated that haplotypes of three single nucleotide polymorphisms (SNPs) within the first intron of the OCA2 gene are extremely strongly associated with variation in human eye color. In the present work, we describe additional fine association mapping of eye color SNPs in the intergenic region upstream of OCA2 and within the neighboring HERC2 (hect domain and RLD2) gene. We screened an additional 92 SNPs in 300–3000 European individuals and found that a single SNP in intron 86 of HERC2, rs12913832, predicted eye color significantly better (ordinal logistic regression R2 = 0.68, association LOD = 444) than our previous best OCA2 haplotype. Comparison of sequence alignments of multiple species showed that this SNP lies in the center of a short highly conserved sequence and that the blue-eye-associated allele (frequency 78%) breaks up this conserved sequence, part of which forms a consensus binding site for the helicase-like transcription factor (HLTF). We were also able to demonstrate the OCA2 R419Q, rs1800407, coding SNP acts as a penetrance modifier of this new HERC2 SNP for eye color, and somewhat independently, of melanoma risk. We conclude that the conserved region around rs12913832 represents a regulatory region controlling constitutive expression of OCA2 and that the C allele at rs12913832 leads to decreased expression of OCA2, particularly within iris melanocytes, which we postulate to be the ultimate cause of blue eye color.

Sturm, Richard A.; Duffy, David L.; Zhao, Zhen Zhen; Leite, Fabio P.N.; Stark, Mitchell S.; Hayward, Nicholas K.; Martin, Nicholas G.; Montgomery, Grant W.

2008-01-01

23

Inhibition of polyadenylation reduces inflammatory gene induction  

PubMed Central

Cordycepin (3? deoxyadenosine) has long been used in the study of in vitro assembled polyadenylation complexes, because it terminates the poly(A) tail and arrests the cleavage complex. It is derived from caterpillar fungi, which are highly prized in Chinese traditional medicine. Here we show that cordycepin specifically inhibits the induction of inflammatory mRNAs by cytokines in human airway smooth muscle cells without affecting the expression of control mRNAs. Cordycepin treatment results in shorter poly(A) tails, and a reduction in the efficiency of mRNA cleavage and transcription termination is observed, indicating that the effects of cordycepin on 3? processing in cells are similar to those described in in vitro reactions. For the CCL2 and CXCL1 mRNAs, the effects of cordycepin are post-transcriptional, with the mRNA disappearing during or immediately after nuclear export. In contrast, although the recruitment of RNA polymerase II to the IL8 promoter is also unaffected, the levels of nascent transcript are reduced, indicating a defect in transcription elongation. We show that a reporter construct with 3? sequences from a histone gene is unaffected by cordycepin, while CXCL1 sequences confer cordycepin sensitivity to the reporter, demonstrating that polyadenylation is indeed required for the effect of cordycepin on gene expression. In addition, treatment with another polyadenyation inhibitor and knockdown of poly(A) polymerase ? also specifically reduced the induction of inflammatory mRNAs. These data demonstrate that there are differences in the 3? processing of inflammatory and housekeeping genes and identify polyadenylation as a novel target for anti-inflammatory drugs.

Kondrashov, Alexander; Meijer, Hedda A.; Barthet-Barateig, Adeline; Parker, Hannah N.; Khurshid, Asma; Tessier, Sarah; Sicard, Marie; Knox, Alan J.; Pang, Linhua; de Moor, Cornelia H.

2012-01-01

24

Expression of a truncated tomato polygalacturonase gene inhibits expression of the endogenous gene in transgenic plants  

Microsoft Academic Search

Tomato plants were transformed with a chimaeric polygalacturonase (PG) gene, designed to produce a truncated PG transcript constitutively. In these plants expression of the endogenous PG gene was inhibited during ripening, resulting in a substantial reduction in PG mRNA and enzyme accumulation. This inhibition was comparable to that achieved previously using antisense genes. The expression of the truncated gene in

C. J. S. Smith; C. F. Watson; C. R. Bird; J. Ray; W. Schuch; D. Grierson

1990-01-01

25

Increased in vivo inhibition of gene expression by combining RNA interference and U1 inhibition  

PubMed Central

Inhibition of gene expression can be achieved with RNA interference (RNAi) or U1 small nuclear RNA—snRNA—interference (U1i). U1i is based on U1 inhibitors (U1in), U1 snRNA molecules modified to inhibit polyadenylation of a target pre-mRNA. In culture, we have shown that the combination of RNAi and U1i results in stronger inhibition of reporter or endogenous genes than that obtained using either of the techniques alone. We have now used these techniques to inhibit gene expression in mice. We show that U1ins can induce strong inhibition of the expression of target genes in vivo. Furthermore, combining U1i and RNAi results in synergistic inhibitions also in mice. This is shown for the inhibition of hepatitis B virus (HBV) sequences or endogenous Notch1. Surprisingly, inhibition obtained by combining a U1in and a RNAi mediator is higher than that obtained by combining two U1ins or two RNAi mediators. Our results suggest that RNAi and U1i cooperate by unknown mechanisms to result in synergistic inhibitions. Analysis of toxicity and specificity indicates that expression of U1i inhibitors is safe. Therefore, we believe that the combination of RNAi and U1i will be a good option to block damaging endogenous genes, HBV and other infectious agents in vivo.

Blazquez, Lorea; Gonzalez-Rojas, Sandra Jovanna; Abad, Amaya; Razquin, Nerea; Abad, Xabier; Fortes, Puri

2012-01-01

26

Antisense RNA inhibition of polygalacturonase gene expression in transgenic tomatoes  

Microsoft Academic Search

Regulation of expression of specific genes by antisense RNA is a naturally occurring mechanism in bacteria1,2, although gene regulation by this mechanism has not yet been observed in higher eukaryotes. However, antisense RNA has been shown to reduce expression of specific genes when injected into frog oocytes3 and Drosophila embryos4. Inhibition of expression of artificially introduced genes has been demonstrated

C. J. S. Smith; C. F. Watson; J. Ray; C. R. Bird; P. C. Morris; W. Schuch; D. Grierson

1988-01-01

27

Three Genome-wide Association Studies and a Linkage Analysis Identify HERC2 as a Human Iris Color Gene  

PubMed Central

Human iris color was one of the first traits for which Mendelian segregation was established. To date, the genetics of iris color is still not fully understood and is of interest, particularly in view of forensic applications. In three independent genome-wide association (GWA) studies of a total of 1406 persons and a genome-wide linkage study of 1292 relatives, all from the Netherlands, we found that the 15q13.1 region is the predominant region involved in human iris color. There were no other regions showing consistent genome-wide evidence for association and linkage to iris color. Single nucleotide polymorphisms (SNPs) in the HERC2 gene and, to a lesser extent, in the neighboring OCA2 gene were independently associated to iris color variation. OCA2 has been implicated in iris color previously. A replication study within two populations confirmed that the HERC2 gene is a new and significant determinant of human iris color variation, in addition to OCA2. Furthermore, HERC2 rs916977 showed a clinal allele distribution across 23 European populations, which was significantly correlated to iris color variation. We suggest that genetic variants regulating expression of the OCA2 gene exist in the HERC2 gene or, alternatively, within the 11.7 kb of sequence between OCA2 and HERC2, and that most iris color variation in Europeans is explained by those two genes. Testing markers in the HERC2-OCA2 region may be useful in forensic applications to predict eye color phenotypes of unknown persons of European genetic origin.

Kayser, Manfred; Liu, Fan; Janssens, A. Cecile J.W.; Rivadeneira, Fernando; Lao, Oscar; van Duijn, Kate; Vermeulen, Mark; Arp, Pascal; Jhamai, Mila M.; van IJcken, Wilfred F.J.; den Dunnen, Johan T.; Heath, Simon; Zelenika, Diana; Despriet, Dominiek D.G.; Klaver, Caroline C.W.; Vingerling, Johannes R.; de Jong, Paulus T.V.M.; Hofman, Albert; Aulchenko, Yurii S.; Uitterlinden, Andre G.; Oostra, Ben A.; van Duijn, Cornelia M.

2008-01-01

28

Three genome-wide association studies and a linkage analysis identify HERC2 as a human iris color gene.  

PubMed

Human iris color was one of the first traits for which Mendelian segregation was established. To date, the genetics of iris color is still not fully understood and is of interest, particularly in view of forensic applications. In three independent genome-wide association (GWA) studies of a total of 1406 persons and a genome-wide linkage study of 1292 relatives, all from the Netherlands, we found that the 15q13.1 region is the predominant region involved in human iris color. There were no other regions showing consistent genome-wide evidence for association and linkage to iris color. Single nucleotide polymorphisms (SNPs) in the HERC2 gene and, to a lesser extent, in the neighboring OCA2 gene were independently associated to iris color variation. OCA2 has been implicated in iris color previously. A replication study within two populations confirmed that the HERC2 gene is a new and significant determinant of human iris color variation, in addition to OCA2. Furthermore, HERC2 rs916977 showed a clinal allele distribution across 23 European populations, which was significantly correlated to iris color variation. We suggest that genetic variants regulating expression of the OCA2 gene exist in the HERC2 gene or, alternatively, within the 11.7 kb of sequence between OCA2 and HERC2, and that most iris color variation in Europeans is explained by those two genes. Testing markers in the HERC2-OCA2 region may be useful in forensic applications to predict eye color phenotypes of unknown persons of European genetic origin. PMID:18252221

Kayser, Manfred; Liu, Fan; Janssens, A Cecile J W; Rivadeneira, Fernando; Lao, Oscar; van Duijn, Kate; Vermeulen, Mark; Arp, Pascal; Jhamai, Mila M; van Ijcken, Wilfred F J; den Dunnen, Johan T; Heath, Simon; Zelenika, Diana; Despriet, Dominiek D G; Klaver, Caroline C W; Vingerling, Johannes R; de Jong, Paulus T V M; Hofman, Albert; Aulchenko, Yurii S; Uitterlinden, Andre G; Oostra, Ben A; van Duijn, Cornelia M

2008-02-01

29

Antisense Oligodeoxynucleotide Inhibition of HIV Gene Expression.  

National Technical Information Service (NTIS)

The goal of this work is to develop novel, efficacious, injectable, gene-specific therapeutics for treatment of human immunodeficiency virus (HIV) infection. These products will be nuclease resistant, stereospecific antisense inhibitors of human immunodef...

E. Wickstrom

1989-01-01

30

CDK9 inhibition strategy defines distinct sets of target genes  

PubMed Central

Background CDK9 is the catalytic subunit of the Positive Transcription Elongation Factor b (P-TEFb), which phosphorylates the CTD of RNAPII and negative elongation factors enabling for productive elongation after initiation. CDK9 associates with T-type cyclins and cyclin K and its activity is tightly regulated in cells at different levels. CDK9 is also the catalytic subunit of TAK (Tat activating Kinase), essential for HIV1 replication. Because of CDK9?s potential as a therapeutic target in AIDS, cancer, inflammation, and cardiomyophathy it is important to understand the consequences of CDK9 inhibition. A previous gene expression profiling study performed with human glioblastoma T98G cells in which CDK9 activity was inhibited either with a dominant negative mutant form of CDK9 (dnCDK9) or the pharmacological inhibitor Flavopiridol unveiled striking differences in gene expression effects. In the present report we extended these studies by (1) using both immortalized normal human fibroblasts and primary human astrocytes, (2) eliminating potential experimental variability due to transduction methodology and (3) also modulating CDK9 activity with siRNA. Findings Striking differences in the effects on gene expression resulting from the strategy used to inhibit CDK9 activity (dnCDK9 or FVP) remain even when potential variability due to viral transduction is eliminated. siRNA mediated CDK9 knockdown in human fibroblasts and astrocytes efficiently reduced CDK9 expression and led to potent changes in gene expression that exhibit little correlation with the effects of dnCDK9 or FVP. Interestingly, HEXIM1 a validated CDK9 target gene, was found to be potently downregulated by dnCDK9, FVP and siCDK9, but the cluster of genes with expression profiles similar to HEXIM1 was small. Finally, cluster analysis of all treatments revealed higher correlation between treatments than cell type origin. Conclusion The nature of the strategy used to inhibit CDK9 profoundly affects the patterns of gene expression resulting from CDK9 inhibition. These results suggest multiple variables that affect outcome, including kinetics of inhibition, potency, off-target effects, and selectivity issues. This is particularly important when considering CDK9 as a potential target for therapeutic intervention.

2014-01-01

31

Inhibition of cytomegalovirus immediate early gene expression: a therapeutic option?  

PubMed

The replication cycle of the human cytomegalovirus (HCMV) is characterized by the expression of immediate early (IE), early (E), and late (L) gene regions. Current antiviral strategies are directed against the viral DNA polymerase expressed during the early phase of infection. The regulation of the IE-1 and IE-2 gene expression is the key to latency and active replication due to their transactivating and repressing functions. There is growing evidence that the pathogenic features of HCMV are largely due to the abilities of IE-1 and IE-2 to transactivate cellular genes. Consequently, current drugs used to inhibit HCMV infection would have no impact on IE-1 and IE-2-induced effects that are produced before the early phase. Moreover, when HCMV DNA replication is inhibited, IE gene products accumulate in infected cells causing disturbances of host cell functions. This review summarizes the biological functions of HCMV-IE gene expression, their relevance in pathogenesis, as well as efforts to develop novel treatment strategies directed against HCMV-IE expression. PMID:11428240

Scholz, M; Doerr, H W; Cinatl, J

2001-03-01

32

Prostaglandins inhibit lipoprotein lipase gene expression in macrophages.  

PubMed Central

In the present investigation of the effects of prostaglandin E2 (PGE2) on lipoprotein lipase (LPL) gene expression in macrophages, we observed that treatment of macrophages with PGE2 increased the levels of adenosine 3',5'-cyclic monophosphate (cAMP), while the addition of exogenous 5-bromo-cAMP to macrophage cultures resulted in down-regulation of LPL expression. Using indomethacin (INDO), an inhibitor of cyclo-oxygenase and prostaglandins production, we determined that PGE2 acts as a feedback inhibitor of LPL expression. We found that inhibited secretion of LPL protein in lipopolysaccharide (LPS)-treated macrophages could be restored to control levels by the addition of INDO to the medium. In contrast, INDO did not reverse the inhibition of LPL mRNA induced by LPS. Overall, our results have demonstrated that PGE2 is a potent inhibitor of LPL gene expression and indicated that its action may play an important physiological role in the regulation of LPL gene expression during bacterial infections. Images Figure 1 Figure 4 Figure 7

Desanctis, J B; Varesio, L; Radzioch, D

1994-01-01

33

Inhibition of host cell encapsulation through inhibiting immune gene expression by the parasitic wasp venom calreticulin.  

PubMed

Parasitoid wasps inject venom into the host to protect their offspring against host immune responses. In our previous study, we identified a calreticulin (CRT) in Pteromalus puparum venom. In this study, we expressed the wild-type and the coiled-coil domain deletion mutant P. puparum calreticulins (PpCRTs) in Escherichia coli and prepared polyclonal antibody in rabbit against PpCRT. Western blot analysis showed that PpCRT protein was not only present in the venom but also in all the tissues tested. Real time PCR results indicated that PpCRT mRNA was highly expressed in the venom gland. The transcript level of PpCRT in the venom gland was peaked at 2 days post-eclosion, while the PpCRT protein in the venom was maintained at a constant level. Both recombinant wild-type and mutant PpCRT proteins could bind to the surface of P. puparum eggs. Recombinant PpCRT inhibited hemocyte spreading and cellular encapsulation of the host Pieris rapae in vitro, and the coiled-coil domain is important for the inhibitory function of PpCRT. Immunocytochemistry results showed that PpCRT entered P. rapae hemocytes, and the coiled-coil domain played a role in this process. After injection of recombinant PpCRT into P. rapae pupae, real time PCR results showed that PpCRT inhibited transcript levels of host encapsulation-related genes, including calreticulin and scavenger receptor genes. In conclusion, our results suggest that P. puparum venom protects its offspring against host cellular immune responses via its functional component PpCRT to inhibit the expression of host cellular response-related genes. PMID:23933213

Wang, Lei; Fang, Qi; Qian, Cen; Wang, Fei; Yu, Xiao-Qiang; Ye, Gongyin

2013-10-01

34

FAK and HAS Inhibition Synergistically Decrease Colon Cancer Cell Viability and Affect Expression of Critical Genes  

PubMed Central

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?M 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, heat-shock 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.

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

2013-01-01

35

A strategy for disease gene identification through nonsense-mediated mRNA decay inhibition  

Microsoft Academic Search

Premature termination codons (PTCs) have been shown to initiate degradation of mutant transcripts through the nonsense-mediated messenger RNA (mRNA) decay (NMD) pathway. We report a strategy, termed gene identification by NMD inhibition (GINI), to identify genes harboring nonsense codons that underlie human diseases. In this strategy, the NMD pathway is pharmacologically inhibited in cultured patient cells, resulting in stabilization of

Erick N. Noensie; Harry C. Dietz

2001-01-01

36

The Homeobox Gene Gax Inhibits Angiogenesis through Inhibition of Nuclear Factor-k kB-Dependent Endothelial Cell Gene Expression  

Microsoft Academic Search

The growth and metastasis of tumors are heavily dependent on angiogenesis, but much of the transcriptional regulation of vascular endothelial cell gene expression responsible for angiogenesis remains to be elucidated. The homeobox gene Gax is expressed in vascular endothelial cells and inhibits proliferation and tube formation in vitro. We hypothesized that Gax is a negative transcriptional regulator of the endothelial

Sejal Patel; Alejandro D. Leal; David H. Gorski

2005-01-01

37

Cyclo(valine-valine) inhibits Vibrio cholerae virulence gene expression.  

PubMed

Vibrio cholerae has been shown to produce a cyclic dipeptide, cyclo(phenylalanine-proline) (cFP), that functions to repress virulence factor production. The objective of this study was to determine if heterologous cyclic dipeptides could repress V. cholerae virulence factor production. To that end, three synthetic cyclic dipeptides that differed in their side chains from cFP were assayed for virulence inhibitory activity in V. cholerae. The results revealed that cyclo(valine-valine) (cVV) inhibited virulence factor production by a ToxR-dependent process that resulted in the repression of the virulence regulator aphA. cVV-dependent repression of aphA was found to be independent of known aphA regulatory genes. The results demonstrated that V. cholerae was able to respond to exogenous cyclic dipeptides and implicated the hydrophobic amino acid side chains on both arms of the cyclo dipeptide scaffold as structural requirements for inhibitory activity. The results further suggest that cyclic dipeptides have potential as therapeutics for cholera treatment. PMID:24644247

Vikram, Amit; Ante, Vanessa M; Bina, X Renee; Zhu, Qin; Liu, Xinyu; Bina, James E

2014-06-01

38

Gene Transfer Using Micellar Nanovectors Inhibits Choroidal Neovascularization In Vivo  

PubMed Central

Purpose Age-related macular degeneration caused by choroidal neovascularization (CNV) remains difficult to be treated despite the recent advent of several treatment options. In this study, we investigated the in vivo angiogenic control by intravenous injection of polyion complex (PIC) micelle encapsulating plasmid DNA (pDNA) using a mice CNV model. Methods The transfection efficiency of the PIC micelle was investigated using the laser-induced CNV in eight-week-old male C57 BJ/6 mice. Firstly, each mouse received intravenous injection of micelle encapsulating pDNA of Yellow Fluorescent Protein (pYFP) on days 1,3 and 5. The expression of YFP was analyzed using fluorescein microscopy and western blotting analysis. In the next experiments, each mouse received intravenous injection of micelle encapsulating pDNA of soluble Fms-like tyrosine kinase-1 (psFlt-1) 1,3 and 5 days after the induction of CNV and the CNV lesion was analyzed by choroidal flatmounts on day 7. Results Fluorescein microscopy and western blotting analysis revealed that the expression of YFP was confirmed in the CNV area after injection of the PIC micelle, but the expression was not detected neither in mice that received naked pDNA nor those without CNV. Furthermore, the CNV area in the mice that received intravenous injection of the psFlt-1-encapsulated PIC micelle was significantly reduced by 65% compared to that in control mice (p<0.01). Conclusions Transfection of sFlt-1 with the PIC micelle by intravenous injection to mice CNV models showed significant inhibition of CNV. The current results revealed the significant potential of nonviral gene therapy for regulation of CNV using the PIC micelle encapsulating pDNA.

Iriyama, Aya; Oba, Makoto; Ishii, Takehiko; Nishiyama, Nobuhiro; Kataoka, Kazunori; Tamaki, Yasuhiro; Yanagi, Yasuo

2011-01-01

39

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

SciTech Connect

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.

Lee, S.T.; Fukai, K.; Spritz, R.A. [Univ. of Wisconsin School of Medicine, Madison, WI (United States)] [and others] [Univ. of Wisconsin School of Medicine, Madison, WI (United States); and others

1995-03-20

40

Multiple Pigmentation Gene Polymorphisms Account for a Substantial Proportion of Risk of Cutaneous Malignant Melanoma  

Microsoft Academic Search

We have previously described the role of red hair (melanocortin-1 receptor, MC1R) and blue eye (oculocutaneous albinism type II, OCA2) gene polymorphisms in modulating the 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, have identified numerous polymorphisms controlling human hair, eye, and skin color. In

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

2010-01-01

41

Composition and Methods for Inhibiting Expression of Hypoxia-Inducible Genes.  

National Technical Information Service (NTIS)

Methods are provided for interfering with a hypoxia-mediated transcriptional pathway using an agent that binds to a hypoxia response element and inhibits transcription of a hypoxia inducible gene associated therewith. Also provided are methods of treating...

B. Z. Olenyuk P. B. Dervan

2005-01-01

42

Adherens junction formation inhibits lentivirus entry and gene transfer.  

PubMed

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 ?-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

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

2013-01-01

43

Inhibition of ERK and p38 MAP Kinases Inhibits Binding of Nrf2 and Induction of GCS Genes  

Microsoft Academic Search

Genes encoding the catalytic (GCSh) and regulatory (GCSl) subunits of human ?-glutamylcysteine synthetase (?GCS), which catalyzes the rate limiting step in glutathione synthesis, are up-regulated in response to xenobiotics through Electrophile Response Elements (EpREs). Exposure of HepG2 cells to the GCS-inducing agent, Pyrrolidine dithiocarbamate (PDTC), results in ERK and p38 MAP kinase activation. Inhibition of ERK or p38 kinases by

Laurie M. Zipper; R. Timothy Mulcahy

2000-01-01

44

Lymphokine gene expression in vivo is inhibited by cyclosporin A  

PubMed Central

Murine T cells were stimulated in vivo by administering allogeneic cells or mitogens into the foot pads and then examining the draining popliteal lymph nodes. Allogeneic spleen cells induced the expression of IL2 and IFN-gamma mRNAs in a time- and dose-dependent manner. Induction of these transcripts also was detected after administration of Con A and anti-CD3 mAb. An increase in DNA-synthesizing cells was observed by 48 h, and these were shown to be T cells because of their sensitivity to anti-Thy-1 but not anti-B220 mAb and complement, and because of their localization to the T-dependent areas of the lymph node. The in vivo administration of cyclosporin A (CSA) reduced several of these T cell responses. The level of DNA synthesis and the frequency of cells synthesizing DNA were decreased by approximately 75%, while the induction of IL-2 responsiveness was not substantially diminished. IL-2 and IFN-gamma transcripts were inhibited at least 70-90%, as determined by Northern blot and in situ hybridization. Although the inhibition by CSA was not as complete in animals as observed previously in tissue culture, our findings indicate that in both systems, a major site of action of CSA is to inhibit T cell growth by inhibiting lymphokine production.

1990-01-01

45

Nonsteroidal anti-inflammatory drugs inhibit expression of the inducible nitric oxide synthase gene.  

PubMed

Increased nitric oxide production is associated with acute and chronic inflammatory processes. Accordingly, we tested the hypothesis that the therapeutic action of nonsteroidal anti-inflammatory drugs could be attributed at least in part to inhibition of excess nitric oxide production. We report here that sodium salicylate, aspirin, ibuprofen, and indomethacin markedly inhibited the appearance of the inducible inflammatory nitric oxide synthase in rat alveolar macrophages activated with lipopolysaccharide and interferon gamma. We attribute the mechanism of nitric oxide synthase inhibition by nonsteroidal anti-inflammatory drugs to pretranslational control of enzyme expression and not to direct inhibition of enzymatic activity. These observations indicate that the chronic anti-inflammatory action of nonsteroidal anti-inflammatory drugs may be due not only to inhibition of prostaglandin synthesis but also to inhibition of inducible nitric oxide synthase gene expression and nitric oxide synthesis. PMID:7535524

Aeberhard, E E; Henderson, S A; Arabolos, N S; Griscavage, J M; Castro, F E; Barrett, C T; Ignarro, L J

1995-03-28

46

Opposite effects of gene deficiency and pharmacological inhibition of soluble epoxide hydrolase on cardiac fibrosis.  

PubMed

Arachidonic acid-derived epoxyeicosatrienoic acids (EETs) are important regulators of cardiac remodeling; manipulation of their levels is a potentially useful pharmacological strategy. EETs are hydrolyzed by soluble epoxide hydrolase (sEH) to form the corresponding diols, thus altering and reducing the activity of these oxylipins. To better understand the phenotypic impact of sEH disruption, we compared the effect of EPHX2 gene knockout (EPHX2-/-) and sEH inhibition in mouse models. Measurement of plasma oxylipin profiles confirmed that the ratio of EETs/DHETs was increased in EPHX2-/- and sEH-inhibited mice. However, plasma concentrations of 9, 11, 15, 19-HETE were elevated in EPHX2-/- but not sEH-inhibited mice. Next, we investigated the role of this difference in cardiac dysfunction induced by Angiotensin II (AngII). Both EPHX2 gene deletion and inhibition protected against AngII-induced cardiac hypertrophy. Interestingly, cardiac dysfunction was attenuated by sEH inhibition rather than gene deletion. Histochemical staining revealed that compared with pharmacological inhibition, EPHX2 deletion aggravated AngII-induced myocardial fibrosis; the mRNA levels of fibrotic-related genes were increased. Furthermore, cardiac inflammatory response was greater in EPHX2-/- than sEH-inhibited mice with AngII treatment, as evidenced by increased macrophage infiltration and expression of MCP-1 and IL-6. In vitro, AngII-upregulated MCP-1 and IL-6 expression was significantly attenuated by sEH inhibition but promoted by EPHX2 deletion in cardiofibroblasts. Thus, compared with pharmacological inhibition of sEH, EPHX2 deletion caused the shift in arachidonic acid metabolism, which may led to pathological cardiac remodeling, especially cardiac fibrosis. PMID:24718617

Li, Lijuan; Li, Nan; Pang, Wei; Zhang, Xu; Hammock, Bruce D; Ai, Ding; Zhu, Yi

2014-01-01

47

Opposite Effects of Gene Deficiency and Pharmacological Inhibition of Soluble Epoxide Hydrolase on Cardiac Fibrosis  

PubMed Central

Arachidonic acid-derived epoxyeicosatrienoic acids (EETs) are important regulators of cardiac remodeling; manipulation of their levels is a potentially useful pharmacological strategy. EETs are hydrolyzed by soluble epoxide hydrolase (sEH) to form the corresponding diols, thus altering and reducing the activity of these oxylipins. To better understand the phenotypic impact of sEH disruption, we compared the effect of EPHX2 gene knockout (EPHX2?/?) and sEH inhibition in mouse models. Measurement of plasma oxylipin profiles confirmed that the ratio of EETs/DHETs was increased in EPHX2?/? and sEH-inhibited mice. However, plasma concentrations of 9, 11, 15, 19-HETE were elevated in EPHX2?/? but not sEH-inhibited mice. Next, we investigated the role of this difference in cardiac dysfunction induced by Angiotensin II (AngII). Both EPHX2 gene deletion and inhibition protected against AngII-induced cardiac hypertrophy. Interestingly, cardiac dysfunction was attenuated by sEH inhibition rather than gene deletion. Histochemical staining revealed that compared with pharmacological inhibition, EPHX2 deletion aggravated AngII-induced myocardial fibrosis; the mRNA levels of fibrotic-related genes were increased. Furthermore, cardiac inflammatory response was greater in EPHX2?/? than sEH-inhibited mice with AngII treatment, as evidenced by increased macrophage infiltration and expression of MCP-1 and IL-6. In vitro, AngII-upregulated MCP-1 and IL-6 expression was significantly attenuated by sEH inhibition but promoted by EPHX2 deletion in cardiofibroblasts. Thus, compared with pharmacological inhibition of sEH, EPHX2 deletion caused the shift in arachidonic acid metabolism, which may led to pathological cardiac remodeling, especially cardiac fibrosis.

Zhang, Xu; Hammock, Bruce D.; Ai, Ding; Zhu, Yi

2014-01-01

48

Beta globin gene inhibition by antisense RNA transcripts  

Microsoft Academic Search

Sickle cell disease is caused by a mutation in the ? globin gene leading to hemoglobin S (Hb S) production. Several approaches have been explored to prevent Hb S polymerization in red blood cells and the symptoms associated with this disorder. To this end we tested a mammalian expression vector carrying a human ? globin antisense cDNA (pZeo?AS) fragment in

L Xu; A E Ferry; C Monteiro; B S Pace

2000-01-01

49

Inhibition of Stat1-Mediated Gene Activation by PIAS1  

Microsoft Academic Search

STAT (signal transducer and activator of transcription) proteins are latent cytoplasmic transcription factors that become activated by tyrosine phosphorylation in response to cytokine stimulation. Tyrosine phosphorylated STATs dimerize and translocate into the nucleus to activate specific genes. Different members of the STAT protein family have distinct functions in cytokine signaling. Biochemical and genetic analysis has demonstrated that Stat1 is essential

Bin Liu; Jiayu Liao; Xiaoping Rao; Steven A. Kushner; Chan D. Chung; David D. Chang; Ke Shuai

1998-01-01

50

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

SciTech Connect

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.

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

1988-01-01

51

Feedback-Insensitive Mutants of the Gene for the Tyrosine-Inhibited Dahp Synthetase in Yeast  

PubMed Central

A yeast strain lacking the phenylalanine-sensitive DAHP synthetase exhibits a tyrosine-inhibited phenotype. Reversions from this phenotype were selected. They were the result of mutations affecting the tyrosine-sensitive DAHP synthetase which became insensitive to feedback inhibition, as shown by genetic and enzymatic studies. The feedback-insensitive mutations were located at five different sites scattered on the map of the aro4 gene.

Meuris, Pierre

1974-01-01

52

Gene expression profiling analysis of deoxynivalenol-induced inhibition of mouse thymic epithelial cell proliferation.  

PubMed

Deoxynivalenol (DON) is a mycotoxin produced as a secondary metabolite by fungal species. It has been shown that DON has serious toxic effects on many kinds of immune cells. However, the toxic effects on thymic epithelial cells were poorly understood. The purpose of this study is to investigate the gene expression differences for the DON-induced inhibition on the proliferation of mouse thymic epithelial cell line 1 (MTEC1). After the experiments of cell viability, morphological investigation and cell cycle analysis, microarray analysis was carried out. The differentially expressed genes belong to a variety of functional categories, including genes involved in metabolic process, cell cycle, oxidation-reduction process and apoptosis. Our results provide molecular insights into the gene expression differences of DON-induced toxic effects and suggest that p53 signaling pathway may play an important role in the inhibition of MTEC1 cell proliferation. PMID:23827195

Li, Daotong; Ye, Yaqiong; Deng, Li; Ma, Haoran; Fan, Xiaolong; Zhang, Yuan; Yan, Haikuo; Deng, Xianbo; Li, Yugu; Ma, Yongjiang

2013-09-01

53

Isolation by PCR-based methods of a plant antifungal polygalacturonase- inhibiting protein gene  

Microsoft Academic Search

A polygalacturonase-inhibiting protein (pgip) gene from Malus domestica cv Granny Smith apple fruit was cloned by degenerate oligo-primed polymerase chain reaction (PCR) and Inverse PCR. An alignment of the pear and bean PGIP sequences was used to design degenerate PCR primers in highly conserved regions. Degenerate PCR allowed the amplification of a 351bp internal fragment of the pgip gene, termed

Melanie S. Arendse; Ian A. Dubery; David K. Berger

1999-01-01

54

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

PubMed Central

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.

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

2013-01-01

55

Metformin inhibits histone H2B monoubiquitination and downstream gene transcription in human breast cancer cells  

PubMed Central

Metformin, one of the most widely prescribed antihyperglycemic drugs, has recently received increasing attention for its potential effects with regard to cancer prevention and treatment. However, the mechanisms behind the suppression of cancer cell growth by metformin remain far from completely understood. The aim of the present study was to investigate whether metformin could regulate histone modification and its downstream gene transcription, and its potential function in inhibiting breast cancer cell proliferation. A T47D cell proliferation curve was determined by cell counting following metformin treatment with differing doses or time courses. The cell cycle was analyzed by flow cytometry with propidium iodide staining. Histone H2B monoubiquitination was evaluated by western blotting subsequent to histone extraction. The histone H2B monoubiquitination downstream gene expression level was determined by quantitative PCR. The results showed that metformin changed the cell-cycle check-point and inhibited breast cancer cell proliferation in a dose-dependent manner. AMPK was activated and histone H2B monoubiquitination and downstream gene transcription were inhibited following metformin treatment in the T47D cells. The effect of metformin on T47D cell proliferation was dependent on AMPK activity. It was concluded that metformin can suppress breast cancer cell growth by the activation of AMPK and the inhibition of histone H2B monoubiquitination and downstream gene transcription. This study reveals a novel potential mechanism of cancer cell growth suppression by metformin.

DU, YU; ZHENG, HAIYAN; WANG, JIANG; REN, YE; LI, MI; GONG, CHEN; XU, FEI; YANG, CAIHONG

2014-01-01

56

RNAi Mediated Tiam1 Gene Knockdown Inhibits Invasion of Retinoblastoma  

PubMed Central

T lymphoma invasion and metastasis protein (Tiam1) is up-regulated in variety of cancers and its expression level is related to metastatic potential of the type of cancer. Earlier, Tiam1 was shown to be overexpressed in retinoblastoma (RB) and we hypothesized that it was involved in invasiveness of RB. This was tested by silencing Tiam1 in RB cell lines (Y79 and Weri-Rb1) using siRNA pool, targeting different regions of Tiam1 mRNA. The cDNA microarray of Tiam1 silenced cells showed gene regulations altered by Tiam1 were predominantly on the actin cytoskeleton interacting proteins, apoptotic initiators and tumorogenic potential targets. The silenced phenotype resulted in decreased growth and increased apoptosis with non-invasive characteristics. Transfection of full length and N-terminal truncated construct (C1199) clearly revealed membrane localization of Tiam1 and not in the case of C580 construct. F-actin staining showed the interaction of Tiam1 with actin in the membrane edges that leads to ruffling, and also imparts varying invasive potential to the cell. The results obtained from our study show for the first time that Tiam1 modulates the cell invasion, mediated by actin cytoskeleton remodeling in RB.

Biswas, Jyotirmay; Kanwar, Rupinder K.; Kanwar, Jagat R.; Krishnakumar, Subramanian

2013-01-01

57

The conditional inhibition of gene expression in cultured Drosophila cells by antisense RNA.  

PubMed Central

Genes producing antisense RNA are becoming important tools for the selective inhibition of gene expression. Experiments in different biological systems, targeting different mRNAs have yielded diverse results with respect to the success of the technique and its mechanism of action. We have examined the potential of three antisense genes, whose transcription is driven by a Drosophila metallothionein promoter, to inhibit the expression of alcohol dehydrogenase (ADH) or a microtubule associated protein (205K MAP) in cultured Drosophila cells. Expression of ADH was significantly reduced upon induction of the anti-ADH genes. The ADH mRNA does not appear to be destabilized by the presence of antisense RNA but rather exists at similar levels in hybrid form. Hybrids are detected with both spliced and unspliced ADH RNA. In contrast to these results, antisense genes producing antisense RNA in great excess to 205K MAP mRNA, which is itself far less abundant than the ADH mRNA, failed to show any inhibition of 205K MAP expression. Images

Bunch, T A; Goldstein, L S

1989-01-01

58

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

SciTech Connect

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.

Yamaguchi, Kazuki [Department of Dermatology, Fukuoka University School of Medicine, 7-45-1 Nanakuma, Jonan-ku, Fukuoka City 814-0180 (Japan) [Department of Dermatology, Fukuoka University School of Medicine, 7-45-1 Nanakuma, Jonan-ku, Fukuoka City 814-0180 (Japan); Department of Anatomy, Fukuoka University School of Medicine, 7-45-1 Nanakuma, Jonan-ku, Fukuoka City 814-0180 (Japan); Feril, Loreto B., E-mail: ferilism@yahoo.com [Department of Anatomy, Fukuoka University School of Medicine, 7-45-1 Nanakuma, Jonan-ku, Fukuoka City 814-0180 (Japan); Tachibana, Katsuro [Department of Anatomy, Fukuoka University School of Medicine, 7-45-1 Nanakuma, Jonan-ku, Fukuoka City 814-0180 (Japan)] [Department of Anatomy, Fukuoka University School of Medicine, 7-45-1 Nanakuma, Jonan-ku, Fukuoka City 814-0180 (Japan); Takahashi, Akira; Matsuo, Miki; Endo, Hitomi [Department of Dermatology, Fukuoka University School of Medicine, 7-45-1 Nanakuma, Jonan-ku, Fukuoka City 814-0180 (Japan)] [Department of Dermatology, Fukuoka University School of Medicine, 7-45-1 Nanakuma, Jonan-ku, Fukuoka City 814-0180 (Japan); Harada, Yoshimi [Department of Anatomy, Fukuoka University School of Medicine, 7-45-1 Nanakuma, Jonan-ku, Fukuoka City 814-0180 (Japan)] [Department of Anatomy, Fukuoka University School of Medicine, 7-45-1 Nanakuma, Jonan-ku, Fukuoka City 814-0180 (Japan); Nakayama, Juichiro [Department of Dermatology, Fukuoka University School of Medicine, 7-45-1 Nanakuma, Jonan-ku, Fukuoka City 814-0180 (Japan)] [Department of Dermatology, Fukuoka University School of Medicine, 7-45-1 Nanakuma, Jonan-ku, Fukuoka City 814-0180 (Japan)

2011-07-22

59

Dimethyl sulfoxide inhibits the expression of early growth-response genes and arrests fibroblasts at quiescence.  

PubMed

We have previously shown that dimethyl sulfoxide (DMSO) treatment of mouse embryo fibroblasts (MEF) at the early hours of mitogenic stimuli resulted in the inhibition of DNA and protein synthesis; delayed treatment of serum-stimulated cells with DMSO had little effect on the synthesis of these macromolecules. Here, we demonstrate the specific inhibition of expression of early growth response genes by DMSO in serum-stimulated MEF. The expression of interleukin 6, and of oncogenes c-myc and c-fos were inhibited when the cells were treated with 2% DMSO from the beginning of serum-stimulated growth but not after 3 h of mitogenic stimuli. Although the actin gene is an early serum-response gene, its expression was not affected by DMSO. The synthesis of another serum-induced protein, the plasminogen activator inhibitor-1 was blocked during concurrent and delayed (after 3 h of stimulation) treatment of serum-stimulated fibroblasts with DMSO. The expression of glyceraldehyde-3-phosphate dehydrogenase gene was not affected by DMSO. These results indicate that the expression of non-growth-related genes are either not affected or affected nonspecifically both at early and late stages of serum-induced growth of mouse embryo fibroblasts. The serum-induced expression of c-fos gene was abolished by DMSO treatment of MEF while the phorbol 12-myristate 13-acetate-induced expression of fos gene was not, indicating that the PMA signaling pathway was refractory to DMSO. Treatment of cells with medium containing 2% DMSO for 24-48 h prevents them from progression into cell cycle by preventing the expression of genes involved in G0-G1 transition of quiescent cells. PMID:1909967

Srinivas, S; Sironmani, T A; Shanmugam, G

1991-10-01

60

Conditional inhibition of autophagy genes in adult Drosophila impairs immunity without compromising longevity  

PubMed Central

Immune function declines with age in Drosophila and humans, and autophagy is implicated in immune function. In addition, autophagy genes are required for life span extension caused by reduced insulin/IGF1-like signaling and dietary restriction in C. elegans. To test if the autophagy pathway might be limiting for immunity and/or life span in adult Drosophila, the Geneswitch system was used to cause conditional inactivation of the autophagy genes Atg5, Atg7 and Atg12 by RNAi. Conditional inhibition of Atg genes in adult flies reduced lysotracker staining of adult tissues, and reduced resistance to injected E. coli, as evidenced by increased bacterial titers and reduced fly survival. However, survival of uninjected flies was unaffected by Atg gene inactivation. The data indicate that Atg gene activity is required for normal immune function in adult flies, and suggest that neither autophagy nor immune function are limiting for adult life span under typical laboratory conditions.

Ren, Chunli; Finkel, Steven E.; Tower, John

2009-01-01

61

SIRT1 Inhibition Alleviates Gene Silencing in Fragile X Mental Retardation Syndrome  

PubMed Central

Expansion of the CGG•CCG-repeat tract in the 5? UTR of the FMR1 gene to >200 repeats leads to heterochromatinization of the promoter and gene silencing. This results in Fragile X syndrome (FXS), the most common heritable form of mental retardation. The mechanism of gene silencing is unknown. We report here that a Class III histone deacetylase, SIRT1, plays an important role in this silencing process and show that the inhibition of this enzyme produces significant gene reactivation. This contrasts with the much smaller effect of inhibitors like trichostatin A (TSA) that inhibit Class I, II and IV histone deacetylases. Reactivation of silenced FMR1 alleles was accompanied by an increase in histone H3 lysine 9 acetylation as well as an increase in the amount of histone H4 that is acetylated at lysine 16 (H4K16) by the histone acetyltransferase, hMOF. DNA methylation, on the other hand, is unaffected. We also demonstrate that deacetylation of H4K16 is a key downstream consequence of DNA methylation. However, since DNA methylation inhibitors require DNA replication in order to be effective, SIRT1 inhibitors may be more useful for FMR1 gene reactivation in post-mitotic cells like neurons where the effect of the gene silencing is most obvious.

Biacsi, Rea; Kumari, Daman; Usdin, Karen

2008-01-01

62

Arctigenin from Arctium lappa inhibits interleukin-2 and interferon gene expression in primary human T lymphocytes  

PubMed Central

Background Arctium lappa (Niubang), a Chinese herbal medicine, is used to treat tissue inflammation. This study investigates the effects of arctigenin (AC), isolated from A. lappa, on anti-CD3/CD28 Ab-stimulated cell proliferation and cytokine gene expression in primary human T lymphocytes. Methods Cell proliferation was determined with enzyme immunoassays and the tritiated thymidine uptake method. Cytokine production and gene expression were analyzed with reverse transcription-polymerase chain reaction. Results AC inhibited primary human T lymphocytes proliferation activated by anti-CD3/CD28 Ab. Cell viability test indicated that the inhibitory effects of AC on primary human T lymphocyte proliferation were not due to direct cytotoxicity. AC suppressed interleukin-2 (IL-2) and interferon-? (IFN-?) production in a concentration-dependent manner. Furthermore, AC decreased the IL-2 and IFN-? gene expression in primary human T lymphocytes induced by anti-CD3/CD28 Ab. Reporter gene analyses revealed that AC decreased NF-AT-mediated reporter gene expression. Conclusion AC inhibited T lymphocyte proliferation and decreased the gene expression of IL-2, IFN-? and NF-AT.

2011-01-01

63

Antisense inhibition of the Nr gene restores normal ripening to the tomato Never-ripe mutant, consistent with the ethylene receptor-inhibition model.  

PubMed

The hormone ethylene regulates many aspects of plant growth and development, including fruit ripening. In transgenic tomato (Lycopersicon esculentum) plants, antisense inhibition of ethylene biosynthetic genes results in inhibited or delayed ripening. The dominant tomato mutant, Never-ripe (Nr), is insensitive to ethylene and fruit fail to ripen. The Nr phenotype results from mutation of the ethylene receptor encoded by the NR gene, such that it can no longer bind the hormone. NR has homology to the Arabidopsis ethylene receptors. Studies on ethylene perception in Arabidopsis have demonstrated that receptors operate by a "receptor inhibition" mode of action, in which they actively repress ethylene responses in the absence of the hormone, and are inactive when bound to ethylene. In ripening tomato fruit, expression of NR is highly regulated, increasing in expression at the onset of ripening, coincident with increased ethylene production. This expression suggests a requirement for the NR gene product during the ripening process, and implies that ethylene signaling via the tomato NR receptor might not operate by receptor inhibition. We used antisense inhibition to investigate the role of NR in ripening tomato fruit and determine its mode of action. We demonstrate restoration of normal ripening in Nr fruit by inhibition of the mutant Nr gene, indicating that this receptor is not required for normal ripening, and confirming receptor inhibition as the mode of action of the NR protein. PMID:11080285

Hackett, R M; Ho, C W; Lin, Z; Foote, H C; Fray, R G; Grierson, D

2000-11-01

64

Gene expression profiling in equine polysaccharide storage myopathy revealed inflammation, glycogenesis inhibition, hypoxia and mitochondrial dysfunctions  

PubMed Central

Background Several cases of myopathies have been observed in the horse Norman Cob breed. Muscle histology examinations revealed that some families suffer from a polysaccharide storage myopathy (PSSM). It is assumed that a gene expression signature related to PSSM should be observed at the transcriptional level because the glycogen storage disease could also be linked to other dysfunctions in gene regulation. Thus, the functional genomic approach could be conducted in order to provide new knowledge about the metabolic disorders related to PSSM. We propose exploring the PSSM muscle fiber metabolic disorders by measuring gene expression in relationship with the histological phenotype. Results Genotypying analysis of GYS1 mutation revealed 2 homozygous (AA) and 5 heterozygous (GA) PSSM horses. In the PSSM muscles, histological data revealed PAS positive amylase resistant abnormal polysaccharides, inflammation, necrosis, and lipomatosis and active regeneration of fibers. Ultrastructural evaluation revealed a decrease of mitochondrial number and structural disorders. Extensive accumulation of an abnormal polysaccharide displaced and partially replaced mitochondria and myofibrils. The severity of the disease was higher in the two homozygous PSSM horses. Gene expression analysis revealed 129 genes significantly modulated (p < 0.05). The following genes were up-regulated over 2 fold: IL18, CTSS, LUM, CD44, FN1, GST01. The most down-regulated genes were the following: mitochondrial tRNA, SLC2A2, PRKC?, VEGF?. Data mining analysis showed that protein synthesis, apoptosis, cellular movement, growth and proliferation were the main cellular functions significantly associated with the modulated genes (p < 0.05). Several up-regulated genes, especially IL18, revealed a severe muscular inflammation in PSSM muscles. The up-regulation of glycogen synthase kinase-3 (GSK3?) under its active form could be responsible for glycogen synthase (GYS1) inhibition and hypoxia-inducible factor (HIF1?) destabilization. Conclusion The main disorders observed in PSSM muscles could be related to mitochondrial dysfunctions, glycogenesis inhibition and the chronic hypoxia of the PSSM muscles.

Barrey, Eric; Mucher, Elodie; Jeansoule, Nicolas; Larcher, Thibaut; Guigand, Lydie; Herszberg, Berenice; Chaffaux, Stephane; Guerin, Gerard; Mata, Xavier; Benech, Philippe; Canale, Marielle; Alibert, Olivier; Maltere, Peguy; Gidrol, Xavier

2009-01-01

65

The tumor suppressor gene ARHI (DIRAS3) inhibits ovarian cancer cell migration through multiple mechanisms  

PubMed Central

ARHI is an imprinted tumor suppressor gene that is downregulated in > 60% of ovarian cancers, associated with decreased progression-free survival. ARHI encodes a 26 kDa GTPase with homology to Ras. Re-expression of ARHI inhibits ovarian cancer growth, initiates autophagy and induces tumor dormancy. Recent studies have demonstrated that ARHI also plays a particularly important role in ovarian cancer cell migration. Re-expression of ARHI decreases motility of IL-6- and EGF-stimulated SKOv3 and Hey ovarian cancer cells, inhibiting both chemotaxis and haptotaxis. ARHI inhibits cell migration by binding and sequestering STAT3 in the cytoplasm, and preventing STAT3 translocation to the nucleus and localization in focal adhesion complexes. Re-expression of ARHI inhibits FAKY397 phosphorylation, disrupts focal adhesions and blocks FAK-mediated RhoA signaling, resulting in decreased levels of GTP-RhoA. Re-expression of ARHI disrupts formation of actin stress fibers in a FAK- and RhoA-dependent manner. Recent studies indicate that re-expression of ARHI inhibits expression of ?-1 integrin which may also contribute to inhibition of migration, adhesion and invasion.

Lu, Zhen; Bast, Jr., Robert C.

2013-01-01

66

Microarray gene expression profiling reveals antioxidant-like effects of angiotensin II inhibition in atherosclerosis  

PubMed Central

Reactive oxygen species (ROS) is a significant feature of atherosclerosis but the impact of ROS on atherogenesis is not clear since antioxidants such as vitamin E have little effect on atherosclerosis development in vivo. To investigate the role of ROS in atherosclerosis, we used ApoE-deficient mice, and compared the treatment effect of the antioxidant vitamin E with that of the angiotensin-converting enzyme (ACE) inhibitor, captopril, because angiotensin II is a major source of ROS in the vasculature. Dihydroethidium (DHE) staining demonstrated that vitamin E and captopril both prevented the atherosclerosis-induced increase in aortic superoxide content. In contrast, seven months of vitamin E treatment retarded the development of atherosclerotic lesions by only 45.8 ± 11.5% whereas captopril reduced the aortic plaque area by 88.1 ± 7.5%. To discriminate between vitamin E-sensitive and -insensitive effects of ACE inhibition, we performed whole genome microarray gene expression profiling. Gene ontology (GO) and immunohistology analyses showed that vitamin E and captopril prevented atherosclerosis-related changes of aortic intima and media genes. However, vitamin E did not reduce the expression of probe sets detecting the aortic recruitment of pro-inflammatory immune cells while immune cell-specific genes were normalized by captopril treatment. Moreover, vitamin E did not prevent the atherosclerosis-dependent down-regulation of perivascular nerve-specific genes, which were preserved in captopril-treated aortas. Taken together, our study detected antioxidant vitamin E-like effects of angiotensin II inhibition in atherosclerosis treatment regarding preservation of aortic intima and media genes. Additional vitamin E-insensitive effects targeting atherosclerosis-enhancing aortic immune cell recruitment and perivascular nerve degeneration could account for the stronger anti-atherogenic activity of ACE inhibition compared to vitamin E.

Abd Alla, Joshua; el Faramawy, Yasser; Quitterer, Ursula

2013-01-01

67

Light-controlled inhibition of malignant glioma by opsin gene transfer  

PubMed Central

Glioblastomas are aggressive cancers with low survival rates and poor prognosis because of their highly proliferative and invasive capacity. In the current study, we describe a new optogenetic strategy that selectively inhibits glioma cells through light-controlled membrane depolarization and cell death. Transfer of the engineered opsin ChETA (engineered Channelrhodopsin-2 variant) gene into primary human glioma cells or cell lines, but not normal astrocytes, unexpectedly decreased cell proliferation and increased mitochondria-dependent apoptosis, upon light stimulation. These optogenetic effects were mediated by membrane depolarization-induced reductions in cyclin expression and mitochondrial transmembrane potential. Importantly, the ChETA gene transfer and light illumination in mice significantly inhibited subcutaneous and intracranial glioma growth and increased the survival of the animals bearing the glioma. These results uncover an unexpected effect of opsin ion channels on glioma cells and offer the opportunity for the first time to treat glioma using a light-controllable optogenetic approach.

Yang, F; Tu, J; Pan, J-Q; Luo, H-L; Liu, Y-H; Wan, J; Zhang, J; Wei, P-F; Jiang, T; Chen, Y-H; Wang, L-P

2013-01-01

68

Inhibition of thyroid-restricted genes by follicular thyroglobulin involves iodinated degree.  

