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1

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

PubMed Central

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. Images Figure 1 PMID:7762554

Spritz, R A; Fukai, K; Holmes, S A; Luande, J

1995-01-01

2

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

3

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

4

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. PMID:24282555

Parkhurst, Amy; Jeffery, William R.

2013-01-01

5

In Southern Africa, Brown Oculocutaneous Albinism (BOCA) Maps to the OCA2 Locus on Chromosome 15q: P-Gene Mutations Identified  

PubMed Central

In southern Africa, brown oculocutaneous albinism (BOCA) is a distinct pigmentation phenotype. In at least two cases, it has occurred in the same families as tyrosinase-positive oculocutaneous albinism (OCA2), suggesting that it may be allelic, despite the fact that this phenotype was attributed to mutations in the TYRP1 gene in an American individual of mixed ancestry. Linkage analysis in five families mapped the BOCA locus to the same region as the OCA2 locus (maximum LOD 3.07; ?=0 using a six-marker haplotype). Mutation analysis of the human homologue of the mouse pink-eyed dilution gene (P), in 10 unrelated individuals with BOCA revealed that 9 had one copy of the 2.7-kb deletion. No other mutations were identified. Additional haplotype studies, based on closely linked markers (telomere to centromere: D15S1048, D15S1019, D15S1533, P-gene 2.7-kb deletion, D15S219, and D15S156) revealed several BOCA-associated P haplotypes. These could be divided into two core haplotypes, suggesting that a limited number of P-gene mutations give rise to this phenotype. PMID:11179026

Manga, Prashiela; Kromberg, Jennifer G. R.; Turner, Angela; Jenkins, Trefor; Ramsay, Michele

2001-01-01

6

In Southern Africa, brown oculocutaneous albinism (BOCA) maps to the OCA2 locus on chromosome 15q: P-gene mutations identified.  

PubMed

In southern Africa, brown oculocutaneous albinism (BOCA) is a distinct pigmentation phenotype. In at least two cases, it has occurred in the same families as tyrosinase-positive oculocutaneous albinism (OCA2), suggesting that it may be allelic, despite the fact that this phenotype was attributed to mutations in the TYRP1 gene in an American individual of mixed ancestry. Linkage analysis in five families mapped the BOCA locus to the same region as the OCA2 locus (maximum LOD 3.07; theta=0 using a six-marker haplotype). Mutation analysis of the human homologue of the mouse pink-eyed dilution gene (P), in 10 unrelated individuals with BOCA revealed that 9 had one copy of the 2.7-kb deletion. No other mutations were identified. Additional haplotype studies, based on closely linked markers (telomere to centromere: D15S1048, D15S1019, D15S1533, P-gene 2.7-kb deletion, D15S219, and D15S156) revealed several BOCA-associated P haplotypes. These could be divided into two core haplotypes, suggesting that a limited number of P-gene mutations give rise to this phenotype. PMID:11179026

Manga, P; Kromberg, J; Turner, A; Jenkins, T; Ramsay, M

2001-03-01

7

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

PubMed Central

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

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

2009-01-01

8

oca2 Regulation of chromatophore differentiation and number is cell type specific in zebrafish.  

PubMed

We characterized a zebrafish mutant that displays defects in melanin synthesis and in the differentiation of melanophores and iridophores of the skin and retinal pigment epithelium. Positional cloning and candidate gene sequencing link this mutation to a 410-kb region on chromosome 6, containing the oculocutaneous albinism 2 (oca2) gene. Quantification of oca2 mutant melanophores shows a reduction in the number of differentiated melanophores compared with wildtype siblings. Consistent with the analysis of mouse Oca2-deficient melanocytes, zebrafish mutant melanophores have immature melanosomes which are partially rescued following treatment with vacuolar-type ATPase inhibitor/cytoplasmic pH modifier, bafilomycin A1. Melanophore-specific gene expression is detected at the correct time and in anticipated locations. While oca2 zebrafish display unpigmented gaps on the head region of mutants 3 days post-fertilization, melanoblast quantification indicates that oca2 mutants have the correct number of melanoblasts, suggesting a differentiation defect explains the reduced melanophore number. Unlike melanophores, which are reduced in number in oca2 mutants, differentiated iridophores are present at significantly higher numbers. These data suggest distinct mechanisms for oca2 in establishing differentiated chromatophore number in developing zebrafish. PMID:24330346

Beirl, Alisha J; Linbo, Tor H; Cobb, Marea J; Cooper, Cynthia D

2014-03-01

9

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

10

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

PubMed Central

Summary Accumulation of proteins in the endoplasmic reticulum (ER) typically induces stress and initiates the unfolded protein response (UPR) to facilitate recovery. If homeostasis is not restored, apoptosis is induced. However, adaptation to chronic UPR activation can increase resistance to subsequent acute ER stress. We therefore investigated adaptive mechanisms in Oculocutaneous albinism type 2 (Oca2)-null melanocytes where UPR signaling is arrested despite continued tyrosinase accumulation leading to resistance to the chemical ER stressor thapsigargin. Although thapsigargin triggers UPR activation, instead of Perk-mediated phosphorylation of eIF2?, in Oca2-null melanocytes, eIF2? was rapidly dephosphorylated upon treatment. Dephosphorylation was mediated by the Gadd34-PP1? phosphatase complex. Gadd34-complex inhibition blocked eIF2? dephosphorylation and significantly increased Oca2-null melanocyte sensitivity to thapsigargin. Thus, Oca2-null melanocytes adapt to acute ER stress by disruption of proapoptotic Perk signaling, which promotes cell survival. This is the first study to demonstrate rapid eIF2? dephosphorylation as an adaptive mechanism to ER stress. PMID:23962237

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

2013-01-01

11

MC1R Mutations Modify the Classic Phenotype of Oculocutaneous Albinism Type 2 (OCA2)  

PubMed Central

The heterogeneous group of disorders known as oculocutaneous albinism (OCA) shares cutaneous and ocular hypopigmentation associated with common developmental abnormalities of the eye. Mutations of at least 11 loci produce this phenotype. The majority of affected individuals develop some cutaneous melanin; this is predominantly seen as yellow/blond hair, whereas fewer have brown hair. The OCA phenotype is dependent on the constitutional pigmentation background of the family, with more OCA pigmentation found in families with darker constitutional pigmentation, which indicates that other genes may modify the OCA phenotype. Sequence variation in the melanocortin-1 receptor (MC1R) gene is associated with red hair in the normal population, but red hair is unusual in OCA. We identified eight probands with OCA who had red hair at birth. Mutations in the P gene were responsible for classic phenotype of oculocutaneous albinism type 2 (OCA2) in all eight, and mutations in the MC1R gene were responsible for the red (rather than yellow/blond) hair in the six of eight who continued to have red hair after birth. This is the first demonstration of a gene modifying the OCA phenotype in humans. PMID:12876664

King, Richard A.; Willaert, Rebecca K.; Schmidt, Ramona M.; Pietsch, Jacy; Savage, Sarah; Brott, Marcia J.; Fryer, James P.; Summers, C. Gail; Oetting, William S.

2003-01-01

12

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

PubMed Central

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

Gross, J B; Wilkens, H

2013-01-01

13

An intragenic deletion of the P gene is the common mutation causing tyrosinase-positive oculocutaneous albinism in southern African Negroids.  

PubMed Central

Tyrosinase-positive oculocutaneous albinism (OCA2), an autosomal recessive disorder of the melanin biosynthetic pathway, is the most common recessive disorder occurring in southern African Bantu-speaking Negroids, with an overall prevalence of 1/3,900. The OCA2 gene, P, has been mapped to chromosome 15q11-q13, and recently alterations in the P gene have been identified in OCA2 individuals. An intragenic deletion has been described and proposed to be of African origin because of its occurrence in four unrelated African American OCA2 individuals and in two individuals, one from Zaire and the other from Cameroon. This study shows that the intragenic deletion is a common cause of OCA2 in southern African Negroids (114/146 [.78]; OCA2 chromosomes) and is associated with one common haplotype (43/55 [.78]; OCA2 chromosomes), confirming the African origin of this allele. On the basis of haplotype data, it would appear that at least seven additional, less frequent OCA2 mutations occur in this population. PMID:7887411

Stevens, G; van Beukering, J; Jenkins, T; Ramsay, M

1995-01-01

14

Inhibition of gastrin gene expression by somatostatin.  

PubMed Central

Previous studies performed in this laboratory have demonstrated somatostatin-containing cells in close proximity to gastrin cells in antral mucosa and have shown that somatostatin exerts a local regulatory effect on gastrin release. The present studies were directed to determine whether the effects of somatostatin on the antral gastrin cell involve pretranslational events. The effects of somatostatin on gastrin mRNA were determined by dot blot hybridization using a gastrin antisense RNA probe derived from human gastrin cDNA. Inclusion of somatostatin in the incubation medium caused a dose-dependent inhibition of steady-state gastrin mRNA. Conversely, when antral somatostatin was neutralized by the addition of specific somatostatin antibodies to the incubation medium, gastrin mRNA levels increased by 116 +/- 31% over control values (P less than 0.01). Northern blot hybridization of total antral RNA demonstrated a single major band with a molecular size of approximately 620 nucleotides, closely matching the predicted size of gastrin mRNA. The effect of somatostatin on the rate of gastrin gene transcription was examined using nuclear run-off transcription assays. Inclusion of antibodies to somatostatin in the incubation medium resulted in a 33.8 +/- 3.3% increase in gastrin gene transcriptional activity (P less than 0.01). These studies indicate that, in addition to its established effect on peptide release, somatostatin exerts inhibitory effects on antral gastrin cells at the pretranslational level. Although this inhibition appears to occur in part at the gene transcriptional level, the results also indicate that somatostatin may affect posttranscriptional processing of gastrin mRNA. Images PMID:2563264

Karnik, P S; Monahan, S J; Wolfe, M M

1989-01-01

15

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

Microsoft Academic Search

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 mappingof eyecolor SNPsin theintergenic region upstream of OCA2and withinthe neighboringHERC2 (hect domainand RLD2) gene. We screened an additional 92 SNPs in

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

2008-01-01

16

Antiviral effects of inhibiting host gene expression.  