PubMed

Follicular thyroglobulin (TG) reflects the storage of both iodine and thyroid hormone. This is because it is a macromolecular precursor of thyroid hormone and organic iodinated compound in follicular lumen. Thus, it may have an important feedback role in thyroid function. In this study, monolayer cells were cultured and follicles were reconstituted with primary pig thyroid cells in vitro. Reconstituted follicles were treated with iodine and methimazole (MMI), a drug that blocks iodine organification and reduces the degree of TG iodination in follicular lumen. The high degree of iodinated TG in follicular lumen was observed to inhibit thyroid-restricted gene expression. To confirm this finding, monolayer thyroid cells were treated with a different degree of TG iodination at the same concentration. These iodinated TG were extracted from reconstituted follicles of different groups. In this manner, this study provides firsthand evidence suggesting that follicular TG inhibits the expressions of thyroid-restricted genes NIS, TPO, TG, and TSHr. PMID:21308730

Huang, Huibin; Shi, Yaxiong; Lin, Ling; Li, Liangyi; Lin, Xiahong; Li, Xisheng; Xu, Dongming

2011-03-01

69

Gene product 0.4 increases bacteriophage T7 competitiveness by inhibiting host cell division.  

PubMed

Bacteriophages take over host resources primarily via the activity of proteins expressed early in infection. One of these proteins, produced by the Escherichia coli phage T7, is gene product (Gp) 0.4. Here, we show that Gp0.4 is a direct inhibitor of the E. coli filamenting temperature-sensitive mutant Z division protein. A chemically synthesized Gp0.4 binds to purified filamenting temperature-sensitive mutant Z protein and directly inhibits its assembly in vitro. Consequently, expression of Gp0.4 in vivo is lethal to E. coli and results in bacteria that are morphologically elongated. We further show that this inhibition of cell division by Gp0.4 enhances the bacteriophage's competitive ability. This division inhibition is thus a fascinating example of a strategy in bacteriophages to maximize utilization of their hosts' cell resources. PMID:24218612

Kiro, Ruth; Molshanski-Mor, Shahar; Yosef, Ido; Milam, Sara L; Erickson, Harold P; Qimron, Udi

2013-11-26

70

Gene product 0.4 increases bacteriophage T7 competitiveness by inhibiting host cell division  

PubMed Central

Bacteriophages take over host resources primarily via the activity of proteins expressed early in infection. One of these proteins, produced by the Escherichia coli phage T7, is gene product (Gp) 0.4. Here, we show that Gp0.4 is a direct inhibitor of the E. coli filamenting temperature-sensitive mutant Z division protein. A chemically synthesized Gp0.4 binds to purified filamenting temperature-sensitive mutant Z protein and directly inhibits its assembly in vitro. Consequently, expression of Gp0.4 in vivo is lethal to E. coli and results in bacteria that are morphologically elongated. We further show that this inhibition of cell division by Gp0.4 enhances the bacteriophage’s competitive ability. This division inhibition is thus a fascinating example of a strategy in bacteriophages to maximize utilization of their hosts’ cell resources.

Kiro, Ruth; Molshanski-Mor, Shahar; Yosef, Ido; Milam, Sara L.; Erickson, Harold P.; Qimron, Udi

2013-01-01

71

The agouti gene product inhibits lipolysis in human adipocytes via a Ca2\\/-dependent mechanism  

Microsoft Academic Search

Overexpression of the murine agouti gene results in obesity. The human homologue of agouti is expressed primarily in human adipocytes, and we have shown recombinant agouti protein to increase adipocyte intracellular Ca2\\/((Ca2\\/)i) and thereby stimulate lipogenesis. However, since recent data demonstrate that increasing adipocyte (Ca2\\/)i may also inhibit lipolysis, we have investigated the role of agouti-induced (Ca2\\/)i increases in regulating

BINGZHONG XUE; NAIMA MOUSTAID-MOUSSA; WILLIAM O. WILKISON; MICHAEL B. ZEMEL

72

Ingestion of bacterially expressed double-stranded RNA inhibits gene expression in planarians  

Microsoft Academic Search

population that is present in the adult planarian. The study of these organisms, classic experimental models for investigating metazoan regeneration, has been revitalized by the application of modern molecular biological approaches. The identification of thousands of unique planarian ESTs, coupled with large-scale whole-mount in situ hybridization screens, and the ability to inhibit planarian gene expression through double-stranded RNA-mediated genetic inter-

Phillip A. Newmark; Peter W. Reddien; Francesc Cebria; Alejandro Sanchez Alvarado

2003-01-01

73

Ibuprofen-induced inhibition of cyclooxygenase isoform gene expression and regression of rat mammary carcinomas  

Microsoft Academic Search

A single dose of 75 mg\\/kg 7,12 dimethylbenz[a]anthracene was administered to 50-day-old virgin female Sprague–Dawley rats and 100 days later, animals were randomized and provided with Teklad rodent chow mixed with a dose of 25 mg\\/rat\\/day ibuprofen for 35 days. Ibuprofen treatment reduced tumor volume (P<0.05) and significantly inhibited gene expression of both cyclooxygenase-1 and cyclooxygenase-2 (P<0.02). These results indicate

Fredika M Robertson; Michelle L Parrett; Farahnaz S Joarder; Mary Ross; Hussein M Abou-Issa; Galal Alshafie; Randall E Harris

1998-01-01

74

Inhibition of reporter gene expression in mammalian cells. Effects of distinct carcinogen lesions in DNA.  

PubMed

The effect of UV photoproducts or benzo[a]pyrene-diol-epoxide-I (BPDE-I) adducts in DNA on the transient expression of a reporter gene was measured in mammalian cells. The plasmid pRSVCAT was UV irradiated or treated with BPDE-I in vitro and co-transfected with undamaged pRSVBGAL into mouse and human fibroblasts. Variations in transfection efficiency among different cell lines were corrected by adjusting the volumes of cell extracts used in the chloramphenicol acetyl transferase (CAT) assays to contain equal beta-galactosidase (BGAL) activity. The expression of the CAT gene was found to decrease exponentially after transfection of pRSVCAT containing increasing numbers of DNA lesions per molecule. The average number of BPDE-I adducts per plasmid molecule was measured by ELISA; the average number of pyrimidine dimers was estimated from the dose kinetics for the disappearance of the supercoiled form of irradiated plasmid DNA treated with Micrococcus luteus UV endonuclease. By expressing the inhibition of CAT activity in terms of the average number of lesions per gene, we were able to compare directly the effects of two different carcinogen lesions on transient transcription. We observed comparable kinetics of inhibition of gene expression by BPDE-I adducts and pyrimidine dimers in DNA. D0 values determined by linear regression analysis of dose-response curves for inhibition of CAT activity were 4.9 BPDE-I adducts or 6.6 pyrimidine dimers per gene in excision-proficient human fibroblasts; the corresponding values in mouse cells were 4.4 BPDE-I adducts or 5.5 pyrimidine dimers. Similar threshold densities of BPDE-I adducts and pyrimidine dimers were observed before inhibition of transcription from pRSVCAT was detected. No threshold was observed in experiments with human fibroblasts deficient in excision repair (xeroderma pigmentosum group A); calculated D0 values were 1.2 pyrimidine dimers of 2.1 BPDE-I adducts. Our results permit direct comparisons of the magnitude of inhibition of gene transcription by distinct DNA lesions, and suggest that BPDE-I adducts and UV-induced cyclobutane pyrimidine dimers in template DNA block transcription with similar efficacy. PMID:8200075

Sorscher, D H; Cordeiro-Stone, M

1994-05-01

75

Inhibition of transcription factor-DNA complexes and gene expression by a microgonotropen  

PubMed Central

Developing minor groove-binding drugs to selectively inhibit transcription factor (TF)/DNA interactions and accompanying gene expression is a current goal in drug development studies. Equipping minor groove-binding agents with positively charged, major groove-contacting side chains yields microgonotropens (MGTs). Previously, we demonstrated that MGTs were superior inhibitors of TF/DNA complexes in cell-free assays compared with “classical” groove binders, but MGTs showed limited ability to inhibit gene expression. To determine what chemical characteristics contribute to or improve activity, we evaluate five MGTs for their effectiveness in inhibiting TF complex formation and resultant transcription by using the c-fos serum response element (SRE) as a target. MGT L1 binds DNA via a bisbenzimidazole equipped with a tripyrrole moiety. It is compared with analog L2, which has been functionalized with propylamines on each of the three pyrroles. L2, which binds DNA at subpicomolar concentrations, was at least three orders of magnitude more potent than L1 at inhibiting TF binding to the c-fos SRE in cell-free assays. Unlike L1 and previous MGTs, L2 also inhibited endogenous c-fos expression in NIH 3T3 cells at micromolar levels. Structure/activity relationships suggest that, although the tripyrrole/polyamine functional group of L2 may be largely responsible for its inhibition of TF complexes in cell-free assays, its bisbenzimidazole moiety appears to impart improved cellular uptake and activity. These findings make L2 a promising lead candidate for future, rational MGT design.

White, Christine M.; Satz, Alexander L.; Bruice, Thomas C.; Beerman, Terry A.

2001-01-01

76

Excitation/inhibition balance and learning are modified by Dyrk1a gene dosage.  

PubMed

Cognitive deficits in Down syndrome (DS) have been linked to increased synaptic inhibition, leading to an imbalance of excitation/inhibition (E/I). Various mouse models and studies from human brains have implicated an HSA21 gene, the serine/threonine kinase DYRK1A, as a candidate for inducing cognitive dysfunction. Here, consequences of alterations in Dyrk1a dosage were assessed in mouse models with varying copy numbers of Dyrk1a: mBACtgDyrk1a, Ts65Dn and Dp(16)1Yey (with 3 gene copies) and Dyrk1a(+/-) (one functional copy). Molecular (i.e. immunoblotting/immunohistochemistry) and behavioral analyses (e.g., rotarod, Morris water maze, Y-maze) were performed in mBACtgDyrk1a mice. Increased expression of DYRK1A in mBACtgDyrk1a induced molecular alterations in synaptic plasticity pathways, particularly expression changes in GABAergic and glutaminergic related proteins. Similar alterations were observed in models with partial trisomy of MMU16, Ts65Dn and Dp(16)1Yey, and were reversed in the Dyrk1a(+/-) model. Dyrk1a overexpression produced an increased number and signal intensity of GAD67 positive neurons, indicating enhanced inhibition pathways in three different models: mBACtgDyrk1a, hYACtgDyrk1a and Dp(16)1Yey. Functionally, Dyrk1a overexpression protected mice from PTZ-induced seizures related to GABAergic neuron plasticity. Our study shows that DYRK1A overexpression affects pathways involved in synaptogenesis and synaptic plasticity and influences E/I balance toward inhibition. Inhibition of DYRK1A activity offers a therapeutic target for DS, but its inhibition/activation may also be relevant for other psychiatric diseases with E/I balance alterations. PMID:24801365

Souchet, Benoit; Guedj, Fayçal; Sahún, Ignasi; Duchon, Arnaud; Daubigney, Fabrice; Badel, Anne; Yanagawa, Yuchio; Barallobre, Maria Jose; Dierssen, Mara; Yu, Eugene; Herault, Yann; Arbones, Mariona; Janel, Nathalie; Créau, Nicole; Delabar, Jean Maurice

2014-09-01

77

The human cytomegalovirus gene product US6 inhibits ATP binding by TAP  

PubMed Central

Human cytomegalovirus (HCMV) encodes several genes that disrupt the major histocompatibility complex (MHC) class I antigen presentation pathway. We recently described the HCMV-encoded US6 gene product, a 23 kDa endoplasmic reticulum (ER)-resident type I integral membrane protein that binds to the transporter associated with antigen processing (TAP), inhibits peptide translocation and prevents MHC class I assembly. The functional consequence of this inhibition is to prevent the cell surface expression of class I bound viral peptides and their recognition by HCMV-specific cytotoxic T cells. Here we describe a novel mechanism of action for US6. We demonstrate that US6 inhibits the binding of ATP by TAP1. This is a conformational effect, as the ER lumenal domain of US6 is sufficient to inhibit ATP binding by the cytosolic nucleotide binding domain of TAP1. US6 also stabilizes TAP at 37°C and prevents conformational rearrangements induced by peptide binding. Our findings suggest that the association of US6 with TAP stabilizes a conformation in TAP1 that prevents ATP binding and subsequent peptide translocation.

Hewitt, Eric W.; Gupta, Soma Sen; Lehner, Paul J.

2001-01-01

78

Inhibition of leptin and leptin receptor gene expression by silibinin-curcumin combination.  

PubMed

Leptin and its receptor are involved in breast carcinogenesis as mitogenic factors. Therefore, they could be considered as targets for breast cancer therapy. Expression of the leptin receptor gene could be modulated by leptin secretion. Silibinin and curcumin are herbal compounds with anti-cancer activity against breast cancer. The aim of this study was to assess their potential to inhibit of expression of the leptin gene and its receptor and leptin secretion. Cytotoxic effects of the two agents on combination on T47D breast cancer cells was investigated by MTT assay test after 24h treatment. With different concentrations the levels of leptin, leptin receptor genes expression were measured by reverse-transcription real-time PCR. Amount of secreted leptin in the culture medium was determined by ELISA. Data were statistically analyzed by one-way ANOVA test. The silibinin and curcumin combination inhibited growth of T47D cells in a dose dependent manner. There were also significant difference between control and treated cells in leptin expression and the quantity of secreted leptin with a relative decrease in leptin receptor expression. In conclusion, these herbal compounds inhibit the expression and secretion of leptin and it could probably be used as drug candidates for breast cancer therapy through leptin targeting in the future. PMID:24377502

Nejati-Koshki, Kazem; Akbarzadeh, Abolfazl; Pourhasan-Moghaddam, Mohammad; Abhari, Alireza; Dariushnejad, Hassan

2013-01-01

79

Systemic Myostatin Inhibition via Liver-Targeted Gene Transfer in Normal and Dystrophic Mice  

PubMed Central

Background Myostatin inhibition is a promising therapeutic strategy to maintain muscle mass in a variety of disorders, including the muscular dystrophies, cachexia, and sarcopenia. Previously described approaches to blocking myostatin signaling include injection delivery of inhibitory propeptide domain or neutralizing antibodies. Methodology/Principal Findings Here we describe a unique method of myostatin inhibition utilizing recombinant adeno-associated virus to overexpress a secretable dominant negative myostatin exclusively in the liver of mice. Systemic myostatin inhibition led to increased skeletal muscle mass and strength in control C57 Bl/6 mice and in the dystrophin-deficient mdx model of Duchenne muscular dystrophy. The mdx soleus, a mouse muscle more representative of human fiber type composition, demonstrated the most profound improvement in force production and a shift toward faster myosin-heavy chain isoforms. Unexpectedly, the 11-month-old mdx diaphragm was not rescued by long-term myostatin inhibition. Further, mdx mice treated for 11 months exhibited cardiac hypertrophy and impaired function in an inhibitor dose–dependent manner. Conclusions/Significance Liver-targeted gene transfer of a myostatin inhibitor is a valuable tool for preclinical investigation of myostatin blockade and provides novel insights into the long-term effects and shortcomings of myostatin inhibition on striated muscle.

Morine, Kevin J.; Bish, Lawrence T.; Pendrak, Klara; Sleeper, Meg M.; Barton, Elisabeth R.; Sweeney, H. Lee

2010-01-01

80

Antisense gene inhibition by phosphorothioate antisense oligonucleotide in Arabidopsis pollen tubes.  

PubMed

Sexual reproduction is an essential biological event for proliferation of plants. The pollen tube (PT) that contained male gametes elongates and penetrates into the pistils for successful fertilization. However, the molecular mechanisms of plant fertilization remain largely unknown. Here, we report a transient inhibition of gene function using phosphorothioate antisense oligodeoxynucleotides (AS-ODNs) without cytofectin, which is a simple way to study gene function in Arabidopsis thaliana PTs. The PTs treated with AS-ODNs against both ANX1 and ANX2 showed short, knotted, and ruptured morphology in vitro/semi-in vitro, whereas normal PT growth was shown in its sense control in vitro/semi-in vitro. PT growth was impaired in a manner dependent on the dose of AS-ODNs against both ANX1 and ANX2 above 10 ?m. The treatment with AS-ODNs against ROP1 and CalS5 resulted in waving PTs and in short PTs with a few callose plugs, respectively. The expression levels of the target genes in PTs treated with their AS-ODNs were lower than or similar to those in the sense control, indicating that the inhibition was directly or indirectly related to the expression of each mRNA. The AS-ODN against fluorescent protein (sGFP) led to reduced sGFP expression, suggesting that the AS-ODN suppressed protein expression. This method will enable the identification of reproductively important genes in Arabidopsis PTs. PMID:24495108

Mizuta, Yoko; Higashiyama, Tetsuya

2014-05-01

81

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

PubMed Central

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 IC50 of ?164±12 ?g/ml. ?-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 ?g/ml, respectively. MCF-10A normal breast epithelial cells showed a higher IC50 ?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.

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

2014-01-01

82

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

PubMed

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 µg/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 µg/ml, respectively. MCF-10A normal breast epithelial cells showed a higher IC?? ~464 µg/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 µg/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

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

2014-04-01

83

In vitro infection with classical swine fever virus inhibits the transcription of immune response genes  

PubMed Central

Background Classical swine fever virus (CSFV) can evade the immune response and establish chronic infection under natural and experimental conditions. Some genes related to antigen processing and presentation and to cytokine regulation are known to be involved in this response, but the precise mechanism through which each gene responds to CSFV infection remains unclear. Results In this study, the amplification standard curve and corresponding linear regression equations for the genes SLA-2, TAP1, SLA-DR, Ii, CD40, CD80, CD86, IFN-?, and IFN-? were established successfully. Real-time RT-PCR was used to quantify the immune response gene transcription in PK-15 cells post CSFV infection. Results showed that: (1) immune response genes were generally down-regulated as a result of CSFV infection, and (2) the expression of SLA-2, SLA-DR, Ii and CD80 was significantly decreased (p<0.001). Conclusion We conclude that in vitro infection with CSFV inhibits the transcription of host immune response genes. These findings may facilitate the development of effective strategies for controlling CSF.

2012-01-01

84

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

PubMed Central

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.

Oduro, Jennifer D.; Uecker, Ralf

2012-01-01

85

The Transcriptional Co-Repressor Myeloid Translocation Gene 16 Inhibits Glycolysis and Stimulates Mitochondrial Respiration  

PubMed Central

The myeloid translocation gene 16 product MTG16 is found in multiple transcription factor–containing complexes as a regulator of gene expression implicated in development and tumorigenesis. A stable Tet-On system for doxycycline–dependent expression of MTG16 was established in B-lymphoblastoid Raji cells to unravel its molecular functions in transformed cells. A noticeable finding was that expression of certain genes involved in tumor cell metabolism including 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 and 4 (PFKFB3 and PFKFB4), and pyruvate dehydrogenase kinase isoenzyme 1 (PDK1) was rapidly diminished when MTG16 was expressed. Furthermore, hypoxia–stimulated production of PFKFB3, PFKFB4 and PDK1 was inhibited by MTG16 expression. The genes in question encode key regulators of glycolysis and its coupling to mitochondrial metabolism and are commonly found to be overexpressed in transformed cells. The MTG16 Nervy Homology Region 2 (NHR2) oligomerization domain and the NHR3 protein–protein interaction domain were required intact for inhibition of PFKFB3, PFKFB4 and PDK1 expression to occur. Expression of MTG16 reduced glycolytic metabolism while mitochondrial respiration and formation of reactive oxygen species increased. The metabolic changes were paralleled by increased phosphorylation of mitogen–activated protein kinases, reduced levels of amino acids and inhibition of proliferation with a decreased fraction of cells in S-phase. Overall, our findings show that MTG16 can serve as a brake on glycolysis, a stimulator of mitochondrial respiration and an inhibitor of cell proliferation. Hence, elevation of MTG16 might have anti–tumor effect.

Kumar, Parveen; Sharoyko, Vladimir V.; Spegel, Peter; Gullberg, Urban; Mulder, Hindrik; Olsson, Inge; Ajore, Ram

2013-01-01

86

Folded RNA from an intron of one gene might inhibit expression of a counteracting gene  

Microsoft Academic Search

Homeostatic maintenance of mRNA levels including prompt availability of mRNAs for translation in response to changing protein demands might be partly enabled by a system of combinatorial controls involving noncoding RNA blocking agents. This article proposes a specific version of that control mechanism, namely, a double-stranded RNA folding from transcription of an intron of one gene might and leading to

Clark Jeffries; Michael Jarstfer; Diana O. Perkins

2005-01-01

87

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)

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.

Ikuzawa, Masayuki; Akiduki, Saori; Asashima, Makoto

88

Evolution of feedback-inhibited ?/? barrel isoenzymes by gene duplication and a single mutation  

PubMed Central

The ?/? barrel is the common protein fold of numerous enzymes and was proposed recently to be the result of gene duplication and fusion of an ancient half-barrel. The initial enzyme of shikimate biosynthesis possesses the additional feature of feedback regulation. The crystal structure and kinetic studies on chimera and mutant proteins of yeast 3-deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) synthase from Saccharomyces cerevisiae inhibited by phenylalanine (Aro3p) and DAHP synthase S. cerevisiae inhibited by tyrosine (Aro4p) give insight into important regions for regulation in the enzyme: The loop, which is connecting the two half-barrels, and structural elements added to the barrel are prerequisites for regulation and form a cavity on the N-terminal side of the ?/? barrel. In the cavity of Aro4p at position 226 is a glycine residue, which is highly conserved in all other tyrosine-regulated DAHP synthases as well. Sequence alignments with phenylalanine-regulated DAHP synthases including Aro3p show a highly conserved serine residue at this position. An exchange of glycine to serine and vice versa leads to a complete change in the regulation pattern. Therefore the evolution of these differently feedback-inhibited isoenzymes required gene duplication and a single mutation within the internal extra element. Numerous additional amino acid substitutions present in the contemporary isoenzymes are irrelevant for regulation and occurred independently.

Hartmann, Markus; Schneider, Thomas R.; Pfeil, Andrea; Heinrich, Gabriele; Lipscomb, William N.; Braus, Gerhard H.

2003-01-01

89

The p53-regulated Sestrin gene products inhibit mTOR signaling  

PubMed Central

Summary Tumor suppressor p53 is activated upon genotoxic and oxidative stress and in turn inhibits cell proliferation and growth through induction of specific target genes. Cell growth is positively regulated by mTOR whose activity is controlled by the TSC1:TSC2 complex, a GTPase activating protein (GAP) for Rheb, the activator of mTOR. However, the mechanism by which p53 and genotoxic stress negatively control cell growth via the TSC1:TSC2-mTOR axis is not firmly established. We now demonstrate that the products of two p53 target genes, Sestrin1 and Sestrin2, activate the AMP-responsive protein kinase (AMPK) and target it to phosphorylate TSC2 and stimulate its GAP activity, thereby inhibiting mTOR. Correspondingly, Sestrin2-deficient mice fail to inhibit mTOR signaling upon genotoxic challenge. Sestrin1 and 2 therefore provide an important link between genotoxic stress, p53 and the mTOR signaling pathway.

Budanov, Andrei V.; Karin, Michael

2009-01-01

90

Human Cytomegalovirus Inhibition by Cardiac Glycosides: Evidence for Involvement of the hERG Gene  

PubMed Central

Infection with human cytomegalovirus (HCMV) continues to be a major threat for pregnant women and the immunocompromised population. Although several anti-HCMV therapies are available, the development of new anti-HCMV agents is highly desired. There is growing interest in identifying compounds that might inhibit HCMV by modulating the cellular milieu. Interest in cardiac glycosides (CG), used in patients with congestive heart failure, has increased because of their established anticancer and their suggested antiviral activities. We report that the several CG—digoxin, digitoxin, and ouabain—are potent inhibitors of HCMV at nM concentrations. HCMV inhibition occurred prior to DNA replication, but following binding to its cellular receptors. The levels of immediate early, early, and late viral proteins and cellular NF-?B were significantly reduced in CG-treated cells. The activity of CG in infected cells correlated with the expression of the potassium channel gene, hERG. CMV infection upregulated hERG, whereas CG significantly downregulated its expression. Infection with mouse CMV upregulated mouse ERG (mERG), but treatment with CG did not inhibit virus replication or mERG transcription. These findings suggest that CG may inhibit HCMV by modulating human cellular targets associated with hERG and that these compounds should be studied for their antiviral activities.

Kapoor, Arun; Cai, Hongyi; Forman, Michael; He, Ran; Shamay, Meir

2012-01-01

91

Prediction on the inhibition ratio of pyrrolidine derivatives on matrix metalloproteinase based on gene expression programming.  

PubMed

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 (R (2)) 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

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

2014-01-01

92

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

PubMed Central

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.

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

2014-01-01

93

Relief of amplification inhibition in PCR with bovine serum albumin or T4 gene 32 protein  

SciTech Connect

The benefits of adding bovine serum albumin (BSA) or T4 gene 32 proteins (gp32) to PCR were evaluated with reaction mixtures containing substances that inhibit amplification. Whereas 10- to 1,000-fold more FeCl{sub 3}, hemin, fulvic acids, humic acids, tannic acids, or extracts from feces, freshwater, or marine water were accommodated in PCR when either 400 ng of BSA per {mu}l was included in the reactions, neither BSA nor gp32 relieved interference significantly when minimum inhibitory levels of bile salts, bilirubin, EDTA, NaCl, sodium dodecyl sulfate, or Triton X-100 were present. Use of BSA and gp32 together offered no more relief of inhibition than either alone at its optimal level, and neither protein had any noticeable effect on amplification in the absence of inhibitors. 21 refs., 3 figs.

Kreader, C.A. [Environmental Protection Agency, Cincinnati, OH (United States)

1996-03-01

94

Vaccinia Virus Inhibits NF-?B-Dependent Gene Expression Downstream of p65 Translocation  

PubMed Central

The transcription factor nuclear factor kappa light-chain enhancer of activated B cells (NF-?B) plays a critical role in host defense against viral infection by inducing the production of proinflammatory mediators and type I interferon. Consequently, viruses have evolved many mechanisms to block its activation. The poxvirus vaccinia virus (VACV) encodes numerous inhibitors of NF-?B activation that target multiple points in the signaling pathway. A derivative of VACV strain Copenhagen, called vv811, lacking 55 open reading frames in the left and right terminal regions of the genome was reported to still inhibit NF-?B activation downstream of tumor necrosis factor alpha (TNF-?) and interleukin-1? (IL-1?), suggesting the presence of one or more additional inhibitors. In this study, we constructed a recombinant vv811 lacking the recently described NF-?B inhibitor A49 (vv811?A49), yielding a virus that lacked all currently described inhibitors downstream of TNF-? and IL-1?. Unlike vv811, vv811?A49 no longer inhibited degradation of the phosphorylated inhibitor of ?B? and p65 translocated into the nucleus. However, despite this translocation, vv811?A49 still inhibited TNF-?- and IL-1?-induced NF-?B-dependent reporter gene expression and the transcription and production of cytokines induced by these agonists. This inhibition did not require late viral gene expression. These findings indicate the presence of another inhibitor of NF-?B that is expressed early during infection and acts by a novel mechanism downstream of p65 translocation into the nucleus.

Sumner, Rebecca P.; Maluquer de Motes, Carlos; Veyer, David L.

2014-01-01

95

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

PubMed

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

Manohar, Senthilvelan; Jamesdaniel, Samson; Salvi, Richard

2014-05-01

96

The extract of Elaeocarpus sylvestris inhibits human cytomegalovirus immediate early gene expression and replication in vitro.  

PubMed

Human cytomegalovirus (HCMV) is a major cause of morbidity and mortality in newborn infants, immunocompromised individuals with HIV/AIDS and organ transplant recipients. In order to identify a novel antiviral candidate for HCMV-related diseases, crude ethanol extracts from plants were screened for their potential inhibitory activity on HCMV replication in vitro. Ethanol (70%) extract of Elaeocarpus sylvestris leaves (ESE) markedly inhibited the replication of the HCMV Towne strain without exhibiting any significant adverse effects on the viability of human foreskin fibroblasts (HFF). In addition, ESE significantly downregulated HCMV immediate early (IE) gene expression. Taken together, this is the first study, to the best of our knowledge, demonstrating that ESE has a potent antiviral activity against HCMV by downregulating HCMV IE gene expression and replication. PMID:24270403

To, Kim Phuong; Kang, Se Chan; Song, Yoon-Jae

2014-02-01

97

Rev-Erbs repress macrophage gene expression by inhibiting enhancer-directed transcription  

PubMed Central

Rev-Erb? and Rev-Erb? are nuclear receptors that regulate the expression of genes involved in the control of circadian rhythm1,2, metabolism3,4, and inflammatory responses5. Rev-Erbs function as transcriptional repressors by recruiting NCoR/HDAC3 co-repressor complexes to Rev-Erb response elements in enhancers and promoters of target genes6-8, but the molecular basis for cell-specific programs of repression is not known. Here, we present evidence that in macrophages, Rev-Erbs regulate target gene expression by inhibiting the functions of distal enhancers that are selected by macrophage lineage-determining factors, thereby establishing a macrophage-specific program of repression. Remarkably, the repressive functions of Rev-Erbs are associated with their ability to inhibit the transcription of enhancer-derived RNAs (eRNAs). Furthermore, targeted degradation of eRNAs at two enhancers subject to negative regulation by Rev-Erbs resulted in reduced expression of nearby mRNAs, implying a direct role of these eRNAs in enhancer function. By precisely defining eRNA start sites using a method that quantifies nascent 5? ends (5?-GRO-Seq), we show that transfer of full enhancer activity to a target promoter requires both the sequences mediating transcription factor binding and the specific sequences encoding the eRNA transcript. These studies provide evidence for direct roles of eRNAs in contributing to enhancer functions and suggest that Rev-Erbs act to suppress gene expression at a distance by repressing eRNA transcription.

Lam, Michael T.Y.; Cho, Han; Lesch, Hanna P.; Gosselin, David; Heinz, Sven; Tanaka-Oishi, Yumiko; Benner, Christopher; Kaikkonen, Minna U.; Kim, Aneeza S.; Kosaka, Mika; Lee, Cindy Y.; Watt, Andy; Grossman, Tamar R.; Rosenfeld, Michael G.; Evans, Ronald M.; Glass, Christopher K.

2013-01-01

98

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

PubMed

We have previously described the role of red hair (melanocortin-1 receptor, MC1R) and blue eye (oculocutaneous albinism type II, OCA2) gene polymorphisms in modulating the 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, have identified numerous polymorphisms controlling human hair, eye, and skin color. In this paper, we test a selected set of polymorphisms in pigmentation loci (ASIP (Agouti signalling protein, nonagouti homolog (mouse) gene), TYR (tyrosinase), TYRP1 (tyrosinase-related protein 1), MC1R, OCA2, IRF4 (interferon regulatory factor 4), SLC24A4 (solute carrier family 24, member 4), and SLC45A2 (solute carrier family 45, member 2)) 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 single nucleotide polymorphisms (rs28777, rs35391, and 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

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

2010-02-01

99

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

PubMed

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

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

2014-07-01

100

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

PubMed

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

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

2014-07-22

101

Fluoride decreased osteoclastic bone resorption through the inhibition of NFATc1 gene expression.  

PubMed

Over the past two decades, fluoride effects on osteoclasts have been evaluated; however, its molecular mechanisms remain unclear. In this study, we investigated the effect of fluoride on osteoclast formation, function, and regulation using osteoclasts formed from mice bone marrow macrophages treated with the receptor activator of NF-?B ligand and macrophage colony-stimulating factor. Our data showed that fluoride levels ? 8 mg/L had no effect on osteoclast formation; however, it significantly reduced osteoclast resorption at 0.5 mg/L. Fluoride activity on bone resorption occurred through the inhibition of nuclear factor of active T cells (NFAT) c1 expression. Furthermore, the expression of its downstream genes, including the dendritic cell-specific transmembrane protein, c-Src, the d2 isoform of vacuolar (H+) ATPase v0 domain, matrix metalloproteinase 9, and cathepsin K were decreased, leading to impaired osteoclast acidification, reduced secretion of proteolytic enzymes, and decreased bone resorption. In summary, our results suggested that fluoride has different roles in osteoclast formation and function. Fluoride ? 8 mg/L did not impact osteoclast formation; however, it significantly decreased the resorption activity of newly formed osteoclasts. The molecular mechanism of fluoride action may involve inhibition of NFATc1 and its downstream genes. © 2012 Wiley Periodicals, Inc. Environ Toxicol 29: 588-595, 2014. PMID:22610969

Pei, Junrui; Li, Bingyun; Gao, Yanhui; Wei, Yudan; Zhou, Lingwang; Yao, Hongju; Wang, Jing; Sun, Dianjun

2014-05-01

102

Adenovirus-mediated transfer of viral IL-10 gene inhibits murine collagen-induced arthritis.  

PubMed

IL-10 is a potent anti-inflammatory cytokine that has received growing attention for its therapeutic potential. We examined the efficiency of adenoviral-mediated gene transfer of IL-10 on the incidence and severity of murine collagen-induced arthritis (CIA). Male DBA1 mice immunized with collagen II were treated by systemic administration of 10(9) plaque-forming units of replication-defective adenoviral vector expressing viral IL-10 (vIL-10) on day 30, when clinical symptoms of arthritis start. The transgene was shown to inhibit the onset of CIA, to decrease severity, and profoundly suppress the overall joint histopathology of the experimental arthritis. Significant IL-10 concentrations were obtained in the serum of injected animals for 7 days. Inhibition of arthritis was enhanced by administration of increasing doses of adenovirus-vIL-10. In addition, the local immunosuppressive effect of gene-delivered vIL-10 could be neutralized by a monoclonal anti-vIL-10 Ab. The CIA symptoms in the group treated with the same construct expressing inactive vIL-10 (vIL-10 mut) were similar to those in untreated animals. Our data indicate that a single systemic administration of an adenoviral vector encoding vIL-10 may be a good candidate to suppress arthritis. PMID:9605116

Apparailly, F; Verwaerde, C; Jacquet, C; Auriault, C; Sany, J; Jorgensen, C

1998-06-01

103

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

SciTech Connect

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.

Fan, Qi; Wang, Xuedi; Zhang, Hanguang; Li, Chuanwei [Department of Hepatobiliary and Vascular Surgery, The First Affiliated Hospital of Guangxi Medical University, Nanning 530021 (China)] [Department of Hepatobiliary and Vascular Surgery, The First Affiliated Hospital of Guangxi Medical University, Nanning 530021 (China); Fan, Junhua [Department of Gastroenterology, The First Affiliated Hospital of Guangxi Medical University, Nanning 530021 (China)] [Department of Gastroenterology, The First Affiliated Hospital of Guangxi Medical University, Nanning 530021 (China); Xu, Jing, E-mail: jxuapr@yahoo.com.cn [Department of Hepatobiliary and Vascular Surgery, The First Affiliated Hospital of Guangxi Medical University, Nanning 530021 (China)] [Department of Hepatobiliary and Vascular Surgery, The First Affiliated Hospital of Guangxi Medical University, Nanning 530021 (China)

2012-09-07

104

AAV-Mediated Gene Targeting Is Significantly Enhanced by Transient Inhibition of Nonhomologous End Joining or the Proteasome In Vivo  

PubMed Central

Abstract Recombinant adeno-associated virus (rAAV) vectors have clear potential for use in gene targeting but low correction efficiencies remain the primary drawback. One approach to enhancing efficiency is a block of undesired repair pathways like nonhomologous end joining (NHEJ) to promote the use of homologous recombination. The natural product vanillin acts as a potent inhibitor of NHEJ by inhibiting DNA-dependent protein kinase (DNA-PK). Using a homology containing rAAV vector, we previously demonstrated in vivo gene repair frequencies of up to 0.1% in a model of liver disease hereditary tyrosinemia type I. To increase targeting frequencies, we administered vanillin in combination with rAAV. Gene targeting frequencies increased up to 10-fold over AAV alone, approaching 1%. Fah?/?Ku70?/? double knockout mice also had increased gene repair frequencies, genetically confirming the beneficial effects of blocking NHEJ. A second strategy, transient proteasomal inhibition, also increased gene-targeting frequencies but was not additive to NHEJ inhibition. This study establishes the benefit of transient NHEJ inhibition with vanillin, or proteasome blockage with bortezomib, for increasing hepatic gene targeting with rAAV. Functional metabolic correction of a clinically relevant disease model was demonstrated and provided evidence for the feasibility of gene targeting as a therapeutic strategy.

Paulk, Nicole K.; Loza, Laura Marquez; Finegold, Milton J.

2012-01-01

105

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

SciTech Connect

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.

Peng Guiqing [State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072 (China); Zhang Fengmin [College of Basic Medical Science, Harbin Medical University, Harbin 150081 (China); Zhang Qi; Wu Kailang; Zhu Fan [State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072 (China); Wu Jianguo [State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072 (China)], E-mail: jwu@edu.edu.cn

2007-09-30

106

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

SciTech Connect

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.

Sun, Zhichao [Department of Biochemistry and Molecular Biology, Shanghai Medical College, Fudan University, Shanghai (China)] [Department of Biochemistry and Molecular Biology, Shanghai Medical College, Fudan University, Shanghai (China); Yu, Xuemei [Department of Endocrinology, Fengxian Central Hospital, Shanghai (China)] [Department of Endocrinology, Fengxian Central Hospital, Shanghai (China); Wu, Weibin; Jia, Dongwei; Chen, Yinle; Ji, Lingling; Liu, Xijun; Peng, Xiaomin [Department of Biochemistry and Molecular Biology, Shanghai Medical College, Fudan University, Shanghai (China)] [Department of Biochemistry and Molecular Biology, Shanghai Medical College, Fudan University, Shanghai (China); Li, Yintao [Institute of Endocrinology and Diabetology, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai (China)] [Institute of Endocrinology and Diabetology, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai (China); Yang, Lili [Department of Endocrinology, Fengxian Central Hospital, Shanghai (China)] [Department of Endocrinology, Fengxian Central Hospital, Shanghai (China); Ruan, Yuanyuan; Gu, Jianxin [Department of Biochemistry and Molecular Biology, Shanghai Medical College, Fudan University, Shanghai (China)] [Department of Biochemistry and Molecular Biology, Shanghai Medical College, Fudan University, Shanghai (China); Ren, Shifang, E-mail: renshifang@fudan.edu.cn [Department of Biochemistry and Molecular Biology, Shanghai Medical College, Fudan University, Shanghai (China)] [Department of Biochemistry and Molecular Biology, Shanghai Medical College, Fudan University, Shanghai (China); Zhang, Songwen, E-mail: songwenzhang@fudan.edu.cn [Department of Biochemistry and Molecular Biology, Shanghai Medical College, Fudan University, Shanghai (China)] [Department of Biochemistry and Molecular Biology, Shanghai Medical College, Fudan University, Shanghai (China)

2012-07-13

107

B?cell translocation 1 gene inhibits cellular metastasis?associated behavior in breast cancer.  

PubMed

B?cell translocation gene 1 (BTG1) is a member of the BTG/transducer of ERBB2 family, which regulates cell cycle progression in a variety of cell types and may have a role in inhibiting proliferation, promoting apoptosis and stimulating cellular differentiation in numerous cell types. However, the role of BTG1 in cancer metastasis is yet to be elucidated. In the present study, analysis of clinical specimens revealed that BTG1 mRNA levels were lower in lymph node metastases than those in benign breast tumors and normal human breast tissue. The effect of BTG1 on the metastatic behavior of breast cancer cells following stable transfection with a BTG1 expression vector was also investigated. The overexpression of BTG1 was observed to inhibit cell adhesion, migration and invasion. Furthermore, the overexpression of BTG1 was found to be involved in the inhibition of the metastasis?related proteins matrix metalloproteinase?2 and ?9, as well as the promotion of the cell?cell adhesion?associated protein E?cadherin. In syngeneic nude mice breast tumor models, hepatic metastasis and angiogenesis were observed in the mice injected with the control cells, but not in those injected with pcDNA3?BTG1 cells. Immunohistochemistry revealed that overexpression of BTG1 decreased vascular endothelial growth factor expression in tumors. To the best of our knowledge, this is the first study to show that BTG1 overexpression decreases migration and invasion of breast cancer cells and thereby inhibits distant metastasis in mice breast tumor models. PMID:24714932

Li, Wei; Zou, Shi-Tao; Zhu, Ran; Wan, Jian-Mei; Xu, Yan; Wu, Hao-Rong

2014-06-01

108

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

PubMed Central

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

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

1995-01-01

109

BMP inhibition initiates neural induction via FGF signaling and Zic genes.  

PubMed

Neural induction is the process that initiates nervous system development in vertebrates. Two distinct models have been put forward to describe this phenomenon in molecular terms. The default model states that ectoderm cells are fated to become neural in absence of instruction, and do so when bone morphogenetic protein (BMP) signals are abolished. A more recent view implicates a conserved role for FGF signaling that collaborates with BMP inhibition to allow neural fate specification. Using the Xenopus embryo, we obtained evidence that may unite the 2 views. We show that a dominant-negative R-Smad, Smad5-somitabun-unlike the other BMP inhibitors used previously-can trigger conversion of Xenopus epidermis into neural tissue in vivo. However, it does so only if FGF activity is uncompromised. We report that this activity may be encoded by FGF4, as its expression is activated upon BMP inhibition, and its knockdown suppresses endogenous, as well as ectopic, neural induction by Smad5-somitabun. Supporting the importance of FGF instructive activity, we report the isolation of 2 immediate early neural targets, zic3 and foxD5a. Conversely, we found that zic1 can be activated by BMP inhibition in the absence of translation. Finally, Zic1 and Zic3 are required together for definitive neural fate acquisition, both in ectopic and endogenous situations. We propose to merge the previous models into a unique one whereby neural induction is controlled by BMP inhibition, which activates directly, and, via FGF instructive activity, early neural regulators such as Zic genes. PMID:19805078

Marchal, Leslie; Luxardi, Guillaume; Thomé, Virginie; Kodjabachian, Laurent

2009-10-13

110

Von Willebrand Factor permeates small vessels in CADASIL and inhibits smooth muscle gene expression  

PubMed Central

Background and Purpose CADASIL (cerebral autosomal dominant arteriopathy subcortical infarcts and leukoencephalopathy) is a genetic disorder hallmarked by ischemic stroke and vascular dementia. Characteristic pathological changes in the vasculature include thickening of small arteries and accumulation of heterogeneous material within the vessel wall. We tested whether endothelial von Willebrand factor (vWF) accumulates in CADASIL vessels and whether exposure of smooth muscle cells to vWF alters the expression of smooth muscle gene expression. Methods Brain sections obtained at autopsy from six North American CADASIL patients were examined using immunohistochemistry for vWF and IgG. Rat aortic smooth muscle cells (A7R5 cells) were tested for binding to infrared-tag labeled vWF. Finally, A7R5 cells were exposed to vWF, and expression of mature smooth muscle marker genes was analyzed by quantitative reverse transcriptase PCR. Results vWF is expressed in the penetrating arterial walls in all CADASIL samples. IgG, a marker of serum extravasation, was present only in a minority of arterial walls. vWF binds to smooth muscle cells in vitro, and low concentrations of vWF rapidly activate c-fos, EGR, TSP1, and c-myc while specifically inhibiting RNA encoding smooth muscle actin, calponin, and SM22. Conclusions These data demonstrate that vWF, likely produced by the endothelium, permeates the vessel wall of CADASIL brains. Exposure of smooth muscle cells to vWF results in reduction of specific RNAs required for normal vascular homeostasis. This is the first report of accumulation of a protein within CADASIL vessels that inhibits vascular gene expression and implicates a role for vWF beyond hemostasis.

Zhang, Xiaojie; Meng, He; Blaivas, Mila; Rushing, Elisabeth J.; Moore, Brian E.; Schwartz, Jessica; Lopes, M. Beatriz S.; Worrall, Bradford B.; Wang, Michael M.

2011-01-01

111

Clustering of mandibular organ-inhibiting hormone and moult-inhibiting hormone genes in the crab, Cancer pagurus, and implications for regulation of expression.  

PubMed

Development and reproduction of crustaceans is regulated by a combination of neuropeptide hormones, ecdysteroids (moulting hormones) and the isoprenoid, methyl farnesoate (MF), the unepoxidised analogue of insect juvenile hormone-III (JH-III). MF and the ecdysteroids are respectively synthesised under the negative control of the sinus gland-derived mandibular organ-inhibiting hormones (MO-IHs) and moult-inhibiting hormone (MIH) that are produced in eyestalk neural ganglia. Previous work has demonstrated the existence of two isoforms of MO-IH, called MO-IH-1 and -2, that differ by a single amino acid in the mature peptide and one in the putative signal peptide. To study the structural organisation of the crab MIH and MO-IH genes, a genomic DNA library was constructed from DNA of an individual female crab and screened with both MO-IH and MIH probes. The results from genomic Southern blot analysis and library screening indicated that the Cancer pagurus genome contains at least two copies of the MIH gene and three copies of the MO-IH genes. Upon screening, two types of overlapping genomic clone were isolated. Each member of one type of genomic clone contains a single copy of each of the convergently transcribed MO-IH-1 and MIH genes clustered within 6.5kb. The other type contains only the MO-IH-2 gene, which is not closely linked to an MIH gene. There are three exons and two introns in all MIH and MO-IH genes analysed. The exon-intron boundary of the crab MIH and MO-IH genes follows Chambon's rule (GT-AG) for the splice donor and acceptor sites. The first intron occurs within the signal peptide region and the second intron occurs in the coding region of the mature peptide. Sequence analysis of upstream regions of MO-IH and MIH genes showed that they contained promoter elements with characteristics similar to other eukaryotic genes. These included sequences with high degrees of similarity to the arthropod initiator, TATA box and cAMP response element binding protein. Additionally, putative CF1/USP and Broad Complex Z2 transcription factor elements were found in the upstream regions of MIH and MO-IH genes respectively. The implications of the presence of the latter two putative transcription factor binding-elements for control of expression of MIH and MO-IH genes is discussed. Phylogenetic analysis and gene organisation show that MO-IH and MIH genes are closely related. Their relationship suggests that they represent an example of evolutionary divergence of crustacean hormones. PMID:10940557

Lu, W; Wainwright, G; Webster, S G; Rees, H H; Turner, P C

2000-08-01

112

The Rb1 gene inhibits the viability of retinoblastoma cells by regulating homologous recombination.  

PubMed

Retinoblastoma is a childhood ocular tumor caused by the inactivation of both alleles of the retinoblastoma gene (Rb1). Without Rb1 gene function, chromosomal aberrations are observed in retinoblastoma cells. The instability of the genome is closely associated with the repair of DNA double-strand breaks (DSBs). However, the precise molecular mechanism of action of Rb1 in DNA DSB repair remains unclear. Thus, in this study, we aimed to investigate whether the Rb1 gene affects DNA stability by assaying DNA DSB repair and also whether it regulates the proliferation of retinoblastoma cells. Rb1 immunofluorescence and RT-PCR were performed, demonstrating that the Rb1 gene is silenced in SO-Rb50 retinoblastoma cells, and the karyotype analysis of SO-Rb50 cells indicated that the loss of Rb1 function led to genomic instability; both numerical and structural chromosomal aberrations were observed in our study. In addition, the DNA DSB repair efficiency of the SO-Rb50 cells was measured by ?-H2AX immunofluorescence, a commonly used in situ marker of DNA DSBs, following exposure to ionizing radiation (IR) (2.5 and 5.0 Gy). We found that the DNA repair efficiency was significantly increased following IR-induced damage (P<0.01). However, there was no significant difference in DNA repair efficiency between the cells expressing exogenous Rb1 and the control (P>0.05). The assay for the screening of the effect of Rb1 on the sub-pathway of DNA DSB repair, non-homologous end joining (NHEJ) and homologous recombination (HR), indicated that Rb1 did not affect NHEJ activity, although it significantly promoted the HR pathway (HR levels increased by 2.46-fold) compared with the control (P<0.01). Furthermore, we found that the cell viability of the SO-Rb50 cells transfected with exogenous Rb1 was significantly inhibited (P<0.01) and cell cycle assay indicated that exogenous Rb1 induced S phase arrest (P<0.001) which also inhibited the proliferation of retinoblastoma cells (SO-Rb50) in vitro. Therefore, this study provides new insight into the mechanisms of action of the Rb1 gene in regulating the proliferation of retinoblastoma cells. PMID:23670186

Yang, Ying; Tian, Sijia; Brown, Bryce; Chen, Pei; Hu, Huan; Xia, Lei; Zhang, Jing; Cai, Xiaoxiao; Chen, Zhao; Pan, Xueke; Ge, Jian; Yu, Keming; Zhuang, Jing

2013-07-01

113

Selective inhibition of interleukin 2 gene function following thymocyte antigen/major histocompatibility complex receptor crosslinking: possible thymic selection mechanism.  

PubMed Central

Considerable evidence now exists to support the notion that the 50-kDa sheep erythrocyte-binding protein, T11, represents an essential cell surface component of a human T-cell lineage activation pathway. Furthermore, it is known that the human T3-Ti T-cell antigen/major histocompatibility complex receptor complex is capable of regulating cell growth mediated by the T11 structure. Here we show that, within the T3+ thymocyte compartment, T3-Ti crosslinking rapidly inhibits T11-initiated interleukin 2 (IL-2) gene transcription and translation. This inhibition is restricted to the IL-2 gene (IL2) as transcription of both the IL-2-receptor gene (IL2R) and the Ti beta-chain gene (TCRB) are not affected (human gene designations are in parentheses). Perhaps more importantly, T3-Ti-mediated IL-2 inhibition of this type is not operational in peripheral T lymphocytes. The results imply that the majority of T3+ thymocytes are functionally distinct from peripheral T lymphocytes despite their T3+ phenotype and must possess a unique endogenous regulatory component for suppressing IL-2 gene activity. Moreover, since IL-2 is likely rate-limiting for growth within the thymus, the findings provide one plausible mechanism for thymic selection--namely, T3-Ti crosslinking of thymocytes upon interaction with self-major histocompatibility complex inhibits clonal expansion of high-affinity autoreactive cells. Images

Ramarli, D; Fox, D A; Milanese, C; Reinherz, E L

1986-01-01

114

Adenovirus-mediated gene transfer of interferon alpha inhibits hepatitis C virus replication in hepatocytes.  

PubMed

Recently we reported that on-site interferon (IFN)-alpha production in the liver using an adenovirus vector can achieve a substantial confinement of IFN-alpha in the target organ and can improve liver fibrosis in a rat liver cirrhosis model. However, the major therapeutic effect of IFN for hepatitis C virus (HCV)-associated liver diseases is its antiviral effect on HCV. As a prelude to the in vivo HCV infection experiment using a primate animal model, here we examined the antiviral effect of IFN-alpha gene transfer into HCV-positive hepatocytes in vitro. The non-neoplastic human hepatocyte cell line PH5CH8 was inoculated with HCV-positive serum. Successful in vitro HCV replication and thus the validity of this model was confirmed by a strong selection for HCV variants determined by sequence analysis of the hypervariable region and an increase of HCV RNA estimated by real time TaqMan RT-PCR. One day after the inoculation of HCV, PH5CH8 cells were infected with adenoviral vectors encoding human IFN-alpha cDNA. HCV completely disappeared 9 days after the adenoviral infection, which is linked to the increase of 2('),5(')-oligoadenylate synthetase activity, suggesting that IFN-alpha produced by gene transfer effectively inhibits HCV replication in hepatocytes. This study supports the development of IFN-alpha gene therapy for HCV-associated liver diseases. PMID:12878183

Suzuki, Koichi; Aoki, Kazunori; Ohnami, Shumpei; Yoshida, Kimiko; Kazui, Teruhisa; Kato, Nobuyuki; Inoue, Kazuaki; Kohara, Michinori; Yoshida, Teruhiko

2003-08-01

115

Exonic microdeletions of the gephyrin gene impair GABAergic synaptic inhibition in patients with idiopathic generalized epilepsy.  

PubMed

Gephyrin is a postsynaptic scaffolding protein, essential for the clustering of glycine and ?-aminobutyric acid type-A receptors (GABAARs) at inhibitory synapses. An impairment of GABAergic synaptic inhibition represents a key pathway of epileptogenesis. Recently, exonic microdeletions in the gephyrin (GPHN) gene have been associated with neurodevelopmental disorders including autism spectrum disorder, schizophrenia and epileptic seizures. Here we report the identification of novel exonic GPHN microdeletions in two patients with idiopathic generalized epilepsy (IGE), representing the most common group of genetically determined epilepsies. The identified GPHN microdeletions involve exons 5-9 (?5-9) and 2-3 (?2-3), both affecting the gephyrin G-domain. Molecular characterization of the GPHN ?5-9 variant demonstrated that it perturbs the clustering of regular gephyrin at inhibitory synapses in cultured mouse hippocampal neurons in a dominant-negative manner, resulting in a significant loss of ?2-subunit containing GABAARs. GPHN ?2-3 causes a frameshift resulting in a premature stop codon (p.V22Gfs*7) leading to haplo-insufficiency of the gene. Our results demonstrate that structural exonic microdeletions affecting the GPHN gene constitute a rare genetic risk factor for IGE and other neuropsychiatric disorders by an impairment of the GABAergic inhibitory synaptic transmission. PMID:24561070

Dejanovic, Borislav; Lal, Dennis; Catarino, Claudia B; Arjune, Sita; Belaidi, Abdel A; Trucks, Holger; Vollmar, Christian; Surges, Rainer; Kunz, Wolfram S; Motameny, Susanne; Altmüller, Janine; Köhler, Anna; Neubauer, Bernd A; Epicure Consortium; Nürnberg, Peter; Noachtar, Soheyl; Schwarz, Günter; Sander, Thomas

2014-07-01

116

Inhibition of p-enolpyruvate carboxykinase gene expression: role of insulin, phorbol esters and/or calcium ionophore  

SciTech Connect

Both insulin and phorbol esters are known to inhibit the transcription of the gene for P-enolpyruvate carboxykinase (GTP) (PEPCK). The promoter-regulatory region of the PEPCK gene was analyzed for responsive sequences, using a series of chimeric genes constructed by fusing segments of the 5'-flanking region of the PEPCK gene (containing a graded set of deletions) to the structural gene for Herpes virus thymidine kinase (TK). These chimeric genes were transfected into TK-deficient rat hepatoma cells and their level of expression and regulation by insulin, phorbol ester and calcium ionophore was tested. A segment of the PEPCK promoter-regulatory region spanning 2 kb was shown to be sensitive to regulation by cAMP and glucocorticoids but not to insulin. In these same cells, insulin effectively inhibited the expression of the endogenous PEPCK gene. In a parallel set of experiments, they showed drastic alterations in the expression of the chimeric PEPCK-TK gene when cells were exposed to phorbol ester and/or calcium ionophore. These findings suggest that functional sequences required for insulin regulation of the PEPCK gene expression are 3' to the promoter-regulatory region of the gene.

Park, E.; Vandenbark, G.R.; Wong, S.S.C.; Wynshaw-Boris, A.; Hanson, R.W.

1986-05-01

117

Quercetin, a bioflavonoid, inhibits the increase of human multidrug resistance gene (MDR1) expression caused by arsenite.  

PubMed

Expression of the MDR1 gene, which encodes P-glycoprotein, is increased under some stress conditions. We have reported that quercetin, a bioflavonoid, inhibits the expression of heat-shock proteins. We have identified the effects of quercetin on the MDR1 gene expression in the human hepatocarcinoma cells line, HepG2. The increase of P-glycoprotein synthesis and MDR1 mRNA accumulation caused by exposure to arsenite were inhibited by quercetin. The CAT assay suggested that quercetin suppressed the transcriptional activation of the MDR1 gene after exposure to arsenite. Although many drugs that prevent the P-glycoprotein function have been reported, this is the first report to describe the inhibition of MDR1 expression by a reagent. PMID:1349537

Kioka, N; Hosokawa, N; Komano, T; Hirayoshi, K; Nagata, K; Ueda, K

1992-04-27

118

psi , a plasmid-linked Rhizobium phaseoli gene that inhibits exopolysaccharide production and which is required for symbiotic nitrogen fixation  

Microsoft Academic Search

A strain of R. phaseoli cured of its symbiotic plasmid, pRP2JI, retained the ability to make exopolysaccharide (EPS). However, a region of pRP2JI, when cloned at an increased copy number in wide host-range vectors and transferred to this and other strains of Rhizobium, inhibited EPS synthesis. The gene responsible was termed psi (polysaccharide inhibition) and was located in a region

D. Borthakur; J. A. Downie; A. W. B. Johnston; J. W. Lamb

1985-01-01

119

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

SciTech Connect

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 t

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

2008-01-15

120

Distinctive anatomical patterns of gene expression for cGMP-inhibited cyclic nucleotide phosphodiesterases.  

PubMed Central

Type III cGMP-inhibited phosphodiesterases (PDE3s) play important roles in hormonal regulation of lipolysis, platelet aggregation, myocardial contractility, and smooth muscle relaxation. We have recently characterized two PDE3 subtypes (PDE3A and PDE3B) as products of distinct but related genes. To elucidate their biological roles, in this study we compare cellular patterns of gene expression for these two enzymes during rat embryonic and postnatal development using in situ hybridization. PDE3B [corrected] mRNA is abundant in adipose tissue and is also expressed in hepatocytes throughout development. This mRNA is also highly abundant in embryonic neuroepithelium including the neural retina, but expression is greatly reduced in the mature nervous system. Finally, PDE3B [corrected] mRNA is localized in spermatocytes and renal collecting duct epithelium in adult rats. PDE3B mRNA is highly expressed in the cardiovascular system, including myocardium and arterial and venous smooth muscle, throughout development. It is also abundant in bronchial, genitourinary and gastrointestinal smooth muscle and epithelium, megakaryocytes, and oocytes. PDE3A [corrected] mRNA demonstrates a complex, developmentally regulated pattern of gene expression in the central nervous system. In summary, the two different PDE3s show distinctive tissue-specific patterns of gene expression suggesting that PDE3B [corrected] is involved in hormonal regulation of lipolysis and glycogenolysis, while regulation of myocardial and smooth muscle contractility appears to be a function of PDE3A [corrected]. In addition, the present findings suggest previously unsuspected roles for these enzymes in gametogenesis and neural development. Images

Reinhardt, R R; Chin, E; Zhou, J; Taira, M; Murata, T; Manganiello, V C; Bondy, C A

1995-01-01

121

Gene Delivery of Sarcoplasmic Reticulum Calcium ATPase Inhibits Ventricular Remodeling in Ischemic Mitral Regurgitation  

PubMed Central

Background Mitral regurgitation (MR) doubles mortality following myocardial infarction (MI). We have demonstrated that MR worsens remodeling after MI, and that early correction reverses remodeling. SERCA2a is downregulated in this process. We hypothesized that upregulating SERCA2a may inhibit remodeling in a surgical model of apical MI (no intrinsic MR) with independent MR-type flow. Methods and Results In 12 sheep, percutaneous gene delivery was performed using a validated protocol to perfuse both LAD and circumflex coronary arteries with occlusion of venous drainage. We administered adeno-associated virus 6 (AAV6) carrying SERCA2a under CMV promoter control in 6 sheep, and a reporter gene in 6 controls. After 2 weeks, standardized apical MI was created, and a shunt implanted between the LV and LA, producing regurgitant fractions of ~30%. Animals were compared at baseline, 1 and 3 months using 3D echo, Millar hemodynamics and biopsies. The SERCA2a group had well-maintained preload-recruitable stroke work at 3 months (decrease by 8±10% vs. 42±12% with reporter gene controls (p<0.001)). LV dP/dt followed the same pattern (no change vs. 55% decrease, p<0.001). LVESV was lower with SERCA2a (82.6±9 6 vs 99.4±9.7 ml, p=0.03); LVEDV, reflecting volume overload, was not significantly different (127.8±6.2 vs 134.3±9.4 ml). SERCA2a sheep showed 15% rise in anti-apoptotic pAkt vs. 30% reduction with reporter gene (P<0.001). Pro-hypertrophic activated STAT3 was also 41% higher with SERCA2a than in controls (p<0.001). Pro-apoptotic activated caspase-3 rose over 5-fold over 1 month in both SERCA2a and controls (p=NS), and decreased by 19% at 3 months, remaining elevated in both groups. Conclusions In this controlled model, upregulating SERCA2a induces better function and lesser remodeling, with improved contractility, smaller volume and activation of pro-hypertrophic/anti-apoptotic pathways. Although caspase-3 remains activated in both arms, SERCA2a sheep had increased molecular anti-remodeling “tone”. We therefore conclude that upregulating SERCA2a inhibits MR-induced post-MI remodeling in this model, and thus may constitute a useful approach to reduce the vicious cycle of remodeling in ischemic MR.

Beeri, Ronen; Chaput, Miguel; Guerrero, J. Luis; Kawase, Yoshiaki; Yosefy, Chaim; Abedat, Suzan; Karakikes, Ioannis; Morel, Charlotte; Tisosky, Ashley; Sullivan, Suzanne; Handschumacher, Mark; Gilon, Dan; Vlahakes, Gus J.; Hajjar, Roger J.; Levine, Robert A.

2010-01-01

122

Pharmacologic- and gene therapeutic-based inhibition of PKC?/? enhances cardiac contractility and attenuates heart failure  

PubMed Central

Background The conventional protein kinase C (PKC) isoform ? functions as a proximal regulator of Ca2+ handling in cardiac myocytes (Braz et al., 2004, Nat. Med. 10:248). Deletion of PKC? in the mouse resulted in augmented sarcoplasmic reticulum Ca2+ loading, enhanced Ca2+ transients, and augmented contractility, while overexpression of PKC? in the heart blunted contractility. Mechanistically, PKC? directly regulates Ca2+ handling by altering the phosphorylation status of inhibitor-1, which in turn suppresses protein phosphatase-1 activity, thus modulating phospholamban activity and secondarily, the sarcoplasmic reticulum Ca2+ ATPase (SERCA). Methods and Results Here we show that acute inhibition of the conventional PKC isoforms with Ro-32-0432 or Ro-31-8220 significantly augmented cardiac contractility in vivo or in an isolated work performing heart preparation in wildtype mice, but not in PKC? deficient mice. Ro-32-0432 also acutely increased cardiac contractility in two different models of heart failure in vivo. Acute or chronic treatment with Ro-31-8220 in a mouse model of heart failure due to deletion of the muscle lim protein (MLP) gene significantly augmented cardiac contractility and restored pump function. Moreover, adenoviral-mediated gene therapy with a dominant negative PKC? cDNA rescued heart failure in a chronic rat model of post-infarction cardiomyopathy. PKC? was also determined to be the dominant conventional PKC isoform expressed in the adult human heart, providing potential relevance of these findings to human pathophysiology. Conclusions Pharmacological inhibition of PKC?, or the conventional isoforms in general, may serve as a novel therapeutic strategy for acutely enhancing cardiac contractility in certain stages of heart failure.

Hambleton, Michael; Hahn, Harvey; Pleger, Sven T.; Kuhn, Matthew C.; Klevitsky, Raisa; Carr, Andrew N.; Kimball, Thomas F.; Hewett, Timothy E.; Dorn, Gerald W.; Koch, Walter J.; Molkentin, Jeffery D.

2009-01-01

123

Inverse PPAR?/? agonists suppress oncogenic signaling to the ANGPTL4 gene and inhibit cancer cell invasion.  

PubMed

Besides its established functions in intermediary metabolism and developmental processes, the nuclear receptor peroxisome proliferator-activated receptor ?/? (PPAR?/?) has a less defined role in tumorigenesis. In the present study, we have identified a function for PPAR?/? in cancer cell invasion. We show that two structurally divergent inhibitory ligands for PPAR?/?, the inverse agonists ST247 and DG172, strongly inhibit the serum- and transforming growth factor ? (TGF?)-induced invasion of MDA-MB-231 human breast cancer cells into a three-dimensional matrigel matrix. To elucidate the molecular basis of this finding, we performed chromatin immunoprecipitation sequencing (ChIP-Seq) and microarray analyses, which identified the gene encoding angiopoietin-like 4 (ANGPTL4) as the major transcriptional PPAR?/? target in MDA-MB-231 cells, previously implicated in TGF?-mediated tumor progression and metastatic dissemination. We show that the induction of ANGPTL4 by TGF? and other oncogenic signals is strongly repressed by ST247 and DG172 in a PPAR?/?-dependent fashion, resulting in the inhibition of ANGPTL4 secretion. This effect is attributable to these ligands' ability to induce a dominant transcriptional repressor complex at the site of transcription initiation that blocks preinitiation complex formation through an histone deacetylase-independent, non-canonical mechanism. Repression of ANGPTL4 transcription by inverse PPAR?/? agonists is functionally linked to the inhibition of cancer cell invasion into a three-dimensional matrix, as (i) invasion of MDA-MB-231 cells is critically dependent on ANGPTL4 expression, (ii) recombinant ANGPTL4 stimulates invasion, and (iii) reverses the inhibitory effect of ST247 and DG172. These findings indicate that a PPAR?/?-ANGPTL4 pathway is involved in the regulation of tumor cell invasion and that its pharmacological manipulation by inverse PPAR?/? agonists is feasible. PMID:23208498

Adhikary, T; Brandt, D T; Kaddatz, K; Stockert, J; Naruhn, S; Meissner, W; Finkernagel, F; Obert, J; Lieber, S; Scharfe, M; Jarek, M; Toth, P M; Scheer, F; Diederich, W E; Reinartz, S; Grosse, R; Müller-Brüsselbach, S; Müller, R

2013-10-31

124

Calcitonin Gene-related Peptide Inhibits Chemokine Production by Human Dermal Microvascular Endothelial Cells  

PubMed Central

This study examined whether the sensory neuropeptide calcitonin gene-related peptide (CGRP) inhibits release of chemokines by dermal microvascular endothelial cells. Dermal blood vessels are associated with nerves containing CGRP, suggesting that CGRP-containing nerves may regulate cutaneous inflammation through effects on vessels. We examined CGRP effects on stimulated chemokine production by a human dermal microvascular endothelial cell line (HMEC-1) and primary human dermal microvascular endothelial cells (pHDMECs). HMEC-1 cells and pHDMECs expressed mRNA for components of the CGRP and adrenomedullin receptors and CGRP inhibited LPS-induced production of the chemokines CXCL8, CCL2, and CXCL1 by both HMEC-1 cells and pHDMECs. The receptor activity-modifying protein (RAMP)1/calcitonin receptor-like receptor (CL)-specific antagonists CGRP8-37 and BIBN4096BS, blocked this effect of CGRP in a dose-dependent manner. CGRP prevented LPS-induced I?B? degradation and NF-?B binding to the promoters of CXCL1, CXCL8 and CCL2 in HMEC-1 cells and Bay 11-7085, an inhibitor of NF-?B activation, suppressed LPS-induced production of CXCL1, CXCL8 and CCL2. Thus, the NF-?B pathway appears to be involved in CGRP-mediated suppression of chemokine production. Accordingly, CGRP treatment of LPS-stimulated HMEC-1 cells inhibited their ability to chemoattract human neutrophils and mononuclear cells. Elucidation of this pathway may suggest new avenues for therapeutic manipulation of cutaneous inflammation.

Huang, Jing; Stohl, Lori L.; Zhou, Xi; Ding, Wanhong; Granstein, Richard D.

2011-01-01

125

Inhibition of xenograft human glioma tumor growth by lentivirus-mediated gene transfer of alphastatin.  

PubMed

Angiogenesis is crucial for the development and metastasis of human brain glioma. Based on our previous successful construction of a lentivirus-mediated alphastatin (an endogenous angiogenesis inhibitor) gene transfer system and our findings that alphastatin exhibited potent inhibitory effects on the migration and differentiation of human umbilical vein endothelial cell lines (HUVECs) induced by vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF) in vitro, here, we investigated the effect of using lentiviral vectors to overexpress alphastatin in human glioma cells to show whether sustained long-term expression of alphastatin diminishes tumor growth in a xenograft glioma model. We found that the transduced glioma cells sustainedly secreted alphastatin, which did not affect the proliferative ability of the glioma cells. Furthermore, tumor xenografts treated with the recombinant lentivirus were significantly smaller compared to the control xenografts and vascularity within the treated tumors was evidently decreased. Our data suggest that stable expression of alphastatin inhibits human glioma growth by inhibiting angiogenesis, with a probable mechanism of suppressing the turnover of VE-cadherin membrane molecules. PMID:23242200

Che, Hongmin; Song, Jianrong; Guo, Shiwen; Wang, Weiwen; Gao, Guodong

2013-03-01

126

ADVANCING AGE IS ASSOCIATED WITH GENE EXPRESSION CHANGES RESEMBLING mTOR INHIBITION: EVIDENCE FROM TWO HUMAN POPULATIONS  

PubMed Central

Interventions which inhibit TOR activity (including rapamycin and caloric restriction) lead to downstream gene expression changes and increased lifespan in laboratory models. However, the role of mTOR signaling in human aging is unclear. We tested the expression of mTOR-related transcripts in two independent study cohorts; the InCHIANTI population study of aging and the San Antonio Family Heart Study (SAFHS). Expression of 27/56 (InCHIANTI) and 19/44 (SAFHS) genes were associated with age after correction for multiple testing. 8 genes were robustly associated with age in both cohorts. Genes involved in insulin signaling (PTEN, PI3K, PDK1), ribosomal biogenesis (S6K), lipid metabolism (SREBF1), cellular apoptosis (SGK1), angiogenesis (VEGFB), insulin production and sensitivity (FOXO), cellular stress response (HIF1A) and cytoskeletal remodeling (PKC) were inversely correlated with age, whereas genes relating to inhibition of ribosomal components (4EBP1) and inflammatory mediators (STAT3) were positively associated with age in one or both datasets. We conclude that the expression of mTOR-related transcripts is associated with advancing age in humans. Changes seen are broadly similar to mTOR inhibition interventions associated with increased lifespan in animals. Work is needed to establish whether these changes are predictive of human longevity and whether further mTOR inhibition would be beneficial in older people.

Harries, Lorna W; Fellows, Alexander D.; Pilling, Luke C.; Hernandez, Dena; Singleton, Andrew; Bandinelli, Stefania; Guralnik, Jack; Powell, Jonathan; Ferruci, Luigi; Melzer, David

2014-01-01

127

Haplotype Polymorphism in the Alpha-2B-Adrenergic Receptor Gene Influences Response Inhibition in a Large Chinese Sample  

PubMed Central

Response inhibition refers to the suppression of inappropriate or irrelevant responses. It has a central role in executive functions, and has been linked to a wide spectrum of prevalent neuropsychiatric disorders. Increasing evidence from neuropharmacological studies has suggested that gene variants in the norepinephrine neurotransmission system make specific contributions to response inhibition. This study genotyped five tag single-nucleotide polymorphisms covering the whole alpha-2B-adrenergic receptor (ADRA2B) gene and investigated their associations with response inhibition in a relatively large healthy Chinese sample (N=421). The results revealed significant genetic effects of the ADRA2B conserved haplotype polymorphisms on response inhibition as measured by stop-signal reaction time (SSRT) (F(2,?418)=5.938, p=0.003). Individuals with the AAGG/AAGG genotype (n=89; mean SSRT=170.2?ms) had significantly shorter SSRTs than did those with either the CCAC/AAGG genotype (n=216; mean SSRT=182.4?ms; uncorrected p=0.03; corrected p=0.09) or the CCAC/CCAC genotype (n=116; mean SSRT=195.8?ms; corrected p<0.002, Cohen's d=0.51). This finding provides the first evidence from association research in support of a critical role of the norepinephrine neurotransmission system in response inhibition. A better understanding of the genetic basis of response inhibition would allow us to develop more effective diagnosis, treatment, and prevention of deficient or underdeveloped response inhibition as well as its related prevalent neuropsychiatric disorders.

Lei, Xuemei; Chen, Chuansheng; He, Qinghua; Moyzis, Robert; Xue, Gui; Chen, Chunhui; Cao, Zhongyu; Li, Jin; Li, He; Zhu, Bi; Zhang, Mingxia; Li, Jun; Dong, Qi

2012-01-01

128

L-carnosine inhibits metastasis of SK-Hep-1 cells by inhibition of matrix metaoproteinase-9 expression and induction of an antimetastatic gene, nm23-H1.  

PubMed

Antioxidants have been suggested to inhibit the expression of matrix metalloproteinases (MMPs), especially MMP-9, which plays a critical role in tumor metastasis. Because of its antioxidant activity and the ability to chelate divalent cations, L-carnosine (LC) was tested for inhibition of MMP-9 in a highly invasive hepatocarcinoma, SK-Hep-1 cells. We found that LC (50-1,000 microM) did not directly inhibit the activity of MMP-9 in a cell-free system. However, LC significantly inhibited the expression and activity of MMP-9 protein in SK-Hep-1 cells [inhibitory concentration of 50% (IC(50))| = 105 and 63 muM, respectively). Whereas LC did not inhibit the viability of SK-Hep-1 cells at concentrations up to 1,000 microM within 3 days of incubation, this dipeptide significantly inhibited cell migration (IC(50) = 82 microM) and invasion (IC(50) = 113 microM). LC significantly (P < 0.05) and dose dependently enhanced the expression of an antimetastatic gene, nonmetastatic cells 1, protein (nm23)-H1, at both protein and messenger ribonucleic acid (mRNA) levels. MMP-9 activity inversely correlated significantly with the expression of protein (r(2) = 0.77, P < 0.001) and mRNA (r(2) = 0.65, P < 0.001) of nm23-H1 in LC-treated cells. Thus, LC can inhibit the migration and invasion of SK-Hep-1 cells, and the effect is likely associated with upregulation of nm23-H1 and downregulation of MMP-9 expression. PMID:18584487

Chuang, Cheng-Hung; Hu, Miao-Lin

2008-01-01

129

Inhibition of Virulence Gene Expression in Staphylococcus aureus by Novel Depsipeptides from a Marine Photobacterium  

PubMed Central

During a global research expedition, more than five hundred marine bacterial strains capable of inhibiting the growth of pathogenic bacteria were collected. The purpose of the present study was to determine if these marine bacteria are also a source of compounds that interfere with the agr quorum sensing system that controls virulence gene expression in Staphylococcus aureus. Using a gene reporter fusion bioassay, we recorded agr interference as enhanced expression of spa, encoding Protein A, concomitantly with reduced expression of hla, encoding ?-hemolysin, and rnaIII encoding RNAIII, the effector molecule of agr. A marine Photobacterium produced compounds interfering with agr in S. aureus strain 8325-4, and bioassay-guided fractionation of crude extracts led to the isolation of two novel cyclodepsipeptides, designated solonamide A and B. Northern blot analysis confirmed the agr interfering activity of pure solonamides in both S. aureus strain 8325-4 and the highly virulent, community-acquired strain USA300 (CA-MRSA). To our knowledge, this is the first report of inhibitors of the agr system by a marine bacterium.

Mansson, Maria; Nielsen, Anita; Kjaerulff, Louise; Gotfredsen, Charlotte H.; Wietz, Matthias; Ingmer, Hanne; Gram, Lone; Larsen, Thomas O.

2011-01-01

130

Von Willebrand Factor Inhibits Mature Smooth Muscle Gene Expression through Impairment of Notch Signaling  

PubMed Central

Von Willebrand factor (vWF), a hemostatic protein normally synthesized and stored by endothelial cells and platelets, has been localized beyond the endothelium in vascular disease states. Previous studies have implicated potential non-hemostatic functions of vWF, but signaling mechanisms underlying its effects are currently undefined. We present evidence that vWF breaches the endothelium and is expressed in a transmural distribution pattern in cerebral small vessel disease (SVD). To determine the potential molecular consequences of vWF permeation into the vessel wall, we also tested whether vWF impairs Notch regulation of key smooth muscle marker genes. In a co-culture system using Notch ligand expressing cells to stimulate Notch in A7R5 cells, vWF strongly inhibited both the Notch pathway and the activation of mature smooth muscle gene promoters. Similar repressive effects were observed in primary human cerebral vascular smooth muscle cells. Expression of the intracellular domain of NOTCH3 allowed cells to bypass the inhibitory effects of vWF. Moreover, vWF forms molecular complexes with all four mammalian Notch ectodomains, suggesting a novel function of vWF as an extracellular inhibitor of Notch signaling. In sum, these studies demonstrate vWF in the vessel wall as a common feature of cerebral SVD; furthermore, we provide a plausible mechanism by which non-hemostatic vWF may play a novel role in the promotion of vascular disease.

Lee, Soo Jung; Wang, Michael M.

2013-01-01

131

Signaling by vitamin A and retinol-binding protein regulates gene expression to inhibit insulin responses  

PubMed Central

It currently is believed that vitamin A, retinol, functions through active metabolites: the visual chromophore 11-cis-retinal, and retinoic acids, which regulate gene transcription. Retinol circulates in blood bound to retinol-binding protein (RBP) and is transported into cells by a membrane protein termed “stimulated by retinoic acid 6” (STRA6). We show here that STRA6 not only is a vitamin A transporter but also is a cell-surface signaling receptor activated by the RBP–retinol complex. Association of RBP-retinol with STRA6 triggers tyrosine phosphorylation, resulting in recruitment and activation of JAK2 and the transcription factor STAT5. The RBP–retinol/STRA6/JAK2/STAT5 signaling cascade induces the expression of STAT target genes, including suppressor of cytokine signaling 3 (SOCS3), which inhibits insulin signaling, and peroxisome proliferator-activated receptor gamma (PPAR?), which enhances lipid accumulation. These observations establish that the parental vitamin A molecule is a transcriptional regulator in its own right, reveal that the scope of biological functions of the vitamin is broader than previously suspected, and provide a rationale for understanding how RBP and retinol regulate energy homeostasis and insulin responses.

Berry, Daniel C.; Jin, Hui; Majumdar, Avijit; Noy, Noa

2011-01-01

132

PETAL LOSS is a boundary gene that inhibits growth between developing sepals in Arabidopsis thaliana.  

PubMed

Flower primordia are partitioned by boundaries during their early development. Such boundaries occur between whorls of organs, and also between organs within whorls. PETAL LOSS (PTL) is a trihelix transcription factor gene that is expressed in boundaries between sepal primordia in the outer whorl. Over-expression of PTL results in growth suppression suggesting that PTL normally inhibits growth between newly arising sepals. We have tested this by examining the consequences of loss of PTL function using confocal imaging. The size of the inter-sepal zone in stage 4 buds expands radially by 35-40% in ptl-1 mutants as a consequence of additional cell proliferation. There is no change in the size of PTL-expressing cells. PTL expression does not overlap with the sites of petal initiation identified using the DR5 auxin response reporter. The latter are closer to the centre of the flower. Thus the consequence of loss of PTL function on petal initiation is indirect, perhaps through interference with a mobile petal-initiation signal or movement of the PTL protein. CUP-SHAPED COTYLEDON (CUC) genes are also involved in defining inter-sepal boundaries. However, genetic studies combining ptl with loss of cuc1 function, and gain of CUC function in extra early petals-1 (miR164c) mutants, have revealed that CUC and PTL act differently. CUC suppresses growth of sepal tissues from the boundary region whereas PTL acts to keep the size of the boundary in check. PMID:22507233

Lampugnani, Edwin R; Kilinc, Aydin; Smyth, David R

2012-09-01

133

Engineered RNase P Ribozymes Effectively Inhibit Human Cytomegalovirus Gene Expression and Replication  

PubMed Central

RNase P ribozyme can be engineered to be a sequence-specific gene-targeting agent with promising application in both basic research and clinical settings. By using an in vitro selection system, we have previously generated RNase P ribozyme variants that have better catalytic activity in cleaving an mRNA sequence than the wild type ribozyme. In this study, one of the variants was used to target the mRNA encoding human cytomegalovirus (HCMV) essential transcription factor immediate-early protein 2 (IE2). The variant was able to cleave IE2 mRNA in vitro 50-fold better than the wild type ribozyme. A reduction of about 98% in IE2 expression and a reduction of 3500-fold in viral production was observed in HCMV-infected cells expressing the variant compared to a 75% reduction in IE2 expression and a 100-fold reduction in viral production in cells expressing the ribozyme derived from the wild type sequence. These results suggest that ribozyme variants that are selected to be highly active in vitro are also more effective in inhibiting the expression of their targets in cultured cells. Our study demonstrates that RNase P ribozyme variants are efficient in reducing HCMV gene expression and growth and are potentially useful for anti-viral therapeutic application.

Yang, Zhu; Vu, Gia-Phong; Qian, Hua; Chen, Yuan-Chuan; Wang, Yu; Reeves, Michael; Zen, Ke; Liu, Fenyong

2014-01-01

134

Changes in rice allelopathy and rhizosphere microflora by inhibiting rice phenylalanine ammonia-lyase gene expression.  

PubMed

Gene expression of phenylalanine ammonia-lyase (PAL) in allelopathic rice PI312777 was inhibited by RNA interference (RNAi). Transgenic rice showed lower levels of PAL gene expression and PAL activity than wild type rice (WT). The concentrations of phenolic compounds were lower in the root tissues and root exudates of transgenic rice than in those of wild type plants. When barndyardgrass (BYG) was used as the receiver plant, the allelopathic potential of transgenic rice was reduced. The sizes of the bacterial and fungal populations in rice rhizospheric soil at the 3-, 5-, and 7-leaf stages were estimated by using quantitative PCR (qPCR), which showed a decrease in both populations at all stages of leaf development analyzed. However, PI312777 had a larger microbial population than transgenic rice. In addition, in T-RFLP studies, 14 different groups of bacteria were detected in WT and only 6 were detected in transgenic rice. This indicates that there was less rhizospheric bacterial diversity associated with transgenic rice than with WT. These findings collectively suggest that PAL functions as a positive regulator of rice allelopathic potential. PMID:23385369

Fang, Changxun; Zhuang, Yuee; Xu, Tiecheng; Li, Yingzhe; Li, Yue; Lin, Wenxiong

2013-02-01

135

Histone Deacetylation of RB-Responsive Promoters: Requisite for Specific Gene Repression but Dispensable for Cell Cycle Inhibition  

Microsoft Academic Search

The retinoblastoma tumor suppressor protein (RB) is targeted for inactivation in the majority of human tumors, underscoring its critical role in attenuating cellular proliferation. RB inhibits proliferation by re- pressing the transcription of genes that are essential for cell cycle progression. To repress transcription, RB assembles multiprotein complexes containing chromatin-modifying enzymes, including histone deacetylases (HDACs). However, the extent to which

Hasan Siddiqui; David A. Solomon; Ranjaka W. Gunawardena; Ying Wang; Erik S. Knudsen

2003-01-01

136

In vivo transcription of a progesterone-responsive gene is specifically inhibited by a triplex-forming oligonucleotide.  

PubMed Central

Oligonucleotides provide novel reagents for inhibition of gene expression because of their high affinity binding to specific nucleotide sequences. We describe a 38 base, single-stranded DNA that forms a triple helix or 'triplex' on progesterone response elements of a target gene. This triplex-forming oligonucleotide binds with a Kd = 100 nM at 37 degrees C and physiological pH, and blocks binding of progesterone receptors to the target. Furthermore, it completely inhibited progesterone receptor-dependent transcription in vitro. To approach in vivo conditions, triplex-forming oligonucleotides were tested in cell transfection studies. The derivation of the oligonucleotides with cholesterol enhanced their cellular uptake and nuclear concentration by at least four-fold. The cholesterol-derivatized triplex-forming oligonucleotide specifically inhibited transcription of the PRE-containing reporter gene in cells when applied to the medium at micromolar concentrations. This is the first demonstration of steroid-responsive gene inhibition by triplex formation and joins the growing body of evidence indicating that oligonucleotides have therapeutic potential. Images

Ing, N H; Beekman, J M; Kessler, D J; Murphy, M; Jayaraman, K; Zendegui, J G; Hogan, M E; O'Malley, B W; Tsai, M J

1993-01-01

137

Inhibition of LINE-1 retrotransposon-encoded reverse transcriptase modulates the expression of cell differentiation genes in breast cancer cells.  

PubMed

Long Interspersed Elements (L1 elements) are biologically active retrotransposons that are capable of autonomous replication using their own reverse transcriptase (RT) enzyme. Expression of the normally repressed RT has been implicated in cancer cell growth. However, at present, little is known about the expression of L1-encoded RT activity or the molecular changes that are associated with RT activity in the development of breast cancer. Here, we report that RT activity is widespread in breast cancer cells. The expression of RT protein decreased markedly in breast cancer cells after treatment with the antiretroviral drug, efavirenz. While the majority of cells showed a significant reduction in proliferation, inhibition of RT was also accompanied by cell-specific differences in morphology. MCF7 cells displayed elongated microtubule extensions that adhered tightly to their substrate, while a large fraction of the T47D cells that we studied formed long filopodia projections. These morphological changes were reversible upon cessation of RT inhibition, confirming their dependence on RT activity. We also carried out gene expression profiling with microarrays and determined the genes that were differentially expressed during the process of cellular differentiation. Genes involved in proliferation, cell migration, and invasive activity were repressed in RT-inhibited cells. Concomitantly, genes involved in cell projection, formation of vacuolar membranes, and cell-to-cell junctions were significantly upregulated in RT-inhibited cells. qRT-PCR examination of the mRNA expression of these genes in additional cell lines yielded close correlation between their differential expression and the degree of cellular differentiation. Our study demonstrates that the inhibition of L1-encoded RT can reduce the rate of proliferation and promote differentiation of breast cancer cells. Together, these results provide a direct functional link between the expression of L1 retrotransposons and the development of breast cancer. PMID:24337508

Patnala, Radhika; Lee, Sung-Hun; Dahlstrom, Jane E; Ohms, Stephen; Chen, Long; Dheen, S Thameem; Rangasamy, Danny

2014-01-01

138

Optically Pure Abscisic Acid Analogs--Tools for Relating Germination Inhibition and Gene Expression in Wheat Embryos 1  

PubMed Central

We report an examination of the structural requirements of the abscisic acid (ABA) recognition response in wheat dormant seed embryos using optically pure isomers of ABA analogs. These compounds include permutations to the ABA structure with either an acetylene or a trans bond at C-4 C-5, and either a single or double bond at the C-2? C-3? double bond. (R)-ABA and the three isomers with the same configuration at C-1? as natural ABA were found to be effective germination inhibitors. The biologically active ABA analogs exhibited differential effects on ABA-responsive gene expression. All the ABA analogs that inhibited germination induced two ABA-responsive genes, wheat group 3 lea and dhn (rab). However, (R)-ABA and (S)-dihydroABA were less effective in inducing the ABA-responsive gene Em within the time that embryonic germination was inhibited. ImagesFigure 3Figure 4

Walker-Simmons, M. K.; Anderberg, Robert J.; Rose, Patricia A.; Abrams, Suzanne R.

1992-01-01

139

Dual inhibition of ?-oryzanol on cellular melanogenesis: inhibition of tyrosinase activity and reduction of melanogenic gene expression by a protein kinase A-dependent mechanism.  

PubMed

The in vitro effects on melanogenesis of ?-oryzanol (1), a rice bran-derived phytosterol, were investigated. The melanin content in B16F1 cells was significantly and dose-dependently reduced (-13% and -28% at 3 and 30 ?M, respectively). Tyrosinase enzyme activity was inhibited by 1 both in a cell-free assay and when analyzed based on the measurement of cellular tyrosinase activity. Transcriptome analysis was performed to investigate the biological pathways altered by 1, and it was found that gene expression involving protein kinase A (PKA) signaling was markedly altered. Subsequent analyses revealed that 1 stimulation in B16 cells reduced cytosolic cAMP concentrations, PKA activity (-13% for cAMP levels and -40% for PKA activity), and phosphorylation of the cAMP-response element binding protein (-57%), which, in turn, downregulated the expression of microphthalmia-associated transcription factor (MITF; -59% for mRNA and -64% for protein), a key melanogenic gene transcription factor. Accordingly, tyrosinase-related protein 1 (TRP-1; -69% for mRNA and -82% for protein) and dopachrome tautomerase (-51% for mRNA and -92% for protein) in 1-stimulated B16F1 cells were also downregulated. These results suggest that 1 has dual inhibitory activities for cellular melanogenesis by inhibiting tyrosinase enzyme activity and reducing MITF and target genes in the PKA-dependent pathway. PMID:23031087

Jun, Hee-jin; Lee, Ji Hae; Cho, Bo-Ram; Seo, Woo-Duck; Kang, Hang-Won; Kim, Dong-Woo; Cho, Kang-Jin; Lee, Sung-Joon

2012-10-26

140

RNA polymerase II transcription inhibits DNA repair by photolyase in the transcribed strand of active yeast genes.  

PubMed Central

Yeast uses nucleotide excision repair (NER) and photolyase (photoreactivation) to repair cyclobutane pyrimidine dimers (CPDs) generated by ultraviolet light. In active genes, NER preferentially repairs the transcribed strand (TS). In contrast, we recently showed that photolyase preferentially repairs the non-transcribed strands (NTS) of the URA3 and HIS3 genes in minichromosomes. To test whether photoreactivation depends on transcription, repair of CPDs was investigated in the transcriptionally regulated GAL10 gene in a yeast strain deficient in NER [AMY3 (rad1Delta)]. In the active gene (cells grown in galactose), photoreactivation was fast in the NTS and slow in the TS demonstrating preferential repair of the NTS. In the inactive gene (cells grown in glucose), both strands were repaired at similar rates. This suggests that RNA polymerases II blocked at CPDs inhibit accessibility of CPDs to photolyase. In a strain in which both pathways are operational [W303-1a (RAD1)], no strand bias was observed either in the active or inactive gene, demonstrating that photoreactivation of the NTS compensates preferential repair of the TS by NER. Moreover, repair of the NTS was more quickly in the active gene than in the repressed gene indicating that transcription dependent disruption of chromatin facilitates repair of an active gene.

Livingstone-Zatchej, M; Meier, A; Suter, B; Thoma, F

1997-01-01

141

Effective inhibition of Japanese encephalitis virus replication by shRNAs targeting various viral genes in vitro and in vivo.  

PubMed

Japanese encephalitis virus (JEV) is a serious mosquito-borne flavivirus that causes acute encephalitis in humans and many animals, with a high fatality rate. RNA interference (RNAi) is an evolutionarily conserved mechanism for the specific suppression of gene expression, which can be used as a reasonable antiviral strategy. In this study, 10 shRNAs targeting different regions of the JEV genome were designed, and their inhibition of viral replication in vitro and in vivo was determined. Treatment with these shRNAs significantly inhibited JEV replication in BHK-21 and SK-N-SH cells. An immunohistochemical analysis of suckling mice showed that brain sections pretreated with pGP-JE-1, pGP-JE-2 or pGP-JE-3 lacked viral particles. The survival of BALB/c mice challenged with 20 LD50 of the JEV NJ2008 strain at 24h post-injection or simultaneously with pGP-JE-2 was 83.3% and 66.7%, respectively. The results demonstrated that the efficiency of gene silencing and virus inhibition varied between shRNAs to different target genes and sites. Meanwhile, the shRNA-mediated antiviral effect was dose- and time-dependent, including prophylactic and therapeutic effect on virus infection both in vitro and in vivo. The whole results indicate that these shRNAs can inhibit JEV infection sufficiently in vitro and in vivo and might be a potential new tool for controlling JEV infection. PMID:24725931

Shen, Ting; Liu, Ke; Miao, Denian; Cao, Ruibing; Chen, Puyan

2014-04-01

142

Effect of shRNA-mediated inhibition of Nanog gene expression on the behavior of human gastric cancer cells  

PubMed Central

The aim of the present study was to employ RNA interference (RNAi) technology to construct and select shRNA-Nanog recombinant plasmids for the inhibition of Nanog gene expression and transfer these plasmids into the human gastric cancer cell line, SGC-7901, as well as to detect the expression of Nanog and the effects on the proliferation, migration, invasion, cell cycle and apoptosis of SGC-7901 cells. The pshRNA-Nanog interference plasmids were constructed and used to transfect SGC-7901 cells using lipofectamine. The expression of the Nanog gene was detected by fluorescence microscopy, RT-PCR and western blotting, and the most markedly inhibited group was identified. The SGC-7901 cells were transfected with recombinant shRNA-Nanog plasmids from the most markedly inhibited group using lipofectamine and the effect on proliferation was determined by CCK-8 assay. The migration and invasion of the SGC-7901 cells was determined by Transwell assays, while the cell cycle and apoptosis were analyzed by flow cytometry. The group with the highest inhibition rate was successfully constructed and identified. It was observed that the proliferation, invasion and migration capacity of the cells was reduced, that the cell cycle was arrested at the S phase and that apoptosis was significantly increased. The Nanog gene in gastric cancer cells is closely associated with cell proliferation, the cell cycle, apoptosis and migration and invasion abilities. The present study establishes the foundations for a novel approach for the genetic treatment of gastric cancer.

JI, WEN; JIANG, ZHENG

2013-01-01

143

Chromosomal-gene-mediated inhibition of intestinal and foodborne pathogens by Lactobacillus acidophilus AA11.  

PubMed

Approximately 63 strains of Lactobacillus acidophilus were isolated from Egyptian home-made cheese and examined for production of antagonism. Only eight strains demonstrated inhibitory activity against spoilage microorganisms (i.e. Staphylococcus aureus and Bacillus cereus) and pathogens (i.e. E. coli, Salmonella sp. and Shigella sp.). Lactobacillus acidophilus AA11 produced a more antimicrobial activity with a wide range of inhibition. The agent AA11 was sensitive to proteolytic enzymes and retained full activity after 30 min at 100 degrees C. Activity against sensitive cells was bactericidal but not bacteriolytic. The compound was produced during growth phase and can be extracted from the culture supernatant fluids with n-Butanol. 12 % SDS-PAGE analysis of 40% ammonium sulphate precipitated agent showed two peptides with molecular weights of approximately 36 kDa and approximately 29 kDa. No plasmid was identified in Lactobacillus acidophilus AA11 indicating that the genes encoding the inhibitory agent located on the chromosome. These characteristics identify the inhibitory substance as a bacteriocin, designated acidocin AA11 and confer the agent an application potential as a biopreservative. PMID:17357571

Abo-Amer, Aly E

2006-01-01

144

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

PubMed Central

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.

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 ?strup; Moser, Claus; Nielsen, Kristian Fog; Eberl, Leo; Larsen, Thomas Ostenfeld; Tanner, David; H?iby, Niels; Bjarnsholt, Thomas

2012-01-01

145

Calcitonin gene-related peptide inhibits Langerhans cell-mediated HIV-1 transmission  

PubMed Central

Upon its mucosal entry, human immunodeficiency virus type 1 (HIV-1) is internalized by Langerhans cells (LCs) in stratified epithelia and transferred locally to T cells. In such epithelia, LCs are in direct contact with peripheral neurons secreting calcitonin gene–related peptide (CGRP). Although CGRP has immunomodulatory effects on LC functions, its potential influence on the interactions between LCs and HIV-1 is unknown. We show that CGRP acts via its receptor expressed by LCs and interferes with multiple steps of LC-mediated HIV-1 transmission. CGRP increases langerin expression, decreases selected integrins, and activates NF-?B, resulting in decreased HIV-1 intracellular content, limited formation of LC–T cell conjugates, and elevated secretion of the CCR5-binding chemokine CCL3/MIP-1?. These mechanisms cooperate to efficiently inhibit HIV-1 transfer from LCs to T cells and T cell infection. In vivo, HIV-1 infection decreases CGRP plasma levels in both vaginally SHIV-challenged macaques and HIV-1–infected individuals. CGRP plasma levels return to baseline after highly active antiretroviral therapy. Our results reveal a novel path by which a peripheral neuropeptide acts at the molecular and cellular levels to limit mucosal HIV-1 transmission and suggest that CGRP receptor agonists might be used therapeutically against HIV-1.

Ganor, Yonatan; Drillet-Dangeard, Anne-Sophie; Lopalco, Lucia; Tudor, Daniela; Tambussi, Giuseppe; Delongchamps, Nicolas Barry; Zerbib, Marc

2013-01-01

146

Gene Delivery to Overcome Astrocyte Inhibition of Axonal Growth: An In Vitro Model of the Glial Scar  

PubMed Central

After injury to the central nervous system, a glial scar develops that physically and biochemically inhibits axon growth. In the scar, activated astrocytes secrete inhibitory extracellular matrix, of which chondroitin sulfate proteoglycans (CSPGs) are considered the major inhibitory component. An inhibitory interface of CSPGs forms around the lesion and prevents axons from traversing the injury, and decreasing CSPGs can enhance axon growth. In this report, we established an in vitro interface model of activated astrocytes and subsequently investigated gene delivery as a means to reduce CSPG levels and enhance axon growth. In the model, a continuous interface of CSPG producing astrocytes was created with neurons seeded opposite the astrocytes, and neurite crossing, stopping, and turning were evaluated as they approached the interface. We investigated the efficacy of lentiviral delivery to degrade or prevent the synthesis of CSPGs, thereby removing CSPG inhibition of neurite growth. Lentiviral delivery of RNAi targeting two key CSPG synthesis enzymes, chondroitin polymerizing factor and chondroitin synthase-1, decreased CSPGs, and reduced inhibition by the interface. Degradation of CSPGs by lentiviral delivery of chondroitinase also resulted in less inhibition and more neurites crossing the interface. These results indicate that the interface model provides a tool to investigate interventions that reduce inhibition by CSPGs, and that gene delivery can be effective in promoting neurite growth across an interface of CSPG producing astrocytes.

Tuinstra, Hannah M.; Ducommun, Melissa M.; Briley, William E.; Shea, Lonnie D.

2014-01-01

147

Effect of myeloperoxidase inhibition on gene expression profiles in HL-60 cells exposed to 1,2,4,-benzenetriol.  

PubMed

While it is known that benzene induces myeloid leukemia in humans, the mechanism has yet to be clarified. Previously, we suggested that myeloperoxidase (MPO) was the key enzyme because it promotes generation of powerful oxidant hypochlorous acid (HOCl) which, reacting with DNA, causes leukemogenesis. In this study, using a whole-human-genome oligonucleotide microarray to clarify the relationships between myelotoxicity of benzene and MPO, we analyzed the genome-wide expression profiles of HL-60 human promyelocytic cell lines exposed to 1,2,4-benzenetriol (BT) with or without MPO inhibition. The microarray analysis revealed that short (1 h) and longer (4 h) exposure to BT changed the expression in HL-60 cells of 1,213 or 1,214 genes associated with transcription, RNA metabolic processes, immune response, apoptosis, cell death, and biosynthetic processes (|Z-score|> 2.0), and that these changes were dramatically lessened by MPO-specific inhibition. The presence of functionally important genes and, specifically, genes related to apoptosis, carcinogenesis, regulation of transcription, immune responses, oxidative stress, and cell-cycle regulation were further validated by real-time RT-PCR. Gene expression profiles along with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation analysis suggest that BT-induced DNA halogenation by MPO is a primary reaction in the leukemogenesis associated with benzene. PMID:24530881

Miyahara, Emiko; Nishikawa, Takuro; Takeuchi, Toru; Yasuda, Kaori; Okamoto, Yasuhiro; Kawano, Yoshifumi; Horiuchi, Masahisa

2014-03-20

148

Reversal of Cancer Phenotype by Inhibiting Expression of Prostate Tumor Inducing Gene.  

National Technical Information Service (NTIS)

This invention provides a method for reversing cancer phenotype of a cancer cell which comprises introducing an exogenous material which is capable of specifically recognizing either a Prostate Tumor Inducing Gene, RNA of said gene or gene product of said...

P. B. Fisher

2004-01-01

149

Methods and Compositions for the Specific Inhibition of Gene Expression by Double-Stranded RNA.  

National Technical Information Service (NTIS)

The invention provides compositions and methods for selectively reducing the expression of a gene product from a desired target gene, as well as treating diseases caused by expression of the gene. The method involves introducing into the environment of a ...

D. Kim J. J. Rossi M. A. Behlke

2005-01-01

150

Inhibition of interferon-inducible gene expression by adenovirus E1A proteins: Block in transcriptional complex formation  

SciTech Connect

Infection with wild-type adenovirus 5, but not with a mutant lacking the E1A gene, prevented the induction by interferon (IFN) {alpha} of chloramphenicol acetyltransferase (CAT) activity in HeLaM cell lines that had been permanently transfected with chimeric CAT reporter genes driven by the transcriptional regulatory regions of the IFN-inducible CAT activity was observed in cells that were cotransfected with the same reporter genes and plasmids expressing either the E1A 289- or 243-amino acid protein. These proteins also prevented the induction of CAT activity by IFN-{gamma} from a cotransfected HLA-DR{alpha}-CAT gene. Experiments with E1A mutants mapped the inhibitory activity to amino acid residues 38-65 of these proteins. In a HeLa cell line permanently expressing the E1A 289-amino acid protein, the replication of vesicular stomatitis virus and encephalomyocarditis virus was not inhibited by IFN-{alpha}, suggesting a global blockade of IFN responses. The observed transcriptional inhibition could be attributed to the lack of formation of the crucial IFN-stimulated gene factor 3 (ISGF3) transcriptional complex. As shown by mobility shift assays, this complex was not formed in the nuclear extracts of IFN-treated adenovirus-infected cells or IFN-treated E1A-producing cells. These nuclear extracts were deficient in both ISGF3{alpha} and ISGF3{gamma} subunits. However, they did not block the formation of ISGF3 complex from exogenously added components.

Kalvakolanu, D.V.R.; Bandyopadhyay, S.K.; Harter, M.L.; Sen, G.C. (Cleveland Clinic Foundation, OH (United States))

1991-09-01

151

Inhibition of expression of the chromatin remodeling gene, SNF2L, selectively leads to DNA damage, growth inhibition, and cancer cell death.  

PubMed

SNF2L, a chromatin remodeling gene expressed in diverse tissues, cancers, and derived cell lines, contributes to the chromatin remodeling complex that facilitates transcription. Because of this wide expression, it has not been exploited as a cancer therapeutic target. However, based on our present studies, we find that cancer cells, although expressing SNF2L at similar levels as their normal counterparts, are sensitive to its knockdown. This is not observed when its imitation SWI ortholog, SNF2H, is inhibited. SNF2L siRNA inhibition using two different siRNAs separately reduced SNF2L transcript levels and protein in both normal and cancer lines, but only the cancer lines showed significant growth inhibition, DNA damage, a DNA damage response, and phosphorylation of checkpoint proteins and marked apoptosis. DNA damage and the damage response preceded apoptosis rather than being consequences of it. The damage response consisted of increased phosphorylation of multiple substrates including ATR, BRCA1, CHK1, CHK2, and H2AX. Both the total and phosphorylated levels of p53 increased. The downstream targets of p53, p21, GADD45A, and 14-3-3sigma, were also upregulated. The alterations in checkpoint proteins included increased phosphorylated cdc2 but not Rb, which resulted in a modest G(2)-M arrest. Although apoptosis may be mediated by Apaf-1/caspase 9, other caspases could be involved. Other members of the chromatin remodeling or SWI/SNF gene families exhibited overall reduced levels of expression in the cancer lines compared with the normal lines. This raised the hypothesis that cancers are sensitive to SNF2L knockdown because, unlike their normal counterparts, they lack sufficient compensation from other family members. PMID:19996304

Ye, Yin; Xiao, Yi; Wang, Wenting; Wang, Qien; Yearsley, Kurtis; Wani, Altaf A; Yan, Qintao; Gao, Jian-Xin; Shetuni, Brandon S; Barsky, Sanford H

2009-12-01

152

Sodium arsenite induces orphan nuclear receptor SHP gene expression via AMP-activated protein kinase to inhibit gluconeogenic enzyme gene expression.  