PubMed

RNA interference (RNAi) has been used to probe the virus-host interface to understand the requirements for host-gene expression needed for virus replication. The availability of arrayed siRNA libraries has enabled a genome-scale, high-throughput analysis of gene pathways usurped for virus replication. Results from these and related screens have led to the discovery of new host factors that regulate virus replication. While effective delivery continues to limit development of RNAi-based drugs, RNAi-based genome discovery has led to identification of druggable targets. These validated targets enable rational development of novel antiviral drugs, including the rescue and repurposing of existing, approved drugs. Existing drugs with known cytotoxicity and mechanisms of action can potentially be re-targeted to regulate host genes and gene products needed by influenza to replicate. Drug repositioning is more cost-effective, less time-consuming, and more effective for anti-influenza virus drug discovery than traditional methods. In this chapter, a general overview of RNAi screening methods, host-gene discovery, and drug repurposing is examined with emphasis on utilizing RNAi to identify druggable genes that can be targeted for drug development or repurposing. PMID:25007848

Tripp, Ralph A; Mark Tompkins, S

2015-01-01

17

ALLOANTISERUM-INDUCED INHIBITION OF IMMUNE RESPONSE GENE PRODUCT FUNCTION  

PubMed Central

It has been previously demonstrated that alloantisera can specifically block the activation of T lymphocytes by antigens, the response to which is linked to the presence of histocompatibility (H) types against which the alloantisera are directed. Thus, strain 13 anti-2 serum can inhibit the activation of (2 x 13)F1 T lymphocytes by a DNP derivative of a copolymer of L-glutamic acid and L-lysine (DNP-GL), an antigen the response to which is controlled by a 2-linked Ir gene. It was proposed that alloantisera can inhibit T-lymphocyte antigen recognition through interference with the activity of immune response (Ir) gene products. In order to further study whether the inhibitory antibodies within the alloantisera are directed against H antigens or against the products of the Ir genes, we have examined whether the anti-2 serum can inhibit the function of an Ir gene (the L-glutamic acid and L-alanine [GA] gene), which is normally linked to strain 2 H genes when this gene occurs in an outbred animal lacking strain 2 H genes. In the majority of cases, the anti-2 serum was capable of inhibiting the in vitro proliferative response to GA of T cells derived from animals that were GA+2+, but the serum had little if any effect on the GA response of T cells from GA+2- animals. Furthermore, an antiserum prepared in strain 13 animals against the lymphoid cells of a GA+2- outbred animal was devoid of inhibitory activity on the GA response of cells from a (2 x 13)F1, while an antiserum prepared in strain 13 animals against the lymphoid cells of a GA+2+ outbred animal was capable of specifically inhibiting the response to GA. It thus appears that the inhibition of the GA response by the anti-2 serum is primarily mediated via antibodies directed toward strain 2 H antigens rather than antibodies specific for the product of the GA Ir gene. The mechanism of alloantiserum induced suppression of Ir gene function would then be by steric interference with the Ir gene product on the cell surface, rather than by direct binding to it. This conclusion implies that the products of both the H genes and the Ir genes are physically related on the cell surface. The implications of such a relationship in terms of the fluid-mosaic model of the lymphocyte surface are discussed. PMID:4591175

Shevach, Ethan M.; Green, Ira; Paul, William E.

1974-01-01

18

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. 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.

2008-01-01

19

Effect of PPARgamma inhibition on pulmonary endothelial cell gene expression: gene profiling in pulmonary hypertension.  

PubMed

Peroxisome proliferator-activated receptor type gamma (PPARgamma) is a subgroup of the PPAR transcription factor family. Recent studies indicate that loss of PPARgamma is associated with the development of pulmonary hypertension (PH). We hypothesized that the endothelial dysfunction associated with PPARgamma inhibition may play an important role in the disease process by altering cellular gene expression and signaling cascades. We utilized microarray analysis to determine if PPARgamma inhibition induced changes in gene expression in pulmonary arterial endothelial cells (PAEC). We identified 100 genes and expressed sequence tags (ESTs) that were upregulated by >1.5-fold and 21 genes and ESTs that were downregulated by >1.3-fold (P < 0.05) by PPARgamma inhibition. The upregulated genes can be broadly classified into four functional groups: cell cycle, angiogenesis, ubiquitin system, and zinc finger proteins. The genes with the highest fold change in expression: hyaluronan-mediated motility receptor (HMMR), VEGF receptor 2 (Flk-1), endothelial PAS domain protein 1 (EPAS1), basic fibroblast growth factor (FGF-2), and caveolin-1 in PAEC were validated by real time RT-PCR. We further validated the upregulation of HMMR, Flk-1, FGF2, and caveolin-1 by Western blot analysis. In keeping with the microarray results, PPARgamma inhibition led to re-entry of cell cycle at G(1)/S phase and cyclin C upregulation. PPARgamma inhibition also exacerbated VEGF-induced endothelial barrier disruption. Finally we confirmed the downregulation of PPARgamma and the upregulation of HMMR, Flk-1, FGF2, and Cav-1 proteins in the peripheral lung tissues of an ovine model of PH. In conclusion, we have identified an array of endothelial genes modulated by attenuated PPARgamma signaling that may play important roles in the development of PH. PMID:19825830

Tian, Jing; Smith, Anita; Nechtman, John; Podolsky, Robert; Aggarwal, Saurabh; Snead, Connie; Kumar, Sanjiv; Elgaish, Manal; Oishi, Peter; Göerlach, Agnes; Fratz, Sohrab; Hess, John; Catravas, John D; Verin, Alexander D; Fineman, Jeffrey R; She, Jin-Xiong; Black, Stephen M

2009-12-30

20

In silico analysis of miRNA-mediated gene regulation in OCA and OA genes.  

PubMed

Albinism is an autosomal recessive genetic disorder due to low secretion of melanin. The oculocutaneous albinism (OCA) and ocular albinism (OA) genes are responsible for melanin production and also act as a potential targets for miRNAs. The role of miRNA is to inhibit the protein synthesis partially or completely by binding with the 3'UTR of the mRNA thus regulating gene expression. In this analysis, we predicted the genetic variation that occurred in 3'UTR of the transcript which can be a reason for low melanin production thus causing albinism. The single nucleotide polymorphisms (SNPs) in 3'UTR cause more new binding sites for miRNA which binds with mRNA which leads to inhibit the translation process either partially or completely. The SNPs in the mRNA of OCA and OA genes can create new binding sites for miRNA which may control the gene expression and lead to hypopigmentation. We have developed a computational procedure to determine the SNPs in the 3'UTR region of mRNA of OCA (TYR, OCA2, TYRP1 and SLC45A2) and OA (GPR143) genes which will be a potential cause for albinism. We identified 37 SNPs in five genes that are predicted to create 87 new binding sites on mRNA, which may lead to abrogation of the translation process. Expression analysis confirms that these genes are highly expressed in skin and eye regions. It is well supported by enrichment analysis that these genes are mainly involved in eye pigmentation and melanin biosynthesis process. The network analysis also shows how the genes are interacting and expressing in a complex network. This insight provides clue to wet-lab researches to understand the expression pattern of OCA and OA genes and binding phenomenon of mRNA and miRNA upon mutation, which is responsible for inhibition of translation process at genomic levels. PMID:25060099

Kamaraj, Balu; Gopalakrishnan, Chandrasekhar; Purohit, Rituraj

2014-12-01

21

Protein inhibitor of activated STAT3 inhibits adipogenic gene expression  

SciTech Connect

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

Deng Jianbei [Department of Nutrition, CB 7461, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 (United States); Hua Kunjie [Department of Nutrition, CB 7461, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 (United States); Caveney, Erica J. [Department of Medicine, CB 7005, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 (United States); Takahashi, Nobuyuki [Department of Pathology and Laboratory Medicine, CB 7525, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 (United States); Harp, Joyce B. [Department of Nutrition, CB 7461, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 (United States)]. E-mail: jharp@unc.edu

2006-01-20

22

Opposite Effects of Gene Deficiency and Pharmacological Inhibition of Soluble Epoxide Hydrolase  

E-print Network

Opposite Effects of Gene Deficiency and Pharmacological Inhibition of Soluble Epoxide Hydrolase of cardiac remodeling; manipulation of their levels is a potentially useful pharmacological strategy. EETs staining revealed that compared with pharmacological inhibition, EPHX2 deletion aggravated Ang

Hammock, Bruce D.

23

Gene Therapy Inhibiting Neointimal Vascular Lesion: In vivo Transfer of Endothelial Cell Nitric Oxide Synthase Gene  

NASA Astrophysics Data System (ADS)

It is postulated that vascular disease involves a disturbance in the homeostatic balance of factors regulating vascular tone and structure. Recent developments in gene transfer techniques have emerged as an exciting therapeutic option to treat vascular disease. Several studies have established the feasibility of direct in vivo gene transfer into the vasculature by using reporter genes such as ?-galactosidase or luciferase. To date no study has documented therapeutic effects with in vivo gene transfer of a cDNA encoding a functional enzyme. This study tests the hypothesis that endothelium-derived nitric oxide is an endogenous inhibitor of vascular lesion formation. After denudation by balloon injury of the endothelium of rat carotid arteries, we restored endothelial cell nitric oxide synthase (ec-NOS) expression in the vessel wall by using the highly efficient Sendai virus/liposome in vivo gene transfer technique. ec-NOS gene transfection not only restored NO production to levels seen in normal untreated vessels but also increased vascular reactivity of the injured vessel. Neointima formation at day 14 after balloon injury was inhibited by 70%. These findings provide direct evidence that NO is an endogenous inhibitor of vascular lesion formation in vivo (by inhibiting smooth muscle cell proliferation and migration) and suggest the possibility of ec-NOS transfection as a potential therapeutic approach to treat neointimal hyperplasia.

von der Leyen, Heiko E.; Gibbons, Gary H.; Morishita, Ryuichi; Lewis, Neil P.; Zhang, Lunan; Nakajima, Masatoshi; Kaneda, Yasufumi; Cooke, John P.; Dzau, Victor J.

1995-02-01

24

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

PubMed

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

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

2015-05-01

25

Gene therapeutic approaches to inhibit hepatitis B virus replication.  

PubMed

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

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

2015-02-27

26

Gene therapeutic approaches to inhibit hepatitis B virus replication  

PubMed Central

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

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

2015-01-01

27

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

28

Efficient shRNA-Mediated Inhibition of Gene Expression in Zebrafish  

E-print Network

Despite the broad repertoire of loss of function (LOF) tools available for use in the zebrafish, there remains a need for a simple and rapid method that can inhibit expression of genes at later stages. RNAi would fulfill ...

Sive, Hazel L.

29

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

E-print Network

This study examined the effects of BMP7 gene transfer on corneal wound healing and fibrosis inhibition in vivo using a rabbit model. Corneal haze in rabbits was produced with the excimer laser performing -9 diopters ...

Tandon, Ashish

30

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

E-print Network

Gene product 0.4 increases bacteriophage T7 competitiveness by inhibiting host cell division Ruth for review July 30, 2013) Bacteriophages take over host resources primarily via the activity of proteins that this inhibition of cell division by Gp0.4 enhances the bacteriophage's competitive ability. This division in

Erickson, Harold P.

31

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

PubMed Central

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

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

2012-01-01

32

Rapamycin, FK506 and cyclosporin A inhibit human prolactin gene expression  

Microsoft Academic Search

In this work we demonstrate that transcription of the human prolactin gene is inhibited by the immunosuppressants FK506 (IC50 = 25 NM), cyclosporin A (IC50 = 190 NM) and rapamycin (IC50 = 25 NM). Whereas the effect of FK506 and cyclosporin A is specific for prolactin gene transcription, rapamycin has a more general effect on transcription and\\/or translation in pituitary

Stefaan Wera; Alexandra Belayew; Joseph A. Martial

1995-01-01

33

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

34

Antidepressant drugs inhibit glucocorticoid receptor-mediated gene transcription – a possible mechanism  

PubMed Central

Antidepressant drugs are known to inhibit some changes evoked by glucocorticoids, as well as a hyperactivity of hypothalamic-pituitary-adrenal (HPA) axis, often observed in depression.The aim of present study was to investigate effects of various antidepressant drugs on the glucocorticoid-mediated gene transcription in fibroblast cells, stably transfected with an MMTV promoter (LMCAT cells).The present study have shown that antidepressants (imipramine, amitriptyline, desipramine, fluoxetine, tianeptine, mianserin and moclobemide), but not cocaine, inhibit the corticosterone-induced gene transcription in a concentration- and a time-dependent manner.Drugs which are known to augment clinical effects of medication in depressed patients (lithium chloride, amantadine, memantine), do not affect the inhibitory effects of imipramine on the glucocorticoid receptor (GR)-mediated gene transcription.Inhibitors of phospholipase C (PLC), protein kinase C (PKC), Ca2+/calmodulin-dependent protein kinase (CaMK) and antagonists of the L-type Ca2+ channel also inhibit the corticosterone-induced gene transcription.Inhibitors of protein kinase A (PKA) and protein kinase G (PKG) are without effect on the GR-induced gene transcription.Phorbol ester (an activator of PKC) attenuates the inhibitory effect of imipramine on the GR-induced gene transcription.Imipramine decreases binding of corticosterone-receptor complex to DNA.It is concluded that antidepressant drugs inhibit the corticosterone-induced gene transcription, and that the inhibitory effect of imipramine depends partly on the PLC/PKC pathway. PMID:10903980

Budziszewska, Bogus?awa; Jaworska-Feil, Lucylla; Kajta, Ma?gorzata; Laso?, W?adys?aw

2000-01-01

35

Inhibition of the mutant p53 gene in transformation assays.  

PubMed

Mutation of the p53 gene is a key element in the development of several human cancers. Intron 4, a noncoding region of the p53 gene, is required for optimal expression of that gene. We have previously shown that nuclear protein binds intron 4 and have defined the protein-binding site. In this paper we address the question, "Does the mutant p53 gene's ability to transform cells to the malignant phenotype depend on protein binding to intron 4?" Using an in vitro assay in which the mutant p53 gene and Ha-ras oncogene cooperate in transformation of cells to the malignant phenotype, we determined the ability of mutant mouse p53 gene constructs, with and without two base pair substitutions at the intron 4 protein-binding site, to participate in malignant transformation. On Day 1, 5 x 10(5) rat embryo fibroblasts were transfected by the calcium phosphate procedure with 10 micrograms of both a mutant p53 gene construct and Ha-ras oncogene. Malignant transformation was evidenced by the formation of discrete foci of heaped-up cells. After 14 days of incubation at 37 degrees C in DMEM and 10% fetal calf serum (8% CO2), the cells were stained with cresyl violet and the foci counted. In three separate experiments, the presence of two base pair substitutions at the intron 4 protein-binding site caused a significant decrease in the number of foci formed (P less than 0.05). PMID:1593878

Beenken, S W; Raycroft, L; Lozano, G

1992-04-01

36

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

37

Tumor suppressor gene PDCD4 negatively regulates autophagy by inhibiting the expression of autophagy-related gene ATG5  

PubMed Central

PDCD4 (programmed cell death protein 4), a suppressor of gene transcription and translation, plays a crucial inhibitory role in several types of human tumors. However, its underlying mechanisms remain unclear. Autophagy, an evolutionarily conserved catabolic process, maintains cellular homeostasis under stress conditions such as starvation and plays a crucial role in tumor initiation and progression. We report here that PDCD4 inhibits autophagy in multiple cell types both in vitro and in vivo, which in turn contributes to its tumor suppressor activity. Importantly, PDCD4 inhibits the expression of an essential autophagy related gene, ATG5 and the formation of an ATG12–ATG5 complex, and its ma3 domains are required for PDCD4-mediated inhibition of autophagy. Unlike most tumor suppressors that act as positive or dual regulators of autophagy, our findings indicate that PDCD4 negatively regulates autophagy by targeting ATG5, which provides a novel mechanism of tumor suppression. PMID:23486359

Song, Xingguo; Zhang, Xia; Wang, Xiaoyan; Zhu, Faliang; Guo, Chun; Wang, Qun; Shi, Yongyu; Wang, Jianing; Chen, Youhai; Zhang, Lining

2013-01-01

38

Thiazolidinediones inhibit REG I{alpha} gene transcription in gastrointestinal cancer cells  

SciTech Connect

REG (Regenerating gene) I{alpha} protein functions as a growth factor for gastrointestinal cancer cells, and its mRNA expression is strongly associated with a poor prognosis in gastrointestinal cancer patients. We here demonstrated that PPAR{gamma}-agonist thiazolidinediones (TZDs) inhibited cell proliferation and REG I{alpha} protein/mRNA expression in gastrointestinal cancer cells. TZDs inhibited the REG I{alpha} gene promoter activity, via its cis-acting element which lacked PPAR response element and could not bind to PPAR{gamma}, in PPAR{gamma}-expressing gastrointestinal cancer cells. The inhibition was reversed by co-treatment with a specific PPAR{gamma}-antagonist GW9662. Although TZDs did not inhibit the REG I{alpha} gene promoter activity in PPAR{gamma}-non-expressing cells, PPAR{gamma} overexpression in the cells recovered their inhibitory effect. Taken together, TZDs inhibit REG I{alpha} gene transcription through a PPAR{gamma}-dependent pathway. The TZD-induced REG I{alpha} mRNA reduction was abolished by cycloheximide, indicating the necessity of novel protein(s) synthesis. TZDs may therefore be a candidate for novel anti-cancer drugs for patients with gastrointestinal cancer expressing both REG I{alpha} and PPAR{gamma}.

Yamauchi, Akiyo [Department of Advanced Biological Sciences for Regeneration (Kotobiken Medical Laboratories), Tohoku University Graduate School of Medicine, Sendai 980-8575 (Japan); Laboratory of Molecular Genetics, Tohoku University Graduate School of Pharmaceutical Sciences, Sendai 980-8578 (Japan); Department of Biochemistry, Nara Medical University, Kashihara 634-8521 (Japan); Takahashi, Iwao [Department of Advanced Biological Sciences for Regeneration (Kotobiken Medical Laboratories), Tohoku University Graduate School of Medicine, Sendai 980-8575 (Japan); Takasawa, Shin [Department of Advanced Biological Sciences for Regeneration (Kotobiken Medical Laboratories), Tohoku University Graduate School of Medicine, Sendai 980-8575 (Japan); Department of Biochemistry, Nara Medical University, Kashihara 634-8521 (Japan); Nata, Koji; Noguchi, Naoya; Ikeda, Takayuki; Yoshikawa, Takeo; Shervani, Nausheen J. [Department of Advanced Biological Sciences for Regeneration (Kotobiken Medical Laboratories), Tohoku University Graduate School of Medicine, Sendai 980-8575 (Japan); Suzuki, Iwao [Laboratory of Molecular Genetics, Tohoku University Graduate School of Pharmaceutical Sciences, Sendai 980-8578 (Japan); Uruno, Akira [Department of Advanced Biological Sciences for Regeneration (Kotobiken Medical Laboratories), Tohoku University Graduate School of Medicine, Sendai 980-8575 (Japan); Unno, Michiaki [Department of Surgery, Tohoku University Graduate School of Medicine, Sendai 980-8574 (Japan); Okamoto, Hiroshi [Department of Advanced Biological Sciences for Regeneration (Kotobiken Medical Laboratories), Tohoku University Graduate School of Medicine, Sendai 980-8575 (Japan)], E-mail: okamotoh@mail.tains.tohoku.ac.jp; Sugawara, Akira [Department of Advanced Biological Sciences for Regeneration (Kotobiken Medical Laboratories), Tohoku University Graduate School of Medicine, Sendai 980-8575 (Japan)], E-mail: akiras2i@mail.tains.tohoku.ac.jp

2009-02-13

39

Modulation of oestrogen action by receptor gene inhibition  

Microsoft Academic Search

Selective oestrogen receptor downregulators (SERDs) are a class of highly effective steroidal antitumour agents that reduce cellular levels of the oestrogen receptor (ER). In this study, we compared the efficacy by which three novel molecular approaches: (1) antisense oligonucleotides; (2) antisense RNA; and (3) dominant negative mutants are able to act as SERDs. Using transient and, where appropriate, stable gene

T. A. Madden; D. Barrow; R. A. McClelland; J. M. W. Gee; R. I. Nicholson

2000-01-01

40

Curcumin inhibits ultraviolet light induced human immunodeficiency virus gene expression  

Microsoft Academic Search

Recently, we reported that the herbal drug St. John's Wort is a potent inhibitor of UV-induced HIV-LTR activation in stably transfected HIVcat\\/HeLa cells [35]. Our previous studies have demonstrated that the activation of p38 MAP kinase (stress-activated protein kinase-2) and NF-?B are both required for a full UV-induced HIV gene expression response. In this study we have investigated the mechanism

Mohiuddin M. Taher; Guido Lammering; Chad Hershey; Kristoffer Valerie

2003-01-01

41

Antitumor Molecular Mechanism of Chlorogenic Acid on Inducting Genes GSK-3? and APC and Inhibiting Gene ?-Catenin  

PubMed Central

Objective. Inhibiting gene ?-catenin and inducting genes GSK-3? and APC, promoting the tumor cell apoptosis in Wnt pathway, by chlorogenic acid were discussed (CGA). Method. The different genes were scanned by the 4?44K mouse microarray chips. The effect of the three genes was confirmed by RT-PCR technique with CGA dosage of 5, 10, and 20?mg/kg. Result. The expression of GSK-3? and APC was upregulated in group of 20?mg/kg dosage (P < 0.05) and the expression of ?-catenin was downregulated in the same dosage (P < 0.05). Conclusion. The results infer that the multimeric protein complex of ?-catenin could be increased by CGA upregulated genes GSK-3? and APC, which could inhibit the free ?-catenin into the nucleus to connect with TCF. So the transcriptional expression of the target genes will be cut to abnormal cell proliferation. It is probably one of the ways that can stop the tumor increase by CGA. PMID:23844319

Xu, Ruoshi; Kang, Qiumei; Ren, Jie; Li, Zukun; Xu, Xiaoping

2013-01-01

42

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

43

Modulation of oestrogen action by receptor gene inhibition.  

PubMed

Selective oestrogen receptor downregulators (SERDs) are a class of highly effective steroidal antitumour agents that reduce cellular levels of the oestrogen receptor (ER). In this study, we compared the efficacy by which three novel molecular approaches: (1) antisense oligonucleotides; (2) antisense RNA; and (3) dominant negative mutants are able to act as SERDs. Using transient and, where appropriate, stable gene transfection experiments we found that constitutive overexpression of ER antisense RNA and a hormone-binding domain compromised dominant-negative ER mutant (DNER-1), were most effective at downregulating ER expression and/or activity in vitro. PMID:11056309

Madden, T A; Barrow, D; McClelland, R A; Gee, J M; Nicholson, R I

2000-09-01

44

Curcumin, the active constituent of turmeric, inhibits amyloid peptide-induced cytochemokine gene expression  

E-print Network

Curcumin, the active constituent of turmeric, inhibits amyloid peptide-induced cytochemokine gene-a and IL-1b) and chemokines (MIP-1b, MCP-1 and IL-8) in monocytes. We determined whether curcumin expression of cytochemokines. We show that curcumin (12.5­25 lM) sup- presses the activation of Egr-1 DNA

Giri, Ranjit K.