PubMed

Sodium arsenite has been demonstrated to alter the expression of genes associated with glucose homeostasis in tissues involved in the pathogenesis of type 2 diabetes; however, the underlying molecular mechanism has not been fully elucidated yet. In this study, we report that the sodium arsenite-induced gene expression of the small heterodimer partner (SHP; NR0B2), an atypical orphan nuclear receptor, regulates the expression of hepatic gluconeogenic genes. Sodium arsenite augments hepatic SHP mRNA levels in an AMP-activated protein kinase (AMPK)-dependent manner. Sodium arsenite activated AMPK and was shown to perturb cellular ATP levels. The arsenite-induced SHP mRNA level was blocked by adenoviral overexpression of dominant negative AMPK (Ad-dnAMPKalpha) or by the AMPK inhibitor compound C in hepatic cell lines. We demonstrated the dose-dependent induction of SHP mRNA levels by sodium arsenite and repressed the forskolin/dexamethasone-induced gene expression of the key hepatic gluconeogenic genes phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase). Ad-dnAMPKalpha blocked the repressive effects of arsenite-induced SHP on PEPCK and G6Pase. Sodium arsenite inhibited the promoter activity of PEPCK and G6Pase, and this repression was abolished by small interfering (si)RNA SHP treatments. The knockdown of SHP expression by oligonucleotide siRNA SHP or adenoviral siRNA SHP released the sodium arsenite-mediated repression of forskolin/dexamethasone-stimulated PEPCK and G6Pase gene expression in a variety of hepatic cell lines. Results from our study suggest that sodium arsenite induces SHP via AMPK to inhibit the expression of hepatic gluconeogenic genes and also provide us with a novel molecular mechanism of arsenite-mediated regulation of hepatic glucose homeostasis. PMID:18505831

Chanda, Dipanjan; Kim, Sung-Jin; Lee, In-Kyu; Shong, Minho; Choi, Hueng-Sik

2008-08-01

153

Genomic targeting and mapping of tiller inhibition gene ( tin3 ) of wheat using ESTs and synteny with rice  

Microsoft Academic Search

Changes in plant architecture have been central to the domestication of wild species. Tillering or the degree of branching\\u000a determines shoot architecture and is a key component of grain yield and\\/or biomass. Previously, a tiller inhibition mutant with monoculm phenotype was isolated and the mutant gene (tin3) was mapped in the distal region of chromosome arm 3AmL of Triticum monococcum.

Vasu Kuraparthy; Shilpa Sood; Bikram S. Gill

2008-01-01

154

Inhibitors of histone demethylation and histone deacetylation cooperate in regulating gene expression and inhibiting growth in human breast cancer cells  

PubMed Central

Abnormal activities of histone lysine demethylases (KDMs) and lysine deacetylases (HDACs) are associated with aberrant gene expression in breast cancer development. However, the precise molecular mechanisms underlying the crosstalk between KDMs and HDACs in chromatin remodeling and regulation of gene transcription are still elusive. In this study, we showed that treatment of human breast cancer cells with inhibitors targeting the zinc cofactor dependent class I/II HDAC, but not NAD+ dependent class III HDAC, led to significant increase of H3K4me2 which is a specific substrate of histone lysine-specific demethylase 1 (LSD1) and a key chromatin mark promoting transcriptional activation. We also demonstrated that inhibition of LSD1 activity by a pharmacological inhibitor, pargyline, or siRNA resulted in increased acetylation of H3K9 (AcH3K9). However, siRNA knockdown of LSD2, a homolog of LSD1, failed to alter the level of AcH3K9, suggesting that LSD2 activity may not be functionally connected with HDAC activity. Combined treatment with LSD1 and HDAC inhibitors resulted in enhanced levels of H3K4me2 and AcH3K9, and exhibited synergistic growth inhibition of breast cancer cells. Finally, microarray screening identified a unique subset of genes whose expression was significantly changed by combination treatment with inhibitors of LSD1 and HDAC. Our study suggests that LSD1 intimately interacts with histone deacetylases in human breast cancer cells. Inhibition of histone demethylation and deacetylation exhibits cooperation and synergy in regulating gene expression and growth inhibition, and may represent a promising and novel approach for epigenetic therapy of breast cancer.

Vasilatos, Shauna N.; Boric, Lamia; Shaw, Patrick G.; Davidson, Nancy E.

2013-01-01

155

Inhibition of human insulin gene transcription and MafA transcriptional activity by the dual leucine zipper kinase.  

PubMed

Insulin biosynthesis is an essential ?-cell function and inappropriate insulin secretion and biosynthesis contribute to the pathogenesis of diabetes mellitus type 2. Previous studies showed that the dual leucine zipper kinase (DLK) induces ?-cell apoptosis. Since ?-cell dysfunction precedes ?-cell loss, in the present study the effect of DLK on insulin gene transcription was investigated in the HIT-T15 ?-cell line. Downregulation of endogenous DLK increased whereas overexpression of DLK decreased human insulin gene transcription. 5'- and 3'-deletion human insulin promoter analyses resulted in the identification of a DLK responsive element that mapped to the DNA binding-site for the ?-cell specific transcription factor MafA. Overexpression of DLK wild-type but not its kinase-dead mutant inhibited MafA transcriptional activity conferred by its transactivation domain. Furthermore, in the non-?-cell line JEG DLK inhibited MafA overexpression-induced human insulin promoter activity. Overexpression of MafA and DLK or its kinase-dead mutant into JEG cells revealed that DLK but not its mutant reduced MafA protein content. Inhibition of the down-stream DLK kinase c-Jun N-terminal kinase (JNK) by SP600125 attenuated DLK-induced MafA loss. Furthermore, mutation of the serine 65 to alanine, shown to confer MafA protein stability, increased MafA-dependent insulin gene transcription and prevented DLK-induced MafA loss in JEG cells. These data suggest that DLK by activating JNK triggers the phosphorylation and degradation of MafA thereby attenuating insulin gene transcription. Given the importance of MafA for ?-cell function, the inhibition of DLK might preserve ?-cell function and ultimately retard the development of diabetes mellitus type 2. PMID:24726898

Stahnke, Marie-Jeannette; Dickel, Corinna; Schröder, Sabine; Kaiser, Diana; Blume, Roland; Stein, Roland; Pouponnot, Celio; Oetjen, Elke

2014-09-01

156

Profiling of Aortic Smooth Muscle Cell Gene Expression in Response to Chronic Inhibition of Nitric Oxide Synthase in Rats  

Microsoft Academic Search

Background—Chronic inhibition of nitric oxide (NO) synthesis by N-nitro-L-arginine methyl ester (L-NAME) induces hypertension associated with remodeling of the arterial wall. In this study, we aimed at identifying genes and pathways involved in this process in aortic smooth muscle cells from Fischer 344 rats, which exhibit an accelerated hypertension after administration of L-NAME. Methods and Results—We studied the transcriptional profile

Morgan Dupuis; Florent Soubrier; Isabelle Brocheriou; Ségolène Raoux; Mounsif Haloui; Liliane Louedec; Jean-Baptiste Michel; Sophie Nadaud

2010-01-01

157

Role of protein kinase C in induction of gene expression and inhibition of cell proliferation by interferon alpha.  

PubMed

Recent studies have suggested that protein kinase C (PKC) may be involved in the mechanism of signal transduction by which members of the interferon (IFN) family regulate gene expression and cell phenotype. We have investigated the role of PKC in the control of cell growth and gene expression by IFN alpha in Daudi cells. Treatment of these cells with two analogues of staurosporine, which are potent inhibitors of PKC, completely blocked the induction by IFN alpha of the mRNA for 2',5'-oligoadenylate synthetase and the 6-16 gene. These compounds also inhibited cell proliferation and thymidine incorporation in this system. In contrast, the protein kinase inhibitor 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine (H7) did not significantly inhibit the induction of these genes by IFN alpha and had no effect on Daudi cell growth or thymidine incorporation in the presence or absence of IFN alpha. No effect of IFN alpha on total PKC activity could be observed, and there were no significant changes in the overall levels of individual PKC isoforms or their mRNA following IFN alpha treatment. In contrast, treatment of Daudi cells with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate, which also inhibits cell proliferation, strongly down-regulated PKC. These data suggest that the activity of a PKC species, or a closely related enzyme, may be required both for continued cell proliferation and the response to IFN alpha in Daudi cells, but that IFN-induced growth inhibition does not involve overall down-regulation or change in activity of PKC. PMID:1425689

James, R I; Menaya, J; Hudson, K; Devalia, V; Ryves, J; Evans, F J; Thomas, S; Clemens, M J

1992-11-01

158

Inhibitors of histone demethylation and histone deacetylation cooperate in regulating gene expression and inhibiting growth in human breast cancer cells.  

PubMed

Abnormal activities of histone lysine demethylases (KDMs) and lysine deacetylases (HDACs) are associated with aberrant gene expression in breast cancer development. However, the precise molecular mechanisms underlying the crosstalk between KDMs and HDACs in chromatin remodeling and regulation of gene transcription are still elusive. In this study, we showed that treatment of human breast cancer cells with inhibitors targeting the zinc cofactor dependent class I/II HDAC, but not NAD(+) dependent class III HDAC, led to significant increase of H3K4me2 which is a specific substrate of histone lysine-specific demethylase 1 (LSD1) and a key chromatin mark promoting transcriptional activation. We also demonstrated that inhibition of LSD1 activity by a pharmacological inhibitor, pargyline, or siRNA resulted in increased acetylation of H3K9 (AcH3K9). However, siRNA knockdown of LSD2, a homolog of LSD1, failed to alter the level of AcH3K9, suggesting that LSD2 activity may not be functionally connected with HDAC activity. Combined treatment with LSD1 and HDAC inhibitors resulted in enhanced levels of H3K4me2 and AcH3K9, and exhibited synergistic growth inhibition of breast cancer cells. Finally, microarray screening identified a unique subset of genes whose expression was significantly changed by combination treatment with inhibitors of LSD1 and HDAC. Our study suggests that LSD1 intimately interacts with histone deacetylases in human breast cancer cells. Inhibition of histone demethylation and deacetylation exhibits cooperation and synergy in regulating gene expression and growth inhibition, and may represent a promising and novel approach for epigenetic therapy of breast cancer. PMID:21452019

Huang, Yi; Vasilatos, Shauna N; Boric, Lamia; Shaw, Patrick G; Davidson, Nancy E

2012-02-01

159

Niacin inhibits vascular oxidative stress, redox-sensitive genes, and monocyte adhesion to human aortic endothelial cells  

Microsoft Academic Search

In pharmacological doses, nicotinic acid (niacin) reduces myocardial infarction, stroke and atherosclerosis. The beneficial effects of niacin on lipoproteins are thought to mediate these effects. We hypothesized that niacin inhibits oxidative stress and redox-sensitive inflammatory genes that play a critical role in early atherogenesis. In cultured human aortic endothelial cells (HAEC), niacin increased nicotinamide adenine dinucleotide phosphate (NAD(P)H) levels by

Shobha H. Ganji; Shucun Qin; Linhua Zhang; Vaijinath S. Kamanna; Moti L. Kashyap

2009-01-01

160

Prospects for inhibiting the post-transcriptional regulation of gene expression in hepatitis B virus  

PubMed Central

There is a continuing need for novel antivirals to treat hepatitis B virus (HBV) infection, as it remains a major health problem worldwide. Ideally new classes of antivirals would target multiple steps in the viral lifecycle. In this review, we consider the steps in which HBV RNAs are processed, exported from the nucleus and translated. These are often overlooked steps in the HBV life-cycle. HBV, like retroviruses, incorporates a number of unusual steps in these processes, which use a combination of viral and host cellular machinery. Some of these unusual steps deserve a closer scrutiny. They may provide alternative targets to existing antiviral therapies, which are associated with increasing drug resistance. The RNA post-transcriptional regulatory element identified 20 years ago promotes nucleocytoplasmic export of all unspliced HBV RNAs. There is evidence that inhibition of this step is part of the antiviral action of interferon. Similarly, the structured RNA epsilon element situated at the 5’ end of the polycistronic HBV pregenomic RNA also performs key roles during HBV replication. The pregenomic RNA, which is the template for translation of both the viral core and polymerase proteins, is also encapsidated and used in replication. This complex process, regulated at the epsilon element, also presents an attractive antiviral target. These RNA elements that mediate and regulate gene expression are highly conserved and could be targeted using novel strategies employing RNAi, miRNAs or aptamers. Such approaches targeting these functionally constrained genomic regions should avoid escape mutations. Therefore understanding these regulatory elements, along with providing potential targets, may also facilitate the development of other new classes of antiviral drugs.

Chen, Augustine; Panjaworayan T-Thienprasert, Nattanan; Brown, Chris M

2014-01-01

161

The effects of PPAR-? inhibition on gene expression and the progression of induced osteogenic differentiation of human mesenchymal stem cells.  

PubMed

Abstract Mesenchymal stem cells (MSCs) can differentiate into several cell types, such as osteoblasts and adipocytes, both in vitro and in vivo. Although these two differentiation pathways are distinct from each other, cross-communication between cells of the two lineages exists both systemically and peripherally in the tissue. The transcription factor PPAR-?, the main switch in adipogenic differentiation of MSCs, has previously been described to have a negative effect on osteogenic differentiation. The aim of this study was to investigate the effect of PPAR-? inhibition on osteogenic differentiation of human MSCs, in vitro. Extracellular matrix analysis and quantification of osteogenic markers, revealed how these cells respond when the adipogenic differentiation pathway is blocked during induction of osteogenic differentiation. The inhibition leads to a significant increase in mineralization of the extracellular matrix, as well as an increased activity or up-regulated gene expression of alkaline phosphatase, the key enzyme involved in matrix mineralization. Furthermore, it was also demonstrated by microarray analysis, that PPAR-? inhibition during osteogenic induction leads to a significant up-regulation of a number of genes related to both osteogenesis and adipogenesis such as c10orf10, leptin, GDF5 and KLF15. In conclusion, inhibition of PPAR-? during induction of osteogenesis leads to increased osteogenic differentiation of human MSCs. PMID:24708348

Granéli, Cecilia; Karlsson, Camilla; Brisby, Helena; Lindahl, Anders; Thomsen, Peter

2014-08-01

162

Chlorophyll revisited: anti-inflammatory activities of chlorophyll a and inhibition of expression of TNF-? gene by the same.  

PubMed

In view of the folklore use of green leaves to treat inflammation, the anti-inflammatory property of chlorophylls and their degradation products were studied. Chlorophyll a and pheophytin a (magnesium-free chlorophyll a) from fresh leaves showed potent anti-inflammatory activity against carrageenan-induced paw edema in mice and formalin-induced paw edema in rats. Chlorophyll a inhibited bacterial lipopolysaccharide-induced TNF-? (a pro-inflammatory cytokine) gene expression in HEK293 cells, but it did not influence the expression of inducible nitric acid synthase and cyclooxygenase-2 genes. Chlorophyll b only marginally inhibited both inflammation and TNF-? gene expression. But both chlorophyll a and chlorophyll b showed the same level of marginal inhibition on 12-O-tetradecanoyl-phorbol-13-acetate-induced NF-?B activation. Chlorophylls and pheophytins showed in vitro anti-oxidant activity. The study shows that chlorophyll a and its degradation products are valuable and abundantly available anti-inflammatory agents and promising for the development of phytomedicine or conventional medicine to treat inflammation and related diseases. PMID:22038065

Subramoniam, Appian; Asha, Velikkakathu V; Nair, Sadasivan Ajikumaran; Sasidharan, Sreejith P; Sureshkumar, Parameswaran K; Rajendran, Krishnan Nair; Karunagaran, Devarajan; Ramalingam, Krishnan

2012-06-01

163

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

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

Zhang, J; Ou, J; Bashmakov, Y; Horton, J D; Brown, M S; Goldstein, J L

2001-03-27

164

Gene- and Protein-Delivered Zinc Finger-Staphylococcal Nuclease Hybrid for Inhibition of DNA Replication of Human Papillomavirus  

PubMed Central

Previously, we reported that artificial zinc-finger proteins (AZPs) inhibited virus DNA replication in planta and in mammalian cells by blocking binding of a viral replication protein to its replication origin. However, the replication mechanisms of viruses of interest need to be disentangled for the application. To develop more widely applicable methods for antiviral therapy, we explored the feasibility of inhibition of HPV-18 replication as a model system by cleaving its viral genome. To this end, we fused the staphylococcal nuclease cleaving DNA as a monomer to an AZP that binds to the viral genome. The resulting hybrid nuclease (designated AZP–SNase) cleaved its target DNA plasmid efficiently and sequence-specifically in vitro. Then, we confirmed that transfection with a plasmid expressing AZP–SNase inhibited HPV-18 DNA replication in transient replication assays using mammalian cells. Linker-mediated PCR analysis revealed that the AZP–SNase cleaved an HPV-18 ori plasmid around its binding site. Finally, we demonstrated that the protein-delivered AZP–SNase inhibited HPV-18 DNA replication as well and did not show any significant cytotoxicity. Thus, both gene- and protein-delivered hybrid nucleases efficiently inhibited HPV-18 DNA replication, leading to development of a more universal antiviral therapy for human DNA viruses.

Mino, Takashi; Mori, Tomoaki; Aoyama, Yasuhiro; Sera, Takashi

2013-01-01

165

U94 alters FN1 and ANGPTL4 gene expression and inhibits tumorigenesis of prostate cancer cell line PC3  

PubMed Central

Background Insensitivity of advanced-stage prostate cancer to androgen ablation therapy is a serious problem in clinical practice because it is associated with aggressive progression and poor prognosis. Targeted therapeutic drug discovery efforts are thwarted by lack of adequate knowledge of gene(s) associated with prostate tumorigenesis. Therefore there is the need for studies to provide leads to targeted intervention measures. Here we propose that stable expression of U94, a tumor suppressor gene encoded by human herpesvirus 6A (HHV-6A), could alter gene expression and thereby inhibit the tumorigenicity of PC3 cell line. Microarray gene expression profiling on U94 recombinant PC3 cell line could reveal genes that would elucidate prostate cancer biology, and hopefully identify potential therapeutic targets. Results We have shown that stable expression of U94 gene in PC3 cell line inhibited its focus formation in culture, and tumorigenesis in nude mice. Moreover gene expression profiling revealed dramatic upregulation of FN 1 (fibronectin, 91 ± 16-fold), and profound downregulation of ANGPTL 4 (angiopoietin-like-4, 20 ± 4-fold) in U94 recombinant PC3 cell line. Quantitative real-time polymerase chain reaction (QRT-PCR) analysis showed that the pattern of expression of FN 1 and ANGPTL 4 mRNA were consistent with the microarray data. Based on previous reports, the findings in this study implicate upregulation of FN 1 and downregulation of ANGPTL 4 in the anti tumor activity of U94. Genes with cancer inhibitory activities that were also upregulated include SERPINE 2 (serine/cysteine protease inhibitor 2, 7 ± 1-fold increase) and ADAMTS 1 (a disintegrin-like and metalloprotease with thrombospondin type 1 motif, 7 ± 2-fold increase). Additionally, SPUVE 23 (serine protease 23) that is pro-tumorigenic was significantly downregulated (10 ± 1-fold). Conclusion The dramatic upregulation of FN 1 and downregulation of ANGPTL 4 genes in PC3 cell line stably expressing U94 implicate up-regulation of FN 1 and downregulation of ANGPTL 4 in anti tumor activity of U94. Further studies are necessary to determine functional roles of differentially expressed genes in U94 recombinant PC3 cell line, and hopefully provide leads to potential therapeutic targets in prostate cancer.

Ifon, Ekwere T; Pang, Alan LY; Johnson, Warren; Cashman, Kathleen; Zimmerman, Sharon; Muralidhar, Sumitra; Chan, Wai-Yee; Casey, John; Rosenthal, Leonard Jason

2005-01-01

166

Silkworm apolipophorin protein inhibits hemolysin gene expression of Staphylococcus aureus via binding to cell surface lipoteichoic acids.  

PubMed

We previously reported that a silkworm hemolymph protein, apolipophorin (ApoLp), binds to the cell surface of Staphylococcus aureus and inhibits expression of the saePQRS operon encoding a two-component system, SaeRS, and hemolysin genes. In this study, we investigated the inhibitory mechanism of ApoLp on S. aureus hemolysin gene expression. ApoLp bound to lipoteichoic acids (LTA), an S. aureus cell surface component. The addition of purified LTA to liquid medium abolished the inhibitory effect of ApoLp against S. aureus hemolysin production. In an S. aureus knockdown mutant of ltaS encoding LTA synthetase, the inhibitory effects of ApoLp on saeQ expression and hemolysin production were attenuated. Furthermore, the addition of anti-LTA monoclonal antibody to liquid medium decreased the expression of S. aureus saeQ and hemolysin genes. In S. aureus strains expressing SaeS mutant proteins with a shortened extracellular domain, ApoLp did not decrease saeQ expression. These findings suggest that ApoLp binds to LTA on the S. aureus cell surface and inhibits S. aureus hemolysin gene expression via a two-component regulatory system, SaeRS. PMID:23873929

Omae, Yosuke; Hanada, Yuichi; Sekimizu, Kazuhisa; Kaito, Chikara

2013-08-30

167

Nonsteroidal Anti-inflammatory Drugs Inhibit Expression of the Inducible Nitric Oxide Synthase Gene  

Microsoft Academic Search

Increased nitric oxide production is associated with acute and chronic inflammatory processes. Accordingly, we tested the hypothesis that the therapeutic action of nonsteroidal anti-inflammatory drugs could be attributed at least in part to inhibition of excess nitric oxide production. We report here that sodium salicylate, aspirin, ibuprofen, and indomethacin markedly inhibited the appearance of the inducible inflammatory nitric oxide synthase

E. E. Aeberhard; S. A. Henderson; N. S. Arabolos; J. M. Griscavage; F. E. Castro; C. T. Barrett; L. J. Ignarro

1995-01-01

168

Recent transcription-induced histone H3 lysine 4 (H3K4) methylation inhibits gene reactivation.  

PubMed

Recent transcription of GAL genes transiently leaves an H3K4 methylation mark at their promoters, providing an epigenetic memory for the recent transcriptional activity. However, the physiological significance of this mark is enigmatic. In our study, we show that the transient H3K4 di- and trimethylation at recently transcribed GAL1 inhibited the reinduction of GAL1. The H3K4 methylation functioned by recruiting the Isw1 ATPase onto GAL1 and thereby limiting the action of RNA polymerase II during GAL1 reactivation. Strikingly, the H3K4 methylation was also observed at the promoters of inositol- and fatty acid-responsive genes after recent transcription and played a negative role in their reinduction. Taken together, our data present a new mechanism by which H3K4 methylation regulates gene transcription. PMID:21849496

Zhou, Bo O; Zhou, Jin-Qiu

2011-10-01

169

Anacardic acid inhibits estrogen receptor alpha-DNA binding and reduces target gene transcription and breast cancer cell proliferation  

PubMed Central

Anacardic acid (2-hydroxy-6-alkylbenzoic acid) is a dietary and medicinal phytochemical with established anticancer activity in cell and animal models. The mechanisms by which anacardic acid inhibits cancer cell proliferation remain undefined. Anacardic acid 24:1?5 (AnAc 24:1?5) was purified from geranium (Pelargonium × hortorum) and shown to inhibit the proliferation of estrogen receptor ? (ER?)-positive MCF-7 and endocrine-resistant LCC9 and LY2 breast cancer cells with greater efficacy than ER?-negative primary human breast epithelial cells, MCF-10A normal breast epithelial cells, and MDA-MB-231 basal-like breast cancer cells. AnAc 24:1?5 inhibited cell cycle progression and induced apoptosis in a cell-specific manner. AnAc 24:1?5 inhibited estradiol (E2)-induced estrogen response element (ERE) reporter activity and transcription of the endogenous E2-target genes: pS2, cyclin D1, and cathepsin D in MCF-7 cells. AnAc 24:1?5 did not compete with E2 for ER? or ER? binding, nor did AnAc 24:1?5 reduce ER? or ER? steady state protein levels in MCF-7 cells; rather, AnAc 24:1?5 inhibited ER-ERE binding in vitro. Virtual Screening with the molecular docking software Surflex evaluated AnAc 24:1?5 interaction with ER? ligand binding and DNA binding domains (LBD and DBD) in conjunction with experimental validation. Molecular modeling revealed AnAc 24:1?5 interaction with the ER? DBD but not the LBD. Chromatin immunoprecipitation (ChIP) experiments revealed that AnAc 24:1?5 inhibited E2-ER? interaction with the endogenous pS2 gene promoter region containing an ERE. These data indicate that AnAc 24:1?5 inhibits cell proliferation, cell cycle progression and apoptosis in an ER-dependent manner by reducing ER-DNA interaction and inhibiting ER-mediated transcriptional responses.

Schultz, David J.; Wickramasinghe, Nalinie S.; Ivanova, Margarita M.; Isaacs, Susan M.; Dougherty, Susan M.; Imbert-Fernandez, Yoannis; Cunningham, Albert R.; Chen, Chunyuan; Klinge, Carolyn M.

2010-01-01

170

Gene Silencing of 4-1BB by RNA Interference Inhibits Acute Rejection in Rats with Liver Transplantation  

PubMed Central

The 4-1BB signal pathway plays a key role in organ transplantation tolerance. In this study, we have investigated the effect of gene silencing of 4-1BB by RNA interference (RNAi) on the acute rejection in rats with liver transplantation. The recombination vector of lentivirus that contains shRNA targeting the 4-1BB gene (LV-sh4-1BB) was constructed. The liver transplantation was performed using the two-cuff technique. Brown-Norway (BN) recipient rats were infected by the recombinant LVs. The results showed that gene silencing of 4-1BB by RNAi downregulated the 4-1BB gene expression of the splenic lymphocytes in vitro, and the splenic lymphocytes isolated from the rats with liver transplantation. LV-sh4-1BB decreased the plasma levels of liver injury markers including AST, ALT, and BIL and also decreased the level of plasma IL-2 and IFN-? in recipient rats with liver transplantation. Lentivirus-mediated delivery of shRNA targeting 4-1BB gene prolonged the survival time of recipient and alleviated the injury of liver morphology in recipient rats with liver transplantation. In conclusion, our results demonstrate that gene silencing of 4-1BB by RNA interference inhibits the acute rejection in rats with liver transplantation.

Shi, Yang; Hu, Shuqun; Song, Qingwei; Yu, Shengcai; Zhou, Xiaojun; Yin, Jun; Qin, Lei; Qian, Haixin

2013-01-01

171

Inhibition of lysine-specific demethylase 1 by polyamine analogues results in reexpression of aberrantly silenced genes.  

PubMed

Epigenetic chromatin modification is a major regulator of eukaryotic gene expression, and aberrant epigenetic silencing of gene expression contributes to tumorigenesis. Histone modifications include acetylation, phosphorylation, and methylation, resulting in a combination of histone marks known collectively as the histone code. The chromatin marks at a given promoter determine, in part, whether specific promoters are in an open/active conformation or closed/repressed conformation. Dimethyl-lysine 4 histone H3 (H3K4me2) is a transcription-activating chromatin mark at gene promoters, and demethylation of this mark by the lysine-specific demethylase 1 (LSD1), a homologue of polyamine oxidases, may broadly repress gene expression. We now report that novel biguanide and bisguanidine polyamine analogues are potent inhibitors of LSD1. These analogues inhibit LSD1 in human colon carcinoma cells and affect a reexpression of multiple, aberrantly silenced genes important in the development of colon cancer, including members of the secreted frizzle-related proteins (SFRPs) and the GATA family of transcription factors. Furthermore, we demonstrate by chromatin immunoprecipitation analysis that the reexpression is concurrent with increased H3K4me2 and acetyl-H3K9 marks, decreased H3K9me1 and H3K9me2 repressive marks. We thus define important new agents for reversing aberrant repression of gene transcription. PMID:17463086

Huang, Yi; Greene, Eriko; Murray Stewart, Tracy; Goodwin, Andrew C; Baylin, Stephen B; Woster, Patrick M; Casero, Robert A

2007-05-01

172

cDNA Microarray Gene Expression Profiling of Hedgehog Signaling Pathway Inhibition in Human Colon Cancer Cells  

PubMed Central

Background Hedgehog (HH) signaling plays a critical role in normal cellular processes, in normal mammalian gastrointestinal development and differentiation, and in oncogenesis and maintenance of the malignant phenotype in a variety of human cancers. Increasing evidence further implicates the involvement of HH signaling in oncogenesis and metastatic behavior of colon cancers. However, genomic approaches to elucidate the role of HH signaling in cancers in general are lacking, and data derived on HH signaling in colon cancer is extremely limited. Methodology/Principal Findings To identify unique downstream targets of the GLI genes, the transcriptional regulators of HH signaling, in the context of colon carcinoma, we employed a small molecule inhibitor of both GLI1 and GLI2, GANT61, in two human colon cancer cell lines, HT29 and GC3/c1. Cell cycle analysis demonstrated accumulation of GANT61-treated cells at the G1/S boundary. cDNA microarray gene expression profiling of 18,401 genes identified Differentially Expressed Genes (DEGs) both common and unique to HT29 and GC3/c1. Analyses using GenomeStudio (statistics), Matlab (heat map), Ingenuity (canonical pathway analysis), or by qRT-PCR, identified p21Cip1 (CDKN1A) and p15Ink4b (CDKN2B), which play a role in the G1/S checkpoint, as up-regulated genes at the G1/S boundary. Genes that determine further cell cycle progression at G1/S including E2F2, CYCLIN E2 (CCNE2), CDC25A and CDK2, and genes that regulate passage of cells through G2/M (CYCLIN A2 [CCNA2], CDC25C, CYCLIN B2 [CCNB2], CDC20 and CDC2 [CDK1], were down-regulated. In addition, novel genes involved in stress response, DNA damage response, DNA replication and DNA repair were identified following inhibition of HH signaling. Conclusions/Significance This study identifies genes that are involved in HH-dependent cellular proliferation in colon cancer cells, and following its inhibition, genes that regulate cell cycle progression and events downstream of the G1/S boundary.

Shi, Ting; Mazumdar, Tapati; DeVecchio, Jennifer; Duan, Zhong-Hui; Agyeman, Akwasi; Aziz, Mohammad; Houghton, Janet A.

2010-01-01

173

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

Microsoft Academic Search

Abstract Abstract Abstract Abstract Abstract 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-relatedand transcription-related genes. METHODS:We,compared,the gene,expression profiles

Hae-Jeong Park; Seo-Hyun Yoon; Long-Shan Han; Long-Tai Zheng; Kyung-Hee Jung; Yoon-Kyung Uhm; Je-Hyun Lee; Ji-Seon Jeong; Woo-Sang Joo; Sung-Vin Yim; Joo-Ho Chung; Seon-Pyo Hong; Park HJ; Yoon SH; Zheng LT; Jung KH; Uhm YK; Lee JH; Jeong JS; Joo WS; Yim SV; Chung JH

2005-01-01

174

Methods and Compositions for the Specific Inhibition of Gene Expression by Double-Stranded RNA.  

National Technical Information Service (NTIS)

The invention is directed to compositions and methods for selectively reducing the expression of a gene product from a desired target gene in a cell, as well as for treating diseases caused by the expression of the gene. More particularly, the invention i...

J. J. Rossi M. A. Behike D. Kim

2005-01-01

175

NMD inhibition fails to identify tumour suppressor genes in microsatellite stable gastric cancer cell lines  

Microsoft Academic Search

BACKGROUND: Gastric cancers frequently show chromosomal alterations which can cause activation of oncogenes, and\\/or inactivation of tumour suppressor genes. In gastric cancer several chromosomal regions are described to be frequently lost, but for most of the regions, no tumour suppressor genes have been identified yet. The present study aimed to identify tumour suppressor genes inactivated by nonsense mutation and deletion

Tineke E Buffart; Marianne Tijssen; Jamila El-Bchiri; Alex Duval; Mark Wiel; Bauke Ylstra; Gerrit A Meijer; Beatriz Carvalho

2009-01-01

176

In vitro Methylation of the Hamster Adenine Phosphoribosyltransferase Gene Inhibits Its Expression in Mouse L Cells  

Microsoft Academic Search

The effect of DNA methylation on the expression of the hamster adenine phosphoribosyltransferase (aprt) gene in mouse cells has been examined. This gene was methylated in vitro at all of its C-C-G-G sites by using Hpa II methylase and was inserted into mouse Ltk- aprt- L cells by cotransformation, with the herpes virus thymidine kinase gene as a selectable vector.

R. Stein; A. Razin; H. Cedar

1982-01-01

177

Polyunsaturated fatty acids suppress glycolytic and lipogenic genes through the inhibition of ChREBP nuclear protein translocation.  

PubMed

Dietary polyunsaturated fatty acids (PUFAs) are potent inhibitors of hepatic glycolysis and lipogenesis. Recently, carbohydrate-responsive element-binding protein (ChREBP) was implicated in the regulation by glucose of glycolytic and lipogenic genes, including those encoding L-pyruvate kinase (L-PK) and fatty acid synthase (FAS). The aim of our study was to assess the role of ChREBP in the control of L-PK and FAS gene expression by PUFAs. We demonstrated in mice, both in vivo and in vitro, that PUFAs [linoleate (C18:2), eicosapentanoic acid (C20:5), and docosahexaenoic acid (C22:6)] suppressed ChREBP activity by increasing ChREBP mRNA decay and by altering ChREBP translocation from the cytosol to the nucleus, independently of an activation of the AMP-activated protein kinase, previously shown to regulate ChREBP activity. In contrast, saturated [stearate (C18)] and monounsaturated fatty acids [oleate (C18:1)] had no effect. Since glucose metabolism via the pentose phosphate pathway is determinant for ChREBP nuclear translocation, the decrease in xylulose 5-phosphate concentrations caused by a PUFA diet favors a PUFA-mediated inhibition of ChREBP translocation. In addition, overexpression of a constitutive nuclear ChREBP isoform in cultured hepatocytes significantly reduced the PUFA inhibition of both L-PK and FAS gene expression. Our results demonstrate that the suppressive effect of PUFAs on these genes is primarily caused by an alteration of ChREBP nuclear translocation. In conclusion, we describe a novel mechanism to explain the inhibitory effect of PUFAs on the genes encoding L-PK and FAS and demonstrate that ChREBP is a pivotal transcription factor responsible for coordinating the PUFA suppression of glycolytic and lipogenic genes. PMID:16184193

Dentin, Renaud; Benhamed, Fadila; Pégorier, Jean-Paul; Foufelle, Fabienne; Viollet, Benoit; Vaulont, Sophie; Girard, Jean; Postic, Catherine

2005-10-01

178

Alterations in tumour suppressor gene p53 correlate with inhibition of thrombospondin-1 gene expression in colon cancer cells  

Microsoft Academic Search

If activation of the p53 gene is involved in the progression or metastasis of colon cancer, it may affect the angiogenic phenotype in vivo. To verify this hypothesis, we studied the correlation between p53 accumulation and expression of thrombospondin-1 (TSP1) in colon cancer specimens. Levels of TSP1 gene expression were estimated by Northern blotting in 65 colon cancers. Accumulation of

Tetsuji Tokunaga; M. Nakamura; Yoshiro Oshika; Takashi Tsuchida; Michitake Kazuno; Yoshitaka Fukushima; Kenji Kawai; Yoshiyuki Abe; Hiroshi Kijima; Hitoshi Yamazaki; Norikazu Tamaoki; Yoshito Ueyama

1998-01-01

179

Regulation of human insulin gene transcription by the immunosuppressive drugs cyclosporin A and tacrolimus at concentrations that inhibit calcineurin activity and involving the transcription factor CREB  

Microsoft Academic Search

Cyclosporin A and tacrolimus are important immunosuppressive drugs. They share a diabetogenic action as one of their most serious adverse effects. In a single study, tacrolimus (100 nM) inhibited human insulin gene transcription in the g-cell line HIT. Using transfections of a human insulin-reporter gene into HIT cells, the present study shows that this inhibition is seen only at high

Elke Oetjen; Daniela Grapentin; Roland Blume; Michael Seeger; Doris Krause; Anke Eggers; Willhart Knepel

2003-01-01

180

Transcriptional inhibition of hypertrophic scars by a gene silencer, pyrrole-imidazole polyamide, targeting the TGF-?1 promoter.  

PubMed

Synthetic pyrrole-imidazole (PI) polyamides bind to the minor groove of double-helical DNA with high affinity and specificity, and inhibit the transcription of corresponding genes. We examined the effects of a transforming growth factor (TGF)-?1-targeted PI polyamide (Polyamide) on hypertrophic skin scars in rats. Hypertrophic scars were created dorsally in rats by incisions. FITC-labeled Polyamide was injected to investigate its distribution in the skin. Expression of TGF-?1, connective tissue growth factor (CTGF), collagen type1, and fibronectin mRNAs was evaluated by reverse transcription PCR analysis. The extent of fibrosis and the expression of TGF-?1 were evaluated histologically and immunohistochemically. Polyamide was distributed in almost all nuclei of skin cells. Expression of TGF-?1 mRNA reached a peak at 3 days after skin incision. Expression of CTGF and extracellular matrix mRNAs was increased continuously even after the peak induction of TGF-?1 mRNA. Injection of Polyamide completely inhibited both the development of scars and the induction of growth factors and extracellular matrix mRNAs. The treatment also markedly inhibited fibrotic changes and reduced the numbers of vimentin-positive spindle-shaped fibroblasts. Injection of Polyamide also reduced established hypertrophic scars in rats. Thus, TGF-?1-targeted PI polyamide should be a feasible gene silencer for hypertrophic scars and keloids. PMID:21654833

Washio, Hisayo; Fukuda, Noboru; Matsuda, Hiroyuki; Nagase, Hiroki; Watanabe, Takayoshi; Matsumoto, Yoshiaki; Terui, Tadashi

2011-10-01

181

The latency-related gene of bovine herpesvirus 1 encodes a product which inhibits cell cycle progression.  

PubMed Central

Bovine herpesvirus 1 (BHV-1) establishes a latent infection in the sensory ganglionic neurons of cattle. The exclusive viral RNA expressed in a latent infection is the latency-related (LR) RNA, suggesting that it regulates some aspect of a latent infection. During the course of a productive infection, alphaherpesviruses induce certain events which occur during cell cycle progression. Consequently, we hypothesized that a BHV-1 infection might induce events in neurons which occur during cell cycle progression. In agreement with this hypothesis, cyclin A was detected in neurons of trigeminal ganglia when rabbits were infected. Neuronal cell cycle progression or inappropriate expression of cyclin A leads to apoptosis, suggesting that a viral factor inhibits the deleterious effects of cyclin A expression. The BHV-1 LR gene inhibited cell cycle progression and proliferation of human osteosarcoma cells. Antibodies directed against cyclin A or the LR protein coprecipitated the LR protein or cyclin A, respectively, suggesting that the two proteins interact with each other. We conclude that LR gene products inhibit cell cycle progression and hypothesize that this activity enhances the survival of infected neurons.

Schang, L M; Hossain, A; Jones, C

1996-01-01

182

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

PubMed

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

Nakagami, Yuki; Watakabe, Akiya; Yamamori, Tetsuo

2013-01-01

183

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

PubMed Central

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.

Nakagami, Yuki; Watakabe, Akiya; Yamamori, Tetsuo

2013-01-01

184

Inhibition of TNF-?-induced MUC5AC mucin gene expression and production by wogonin through the inactivation of NF-?B signaling in airway epithelial cells.  

PubMed

In this study, we investigated whether wogonin significantly affects MUC5AC mucin gene expression and production in human airway epithelial cells. Confluent NCI-H292 cells were pretreated with wogonin for 30 min and then stimulated with tumor necrosis factor-? (TNF-?) for 24 h or the indicated periods. The MUC5AC mucin gene expression and mucin protein production were measured by RT-PCR and ELISA, respectively. We found that incubation of NCI-H292 cells with wogonin significantly inhibited mucin production and down-regulated MUC5AC gene expression induced by TNF-? in a dose-dependent fashion. To elucidate the action mechanism of wogonin, effect of wogonin on TNF-?-induced NF-?B signaling pathway was investigated by western blot analysis. Wogonin inhibited NF-?B activation induced by TNF-?. Inhibition of IKK by wogonin led to the suppression of I?B phosphorylation and degradation, p65 nuclear translocation and NF-?B-regulated gene expression. This, in turn, led to the down-regulation of MUC5AC protein production in NCI-H292 cells. Wogonin also inhibited the gene products involved in cell survival (Bcl-2) and proliferation (cyclooxygenase-2). These results suggest that wogonin inhibits the NF-?B signaling pathway, which may explain its role in the inhibition of MUC5AC mucin gene expression and production. PMID:23463646

Sikder, Md Asaduzzaman; Lee, Hyun Jae; Mia, Md Zakaria; Park, Su Hyun; Ryu, Jiho; Kim, Jang-Hyun; Min, Sang Yeon; Hong, Jang-Hee; Seok, Jeong Ho; Lee, Choong Jae

2014-01-01

185

Inhibition of hepatitis B virus (HBV) gene expression and replication by HBx gene silencing in a hydrodynamic injection mouse model with a new clone of HBV genotype B  

PubMed Central

Background It has been suggested that different hepatitis B virus (HBV) genotypes may have distinct virological characteristics that correlate with clinical outcomes during antiviral therapy and the natural course of infection. Hydrodynamic injection (HI) of HBV in the mouse model is a useful tool for study of HBV replication in vivo. However, only HBV genotype A has been used for studies with HI. Methods We constructed 3 replication-competent clones containing 1.1, 1.2 and 1.3 fold overlength of a HBV genotype B genome and tested them both in vitro and in vivo. Moreover, A HBV genotype B clone based on the pAAV-MCS vector was constructed with the 1.3 fold HBV genome, resulting in the plasmid pAAV-HBV1.3B and tested by HI in C57BL/6 mice. Application of siRNA against HBx gene was tested in HBV genotype B HI mouse model. Results The 1.3 fold HBV clone showed higher replication and gene expression than the 1.1 and 1.2 fold HBV clones. Compared with pAAV-HBV1.2 (genotype A), the mice HI with pAAV-HBV1.3B showed higher HBsAg and HBeAg expression as well as HBV DNA replication level but a higher clearance rate. Application of two plasmids pSB-HBxi285 and pSR-HBxi285 expressing a small/short interfering RNA (siRNA) to the HBx gene in HBV genotype B HI mouse model, leading to an inhibition of HBV gene expression and replication. However, HBV gene expression may resume in some mice despite an initial delay, suggesting that transient suppression of HBV replication by siRNA may be insufficient to prevent viral spread, particularly if the gene silencing is not highly effective. Conclusions Taken together, the HI mouse model with a HBV genotype B genome was successfully established and showed different characteristics in vivo compared with the genotype A genome. The effectiveness of gene silencing against HBx gene determines whether HBV replication may be sustainably inhibited by siRNA in vivo.

2013-01-01

186

p27-p16 Fusion Gene Inhibits Angioplasty-Induced Neointimal Hyperplasia and Coronary Artery Occlusion  

Microsoft Academic Search

Inhibition of proliferative neointima formed by vascular smooth muscle cells is a potential target in preventing angioplasty-induced restenosis. We have created a potent antiproliferative by fusing the active regions of the p27 and p16 cell cycle inhibitors. Intravascular delivery of a replication-deficient adenoviral vector (AV) encoding this p27-p16 fusion protein, named W9, inhibited balloon injury-induced neointimal hyperplasia in rabbit carotid

Lisa V. Tsui; Allan Camrud; Jean Mondesire; Paula Carlson; Nathalie Zayek; LaDonna Camrud; Brian Donahue; Scott Bauer; Andy Lin; David Frey; Marianne Rivkin; Ajit Subramanian; Robert Falotico; Jeno Gyuris; Robert Schwartz; James G. McArthur

187

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

PubMed

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

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

2014-06-01

188

Histone deacetylation of RB-responsive promoters: requisite for specific gene repression but dispensable for cell cycle inhibition.  

PubMed

The retinoblastoma tumor suppressor protein (RB) is targeted for inactivation in the majority of human tumors, underscoring its critical role in attenuating cellular proliferation. RB inhibits proliferation by repressing the transcription of genes that are essential for cell cycle progression. To repress transcription, RB assembles multiprotein complexes containing chromatin-modifying enzymes, including histone deacetylases (HDACs). However, the extent to which HDACs participate in transcriptional repression and are required for RB-mediated repression has not been established. Here, we investigated the role of HDACs in RB-dependent cell cycle inhibition and transcriptional repression. We find that active RB mediates histone deacetylation on cyclin A, Cdc2, topoisomerase IIalpha, and thymidylate synthase promoters. We also demonstrate that this deacetylation is HDAC dependent, since the HDAC inhibitor trichostatin A (TSA) prevented histone deacetylation at each promoter. However, TSA treatment blocked RB repression of only a specific subset of genes, thereby demonstrating that the requirement of HDACs for RB-mediated transcriptional repression is promoter specific. The HDAC-independent repression was not associated with DNA methylation or gene silencing but was readily reversible. We show that this form of repression resulted in altered chromatin structure and was dependent on SWI/SNF chromatin remodeling activity. Importantly, we find that cell cycle inhibitory action of RB is not intrinsically dependent on the ability to recruit HDAC activity. Thus, while HDACs do play a major role in RB-mediated repression, they are dispensable for the repression of critical targets leading to cell cycle arrest. PMID:14560017

Siddiqui, Hasan; Solomon, David A; Gunawardena, Ranjaka W; Wang, Ying; Knudsen, Erik S

2003-11-01

189

Overexpression of Inducible Cyclic AMP Early Repressor Inhibits Transactivation of Genes and Cell Proliferation in Pancreatic ? Cells  

PubMed Central

Transcriptional control mediated by the cyclic AMP-responsive element (CRE) represents an important mechanism of gene regulation. To test our hypothesis that increased inducible cyclic AMP early repressor (ICER) I? inhibits function of CRE-binding proteins and thus disrupts CRE-mediated transcription in pancreatic ? cells, we generated transgenic mice with ?-cell-directed expression of ICER I?, a powerful repressor that is greatly increased in diabetes. Three transgenic lines clearly show that increased ICER I? expression in ? cells results in early severe diabetes. From birth islets were severely disorganized with a significantly increased proportion of ? cells throughout the islet. Diabetes results from the combined effects of impaired insulin expression and a decreased number of ? cells. The decrease in ? cells appears to result from impaired proliferation rather than from increased apoptosis after birth. Cyclin A gene expression is impaired by the strong inhibition of ICER; the suppression of cyclin A results in a substantially decreased proliferation of ? cells in the postnatal period. These results suggest that CRE and CRE-binding factors have an important role in pancreatic ?-cell physiology not only directly by regulation of gene trans-activation but also indirectly by regulation of ?-cell mass.

Inada, Akari; Hamamoto, Yoshiyuki; Tsuura, Yoshiyuki; Miyazaki, Jun-ichi; Toyokuni, Shinya; Ihara, Yu; Nagai, Koichiro; Yamada, Yuichiro; Bonner-Weir, Susan; Seino, Yutaka

2004-01-01

190

Histone Deacetylase Inhibition Decreases Cholesterol Levels in Neuronal Cells by Modulating Key Genes in Cholesterol Synthesis, Uptake and Efflux  

PubMed Central

Cholesterol is an essential component of the central nervous system and increasing evidence suggests an association between brain cholesterol metabolism dysfunction and the onset of neurodegenerative disorders. Interestingly, histone deacetylase inhibitors (HDACi) such as trichostatin A (TSA) are emerging as promising therapeutic approaches in neurodegenerative diseases, but their effect on brain cholesterol metabolism is poorly understood. We have previously demonstrated that HDACi up-regulate CYP46A1 gene transcription, a key enzyme in neuronal cholesterol homeostasis. In this study, TSA was shown to modulate the transcription of other genes involved in cholesterol metabolism in human neuroblastoma cells, namely by up-regulating genes that control cholesterol efflux and down-regulating genes involved in cholesterol synthesis and uptake, thus leading to an overall decrease in total cholesterol content. Furthermore, co-treatment with the amphipathic drug U18666A that can mimic the intracellular cholesterol accumulation observed in cells of Niemman-Pick type C patients, revealed that TSA can ameliorate the phenotype induced by pathological cholesterol accumulation, by restoring the expression of key genes involved in cholesterol synthesis, uptake and efflux and promoting lysosomal cholesterol redistribution. These results clarify the role of TSA in the modulation of neuronal cholesterol metabolism at the transcriptional level, and emphasize the idea of HDAC inhibition as a promising therapeutic tool in neurodegenerative disorders with impaired cholesterol metabolism.

Nunes, Maria Joao; Moutinho, Miguel; Gama, Maria Joao; Rodrigues, Cecilia M. P.; Rodrigues, Elsa

2013-01-01

191

SIRT1 Inhibition Alleviates Gene Silencing in Fragile X Mental Retardation Syndrome  

Microsoft Academic Search

Expansion of the CGG•CCG-repeat tract in the 5? UTR of the FMR1 gene to >200 repeats leads to heterochromatinization of the promoter and gene silencing. This results in Fragile X syndrome (FXS), the most common heritable form of mental retardation. The mechanism of gene silencing is unknown. We report here that a Class III histone deacetylase, SIRT1, plays an important

Rea Biacsi; Daman Kumari; Karen Usdin

2008-01-01

192

A genomic screen for genes upregulated by demethylation and histone deacetylase inhibition in human colorectal cancer  

Microsoft Academic Search

Aberrant hypermethylation of gene promoters is a major mechanism associated with inactivation of tumor-suppressor genes in cancer. We previously showed this transcriptional silencing to be mediated by both methylation and histone deacetylase activity, with methylation being dominant. Here, we have used cDNA microarray analysis to screen for genes that are epigenetically silenced in human colorectal cancer. By screening over 10,000

Hiromu Suzuki; Edward Gabrielson; Wei Chen; Ramaswamy Anbazhagan; Manon van Engeland; Matty P. Weijenberg; James G. Herman; Stephen B. Baylin

2002-01-01

193

Adenovirus-Mediated Prothymosin ? Gene Transfer Inhibits the Development of Atherosclerosis in Apoe-Deficient Mice  

PubMed Central

Prothymosin ? (ProT) is involved in regulating expression of the oxidative stress-protective genes and it also exerts immunomodulatory activities. In this study, we investigated the therapeutic effects of ProT gene transfer on atherosclerosis in endothelial cells and in ApoE-deficient mice. Adenoviruses encoding mouse ProT (AdProT) were used for the management of atherosclerosis. In vitro, the effects of ProT on antioxidant gene expressions and the protection effect against oxidant-mediated injury in endothelial cells were examined. In vivo, AdProT were administered intraventricularly into the heart of ApoE-/- mice. Histopathological and immunohistochemical assessments of the aortic tissues were performed. Expressions of HO-1 and antioxidant genes in the aortic tissues were also determined. Our results demonstrated that ProT gene transfer increased antioxidant gene expressions, eNOS expression and NO release, as well as reduced the reactive oxygen species production in endothelial cells. Intraventricular administration of AdProT reduced the lesion formation, increased expressions of HO-1 and SOD genes, and reduced infiltrating macrophages in the aorta of ApoE-/- mice. This study suggests that ProT gene transfer may have the therapeutic potential for the management of atherosclerosis via inducing antioxidant gene expressions, eNOS expression and NO release, reducing ROS production and macrophage infiltration in endothelium.

Chang, Meng-Ya; Yang, Yu-Shan; Tsai, Mei-Ling; Lee, Che-Hsin; Chang, Chih-Jui; Shiau, Ai-Li; Wu, Chao-Liang

2014-01-01

194

Identification of Functional Toxin/Immunity Genes Linked to Contact-Dependent Growth Inhibition (CDI) and Rearrangement Hotspot (Rhs) Systems  

PubMed Central

Bacterial contact-dependent growth inhibition (CDI) is mediated by the CdiA/CdiB family of two-partner secretion proteins. Each CdiA protein exhibits a distinct growth inhibition activity, which resides in the polymorphic C-terminal region (CdiA-CT). CDI+ cells also express unique CdiI immunity proteins that specifically block the activity of cognate CdiA-CT, thereby protecting the cell from autoinhibition. Here we show that many CDI systems contain multiple cdiA gene fragments that encode CdiA-CT sequences. These “orphan” cdiA-CT genes are almost always associated with downstream cdiI genes to form cdiA-CT/cdiI modules. Comparative genome analyses suggest that cdiA-CT/cdiI modules are mobile and exchanged between the CDI systems of different bacteria. In many instances, orphan cdiA-CT/cdiI modules are fused to full-length cdiA genes in other bacterial species. Examination of cdiA-CT/cdiI modules from Escherichia coli EC93, E. coli EC869, and Dickeya dadantii 3937 confirmed that these genes encode functional toxin/immunity pairs. Moreover, the orphan module from EC93 was functional in cell-mediated CDI when fused to the N-terminal portion of the EC93 CdiA protein. Bioinformatic analyses revealed that the genetic organization of CDI systems shares features with rhs (rearrangement hotspot) loci. Rhs proteins also contain polymorphic C-terminal regions (Rhs-CTs), some of which share significant sequence identity with CdiA-CTs. All rhs genes are followed by small ORFs representing possible rhsI immunity genes, and several Rhs systems encode orphan rhs-CT/rhsI modules. Analysis of rhs-CT/rhsI modules from D. dadantii 3937 demonstrated that Rhs-CTs have growth inhibitory activity, which is specifically blocked by cognate RhsI immunity proteins. Together, these results suggest that Rhs plays a role in intercellular competition and that orphan gene modules expand the diversity of toxic activities deployed by both CDI and Rhs systems.

Poole, Stephen J.; Diner, Elie J.; Aoki, Stephanie K.; Braaten, Bruce A.; t'Kint de Roodenbeke, Claire; Low, David A.; Hayes, Christopher S.

2011-01-01

195

Pharmacologic inhibition of ROR?t regulates Th17 signature gene expression and suppresses cutaneous inflammation in vivo.  

PubMed

IL-17-producing CD4(+)Th17 cells, CD8(+)Tc17 cells, and ?? T cells play critical roles in the pathogenesis of autoimmune psoriasis. ROR?t is required for the differentiation of Th17 cells and expression of IL-17. In this article, we describe a novel, potent, and selective ROR?t inverse agonist (TMP778), and its inactive diastereomer (TMP776). This chemistry, for the first time to our knowledge, provides a unique and powerful set of tools to probe ROR?t-dependent functions. TMP778, but not TMP776, blocked human Th17 and Tc17 cell differentiation and also acutely modulated IL-17A production and inflammatory Th17-signature gene expression (Il17a, Il17f, Il22, Il26, Ccr6, and Il23) in mature human Th17 effector/memory T cells. In addition, TMP778, but not TMP776, inhibited IL-17A production in both human and mouse ?? T cells. IL-23-induced IL-17A production was also blocked by TMP778 treatment. In vivo targeting of ROR?t in mice via TMP778 administration reduced imiquimod-induced psoriasis-like cutaneous inflammation. Further, TMP778 selectively regulated Th17-signature gene expression in mononuclear cells isolated from both the blood and affected skin of psoriasis patients. In summary, to our knowledge, we are the first to demonstrate that ROR?t inverse agonists: 1) inhibit Tc17 cell differentiation, as well as IL-17 production by ?? T cells and CD8(+) Tc17 cells; 2) block imiquimod-induced cutaneous inflammation; 3) inhibit Th17 signature gene expression by cells isolated from psoriatic patient samples; and 4) block IL-23-induced IL-17A expression. Thus, ROR?t is a tractable drug target for the treatment of cutaneous inflammatory disorders, which may afford additional therapeutic benefit over existing modalities that target only IL-17A. PMID:24516202

Skepner, Jill; Ramesh, Radha; Trocha, Mark; Schmidt, Darby; Baloglu, Erkan; Lobera, Mercedes; Carlson, Thaddeus; Hill, Jonathan; Orband-Miller, Lisa A; Barnes, Ashley; Boudjelal, Mohamed; Sundrud, Mark; Ghosh, Shomir; Yang, Jianfei

2014-03-15

196

Alivin 1, a novel neuronal activity-dependent gene, inhibits apoptosis and promotes survival of cerebellar granule neurons.  

PubMed

Neurons require Ca2+-dependent gene transcription for their activity-dependent survival, the mechanisms of which have not been fully elucidated yet. Here, we demonstrate that a novel primary response gene, alivin 1 (ali1), is an activity-dependent gene and promotes survival of neurons. Sequence analyses reveal that rat, mouse, and human Ali1 proteins contain seven leucine-rich repeats, one IgC2-like loop and a transmembrane domain, and display homology to Kek and Trk families. Expression of ali1 mRNA in cultured cerebellar granule neurons is rigidly regulated by KCl and/or NMDA concentrations in the culture medium and tightly correlated to depolarization-dependent survival and/or NMDA-dependent survival of the granule neuron. ali1 mRNA expression was regulated at the transcriptional step by the Ca2+ influx through voltage-dependent L-type Ca2+ channels when the cells were stimulated by 25 mm KCl. Expression of ali1 mRNA in cultured cortical neurons was inhibited when their spontaneous electrical activity was blocked by tetrodotoxin. Thus, the expression is neuronal activity dependent. Overexpression of Ali1 in cerebellar granule neurons inhibited apoptosis that was induced by the medium containing 5 mm KCl. The addition of anti-Ali1 antiserum or the soluble putative extracellular Ali1 domain to the 25 mm KCl-supported culture inhibited the survival of the granule neuron. These results suggest that expression of ali1 promotes depolarization-dependent survival of the granule neuron. Mouse ali1 was mapped to a locus approximately 55.3 cM from the centromere on chromosome 15 that is syntenic to positional candidate loci for familial Alzheimer's disease type 5 and Parkinson's disease 8 on human chromosome 12. PMID:12843293

Ono, Tomio; Sekino-Suzuki, Naoko; Kikkawa, Yoshiaki; Yonekawa, Hiromichi; Kawashima, Seiichi

2003-07-01

197

Cryptopleurine Targets NF-?B Pathway, Leading to Inhibition of Gene Products Associated with Cell Survival, Proliferation, Invasion, and Angiogenesis  

PubMed Central

Background Cryptopleurine, a phenanthroquinolizidine alkaloid, was known to exhibit anticancer activity; however, the underlying mechanism is poorly understood. Because the nuclear factor-?B (NF-?B) transcription factors control many physiological processes including inflammation, immunity, and development and progression of cancer, we investigated the effects of cryptopleurine on tumor necrosis factor alpha (TNF-?)-induced NF-?B activation pathway and on the expression of NF-?B-regulated gene products associated with many pathophysiological processes. Methodology and Principal Finding MDA-MB231, MDA-MB435, MCF-7, HEK293, RAW264.7 and Hep3B cells were used to examine cryptopleurine's effect on the NF-?B activation pathway. Major assays were promoter-reporter gene assay, electrophoretic mobility shift assay (EMSA), in vitro immune complex kinase assay, real-time PCR, Western blot analysis, and Matrigel invasion assay. Experiments documenting cell proliferation and apoptosis were analyzed by MTT method and flow cytometry, respectively. The results indicated that cryptopleurine suppressed the NF-?B activation through the inhibition of I?B kinase (IKK) activation, thereby blocking the phosphorylation and degradation of the inhibitor of NF-?B alpha (I?B?) and the nuclear translocation and DNA-binding activity of p65. The suppression of NF-?B by cryptopleurine led to the down-regulation of gene products involved in inflammation, cell survival, proliferation, invasion, and angiogenesis. Conclusions and Significance Our results show that cryptopleurine inhibited NF-?B activation pathway, which leads to inhibition of inflammation, proliferation, and invasion, as well as potentiation of apoptosis. Our findings provide a new insight into the molecular mechanisms and a potential application of cryptopleurine for inflammatory diseases as well as certain cancers associated with abnormal NF-?B activation.

Cai, Xing Fu; Li, Donghao; Wu, Xue; Nan, Ji Xing; Lee, Jung Joon; Jin, Xuejun

2012-01-01

198

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

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…

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

199

Expressed copies of the MN7 (D15F37) gene family map close to the common deletion breakpoints in the Prader-Willi/Angelman syndromes.  

PubMed

Approximately 70% of patients with Prader-Willi syndrome or Angelman syndrome have a similar sized de novo deletion of 3-4 Mb in the proximal region of 15q. The distal breakpoints appear to cluster between the P gene (OCA2) and D15S24, whereas two deletion breakpoint clusters have been identified on the proximal side (one centromeric to D15S541 and one between D15S541 and D15S9). Based on the identification of a gene family in 15q11-->q13 (MN7, D15F37), we have previously proposed that the presence of multiple copies of this sequence may be related to the instability of this region. Using fluorescence in situ hybridization and YAC mapping, we have found that at least one D15F37 locus is centromeric to D15S9 and at least two are between OCA2 and D15S24. As determined by cDNA cloning and sequence analysis, each of the individual loci is expressed. The close proximity of the D15F37 loci and the deletion breakpoints suggests that the common deletions arise by unequal crossover events at or near these loci. PMID:9730612

Buiting, K; Gross, S; Ji, Y; Senger, G; Nicholls, R D; Horsthemke, B

1998-01-01

200

A microRNA encoded by HSV-1 inhibits a cellular transcriptional repressor of viral immediate early and early genes.  

PubMed

Viral microRNAs are one component of the RNA interference phenomenon generated during viral infection. They were first identified in the Herpesviridae family, where they were found to regulate viral mRNA translation. In addition, prior work has suggested that Kaposi's sarcoma-associated herpesvirus (KSHV) is capable of regulating cellular gene transcription by miRNA. We demonstrate that a miRNA, hsv1-mir-H27, encoded within the genome of herpes simplex virus 1 (HSV-1), targets the mRNA of the cellular transcriptional repressor Kelch-like 24 (KLHL24) that inhibits transcriptional efficiency of viral immediate-early and early genes. The viral miRNA is able to block the expression of KLHL24 in cells infected by HSV-1. Our discovery reveals an effective viral strategy for evading host cell defenses and supporting the efficient replication and proliferation of HSV-1. PMID:23512275

Wu, Wenjuan; Guo, Zhongping; Zhang, Xuemei; Guo, Lei; Liu, Longding; Liao, Yun; Wang, Jingjing; Wang, Lichun; Li, Qihan

2013-04-01

201

Clara Cell 10-kDa Protein Gene Transfection Inhibits NF-?B Activity in Airway Epithelial Cells  

PubMed Central

Background Clara cell 10-kDa protein (CC10) is a multifunctional protein with anti-inflammatory and immunomodulatory effects. Induction of CC10 expression by gene transfection may possess potential therapeutic effect. Nuclear factor ?B (NF-?B) plays a key role in the inflammatory processes of airway diseases. Method/Results To investigate potential therapeutic effect of CC10 gene transfection in controlling airway inflammation and the underlying intracellular mechanisms, in this study, we constructed CC10 plasmid and transfected it into bronchial epithelial cell line BEAS-2B cells and CC10 knockout mice. In BEAS-2B cells, CC10's effect on interleukin (IL)-1? induced IL-8 expression was explored by means of RT-PCR and ELISA and its effect on NF-?B classical signaling pathway was studied by luciferase reporter, western blot, and immunoprecipitation assay. The effect of endogenous CC10 on IL-1? evoked IL-8 expression was studied by means of nasal explant culture. In mice, CC10's effect on IL-1? induced IL-8 and nuclear p65 expression was examined by immunohistochemistry. First, we found that the CC10 gene transfer could inhibit IL-1? induced IL-8 expression in BEAS-2B cells. Furthermore, we found that CC10 repressed IL-1? induced NF-?B activation by inhibiting the phosphorylation of I?B-? but not I?B kinase-?/? in BEAS-2B cells. Nevertheless, we did not observe a direct interaction between CC10 and p65 subunit in BEAS-2B cells. In nasal explant culture, we found that IL-1? induced IL-8 expression was inversely correlated with CC10 levels in human sinonasal mucosa. In vivo study revealed that CC10 gene transfer could attenuate the increase of IL-8 and nuclear p65 staining in nasal epithelial cells in CC10 knockout mice evoked by IL-1? administration. Conclusion These results indicate that CC10 gene transfer may inhibit airway inflammation through suppressing the activation of NF-?B, which may provide us a new consideration in the therapy of airway inflammation.

Long, Xiao-Bo; Hu, Shuang; Wang, Nan; Zhen, Hong-Tao; Cui, Yong-Hua; Liu, Zheng

2012-01-01

202

Mithramycin selectively inhibits collagen-alpha 1(I) gene expression in human fibroblast.  

PubMed Central

The products of the collagen-alpha 1(I) and -alpha 2(I) genes form the triple helical molecule collagen type I, which constitutes the major ECM protein in tissue fibrosis. The collagen-alpha 1(I) gene is mainly transcriptionally regulated, and its promoter activity depends on the interaction of the transcription factors NF-I and Sp1 with a tandem repeat of evolutionary conserved NF-I/Sp1 switch elements. An increased affinity of Sp1 to these elements has been observed in experimental liver fibrosis. Here, we demonstrate that the DNA binding drug mithramycin displays a high affinity binding to the GC-rich elements in the collagen-alpha 1(I) promoter as measured by DNAse I protection and gel retardation assays. Mithramycin interferes with Sp1 but not with NF-I binding to these sites. At a concentration of 100 nM, mithramycin efficiently reduces basal and TGF-beta-stimulated alpha 1(I) gene expression in human primary fibroblasts. The transcriptional activity and mRNA steady state levels of other genes, including the collagenase gene, as well as the growth rate of fibroblasts remained unchanged on exposure to this drug. Taken together, our results indicate that the transcriptional activity of the type I collagen gene highly depends on its GC-rich regulatory elements, and further, that these elements can be differentially blocked, thereby changing the balance between ECM structural and degrading gene activities in human fibroblasts. Images

Nehls, M C; Brenner, D A; Gruss, H J; Dierbach, H; Mertelsmann, R; Herrmann, F

1993-01-01

203

Downregulation of Homologous Recombination DNA Repair Genes by HDAC Inhibition in Prostate Cancer Is Mediated through the E2F1 Transcription Factor  

PubMed Central

Background Histone deacetylase inhibitors (HDACis) re-express silenced tumor suppressor genes and are currently undergoing clinical trials. Although HDACis have been known to induce gene expression, an equal number of genes are downregulated upon HDAC inhibition. The mechanism behind this downregulation remains unclear. Here we provide evidence that several DNA repair genes are downregulated by HDAC inhibition and provide a mechanism involving the E2F1 transcription factor in the process. Methodology/Principal Findings Applying Analysis of Functional Annotation (AFA) on microarray data of prostate cancer cells treated with HDACis, we found a number of genes of the DNA damage response and repair pathways are downregulated by HDACis. AFA revealed enrichment of homologous recombination (HR) DNA repair genes of the BRCA1 pathway, as well as genes regulated by the E2F1 transcription factor. Prostate cancer cells demonstrated a decreased DNA repair capacity and an increased sensitization to chemical- and radio-DNA damaging agents upon HDAC inhibition. Recruitment of key HR repair proteins to the site of DNA damage, as well as HR repair capacity was compromised upon HDACi treatment. Based on our AFA data, we hypothesized that the E2F transcription factors may play a role in the downregulation of key repair genes upon HDAC inhibition in prostate cancer cells. ChIP analysis and luciferase assays reveal that the downregulation of key repair genes is mediated through decreased recruitment of the E2F1 transcription factor and not through active repression by repressive E2Fs. Conclusions/Significance Our study indicates that several genes in the DNA repair pathway are affected upon HDAC inhibition. Downregulation of the repair genes is on account of a decrease in amount and promoter recruitment of the E2F1 transcription factor. Since HDAC inhibition affects several pathways that could potentially have an impact on DNA repair, compromised DNA repair upon HDAC inhibition could also be attributed to several other pathways besides the ones investigated in this study. However, our study does provide insights into the mechanism that governs downregulation of HR DNA repair genes upon HDAC inhibition, which can lead to rationale usage of HDACis in the clinics.

Kachhap, Sushant K.; Rosmus, Nadine; Collis, Spencer J.; Kortenhorst, Madeleine S. Q.; Wissing, Michel D.; Hedayati, Mohammad; Shabbeer, Shabana; Mendonca, Janet; Deangelis, Justin; Marchionni, Luigi; Lin, Jianqing; Hoti, Naseruddin; Nortier, Johan W. R.; DeWeese, Theodore L.; Hammers, Hans; Carducci, Michael A.

2010-01-01

204

Short-Chain Fatty Acids Inhibit Growth Hormone and Prolactin Gene Transcription via cAMP/PKA/CREB Signaling Pathway in Dairy Cow Anterior Pituitary Cells  

PubMed Central

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.

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

205

The endogenous retroviral insertion in the human complement C4 gene modulates the expression of homologous genes by antisense inhibition  

Microsoft Academic Search

Intron 9 contains the complete endogenous retrovirus HERV-K(C4) as a 6.4-kb insertion in 60% of human C4 genes. The retroviral insertion is in reverse orientation to the C4 coding sequence. Therefore, expression of C4 could lead to the transcription of an antisense RNA, which might protect against exogenous retroviral infections. To test this hypothesis, open reading frames from the HERV

P. M. Schneider; K. Witzel-Schlömp; C. Rittner; L. Zhang

2001-01-01

206

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

PubMed Central

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.

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

2010-01-01

207

An apoptosis-inhibiting gene from a nuclear polyhedrosis virus encoding a polypeptide with Cys/His sequence motifs.  

PubMed Central

Two different baculovirus genes are known to be able to block apoptosis triggered upon infection of Spodoptera frugiperda cells with p35 mutants of the insect baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV):p35 (P35-encoding gene) of AcMNPV (R. J. Clem, M. Fechheimer, and L. K. Miller, Science 254:1388-1390, 1991) and iap (inhibitor of apoptosis gene) of Cydia pomonella granulosis virus (CpGV) (N. E. Crook, R. J. Clem, and L. K. Miller, J. Virol. 67:2168-2174, 1993). Using a genetic complementation assay to identify additional genes which inhibit apoptosis during infection with a p35 mutant, we have isolated a gene from Orgyia pseudotsugata NPV (OpMNPV) that was able to functionally substitute for AcMNPV p35. The nucleotide sequence of this gene, Op-iap, predicted a 30-kDa polypeptide product with approximately 58% amino acid sequence identity to the product of CpGV iap, Cp-IAP. Like Cp-IAP, the predicted product of Op-iap has a carboxy-terminal C3HC4 zinc finger-like motif. In addition, a pair of additional cysteine/histidine motifs were found in the N-terminal regions of both polypeptide sequences. Recombinant p35 mutant viruses carrying either Op-iap or Cp-iap appeared to have a normal phenotype in S. frugiperda cells. Thus, Cp-IAP and Op-IAP appear to be functionally analogous to P35 but are likely to block apoptosis by a different mechanism which may involve direct interaction with DNA. Images

Birnbaum, M J; Clem, R J; Miller, L K

1994-01-01

208

Modifying metabolically sensitive histone marks by inhibiting glutamine metabolism affects gene expression and alters cancer cell phenotype  

PubMed Central

The interplay of metabolism and epigenetic regulatory mechanisms has become a focal point for a better understanding of cancer development and progression. In this study, we have acquired data supporting previous observations that demonstrate glutamine metabolism affects histone modifications in human breast cancer cell lines. Treatment of non-invasive epithelial (T-47D and MDA-MB-361) and invasive mesenchymal (MDA-MB-231 and Hs-578T) breast cancer cell lines with the glutaminase inhibitor, Compound 968, resulted in cytotoxicity in all cell lines, with the greatest effect being observed in MDA-MB-231 breast cancer cells. Compound 968-treatment induced significant downregulation of 20 critical cancer-related genes, the majority of which are anti-apoptotic and/or promote metastasis, including AKT, BCL2, BCL2L1, CCND1, CDKN3, ERBB2, ETS1, E2F1, JUN, KITLG, MYB, and MYC. Histone H3K4me3, a mark of transcriptional activation, was reduced at the promoters of all but one of these critical cancer genes. The decrease in histone H3K4me3 at global and gene-specific levels correlated with reduced expression of SETD1 and ASH2L, genes encoding the histone H3K4 methyltransferase complex. Further, the expression of other epigenetic regulatory genes, known to be downregulated during apoptosis (e.g., DNMT1, DNMT3B, SETD1 and SIRT1), was also downregulated by Compound 968. These changes in gene expression and histone modifications were accompanied by the activation of apoptosis, and decreased invasiveness and resistance of MDA-MB-231 cells to chemotherapeutic drug doxorubicin. The results of this study provide evidence to a link between cytotoxicity caused by inhibiting glutamine metabolism with alterations of the epigenome of breast cancer cells and suggest that modification of intracellular metabolism may enhance the efficiency of epigenetic therapy.

Simpson, Natalie E.; Tryndyak, Volodymyr P.; Pogribna, Marta; Beland, Frederick A.; Pogribny, Igor P.

2012-01-01

209

Can biological nitrification inhibition (BNI) genes from perennial Leymus racemosus ( Triticeae ) combat nitrification in wheat farming?  

Microsoft Academic Search

Using a recombinant luminescent Nitrosomonas europaea assay to quantify biological nitrification inhibition (BNI), we found that a wild relative of wheat (Leymus racemosus (Lam.) Tzvelev) had a high BNI capacity and releases about 20 times more BNI compounds (about 30 ATU g?1 root dry weight 24 h?1) than Triticum aestivum L. (cultivated wheat). The root exudate from cultivated wheat has no inhibitory

G. V. Subbarao; Ban Tomohiro; Kishii Masahiro; Ito Osamu; H. Samejima; H. Y. Wang; S. J. Pearse; S. Gopalakrishnan; K. Nakahara; A. K. M. Zakir Hossain; H. Tsujimoto; W. L. Berry

2007-01-01

210

Efficient delivery of siRNA for inhibition of gene expression in postnatal mice.  

PubMed

It has recently been shown that RNA interference can be induced in cultured mammalian cells by delivery of short interfering RNAs (siRNAs). Here we describe a method for efficient in vivo delivery of siRNAs to organs of postnatal mice and demonstrate effective and specific inhibition of transgene expression in a variety of organs. PMID:12145662

Lewis, David L; Hagstrom, James E; Loomis, Aaron G; Wolff, Jon A; Herweijer, Hans

2002-09-01

211

Transcriptional responses of bacterial amoA gene to dimethyl sulfide inhibition in complex microbial communities.  

PubMed

This study presented an approach by combining the real-time reverse transcription polymerase chain reaction with the terminal restriction fragment length polymorphism (T-RFLP) to investigate transcriptional responses of ammonia-oxidizing bacteria (AOB) to dimethyl sulfide (DMS) inhibition. Batch experiments with added ammonium and DMS were conducted using three activated sludges and Nitrosomonas europaea, and the transcriptional responses of the amo subunit A (amoA) mRNA were evaluated. It was found that DMS inhibited ammonium oxidation and amoA mRNA expression in all batch experiments but the inhibition degree observed was different for different sludges examined. It is likely that the different inhibitory effects of DMS on ammonium oxidation and amoA mRNA expression stemmed from different dominant AOB populations in the sludges. The T-RFLP results for amoA mRNA suggested that inhibition of ammonium oxidation by DMS to Nm. europaea-like AOB with T-RF 219/270 is relatively minor compared to other AOB populations in the examined sludges, such as Nm. europaea-like AOB with T-RF 491/491. PMID:24666625

Fukushima, Toshikazu; Whang, Liang-Ming; Lee, Ya-Ching; Putri, Dyah Wulandari; Chen, Po-Chun; Wu, Yi-Ju

2014-08-01

212

Calcitonin gene-related peptide inhibits osteoclastic bone resorption: A comparative study  

Microsoft Academic Search

Summary  Besides the calcitonin (CT) precursor, the calcitonin gene also encodes another peptide—calcitonin gene-related peptide (CGRP).\\u000a We have previously reported that CGRP lowers plasma calcium in the rat. In the present study we have evaluated the effect\\u000a of CGRP on resorption of bone by isolated rat osteoclasts and have compared these effects to those produced by calcitonins\\u000a from three species (salmon,

Mone Zaidi; Karen Fuller; Peter J. R. Bevis; Rose E. GainesDas; Timothy J. Chambers; Iain MacIntyre

1987-01-01

213

Nitric oxide decreases expression of osmoprotective genes via direct inhibition of TonEBP transcriptional activity  

Microsoft Academic Search

During antidiuresis, renal medullary cells adapt to the hyperosmotic interstitial environment by increased expression of osmoprotective\\u000a genes, which is driven by a common transcriptional activator, tonicity-responsive enhancer binding protein (TonEBP). Because\\u000a nitric oxide (NO) is abundantly produced in the renal medulla, the present studies addressed the effect of NO on expression\\u000a of osmoprotective genes and TonEBP activation in MDCK cells.

Wolfgang Neuhofer; Maria-Luisa Fraek; Franz-X. Beck

2009-01-01

214

PUVA Inhibits DNA Replication, but not Gene Transcription at Nonlethal Dosages  

Microsoft Academic Search

The combination of psoralens and UVA radiation (PUVA photochemotherapy) is an established treatment for many skin disorders. UVA-induced psoralen–DNA interactions are assumed to contribute to the cutaneous anti-inflammatory and anti-proliferative effects of PUVA. PUVA-induced DNA modifications might interfere not only with DNA replication, but also with gene transcription of proinflammatory genes. We therefore studied the effect of PUVA on cell

Matthias Lüftl; Martin Röcken; Gerd Plewig; Klaus Degitz

1998-01-01

215

The response of early neural genes to FGF signaling or inhibition of BMP indicate the absence of a conserved neural induction module  

PubMed Central

Background The molecular mechanism that initiates the formation of the vertebrate central nervous system has long been debated. Studies in Xenopus and mouse demonstrate that inhibition of BMP signaling is sufficient to induce neural tissue in explants or ES cells respectively, whereas studies in chick argue that instructive FGF signaling is also required for the expression of neural genes. Although additional signals may be involved in neural induction and patterning, here we focus on the roles of BMP inhibition and FGF8a. Results To address the question of necessity and sufficiency of BMP inhibition and FGF signaling, we compared the temporal expression of the five earliest genes expressed in the neuroectoderm and determined their requirements for induction at the onset of neural plate formation in Xenopus. Our results demonstrate that the onset and peak of expression of the genes vary and that they have different regulatory requirements and are therefore unlikely to share a conserved neural induction regulatory module. Even though all require inhibition of BMP for expression, some also require FGF signaling; expression of the early-onset pan-neural genes sox2 and foxd5? requires FGF signaling while other early genes, sox3, geminin and zicr1 are induced by BMP inhibition alone. Conclusions We demonstrate that BMP inhibition and FGF signaling induce neural genes independently of each other. Together our data indicate that although the spatiotemporal expression patterns of early neural genes are similar, the mechanisms involved in their expression are distinct and there are different signaling requirements for the expression of each gene.

2011-01-01

216

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

PubMed Central

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.

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

2013-01-01

217

Nitric oxide regulates nitric oxide synthase-2 gene expression by inhibiting NF-kappaB binding to DNA.  

PubMed Central

Treatment of astroglial cells with interleukin 1beta and interferon gamma transcriptionally activates the nitric oxide synthase (NOS)-2 gene. The duration of mRNA expression is brief because of transcript instability. In addition, NO donors reduce the expression of NOS-2 mRNA dramatically by reducing the rate of transcription. In this study we observed that the NO donor, spermine NONOate did not inhibit the activation and translocation of NF-kappaB, a key transcription factor in the induction of NOS-2, but inhibited formation of the NF-kappaB-DNA complex. This effect was reversed by methaemoglobin (acting as an NO trap) and by the reducing agent dithiothreitol. Formation of the interferon-regulatory factor-DNA complex was unaffected by NO. These results suggest that NO can modulate its own production by interfering with NF-kappaB interaction with the promoter region of the NOS gene, a negative feedback effect that may be important for limiting NO production in vivo.

Park, S K; Lin, H L; Murphy, S

1997-01-01

218

RNAi silencing of the HaHMG-CoA reductase gene inhibits oviposition in the Helicoverpa armigera cotton bollworm.  

PubMed

RNA interference (RNAi) has considerable promise for developing novel pest control techniques, especially because of the threat of the development of resistance against current strategies. For this purpose, the key is to select pest control genes with the greatest potential for developing effective pest control treatments. The present study demonstrated that the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase; HMGR) gene is a potential target for insect control using RNAi. HMGR is a key enzyme in the mevalonate pathway in insects. A complete cDNA encoding full length HMGR (encoding an 837-aa protein) was cloned from Helicoverpa armigera (Lepidoptera: Noctuidae). The HaHMGR (H. armigera HMGR) knockdown using systemic RNAi in vivo inhibited the fecundity of the females, effectively inhibited ovipostion, and significantly reduced vitellogenin (Vg) mRNA levels. Moreover, the oviposition rate of the female moths was reduced by 98% by silencing HaHMGR compared to the control groups. One-pair experiments showed that both the proportions of valid mating and fecundity were zero. Furthermore, the HaHMGR-silenced females failed to lay eggs (approximate 99% decrease in oviposition) in the semi-field cage performance. The present study demonstrated the potential implications for developing novel pest management strategies using HaHMGR RNAi in the control of H. armigera and other insect pests. PMID:23844078

Wang, Zhijian; Dong, Yongcheng; Desneux, Nicolas; Niu, Changying

2013-01-01

219

Knockdown of astrocyte elevated gene-1 inhibits proliferation and enhancing chemo-sensitivity to cisplatin or doxorubicin in neuroblastoma cells  

PubMed Central

Background Astrocyte elevated gene-1 (AEG-1) was originally characterized as a HIV-1-inducible gene in primary human fetal astrocyte. Recent studies highlight a potential role of AEG-1 in promoting tumor progression and metastasis. The aim of this study was to investigate if AEG-1 serves as a potential therapeutic target of human neuroblastoma. Methods We employed RNA interference to reduce AEG-1 expression in human neuroblastoma cell lines and analyzed their phenotypic changes. Results We found that the knockdown of AEG-1 expression in human neuroblastoma cells significantly inhibited cell proliferation and apoptosis. The specific downregulation induced cell arrest in the G0/G1 phase of cell cycle. In the present study, we also observed a significant enhancement of chemo-sensitivity to cisplatin and doxorubicin by knockdown of AEG-1. Conclusion Our study suggests that overexpressed AEG-1 enhance the tumorogenic properties of neuroblastoma cells. The inhibition of AEG-1 expression could be a new adjuvant therapy for neuroblastoma.

Liu, Haiyan; Song, Xianrang; Liu, Chunxi; Xie, Li; Wei, Ling; Sun, Ruopeng

2009-01-01

220

Impact of the 3D microenvironment on phenotype, gene expression, and EGFR inhibition of colorectal cancer cell lines.  

PubMed

Three-dimensional (3D) tumor cell cultures grown in laminin-rich-extracellular matrix (lrECM) are considered to reflect human tumors more realistic as compared to cells grown as monolayer on plastic. Here, we systematically investigated the impact of ECM on phenotype, gene expression, EGFR signaling pathway, and on EGFR inhibition in commonly used colorectal cancer (CRC) cell lines. LrECM on-top (3D) culture assays were performed with the CRC cell lines SW-480, HT-29, DLD-1, LOVO, CACO-2, COLO-205 and COLO-206F. Morphology of lrECM cultivated CRC cell lines was determined by phase contrast and confocal laser scanning fluorescence microscopy. Proliferation of cells was examined by MTT assay, invasive capacity of the cell lines was assayed using Matrigel-coated Boyden chambers, and migratory activity was determined employing the Fence assay. Differential gene expression was analyzed at the transcriptional level by the Agilent array platform. EGFR was inhibited by using the specific small molecule inhibitor AG1478. A specific spheroid growth pattern was observed for all investigated CRC cell lines. DLD-1, HT-29 and SW-480 and CACO-2 exhibited a clear solid tumor cell formation, while LOVO, COLO-205 and COLO-206F were characterized by forming grape-like structures. Although the occurrence of a spheroid morphology did not correlate with an altered migratory, invasive, or proliferative capacity of CRC cell lines, gene expression was clearly altered in cells grown on lrECM as compared to 2D cultures. Interestingly, in KRAS wild-type cell lines, inhibition of EGFR was less effective in lrECM (3D) cultures as compared to 2D cell cultures. Thus, comparing both 2D and 3D cell culture models, our data support the influence of the ECM on cancer growth. Compared to conventional 2D cell culture, the lrECM (3D) cell culture model offers the opportunity to investigate permanent CRC cell lines under more physiological conditions, i.e. in the context of molecular therapeutic targets and their pharmacological inhibition. PMID:23555746

Luca, Anna C; Mersch, Sabrina; Deenen, René; Schmidt, Stephan; Messner, Isabelle; Schäfer, Karl-Ludwig; Baldus, Stephan E; Huckenbeck, Wolfgang; Piekorz, Roland P; Knoefel, Wolfram T; Krieg, Andreas; Stoecklein, Nikolas H

2013-01-01

221

Impact of the 3D Microenvironment on Phenotype, Gene Expression, and EGFR Inhibition of Colorectal Cancer Cell Lines  

PubMed Central

Three-dimensional (3D) tumor cell cultures grown in laminin-rich-extracellular matrix (lrECM) are considered to reflect human tumors more realistic as compared to cells grown as monolayer on plastic. Here, we systematically investigated the impact of ECM on phenotype, gene expression, EGFR signaling pathway, and on EGFR inhibition in commonly used colorectal cancer (CRC) cell lines. LrECM on-top (3D) culture assays were performed with the CRC cell lines SW-480, HT-29, DLD-1, LOVO, CACO-2, COLO-205 and COLO-206F. Morphology of lrECM cultivated CRC cell lines was determined by phase contrast and confocal laser scanning fluorescence microscopy. Proliferation of cells was examined by MTT assay, invasive capacity of the cell lines was assayed using Matrigel-coated Boyden chambers, and migratory activity was determined employing the Fence assay. Differential gene expression was analyzed at the transcriptional level by the Agilent array platform. EGFR was inhibited by using the specific small molecule inhibitor AG1478. A specific spheroid growth pattern was observed for all investigated CRC cell lines. DLD-1, HT-29 and SW-480 and CACO-2 exhibited a clear solid tumor cell formation, while LOVO, COLO-205 and COLO-206F were characterized by forming grape-like structures. Although the occurrence of a spheroid morphology did not correlate with an altered migratory, invasive, or proliferative capacity of CRC cell lines, gene expression was clearly altered in cells grown on lrECM as compared to 2D cultures. Interestingly, in KRAS wild-type cell lines, inhibition of EGFR was less effective in lrECM (3D) cultures as compared to 2D cell cultures. Thus, comparing both 2D and 3D cell culture models, our data support the influence of the ECM on cancer growth. Compared to conventional 2D cell culture, the lrECM (3D) cell culture model offers the opportunity to investigate permanent CRC cell lines under more physiological conditions, i.e. in the context of molecular therapeutic targets and their pharmacological inhibition.

Deenen, Rene; Schmidt, Stephan; Messner, Isabelle; Schafer, Karl-Ludwig; Baldus, Stephan E.; Huckenbeck, Wolfgang; Piekorz, Roland P.; Knoefel, Wolfram T.; Krieg, Andreas; Stoecklein, Nikolas H.

2013-01-01

222

[Prosapogenin A inhibits cell growth of MCF7 via downregulating STAT3 and glycometabolism-related gene].  

PubMed

This study is to investigate the inhibitory effect and mechanism of prosapogenin A (PSA) on MCF7. MTT assay was performed to determine the inhibitory effect of PSA on MCF7 cells. PI/Hoechst 33342 double staining was used to detect cell apoptosis. RT-PCR was used to test the mRNA levels of STAT3, GLUT1, HK and PFKL. Western blotting was performed to determine the expression of STAT3 and pSTAT3 protein in MCF7 cells. The results showed that PSA could dose-dependently inhibit cell growth of MCF7 followed by IC50 of 9.65 micrmol x L(-1) and promote cell apoptosis of MCF7. Reduced mRNA levels of STAT3, HK and PFKL were observed in MCF7 cells treated with 5 micromol x L(-1) of PSA. PSA also decreased the level of pSTAT3 protein. STAT3 siRNA caused decrease of mRNA of GLUT1, HK and PFKL which indicated STAT3 could regulate the expressions of GLUT1, HK and PFKL. The results suggested that PSA could inhibit cell growth and promote cell apoptosis of MCF7 via inhibition of STAT3 and glycometabolism-related gene. PMID:24358789

Wang, Tian-xiao; Shi, Xiao-yan; Cong, Yue; Zhang, Zhong-qing; Liu, Ying-hua

2013-09-01

223

HIF-1 Regulates Iron Homeostasis in Caenorhabditis elegans by Activation and Inhibition of Genes Involved in Iron Uptake and Storage  

PubMed Central

Caenorhabditis elegans ftn-1 and ftn-2, which encode the iron-storage protein ferritin, are transcriptionally inhibited during iron deficiency in intestine. Intestinal specific transcription is dependent on binding of ELT-2 to GATA binding sites in an iron-dependent enhancer (IDE) located in ftn-1 and ftn-2 promoters, but the mechanism for iron regulation is unknown. Here, we identify HIF-1 (hypoxia-inducible factor -1) as a negative regulator of ferritin transcription. HIF-1 binds to hypoxia-response elements (HREs) in the IDE in vitro and in vivo. Depletion of hif-1 by RNA interference blocks transcriptional inhibition of ftn-1 and ftn-2 reporters, and ftn-1 and ftn-2 mRNAs are not regulated in a hif-1 null strain during iron deficiency. An IDE is also present in smf-3 encoding a protein homologous to mammalian divalent metal transporter-1. Unlike the ftn-1 IDE, the smf-3 IDE is required for HIF-1–dependent transcriptional activation of smf-3 during iron deficiency. We show that hif-1 null worms grown under iron limiting conditions are developmentally delayed and that depletion of FTN-1 and FTN-2 rescues this phenotype. These data show that HIF-1 regulates intestinal iron homeostasis during iron deficiency by activating and inhibiting genes involved in iron uptake and storage.

Romney, Steven Joshua; Newman, Ben S.; Thacker, Colin; Leibold, Elizabeth A.

2011-01-01

224

HIF-1 regulates iron homeostasis in Caenorhabditis elegans by activation and inhibition of genes involved in iron uptake and storage.  

PubMed

Caenorhabditis elegans ftn-1 and ftn-2, which encode the iron-storage protein ferritin, are transcriptionally inhibited during iron deficiency in intestine. Intestinal specific transcription is dependent on binding of ELT-2 to GATA binding sites in an iron-dependent enhancer (IDE) located in ftn-1 and ftn-2 promoters, but the mechanism for iron regulation is unknown. Here, we identify HIF-1 (hypoxia-inducible factor -1) as a negative regulator of ferritin transcription. HIF-1 binds to hypoxia-response elements (HREs) in the IDE in vitro and in vivo. Depletion of hif-1 by RNA interference blocks transcriptional inhibition of ftn-1 and ftn-2 reporters, and ftn-1 and ftn-2 mRNAs are not regulated in a hif-1 null strain during iron deficiency. An IDE is also present in smf-3 encoding a protein homologous to mammalian divalent metal transporter-1. Unlike the ftn-1 IDE, the smf-3 IDE is required for HIF-1-dependent transcriptional activation of smf-3 during iron deficiency. We show that hif-1 null worms grown under iron limiting conditions are developmentally delayed and that depletion of FTN-1 and FTN-2 rescues this phenotype. These data show that HIF-1 regulates intestinal iron homeostasis during iron deficiency by activating and inhibiting genes involved in iron uptake and storage. PMID:22194696

Romney, Steven Joshua; Newman, Ben S; Thacker, Colin; Leibold, Elizabeth A

2011-12-01

225

Hepatocyte nuclear factor-1alpha inhibits insulin promoter factor 1-dependent transactivation of the human insulin gene.  

PubMed

To investigate the regulational interaction of hepatocyte nuclear factor-1alpha (HNF-1alpha) and insulin promoter factor 1 (IPF1) on insulin gene expression, either or both of the expression vectors carrying each transcription factor were transiently transfected into HeLa cells, RINm5F cells and MIN6 cells together with the luciferase reporter construct driven by a human preproinsulin gene promoter (-1998 to +237) designated as, pINS-1998/luc. IPF1-transfection into HeLa cells strongly stimulated the luciferase activity to 725 fold that of the basal level. In contrast, HNF-1alpha-transfection resulted in only a 6.7 fold increase. In co-transfection experiments, increasing the amount of HNF-1alpha resulted in an 84.5% and 74.4% decrease in IPF1-stimulated luciferase activity in HeLa and RINm5F cells, respectively. Deletion constructs designated as pINS-248/luc, pINS-213/luc and pINS-185/luc were transfected into RINm5F cells to determine the role of the A3 element and its 5' flanking sequence in the inhibitory effect of HNF-1alpha. The results showed that the inhibiting effects of HNF-1alpha with pINS-213/luc and pINS-185/luc were significantly smaller than those with both pINS-1998/luc and pINS-248/luc. Transfection into MN6 cells with pINS-1998/luc in the absence of IPF1 resulted in constitutional transactivation of the insulin gene, and this transactivation was abolished by the co-transfection with HNF-1alpha. The present data indicate that IPF1 rather than HNF-1alpha predominantly transactivates the insulin gene, and that HNF-1alpha inhibits IPF1-dependent insulin gene transactivation mediated through the 5' flanking sequence of the A3 element. It is suggested that HNF-1alpha may be involved in insulin gene expression as a negative regulator. PMID:11428722

Yamakawa, K; Yamasaki, H; Ozaki, M; Yamauchi, M D; Fujita, N; Abe, T; Miyazoe, H; Sera, Y; Uotani, S; Kawasaki, E; Takino, H; Yamaguchi, Y; Eguchi, K

2001-01-01

226

The Burkholderia bcpAIOB Genes Define Unique Classes of Two-Partner Secretion and Contact Dependent Growth Inhibition Systems  

PubMed Central

Microbes have evolved many strategies to adapt to changes in environmental conditions and population structures, including cooperation and competition. One apparently competitive mechanism is contact dependent growth inhibition (CDI). Identified in Escherichia coli, CDI is mediated by Two–Partner Secretion (TPS) pathway proteins, CdiA and CdiB. Upon cell contact, the toxic C-terminus of the TpsA family member CdiA, called the CdiA-CT, inhibits the growth of CDI? bacteria. CDI+ bacteria are protected from autoinhibition by an immunity protein, CdiI. Bioinformatic analyses indicate that CDI systems are widespread amongst ?, ?, and ? proteobacteria and that the CdiA-CTs and CdiI proteins are highly variable. CdiI proteins protect against CDI in an allele-specific manner. Here we identify predicted CDI system-encoding loci in species of Burkholderia, Ralstonia and Cupriavidus, named bcpAIOB, that are distinguished from previously-described CDI systems by gene order and the presence of a small ORF, bcpO, located 5? to the gene encoding the TpsB family member. A requirement for bcpO in function of BcpA (the TpsA family member) was demonstrated, indicating that bcpAIOB define a novel class of TPS system. Using fluorescence microscopy and flow cytometry, we show that these genes are expressed in a probabilistic manner during culture of Burkholderia thailandensis in liquid medium. The bcpAIOB genes and extracellular DNA were required for autoaggregation and adherence to an abiotic surface, suggesting that CDI is required for biofilm formation, an activity not previously attributed to CDI. By contrast to what has been observed in E. coli, the B. thailandensis bcpAIOB genes only mediated interbacterial competition on a solid surface. Competition occurred in a defined spatiotemporal manner and was abrogated by allele-specific immunity. Our data indicate that the bcpAIOB genes encode distinct classes of CDI and TPS systems that appear to function in sociomicrobiological community development.

Anderson, Melissa S.; Garcia, Erin C.; Cotter, Peggy A.

2012-01-01

227

IL-10 Gene Modified Dendritic Cells Inhibit T Helper Type 1-Mediated Alloimmune Responses and Promote Immunological Tolerance in Diabetes  

PubMed Central

Dendritic cells (DCs) have the potency to regulate the outcome of autoimmunity through the modulation of immune responses. The induction of antigen specific tolerance is critical for prevention and treatment of allograft rejection. In the present study, we transfected IL-10 gene into DCs and investigated their effect on inhibition of lymphocyte activity in vitro and induction of immune tolerance on islet allograft in mice. An IDDM C57BL/6 mouse model was induced by streptozotocin. The islet cells isolated from the BALB/c mice were transplanted into the kidney capules of the model mice followed by injection of IL-10 modified DCs (mDCs). The results showed that mDCs could significantly inhibit T lymphocyte proliferation mediated by allotype cells and induce its apoptosis, whereas, unmodified DCs (umDCs) could promote the murine lymphocyte proliferation markedly. The injection of mDCs could prolong the survival of allotype islet transplanted IDDM mice. The average plasma glucose (PG) level in mDCs treated mice returned to normal within 3 days and lasted for about 2 weeks. The rejection response in control mice occurred for 5 days after transplantation. The level of IFN-? was lower while IL-4 was higher in mDCs treated mice than that in umDCs treated mice, which indicated that Th1/Th2 deviation occurred. Our studies suggest that IL-10 gene modified DCs can induce the immune tolerance to islet graft and prolong survival of the recipients by the inhibiting of T cell proliferation in allotype mice.

Zhu, Huifen; Qiu, Wenhong; Lei, Ping; Zhou, Wei; Wen, Xue; He, Fengrong; Li, Li; Dai, Hong; Shen, Guanxin; Gong, Feili

2008-01-01

228

Tetradecylthioacetic acid inhibits proliferation of human SW620 colon cancer cells - gene expression profiling implies endoplasmic reticulum stress  

PubMed Central

Background Previous reports have shown an antiproliferative effect of the synthetic, 3-thia fatty acid tetradecylthioacetic acid (TTA) on different cancer cells in vitro and in vivo. The mechanisms behind the observed effects are poorly understood. We therefore wanted to explore the molecular mechanisms involved in TTA-induced growth inhibition of the human colon cancer cell line SW620 by gene expression profiling. Methods An antiproliferative effect of TTA on SW620 cells in vitro was displayed in real time using the xCELLigence System (Roche). Affymetrix gene expression profiling was performed to elucidate the molecular mechanisms behind the antiproliferative effect of TTA. Changes in gene expression were verified at protein level by western blotting. Results TTA reduced SW620 cell growth, measured as baseline cell index, by 35% and 55% after 48 h and 72 h, respectively. We show for the first time that TTA induces an endoplasmic reticulum (ER) stress response in cancer cells. Gene expression analysis revealed changes related to ER stress and unfolded protein response (UPR). This was verified at protein level by phosphorylation of eukaryote translation initiation factor 2 alpha (eIF2?) and downstream up-regulation of activating transcription factor 4 (ATF4). Transcripts for positive and negative cell cycle regulators were down- and up-regulated, respectively. This, together with a down-regulation of Cyclin D1 at protein level, indicates inhibition of cell cycle progression. TTA also affected transcripts involved in calcium homeostasis. Moreover, mRNA and protein level of the ER stress inducible C/EBP-homologous protein (CHOP), Tribbles homolog 3 (Drosophila) (TRIB3) and CCAAT/enhancer binding protein beta (C/EBP?) were enhanced, and the C/EBP? LIP/LAP ratio was significantly increased. These results indicate prolonged ER stress and a possible link to induction of cell death. Conclusion We find that TTA-induced growth inhibition of SW620 cells seems to be mediated through induction of ER stress and activation of the UPR pathway.

2011-01-01

229

Inhibition of proliferation and gene expression regulation by (-)-epigallocatechin-3-gallate in human synovial sarcoma cells.  

PubMed

Synovial sarcoma is an aggressive soft-tissue malignancy with poor prognosis and lack of response to conventional cytotoxic chemotherapy. The regulatory mechanisms for the rapid proliferation of synovial sarcoma cells and the particular aggressiveness of this sarcoma remain poorly understood. In this study, we investigate the effect of epigallocatechin-3-gallate (EGCG) on growth and apoptosis of chondrosarcoma cells. The MTT assay and DAPI staining indicated that EGCG effectively inhibited cellular proliferation and induces apoptosis of the synovial sarcoma cells and induced apoptosis as confirmed by flow cytometry. Furthermore, Bcl-2 and Mcl-1 levels significantly decreased, Bax levels significantly increased, whereas expression levels of the proteins Bcl-XL were unchanged in response to EGCG treatment in SW982. In conclusion, our findings demonstrate that EGCG is effective for growth inhibition of synovial sarcoma cell lines in vitro and suggest that EGCG may be a new therapeutic option for patients with synovial sarcoma. PMID:20480267

Sun, Yongming; Wang, Haibin; Lin, Fanguo; Hua, Jun; Zhou, Gaoli

2011-12-01

230

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

PubMed

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 five 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 dephosphorylation of ERK, a downstream RAS target that may be critical for mutant RAS-induced cell growth. In addition, PTPRG expression is upregulated 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

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

2014-09-01

231

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

PubMed Central

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 ?1.6–1.8 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.

Lim, Chiat Koo; Kelley, Richard L.

2013-01-01

232

Gene silencing of ?-galactosamide ?-2,6-sialyltransferase 1 inhibits human influenza virus infection of airway epithelial cells  

PubMed Central

Background Human influenza virus hemagglutinin prefers to use sialic acid (SA) receptors via ?-2,6 linkages. The ?-galactoside ?-2,6-sialyltransferase I (ST6Gal I) protein is encoded by the ST6GAL1 gene and is responsible for the addition of ?-2,6 linked SA to the Gal?1-4GlcNAc disaccharide of glycans and glycoproteins found on the cellular surface. Therefore, ST6GAL1 could be a potential target for anti-influenza therapeutics. We used specific small interfering RNAs (siRNAs) to block expression of ST6GAL1 and limit distribution of SA receptors on the surface of airway epithelial cells. Results The siRNA duplexes we used inhibited ST6GAL1 mRNA expression and subsequent expression of the encoding protein. As a result, synthesis of ?-2,6 SA galactose was inhibited. Adsorption of influenza virus particles to the surface of cells transfected with appropriate specific siRNAs was significantly reduced. Intracellular viral genome copy number and virus titer within the supernatant of cells transfected with siRNAs was significantly reduced in a dose-dependent manner compared with those for untransfected cells and cells transfected with non-specific siRNAs. Conclusions We used siRNAs targeting ST6GAL1 to inhibit the expression of certain cell surface receptors, thereby preventing virus adsorption. This resulted in the inhibition of human influenza virus infection. Our findings are a significant development in the identification of potential new anti-influenza drug targets.

2014-01-01

233

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

SciTech Connect

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.

Ku, Bo Mi [Department of Anatomy and Neurobiology, Institute of Health Sciences, School of Medicine, Gyeongsang National University, Jinju 660-751 (Korea, Republic of)] [Department of Anatomy and Neurobiology, Institute of Health Sciences, School of Medicine, Gyeongsang National University, Jinju 660-751 (Korea, Republic of); Ryu, Hyung Won [Division of Applied Life Science (BK21 Program), EB-NCRC, Institute of Agriculture Life Science, Graduate School of Gyeongsang National University, Jinju 660-701 (Korea, Republic of)] [Division of Applied Life Science (BK21 Program), EB-NCRC, Institute of Agriculture Life Science, Graduate School of Gyeongsang National University, Jinju 660-701 (Korea, Republic of); Lee, Yeon Kyung; Ryu, Jinhyun; Jeong, Joo Yeon; Choi, Jungil [Department of Anatomy and Neurobiology, Institute of Health Sciences, School of Medicine, Gyeongsang National University, Jinju 660-751 (Korea, Republic of)] [Department of Anatomy and Neurobiology, Institute of Health Sciences, School of Medicine, Gyeongsang National University, Jinju 660-751 (Korea, Republic of); Cho, Hee Jun [Department of Microbiology, Research Institute of Life Science, College of Natureal Sciences, Gyeongsang National University, Jinju 660-701 (Korea, Republic of)] [Department of Microbiology, Research Institute of Life Science, College of Natureal Sciences, Gyeongsang National University, Jinju 660-701 (Korea, Republic of); Park, Ki Hun, E-mail: khpark@gnu.ac.kr [Division of Applied Life Science (BK21 Program), EB-NCRC, Institute of Agriculture Life Science, Graduate School of Gyeongsang National University, Jinju 660-701 (Korea, Republic of); Kang, Sang Soo, E-mail: kangss@gnu.ac.kr [Department of Anatomy and Neurobiology, Institute of Health Sciences, School of Medicine, Gyeongsang National University, Jinju 660-751 (Korea, Republic of)

2010-11-19

234

A Proteasome Inhibitor, Bortezomib, Inhibits Breast Cancer Growth and Reduces Osteolysis by Downregulating Metastatic Genes  

PubMed Central

Purpose The incidence of bone metastasis in advanced breast cancer exceeds 70%. Bortezomib (Bzb), a proteasome inhibitor used for the treatment of multiple myeloma, also promotes bone formation. We tested the hypothesis that proteasome inhibitors can ameliorate breast cancer osteolytic disease. Experimental Design To address the potentially beneficial effect of Bzb in reducing tumor growth in the skeleton and counteracting bone osteolysis, human MDA-MB-231 breast cancer (BrCa) cells were injected into the tibia of mice to model bone tumor growth for in vivo assessment of treatment regimens pre- and post-tumor growth. Results Controls exhibited tumor growth destroying trabecular and cortical bone and invading muscle. Bzb treatment initiated following inoculation of tumor cells strikingly reduced tumor growth, restricted tumor cells mainly to the marrow cavity, and almost completely inhibited osteolysis in the bone microenvironment over a 3–4 week period demonstrated by 18F-FDG PET, micro-CT scanning, radiography, and histology. Thus, proteasome inhibition is effective in killing tumor cells within bone. Pre-treatment with Bzb for 3 weeks prior to inoculation of tumor cells was also effective in reducing osteolysis. Our in vitro and in vivo studies indicate mechanisms by which Bzb inhibits tumor growth and reduces osteolysis result from inhibited cell proliferation, necrosis and decreased expression of factors that promote BrCa tumor progression in bone. Conclusion These findings provide a basis for a novel strategy to treat patients with breast cancer osteolytic lesions, and represent an approach for protecting the entire skeleton from metastatic bone disease.