45

Licznar et al. Identification of genes involved in growth inhibition of breast  

E-print Network

Licznar et al. - 1 - Identification of genes involved in growth inhibition of breast cancer cells Vignon and Gwendal Lazennec ¶ INSERM U540 "Molecular and Cellular Endocrinology of Cancers", 60, rue de Estrogen receptor alpha (ER)-negative breast cancer cells display an aggressive phenotype. We previously

Boyer, Edmond

46

Prostaglandin E2 Stimulates Amyloid Precursor Protein Gene Expression: Inhibition by Immunosuppressants  

E-print Network

Prostaglandin E2 Stimulates Amyloid Precursor Protein Gene Expression: Inhibition dysfunction in transgenic mice. We now report that activation of prostaglandin E2 (PGE2 ) receptors increases. These results suggest that prostaglandins pro- duced by brain injury or inflammation can activate APP tran

Wurtman, Richard

47

Planar polarity genes and inhibition of supernumerary neurites.  

PubMed

Planar cell polarity (PCP) genes have recently emerged as important players in sculpting neuronal connections. The bipolar VC neurons display stereotypical differences in axon extension along the anterior-posterior (AP) body axis: VC1-3 and VC6 polarize along the AP axis while VC4 and VC5 polarize along the orthogonal left-right (LR) axis generated by the developing vulva. vang-1 and prkl-1, the worm orthologs of Van Gogh and Prickle, are required to restrict the polarity of neurite emergence to a specific tissue axis. vang-1 and prkl-1 loss results in ectopic VC4 and VC5 neurites extending inappropriately along the AP axis. Conversely, prkl-1 overexpression in VC neurons suppresses neurite formation. These findings suggest that a PCP-like pathway acts to silence or antagonize neuronal responses to polarity cues that would otherwise be permissive for neurite growth. PMID:24058835

Colavita, Antonio

2012-04-01

48

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

49

Neuropeptide Y gene transfection inhibits post-epileptic hippocampal synaptic reconstruction?  

PubMed Central

Exogenous neuropeptide Y has antiepileptic effects; however, the underlying mechanism and optimal administration method for neuropeptide Y are still unresolved. Previous studies have used intracerebroventricular injection of neuropeptide Y into animal models of epilepsy. In this study, a recombinant adeno-associated virus expression vector carrying the neuropeptide Y gene was injected into the lateral ventricle of rats, while the ipsilateral hippocampus was injected with kainic acid to establish the epileptic model. After transfection of neuropeptide Y gene, mossy fiber sprouting in the hippocampal CA3 region of epileptic rats was significantly suppressed, hippocampal synaptophysin (p38) mRNA and protein expression were inhibited, and epileptic seizures were reduced. These experimental findings indicate that a recombinant adeno-associated virus expression vector carrying the neuropeptide Y gene reduces mossy fiber sprouting and inhibits abnormal synaptophysin expression, thereby suppressing post-epileptic synaptic reconstruction. PMID:25206456

Zhang, Fan; Zhao, Wenqing; Li, Wenling; Dong, Changzheng; Zhang, Xinying; Wu, Jiang; Li, Na; Liang, Chuandong

2013-01-01

50

Inhibition of 13-cis retinoic acid-induced gene expression of homeobox B7 by thalidomide.  

PubMed

Thalidomide and 13-cis retinoic acid (RA) show anticancer effects as sole agents or in combination with other drugs. However, induction of homeobox (HOX) gene expression by 13-cis RA may contribute to tumor progression thereby potentially limiting its efficacy. The purpose was to test if thalidomide can inhibit 13-cis RA-induced HOXB7 expression and whether thalidomide may enhance the antiproliferative effect of 13-cis RA in U343MG glioblastoma cells. Quantitative real-time PCR showed significant inhibition of 13-cis RA-induced HOXB7 expression by thalidomide with IC(50) approximately 0.1-0.2 microg/ml when given simultaneously with 13-cis RA but not when administered 18 h later (p < 0.0001). 13-cis RA alone inhibited proliferation and colony formation in a concentration-dependent manner whereas growth inhibition by thalidomide alone at 5-100 microg/ml was constant at 80-90% of controls. At 10% serum concentration, growth inhibition by a combination of the 2 drugs was additive but at 1% serum, growth inhibition was synergistic. It is concluded that thalidomide inhibits the RA-induced HOXB7 expression in glioblastoma cells and that 13-cis RA/thalidomide combinations can in principle enhance cytotoxicity. The improved cell kill induced by thalidomide is attributed to downregulation of growth stimulatory factors induced by 13-cis RA. Implications for the modus operandi of thalidomide in embryogenesis are noted. PMID:17514648

Milanovi?, Dusan; Maier, Patrick; Lohr, Frank; Wenz, Frederik; Herskind, Carsten

2007-09-15

51

Resveratrol inhibits LXR?-dependent hepatic lipogenesis through novel antioxidant Sestrin2 gene induction  

SciTech Connect

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

Jin, So Hee; Yang, Ji Hye; Shin, Bo Yeon; Seo, Kyuhwa; Shin, Sang Mi [College of Pharmacy, Chosun University, Gwangju 501-759 (Korea, Republic of); Cho, Il Je, E-mail: skek023@dhu.ac.kr [MRC-GHF, College of Korean Medicine, Daegu Haany University, Gyeongsan, Gyeongsangbukdo 712-715 (Korea, Republic of); Ki, Sung Hwan, E-mail: shki@chosun.ac.kr [College of Pharmacy, Chosun University, Gwangju 501-759 (Korea, Republic of)

2013-08-15

52

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

SciTech Connect

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

Sorscher, E.J.; Kirk, K.L.; Weaver, M.L.; Jilling, T.; Blalock, J.E.; LeBoeuf, R.D. (Univ of Alabama, Birmingham (United States))

1991-09-01

53

Inhibition and gene expression of Nitrosomonas europaea biofilms exposed to phenol and toluene.  

PubMed

Pure culture biofilms of the ammonia-oxidizing bacterium Nitrosomonas europaea were grown in a Drip Flow Biofilm Reactor and exposed to the aromatic hydrocarbons phenol and toluene. Ammonia oxidation rates, as measured by nitrite production in the biofilms, were inhibited 50% when exposed to 56?µM phenol or 100?µM toluene, while 50% inhibition of suspended cells occurred at 8?µM phenol or 20?µM toluene. Biofilm-grown cells dispersed into liquid medium and immediately exposed to phenol or toluene experienced similar inhibition levels as batch grown cells, indicating that mass transfer may be a factor in N. europaea biofilm resistance. Whole genome microarray analysis of gene expression was used to detect genes up-regulated in biofilms during toluene and phenol exposure. Two genes, a putative pirin protein (NE1545) and a putative inner membrane protein (NE1546) were up-regulated during phenol exposure, but no genes were up-regulated during toluene exposure. Using qRT-PCR, up-regulation of NE1545 was detected in biofilms and suspended cells exposed to a range of phenol concentrations and levels of inhibition. In the biofilms, NE1545 expression was up-regulated an average of 13-fold over the range of phenol concentrations tested, and was essentially independent of phenol concentration. However, the expression of NE1545 in suspended cells increased from 20-fold at 7?µM phenol up to 80-fold at 30?µM phenol. This study demonstrates that biofilms of N. europaea are more resistant than suspended cells to inhibition of ammonia oxidation by phenol and toluene, even though the global transcriptional responses to the inhibitors do not differ in N. europaea between the suspended and attached growth states. PMID:21404249

Lauchnor, Ellen G; Radniecki, Tyler S; Semprini, Lewis

2011-04-01

54

Gene delivery of suppressors of cytokine signaling (SOCS) inhibits inflammation and atherosclerosis development in mice.  

PubMed

Chronic activation of Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway contributes to vascular inflammation and atherosclerosis by inducing expression of genes involved in cell proliferation, differentiation and migration. We aimed to investigate whether enforced expression of negative regulators, the suppressors of cytokine signaling (SOCS1 and SOCS3), inhibits harmful JAK/STAT-mediated responses and affects atherosclerosis in apolipoprotein E knockout mice. Adenovirus-mediated SOCS1 transgene expression impaired the onset and progression of atherosclerosis without impact on lipid profile, whereas SOCS3 was only effective on early atherosclerosis. Mechanistically, SOCS gene delivery, primarily SOCS1, attenuated STAT1 and STAT3 activation and reduced the expression of STAT-dependent genes (chemokine/chemokine receptors, adhesion molecules, pro-inflammatory cytokines and scavenger receptors) in aortic tissue. Furthermore, atherosclerotic plaques exhibit a more stable phenotype characterized by lower lipids, T cells and M1 macrophages and higher M2 macrophages and collagen. Atheroprotection was accompanied by a systemic alteration of T helper- and T regulatory-related genes and a reduced activation state of circulating monocytes. In vascular smooth muscle cells and macrophages, SOCS gene delivery inhibited cytokine-induced STAT activation, pro-inflammatory gene expression, cell migration and proliferation. In conclusion, targeting SOCS proteins, predominantly SOCS1, to suppress pathological mechanisms involved in atheroma plaque progression and destabilization could be an interesting anti-atherosclerotic strategy. PMID:25604439

Recio, Carlota; Oguiza, Ainhoa; Mallavia, Beńat; Lazaro, Iolanda; Ortiz-Muńoz, Guadalupe; Lopez-Franco, Oscar; Egido, Jesus; Gomez-Guerrero, Carmen

2015-03-01

55

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. PMID:18369442

Biacsi, Rea; Kumari, Daman; Usdin, Karen

2008-01-01

56

Inhibition of HCV 3a core gene through Silymarin and its fractions  

PubMed Central

Hepatitis C is a major health problem affecting 270 million individuals in world including Pakistan. Current treatment regimen, interferon alpha and ribavirin only cure half of patients due to side effects and high cost. Results In the present study Silybum marianum (Milk thistle) seeds were collected, extracted and analyzed against HCV 3a core gene by transiently transfecting the liver cells with HCV core plasmid. Our results demonstrated that Silymarin (SM) dose dependently inhibit the expression or function of HCV core gene at a non toxic concentration while the GAPDH remained constant. To identify the active ingredient, SM was fractioned by thin layer chromatography (TLC), column chromatography and HPLC. Purified fractions were tested for HCV core gene and western blotting results showed that two factions of SM (S1 and S2) inhibit HCV 3a core expression or function in liver cells Conclusion Our results suggest SM and its fractions (S1 and S2) inhibit HCV core gene of 3a genotype and combination of SM and its fractions with interferon will be a better option to treat HCV infection PMID:21453551

2011-01-01

57

Glioma Cell Growth Inhibition Following Photochemical Internalization Enhanced Non-Viral PTEN Gene Transfection  

PubMed Central

Background and Objective One of many limitations for cancer gene therapy is the inability of the therapeutic gene to transfect a sufficient number of tumor cells. Photochemical internalization (PCI) is a photodynamic therapy-based approach for improving the delivery of macromolecules and genes into the cell cytosol. The utility of PCI for the delivery of the GFP reporter gene on the same plasmid as a tumor suppressor gene (PTEN) was investigated in monolayers of U251 human glioma cells and muticell U87 glioma spheroids. Materials and Methods U251 monolayers or U87 spheroids were incubated in AlPcS2a and non-viral vector polyplexes for 18 hours. In all cases, light treatment was performed with a diode laser at a wavelength of 670 nm. The non-viral transfection agents, branched polyethylenimine (bPEI), or protamine sulfate (PS), were used with the plasmid constructs GFP/PTEN or GFP. Results PS/GFP polyplexes were much less toxic to the glioma cells compared to bPEI/GFP polyplexes but were highly inefficient at gene transfection if used alone. PCI resulted in a 5- to 10-fold increase in GFP protein expression compared to controls. PCI-bPEI/PTEN or PCI-PS/PTEN transfection of either U251 monolayers or U87 spheroids significantly inhibited their growth. but had no effect on MCF-7 cells containing a wild-type PTEN gene. In addition PCI-GFP transfection of gliomas cells had no effect on their growth pattern. Conclusions Collectively, the results suggest that AlPcS2a-mediated PCI can be used to enhance cell growth inhibition via transfection of tumor suppressor genes in glioma cells containing mutant PTEN genes. PMID:23018764

Mathews, Marlon S.; Shih, En-Chung; Zamora, Genesis; Sun, Chung-Ho; Cho, Soo Kyung; Kwon, Young Jik; Hirschberg, Henry

2014-01-01

58

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

PubMed

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

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

2014-09-20

59

Overexpression of the NF2 gene inhibits schwannoma cell proliferation through promoting PDGFR degradation.  