Jones, Marci D.; Liu, Julie C.; Barthel, Thomas K.; Hussain, Sadiq; Lovria, Erik; Cheng, Dengfeng; Schoonmaker, Jesse.A.; Mulay, Sudhanshu; Ayers, David C.; Bouxsein, Mary L.; Stein, Gary S.; Mukherjee, Siddhartha; Lian, Jane B.

2010-01-01

235

Reversible Inhibition of Tomato Fruit Gene Expression at High Temperature (Effects on Tomato Fruit Ripening).  

PubMed

The reversible inhibition of three ripening-related processes by high-temperature treatment (38[deg]C) was examined in tomato (Lycopersicon esculentum L. cv Daniella) fruit. Ethylene production, color development, and softening were inhibited during heating and recovered afterward, whether recovery took place at 20[deg]C or fruit were first held at chilling temperature (2[deg]C) after heating and then placed at 20[deg]C. Ethylene production and color development proceeded normally in heated fruit after 14 d of chilling, whereas the unheated fruit had delayed ethylene production and uneven color development. Levels of mRNA for 1-aminocyclopropane-1-carboxylic acid oxidase, phytoene synthase, and polygalacturonase decreased dramatically during the heat treatment but recovered afterward, whereas the mRNA for HSP17 increased during the high-temperature treatment and then decreased when fruit were removed from heat. As monitored by western blots, the HSP17 protein disappeared from fruit tissue after 3 d at 20[deg]C but remained when fruit were held at 2[deg]C. The persistence of heat-shock proteins at low temperature may be relevant to the protection against chilling injury provided by the heat treatment. Protein levels of 1-aminocyclopropane-1-carboxylic acid oxidase and polygalacturonase also did not closely follow the changes in their respective mRNAs. This implied both differences in relative stability and turnover rates of mRNA compared to protein and nontranslation of the message that accumulated in low temperature. The results suggest that high temperature inhibits ripening by inhibiting the accumulation of ripening-related mRNAs. Ripening processes that depend on continuous protein synthesis including ethylene production, lycopene accumulation, and cell-wall dissolution are thereby diminished. PMID:12226253

Lurie, S.; Handros, A.; Fallik, E.; Shapira, R.

1996-04-01

236

Synergistic re-activation of epigenetically silenced genes by combinatorial inhibition of DNMTs and LSD1 in cancer cells.  

PubMed

Epigenetic gene silencing, mediated by aberrant promoter DNA hypermethylation and repressive histone modifications, is a hallmark of cancer. Although heritable, the dynamic nature and potential reversibility through pharmacological interventions make such aberrations attractive targets. Since cancers contain multiple epigenetic abnormalities, combining therapies that target different defects could potentially enhance their individual efficacies. 5-Aza-2'-deoxycytidine (5-Aza-CdR), FDA-approved drug for the treatment of myelodysplastic syndrome, can inhibit DNA methyltransferases (DNMTs) upon incorporation into the DNA of dividing cells, resulting in global demethylation. More recently, the first histone demethylase, lysine specific demethylase 1 (LSD1), which demethylates both histone and non-histone substrates, has become a new target for epigenetic therapy. Using, clorgyline, an LSD1 inhibitor (LSD1i) to treat cancer cell lines, we show that clorgyline employs two mechanisms of action depending on the cell type: it can either induce global DNA demethylation or inhibit LSD1-driven H3K4me2 and H3K4me1 demethylation to establish an active chromatin configuration. We also investigate the therapeutic efficacy of combining 5-Aza-CdR with clorgyline and determine that this combinatorial treatment has synergistic effects on reactivating aberrantly silenced genes by enriching H3K4me2 and H3K4me1. Many of the reactivated genes are categorized as cancer testis antigens or belong to the interferon-signaling pathway, suggesting potential implications for immunotherapy. Together, our results demonstrate that combinatorial treatment consisting of a DNMT inhibitor (DNMTi) and an LSD1i have enhanced therapeutic values and could improve the efficacy of epigenetic therapy. PMID:24040395

Han, Han; Yang, Xiaojing; Pandiyan, Kurinji; Liang, Gangning

2013-01-01

237

Synergistic Re-Activation of Epigenetically Silenced Genes by Combinatorial Inhibition of DNMTs and LSD1 in Cancer Cells  

PubMed Central

Epigenetic gene silencing, mediated by aberrant promoter DNA hypermethylation and repressive histone modifications, is a hallmark of cancer. Although heritable, the dynamic nature and potential reversibility through pharmacological interventions make such aberrations attractive targets. Since cancers contain multiple epigenetic abnormalities, combining therapies that target different defects could potentially enhance their individual efficacies. 5-Aza-2'-deoxycytidine (5-Aza-CdR), FDA-approved drug for the treatment of myelodysplastic syndrome, can inhibit DNA methyltransferases (DNMTs) upon incorporation into the DNA of dividing cells, resulting in global demethylation. More recently, the first histone demethylase, lysine specific demethylase 1 (LSD1), which demethylates both histone and non-histone substrates, has become a new target for epigenetic therapy. Using, clorgyline, an LSD1 inhibitor (LSD1i) to treat cancer cell lines, we show that clorgyline employs two mechanisms of action depending on the cell type: it can either induce global DNA demethylation or inhibit LSD1-driven H3K4me2 and H3K4me1 demethylation to establish an active chromatin configuration. We also investigate the therapeutic efficacy of combining 5-Aza-CdR with clorgyline and determine that this combinatorial treatment has synergistic effects on reactivating aberrantly silenced genes by enriching H3K4me2 and H3K4me1. Many of the reactivated genes are categorized as cancer testis antigens or belong to the interferon-signaling pathway, suggesting potential implications for immunotherapy. Together, our results demonstrate that combinatorial treatment consisting of a DNMT inhibitor (DNMTi) and an LSD1i have enhanced therapeutic values and could improve the efficacy of epigenetic therapy.

Pandiyan, Kurinji; Liang, Gangning

2013-01-01

238

Inhibition and enhancement of eukaryotic gene expression by potential non-B DNA sequences.  

PubMed

In a transient or constitutive expression assay we have examined the effect of non-B DNA sequences d(CA)40 and d(CAAAAATGCC)n on gene expression in eukaryotic cells. These sequences were cloned adjacent to the weak eukaryotic promoter (CGTATTTATTTG) and located upstream from the coding sequence of galactokinase enzyme. Recombinants were micro-injected in cultured cells (Chinese hamster fibroblasts R1610, mutant gal-K-) and expression levels have been determined. The alternating purine-pyrimidine tract found in d(CA)40 able to assume the Z-DNA conformation shows an inhibitory effect on gene expression. In addition, our results suggest a new potential role of Z-DNA motifs in vivo to stimulate recombination. The sequences d(CAAAAATGCC)n able to adopt another non-B structure, corresponding to curved (or bended) helix conformation, strongly enhance gene expression and this enhancement depends on sequence redundancy. PMID:1755861

Delic, J; Onclercq, R; Moisan-Coppey, M

1991-12-16

239

Arctigenin from Arctium lappa inhibits interleukin-2 and interferon gene expression in primary human T lymphocytes  

Microsoft Academic Search

Background  \\u000a Arctium lappa (Niubang), a Chinese herbal medicine, is used to treat tissue inflammation. This study investigates the effects of arctigenin (AC),\\u000a isolated from A. lappa, on anti-CD3\\/CD28 Ab-stimulated cell proliferation and cytokine gene expression in primary human T lymphocytes.\\u000a \\u000a \\u000a \\u000a \\u000a Methods  Cell proliferation was determined with enzyme immunoassays and the tritiated thymidine uptake method. Cytokine production\\u000a and gene expression were analyzed

Wei-Jern Tsai; Chu-Ting Chang; Guei-Jane Wang; Tzong-Huei Lee; Shwu-Fen Chang; Shao-Chun Lu; Yuh-Chi Kuo

2011-01-01

240

Long-Term Exposure to Hexavalent Chromium Inhibits Expression of Tumor Suppressor Genes in cultured Cells and in Mice  

PubMed Central

We have used mouse hepatoma cells in culture to study acute, short-term high-dose effects of hexavalent chromium on gene regulation directed by the polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP). We find that the mixture engages three major signaling pathways: (i) activation of detoxification genes; (ii) induction of signal transduction effectors; and, (iii) epigenetic modification of chromatin marks. Preliminary results in mice exposed to mixtures of low doses of Cr (VI) plus BaP indicate that all three pathways are likely to be engaged also in long-term effects resulting from exposure to environmentally relevant doses of the mixture that inhibit the expression of tumor suppressor genes. Given the toxicity and carcinogenicity of these mixtures, we expect that a two-way analytical approach, from cells in culture to biological effects in vivo and vice versa, will provide a better understanding of the molecular mechanisms responsible for the biological effects of mixtures. By focusing both the in vivo and the in vitro work into long-term, low-dose, environmentally relevant exposures, we expect to develop much needed information pertinent to the type of diseases found in human populations exposed to mixtures of environmental toxicants.

Fan, Yunxia; Ovesen, Jerald L.; Puga, Alvaro

2012-01-01

241

A cucumber DELLA homolog CsGAIP may inhibit staminate development through transcriptional repression of B class floral homeotic genes.  

PubMed

In hermaphroditic Arabidopsis, the phytohormone gibberellin (GA) stimulates stamen development by opposing the DELLA repression of B and C classes of floral homeotic genes. GA can promote male flower formation in cucumber (Cucumis sativus L.), a typical monoecious vegetable with unisexual flowers, and the molecular mechanism remains unknown. Here we characterized a DELLA homolog CsGAIP in cucumber, and we found that CsGAIP is highly expressed in stem and male flower buds. In situ hybridization showed that CsGAIP is greatly enriched in the stamen primordia, especially during the hermaphrodite stage of flower development. Further, CsGAIP protein is located in nucleus. CsGAIP can partially rescue the plant height, stamen development and fertility phenotypes of Arabidopsis rga-24/gai-t6 mutant, and ectopic expression of CsGAIP in wide-type Arabidopsis results in reduced number of stamens and decreased transcription of B class floral homeotic genes APETALA3 (AP3) and PISTILLATA (PI). Our data suggest that monoecious CsGAIP may inhibit staminate development through transcriptional repression of B class floral homeotic genes in Arabidopsis. PMID:24632777

Zhang, Yan; Liu, Bin; Yang, Sen; An, Jingbo; Chen, Chunhua; Zhang, Xiaolan; Ren, Huazhong

2014-01-01

242

Inhibition of intracellular antiviral defense mechanisms augments lentiviral transduction of human natural killer cells: implications for gene therapy.  

PubMed

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/IKK? 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

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

2012-10-01

243

A molecular insight of Hes5-dependent inhibition of myelin gene expression: old partners and new players  

PubMed Central

This study identifies novel mechanisms of Hes5 function in developmental myelination. We report here upregulation of myelin gene expression in Hes5?/? mice compared to wild-type siblings and downregulation in overexpressing progenitors. This effect was only partially explained by the ability to regulate the levels of Mash1 and bind to N boxes in myelin promoters, as deletion of the DNA-binding domain of Hes5 did not suppress its inhibitory role on myelin gene expression. Novel mechanisms of Hes5 function in the oligodendrocyte lineage include the regulation of feedback loops with the cell-specific transcriptional activator Sox10. In progenitors with low levels of Sox10, Hes5 further decreases the bioavailability of this protein by transcriptional inhibition and direct sequestration of this activator. Increasing levels of Sox10 in progenitors, in turn, bind to Hes5 and titrate out its inhibitory effect by sequestration and displacement of the repressive complexes from myelin promoters. Thus, Hes5-dependent modulation of myelin gene expression involves old players (i.e. Mash1) and novel mechanisms of transcriptional regulation that include cell-specific regulatory loops with transcriptional activators (i.e. Sox10).

Liu, Aixiao; Li, Jiadong; Marin-Husstege, Mireya; Kageyama, Ryochiro; Fan, Yongjun; Gelinas, Celine; Casaccia-Bonnefil, Patrizia

2006-01-01

244

A novel 3p22.3 gene CMTM7 represses oncogenic EGFR signaling and inhibits cancer cell growth.  

PubMed

Deletion of 3p12-22 is frequent in multiple cancer types, indicating the presence of critical tumor-suppressor genes (TSGs) at this region. We studied a novel candidate TSG, CMTM7, located at the 3p22.3 CMTM-gene cluster, for its tumor-suppressive functions and related mechanisms. The three CMTM genes, CMTM6, 7 and 8, are broadly expressed in human normal adult tissues and normal epithelial cell lines. Only CMTM7 is frequently silenced or downregulated in esophageal and nasopharyngeal cell lines, but uncommon in other carcinoma cell lines. Immunostaining of tissue microarrays for CMTM7 protein showed its downregulation or absence in esophageal, gastric, pancreatic, liver, lung and cervix tumor tissues. Promoter CpG methylation and loss of heterozygosity were both found contributing to CMTM7 downregulation. Ectopic expression of CMTM7 in carcinoma cells inhibits cell proliferation, motility and tumor formation in nude mice, but not in immortalized normal cells, suggesting a tumor inhibitory role of CMTM7. The tumor-suppressive function of CMTM7 is associated with its role in G1/S cell cycle arrest, through upregulating p27 and downregulating cyclin-dependent kinase 2 (CDK2) and 6 (CDK6). Moreover, CMTM7 could promote epidermal growth factor receptor (EGFR) internalization, and further suppress AKT signaling pathway. Thus, our findings suggest that CMTM7 is a novel 3p22 tumor suppressor regulating G1/S transition and EGFR/AKT signaling during tumor pathogenesis. PMID:23893243

Li, H; Li, J; Su, Y; Fan, Y; Guo, X; Li, L; Su, X; Rong, R; Ying, J; Mo, X; Liu, K; Zhang, Z; Yang, F; Jiang, G; Wang, J; Zhang, Y; Ma, D; Tao, Q; Han, W

2014-06-12

245

A Cucumber DELLA Homolog CsGAIP May Inhibit Staminate Development through Transcriptional Repression of B Class Floral Homeotic Genes  

PubMed Central

In hermaphroditic Arabidopsis, the phytohormone gibberellin (GA) stimulates stamen development by opposing the DELLA repression of B and C classes of floral homeotic genes. GA can promote male flower formation in cucumber (Cucumis sativus L.), a typical monoecious vegetable with unisexual flowers, and the molecular mechanism remains unknown. Here we characterized a DELLA homolog CsGAIP in cucumber, and we found that CsGAIP is highly expressed in stem and male flower buds. In situ hybridization showed that CsGAIP is greatly enriched in the stamen primordia, especially during the hermaphrodite stage of flower development. Further, CsGAIP protein is located in nucleus. CsGAIP can partially rescue the plant height, stamen development and fertility phenotypes of Arabidopsis rga-24/gai-t6 mutant, and ectopic expression of CsGAIP in wide-type Arabidopsis results in reduced number of stamens and decreased transcription of B class floral homeotic genes APETALA3 (AP3) and PISTILLATA (PI). Our data suggest that monoecious CsGAIP may inhibit staminate development through transcriptional repression of B class floral homeotic genes in Arabidopsis.

Zhang, Yan; Liu, Bin; Yang, Sen; An, Jingbo; Chen, Chunhua; Zhang, Xiaolan; Ren, Huazhong

2014-01-01

246

Gax gene transfer inhibits vascular remodeling induced by adventitial inflammation in rabbits  

Microsoft Academic Search

AimsAdventitial fibroblasts (AFs) and inflammation play an important role in neointimal formation and vascular remodeling. The present study was aimed to investigate the therapeutic effects and underlying mechanisms of transcriptional regulator Gax gene transfection in aortic remodeling induced by adventitial inflammation.

Ping Liu; Cheng Zhang; Yu Xia Zhao; Jin Bo Feng; Chun Xi Liu; Wen Qiang Chen; Gui Hua Yao; Mei Zhang; Xing Li Wang; Yun Zhang

2010-01-01

247

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

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 17?-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.

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

2010-01-01

248

Adenoviral gene transfer of angiostatic ATF-BPTI inhibits tumour growth  

PubMed Central

Background The outgrowth of new vessels – angiogenesis – in the tumour mass is considered to be a limiting factor of tumour growth. To inhibit the matrix lysis that is part of the tumour angiogenesis, we employed the chimeric protein mhATF-BPTI, composed of the receptor binding part of the urokinase (ATF) linked to an inhibitor of plasmin (BPTI). Methods For delivery, recombinant adenovirus encoding the transgene of interest was injected intravenously or locally into the tumour. The anti tumour effect of this compound was compared to that of human endostatin and of mhATF alone in two different rat bronchial carcinomas growing either as subcutaneous implants or as metastases. Results Significant inhibition of the tumour growth and decrease of the number of lung metastasis was achieved when the concentration of mhATF-BPTI at the tumour site was above 400 of ng / g tissue. This concentration could be achieved via production by the liver, only if permissive to the recombinant adenovirus. When the tumour cells could be transduced, local delivery of the vector was enough to obtain a response. In the case of metastasis, the capacity of the lung tissue to concentrate the encoded protein was essential to reach the required therapeutic levels. Further, endostatin or mhATF could not reproduce the effects of mhATF-BPTI, at similar concentrations (mhATF) and even at 10-fold higher concentration (endostatin). Conclusion The ATF-BPTI was shown to inhibit tumour growth of different rat lung tumours when critical concentration was reached. In these tumour models, endostatin or ATF induce almost no tumour response.

Lefesvre, Pierre; Attema, Joline; van Bekkum, Dirk

2002-01-01

249

Inhibition of Rev-mediated HIV-1 expression by an RNA binding protein encoded by the interferon-inducible 9-27 gene  

SciTech Connect

Interferon inhibits expression of human immunodeficiency virus type-1 (HIV-1) through unknown mechanisms. A gene inducible by interferon-[alpha] (IFN-[alpha]) and interferon-[gamma] (IFN-[gamma]) was isolated by screening of a human complementary DNA library for proteins binding to the Rev-responsive element (RRE) of HIV-1. The product of this gene, RBP9-27, was shown to bind RNA in vitro and to inhibit HIV-1 expression after transfection into human cells. RBP9-27 primarily inhibited Rev-dependent posttransscriptional steps of viral gene expression. Thus, RBP9-27 is a cellular factor that antagonizes Rev function. These results suggest an inteferon-induced antiviral mechanism operating through the induction of RNA binding proteins such as RBP9-27. Elucidation of RBP9-27 function may lead to a better understanding of the mechanism of interferon action during HIV-1 infection. 29 refs., 4 figs.

Constantoulakis, P.; Campbell, M.; Felber, B.K.; Nasioulas, G.; Afonina, E.; Pavlakis, G.N. (National Cancer Inst., Frederick, MD (United States))

1993-02-26

250

Resveratrol Inhibits Dioxin-Induced Expression of Human CYP1A1 and CYP1B1 by Inhibiting Recruitment of the Aryl Hydrocarbon Receptor Complex and RNA Polymerase II to the Regulatory Regions of the Corresponding Genes  

PubMed Central

The CYP1A family of cytochrome P450s (CYPs), comprising CYP1A1, CYP1A2, and CYP1B1, plays a role in bioactivation of several procarcinogens to carcinogenic derivatives, and also in detoxification of several xenobiotic compounds. Resveratrol (3,4,5-trihydroxystelbine) is a naturally occurring compound that has been shown in a number of studies to inhibit the induction of CYP1A1 and CYP1B1 by dioxin (2,3,7,8-tetrachloro-dibenzo-p-dioxin), but the mechanism(s) of resveratrol inhibition is controversial. In the current study, 100nM dioxin treatment for 24, 48, and 72 h induced CYP1A1, CYP1A2, and CYP1B1 mRNA levels in the human breast cancer cell line MCF-7, and CYP1A1 and CYP1A2 mRNA levels in the human hepatocellular carcinoma cell line, HepG2. Simultaneous treatment with 10?M resveratrol significantly inhibited dioxin-induced mRNA expression levels of these genes in both cell lines. Our studies are novel in that we used the chromatin immunoprecipitation assay to assay dioxin-induced recruitment of the aryl hydrocarbon receptor (AHR), and aryl hydrocarbon nuclear translocator (ARNT) to the enhancer regions and recruitment of RNA polymerase II to the promoter regions, of the CYP1A1 and CYP1B1 genes in their natural chromosomal settings. These recruitments were significantly inhibited in cells cotreated with resveratrol. Our studies thus indicate that resveratrol inhibits dioxin induction of the CYP1 family members either by directly or indirectly inhibiting the recruitment of the transcription factors AHR and ARNT to the xenobiotic response elements of the corresponding genes. The reduced transcriptional factor binding at their enhancers then results in reduced pol II recruitment at the promoters of these genes.

Beedanagari, Sudheer R.; Bebenek, Ilona; Bui, Peter; Hankinson, Oliver

2009-01-01

251

Resveratrol inhibits dioxin-induced expression of human CYP1A1 and CYP1B1 by inhibiting recruitment of the aryl hydrocarbon receptor complex and RNA polymerase II to the regulatory regions of the corresponding genes.  

PubMed

The CYP1A family of cytochrome P450s (CYPs), comprising CYP1A1, CYP1A2, and CYP1B1, plays a role in bioactivation of several procarcinogens to carcinogenic derivatives, and also in detoxification of several xenobiotic compounds. Resveratrol (3,4,5-trihydroxystelbine) is a naturally occurring compound that has been shown in a number of studies to inhibit the induction of CYP1A1 and CYP1B1 by dioxin (2,3,7,8-tetrachloro-dibenzo-p-dioxin), but the mechanism(s) of resveratrol inhibition is controversial. In the current study, 100nM dioxin treatment for 24, 48, and 72 h induced CYP1A1, CYP1A2, and CYP1B1 mRNA levels in the human breast cancer cell line MCF-7, and CYP1A1 and CYP1A2 mRNA levels in the human hepatocellular carcinoma cell line, HepG2. Simultaneous treatment with 10 microM resveratrol significantly inhibited dioxin-induced mRNA expression levels of these genes in both cell lines. Our studies are novel in that we used the chromatin immunoprecipitation assay to assay dioxin-induced recruitment of the aryl hydrocarbon receptor (AHR), and aryl hydrocarbon nuclear translocator (ARNT) to the enhancer regions and recruitment of RNA polymerase II to the promoter regions, of the CYP1A1 and CYP1B1 genes in their natural chromosomal settings. These recruitments were significantly inhibited in cells cotreated with resveratrol. Our studies thus indicate that resveratrol inhibits dioxin induction of the CYP1 family members either by directly or indirectly inhibiting the recruitment of the transcription factors AHR and ARNT to the xenobiotic response elements of the corresponding genes. The reduced transcriptional factor binding at their enhancers then results in reduced pol II recruitment at the promoters of these genes. PMID:19376845

Beedanagari, Sudheer R; Bebenek, Ilona; Bui, Peter; Hankinson, Oliver

2009-07-01

252

Inhibition of human esophageal squamous cell carcinomas by targeted silencing of tumor enhancer genes: an overview  

PubMed Central

Esophageal cancer has been reported as the ninth most common malignancy and ranks as the sixth most frequent cause of death worldwide. Esophageal cancer treatment involves surgery, chemotherapy, radiation therapy, or combination therapy. Novel strategies are needed to boost the oncologic outcome. Recent advances in the molecular biology of esophageal cancer have documented the role of genetic alterations in tumorigenesis. Oncogenes serve a pivotal function in tumorigenesis. Targeted therapies are directed at the unique molecular signature of cancer cells for enhanced efficacy with low toxicity. RNA interference (RNAi) technology is a powerful tool for silencing endogenous or exogenous genes in mammalian cells. Related results have shown that targeting oncogenes with siRNAs, specifically the mRNA, effectively reduces tumor cell proliferation and induces apoptotic cell death. This article will briefly review studies on silencing tumor enhancer genes related to the induction of esophageal cancer.

Islamian, Jalil Pirayesh; Mohammadi, Mohsen; Baradaran, Behzad

2014-01-01

253

Inhibition of pituitary tumor-transforming gene-1 in thyroid cancer cells by drugs that decrease specificity proteins.  

PubMed

Methyl 2-cyano-3,11-dioxo-18?-olean-1,12-dien-30-oate (CDODA-Me) and the corresponding 2-trifluoromethyl analog (CF(3)DODA-Me) are derived synthetically from the triterpenoid glycyrrhetinic acid, a major component of licorice. CDODA-Me and CF(3)DODA-Me inhibited growth of highly invasive ARO, DRO, K-18, and HTh-74 thyroid cancer cells and this was due, in part, to decreased expression of specificity protein (Sp) transcription factors Sp1, Sp3, and Sp4 that are overexpressed in these cells. CDODA-Me and CF(3)DODA-Me also decreased expression of Sp-dependent genes, such as survivin and vascular endothelial growth factor (VEGF), and induced apoptosis. In addition, pituitary tumor-transforming gene-1 (PTTG-1) protein and mRNA levels were also decreased in thyroid cancer cells treated with CDODA-Me or CF(3)DODA-Me and this was accompanied by decreased expression of PTTG-1-dependent c-Myc and fibroblast growth factor-2 (FGF-2) genes. RNA interference studies against Sp1, Sp3, and Sp4 proteins showed that in thyroid cancer cells, PTTG-1 was an Sp-dependent gene. This study demonstrates for the first time that drugs, such as CDODA-Me and CF(3)DODA-Me, that decrease Sp protein expression also downregulate PTTG-1 in thyroid cancer cells and therefore have potential for clinical treatment of thyroid cancer and other endocrine neoplasias where PTTG-1 is a major pro-oncogenic factor. PMID:21268135

Chintharlapalli, Sudhakar; Papineni, Sabitha; Lee, Syng-Ook; Lei, Ping; Jin, Un Ho; Sherman, Steven I; Santarpia, Libero; Safe, Stephen

2011-09-01

254

INHIBITION OF PITUITARY TUMOR-TRANSFORMING GENE-1 IN THYROID CANCER CELLS BY DRUGS THAT DECREASE SPECIFICITY PROTEINS  

PubMed Central

Methyl 2-cyano-3,11-dioxo-18?-olean-1,12-dien-30-oate (CDODA-Me) and the corresponding 2-trifluoromethyl analog (CF3DODA-Me) are derived synthetically from the triterpenoid glycyrrhetinic acid, a major component of licorice. CDODA-Me and CF3DODA-Me inhibited growth of highly invasive ARO, DRO, K-18 and HTh-74 thyroid cancer cells and this was due, in part, to decreased expression of specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 that are overexpressed in these cells. CDODA-Me and CF3DODA-Me also decreased expression of Sp-dependent genes, such as survivin and vascular endothelial growth factor, and induced apoptosis. In addition, pituitary tumor-transforming gene-1 (PTTG-1) protein and mRNA levels were also decreased in thyroid cancer cells treated with CDODA-Me or CF3DODA-Me and this was accompanied by decreased expression of PTTG-1-dependent c-Myc and fibroblast growth factor 2 genes. RNA interference studies against Sp1, Sp3 and Sp4 proteins showed that in thyroid cancer cells, PTTG-1 was an Sp-dependent gene. This study demonstrates for the first time that drugs, such as CDODA-Me and CF3DODA-Me, that decrease Sp protein expression also downregulate PTTG-1 in thyroid cancer cells and therefore have potential for clinical treatment of thyroid cancer and other endocrine neoplasias where PTTG-1 is a major pro-oncogenic factor.

Chintharlapalli, Sudhakar; Papineni, Sabitha; Lee, Syng-Ook; Lei, Ping; Jin, Un Ho; Sherman, Steven I.; Santarpia, Libero; Safe, Stephen

2011-01-01

255

Cloning and Sequencing of the Gene Encoding Toho-2, a Class A ?-Lactamase Preferentially Inhibited by Tazobactam  

PubMed Central

Escherichia coli TUM1083, which is resistant to ampicillin, carbenicillin, cephaloridine, cephalothin, piperacillin, cefuzonam, and aztreonam while being sensitive to cefoxitin, moxalactam, cefmetazole, ceftazidime, and imipenem, was isolated from the urine of a patient treated with ?-lactam antibiotics. The ?-lactamase (Toho-2) purified from the bacteria hydrolyzed ?-lactam antibiotics such as penicillin G, carbenicillin, cephaloridine, cefoxitin, cefotaxime, ceftazidime, and aztreonam and especially had increased relative hydrolysis rates for cephalothin, cephaloridine, cefotaxime, and ceftizoxime. Different from other extended-spectrum ?-lactamases, Toho-2 was inhibited 16-fold better by the ?-lactamase inhibitor tazobactam than by clavulanic acid. Resistance to ?-lactams was transferred by conjugation from E. coli TUM1083 to E. coli ML4909, and the transferred plasmid was about 54.4 kbp, belonging to the incompatibility group IncFII. The cefotaxime resistance gene for Toho-2 was subcloned from the 54.4-kbp plasmid. The sequence of the gene was determined, and the open reading frame of the gene was found to consist of 981 bases. The nucleotide sequence of the gene (DDBJ accession no. D89862) designated as blatoho was found to have 76.3% identity to class A ?-lactamase CTX-M-2 and 76.2% identity to Toho-1. It has 55.9% identity to SHV-1 ?-lactamase and 47.5% identity to TEM-1 ?-lactamase. Therefore, the newly isolated ?-lactamase designated as Toho-2 produced by E. coli TUM1083 is categorized as an enzyme similar to Toho-1 group ?-lactamases rather than to mutants of TEM or SHV enzymes. According to the amino acid sequence deduced from the DNA sequence, the precursor consisted of 327 amino acid residues. Comparison of Toho-2 with other ?-lactamase (non-Toho-1 group) suggests that the substitutions of threonine for Arg-244 and arginine for Asn-276 are important for the extension of the substrate specificity.

Ma, Ling; Ishii, Yoshikazu; Ishiguro, Masaji; Matsuzawa, Hiroshi; Yamaguchi, Keizo

1998-01-01

256

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

PubMed

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

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

2012-07-01

257

LYRM1, a novel gene promotes proliferation and inhibits apoptosis of preadipocytes  

Microsoft Academic Search

Objective: To characterize a novel gene, Homo sapiens LYR motif containing 1 (LYRM1), that is highly expressed in omental adipose tissue of obese subjects. Methods and results: RT-PCR and western blot analysis confirmed that both mRNA and protein levels of LYRM1 were higher in omental adipose tissue of obese subjects than in normal weight subjects. RT-PCR analysis demonstrated that LYRM1

Jie Qiu; Chun-Lin Gao; Min Zhang; Rong-Hua Chen; Xia Chi; Feng Liu; Chun-Mei Zhang; Chen-Bo Ji; Xiao-Hui Chen; Ya-Ping Zhao; Xiao-Nan Li; Mei-Ling Tong; Yu-Hui Ni; Xi-Rong Guo

2009-01-01

258

NF-?B Regulation of YY1 Inhibits Skeletal Myogenesis through Transcriptional Silencing of Myofibrillar Genes? †  

PubMed Central

NF-?B signaling is implicated as an important regulator of skeletal muscle homeostasis, but the mechanisms by which this transcription factor contributes to muscle maturation and turnover remain unclear. To gain insight into these mechanisms, gene expression profiling was examined in C2C12 myoblasts devoid of NF-?B activity. Interestingly, even in proliferating myoblasts, the absence of NF-?B caused the pronounced induction of several myofibrillar genes, suggesting that NF-?B functions as a negative regulator of late-stage muscle differentiation. Although several myofibrillar promoters contain predicted NF-?B binding sites, functional analysis using the troponin-I2 gene as a model revealed that NF-?B-mediated repression does not occur through direct DNA binding. In the search for an indirect mediator, the transcriptional repressor YinYang1 (YY1) was identified. While inducers of NF-?B stimulated YY1 expression in multiple cell types, genetic ablation of the RelA/p65 subunit of NF-?B in both cultured cells and adult skeletal muscle correlated with reduced YY1 transcripts and protein. NF-?B regulation of YY1 occurred at the transcriptional level, mediated by direct binding of the p50/p65 heterodimer complex to the YY1 promoter. Furthermore, YY1 was found associated with multiple myofibrillar promoters in C2C12 myoblasts containing NF-?B activity. Based on these results, we propose that NF-?B regulation of YY1 and transcriptional silencing of myofibrillar genes represent a new mechanism by which NF-?B functions in myoblasts to modulate skeletal muscle differentiation.

Wang, Huating; Hertlein, Erin; Bakkar, Nadine; Sun, Hao; Acharyya, Swarnali; Wang, Jingxin; Carathers, Micheal; Davuluri, Ramana; Guttridge, Denis C.

2007-01-01

259

Herpes Simplex Virus Gene Products Required for Viral Inhibition of Expression of G1Phase Functions  

Microsoft Academic Search

HSV infection blocks G1 events in the cell cycle and arrests host cell growth in the G1 phase. To further define the mechanism of the effect and determine the viral gene product(s) responsible, we examined various mutant viruses for their effects on cell cycle regulatory proteins (pRb, cyclin D1, and cdk4) and on cell cycle progression into S phase. Unlike

Byeongwoon Song; Kung-Chieh Yeh; Jian Liu; David M. Knipe

2001-01-01

260

Inhibition of retinal neovascularisation by gene transfer of soluble VEGF receptor sFlt-1  

Microsoft Academic Search

Retinal angiogenesis is a central feature of the leading causes of blindness. Current treatments for these conditions are of limited efficacy and cause significant adverse effects. In this study, we evaluated the angiostatic effect of gene transfer of the soluble VEGF receptor sFlt-1 in a mouse model of ischaemia-induced retinal neovascularisation using adenovirus and adeno-associated virus (AAV) vectors. We induced

JWB Bainbridge; A Mistry; M De Alwis; E Paleolog; A Baker; AJ Thrasher; RR Ali

2002-01-01

261

Inhibition of gene expression in mice muscle by in vivo electrically mediated siRNA delivery  

Microsoft Academic Search

Owing to their capacity to induce strong, sequence-specific, gene silencing in cells, short interfering RNAs (siRNAs) represent new potential therapeutic tools. This development requires, however, new safe and efficient in vivo siRNA delivery methods. In the present technical report, we show that electrically mediated siRNA transfer can suppress transgene expression in adult mice muscles. Using electropulsation for siRNA delivery opens

M Golzio; L Mazzolini; P Moller; M P Rols; J Teissié

2005-01-01

262

Cobalt stimulates HIF-1-dependent but inhibits HIF-2-dependent gene expression in liver cancer cells  

PubMed Central

Hypoxia-inducible factors (HIFs) are transcriptional regulators that mediate the cellular response to low oxygen. Although HIF-1 is usually considered as the principal mediator of hypoxic adaptation, several tissues and different cell types express both HIF-1 and HIF-2 isoforms under hypoxia or when treated with hypoxia mimetic chemicals such as cobalt. However, the similarities or differences between HIF-1 and HIF-2, in terms of their tissue- and inducer-specific activation and function, are not adequately characterized. To address this issue, we investigated the effects of true hypoxia and hypoxia mimetics on HIF-1 and HIF-2 induction and specific gene transcriptional activity in two hepatic cancer cell lines, Huh7 and HepG2. Both hypoxia and cobalt caused rapid induction of both HIF-1? and HIF-2? proteins. Hypoxia induced erythropoietin (EPO) expression and secretion in a HIF-2-dependent way. Surprisingly, however, EPO expression was not induced when cells were treated with cobalt. In agreement, both HIF-1- and HIF-2-dependent promoters (of PGK and SOD2 genes, respectively) were activated by hypoxia while cobalt only activated the HIF-1-dependent PGK promoter. Unlike cobalt, other hypoxia mimetics such as DFO and DMOG activated both types of promoters. Furthermore, cobalt impaired the hypoxic stimulation of HIF-2, but not HIF-1, activity and cobalt-induced HIF-2? interacted poorly with USF-2, a HIF-2-specific co-activator. These data show that, despite similar induction of HIF-1? and HIF-2? protein expression, HIF-1 and HIF-2 specific gene activating functions respond differently to different stimuli and suggest the operation of oxygen-independent and gene- or tissue-specific regulatory mechanisms involving additional transcription factors or co-activators.

Befani, Christina; Mylonis, Ilias; Gkotinakou, Ioanna-Maria; Georgoulias, Panagiotis; Hu, Cheng-Jun; Simos, George; Liakos, Panagiotis

2013-01-01

263

Toxoplasma gondii Clonal Strains All Inhibit STAT1 Transcriptional Activity but Polymorphic Effectors Differentially Modulate IFN? Induced Gene Expression and STAT1 Phosphorylation  

PubMed Central

Host defense against the parasite Toxoplasma gondii requires the cytokine interferon-gamma (IFN?). However, Toxoplasma inhibits the host cell transcriptional response to IFN?, which is thought to allow the parasite to establish a chronic infection. It is not known whether all strains of Toxoplasma block IFN?-responsive transcription equally and whether this inhibition occurs solely through the modulation of STAT1 activity or whether other transcription factors are involved. We find that strains from three North American/European clonal lineages of Toxoplasma, types I, II, and III, can differentially modulate specific aspects of IFN? signaling through the polymorphic effector proteins ROP16 and GRA15. STAT1 tyrosine phosphorylation is activated in the absence of IFN? by the Toxoplasma kinase ROP16, but this ROP16-activated STAT1 is not transcriptionally active. Many genes induced by STAT1 can also be controlled by other transcription factors and therefore using these genes as specific readouts to determine Toxoplasma inhibition of STAT1 activity might be inappropriate. Indeed, GRA15 and ROP16 modulate the expression of subsets of IFN? responsive genes through activation of the NF-?B/IRF1 and STAT3/6 transcription factors, respectively. However, using a stable STAT1-specific reporter cell line we show that strains from the type I, II, and III clonal lineages equally inhibit STAT1 transcriptional activity. Furthermore, all three of the clonal lineages significantly inhibit global IFN? induced gene expression.

Rosowski, Emily E.; Saeij, Jeroen P. J.

2012-01-01

264

18?-Glycyrrhetinic Acid Inhibits Methicillin-Resistant Staphylococcus aureus Survival and Attenuates Virulence Gene Expression  

PubMed Central

Methicillin-resistant Staphylococcus aureus (MRSA) has become a major source of infection in hospitals and in the community. Increasing antibiotic resistance in S. aureus strains has created a need for alternative therapies to treat disease. A component of the licorice root Glycyrrhiza spp., 18?-glycyrrhetinic acid (GRA), has been shown to have antiviral, antitumor, and antibacterial activity. This investigation explores the in vitro and in vivo effects of GRA on MRSA pulsed-field gel electrophoresis (PFGE) type USA300. GRA exhibited bactericidal activity at concentrations exceeding 0.223 ?M. Upon exposure of S. aureus to sublytic concentrations of GRA, we observed a reduction in expression of key virulence genes, including saeR and hla. In murine models of skin and soft tissue infection, topical GRA treatment significantly reduced skin lesion size and decreased the expression of saeR and hla genes. Our investigation demonstrates that at high concentrations GRA is bactericidal to MRSA and at sublethal doses it reduces virulence gene expression in S. aureus both in vitro and in vivo.

Long, Danyelle R.; Mead, Julia; Hendricks, Jay M.

2013-01-01

265

Thymoquinone efficiently inhibits the survival of EBV-infected B cells and alters EBV gene expression.  

PubMed

Epstein--Barr virus (EBV) is a human virus with oncogenic potentials that is implicated in various human diseases and malignancies. In this study, the modulator activity of the potent herbal extract drug thymoquinone on EBV was assessed in vitro. Thymoquinone was tested for cytotoxicity on human cells of lymphoblastoid cells, Raji Burkitt's lymphoma, DG-75 Burkitt's lymphoma, peripheral blood mononuclear cells, and periodontal ligament fibroblast. Apoptosis induction was analyzed via TUNEL assay and activity studies of caspase-3. The effect of thymoquinone on EBV gene expression was determined using real-time polymerase chain reaction. We report here, for the first time, a promising selective inhibitory affect of thymoquinone on EBV-infected B cell lines in vitro, compared with lower activity on EBV negative B cell line and very low toxicity on human peripheral blood mononuclear cells and periodontal ligament fibroblasts. Moreover, the drug was found to efficiently suppress the RNA expression of EBNA2, LMP1, and EBNA1 genes. Specifically, EBNA2 expression levels were the most affected indicating that this gene might have a major contribution to thymoquinone potency against EBV infected cells. Overall, our results suggest that thymoquinone has the potential to suppress the growth of EBV-infected B cells efficiently. PMID:23089554

Zihlif, Malek A; Mahmoud, Ismail S; Ghanim, Majd T; Zreikat, Manar S; Alrabadi, Nasr; Imraish, Amer; Odeh, Fadwa; Abbas, Manal A; Ismail, Said I

2013-05-01

266

PROTEASOME INHIBITION UP-REGULATES INFLAMMATORY GENE TRANSCRIPTION INDUCED BY AN ATYPICAL PATHWAY OF NF-?B ACTIVATION  

PubMed Central

Proteasome inhibition has become synonymous with inhibition of NF-?B activity. However, hyperactive NF-?B responses often accompany physiological conditions marked by proteasomal defects, i.e. advancing age, geriatric diseases, and bortezomib resistance. These paradoxical NF-?B responses are likely to be impervious to proteasomal defects because they stem from atypical NF-?B signaling induced by upstream mechanisms which are proteasome-independent. While this atypical pathway does not require proteasome for NF-?B nuclear translocation, a role for proteasome in regulating nuclear NF-?B remains unexplored. We now demonstrate that proteasome stringently controls transcription of inflammatory mediators regulated by this atypical NF-?B pathway. Proteolytic activity of the proteasome mediates the removal of the NF-?B subunit, p65/RelA, from inflammatory genes, thereby terminating atypical NF-?B-dependent transcriptional responses. For the first time, we demonstrate that both 19S and 20S components of the 26S proteasome complex are recruited to an inflammatory gene promoter; additionally, the 19S and 20S complexes appear to play distinct roles in the negative regulation of NF-?B-dependent transcription. By demonstrating that proteasome regulates the termination of atypical NF-?B-dependent transcriptional responses, these studies clearly indicate a novel, regulatory role for proteasome in atypical NF-?B signaling. Moreover, these results signal a potential interplay between lowered proteasomal function and increased inflammation and may explain why inflammation accompanies physiological conditions under which proteasomal function is compromised, such as during advancing age or following bortezomib treatment. Given this role for proteasome in inflammation resolution, restoration of proteasome function may constitute a novel mechanism for intervening in chronic inflammatory diseases.

Cullen, Sarah J.; Ponnappan, Subramaniam; Ponnappan, Usha

2009-01-01

267

Oregonin inhibits lipopolysaccharide-induced iNOS gene transcription and upregulates HO-1 expression in macrophages and microglia  

PubMed Central

Oregonin isolated from Alnus formosana is a diarylheptanoid derivative, which appears to have antioxidative and anti-inflammatory activities. In this study, our data demonstrated inhibitory actions of oregonin on the LPS-induced iNOS protein in RAW264.7 macrophages and BV-2 microglial cells. We also suggested that HO-1 induction by oregonin might contribute to this action. Oregonin is able to dose-dependently reduce NO production, iNOS protein and iNOS promoter activity stimulated by LPS in RAW264.7 and BV-2 cells. Oregonin also showed inhibition of LPS-mediated NF-?B promoter activity and DNA-binding ability, as well as p65 nuclear translocation and phosphorylation. However, oregonin had no effect on IKK activity. AP-1 promoter activity and p38 MAPK activation but not PKC, ERK and JNK activation induced by LPS were attenuated by oregonin. Accompanying with iNOS protein reduction, moreover, we found that oregonin was able to induce HO-1 protein level. Results using a CO donor, [Ru(CO)3Cl2]2 further showed the ability of CO in reduction of iNOS protein level induced by LPS through the blockade of NF-?B and AP-1. Taken together, these results provide new evidences into the anti-inflammatory actions of oregonin, which include the inhibition of iNOS gene transcription via suppressing transcriptional activity of NF-?B and AP-1, as well as the upregulation of anti-inflammatory molecule HO-1. The HO-1-derived CO may also be involved in the suppressive effect on iNOS gene regulation.

Lee, Cheng-Jui; Lee, Shoei-Sheng; Chen, Su-Chung; Ho, Feng-Ming; Lin, Wan-Wan

2005-01-01

268

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

PubMed Central

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-?B) 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.

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

2010-01-01

269

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

PubMed Central

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.

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

2014-01-01

270

Blocking Signaling at the Level of GLI Regulates Downstream Gene Expression and Inhibits Proliferation of Canine Osteosarcoma Cells  

PubMed Central

The Hedgehog-GLI signaling pathway is active in a variety of human malignancies and is known to contribute to the growth and survival of human osteosarcoma cells. In this study, we examined the expression and regulation of GLI transcription factors in multiple canine osteosarcoma cell lines and analyzed the effects of inhibiting GLI with GANT61, a GLI-specific inhibitor. Compared with normal canine osteoblasts, real-time PCR showed that GLI1 and GLI2 were highly expressed in two out of three cell lines and correlated with downstream target gene expression of PTCH1and PAX6. Treatment of canine osteosarcoma cells with GANT61 resulted in decreased expression of GLI1, GLI2, PTCH1, and PAX6. Furthermore, GANT61 inhibited proliferation and colony formation in all three canine osteosarcoma cell lines. The finding that GLI signaling activity is present and active in canine osteosarcoma cells suggests that spontaneously arising osteosarcoma in dogs might serve as a good model for future preclinical testing of GLI inhibitors.

Shahi, Mehdi Hayat; Holt, Roseline; Rebhun, Robert B.

2014-01-01

271

L-Carnosine Inhibits Metastasis of SK-Hep-1 Cells by Inhibition of Matrix Metaoproteinase-9 Expression and Induction of an Antimetastatic Gene, nm23-H1  

Microsoft Academic Search

Antioxidants have been suggested to inhibit the expression of matrix metalloproteinases (MMPs), especially MMP-9, which plays a critical role in tumor metastasis. Because of its antioxidant activity and the ability to chelate divalent cations, L-carnosine (LC) was tested for inhibition of MMP-9 in a highly invasive hepatocarcinoma, SK-Hep-1 cells. We found that LC (50–1,000 ?M) did not directly inhibit the

Cheng-Hung Chuang; Miao-Lin Hu

2008-01-01

272

Inhibition of Ocular Angiogenesis by siRNA Targeting Vascular Endothelial Growth Factor Pathway Genes  

PubMed Central

Ocular neovascularization often results in vision impairment. Frequently vascular endothelial cell growth factors (VEGFs) are mainly responsible for the pathological neovascularization as in the case in neovascularization induced by CpG oligodeoxynucleotides and herpes simplex virus infection in this report. siRNAs targeting either VEGFA, VEGFR1, VEGFR2, or a mix of the three were shown to significantly inhibit neovascularization induced by CpG when given locally or systemically. The efficacy of systemic administration was facilitated by the use of a polymer delivery vehicle. Additional experiments showed a significant inhibitory effect of the siRNAs mix when given either locally or systemically in vehicle against herpes simplex virus-induced angiogenesis as well as against lesions of stromal keratitis. These results indicate that the use of VEGF pathway-specific siRNAs represents a useful therapy against neovascularization-related eye diseases.

Kim, Bumseok; Tang, Qingquan; Biswas, Partha S.; Xu, Jun; Schiffelers, Raymond M.; Xie, Frank Y.; Ansari, Aslam M.; Scaria, Puthupparampil V.; Woodle, Martin C.; Lu, Patrick; Rouse, Barry T.

2004-01-01

273

Inhibition of Proprotein Convertase SKI-1 Blocks Transcription of Key Extracellular Matrix Genes Regulating Osteoblastic Mineralization*  

PubMed Central

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.

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

2011-01-01

274

TBLR1 as an AR coactivator selectively activates AR target genes to inhibit prostate cancer growth  

PubMed Central

Androgen Receptor (AR), a steroid hormone receptor, is critical for prostate cancer growth. However, activation of AR by androgens can also lead to growth suppression and differentiation. Transcriptional cofactors play an important role in this switch between proliferative and anti-proliferative AR target gene programs. TBLR1, a core component of the nuclear receptor corepressor (NCoR) complex, shows both co-repressor and co-activator activities on nuclear receptors, but little is known about its effects on AR and prostate cancer. We characterized TBLR1 as a coactivator of AR in prostate cancer cells and the activation is both phosphorylation and 19S proteosome dependent. We showed that TBLR1 physically interacts with AR and directly occupies the androgen response elements of affected AR target genes in an androgen-dependent manner. TBLR1 is primarily localized in the nucleus in benign prostate cells and nuclear expression is significantly reduced in prostate cancer cells in culture. Similarly, in human tumor samples, the expression of TBLR1 in the nucleus is significantly reduced in the malignant glands compared to the surrounding benign prostatic glands (p<0.005). Stable ectopic expression of nuclear TBLR1 leads to androgen-dependent growth suppression of prostate cancer cells in vitro and in vivo by selective activation of androgen regulated genes associated with differentiation (e.g. KRT18) and growth suppression (e.g. NKX3.1), but not cell proliferation of the prostate. Understanding the molecular switches involved in the transition from AR dependent growth promotion to AR dependent growth suppression will lead to more successful prostate cancer treatments.

Daniels, Garrett; Li, Yirong; Gellert, Lan Lin; Zhou, Albert; Melamed, Jonathan; Wu, Xinyu; Zhang, Xinming; Zhang, David; Meruelo, Daniel; Logan, Susan K.; Basch, Ross; Lee, Peng

2014-01-01

275

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

PubMed

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

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

2011-01-21

276

A threshold neurotoxic amphetamine exposure inhibits parietal cortex expression of synaptic plasticity-related genes.  

PubMed

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 (AMPH) on the parietal cortex, gene expression responses to amphetamine challenge (7.5 mg/kg) were examined in the parietal cortex of rats pretreated for nine days with either saline, non-neurotoxic amphetamine, or neurotoxic AMPH dosing regimens. The neurotoxic AMPH exposure [three doses of 7.5 mg/kg/day AMPH (6 h 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 x 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 pretreatments. 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 is sensitive to disruption specifically in the parietal cortex by threshold neurotoxic AMPH exposures. PMID:17049170

Bowyer, J F; Pogge, A R; Delongchamp, R R; O'Callaghan, J P; Patel, K M; Vrana, K E; Freeman, W M

2007-01-01

277

Polyunsaturated fatty acids inhibit fatty acid synthase and spot-14-protein gene expression in cultured rat hepatocytes by a peroxidative mechanism.  

PubMed Central

In vivo, polyunsaturated fatty acids (PUFA) inhibit the expression of hepatic genes related to the lipogenic process such as fatty acid synthase and spot-14-protein (S14) genes. In vitro studies have suggested that this was a direct transcriptional effect of PUFA. In hepatocytes, the inhibition of the lipogenic rate by PUFA is not specific, but is linked to a cytotoxic effect due to peroxidative mechanisms. We have investigated whether peroxidation could also explain the inhibitory effect of PUFA on gene expression. Rat hepatocytes were cultured for 24 h with mono-unsaturated or PUFA. PUFA inhibited the expression of fatty acid synthase and S14 genes, and this inhibition was directly related to the number of unsaturations. However, the beta-actin and albumin mRNA concentrations were also affected by the most unsaturated fatty acids, suggesting a non-specific effect of PUFA on gene expression. Measurement of lactate dehydrogenase released into the medium indicated a cytotoxicity of PUFA. This was associated with their peroxidation as evaluated by the presence of thiobarbituric acid-reactive substances in the culture medium. The addition of high concentrations of antioxidants abolished lipid peroxidation and lactate dehydrogenase leakage and completely reversed the inhibitory effect of PUFA on gene expression. This suggests (i) that the results obtained previously in cultured hepatocytes in the presence of low concentrations of antioxidants must be interpretated cautiously and (ii) that in vivo, the inhibitory effect of PUFA on lipogenesis-related genes could be indirect through hormonal or metabolic changes or that their effect on gene expression is somehow linked to peroxidative mechanisms.