PubMed

The loss of NF2 gene function leads to vestibular nerve schwannoma formation in humans. The NF2 gene product, Merlin/Schwannomin, has recently been found to interact with the two PDZ domains containing protein EBP50/NHE-RF, which is itself known to interact with the PDGF receptor (PDGFR) in several cell types. In this study, an up-regulation of both PDGFR and EBP50/NHE-RF, and an interaction of both proteins were found in primary human schwannoma tissue. Furthermore, using an adenoviral vector mediated gene transfer technique, changes in the phenotypic characteristics after NF2 gene restoration in a newly established NF2 gene-mutated human schwannoma cell line (HEI 193) were investigated. The overexpression of Merlin/Schwannomin in HEI 193 led to an inhibition of cell proliferation under serum-free conditions. Upon PDGF stimulation in culture, Merlin/Schwannomin appeared to inhibit the activation of the MAPK and PI3K signaling pathways, impinging on the phosphorylation of Erk 1/2 and Akt, respectively. The data also show that PDGFR is more rapidly internalized by the schwannoma cells overexpressing NF2. Therefore, this process is suggested as a model for a mechanism of Merlin/Schwannomin tumor suppressor function, which intermediates acceleration of the cell surface growth factor degradation. PMID:14612918

Fraenzer, Juergen-Theodor; Pan, Huiqi; Minimo, Lauro; Smith, George M; Knauer, Dan; Hung, Gene

2003-12-01

60

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. PMID:24176851

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

61

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. PMID:24218612

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

2013-01-01

62

Subinhibitory Clindamycin Differentially Inhibits Transcription of Exoprotein Genes in Staphylococcus aureus  

Microsoft Academic Search

It has long been known that certain antibiotics, at subinhibitory concentrations, differentially inhibit the synthesis of a-hemolysin and other staphylococcal virulence factors. In this report, we show that subinhibitory clindamycin (SBCL) eliminates production of nearly all exoproteins by Staphylococcus aureus but has virtually no effect on cytoplasmic proteins. The effect was abolished by a gene conferring resistance to macrolides-linco- samides-streptogramin

SILVIA HERBERT; PETER BARRY; RICHARD P. NOVICK

2001-01-01

63

Inhibition of herpes simplex virus 1 gene expression by designer zinc-finger transcription factors  

Microsoft Academic Search

The herpes simplex virus 1 (HSV-1) replicative cycle begins by binding of the viral activator, VP16, to a set of sequences in the immediate-early (IE) gene promoters. With the aim of inhibiting this cycle, we have constructed a number of synthetic zinc-finger DNA-binding peptides by using recently reported methods. Peptides containing either three or six fingers, targeted to a viral

Monika Papworth; Michael Moore; Mark Isalan; Michal Minczuk; Yen Choo; Aaron Klug

2003-01-01

64

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

65

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

66

Inhibition of spermidine synthase gene expression by transforming growth factor-beta 1 in hepatoma cells.  

PubMed Central

We screened genes responsive to transforming growth factor-beta (TGF-beta 1) protein in a human hepatoma cell line (Hep3B) using a PCR-mediated differential display technique, in order to investigate the mechanisms involved in TGF-beta-induced growth suppression. We found a gene that was down-regulated by TGF-beta 1 to be completely identical in an approx. 620 bp segment to the gene for the enzyme spermidine synthase, which mediates the conversion of putrescine into spermidine. Both spermidine synthase mRNA expression and its enzyme activity were decreased after TGF-beta 1 treatment of Hep3B cells. The inhibition of spermidine synthase gene expression by TGF-beta 1 protein was also observed in other hepatoma cell lines. The expression of genes for other biosynthetic enzymes in polyamine metabolism (ornithine decarboxylase and S-adenosylmethionine decarboxylase) was also inhibited to the same extent as for spermidine synthase, while the gene expression of spermidine/spermine N1-acetyltransferase, a catabolic enzyme, was relatively resistant to TGF-beta 1. Spermine levels in Hep3B cells were decreased by TGF-beta 1 treatment, although the levels of spermidine and putrescine were unchanged, probably due to compensation by remaining spermidine/spermine N1-acetyltransferase activity. Exogenously added spermidine or spermine, but not putrescine, partially antagonized the growth-inhibitor effects of TGF-beta 1 on Hep3B cells. Our data suggest that down-regulation of gene expression of the enzymes involved in polyamine metabolism, including spermidine synthase, may be associated with the mechanism of TGF-beta-induced growth suppression. PMID:9020892

Nishikawa, Y; Kar, S; Wiest, L; Pegg, A E; Carr, B I

1997-01-01

67

Inhibition of spermidine synthase gene expression by transforming growth factor-beta 1 in hepatoma cells.  

PubMed

We screened genes responsive to transforming growth factor-beta (TGF-beta 1) protein in a human hepatoma cell line (Hep3B) using a PCR-mediated differential display technique, in order to investigate the mechanisms involved in TGF-beta-induced growth suppression. We found a gene that was down-regulated by TGF-beta 1 to be completely identical in an approx. 620 bp segment to the gene for the enzyme spermidine synthase, which mediates the conversion of putrescine into spermidine. Both spermidine synthase mRNA expression and its enzyme activity were decreased after TGF-beta 1 treatment of Hep3B cells. The inhibition of spermidine synthase gene expression by TGF-beta 1 protein was also observed in other hepatoma cell lines. The expression of genes for other biosynthetic enzymes in polyamine metabolism (ornithine decarboxylase and S-adenosylmethionine decarboxylase) was also inhibited to the same extent as for spermidine synthase, while the gene expression of spermidine/spermine N1-acetyltransferase, a catabolic enzyme, was relatively resistant to TGF-beta 1. Spermine levels in Hep3B cells were decreased by TGF-beta 1 treatment, although the levels of spermidine and putrescine were unchanged, probably due to compensation by remaining spermidine/spermine N1-acetyltransferase activity. Exogenously added spermidine or spermine, but not putrescine, partially antagonized the growth-inhibitor effects of TGF-beta 1 on Hep3B cells. Our data suggest that down-regulation of gene expression of the enzymes involved in polyamine metabolism, including spermidine synthase, may be associated with the mechanism of TGF-beta-induced growth suppression. PMID:9020892

Nishikawa, Y; Kar, S; Wiest, L; Pegg, A E; Carr, B I

1997-01-15

68

Tapentadol inhibits calcitonin gene-related peptide release from rat brainstem in vitro.  

PubMed

We have previously developed an in vitro model of rat brainstem explants. The latter release sizable amounts of calcitonin gene-related peptide (CGRP); basal release can be stimulated by such secretagogues as high KCl concentrations, veratridine or capsaicine. In this paradigm we investigated the activity of the analgesic agent tapentadol; the effects of tapentadol were compared to those of a classical opioid receptor agonist, morphine, and the selective noradrenaline reuptake inhibitor reboxetine. Morphine inhibited basal CGRP release, with statistical significance from 1 nM onward and maximal (-44%) inhibition at 100 ?M. Morphine also inhibited K(+)-stimulated peptide release, with a significant effect from 1 ?M and maximal (-39%) decrease at 100 ?M, but failed to inhibit release stimulated by 10 ?M capsaicin. At variance, reboxetine had no effect on baseline CGRP outflow, but was able to inhibit both K(+)-stimulated [significant inhibition from 1 ?M onward and maximal (-37%) decrease at 100 ?M], and capsaicin-stimulated release [significant effect from 1 ?M and maximal (-31%) decrease at 100 ?M]. Likewise, tapentadol had no effect on baseline CGRP release up to 100 ?M, but decreased secretion stimulated by 56 mM KCl or capsaicin, with significant effects from 0.1 and 1 ?M respectively; maximal inhibition over 56 mM KCl and capsaicin stimuli was -29% and -31%, respectively. Naloxone antagonized the effect of morphine, but not those of reboxetine and tapentadol, on K(+)-stimulated CGRP secretion. In conclusion the present study provides consistent pharmacological evidence that tapentadol acts as a noradrenaline reuptake inhibitor agent in this experimental model. PMID:24662320

Greco, Maria Cristina; Lisi, Lucia; Currň, Diego; Navarra, Pierluigi; Tringali, Giuseppe

2014-06-01

69

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

70

?-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

71

Effective inhibition of Japanese encephalitis virus replication by small interfering RNAs targeting the NS5 gene.  

PubMed

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, causes an acute infection of the central nervous system resulting in encephalitis of humans and many kinds of animals. NS5, the largest and most conserved flavivirus protein, is homologous to methyltransferase and RNA-dependent RNA polymerase. RNA interference is an effective anti-viral strategy to inhibit viral replication in vitro. In this study, four short hairpin RNA (shRNA) expression vectors (pS4.1-NS5-201, pS4.1-NS5-455, pS4.1-NS5-699, and pS4.1-NS5-804) targeting the NS5 gene of JEV were employed to target and destroy JEV transcripts. The four shRNAs expression plasmids were individually co-transfected into 293T cells with the plasmid pNS5-EGFP expressing NS5 fused to enhanced green fluorescent protein. The expression level of NS5 was evaluated by fluorescence microscopy, flow cytometry, real time RT-PCR, and Western blot. The four shRNA expression plasmids were also transfected into BHK-21 cells to examine their inhibition of viral replication by indirect immunofluorescence, real time RT-PCR, and Western blot. The results provided strong evidence that shRNAs targeting the NS5 gene could specifically and efficiently inhibit JEV replication. Three out of four plasmids were highly efficient at inhibiting viral replication, including pS4.1-NS5-455, pS4.1-NS5-699, and pS4.1-NS5-804. This was especially true for pS4.1-NS5-699, which reduced the levels of virus RNA and protein the most. Our data suggest that shRNAs could be used as a tool to inhibit JEV replication in vivo. PMID:18190994

Qi, Wen-Bao; Hua, Rong-Hong; Yan, Li-Ping; Tong, Guang-Zhi; Zhang, Gui-Hong; Ren, Tao; Wu, Dong-Lai; Liao, Ming

2008-03-01

72

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. PMID:22718829

Oduro, Jennifer D.; Uecker, Ralf

2012-01-01

73

The Krüppel-like factor KLF15 inhibits transcription of the adrenomedullin gene in adipocytes.  

PubMed

KLF15 (Krüppel-like factor 15) plays a key role in adipocyte differentiation and glucose transport in adipocytes through activation of its target genes. We have now identified six target genes regulated directly by KLF15 in 3T3-L1 mouse adipocytes with the use of a combination of microarray-based chromatin immunoprecipitation and gene expression analyses. We confirmed the direct regulation by KLF15 of one of these genes, that for adrenomedullin, with the use of a luciferase reporter assay in 3T3-L1 preadipocytes and adipocytes. Such analysis revealed that the most proximal CACCC element in the promoter of the human adrenomedullin gene (located in the region spanning nucleotides -70 and -29) is required for trans-inhibition by KLF15. Furthermore, chromatin immunoprecipitation showed that KLF15 binds to this region of the human adrenomedullin gene promoter in cultured human adipocytes. These results thus implicate KLF15 in the regulation of adrenomedullin expression in adipose tissue. PMID:19094967

Nagare, Tomoki; Sakaue, Hiroshi; Takashima, Mototsugu; Takahashi, Kazuhiro; Gomi, Hideyuki; Matsuki, Yasushi; Watanabe, Eijiro; Hiramatsu, Ryuji; Ogawa, Wataru; Kasuga, Masato

2009-01-30

74

Flurbiprofen inhibits capsaicin induced calcitonin gene related peptide release from rat spinal cord via an endocannabinoid dependent mechanism  

Microsoft Academic Search

Calcitonin gene related peptide (CGRP) is involved in nociceptive transmission and modulation at the spinal level. In the spinal superperfusion model, ?9 tetrahydrocannabinol inhibited capsaicin induced CGRP release in a concentration dependent manner. Similarly, flurbiprofen (3 ?M) inhibited spinal CGRP release. This inhibition was reversed by the CB1 antagonist AM-251 (1 ?M), but not by co-administration of prostaglandin E2 (PGE2;

Kay Seidel; May Hamza; Mehmet Ates; Hans Gühring

2003-01-01

75

Increased MAP kinase inhibition enhances epiblast-specific gene expression in bovine blastocysts.  

PubMed

Mammalian blastocysts comprise three distinct lineages, namely, trophoblast, hypoblast, and epiblast, which develop into fetal placenta, extraembryonic yolk sac, and embryo proper, respectively. Pluripotent embryonic stem cells, capable of forming all adult cell types, can only be derived from the epiblast. In mouse and rat, this process is promoted by the double inhibition (2i) of mitogen-activated protein kinase kinase (MAP2K), which antagonizes FGF signaling, and glycogen synthase kinase 3 (GSK3), which stimulates the WNT pathway. We investigated variations of the 2i treatment on lineage segregation and pluripotency-related gene expression in bovine blastocysts. In vitro-fertilized embryos were cultured either in the presence of inhibitors of GSK3 (3 ?M CHIR) and MAP2K (0.4 vs. 10 ?M PD0325901, designated 2i and 2i+, respectively) or in 2i/2i+ with FGFR inhibitor (0.1 ?M PD173074, designated 3i [2i and PD173074] and 3i+ [2i+ and PD173074]). Compared with 2i, both 2i+ and 3i+ potentiated the improvement in blastocyst morphology. Using an automated platform for multiplexed digital mRNA profiling, we simultaneously counted transcripts of 76 candidate genes in bovine blastocysts treated with multiple kinase inhibitors. We show that 2i+ medium specifically increased FGF4 and NANOG while reducing PDGFRalpha and SOX17 levels. The shift from a hypoblast to an epiblast gene expression signature was confirmed by quantitative PCR. A wide range of functionally related genes, including candidates involved in DNA methylation, were not significantly changed. This well-defined 2i+ effect was not observed after pharmacologically inhibiting FGF receptor or related MAP kinases (p38, JNK, and ERK5). In summary, our data suggest that increased MAP2K inhibition exerts its pluripotency-promoting effects through as yet unidentified signals. PMID:25009207

McLean, Zachariah; Meng, Fanli; Henderson, Harold; Turner, Pavla; Oback, Björn

2014-08-01

76

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

77

Gene silencing of IL-12 in dendritic cells inhibits autoimmune arthritis  

PubMed Central

Background We have previously demonstrated that immune modulation can be accomplished by administration of gene silenced dendritic cells (DC) using siRNA. In this study, we demonstrate the therapeutic utilization of shRNA-modified DC as an antigen-specific tolerogenic vaccine strategy for autoimmune arthritis. Methods A shRNA that specifically targets IL-12 p35 was designed and cloned into a plasmid vectors (IL-12 shRNA). Bone marrow-derived DC from DBA/1 mice were transfected with the IL-12 shRNA construct in vitro. Mice with collagen II (CII)-induced arthritis (CIA) were treated with the modified DCs expressing the shRNA. Recall response and disease progression were assessed. Results After gene silencing of IL-12 in DC, DC were shown to selectively inhibit T cell proliferation on recall responses and in an MLR. In murine CIA, we demonstrated that administration of IL-12 shRNA-expressing DC that were pulsed with CII inhibited progression of arthritis. The therapeutic effects were evidenced by decreased clinical scores, inhibition of inflammatory cell infiltration in the joint, and suppression of T cell and B cell responses to CII. Conclusion We demonstrate a novel tolerance-inducing protocol for the treatment of autoimmune inflammatory joint disease in which the target antigen is known, utilizing DNA-directed RNA interference. PMID:22289162

2012-01-01

78

Antisense oligodeoxynucleotide inhibition as an alternative and convenient method for gene function analysis in pollen tubes.  

PubMed

Antisense oligodeoxynucleotide (A-ODN) inhibition works well in animal cells. However, there have been few successful examples to date of its application in plants, and more specifically whether the technique can be used in pollen tubes as a model of plant cell growth. NtGNL1 plays an important role in pollen tube development and was thus selected as an indicator to assess the biological effects of A-ODN. An A-ODN inhibition technique was used to down-regulate NtGNL1 expression in tobacco pollen tubes and showed that A-ODNs could quickly enter pollen tubes through the thick wall and cell membrane and effectively block NtGNL1 expression. Phenotype analysis revealed that the down-regulation of NtGNL1 by A-ODNs resulted in abnormalities in endocytosis and subsequent vesicle trafficking, similar to the phenotypes of pollen tubes treated with NtGNL1 RNAi. This investigation confirmed that A-ODNs could specifically inhibit target gene expression, and furthermore demonstrated that A-ODN functioned in a concentration- and duration-dependent manner, because A-ODNs could be degraded when incubated with pollen tubes. Thus, the A-ODN technique was successfully used for gene function analysis in pollen tubes and appears to be an alternative and convenient technique when the in vitro pollen tube is used as the study model. This technique will greatly facilitate investigations on the molecular mechanism(s) underlying pollen tube growth. PMID:23527102

Liao, Fanglei; Wang, Lu; Yang, Li-Bo; Zhang, Liyao; Peng, Xiongbo; Sun, Meng-Xiang

2013-01-01

79

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

PubMed Central

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

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

2013-01-01

80

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

PubMed Central

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

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

2013-01-01

81

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

82

Inhibition of major histocompatibility complex class II gene transcription by nitric oxide and antioxidants.  

PubMed

Interferon (IFN)-gamma facilitates cellular immune response, in part, by inducing the expression of major histocompatibility complex class II (MHC-II) molecules. We demonstrate that IFN-gamma induces the expression of HLA-DRA in vascular endothelial cells via mechanisms involving reactive oxygen species. IFN-gamma-induced HLA-DRA expression was inhibited by nitric oxide (NO) and antioxidants such as superoxide dismutase, catalase, pyrrolidine dithiocarbamate, and N-acetylcysteine. Nuclear run-on assays demonstrated that NO and antioxidants inhibited IFN-gamma-induced HLA-DRA gene transcription. Transient transfection studies using a fully functional HLA-DRA promoter construct ([-300]DR alpha.CAT) showed that inhibition of endogenous NO synthase activity by N(omega)-monomethyl-l-arginine or addition of exogenous hydrogen peroxide (H(2)O(2)) augmented basal and IFN-gamma-stimulated [-300]DR alpha.CAT activity. However, H(2)O(2) and N(omega)-monomethyl-l-arginine could induce HLA-DRA expression suggesting that H(2)O(2) is a necessary but not a sufficient mediator of IFN-gamma-induced HLA-DRA expression. Electrophoretic mobility shift assay and Western blotting demonstrated that NO and antioxidants had little or no effect on IFN-gamma-induced IRF-1 activation or MHC-II transactivator (CIITA) expression but did inhibit IFN-gamma-induced activation of STAT1 alpha (p91) and Y box transcription factors, NF-Y(A) and NF-Y(B). These results indicate that NO and antioxidants may attenuate vascular inflammation by antagonizing the effects of intracellular reactive oxygen species generation by IFN-gamma, which is necessary for MHC-II gene transcription. PMID:12006557

Grimm, Michael; Spiecker, Martin; De Caterina, Raffaele; Shin, Wee Soo; Liao, James K

2002-07-19

83

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. PMID:24371075

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

2014-01-01

84

Vaccinia virus inhibits NF-?B-dependent gene expression downstream of p65 translocation.  

PubMed

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. PMID:24371075

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

2014-03-01

85

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

PubMed Central

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

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

1980-01-01

86

Overexpression of Transcription Factor Sp1 Leads to Gene Expression Perturbations and Cell Cycle Inhibition  

PubMed Central

Background The ubiquitous transcription factor Sp1 regulates the expression of a vast number of genes involved in many cellular functions ranging from differentiation to proliferation and apoptosis. Sp1 expression levels show a dramatic increase during transformation and this could play a critical role for tumour development or maintenance. Although Sp1 deregulation might be beneficial for tumour cells, its overexpression induces apoptosis of untransformed cells. Here we further characterised the functional and transcriptional responses of untransformed cells following Sp1 overexpression. Methodology and Principal Findings We made use of wild-type and DNA-binding-deficient Sp1 to demonstrate that the induction of apoptosis by Sp1 is dependent on its capacity to bind DNA. Genome-wide expression profiling identified genes involved in cancer, cell death and cell cycle as being enriched among differentially expressed genes following Sp1 overexpression. In silico search to determine the presence of Sp1 binding sites in the promoter region of modulated genes was conducted. Genes that contained Sp1 binding sites in their promoters were enriched among down-regulated genes. The endogenous sp1 gene is one of the most down-regulated suggesting a negative feedback loop induced by overexpressed Sp1. In contrast, genes containing Sp1 binding sites in their promoters were not enriched among up-regulated genes. These results suggest that the transcriptional response involves both direct Sp1-driven transcription and indirect mechanisms. Finally, we show that Sp1 overexpression led to a modified expression of G1/S transition regulatory genes such as the down-regulation of cyclin D2 and the up-regulation of cyclin G2 and cdkn2c/p18 expression. The biological significance of these modifications was confirmed by showing that the cells accumulated in the G1 phase of the cell cycle before the onset of apoptosis. Conclusion This study shows that the binding to DNA of overexpressed Sp1 induces an inhibition of cell cycle progression that precedes apoptosis and a transcriptional response targeting genes containing Sp1 binding sites in their promoter or not suggesting both direct Sp1-driven transcription and indirect mechanisms. PMID:19753117

Deniaud, Emmanuelle; Baguet, Joël; Chalard, Roxane; Blanquier, Bariza; Brinza, Lilia; Meunier, Julien; Michallet, Marie-Cécile; Laugraud, Aurélie; Ah-Soon, Claudette; Wierinckx, Anne; Castellazzi, Marc; Lachuer, Joël; Gautier, Christian

2009-01-01

87

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. PMID:23728303

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

88

A novel Capsicum gene inhibits host-specific disease resistance to Phytophthora capsici.  