Foretz, M; Foufelle, F; Ferre, P

1999-01-01

278

The promoter of a gene encoding a polygalacturonase-inhibiting protein of Phaseolus vulgaris L. is activated by wounding but not by elicitors or pathogen infection  

Microsoft Academic Search

.   Polygalacturonase-inhibiting proteins (PGIPs), leucine-rich repeat (LRR) proteins evolutionarily related to several plant\\u000a resistance genes, bind to and regulate the action of fungal endopolygalacturonases. In Phaseolus vulgaris L., PGIPs are encoded by a gene family comprising at least five members. As a start for a systematic analysis of the regulation\\u000a of the pgip family, we have analysed the ability of

Alessandra Devoto; Fiona Leckie; Elisabetta Lupotto; Felice Cervone; Giulia de Lorenzo

1998-01-01

279

Polygonum cuspidatum, compared with baicalin and berberine, inhibits inducible nitric oxide synthase and cyclooxygenase-2 gene expressions in RAW 264.7 macrophages.  

PubMed

Polygonum cuspidatum water extract (PCWE) was shown to be a potent inhibitor of lipopolysaccharide (LPS)-induced expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). PCWE was compared to baicalin isolated from Scutellaria baicalensis Georgi and berberine of Coptidis rhizoma and Phellodendri cortex, for their effects on LPS-induced nitric oxide (NO) production and iNOS and COX-2 gene expressions in RAW 264.7 macrophages. Both PCWE and the compounds inhibited LPS-induced NO production in a concentration-dependent manner without a cytotoxicity. The decrease in NO production was in parallel with the inhibition of LPS-induced iNOS gene expression by PCWE and the compounds. In contrast, iNOS enzyme activity was not inhibited by PCWE and two agents. In addition, only PCWE inhibited LPS-induced prostaglandin E2 (PGE2) production and COX-2 gene expression without affecting COX-2 enzyme activity, while baicalin or berberine did not. Furthermore, N-nitro-L-arginine (NLA) and N-nitro-L-arginine methyl ester (L-NAME) pretreatment enhanced LPS-induced iNOS protein expression, which was inhibited by these PCWE and two agents, although LPS-induced COX-2 protein expression was not affected by NLA and L-NAME. PCWE inhibited PGE2 production and COX-2 protein expression in NLA/LPS or L-NAME/LPS-co-treated RAW 264.7 cell, however, baicalin or berberine did not. From the results, it was concluded that co-treatment with NOS inhibitors and PCWE effectively blocks acute production of NO and inhibits expression of iNOS and COX-2 genes. PMID:17553752

Kim, Kyung-Woon; Ha, Ki-Tai; Park, Cheol-Soo; Jin, Un-Ho; Chang, Hyen Wook; Lee, In-Seon; Kim, Cheorl-Ho

2007-01-01

280

Shengjie Tongyu Granule Inhibits Vascular Remodeling in ApoE-Gene-Knockout Mice  

PubMed Central

The aim of the present paper was to investigate the effect of Shengjie Tongyu granule on vascular remodeling in atherosclerotic mice and the relevant underlying mechanism. Sixty male ApoE-gene-knockout mice, fed a high-fat diet from 6 weeks of age, were randomized into a Shengjie Tongyu granule group (4.00?g/kg/d), a simvastatin group (9.01?mg/kg/d), and a control group (normal saline: 0.2?mL/d). At the ages of 30 and 40 weeks, we sacrificed the mice for various measurements. The results show that treatment with Shengjie Tongyu granule and simvastatin significantly decreased lumen and plaque areas in the aortic root at 30 and 40 weeks of age, decreased grade II elastic fiber lesions in the ascending aorta at 30 weeks of age, and decreased both grade II and III lesions at 40 weeks of age, compared to controls. The content of superoxide anions, and expression of MOMA-2, plasma ICAM-1, and NF?B p50 in 30- and 40-week-old mice in the Shengjie Tongyu granule and simvastatin groups were also significantly reduced compared to the control group. In conclusion, Shengjie Tongyu granule has a clear inhibitory effect on vascular remodeling and on inflammatory pathways in ApoE-gene-knockout mice.

Chen, Min; Cheng, Wenli; Shi, Zaixiang; Tatsukazu, Nishihara; Zhou, Tongliang; Li, Hong; Guo, Jian

2012-01-01

281

Inhibition of T lymphocyte activation in mice heterozygous for loss of the IMPDH II gene  

PubMed Central

Inosine 5?-monophosphate dehydrogenase (IMPDH) is the rate-limiting enzyme in the de novo synthesis of guanine nucleotides, which are also synthesized from guanine by a salvage reaction catalyzed by the X chromosome–linked enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT). Since inhibitors of IMPDH are in clinical use as immunosuppressive agents, we have examined the consequences of knocking out the IMPDH type II enzyme by gene targeting in a mouse model. Loss of both alleles of the gene encoding this enzyme results in very early embryonic lethality despite the presence of IMPDH type I and HPRT activities. Lymphocytes from IMPDH II+/– heterozygous mice are normal with respect to subpopulation distribution and respond normally to a variety of mitogenic stimuli. However, mice with an IMPDH II+/–, HPRT–/o genotype demonstrate significantly decreased lymphocyte responsiveness to stimulation with anti-CD3 and anti-CD28 antibodies and show a 30% mean reduction in GTP levels in lymphocytes activated by these antibodies. Furthermore, the cytolytic activity of their T cells against allogeneic target cells is significantly impaired. These results demonstrate that a moderate decrease in the ability of murine lymphocytes to synthesize guanine nucleotides during stimulation results in significant impairment in T-cell activation and function.

Gu, Jing Jin; Stegmann, Sander; Gathy, Karen; Murray, Robert; Laliberte, Josee; Ayscue, Lanier; Mitchell, Beverly S.

2000-01-01

282

Inhibition of MDR1 gene expression by chimeric HNA antisense oligonucleotides  

PubMed Central

Hexitol nucleic acids (HNAs) are nuclease resistant and provide strong hybridization to RNA. However, there is relatively little information on the biological properties of HNA antisense oligonucleotides. In this study, we compared the antisense effects of a chimeric HNA ‘gapmer’ oligonucleotide comprising a phosphorothioate central sequence flanked by 5? and 3? HNA sequences to conventional phosphorothioate oligonucleotides and to a 2?-O-methoxyethyl (2?-O-ME) phosphorothioate ‘gapmer’. The antisense oligomers each targeted a sequence bracketing the start codon of the message of MDR1, a gene involved in multi-drug resistance in cancer cells. Antisense and control oligonucleotides were delivered to MDR1-expressing cells using transfection with the cationic lipid Lipofectamine 2000. The anti-MDR1 HNA gapmer was substantially more potent than a phosphorothioate oligonucleotide of the same sequence in reducing expression of P-glycoprotein, the MDR1 gene product. HNA and 2?-O-ME gapmers displayed similar potency, but a pure HNA antisense oligonucleotide (lacking the phosphorothioate ‘gap’) was ineffective, indicating that RNase H activity was likely required. Treatment with anti-MDR1 HNA gapmer resulted in increased cellular accumulation of the drug surrogate Rhodamine 123 that correlated well with the reduced cell surface expression of P-glycoprotein. Thus, HNA gapmers may provide a valuable additional tool for antisense-based investigations and therapeutic approaches.

Kang, Hyunmin; Fisher, Michael H.; Xu, Dong; Miyamoto, Yuko J.; Marchand, Arnaud; Van Aerschot, Arthur; Herdewijn, Piet; Juliano, Rudolph L.

2004-01-01

283

Inhibition of MDR1 gene expression by chimeric HNA antisense oligonucleotides.  

PubMed

Hexitol nucleic acids (HNAs) are nuclease resistant and provide strong hybridization to RNA. However, there is relatively little information on the biological properties of HNA antisense oligonucleotides. In this study, we compared the antisense effects of a chimeric HNA 'gapmer' oligonucleotide comprising a phosphorothioate central sequence flanked by 5' and 3' HNA sequences to conventional phosphorothioate oligonucleotides and to a 2'-O-methoxyethyl (2'-O-ME) phosphorothioate 'gapmer'. The antisense oligomers each targeted a sequence bracketing the start codon of the message of MDR1, a gene involved in multi-drug resistance in cancer cells. Antisense and control oligonucleotides were delivered to MDR1-expressing cells using transfection with the cationic lipid Lipofectamine 2000. The anti-MDR1 HNA gapmer was substantially more potent than a phosphorothioate oligonucleotide of the same sequence in reducing expression of P-glycoprotein, the MDR1 gene product. HNA and 2'-O-ME gapmers displayed similar potency, but a pure HNA antisense oligonucleotide (lacking the phosphorothioate 'gap') was ineffective, indicating that RNase H activity was likely required. Treatment with anti-MDR1 HNA gapmer resulted in increased cellular accumulation of the drug surrogate Rhodamine 123 that correlated well with the reduced cell surface expression of P-glycoprotein. Thus, HNA gapmers may provide a valuable additional tool for antisense-based investigations and therapeutic approaches. PMID:15316104

Kang, Hyunmin; Fisher, Michael H; Xu, Dong; Miyamoto, Yuko J; Marchand, Arnaud; Van Aerschot, Arthur; Herdewijn, Piet; Juliano, Rudolph L

2004-01-01

284

NADPH inhibits transcription of the Escherichia coli manganese superoxide dismutase gene (sodA) in vitro.  

PubMed

We have previously reported that the thiols glutathione, dithiothreitol, and beta-mercaptoethanol suppress transcription of the Escherichia coli manganese-containing superoxide dismutase gene (sodA) in an in vitro coupled transcription plus translation system (Gardner, P. R., and Fridovich, I. (1987) J. Biol. Chem. 262, 17591-17595). We now report that NADPH, but not NADH, selectively decreases transcription of sodA in vitro and that an NADPH generating system utilizing glucose 6-phosphate and the corresponding dehydrogenase markedly augments this suppressive effect. A redox buffer containing various ratios of oxidized and reduced glutathione also modulated transcription of sodA thus demonstrating the existence of a redox-sensitive mechanism controlling sodA transcription. Fusion of a 120-base pair fragment, containing 90 base pairs of DNA upstream of the sodA transcription initiation site, to a promoterless galactokinase gene (galK) conferred redox-sensitivity to GalK synthesis. We propose that these redox effects act through a redox-sensitive regulator of sodA and that the anabolic reduction charge, [NADPH]/([NADPH]+[NADP+]), is one cellular signal controlling sodA transcription. PMID:8509428

Gardner, P R; Fridovich, I

1993-06-15

285

Amorfrutin A inhibits TNF-?-induced NF-?B activation and NF-?B-regulated target gene products.  

PubMed

The nuclear factor-?B (NF-?B) transcription factors control many physiological processes including inflammation, immunity, apoptosis, and angiogenesis. In our search for NF-?B inhibitors from natural resources, we identified amorfrutin A as an inhibitor of NF-?B activation from the fruits of Amorpha fruticosa L. In present study, this compound significantly inhibited the TNF-?-induced expression of NF-?B reporter gene. Further analysis revealed that amorfrutin A was a potent inhibitor of NF-?B activation by the suppression of TNF-?-induced inhibitor of ?B? (I?B?) degradation, p65 nuclear translocation, and DNA-binding activity of NF-?B. We also demonstrated that pretreatment of cells with this compound prevented the TNF-?-induced expression of NF-?B target genes, such as antiapoptosis (cIAP-1 and FLIP), proliferation (COX-2 and cyclinD1), invasion (MMP-9), angiogenesis (VEGF), and major inflammatory cytokines (TNF-?, IL-8, and MCP1). Furthermore, our results suggest that amorfrutin A potentiates TNF-?-induced apoptosis. Taken together, amorfrutin A could be a valuable candidate for the intervention of NF-?B-dependent pathological conditions such as inflammation. PMID:24785328

Shi, Hui; Ma, Juan; Mi, Chunliu; Li, Jing; Wang, Fei; Lee, Jung Joon; Jin, Xuejun

2014-07-01

286

Signal-dependent regulation of gene expression as a target for cancer treatment: inhibiting p38alpha in colorectal tumors.  

PubMed

In the last year, several evidences indicated that pharmacological manipulation of relevant signaling pathways could selectively affect gene expression to influence cell fate. These findings render of extreme importance the elucidation of how external stimuli are transduced to mediate chromatin modifications, resulting in a permissive or repressive environment for gene expression. These signaling cascades activate or repress the function of chromatin binding proteins that represent attractive pharmacological targets for human diseases. Actually, the closer the target is to chromatin, the more the transcriptional effect will be selective. Recent studies suggest that pharmacological manipulation of signaling pathways to modulate cell fate is indeed possible and that chromatin-associated kinases could represent an optimal target. The p38 MAPK is the prototype of this class of enzymes and its central role in the transcription process is evolutionary conserved. In this review we will focus on the possibility to inhibit p38alpha in colorectal cancer to arrest tumor progression and induce autophagic cell death. PMID:18395970

Chiacchiera, Fulvio; Simone, Cristiano

2008-06-28

287

Constitutive S-adenosylmethionine decarboxylase gene expression increases drought tolerance through inhibition of reactive oxygen species accumulation in Arabidopsis.  

PubMed

Using subtractive hybridization analysis, the S-adenosylmethionine decarboxylase (SAMDC) gene from Capsicum annuum was isolated and renamed CaSAMDC. We generated independent transgenic Arabidopsis (Arabidopsis thaliana) lines constitutively expressing a 35S::CaSAMDC construct. Drought tolerance was significantly enhanced in Arabidopsis T4 transgenic homozygous lines as compared to wild-type (WT) plants. The levels of main polyamines (PAs) were more significantly increased in CaSAMDC-overexpressing transgenic plants after 6 h of drought stress as compared to stressed WT plants. Basal transcription of polyamine oxidase (PAO) showed at a much higher level in unstressed-transgenic plants as compared to unstressed WT plants. However, the difference in PAO transcription level between WT and transgenic plants was reduced after drought stress. Cellular accumulation of reactive oxygen species (ROS) was significantly reduced following drought stress in transgenic Arabidopsis plants as compared to WT plants. These results were in agreement with additional observations that stress-induced ROS generation, as determined by qRT-PCR analysis of NADPH oxidase (RbohD and RbohF), was significantly suppressed while transcription of ROS-detoxifying enzymes was notably elevated in transgenic lines in response to drought stress. Further, ROS-induced transcription of the metacaspase II gene was remarkably inhibited in transgenic plants. Collectively, these results suggest that drought stress tolerance due to reduction of ROS production and enhancement of ROS detoxification can be attributed to elevation of PAs. PMID:24477528

Wi, Soo Jin; Kim, Soo Jin; Kim, Woo Taek; Park, Ky Young

2014-05-01

288

ss-siRNAs allele selectively inhibit ataxin-3 expression: multiple mechanisms for an alternative gene silencing strategy  

PubMed Central

Single-stranded silencing RNAs (ss-siRNAs) provide an alternative approach to gene silencing. ss-siRNAs combine the simplicity and favorable biodistribution of antisense oligonucleotides with robust silencing through RNA interference (RNAi). Previous studies reported potent and allele-selective inhibition of human huntingtin expression by ss-siRNAs that target the expanded CAG repeats within the mutant allele. Mutant ataxin-3, the genetic cause of Machado–Joseph Disease, also contains an expanded CAG repeat. We demonstrate here that ss-siRNAs are allele-selective inhibitors of ataxin-3 expression and then redesign ss-siRNAs to optimize their selectivity. We find that both RNAi-related and non-RNAi-related mechanisms affect gene expression by either blocking translation or affecting alternative splicing. These results have four broad implications: (i) ss-siRNAs will not always behave similarly to analogous RNA duplexes; (ii) the sequences surrounding CAG repeats affect allele-selectivity of anti-CAG oligonucleotides; (iii) ss-siRNAs can function through multiple mechanisms and; and (iv) it is possible to use chemical modification to optimize ss-siRNA properties and improve their potential for drug discovery.

Liu, Jing; Yu, Dongbo; Aiba, Yuichiro; Pendergraff, Hannah; Swayze, Eric E.; Lima, Walt F.; Hu, Jiaxin; Prakash, Thazha P.; Corey, David R.

2013-01-01

289

The mengovirus leader protein blocks interferon-alpha/beta gene transcription and inhibits activation of interferon regulatory factor 3.  

PubMed

Viral infection of mammalian cells triggers the synthesis and secretion of type I interferons (i.e. IFN-alpha/beta), which induce the transcription of genes that cause cells to adopt an antiviral state. Many viruses have adapted mechanisms to evade IFN-alpha/beta-mediated responses. The leader protein of mengovirus, a picornavirus, has been implicated as an IFN-alpha/beta antagonist. Here, we show that the leader inhibits the transcription of IFN-alpha/beta and that both the presence of a zinc finger motif in its N-terminus and phosphorylation of threonine-47 are required for this function. Transcription of IFN-alpha/beta genes relies on the activity of a number of transcription factors, including interferon regulatory factor 3 (IRF-3). We show that the leader interferes with the transactivation activity of IRF-3 by interfering with its dimerization. Accordingly, mutant viruses with a disturbed leader function were impaired in their ability to suppress IFN-alpha/beta transcription in vivo. By consequence, the leader mutant viruses had an impaired ability to replicate and spread in normal mice but not in IFNAR-KO mice, which are incapable of mounting an IFN-alpha/beta-dependent antiviral response. These results suggest that the leader, by suppressing IRF3-mediated IFN-alpha/beta production, plays an important role in replication and dissemination of mengovirus in its host. PMID:17991048

Hato, Stanleyson V; Ricour, Celine; Schulte, Barbara M; Lanke, Kjerstin H W; de Bruijni, Mike; Zoll, Jan; Melchers, Willem J G; Michiels, Thomas; van Kuppeveld, Frank J M

2007-12-01

290

Salvianolate inhibits cytokine gene expression in small intestine of cirrhotic rats  

PubMed Central

AIM: To study the effect of salvianolate on expression of tumor necrosis factor (TNF)-? and interleukin (IL)-6 mRNA in small intestine of cirrhotic rats. METHODS: Cirrhosis in rats was induced using CCl4 (0.3 mL/kg). Rats were randomly divided into non-treatment group, low-dose salvianolate (12 mg/kg) treatment group, medium-dose salvianolate (24 mg/kg) treatment group, and high-dose salvianolate (48 mg/kg) treatment group, and treated for 2 wk. Another 10 healthy rats served as a normal control group. Mortality of cirrhotic rats in each group was evaluated after treatment with salvianolate. Serum samples were taken from portal vein for the detection of endotoxin. Morphological changes in tissue samples from the ileocecum were observed under a light microscope. Expression of TNF-? and IL-6 mRNA in the small intestine of rats was analyzed by real-time reverse-transcriptase polymerase chain reaction. RESULTS: The mortality of cirrhotic rats in the non-treatment group was 37.5%. No cirrhotic rat died in the high-dose salvianolate treatment group. The serum endotoxin level was significantly higher in the non-treatment group than in the salvianolate treatment and normal control groups. The intestinal mucosal and villous atrophy, necrosis and shedding of the intestinal mucosal epithelium, observed in the non-treatment group, were reversed in different salvianolate treatment groups. The TNF-? and IL-6 mRNA expression levels in small intestine were significantly lower in different salvianolate treatment groups than in the non-treatment group. CONCLUSION: Salvianolate can reduce the endotoxin level, ameliorate the injury of intestinal mucosa, and inhibit the expression of TNF-? and IL-6 mRNA in small intestine of cirrhotic rats.

Yang, Dan-Hong; Ye, Zai-Yuan; Jin, Bo; He, Xu-Jun; Zhang, Qin; Zhou, Wei-Ming; Xu, Wen-Juan; Lu, Huo-Xiang

2011-01-01

291

The tsr gene-coding plasmid pIJ702 prevents thiopeptin from inhibiting ppGpp synthesis in Streptomyces lividans.  

PubMed

Streptomyces lividans normally accumulated high levels of ppGpp during nutritional shift-down. Its accumulation was, however, severely inhibited when a small amount of thiopeptin (an analogue of thiostrepton) was included in the transfer medium. In contrast, a S. lividans strain, which harbours the plasmid pIJ702 carrying the tsr gene resist to thiopeptin through methylation of the 23S rRNA, was still capable of accumulating ppGpp in the presence of large amounts of thiopeptin. These results indicate that the rRNA methylation resulting from the action of tsr gene prevents thiopeptin not only from inhibiting cell-growth but also from inhibiting ppGpp synthesis. The results also indicate that the observed accumulation of ppGpp during nutritional shift-down was associated with ribosomal function, as has been shown in E. coli and B. subtilis. PMID:2599357

Ochi, K

1989-10-01

292

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

PubMed Central

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 ? (PPAR?) 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.

2010-01-01

293

Inhibition of storage pathology in prenatal CLN5-deficient sheep neural cultures by lentiviral gene therapy.  

PubMed

The neuronal ceroid lipofuscinoses (NCLs, Batten disease) are inherited neurodegenerative lysosomal storage diseases caused by mutations in several different genes. Mutations in CLN5 cause a variant late-infantile human disease and some cases of juvenile and adult clinical disease. NCLs also occur in animals, and a flock of New Zealand Borderdale sheep with a CLN5 splice-site mutation has been developed for model studies. Dissociated mixed neural cells from CLN5-deficient foetal sheep brains contained no obvious storage bodies at plating but these accumulated rapidly in culture, mainly in microglial cells and also in neurons and astrocytes. Accumulation was very obvious after a week, as monitored by fluorescent microscopy and immunostaining for subunit c of mitochondrial ATP synthase. Photography at intervals revealed the dynamic nature of the cultures and a flow of storage bodies between cells, specifically the phagocytosis of storage-body containing cells by microglia and incorporation of the storage bodies into the host cells. No storage was observed in cultured control cells. Transduction of cell cultures with a lentiviral vector expressing a C-terminal Myc tagged CLN5 resulted in secretion of post-translationally glycosylated and processed CLN5. Transduction of CLN5-deficient cultures with this construct rapidly reversed storage body accumulation, to less than half in only six days. These results show that storage body accumulation is reversible with enzyme correction and support the use of these cultures for testing of therapeutics prior to whole animal studies. PMID:24269732

Hughes, Stephanie M; Hope, Katie M; Xu, Janet Boyu; Mitchell, Nadia L; Palmer, David N

2014-02-01

294

MUTATIONS IN THE GABRB1 GENE PROMOTE ALCOHOL CONSUMPTION THROUGH INCREASED TONIC INHIBITION  

PubMed Central

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 ?-aminobutyric acidA receptor (GABAAR) ?1 subunit gene (Gabrb1). Using N-ethyl-N-nitrosourea mutagenesis on an alcohol-averse background (F1 BALB/cAnN × 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.

Anstee, Quentin M.; Knapp, Susanne; Maguire, Edward P.; 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

295

Cloning, primary structure and regulation of the ARO4 gene, encoding the tyrosine-inhibited 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Saccharomyces cerevisiae.  

PubMed

In Saccharomyces cerevisiae, two differently regulated 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase (DAHPS; EC 4.1.2.15) isoenzymes carry out the first step in the shikimate pathway. Mutations in both genes are necessary to cause aromatic amino acid (aa) auxotrophy, since one isoenzyme alone is sufficient to produce enough DAHP for normal growth of the cells. The phenylalanine-inhibited DAHPS is encoded by the previously isolated and characterized ARO3 gene. Here, we report the cloning and characterization of the ARO4 gene, encoding the second DAHPS, which is inhibited by tyrosine. The aa sequence of the ARO4 gene product reveals 76% similarity to the ARO3-encoded isoenzyme and 66 and 73% to the three DAHPS isoenzymes from Escherichia coli. ARO4 gene expression is regulated by the general control system of aa biosynthesis. As in the case of the ARO3 gene, a single GCN4-recognition element in the promoter is responsible for derepression of the ARO4 gene under aa starvation conditions. However, in contrast to the situation in the isogene, ARO3, GCN4 does not contribute to the basal level of ARO4 transcription under nonderepressing conditions. PMID:1348717

Künzler, M; Paravicini, G; Egli, C M; Irniger, S; Braus, G H

1992-04-01

296

Luteolin Inhibits the Activity, Secretion and Gene Expression of MMP-3 in Cultured Articular Chondrocytes and Production of MMP-3 in the Rat Knee  

PubMed Central

We investigated whether luteolin affects the gene expression, secretion and activity of matrix metalloproteinase-3 (MMP-3) in primary cultured rabbit articular chondrocytes, as well as production of MMP-3 in the rat knee to evaluate the potential chondro-protective effects of luteolin. Rabbit articular chondrocytes were cultured in a monolayer and IL-1?-induced gene expression levels of MMP-3, MMP-1, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4), ADAMTS-5 and type II collagen were measured by reverse transcription - polymerase chain reaction (RT-PCR). Effects of luteolin on interleukin-1? (IL-1?)-induced secretion and enzyme activity of MMP-3 in rabbit articular chondrocytes were investigated by western blot analysis and casein zymography, respectively. The effect of luteolin on MMP-3 protein production was also examined in vivo. The results were as follows: (1) luteolin inhibited the gene expression levels of MMP-3, MMP-1, MMP-13, ADAMTS-4 and ADAMTS-5. However, it increased the gene expression level of collagen in rabbit articular chondrocytes; (2) luteolin inhibited the secretion and activity of MMP-3; (3) luteolin inhibited in vivo production of MMP-3 protein. These results suggest that luteolin can regulate the gene expression, secretion and activity of MMP-3, by directly acting on articular chondrocytes.

Kang, Bun-Jung; Ryu, Jiho; Lee, Choong Jae; Hwang, Sun-Chul

2014-01-01

297

Adiponectin gene expression and secretion is inhibited by interleukin-6 in 3T3-L1 adipocytes.  

PubMed

Recently, it has been shown that adiponectin is an important insulin-sensitizing fat-derived protein which is downregulated in insulin resistance and obesity, and replenishment of which improves insulin sensitivity. In contrast, interleukin (IL)-6 appears as an adipocytokine serum concentrations of which are elevated in these states. However, it has not been determined whether IL-6 might impact on expression and secretion of adiponectin. To clarify this, 3T3-L1 adipocytes were treated with different concentrations of IL-6 for various periods of time. Adiponectin mRNA was measured by quantitative real-time reverse transcription-polymerase chain reaction and secretion was determined by radioimmunoassays. Interestingly, treatment of 3T3-L1 cells with 30 ng/ml IL-6 significantly decreased adiponectin secretion to 75% of control levels. Adiponectin secretion was also inhibited between 25% and 45% by chronic treatment with forskolin (50 microM), tumor necrosis factor alpha (100 ng/ml), and dexamethasone (100 nM). Furthermore, adiponectin mRNA expression was downregulated by up to 50% in a time- and dose-dependent manner, with significant inhibition detectable at concentrations as low as 3 ng/ml IL-6 and as early as 8h after effector addition. The inhibitory effect of IL-6 was partially reversed by pretreatment of 3T3-L1 cells with pharmacological inhibitors of a p44/42 mitogen-activated protein (MAP) kinase. Moreover, the negative effect of IL-6 on adiponectin mRNA expression could be reversed by withdrawal of the hormone for 24h. Taken together, our results suggest that adiponectin gene expression is reversibly downregulated by IL-6 and support the concept of adiponectin being an important selectively controlled modulator of insulin sensitivity. PMID:12589818

Fasshauer, Mathias; Kralisch, Susan; Klier, Margit; Lossner, Ulrike; Bluher, Matthias; Klein, Johannes; Paschke, Ralf

2003-02-21

298

Silencing of the IKK? gene by siRNA inhibits invasiveness and growth of breast cancer cells  

PubMed Central

Introduction I?B kinase ? (IKK?) is a member of the IKK family that plays an important role in the activation of NF-?B. Overexpressed in more than 30% of breast cancers, IKK? has been recently identified as a potential breast cancer oncogene. The purpose of the present study is to examine the therapeutic potential of IKK? siRNA on human breast cancer cells. Methods Eight siRNAs targeting different regions of the IKK? mRNA were designed, and the silencing effect was screened by quantitative real-time RT-PCR. The biological effects of synthetic siRNAs on human breast cancer cells were investigated by examining the cell proliferation, migration, invasion, focus formation, anchorage-independent growth (via soft agar assay), cell cycle arrest, apoptosis (via annexing binding), NF-?B basal level, and NF-?B-related gene expressions upon the IKK? silencing. Results Silencing of IKK? in human breast cancer cells resulted in a decrease of focus formation potential and clonogenicity as well as in vitro cell migration/invasion capabilities. Moreover, knockdown of IKK? suppressed cell proliferation. Cell cycle assay showed that the anti-proliferation effect of IKK? siRNA was mediated by arresting cells in the G0/G1 phase, which was caused by downregulation of cyclin D1. Furthermore, we demonstrated that silencing of IKK? inhibited the NF-?B basal activity as well as the Bcl-2 expression. Significant apoptosis was not observed in breast cancer cells upon the silencing of IKK?. The present study provided the first evidence that silencing IKK? using synthetic siRNA can inhibit the invasiveness properties and proliferation of breast cancer cells. Conclusions Our results suggested that silencing IKK? using synthetic siRNA may offer a novel therapeutic strategy for breast cancer.

2010-01-01

299

Inhibition of transcription of cytosine-containing DNA in vitro by the alc gene product of bacteriophage T4  

SciTech Connect

The alc gene product (gpalc) of bacteriophage T4 inhibits the transcription of cytosine-containing DNA in vivo. The authors examined its effect on transcription in vitro by comparing RNA polymerase isolated from Escherichia coli infected with either wild-type T4D{sup +} or alc mutants. A 50 to 60% decline in RNA polymerase activity, measured on phage T7 DNA, was observed by 1 min after infection with either T4D{sup +} or alc mutants; this did not occur when the infecting phage lacked gpalt. In the case of the T4D{sup +} strain but not alc mutants, this was followed by a further decrease. By 5 min after infection the activity of alc mutants was 1.5 to 2.5 times greater than that of the wild type on various cytosine-containing DNA templates, whereas there was little or no difference in activity on T4 HMdC-DNA, in agreement with the in vivo specificity. Effects on transcript initiation and elongation were distinguished by using a T7 phage DNA template. Rifampin challenge, end-labeling with ({gamma}-{sup 32}P)ATP, and selective initiation with a dinucleotide all indicate that the decreased in vitro activity of the wild-type polymerase relative to that of the alc mutants was due to inhibition of elongation, not to any difference in initiation rates. Wild-type (but not mutated) gpalc copurified with RNA polymerase on heparin agarose but not in subsequent steps. Immunoprecipitation of modified RNA polymerase also indicated that gpalc was not tightly bound to RNA polymerase intracellularly.

Drivdahl, R.H.; Kutter, E.M. (Evergreen State College, Olympia, WA (USA))

1990-05-01

300

Knocking down gene expression for growth hormone-releasing hormone inhibits proliferation of human cancer cell lines.  

PubMed

Splice Variant 1 (SV-1) of growth hormone-releasing hormone (GHRH) receptor, found in a wide range of human cancers and established human cancer cell lines, is a functional receptor with ligand-dependent and independent activity. In the present study, we demonstrated by western blots the presence of the SV1 of GHRH receptor and the production of GHRH in MDA-MB-468, MDA-MB-435S and T47D human breast cancer cell lines, LNCaP prostate cancer cell line as well as in NCI H838 non-small cell lung carcinoma. We have also shown that GHRH produced in the conditioned media of these cell lines is biologically active. We then inhibited the intrinsic production of GHRH in these cancer cell lines using si-RNA, specially designed for human GHRH. The knocking down of the GHRH gene expression suppressed the proliferation of T47D, MDA-MB-435S, MDA-MB-468 breast cancer, LNCaP prostate cancer and NCI H838 non-SCLC cell lines in vitro. However, the replacement of the knocked down GHRH expression by exogenous GHRH (1-29)NH(2) re-established the proliferation of the silenced cancer cell lines. Furthermore, the proliferation rate of untransfected cancer cell lines could be stimulated by GHRH (1-29)NH(2) and inhibited by GHRH antagonists MZ-5-156, MZ-4-71 and JMR-132. These results extend previous findings on the critical function of GHRH in tumorigenesis and support the role of GHRH as a tumour growth factor. PMID:18506184

Barabutis, N; Schally, A V

2008-06-01

301

Knocking down gene expression for growth hormone-releasing hormone inhibits proliferation of human cancer cell lines  

PubMed Central

Splice Variant 1 (SV-1) of growth hormone-releasing hormone (GHRH) receptor, found in a wide range of human cancers and established human cancer cell lines, is a functional receptor with ligand-dependent and independent activity. In the present study, we demonstrated by western blots the presence of the SV1 of GHRH receptor and the production of GHRH in MDA-MB-468, MDA-MB-435S and T47D human breast cancer cell lines, LNCaP prostate cancer cell line as well as in NCI H838 non-small cell lung carcinoma. We have also shown that GHRH produced in the conditioned media of these cell lines is biologically active. We then inhibited the intrinsic production of GHRH in these cancer cell lines using si-RNA, specially designed for human GHRH. The knocking down of the GHRH gene expression suppressed the proliferation of T47D, MDA-MB-435S, MDA-MB-468 breast cancer, LNCaP prostate cancer and NCI H838 non-SCLC cell lines in vitro. However, the replacement of the knocked down GHRH expression by exogenous GHRH (1–29)NH2 re-established the proliferation of the silenced cancer cell lines. Furthermore, the proliferation rate of untransfected cancer cell lines could be stimulated by GHRH (1–29)NH2 and inhibited by GHRH antagonists MZ-5-156, MZ-4-71 and JMR-132. These results extend previous findings on the critical function of GHRH in tumorigenesis and support the role of GHRH as a tumour growth factor.

Barabutis, N; Schally, A V

2008-01-01

302

Clustering of mandibular organ-inhibiting hormone and moult-inhibiting hormone genes in the crab, Cancer pagurus, and implications for regulation of expression  

Microsoft Academic Search

Development and reproduction of crustaceans is regulated by a combination of neuropeptide hormones, ecdysteroids (moulting hormones) and the isoprenoid, methyl farnesoate (MF), the unepoxidised analogue of insect juvenile hormone-III (JH-III). MF and the ecdysteroids are respectively synthesised under the negative control of the sinus gland-derived mandibular organ-inhibiting hormones (MO-IHs) and moult-inhibiting hormone (MIH) that are produced in eyestalk neural ganglia.

Weiqun Lu; Geoffrey Wainwright; Simon G. Webster; Huw H. Rees; Philip C. Turner

2000-01-01

303

Inhibition of VEGF mRNA by 2'-O,4'-C-ethylene-bridged nucleic acids (ENA) antisense oligonucleotides and their influence on off-target gene expressions.  

PubMed

We investigated 2'-O,4'-C-ethylene-bridged nucleic acids (ENA) antisense oligonucleotides (AONs) for vascular endothelial growth factor (VEGF) in human lung carcinoma A549 cells. An ENA/DNA gapmer AON with RNase H-mediated activity was virtually stable in rat plasma and exhibited more than 90% inhibition of VEGF mRNA production. Moreover, 22 genes that are likely to bind to the AON were found in the GenBank database by BLAST and CLUSTAL W searches. Three of these genes were actually inhibited by the ENA AON. In shorter ENA AONs with fewer matched sequences of these genes, inhibitiory activities were decreased and off-target effects were improved. These results indicate that ENA AONs act in a sequence-specific manner and could be used as effective antisense drugs. PMID:16838842

Morita, Koji; Yamate, Koji; Kurakata, Shin-ichi; Abe, Koji; Watanabe, Kenji; Koizumi, Makoto; Imanishi, Takeshi

2006-01-01

304

C. elegans FOG-3/Tob can either promote or inhibit germline proliferation, depending on gene dosage and genetic context  

PubMed Central

Vertebrate Tob/BTG proteins inhibit cell proliferation when overexpressed in tissue culture cells, and they can function as tumor suppressors in mice. The single Caenorhabditis elegans Tob/BTG ortholog, FOG-3, by contrast, was identified from its loss-of-function phenotype as a regulator of sperm fate specification. Here we report that FOG-3 also regulates proliferation in the germline tissue. We first demonstrate that FOG-3 is a positive regulator of germline proliferation. Thus, fog-3 null mutants possess fewer germ cells than normal, a modest but reproducible decrease observed for each of two distinct fog-3 null alleles. A similar decrease also occurred in fog-3/+ heterozygotes, again for both fog-3 alleles, revealing a haplo-insufficient effect on proliferation. Therefore, FOG-3 normally promotes proliferation, and two copies of the fog-3 gene are required for this function. We next overexpressed FOG-3 by removal of FBF, the collective term for FBF-1 and FBF-2, two nearly identical PUF RNA-binding proteins. We find that overexpressed FOG-3 blocks proliferation in fbf-1 fbf-2 mutants: whereas germ cells stop dividing and instead differentiate in fbf-1 fbf-2 double mutants, they continue to proliferate in fog-3; fbf-1 fbf-2 triple mutants. Therefore, like its vertebrate Tob/BTG cousins, overexpressed FOG-3 is “antiproliferative”. Indeed, some fog-3; fbf-1 fbf-2 mutants possess small tumors, suggesting that FOG-3 can act as a tumor suppressor. Finally, we show that FOG-3 and FBF work together to promote tumor formation in animals carrying oncogenic Notch mutations. A similar effect was not observed when germline tumors were induced by manipulation of other regulators; therefore this FOG-3 tumor-promoting effect is context-dependent. We conclude that FOG-3 can either promote or inhibit proliferation in a manner that is sensitive to both genetic context and gene dosage. The discovery of these FOG-3 effects on proliferation has implications for our understanding of vertebrate Tob/BTG proteins and their influence on normal development and tumorigenesis.

Snow, Joshua J.; Lee, Myon-Hee; Verheyden, Jamie; Kroll-Conner, Peggy L.; Kimble, Judith

2012-01-01

305

C. elegans FOG-3/Tob can either promote or inhibit germline proliferation, depending on gene dosage and genetic context.  

PubMed

Vertebrate Tob/BTG proteins inhibit cell proliferation when overexpressed in tissue-culture cells, and they can function as tumor suppressors in mice. The single Caenorhabditis elegans Tob/BTG ortholog, FOG-3, by contrast, was identified from its loss-of-function phenotype as a regulator of sperm fate specification. Here we report that FOG-3 also regulates proliferation in the germline tissue. We first demonstrate that FOG-3 is a positive regulator of germline proliferation. Thus, fog-3 null mutants possess fewer germ cells than normal, a modest but reproducible decrease observed for each of two distinct fog-3 null alleles. A similar decrease also occurred in fog-3/+ heterozygotes, again for both fog-3 alleles, revealing a haplo-insufficient effect on proliferation. Therefore, FOG-3 normally promotes proliferation, and two copies of the fog-3 gene are required for this function. We next overexpressed FOG-3 by removal of FBF, the collective term for FBF-1 and FBF-2, two nearly identical PUF RNA-binding proteins. We find that overexpressed FOG-3 blocks proliferation in fbf-1 fbf-2 mutants; whereas germ cells stop dividing and instead differentiate in fbf-1 fbf-2 double mutants, they continue to proliferate in fog-3; fbf-1 fbf-2 triple mutants. Therefore, like its vertebrate Tob/BTG cousins, overexpressed FOG-3 is 'antiproliferative'. Indeed, some fog-3; fbf-1 fbf-2 mutants possess small tumors, suggesting that FOG-3 can act as a tumor suppressor. Finally, we show that FOG-3 and FBF work together to promote tumor formation in animals carrying oncogenic Notch mutations. A similar effect was not observed when germline tumors were induced by manipulation of other regulators; therefore, this FOG-3 tumor-promoting effect is context dependent. We conclude that FOG-3 can either promote or inhibit proliferation in a manner that is sensitive to both genetic context and gene dosage. The discovery of these FOG-3 effects on proliferation has implications for our understanding of vertebrate Tob/BTG proteins and their influence on normal development and tumorigenesis. PMID:22797076

Snow, J J; Lee, M-H; Verheyden, J; Kroll-Conner, P L; Kimble, J

2013-05-23

306

Inhibition of gene expression inside cells by peptide nucleic acids: effect of mRNA target sequence, mismatched bases, and PNA length.  

PubMed

Genome sequencing has revealed thousands of novel genes, placing renewed emphasis on chemical approaches for controlling gene expression. Antisense oligomers designed directly from the information generated by sequencing are one option for achieving this control. Here we explore the rules governing the inhibition of gene expression by peptide nucleic acids (PNAs) inside cells. PNAs are a DNA/RNA mimic in which the phosphate deoxyribose backbone has been replaced by uncharged linkages. Binding to complementary sequences is not hindered by electrostatic repulsion and is characterized by high rates of association and elevated affinities. Here we test the hypothesis that the favorable properties of PNAs offer advantages for recognition of mRNA and antisense inhibition of gene expression in vivo. We have targeted 27 PNAs to 18 different sites throughout the 5'-untranslated region (5'-UTR), start site, and coding regions of luciferase mRNA. PNAs were introduced into living cells in culture as PNA-DNA-lipid complexes, providing a convenient high throughput method for cellular delivery. We find that PNAs targeted to the terminus of the 5'-UTR are potent and sequence-specific antisense agents. PNAs fifteen to eighteen bases in length were optimal inhibitors. The introduction of one or two mismatches abolished inhibition, and complementary PNAs targeted to the sense strand were also inactive. In striking contrast to effective inhibition by PNAs directed to the terminal region, PNAs complementary to other sites within the 5'-UTR do not inhibit gene expression. We also observe no inhibition by PNAs complementary to the start site or rest of the coding region, nor do we detect inhibition by PNAs that are highly C/G rich and possess extremely high affinities for their target sequences. Our results suggest that PNAs can block binding of the translation machinery but are less able to block the progress of the ribosome along mRNA. The high specificity of antisense inhibition by PNAs emphasizes both the promise and the challenges for PNAs as antisense agents and provides general guidelines for using PNAs to probe the molecular recognition of biological targets inside cells. PMID:11141056

Doyle, D F; Braasch, D A; Simmons, C G; Janowski, B A; Corey, D R

2001-01-01

307

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

SciTech Connect

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.

Sakaeda, Yoshiichi [Division of Microbiology and Immunology, Department of Oral Biology and Tissue Engineering, Meikai University School of Dentistry, 1-1 Keyakidai, Sakado, Saitama 350-0283 (Japan); Hiroi, Miki [Division of Microbiology and Immunology, Department of Oral Biology and Tissue Engineering, Meikai University School of Dentistry, 1-1 Keyakidai, Sakado, Saitama 350-0283 (Japan); Shimojima, Takahiro [Division of Oral Rehabilitation, Meikai University School of Dentistry, 1-1 Keyakidai, Sakado, Saitama 350-0283 (Japan); Iguchi, Mayumi [Division of Orthodontics, Meikai University School of Dentistry, 1-1 Keyakidai, Sakado, Saitama 350-0283 (Japan); Kanegae, Haruhide [Division of Orthodontics, Meikai University School of Dentistry, 1-1 Keyakidai, Sakado, Saitama 350-0283 (Japan); Ohmori, Yoshihiro [Division of Microbiology and Immunology, Department of Oral Biology and Tissue Engineering, Meikai University School of Dentistry, 1-1 Keyakidai, Sakado, Saitama 350-0283 (Japan)]. E-mail: ohmori@dent.meikai.ac.jp

2006-11-17

308

Inhibition of lymphogenous metastasis using adeno-associated virus-mediated gene transfer of a soluble VEGFR-3 decoy receptor.  

PubMed

The presence of metastases in regional lymph nodes is a strong indicator of poor patient survival in many types of cancer. It has recently been shown that the lymphangiogenic growth factor, vascular endothelial growth factor-C (VEGF-C), and its receptor, VEGF receptor-3 (VEGFR3), may play a pivotal role in the promotion of metastasis to regional lymph nodes. In this study, human prostate and melanoma tumor models that preferentially metastasize to the lymph nodes following s.c. tumor cell implantation were established from lymph node metastases via in vivo selection. Melanoma tumor cell sublines established from lymph node metastasis express higher amounts of VEGF-C than the parental tumor cells. The inhibition of tumor-derived VEGF-C with a soluble VEGFR3 decoy receptor, sVEGFR3-Fc, expressed via a recombinant adeno-associated viral vector, potently blocks tumor-associated lymphangiogenesis and tumor metastasis to the lymph nodes, when the treatment was initiated before the tumor implantation. In addition, sVEGFR3-Fc serum levels required for efficient blockade of lymph node metastases are strictly dependent on the VEGF-C levels generated by the primary tumor. Recombinant adeno-associated virus-mediated gene transfer of sVEGFR3-Fc may represent a feasible therapeutic strategy for blockade of lymphogenous metastasis. PMID:16061674

Lin, JianMin; Lalani, Alshad S; Harding, Thomas C; Gonzalez, Melissa; Wu, Wei-Wei; Luan, Bo; Tu, Guang Huan; Koprivnikar, Kathryn; VanRoey, Melinda J; He, Yulong; Alitalo, Kari; Jooss, Karin

2005-08-01

309

Inhibition of the human ether-a-go-go-related gene (HERG) K+ channels by Lindera erythrocarpa.  

PubMed

Lindera erythrocarpa Makino (Lauraceae) is used as a traditional medicine for analgesic, antidote, and antibacterial purposes and shows anti-tumor activity. We studied the effects of Lindera erythrocarpa on the human ether-a-go-go-related gene (HERG) channel, which appears of importance in favoring cancer progression in vivo and determining cardiac action potential duration. Application of MeOH extract of Lindera erythrocarpa showed a dose-dependent decrease in the amplitudes of the outward currents measured at the end of the pulse (I(HERG)) and the tail currents of HERG (I(tail)). When the BuOH fraction and H(2)O fraction of Lindera erythrocarpa were added to the perfusate, both I(HERG) and I(tail) were suppressed, while the hexane fraction, CHCl(3) fraction, and EtOAc fraction did not inhibit either I(HERG) or I(tail). The potential required for half-maximal activation caused by EtOAc fraction, BuOH fraction, and H(2)O fraction shifted significantly. The BuOH fraction and H(2)O fraction (100 microg/mL) decreased g(max) by 59.6% and 52.9%, respectively. The H(2)O fraction- and BuOH fraction-induced blockades of I(tail) progressively decreased with increasing depolarization, showing the voltage-dependent block. Our findings suggest that Lindera erythrocarpa, a traditional medicine, blocks HERG channel, which could contribute to its anticancer and cardiac arrhythmogenic effect. PMID:19949665

Hong, Hee-Kyung; Yoon, Weon-Jong; Kim, Young Ho; Yoo, Eun-Sook; Jo, Su-Hyun

2009-12-01

310

Inhibition of the Human Ether-a-go-go-related Gene (HERG) K+ Channels by Lindera erythrocarpa  

PubMed Central

Lindera erythrocarpa Makino (Lauraceae) is used as a traditional medicine for analgesic, antidote, and antibacterial purposes and shows anti-tumor activity. We studied the effects of Lindera erythrocarpa on the human ether-a-go-go-related gene (HERG) channel, which appears of importance in favoring cancer progression in vivo and determining cardiac action potential duration. Application of MeOH extract of Lindera erythrocarpa showed a dose-dependent decrease in the amplitudes of the outward currents measured at the end of the pulse (IHERG) and the tail currents of HERG (Itail). When the BuOH fraction and H2O fraction of Lindera erythrocarpa were added to the perfusate, both IHERG and Itail were suppressed, while the hexane fraction, CHCl3 fraction, and EtOAc fraction did not inhibit either IHERG or Itail. The potential required for half-maximal activation caused by EtOAc fraction, BuOH fraction, and H2O fraction shifted significantly. The BuOH fraction and H2O fraction (100 µg/mL) decreased gmax by 59.6% and 52.9%, respectively. The H2O fraction- and BuOH fraction-induced blockades of Itail progressively decreased with increasing depolarization, showing the voltage-dependent block. Our findings suggest that Lindera erythrocarpa, a traditional medicine, blocks HERG channel, which could contribute to its anticancer and cardiac arrhythmogenic effect.

Hong, Hee-Kyung; Yoon, Weon-Jong; Kim, Young Ho; Yoo, Eun-Sook

2009-01-01

311

Gene Expression Analysis Reveals Inhibition of Radiation-Induced TGF?-Signaling by Hyperbaric Oxygen Therapy in Mouse Salivary Glands  

PubMed Central

A side effect of radiation therapy in the head and neck region is injury to surrounding healthy tissues such as irreversible impaired function of the salivary glands. Hyperbaric oxygen therapy (HBOT) is clinically used to treat radiation-induced damage but its mechanism of action is largely unknown. In this study, we investigated the molecular pathways that are affected by HBOT in mouse salivary glands two weeks after radiation therapy by microarray analysis. Interestingly, HBOT led to significant attenuation of the radiation-induced expression of a set of genes and upstream regulators that are involved in processes such as fibrosis and tissue regeneration. Our data suggest that the TGF?-pathway, which is involved in radiation-induced fibrosis and chronic loss of function after radiation therapy, is affected by HBOT. On the longer term, HBOT reduced the expression of the fibrosis-associated factor ?-smooth muscle actin in irradiated salivary glands. This study highlights the potential of HBOT to inhibit the TGF?-pathway in irradiated salivary glands and to restrain consequential radiation induced tissue injury.

Spiegelberg, Linda; Swagemakers, Sigrid MA; van IJcken, Wilfred FJ; Oole, Edwin; Wolvius, Eppo B; Essers, Jeroen; Braks, Joanna AM

2014-01-01

312

ROS mediate the hypoxic repression of the hepcidin gene by inhibiting C/EBPalpha and STAT-3.  

PubMed

Hepcidin, a liver peptide, systemically inhibits iron utilization and is downregulated under hypoxic conditions. However, little is known about the mechanism underlying the hypoxic suppression of hepcidin. Here, we tested the possibility that HIF-1 and ROS are involved in hepcidin regulation. Hepcidin mRNA, pre-mRNA, and protein levels were reduced in mouse livers and in HepG2 cells after hypoxic incubation, and HIF-1 overexpression and knock-down studies showed that hepcidin regulation is independent of HIF-1. On the other hand, ROS levels were significantly elevated in hypoxic HepG2 cells, and anti-oxidants prevented the hypoxic down-regulation of hepcidin. Conversely, a prooxidant, H(2)O(2), suppressed hepcidin expression in these cells even in normoxia. Of the various transcription factors examined, C/EBPalpha and STAT-3 were found to dissociate from hepcidin promoter under hypoxia, but to become fully engaged after anti-oxidant treatment. These results suggest that ROS repress the hepcidin gene by preventing C/EBPalpha and STAT-3 binding to hepcidin promoter during hypoxia. PMID:17349976

Choi, Si-On; Cho, Young-Suk; Kim, Hye-Lim; Park, Jong-Wan

2007-04-27

313

Polycomb group gene BMI1 controls invasion of medulloblastoma cells and inhibits BMP-regulated cell adhesion  

PubMed Central

Background Medulloblastoma is the most common intracranial childhood malignancy and a genetically heterogeneous disease. Despite recent advances, current therapeutic approaches are still associated with high morbidity and mortality. Recent molecular profiling has suggested the stratification of medulloblastoma from one single disease into four distinct subgroups namely: WNT Group (best prognosis), SHH Group (intermediate prognosis), Group 3 (worst prognosis) and Group 4 (intermediate prognosis). BMI1 is a Polycomb group repressor complex gene overexpressed across medulloblastoma subgroups but most significantly in Group 4 tumours. Bone morphogenetic proteins are morphogens belonging to TGF-? superfamily of growth factors, known to inhibit medulloblastoma cell proliferation and induce apoptosis. Results Here we demonstrate that human medulloblastoma of Group 4 characterised by the greatest overexpression of BMI1, also display deregulation of cell adhesion molecules. We show that BMI1 controls intraparenchymal invasion in a novel xenograft model of human MB of Group 4, while in vitro assays highlight that cell adhesion and motility are controlled by BMI1 in a BMP dependent manner. Conclusions BMI1 controls MB cell migration and invasion through repression of the BMP pathway, raising the possibility that BMI1 could be used as a biomarker to identify groups of patients who may benefit from a treatment with BMP agonists.