PubMed

A novel disease resistance inhibitor gene (inhibitor of P. capsici resistance [Ipcr]), found in the chile pepper (Capsicum annuum) variety 'New Mexico Capsicum Accession 10399' (NMCA10399), inhibits resistance to Phytophthora capsici but not to other species of Phytophthora. When a highly P. capsici-resistant variety was hybridized with NMCA10399, the resultant F1 populations, when screened, were completely susceptible to P. capsici for root rot and foliar blight disease syndromes, despite the dominance inheritance of P. capsici resistance in chile pepper. The F2 population displayed a 3:13 resistant-to-susceptible (R:S) ratio. The testcross population displayed a 1:1 R:S ratio, and a backcross population to NMCA10399 displayed complete susceptibility. These results demonstrate the presence of a single dominant inhibitor gene affecting P. capsici resistance in chile pepper. Moreover, when lines carrying the Ipcr gene were challenged against six Phytophthora spp., the nonhost resistance was not overcome. Therefore, the Ipcr gene is interfering with host-specific resistance but not the pathogen- or microbe-associated molecular pattern nonhost responses. PMID:23577838

Reeves, Gregory; Monroy-Barbosa, Ariadna; Bosland, Paul W

2013-05-01

89

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

PubMed Central

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

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

2012-01-01

90

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

91

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

92

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

93

HBx Inhibits CYP2E1 Gene Expression via Downregulating HNF4? in Human Hepatoma Cells  

PubMed Central

CYP2E1, one of the cytochrome P450 mixed-function oxidases located predominantly in liver, plays a key role in metabolism of xenobiotics including ethanol and procarcinogens. Recently, down-expression of CYP2E1 was found in hepatocellular carcinoma (HCC) with the majority to be chronic hepatitis B virus (HBV) carriers. In this study, we tested a hypothesis that HBx may inhibit CYP2E1 gene expression via hepatocyte nuclear factor 4? (HNF4?). By enforced HBx gene expression in cultured HepG2 cells, we determined the effect of HBx on CYP2E1 mRNA and protein expression. With a bioinformatics analysis, we found a consensus HNF-4? binding sequence located on ?318 to ?294 bp upstream of human CYP2E1 promoter. Using reporter gene assay and site-directed mutagenesis, we have shown that mutation of this site dramatically decreased CYP2E1 promoter activity. By silencing endogenous HNF-4?, we have further validated knockdown of HNF-4? significantly decreased CYP2E1expression. Ectopic overexpression of HBx in HepG2 cells inhibits HNF-4? expression, and HNF-4? levels were inversely correlated with viral proteins both in HBV-infected HepG2215 cells and as well as HBV positive HCC liver tissues. Moreover, the HBx-induced CYP2E1 reduction could be rescued by ectopic supplement of HNF4? protein expression. Furthermore, human hepatoma cells C34, which do not express CYP2E1, shows enhanced cell growth rate compared to E47, which constitutively expresses CYP2E1. In addition, the significantly altered liver proteins in CYP2E1 knockout mice were detected with proteomics analysis. Together, HBx inhibits human CYP2E1 gene expression via downregulating HNF4? which contributes to promotion of human hepatoma cell growth. The elucidation of a HBx-HNF4?-CYP2E1 pathway provides novel insight into the molecular mechanism underlining chronic HBV infection associated hepatocarcinogenesis. PMID:25238230

Li, Chongyi; Wang, Xiaodong; Cederbaum, Arthur I.; Gan, Lixia; Xie, Bin

2014-01-01

94

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 PMID:7543732

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

1995-01-01

95

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

PubMed

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

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

2015-04-01

96

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

PubMed Central

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

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

2012-01-01

97

The NF2 tumor suppressor gene product, merlin, mediates contact inhibition of growth through interactions with CD44  

Microsoft Academic Search

The neurofibromatosis-2 (NF2) gene encodes merlin, an ezrin-radixin-moesin-(ERM)-related protein that functions as a tumor suppressor. We found that merlin mediates contact inhibition of growth through signals from the extracellular matrix. At high cell density, merlin becomes hypo-phosphorylated and inhibits cell growth in response to hyaluronate (HA), a mucopolysaccharide that surrounds cells. Merlin's growth-inhibitoryactivitydepends on specific interaction with the cy toplasmic

Helen Morrison; James Legg; Fatima Banine; Clare Isacke; Carrie A. Haipek; David H. Gutmann; Helmut Ponta; Peter Herrlich

2001-01-01

98

Inhibition of PPAR gamma 2 gene expression by the HIF-1-regulated gene DEC1/Stra13: a mechanism for regulation of adipogenesis by hypoxia.  

PubMed

Cellular differentiation involves transcriptional responses to environmental stimuli. Adipocyte differentiation is inhibited under hypoxic conditions, indicating that oxygen (O(2)) is an important physiological regulator of adipogenesis. Hypoxia inhibits PPAR gamma 2 nuclear hormone receptor transcription, and overexpression of PPAR gamma 2 or C/EBP beta stimulates adipogenesis under hypoxia. Mouse embryonic fibroblasts deficient in hypoxia-inducible transcription factor 1 alpha (HIF-1 alpha) are refractory to hypoxia-mediated inhibition of adipogenesis. The HIF-1-regulated gene DEC1/Stra13, a member of the Drosophila hairy/Enhancer of split transcription repressor family, represses PPAR gamma 2 promoter activation and functions as an effector of hypoxia-mediated inhibition of adipogenesis. These data indicate that an O(2)-sensitive signaling mechanism regulates adipogenesis. Thus, agents that regulate HIF-1 activity or O(2) sensing may be used to inhibit adipogenesis and control obesity. PMID:11879638

Yun, Zhong; Maecker, Heather L; Johnson, Randall S; Giaccia, Amato J

2002-03-01

99

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

100

Antibodies to the ras gene product inhibit adenylate cyclase and accelerate progesterone-induced cell division in Xenopus laevis oocytes.  

PubMed Central

Microinjection of monoclonal antibodies (lines 238, 172, and 259) directed against the ras gene product, p21, into Xenopus laevis oocytes accelerated progesterone-induced germinal vesicle breakdown. Antibody 238 had the greatest effect on the acceleration of progesterone-induced oocyte maturation, and this effect was correlated with in vitro inhibition of adenylate cyclase (EC 4.6.1.1) activity in a concentration-dependent manner. Inhibition of adenylate cyclase by antibody 238 was also measured in membranes prepared from oocytes pretreated with either cholera toxin or pertussis toxin. These results suggest a role for the ras gene product in the regulation of vertebrate cell adenylate cyclase activity. PMID:3537692

Sadler, S E; Schechter, A L; Tabin, C J; Maller, J L

1986-01-01

101

FOXO1 Transcription Factor Inhibits Luteinizing Hormone ? Gene Expression in Pituitary Gonadotrope Cells*  

PubMed Central

Synthesis of luteinizing hormone (LH) is tightly controlled by a complex network of hormonal signaling pathways that can be modulated by metabolic cues, such as insulin. One group of candidate genes that may be regulated by insulin signaling in pituitary gonadotrope cells is the FOXO subfamily of forkhead transcription factors. In this study we investigated whether FOXO1 is expressed in gonadotropes and if it can modulate LH ?-subunit (Lhb) gene expression. We demonstrated that FOXO1 is expressed in murine gonadotrope cells and that insulin signaling increased FOXO1 phosphorylation and cytoplasmic localization in a PI3K-dependent manner. We also showed that FOXO1 repressed basal transcription and gonadotropin-releasing hormone (GnRH) induction of both the murine and human LHB genes in L?T2 cells, suggesting that FOXO1 regulation of LHB transcription may be conserved between rodents and humans. Although we did not detect FOXO1 binding to the proximal Lhb promoter, the FOXO1 DNA binding domain was necessary for the suppression, suggesting that FOXO1 exerts its effect through protein-protein interactions with transcription factors/cofactors required for Lhb gene expression. FOXO1 repression mapped to the proximal Lhb promoter containing steroidogenic factor 1 (SF1), pituitary homeobox 1 (PTX1), and early growth response protein 1 (EGR1) binding elements. Additionally, FOXO1 blocked induction of the Lhb promoter with overexpressed SF1, PTX1, and EGR1, indicating that FOXO1 repression occurs via these transcription factors but not through regulation of their promoters. In summary, we demonstrate that FOXO1 phosphorylation and cellular localization is regulated by insulin signaling in gonadotropes and that FOXO1 inhibits Lhb transcription. Our study also suggests that FOXO1 may play an important role in controlling LH levels in response to metabolic cues. PMID:22865884

Arriola, David J.; Mayo, Susan L.; Skarra, Danalea V.; Benson, Courtney A.; Thackray, Varykina G.

2012-01-01

102

FOXO1 transcription factor inhibits luteinizing hormone ? gene expression in pituitary gonadotrope cells.  

PubMed

Synthesis of luteinizing hormone (LH) is tightly controlled by a complex network of hormonal signaling pathways that can be modulated by metabolic cues, such as insulin. One group of candidate genes that may be regulated by insulin signaling in pituitary gonadotrope cells is the FOXO subfamily of forkhead transcription factors. In this study we investigated whether FOXO1 is expressed in gonadotropes and if it can modulate LH ?-subunit (Lhb) gene expression. We demonstrated that FOXO1 is expressed in murine gonadotrope cells and that insulin signaling increased FOXO1 phosphorylation and cytoplasmic localization in a PI3K-dependent manner. We also showed that FOXO1 repressed basal transcription and gonadotropin-releasing hormone (GnRH) induction of both the murine and human LHB genes in L?T2 cells, suggesting that FOXO1 regulation of LHB transcription may be conserved between rodents and humans. Although we did not detect FOXO1 binding to the proximal Lhb promoter, the FOXO1 DNA binding domain was necessary for the suppression, suggesting that FOXO1 exerts its effect through protein-protein interactions with transcription factors/cofactors required for Lhb gene expression. FOXO1 repression mapped to the proximal Lhb promoter containing steroidogenic factor 1 (SF1), pituitary homeobox 1 (PTX1), and early growth response protein 1 (EGR1) binding elements. Additionally, FOXO1 blocked induction of the Lhb promoter with overexpressed SF1, PTX1, and EGR1, indicating that FOXO1 repression occurs via these transcription factors but not through regulation of their promoters. In summary, we demonstrate that FOXO1 phosphorylation and cellular localization is regulated by insulin signaling in gonadotropes and that FOXO1 inhibits Lhb transcription. Our study also suggests that FOXO1 may play an important role in controlling LH levels in response to metabolic cues. PMID:22865884

Arriola, David J; Mayo, Susan L; Skarra, Danalea V; Benson, Courtney A; Thackray, Varykina G

2012-09-28

103

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

PubMed

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

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

2015-01-01

104

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. PMID:21334428

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

2011-01-01

105

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

106

Alzheimer’s Disease Risk Gene CD33 Inhibits Microglial Uptake of Amyloid Beta  

PubMed Central

SUMMARY The transmembrane protein CD33 is a sialic acid-binding immunoglobulin-like lectin that regulates innate immunity but has no known functions in the brain. We have previously shown that the CD33 gene is a risk factor for Alzheimer’s disease (AD). Here, we observed increased expression of CD33 in microglial cells in AD brain. The minor allele of the CD33 SNP rs3865444, which confers protection against AD, was associated with reductions in both CD33 expression and insoluble amyloid beta 42 (A?42) levels in AD brain. Furthermore, the numbers of CD33-immunoreactive microglia were positively correlated with insoluble A?42 levels and plaque burden in AD brain. CD33 inhibited uptake and clearance of A?42 in microglial cell cultures. Finally, brain levels of insoluble A?42 as well as amyloid plaque burden were markedly reduced in APPSwe/PS1?E9/CD33?/? mice. Therefore, CD33 inactivation mitigates A? pathology and CD33 inhibition could represent a novel therapy for AD. PMID:23623698

Griciuc, Ana; Serrano-Pozo, Alberto; Parrado, Antonio R.; Lesinski, Andrea N.; Asselin, Caroline N.; Mullin, Kristina; Hooli, Basavaraj; Choi, Se Hoon; Hyman, Bradley T.; Tanzi, Rudolph E.