2014-01-01

314

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

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 ?M 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.

Donnelly, Christina; Olsen, Arne M.; Lewis, Lionel D.; Eisenberg, Burton L.; Eastman, Alan; Kinlaw, William B.

2010-01-01

315

Kaposi's Sarcoma-Associated Herpesvirus-Induced Angiogenin Plays Roles in Latency via the Phospholipase C? Pathway: Blocking Angiogenin Inhibits Latent Gene Expression and Induces the Lytic Cycle? †  

PubMed Central

During de novo infection of human dermal microvascular endothelial cells (HMVEC-d), Kaposi's sarcoma-associated herpesvirus (KSHV) induced the multifunctional angiogenin (ANG) protein, which entered the nuclei and nucleoli of infected cells and stimulated 45S rRNA gene transcription, proliferation, and tube formation, which were inhibited by blocking ANG nuclear translocation with the antibiotic neomycin (S. Sadagopan et al., J. Virol. 83:3342-3364, 2009). ANG was induced by KSHV latency protein LANA-1 (open reading frame 73 [ORF73]). Here we examined the presence and functions of ANG in KSHV-positive (KSHV+) primary effusion lymphoma (PEL/BCBL) cells. Significant ANG gene expression and secretion were observed in KSHV+ (BCBL-1 and BC-3) and KSHV+ and Epstein-Barr virus-positive (KSHV+ EBV+) (JSC-1) PEL cells and in BJAB-KSHV cells but not in EBV? KSHV? lymphoma cells (Akata, Loukes, Ramos, and BJAB), EBV+ lymphoma cells (Akata-EBV and Raji), and cells from an EBV+ lymphoblastoid cell line, thus suggesting a specific association of ANG in KSHV biology. Inhibition of nuclear translocation of ANG resulted in reduced BCBL-1 and TIVE-LTC (latently infected endothelial) cell survival and proliferation, while EBV? and EBV+ Akata cells were unaffected. Blocking nuclear transport of ANG inhibited latent ORF73 gene expression and increased lytic switch ORF50 gene expression, both during de novo infection and in latently infected cells. A greater quantity of infectious KSHV was detected in the supernatants of neomycin-treated BCBL-1 cells than 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated cells. Neomycin treatment and ANG silencing inhibited phospholipase C? (PLC-?) and AKT phosphorylation, and in contrast, ANG induced ORF73 expression and PLC-? and AKT phosphorylation. Further studies provided evidence that blockage of PLC-? activation by neomycin appears to be mediating the inhibition of latent gene expression, since treatment with the conventional PLC-? inhibitor U73122 also showed similar results. Silencing of ANG also resulted in reduced cell survival, reduced ORF73 gene expression, and lytic gene activation in BCBL-1 and TIVE-LTC cells and during de novo infection. Taken together, these studies suggest that KSHV has evolved to exploit ANG for its advantage via a so-far-unexplored PLC-? pathway for maintaining its latency.

Sadagopan, Sathish; Valiya Veettil, Mohanan; Paudel, Nitika; Bottero, Virginie; Chandran, Bala

2011-01-01

316

Role of Leishmania (Leishmania) chagasi amastigote cysteine protease in intracellular parasite survival: studies by gene disruption and antisense mRNA inhibition  

PubMed Central

Background The parasitic protozoa belonging to Leishmania (L.) donovani complex possess abundant, developmentally regulated cathepsin L-like cysteine proteases. Previously, we have reported the isolation of cysteine protease gene, Ldccys2 from Leishmania (L.) chagasi. Here, we have further characterized this cysteine protease gene and demonstrated its role during infection and survival of Leishmania (L.) chagasi within the U937 macrophage cells. Results The amastigote specific Ldccys2 genes of L. (L.) chagasi and L. (L.) donovani have identical gene organization, as determined by southern blots. In vivo expression analyses by Northern blots showed that Ldccys2 is amastigote specific. Western blot using anti-Ldccys2 antibody confirmed the amastigote specific protein expression. Recombinant expression of Ldccys2, a 30 kDA protein, was functionally active in a gelatin assay. Results from Ldccys2 heterozygous knockout mutants showed its role during macrophage infection and in intra-macrophage survival of the parasites. Since attempts to generate null mutants failed, we used antisense RNA inhibition to regulate Ldcccys2 gene expression. Not surprisingly, the results from antisense studies further confirmed the results from heterozygous knockout mutants, reiterating the importance of amastigote specific cysteine proteases in Leishmania infection and pathogenesis. Conclusions The study shows that Ldccys2 is a developmentally regulated gene and that Ldccys2 is expressed only in infectious amastigote stages of the parasite. The collective results from both the heterozygous knockout mutants and antisense mRNA inhibition studies shows that Ldccys2 helps in infection and survival of L. (L.) chagasi amastigotes within the macrophage cells. Finally, antisense RNA technique can be used as an alternate approach to gene knockout, for silencing gene expression in L. (L.) chagasi, especially in cases such as this, where a null mutant cannot be achieved by homologous recombination.

Mundodi, Vasanthakrishna; Kucknoor, Ashwini S; Gedamu, Lashitew

2005-01-01

317

The Caenorhabditis elegans rhy-1 Gene Inhibits HIF-1 Hypoxia-Inducible Factor Activity in a Negative Feedback Loop That Does Not Include vhl-1  

PubMed Central

Hypoxia-inducible factor (HIF) transcription factors implement essential changes in gene expression that enable animals to adapt to low oxygen (hypoxia). The stability of the C. elegans HIF-1 protein is controlled by the evolutionarily conserved EGL-9/VHL-1 pathway for oxygen-dependent degradation. Here, we describe vhl-1-independent pathways that attenuate HIF-1 transcriptional activity in C. elegans. First, the expression of HIF-1 target genes is markedly higher in egl-9 mutants than in vhl-1 mutants. We show that HIF-1 protein levels are similar in animals carrying strong loss-of-function mutations in either egl-9 or vhl-1. We conclude that EGL-9 inhibits HIF-1 activity, as well as HIF-1 stability. Second, we identify the rhy-1 gene and show that it acts in a novel negative feedback loop to inhibit expression of HIF-1 target genes. rhy-1 encodes a multi-pass transmembrane protein. Although loss-of-function mutations in rhy-1 cause relatively modest increases in hif-1 mRNA and HIF-1 protein expression, some HIF-1 target genes are expressed at higher levels in rhy-1 mutants than in vhl-1 mutants. Animals lacking both vhl-1 and rhy-1 function have a more severe phenotype than either single mutant. Collectively, these data support models in which RHY-1 and EGL-9 function in VHL-1-independent pathway(s) to repress HIF-1 transcriptional activity.

Shen, Chuan; Shao, Zhiyong; Powell-Coffman, Jo Anne

2006-01-01

318

Microsomal Triglyceride Transfer Protein Inhibition Induces Endoplasmic Reticulum Stress and Increases Gene Transcription via Ire1?/cJun to Enhance Plasma ALT/AST*  

PubMed Central

Microsomal triglyceride transfer protein (MTP) is a target to reduce plasma lipids because of its indispensable role in triglyceride-rich lipoprotein biosynthesis. MTP inhibition in Western diet fed mice decreased plasma triglycerides/cholesterol, whereas increasing plasma alanine/aspartate aminotransferases (ALT/AST) and hepatic triglycerides/free cholesterol. Free cholesterol accumulated in the endoplasmic reticulum (ER) and mitochondria resulting in ER and oxidative stresses. Mechanistic studies revealed that MTP inhibition increased transcription of the GPT/GOT1 genes through up-regulation of the IRE1?/cJun pathway leading to increased synthesis and release of ALT1/AST1. Thus, transcriptional up-regulation of GPT/GOT1 genes is a major mechanism, in response to ER stress, elevating plasma transaminases. Increases in plasma and tissue transaminases might represent a normal response to stress for survival.

Josekutty, Joby; Iqbal, Jahangir; Iwawaki, Takao; Kohno, Kenji; Hussain, M. Mahmood

2013-01-01

319

BAY 87-2243, a highly potent and selective inhibitor of hypoxia-induced gene activation has antitumor activities by inhibition of mitochondrial complex I  

PubMed Central

The activation of the transcription factor hypoxia-inducible factor-1 (HIF-1) plays an essential role in tumor development, tumor progression, and resistance to chemo- and radiotherapy. In order to identify compounds targeting the HIF pathway, a small molecule library was screened using a luciferase-driven HIF-1 reporter cell line under hypoxia. The high-throughput screening led to the identification of a class of aminoalkyl-substituted compounds that inhibited hypoxia-induced HIF-1 target gene expression in human lung cancer cell lines at low nanomolar concentrations. Lead structure BAY 87-2243 was found to inhibit HIF-1? and HIF-2? protein accumulation under hypoxic conditions in non-small cell lung cancer (NSCLC) cell line H460 but had no effect on HIF-1? protein levels induced by the hypoxia mimetics desferrioxamine or cobalt chloride. BAY 87-2243 had no effect on HIF target gene expression levels in RCC4 cells lacking Von Hippel–Lindau (VHL) activity nor did the compound affect the activity of HIF prolyl hydroxylase-2. Antitumor activity of BAY 87-2243, suppression of HIF-1? protein levels, and reduction of HIF-1 target gene expression in vivo were demonstrated in a H460 xenograft model. BAY 87-2243 did not inhibit cell proliferation under standard conditions. However under glucose depletion, a condition favoring mitochondrial ATP generation as energy source, BAY 87-2243 inhibited cell proliferation in the nanomolar range. Further experiments revealed that BAY 87-2243 inhibits mitochondrial complex I activity but has no effect on complex III activity. Interference with mitochondrial function to reduce hypoxia-induced HIF-1 activity in tumors might be an interesting therapeutic approach to overcome chemo- and radiotherapy-resistance of hypoxic tumors.

Ellinghaus, Peter; Heisler, Iring; Unterschemmann, Kerstin; Haerter, Michael; Beck, Hartmut; Greschat, Susanne; Ehrmann, Alexander; Summer, Holger; Flamme, Ingo; Oehme, Felix; Thierauch, Karlheinz; Michels, Martin; Hess-Stumpp, Holger; Ziegelbauer, Karl

2013-01-01

320

mda-7/IL-24 expression inhibits breast cancer through upregulation of growth arrest-specific gene 3 (gas3) and disruption of ?1 integrin function.  

PubMed

Melanoma differentiation-associated gene (MDA)-7)/interleukin (IL)-24, a member of the IL-10 family of cytokines, inhibits growth of various human cancer cells, yet the underlying mechanism is largely unknown. Here, we report that mda-7/IL-24 efficiently suppresses the development of rat mammary tumors in vivo. Microarray analysis for genes differentially expressed in rat mammary tumor cells overexpressing MDA-7/IL-24 compared with those that do not express this cytokine identified growth arrest-specific gene-3 (gas3) as a target for mda-7/IL-24. Upregulation of gas3 by mda-7/IL-24 was STAT3 dependent. Induction of gas3 inhibited attachment and proliferation of tumor cells in vitro and in vivo by inhibiting the interaction of ?1 integrin with fibronectin. A mutated GAS3, which is unable to bind ?1 integrin, was also unable to inhibit fibronectin-mediated attachment and cell growth both in adherent and suspension cultures, suggesting that GAS3 exerts its effects through interaction with and regulation of ?1 integrin. Thus, mda-7/IL-24 inhibits breast cancer growth, at least in part, through upregulation of GAS3 and disruption of ?1 integrin function. Importantly, the expression of the mda-7/IL-24 receptor, IL-20R1, is highly correlated with GAS3 expression in human breast cancer (P = 1.02 × 10(-9)), and the incidence of metastases is significantly reduced in patients with HER2(+) breast cancer expressing high-levels of IL-20R1. Together, our results identify a novel MDA-7/IL-24-GAS3-?1integrin-fibronectin signaling pathway that suppresses breast cancer growth and can be targeted for therapy. PMID:23468528

Li, You-Jun; Liu, Guodong; Li, Yanmei; Vecchiarelli-Federico, Laura M; Liu, Jeff C; Zacksenhaus, Eldad; Shan, Sze W; Yang, Burton B; Li, Qi; Dash, Rupesh; Fisher, Paul B; Archer, Michael C; Ben-David, Yaacov

2013-06-01

321

Antigene Oligomers Inhibit Transcription.  

National Technical Information Service (NTIS)

Transcription of a gene in a mammalian cell is methylase-independently inhibited by contacting the cell with a nucleic acid oligomer of 12-28 bases complementary for a partially single-stranded target genomic sequence of the gene.

B. A. Janowski D. R. Corey

2006-01-01

322

5?-Triphosphate-Short Interfering RNA: Potent Inhibition of Influenza A Virus Infection by Gene Silencing and RIG-I Activation  

PubMed Central

Limited protection of current vaccines and antiviral drugs against influenza A virus infection underscores the urgent need for development of novel anti-influenza virus interventions. While short interfering RNA (siRNA) has been shown to be able to inhibit influenza virus infection in a gene-specific manner, activation of the retinoic acid-inducible gene I protein (RIG-I) pathway has an antiviral effect in a non-gene-specific mode. In this study, we designed and tested the anti-influenza virus effect of a short double-stranded RNA, designated 3p-mNP1496-siRNA, that possesses dual functions: an siRNA-targeting influenza NP gene and an agonist for RIG-I activation. This double-stranded siRNA possesses a triphosphate group at the 5? end of the sense strand and is blunt ended. Our study showed that 3p-mNP1496-siRNA could potently inhibit influenza A virus infection both in cell culture and in mice. The strong inhibition effect was attributed to its siRNA function as well as its ability to activate the RIG-I pathway. To the best of our knowledge, this is the first report that the combination of siRNA and RIG-I pathway activation can synergistically inhibit influenza A virus infection. The development of such dual functional RNA molecules will greatly contribute to the arsenal of tools to combat not only influenza viruses but also other important viral pathogens.

Lin, Li; Liu, Qiang; Berube, Nathalie; Detmer, Susan

2012-01-01

323

Inhibition of VEGF mRNA by 2?-O,4??C?Ethylene-Bridgednucleicacids (ENA®) Antisense Oligonucleotides and Their Influence on Off-Target Gene Expressions  

Microsoft Academic Search

We investigated 2 ?-O,4 ?-C-ethylene-bridged nucleic acids (ENA) antisense oligonucleotides (AONs) for vascular endothelial growth factor (VEGF) in human lung carcinoma A549 cells. An ENA\\/DNA gapmer AON with RNase H-mediated activity was virtually stable in rat plasma and exhibited more than 90% inhibition of VEGF mRNA production. Moreover, 22 genes that are likely to bind to the AON were found

Koji Morita; Koji Yamate; Shin-ichi Kurakata; Koji Abe; Kenji Watanabe; Makoto Koizumi; Takeshi Imanishi

2006-01-01

324

Induction of Mammary Differentiation by Mammary-derived Growth Inhibitor related Gene That Interacts with an v-3 Fatty Acid on Growth Inhibition of Breast Cancer Cells1  

Microsoft Academic Search

We previously identified and characterized a novel tumor growth inhibitor and a fatty acid-binding protein in human mammary gland and named it the mammary-derived growth inhibitor-related gene (MRG). Here, the effects of MRG on mammary gland differentiation and its interaction with v-3 polyunsaturated fatty acids (v-3 PUFAs) on growth inhibition were investigated. MRG protein expression was associated with human mammary

Mingsheng Wang; Yiliang E. Liu; Jian Ni; Banu Aygun; Itzhak D. Goldberg; Y. Eric Shi

2000-01-01

325

Regulated Expression of the Escherichia coli lepB Gene as a Tool for Cellular Testing of Antimicrobial Compounds That Inhibit Signal Peptidase I In Vitro  

PubMed Central

Escherichia coli under-expressing lepB was utilized to test cellular inhibition of signal peptidase I (SPase). For the construction of a lepB regulatable strain, the E. coli lepB gene was cloned into pBAD, with expression dependent on l-arabinose. The chromosomal copy of lepB was replaced with a kanamycin resistance gene, which was subsequently removed. SPase production by the lepB regulatable strain in the presence of various concentrations of l-arabinose was monitored by Western blot analysis. At lower arabinose concentrations growth proceeded more slowly, possibly due to a decrease of SPase levels in the cells. A penem SPase inhibitor with little antimicrobial activity against E. coli when tested at 100 ?M was utilized to validate the cell-based system. Under-expression of lepB sensitized the cells to penem, with complete growth inhibition observed at 10 to 30 ?M. Growth was rescued by increasing the SPase levels. The cell-based assay was used to test cellular inhibition of SPase by compounds that inhibit the enzyme in vitro. MD1, MD2, and MD3 are SPase inhibitors with antimicrobial activity against Staphylococcus aureus, although they do not inhibit growth of E. coli. MD1 presented the best spectrum of antimicrobial activity. Both MD1 and MD2 prevented growth of E. coli under-expressing lepB in the presence of polymyxin B nonapeptide, with growth rescue observed when wild-type levels of SPase were produced. MD3 and MD4, a reactive analog of MD3, inhibited growth of E. coli under-expressing lepB. However, growth rescue in the presence of these compounds following increased lepB expression was observed only after prolonged incubation.

Barbosa, Maria D. F. S.; Lin, Siqi; Markwalder, Jay A.; Mills, Jonathan A.; DeVito, Joseph A.; Teleha, Christopher A.; Garlapati, Vasudha; Liu, Charles; Thompson, Andy; Trainor, George L.; Kurilla, Michael G.; Pompliano, David L.

2002-01-01

326

Regulated expression of the Escherichia coli lepB gene as a tool for cellular testing of antimicrobial compounds that inhibit signal peptidase I in vitro.  

PubMed

Escherichia coli under-expressing lepB was utilized to test cellular inhibition of signal peptidase I (SPase). For the construction of a lepB regulatable strain, the E. coli lepB gene was cloned into pBAD, with expression dependent on L-arabinose. The chromosomal copy of lepB was replaced with a kanamycin resistance gene, which was subsequently removed. SPase production by the lepB regulatable strain in the presence of various concentrations of L-arabinose was monitored by Western blot analysis. At lower arabinose concentrations growth proceeded more slowly, possibly due to a decrease of SPase levels in the cells. A penem SPase inhibitor with little antimicrobial activity against E. coli when tested at 100 micro M was utilized to validate the cell-based system. Under-expression of lepB sensitized the cells to penem, with complete growth inhibition observed at 10 to 30 micro M. Growth was rescued by increasing the SPase levels. The cell-based assay was used to test cellular inhibition of SPase by compounds that inhibit the enzyme in vitro. MD1, MD2, and MD3 are SPase inhibitors with antimicrobial activity against Staphylococcus aureus, although they do not inhibit growth of E. coli. MD1 presented the best spectrum of antimicrobial activity. Both MD1 and MD2 prevented growth of E. coli under-expressing lepB in the presence of polymyxin B nonapeptide, with growth rescue observed when wild-type levels of SPase were produced. MD3 and MD4, a reactive analog of MD3, inhibited growth of E. coli under-expressing lepB. However, growth rescue in the presence of these compounds following increased lepB expression was observed only after prolonged incubation. PMID:12384363

Barbosa, Maria D F S; Lin, Siqi; Markwalder, Jay A; Mills, Jonathan A; DeVito, Joseph A; Teleha, Christopher A; Garlapati, Vasudha; Liu, Charles; Thompson, Andy; Trainor, George L; Kurilla, Michael G; Pompliano, David L

2002-11-01

327

Ursolic Acid Inhibits Na+/K+-ATPase Activity and Prevents TNF-?-Induced Gene Expression by Blocking Amino Acid Transport and Cellular Protein Synthesis.  

PubMed

Pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-?, induce the expression of a wide variety of genes, including intercellular adhesion molecule-1 (ICAM-1). Ursolic acid (3?-hydroxy-urs-12-en-28-oic acid) was identified to inhibit the cell-surface ICAM-1 expression induced by pro-inflammatory cytokines in human lung carcinoma A549 cells. Ursolic acid was found to inhibit the TNF-?-induced ICAM-1 protein expression almost completely, whereas the TNF-?-induced ICAM-1 mRNA expression and NF-?B signaling pathway were decreased only partially by ursolic acid. In line with these findings, ursolic acid prevented cellular protein synthesis as well as amino acid uptake, but did not obviously affect nucleoside uptake and the subsequent DNA/RNA syntheses. This inhibitory profile of ursolic acid was similar to that of the Na+/K+-ATPase inhibitor, ouabain, but not the translation inhibitor, cycloheximide. Consistent with this notion, ursolic acid was found to inhibit the catalytic activity of Na+/K+-ATPase. Thus, our present study reveals a novel molecular mechanism in which ursolic acid inhibits Na+/K+-ATPase activity and prevents the TNF-?-induced gene expression by blocking amino acid transport and cellular protein synthesis. PMID:24970122

Yokomichi, Tomonobu; Morimoto, Kyoko; Oshima, Nana; Yamada, Yuriko; Fu, Liwei; Taketani, Shigeru; Ando, Masayoshi; Kataoka, Takao

2011-01-01

328

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

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

Muller-Rober, B; Sonnewald, U; Willmitzer, L

1992-01-01

329

Ursolic Acid Inhibits Na+/K+-ATPase Activity and Prevents TNF-?-Induced Gene Expression by Blocking Amino Acid Transport and Cellular Protein Synthesis  

PubMed Central

Pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-?, induce the expression of a wide variety of genes, including intercellular adhesion molecule-1 (ICAM-1). Ursolic acid (3?-hydroxy-urs-12-en-28-oic acid) was identified to inhibit the cell-surface ICAM-1 expression induced by pro-inflammatory cytokines in human lung carcinoma A549 cells. Ursolic acid was found to inhibit the TNF-?-induced ICAM-1 protein expression almost completely, whereas the TNF-?-induced ICAM-1 mRNA expression and NF-?B signaling pathway were decreased only partially by ursolic acid. In line with these findings, ursolic acid prevented cellular protein synthesis as well as amino acid uptake, but did not obviously affect nucleoside uptake and the subsequent DNA/RNA syntheses. This inhibitory profile of ursolic acid was similar to that of the Na+/K+-ATPase inhibitor, ouabain, but not the translation inhibitor, cycloheximide. Consistent with this notion, ursolic acid was found to inhibit the catalytic activity of Na+/K+-ATPase. Thus, our present study reveals a novel molecular mechanism in which ursolic acid inhibits Na+/K+-ATPase activity and prevents the TNF-?-induced gene expression by blocking amino acid transport and cellular protein synthesis.

Yokomichi, Tomonobu; Morimoto, Kyoko; Oshima, Nana; Yamada, Yuriko; Fu, Liwei; Taketani, Shigeru; Ando, Masayoshi; Kataoka, Takao

2011-01-01

330

N-Acyl-Homoserine Lactone Inhibition of Rhizobial Growth Is Mediated by Two Quorum-Sensing Genes That Regulate Plasmid Transfer  

PubMed Central

The growth of some strains of Rhizobium leguminosarum bv. viciae is inhibited by N-(3-hydroxy-7-cis tetradecenoyl)-l-homoserine lactone (3OH-C14:1-HSL), which was previously known as the small bacteriocin before its characterization as an N-acyl homoserine lactone (AHL). Tn5-induced mutants of R. leguminosarum bv. viciae resistant to 3OH-C14:1-HSL were isolated, and mutations in two genes were identified. These genes, bisR and triR, which both encode LuxR-type regulators required for plasmid transfer, were found downstream of an operon containing trb genes involved in the transfer of the symbiotic plasmid pRL1JI. The first gene in this operon is traI, which encodes an AHL synthase, and the trbBCDEJKLFGHI genes were found between traI and bisR. Mutations in bisR, triR, traI, or trbL blocked plasmid transfer. Using gene fusions, it was demonstrated that bisR regulates triR in response to the presence of 3OH-C14:1-HSL. In turn, triR is then required for the induction of the traI-trb operon required for plasmid transfer. bisR also represses expression of cinI, which is chromosomally located and determines the level of production of 3OH-C14:1-HSL. The cloned bisR and triR genes conferred 3OH-C14:1-HSL sensitivity to strains of R. leguminosarum bv. viciae normally resistant to this AHL. Furthermore, bisR and triR made Agrobacterium tumefaciens sensitive to R. leguminosarum bv. viciae strains producing 3OH-C14:1-HSL. Analysis of patterns of growth inhibition using mutant strains and synthetic AHLs revealed that maximal growth inhibition required, in addition to 3OH-C14:1-HSL, the presence of other AHLs such as N-octanoyl-l-homoserine lactone and/or N-(3-oxo-octanoyl)-l-homoserine lactone. In an attempt to identify the causes of growth inhibition, a strain of R. leguminosarum bv. viciae carrying cloned bisR and triR was treated with an AHL extract containing 3OH-C14:1-HSL. N-terminal sequencing of induced proteins revealed one with significant similarity to the protein translation factor Ef-Ts.

Wilkinson, A.; Danino, V.; Wisniewski-Dye, F.; Lithgow, J. K.; Downie, J. A.

2002-01-01

331

Transcriptional up-regulation of antioxidant genes by PPAR{delta} inhibits angiotensin II-induced premature senescence in vascular smooth muscle cells  

SciTech Connect

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.

Kim, Hyo Jung; Ham, Sun Ah [Department of Pharmacology, Gyeongsang Institute of Health Science, Gyeongsang National University School of Medicine, Jinju (Korea, Republic of)] [Department of Pharmacology, Gyeongsang Institute of Health Science, Gyeongsang National University School of Medicine, Jinju (Korea, Republic of); Paek, Kyung Shin [Department of Nursing, Semyung University, Jechon (Korea, Republic of)] [Department of Nursing, Semyung University, Jechon (Korea, Republic of); 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 [Department of Pharmacology, Gyeongsang Institute of Health Science, Gyeongsang National University School of Medicine, Jinju (Korea, Republic of)] [Department of Pharmacology, Gyeongsang Institute of Health Science, Gyeongsang National University School of Medicine, Jinju (Korea, Republic of); Han, Chang Woo [Department of Internal Medicine, Pusan National University School of Korean Medicine, Yangsan (Korea, Republic of)] [Department of Internal Medicine, Pusan National University School of Korean Medicine, Yangsan (Korea, Republic of); Seo, Han Geuk, E-mail: hgseo@gnu.ac.kr [Department of Pharmacology, Gyeongsang Institute of Health Science, Gyeongsang National University School of Medicine, Jinju (Korea, Republic of)

2011-03-25

332

Polyamine analogues modulate gene expression by inhibiting Lysine-Specific Demethylase 1 (LSD1) and altering chromatin structure in human breast cancer cells  

PubMed Central

Aberrant epigenetic repression of gene expression has been implicated in most cancers, including breast cancer. The nuclear amine oxidase, lysine-specific demethylase 1 (LSD1) has the ability to broadly repress gene expression by removing the activating mono- and di-methylation marks at the lysine 4 residue of histone 3 (H3K4me1 & me2). Additionally, LSD1 is highly expressed in estrogen receptor ? negative (ER?) breast cancer cells. Since epigenetic marks are reversible, they make attractive therapeutic targets. Here we examine the effects of polyamine analogue inhibitors of LSD1 on gene expression, with the goal of targeting LSD1 as a therapeutic modality in the treatment of breast cancer. Exposure of the ER-negative human breast cancer cells, MDA-MB-231, to the LSD1 inhibitors, 2d or PG11144, significantly increases global H3K4me1 and H3K4me2, and alters gene expression. Array analysis indicated that 98 (75 up and 23 down) and 477 (237 up and 240 down) genes changed expression by at least 1.5-fold or greater after treatment with 2d and PG11144, respectively. The expression of twelve up-regulated genes by 2d and fourteen up-regulated genes by PG11144 was validated by quantitative RT-PCR. Quantitative chromatin immunoprecipitation (ChIP) analysis demonstrated that up-regulated gene expression by polyamine analogues is associated with increase of the active histone marks H3K4me1, H3K4me2 and H3K9ac, and decrease of the repressive histone marks H3K9me2 and H3K27me3, in the promoter regions of the relevant target genes. These data indicate that the pharmacologic inhibition of LSD1 can effectively alter gene expression and that this therapeutic strategy has potential.

Zhu, Qingsong; Huang, Yi; Marton, Laurence J.; Woster, Patrick M.; Davidson, Nancy E.; Casero, Robert A.

2011-01-01

333

Oligogalacturonides Prevent Rhizogenesis in rolB-Transformed Tobacco Explants by Inhibiting Auxin-Induced Expression of the rolB Gene.  

PubMed Central

Oligogalacturonides elicit several defense responses and regulate different aspects of growth and development in plants. Many of the development-related effects of oligogalacturonides appear to be amenable to an auxin antagonist activity of these oligosaccharins. To clarify the role of oligogalacturonides in antagonizing auxin, we analyzed their effect on root formation in leaf explants of tobacco harboring the plant oncogene rolB. We show here that oligogalacturonides are capable of inhibiting root morphogenesis driven by rolB in transgenic leaf explants when this process requires exogenous auxin. Because rolB expression is induced by auxin and dramatically alters the response to this hormone in transformed plant cells, the inhibiting effect of oligogalacturonides could be exerted on the induction of rolB and/or at some other auxin-requiring step(s) in rhizogenesis. We show that oligogalacturonides antagonize auxin primarily because they strongly inhibit auxin-regulated transcriptional activation of a rolB-[beta]-glucuronidase gene fusion in both leaf explants and cultured leaf protoplasts. In contrast, oligogalacturonides do not inhibit rhizogenesis when rolB transcriptional activation is made independent of auxin, as shown by the lack of inhibition of root formation in leaf explants containing rolB driven by a tetracycline-inducible promoter.

Bellincampi, D; Cardarelli, M; Zaghi, D; Serino, G; Salvi, G; Gatz, C; Cervone, F; Altamura, MM; Costantino, P; Lorenzo, GD

1996-01-01

334

The major volatile compound 2-phenylethanol from the biocontrol yeast, Pichia anomala, inhibits growth and expression of aflatoxin biosynthetic genes of Aspergillus flavus.  

PubMed

Aspergillus flavus is a ubiquitous saprophyte that is able to produce the most potent natural carcinogenic compound known as aflatoxin B1 (AFB1). This toxin frequently contaminates crops including corn, cotton, peanuts, and tree nuts causing substantial economic loss worldwide. Consequently, more than 100 countries have strict regulations limiting AFB1 in foodstuffs and feedstuffs. Plants and microbes are able to produce volatile compounds that act as a defense mechanism against other organisms. Pichia anomala strain WRL-076 is a biocontrol yeast currently being tested to reduce AF contamination of tree nuts in California. We used the SPME-GC/MS analysis and identified the major volatile compound produced by this strain to be 2-phenylethanol (2-PE). It inhibited spore germination and AF production of A. flavus. Inhibition of AF formation by 2-PE was correlated with significant down regulation of clustering AF biosynthesis genes as evidenced by several to greater than 10,000-fold decrease in gene expression. In a time-course analysis we found that 2-PE also altered the expression patterns of chromatin modifying genes, MYST1, MYST2, MYST3, gcn5, hdaA and rpdA. The biocontrol capacity of P. anomala can be attributed to the production of 2-PE, which affects spore germination, growth, toxin production, and gene expression in A. flavus. PMID:24504634

Hua, Sui Sheng T; Beck, John J; Sarreal, Siov Bouy L; Gee, Wai

2014-05-01

335

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

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.

Yin Huquan [College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, San 56-1, Sillim-dong, Gwanak-gu, Seoul 151-742 (Korea, Republic of); Kim, Youn-Chul [College of Pharmacy, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of); Chung, Young-Suk; Kim, Young-Chul; Shin, Young-Kee [College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, San 56-1, Sillim-dong, Gwanak-gu, Seoul 151-742 (Korea, Republic of); Lee, Byung-Hoon [College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, San 56-1, Sillim-dong, Gwanak-gu, Seoul 151-742 (Korea, Republic of)], E-mail: lee@snu.ac.kr

2009-04-01

336

Arrest of G1-S Progression by the p53-Inducible Gene PC3 Is Rb Dependent and Relies on the Inhibition of Cyclin D1 Transcription  

PubMed Central

The p53-inducible gene PC3 (TIS21, BTG2) is endowed with antiproliferative activity. Here we report that expression of PC3 in cycling cells induced accumulation of hypophosphorylated, growth-inhibitory forms of pRb and led to G1 arrest. This latter was not observed in cells with genetic disruption of the Rb gene, indicating that the PC3-mediated G1 arrest was Rb dependent. Furthermore, (i) the arrest of G1-S transition exerted by PC3 was completely rescued by coexpression of cyclin D1 but not by that of cyclin A or E; (ii) expression of PC3 caused a significant down-regulation of cyclin D1 protein levels, also in Rb-defective cells, accompanied by inhibition of CDK4 activity in vivo; and (iii) the removal from the PC3 molecule of residues 50 to 68, a conserved domain of the PC3/BTG/Tob gene family, which we term GR, led to a loss of the inhibition of proliferation as well as of the down-regulation of cyclin D1 levels. These data point to cyclin D1 down-regulation as the main factor responsible for the growth inhibition by PC3. Such an effect was associated with a decrease of cyclin D1 transcript and of cyclin D1 promoter activity, whereas no effect of PC3 was observed on cyclin D1 protein stability. Taken together, these findings indicate that PC3 impairs G1-S transition by inhibiting pRb function in consequence of a reduction of cyclin D1 levels and that PC3 acts, either directly or indirectly, as a transcriptional regulator of cyclin D1.

Guardavaccaro, Daniele; Corrente, Giuseppina; Covone, Francesca; Micheli, Laura; D'Agnano, Igea; Starace, Giuseppe; Caruso, Maurizia; Tirone, Felice

2000-01-01

337

Structure-activity relationship studies of naphthol AS-E and its derivatives as anticancer agents by inhibiting CREB-mediated gene transcription  

PubMed Central

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.

Li, Bingbing X.; Yamanaka, Kinrin; Xiao, Xiangshu

2012-01-01

338

Thyroid hormone (T3) inhibits ciprofibrate-induced transcription of genes encoding beta-oxidation enzymes: cross talk between peroxisome proliferator and T3 signaling pathways.  

PubMed Central

Peroxisome proliferators cause rapid and coordinated transcriptional activation of genes encoding peroxisomal beta-oxidation system enzymes by activating peroxisome proliferator-activated receptor (PPAR) isoform(s). Since the thyroid hormone (T3; 3,3',5-triiodothyronine) receptor (TR), another member of the nuclear hormone receptor superfamily, regulates a subset of fatty acid metabolism genes shared with PPAR, we examined the possibility of interplay between peroxisome proliferator and T3 signaling pathways. T3 inhibited ciprofibrate-induced luciferase activity as well as the endogenous peroxisomal beta-oxidation enzymes in transgenic mice carrying a 3.2-kb 5'-flanking region of the rat peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase gene fused to the coding region of luciferase. Transfection assays in hepatoma H4-II-E-C3 and CV-1 cells indicated that this inhibition is mediated by TR in a ligand-dependent fashion. Gel shift assays revealed that modulation of PPAR action by TR occurs through titration of limiting amounts of retinoid X receptor (RXR) required for PPAR activation. Increasing amounts of RXR partially reversed the inhibition in a reciprocal manner; PPAR also inhibited TR activation. Results with heterodimerization-deficient TR and PPAR mutants further confirmed that interaction between PPAR and TR signaling systems is indirect. These results suggest that a convergence of the peroxisome proliferator and T3 signaling pathways occurs through their common interaction with the heterodimeric partner RXR. Images Fig. 1 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7

Chu, R; Madison, L D; Lin, Y; Kopp, P; Rao, M S; Jameson, J L; Reddy, J K

1995-01-01

339

Caprylic acid and medium-chain triglycerides inhibit IL-8 gene transcription in Caco-2 cells: comparison with the potent histone deacetylase inhibitor trichostatin A.  

PubMed

1. Medium-chain triglyceride (MCT) is often administered to patients with Crohn's disease (CD) or short-bowel syndrome. However, little is known about the effects of medium-chain fatty acids (MCFAs) and MCT on intestinal inflammation. In this study we examined whether caprylic acid, one of the MCFAs, and MCT suppress IL-8 secretion by differentiated Caco-2 cells. 2. We found for the first time that caprylic acid and MCT suppress IL-8 secretion by Caco-2 cells at the transcriptional level when precultured together for 24 h. We also tried to clarify the mechanism of IL-8 gene inhibition by examining the activation of NF-kappaB and other transcription factors by electrophoretic mobility shift assay (EMSA), and found that caprylic acid did not modulate their activation. 3. The result of dual-luciferase assay using Caco-2 cells transfected with IL-8 promoter/luciferase reporter plasmid revealed that caprylic acid inhibited the activation of IL-8 promoter. 4. Similar results were observed when cells were precultured with the well-known potent histone deacetylase inhibitor trichostatin A (TSA). 5. We examined the state of H4 acetylation in IL-8 promoter using the technique known as chromatin immunoprecipitation (Chr-IP). TSA rapidly induced H4 acetylation in IL-8 promoter chromatin, whereas caprylic acid did not. These results suggest that the inhibition of IL-8 gene transcription induced by caprylic acid and TSA does not necessarily require the marked suppression of transcription factors, and the mechanism of inhibition of IL-8 gene transcription may be different between caprylic acid and TSA. PMID:12010777

Hoshimoto, Aihiro; Suzuki, Yasuo; Katsuno, Tatsuro; Nakajima, Hiroshi; Saito, Yasushi

2002-05-01

340

The UL4 protein of equine herpesvirus 1 is not essential for replication or pathogenesis and inhibits gene expression controlled by viral and heterologous promoters  

PubMed Central

Defective interfering particles (DIP) of equine herpesvirus 1 (EHV-1) inhibit standard virus replication and mediate persistent infection. The DIP genome is comprised of only three genes: UL3, UL4, and a hybrid gene composed of portions of the IR4 (EICP22) and UL5 (EICP27) genes. The hybrid gene is important for DIP interference, but the function(s) of the UL3 and UL4 genes are unknown. Here, we show that UL4 is an early gene activated solely by the immediate early protein. The UL4 protein (UL4P) was detected at 4 hours post-infection, was localized throughout the nucleus and cytoplasm, and was not present in purified virions. EHV-1 lacking UL4P expression was infectious and displayed cell tropism and pathogenic properties in the mouse model similar to those of parental and revertant viruses. Reporter assays demonstrated that the UL4P has a broad inhibitory function, suggesting a potential role in establishing and/or maintaining DIP-mediated persistent infection.

Charvat, Robert A.; Breitenbach, Jonathan E.; Ahn, ByungChul; Zhang, Yunfei; O'Callaghan, Dennis J.

2011-01-01

341

Activity of the Agrobacterium Ti plasmid conjugal transfer regulator TraR is inhibited by the product of the traM gene.  

PubMed Central

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.

Fuqua, C; Burbea, M; Winans, S C

1995-01-01

342

Inhibition of beta-defensin gene expression in airway epithelial cells by low doses of residual oil fly ash is mediated by vanadium.  

PubMed

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

Klein-Patel, Marcia E; Diamond, Gill; Boniotto, Michele; Saad, Sherif; Ryan, Lisa K

2006-07-01

343

Leukemia/lymphoma-related factor, a POZ domain-containing transcriptional repressor, interacts with histone deacetylase-1 and inhibits cartilage oligomeric matrix protein gene expression and chondrogenesis.  

PubMed

Mutations in the human cartilage oligomeric matrix protein (COMP) gene have been linked to the development of pseudoachondroplasia and multiple epiphyseal dysplasia. We previously cloned the promoter region of the COMP gene and delineated a minimal negative regulatory element (NRE) that is both necessary and sufficient to repress its promoter (Issack, P. S., Fang, C. H., Leslie, M. P., and Di Cesare, P. E. (2000) J. Orthop. Res. 18, 345-350; Issack, P. S., Liu, C. J., Prazak, L., and Di Cesare, P. E. (2004) J. Orthop. Res. 22, 751-758). In this study, a yeast one-hybrid screen for proteins that associate with the NRE led to the identification of the leukemia/lymphoma-related factor (LRF), a transcriptional repressor that contains a POZ (poxvirus zinc finger) domain, as an NRE-binding protein. LRF bound directly to the NRE both in vitro and in living cells. Nine nucleotides (GAGGGTCCC) in the 30-bp NRE are essential for binding to LRF. LRF showed dose-dependent inhibition of COMP-specific reporter gene activity, and exogenous overexpression of LRF repressed COMP gene expression in both rat chondrosarcoma cells and bone morphogenetic protein-2-treated C3H10T1/2 progenitor cells. In addition, LRF also inhibited bone morphogenetic protein-2-induced chondrogenesis in high density micromass cultures of C3H10T1/2 cells, as evidenced by lack of expression of other chondrocytic markers, such as aggrecan and collagen types II, IX, X, and XI, and by Alcian blue staining. LRF associated with histone deacetylase-1 (HDAC1), and experiments utilizing the HDAC inhibitor trichostatin A revealed that LRF-mediated repression requires deacetylase activity. LRF is the first transcription factor found to bind directly to the COMP gene promoter, to recruit HDAC1, and to regulate both COMP gene expression and chondrogenic differentiation. PMID:15337766

Liu, Chuan-ju; Prazak, Lisa; Fajardo, Marc; Yu, Shuang; Tyagi, Neetu; Di Cesare, Paul E

2004-11-01

344

c-Jun N-terminal kinase-interacting protein 1 inhibits gene expression in response to hypertrophic agonists in neonatal rat ventricular myocytes.  

PubMed Central

G(q)-coupled receptor agonists, such as endothelin-1 (ET-1) and phenylephrine (PE), initiate a hypertrophic response in cardiac myocytes that is characterized by increased expression of atrial natriuretic factor (ANF), beta-myosin heavy chain (beta-MHC), skeletal muscle alpha-actin (SkalphaA) and ventricular myosin light chain-2 (vMLC2). ET-1 and PE activate both the extracellular signal-regulated kinases and c-Jun N-terminal kinases (JNKs) in cardiac myocytes, but the extent to which each contributes to the hypertrophic response is uncertain. Here we have used the JNK-binding domain of JNK-interacting protein 1 (JIP-1), a cytosolic scaffold protein that binds to JNK and inhibits its signalling when overexpressed, to assess the contribution of JNK activation to the hypertrophic response. Expression of JIP-1 inhibited the increase in ANF, beta-MHC, SkalphaA and vMLC2 reporter gene expression in response to ET-1 (by 45-86%) and PE (by 56-60%). However, activation of these reporter genes by PMA, which does not activate JNK significantly in myocytes, was much less affected by overexpression of JIP-1. JIP-1 also failed to inhibit reporter gene activation in response to constitutively active Ras or Raf, but attenuated reporter gene activation induced by a constitutively active mutant of mitogen-activated protein kinase kinase kinase 1 (MEKK1), an upstream kinase that preferentially activates JNKs, by 50%. Overexpression of JIP-1 also significantly reduced the increase in cell area in response to PE from 63% to 56%, but had no effect on the increase in cell size in response to ET-1 (38%). These results suggest that activation of the JNK pathway contributes to the transcriptional and morphological responses to G(q) receptor-coupled hypertrophic agonists.

Finn, S G; Dickens, M; Fuller, S J

2001-01-01

345

N-Octanoyl Dopamine Inhibits the Expression of a Subset of ?B Regulated Genes: Potential Role of p65 Ser276 Phosphorylation  

PubMed Central

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.

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

346

Penta O-galloyl-?- d-glucose inhibits phorbol myristate acetate-induced intereukin-8 gene expression in human monocytic U937 cells through its inactivation of nuclear factor-?B  

Microsoft Academic Search

We investigated the effects of the gallotannin penta-O-galloyl-?-d-glucose (PGG) on interleukin (IL)-8 gene expression and nuclear factor (NF)-?B activation. PGG inhibited IL-8 production and gene expression in human monocytic U937 cells stimulated with phorbol myristate acetate (PMA), as measured by enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction analysis, respectively. PGG also inhibited PMA-mediated NF-?B activation, as measured by electromobility

G. S. Oh; H. O. Pae; B. M. Choi; H. S. Lee; I. K. Kim; Y. G. Yun; J. D. Kim; H. T. Chung

2004-01-01

347

Growth inhibition of established B16-F10 lung metastases by sequential aerosol delivery of p53 gene and 9-nitrocamptothecin.  

PubMed

Growth inhibition of established tumor metastases in the lungs poses a difficult challenge for most clinical settings in spite of extensive multi-modality approaches. Aerosol delivery of drugs and genes holds promise for the treatment of disseminated lung metastases, since aerosol delivery can target the lungs specifically and uniformly. We previously demonstrated that aerosol delivery of dilauroylphosphatidylcholine liposome formulation of 9-nitrocamptothecin (9NC-DLPC) inhibits B16-F10 melanoma lung metastases. Aerosol delivery of polyethleneimine-p53 DNA (PEI-p53) complexes results in a similar anti-tumor effect in the B16-F10 model. In both these previous studies, the protocols were designed to inhibit development of lung metastases. In this study we demonstrate, using the B16-F10 melanoma lung metastasis model, that sequential aerosol delivery of PEI-p53 and 9NC-DLPC acts additively to inhibit growth of established B16-F10 tumor metastases in the lungs. Mice injected with B16-F10 cells and treated with a combination of 9NC-DLPC (twice weekly) and PEI-p53 (once weekly) aerosol complexes starting on day 11 after tumor inoculation, exhibited a highly significant (P < 0.01) reduction in the number of visible tumor foci as compared with untreated mice or mice treated with either single agent alone, or with a combination of 9NC and a control plasmid. There was a highly significant reduction in the tumor burden, as well as the lung weights for the 9NC and p53 combination group (P < 0.001 as compared with other groups). Moreover, the doses of p53 gene and 9NC in the combination group were reduced at least two-fold as compared with our previous single agent studies, but still achieved significant tumor inhibition. Furthermore, the sequential aerosol delivery of p53 and 9NC lead to a 30-40% increase in the mean survival time of these mice, as compared with animals in different control groups. The data suggest that the combination of 9NC and p53 gene delivered by aerosol is an attractive strategy for growth inhibition of established tumor metastases in the lungs. PMID:11938455

Gautam, A; Waldrep, J C; Densmore, C L; Koshkina, N; Melton, S; Roberts, L; Gilbert, B; Knight, V

2002-03-01

348

Hydrogen sulfide inhibits opioid withdrawal-induced pain sensitization in rats by down-regulation of spinal calcitonin gene-related peptide expression in the spine.  

PubMed

Hyperalgesia often occurs in opioid-induced withdrawal syndrome. In the present study, we found that three hourly injections of DAMGO (a ?-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) ?, 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

Yang, Hai-Yu; Wu, Zhi-Yuan; Bian, Jin-Song

2014-09-01

349

Interaction between SNPs in the RXRA and near ANGPTL3 gene region inhibits apoB reduction after statin-fenofibric acid therapy in individuals with mixed dyslipidemia.  

PubMed

The mixed dyslipidemia phenotype is characterized by elevated triglycerides (TG), low HDL cholesterol (HDL-C), increased ApoB levels, and premature coronary atherosclerosis. Fibrate-statin combination therapy reduces ApoB levels and coronary events in the mixed dyslipidemia population. We sought to identify gene-gene interactions that affect ApoB response to statin-fenofibric acid therapy in the mixed dyslipidemia population. Using a predefined subset of single-nucleotide polymorphisms (SNPs) that were previously associated with TG, VLDL, or HDL-C, we applied gene-gene interaction testing in a randomized, double-blind, clinical trial examining the response to fenofibric acid (FNA) and its combination with statin in 1,865 individuals with mixed dyslipidemia. Of 11,783 possible SNP pairs examined, we detected a single significant interaction between rs12130333, located within the ANGPTL3 gene region, and rs4240705, within the RXRA gene, on ApoB reduction after statin-FNA therapy (P = 4.0 × 10(-6)). ApoB response to therapy gradually reduced with the increasing number of T alleles in the rs12130333 but only in the presence of the GG genotype of rs4240705. Individuals doubly homozygous for the minor alleles at rs12130333 and rs4240705 showed a paradoxical increase of 1.8% in ApoB levels after FNA-statin combination therapy. No gene-gene interaction was identified other than an interaction between SNPs in the ANGPTL3 and RXRA regions, which results in the inhibition of ApoB reduction in response to statin-FNA therapy. Further study is required to examine the clinical applicability of this genetic interaction and its effect on coronary events. PMID:22896670

Ma, Li; Ballantyne, Christie M; Belmont, John W; Keinan, Alon; Brautbar, Ariel

2012-11-01

350

Inhibition of foreign body giant cell formation by 4- hexylresorcinol through suppression of diacylglycerol kinase delta gene expression.  

PubMed

Grafted macromolecules often induce granuloma formation with foreign body giant cell (FBGC) infiltration, and this is the main reason for graft failure. Diacylglycerol kinase (DAGK) is an important intracellular mediator of FBGC formation in macrophages. In this study, 4-hexylresorcinol (4HR) inhibited DAGK? in a macrophage cell line (RAW264.7 cells). As a result of DAGK-? inhibition by 4HR, FBGC formation was significantly inhibited in RAW264.7 cells. Silk fibroin is a well-known natural macromolecule, and when it is grafted into bone defects, it results in granuloma formation with massive FBGC formation. 4HR-incorporating silk graft materials displayed significant reduction of granuloma formation and increases in the extent of new bone formation in a rabbit calvarial defect model. In conclusion, 4HR could inhibit foreign body reaction via a DAGK-mediated pathway. PMID:25023393

Kweon, HaeYong; Kim, Seong-Gon; Choi, Je-Yong

2014-10-01

351

Inhibition of Human Cytomegalovirus DNA Maturation by a Benzimidazole Ribonucleoside Is Mediated through the UL89 Gene Product  

Microsoft Academic Search

2-Bromo-5,6-dichloro-1-b-D-ribofuranosyl benzimidazole (BDCRB) is a member of a new class of benzimid- azole ribonucleosides which inhibit human cytomegalovirus (HCMV) late in the replication cycle without inhibiting viral DNA synthesis. We show here that polygenomic concatemeric HCMV DNA does not mature to unit genome length in the presence of BDCRB. To discover the locus of action, virus resistant to BDCRB was

MARK R. UNDERWOOD; ROBERT J. HARVEY; SYLVIA C. STANAT; MARY LOU HEMPHILL; TERESA MILLER; JOHN C. DRACH; LEROY B. TOWNSEND; KAREN K. BIRON

1998-01-01

352

Activation of the unfolded protein response by 2-deoxy-D-glucose inhibits Kaposi's sarcoma-associated herpesvirus replication and gene expression.  

PubMed

Lytic replication of the Kaposi's sarcoma-associated herpesvirus (KSHV) is essential for the maintenance of both the infected state and characteristic angiogenic phenotype of Kaposi's sarcoma and thus represents a desirable therapeutic target. During the peak of herpesvirus lytic replication, viral glycoproteins are mass produced in the endoplasmic reticulum (ER). Normally, this leads to ER stress which, through an unfolded protein response (UPR), triggers phosphorylation of the ? subunit of eukaryotic initiation factor 2 (eIF2?), resulting in inhibition of protein synthesis to maintain ER and cellular homeostasis. However, in order to replicate, herpesviruses have acquired the ability to prevent eIF2? phosphorylation. Here we show that clinically achievable nontoxic doses of the glucose analog 2-deoxy-d-glucose (2-DG) stimulate ER stress, thereby shutting down eIF2? and inhibiting KSHV and murine herpesvirus 68 replication and KSHV reactivation from latency. Viral cascade genes that are involved in reactivation, including the master transactivator (RTA) gene, glycoprotein B, K8.1, and angiogenesis-regulating genes are markedly decreased with 2-DG treatment. Overall, our data suggest that activation of UPR by 2-DG elicits an early antiviral response via eIF2? inactivation, which impairs protein synthesis required to drive viral replication and oncogenesis. Thus, induction of ER stress by 2-DG provides a new antiherpesviral strategy that may be applicable to other viruses. PMID:2