2013-01-01

107

Inhibition of Corticosteroid-Binding Globulin Gene Expression by Glucocorticoids Involves C/EBP?  

PubMed Central

Corticosteroid-binding globulin (CBG), a negative acute phase protein produced primarily in the liver, is responsible for the transport of glucocorticoids (GCs). It also modulates the bioavailability of GCs, as only free or unbound steroids are biologically active. Fluctuations in CBG levels therefore can directly affect GC bioavailability. This study investigates the molecular mechanism whereby GCs inhibit the expression of CBG. GCs regulate gene expression via the glucocorticoid receptor (GR), which either directly binds to DNA or acts indirectly via tethering to other DNA-bound transcription factors. Although no GC-response elements (GRE) are present in the Cbg promoter, putative binding sites for C/EBP?, able to tether to the GR, as well as HNF3? involved in GR signaling, are present. C/EBP?, but not HNF3?, was identified as an important mediator of DEX-mediated inhibition of Cbg promoter activity by using specific deletion and mutant promoter reporter constructs of Cbg. Furthermore, knockdown of C/EBP? protein expression reduced DEX-induced repression of CBG mRNA, confirming C/EBP?’s involvement in GC-mediated CBG repression. Chromatin immunoprecipitation (ChIP) after DEX treatment indicated increased co-recruitment of C/EBP? and GR to the Cbg promoter, while C/EBP? knockdown prevented GR recruitment. Together, the results suggest that DEX repression of CBG involves tethering of the GR to C/EBP?. PMID:25335188

Verhoog, Nicolette; Allie-Reid, Fatima; Vanden Berghe, Wim; Smith, Carine; Haegeman, Guy; Hapgood, Janet; Louw, Ann

2014-01-01

108

Inhibition of Growth and Gene Expression by PNA-peptide Conjugates in Streptococcus pyogenes.  

PubMed

While Streptococcus pyogenes is consistently susceptible toward penicillin, therapeutic failure of penicillin treatment has been reported repeatedly and a considerable number of patients exhibit allergic reactions to this substance. At the same time, streptococcal resistance to alternative antibiotics, e.g., macrolides, has increased. Taken together, these facts demand the development of novel therapeutic strategies. In this study, S. pyogenes growth was inhibited by application of peptide-conjugated antisense-peptide nucleic acids (PNAs) specific for the essential gyrase A gene (gyrA). Thereby, HIV-1 Tat peptide-coupled PNAs were more efficient inhibitors of streptococcal growth as compared with (KFF)3K-coupled PNAs. Peptide-anti-gyrA PNAs decreased the abundance of gyrA transcripts in S. pyogenes. Growth inhibition by antisense interference was enhanced by combination of peptide-coupled PNAs with protein-level inhibitors. Antimicrobial synergy could be detected with levofloxacin and novobiocin, targeting the gyrase enzyme, and with spectinomycin, impeding ribosomal function. The prospective application of carrier peptide-coupled antisense PNAs in S. pyogenes covers the use as an antimicrobial agent and the employment as a knock-down strategy for the investigation of virulence factor function.Molecular Therapy-Nucleic Acids (2013) 2, e132; doi:10.1038/mtna.2013.62; published online 5 November 2013. PMID:24193033

Patenge, Nadja; Pappesch, Roberto; Krawack, Franziska; Walda, Claudia; Mraheil, Mobarak Abu; Jacob, Anette; Hain, Torsten; Kreikemeyer, Bernd

2013-01-01

109

Cyclosporin A inhibits T-cell growth factor gene expression at the level of mRNA transcription.  

PubMed Central

Cyclosporin A (CsA) is a potent immunosuppressive agent, now gaining wide application in human organ transplantation. The immunosuppressive activity of CsA is at least in part due to inhibition of lymphokine production by activated T lymphocytes. Specifically, inhibition of T-cell growth factor (TCGF; also designated interleukin 2) production appears to be an important pathway by which CsA impairs T-cell function. To define further both the specificity of CsA and the level at which it interferes with lymphokine gene expression, we have studied its effects on TCGF mRNA accumulation as well as TCGF gene transcription. These studies were performed with a cloned human leukemic T-cell line (Jurkat, subclone 32), which can be induced with phytohemagglutinin and phorbol 12-myristate 13-acetate to produce large amounts of TCGF. In these cells, high levels of TCGF mRNA were present in induced but not in uninduced Jurkat cells as judged by hybridization to a cloned human TCGF cDNA probe. CsA completely inhibited induced TCGF mRNA accumulation at concentrations of 0.3-1.0 microgram/ml, whereas low levels of appropriately sized TCGF mRNA were present at 0.01 microgram/ml. In nuclear transcription experiments, CsA inhibited the synthesis of TCGF transcripts in a dose-dependent manner with complete inhibition at a concentration of 1 microgram/ml. In contrast, CsA did not inhibit the expression of two other inducible genes, TCGF receptor and HT-3. Further, HLA gene expression was also less affected than TCGF in CsA-treated cells. These data suggest a relatively selective action of CsA on TCGF gene transcription. Images PMID:6332315

Krönke, M; Leonard, W J; Depper, J M; Arya, S K; Wong-Staal, F; Gallo, R C; Waldmann, T A; Greene, W C

1984-01-01

110

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. PMID:22363239

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

2011-01-01

111

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. PMID:24932966

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

2014-01-01

112

Thyrotrope expression and thyroid hormone inhibition map to different regions of the mouse glycoprotein hormone alpha-subunit gene promoter.  

PubMed

The alpha-subunit gene of the glycoprotein hormones is normally expressed in pituitary thyrotropes and gonadotropes and in placental cells. Thus, this gene must contain elements that mediate expression and hormonal responses in different cell types. The localization of DNA regions important for expression and regulation of the alpha-subunit gene in thyrotrope cells has not previously been reported. In these studies luciferase expression constructs containing 1700 basepairs of 5' flanking DNA derived from the mouse alpha-subunit gene were introduced by electroporation into freshly dispersed cells from TSH-producing mouse pituitary tumors (TtT 97). This promoter functioned with greater efficiency in thyrotropes than in nonthyrotrope pituitary GH4 cells and L-cell fibroblasts. Primer extension confirmed that transcription from the alpha-subunit constructs initiated at the same site as the endogenous gene. Studies using 5' truncations showed a progressive loss of alpha-subunit promoter activity in thyrotropes between -480 and -120, with regions upstream of -254 contributing substantially to expression in thyrotrope cells. Thyroid hormone inhibited alpha-subunit promoter activity in a dose-dependent fashion, although in vivo treatment of tumors with thyroid hormone before transfection was necessary to achieve maximal inhibition. Thyroid hormone inhibition of alpha-subunit promoter activity also occurred in GH4 cells, but no effect was observed in L-cells. Studies using 5' truncations localized a region responsible for thyroid hormone inhibition between -62 and +43, encompassing the TATA sequence and the transcriptional initiation site. When this region was compared to the thyroid hormone inhibitory regions of the alpha-subunit genes from other species and the mouse TSH beta-subunit gene, a 6-basepair motif, 5' (G/A)GTG(G/A)G 3', emerged as a possible consensus sequence for a thyroid hormone inhibitory element. PMID:1696883

Sarapura, V D; Wood, W M; Gordon, D F; Ocran, K W; Kao, M Y; Ridgway, E C

1990-09-01

113

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

PubMed Central

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

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

2014-01-01

114

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

115

Herpes simplex virus gene products required for viral inhibition of expression of G1-phase functions.  

PubMed

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 the wild-type virus, the ICP27 mutant virus was defective for blocking the phosphorylation of pRb proteins, and the normal pRb pattern was restored in cells infected with a rescued virus. The virion host shutoff (vhs) function, DNA replication, and late gene functions were not required for the virus-induced effects on pRb protein. BrdU incorporation in synchronized HSV-infected cells showed that ICP27 was required for blocking the cell cycle in the G1 phase. Furthermore, ICP27, ICP4, ICP0, and vhs were required for blocking the induction of the G1 cell cycle regulators cyclin D1 and cdk4 in HSV-infected cells. Both ICP27 and the vhs function contributed to the reduction of cyclin D1 mRNA levels in HSV-infected cells: These results provide evidence that HSV-1 ICP27 protein is essential for viral inhibition of G1-phase functions and that certain other HSV proteins are required for some of the viral effects on the cell cycle. Finally, these results show that HSV-1 ICP27 and vhs act jointly to reduce host mRNA levels in infected cells. PMID:11883196

Song, B; Yeh, K C; Liu, J; Knipe, D M

2001-11-25

116

Differential expression of calcium transport genes caused by COMT inhibition in the duodenum, kidney and placenta of pregnant mice.  

PubMed

Preeclampsia is a pregnancy-specific disease characterized by concurrent development of hypertension, proteinuria, and oxidative stress in the placenta. Preeclampsia-like genetic models were also developed by modification of preeclampsia-related genes, such as catechol-O-methyltranferase (COMT). In this study, we induced COMT inhibition in mice during pregnancy in order to reproduce physiological conditions associated with preeclampsia. Expression of the gene known as hypoxia biomarker, HIF-1?, was highly induced in the placenta of this model. The over-expression of HIF-1? demonstrates that our experimental conditions were similar to those of preeclampsia. We measured the expression of several calcium transport genes (CTGs; TRPV5, TRPV6, PMCA1 and CaBP-9k) in the placenta, duodenum and kidney after COMT inhibition on gestation day 17.5 (GD 17.5). In addition, we evaluated the calcium transporters in the kidney, duodenum of non-pregnant female mice. Placental TRPV5, TRPV6 and PMCA1 expressions were down-regulated by COMT inhibitor (ro41-0960). In addition, the reduced PMCA1 expression in the placenta was reversed by calcium supplementation. Duodenal expressions of TRPV5, TRPV6, and PMCA1 were decreased in COMT-inhibited mice, and recovered slightly after calcium supplementation. Renal expression of TRPV5, TRPV6, and PMCA1 was also decreased by COMT inhibition, while it was reversed by calcium supplementation to the level of control. Duodenal- and renal calcium transporting genes, TRPV5, TPRV6, PMCA1 and CaBP-9k, were down-regulated by COMT treatment in female mice. Taken together, these results indicate that physiological changes observed in COMT inhibition were similar to symptoms of preeclampsia, which may be related to disturbance of calcium metabolism during pregnancy. PMID:25486511

Yang, Hyun; Ahn, Changhwan; Jeung, Eui-Bae

2015-02-01

117

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. PMID:24081951

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

2013-01-01

118

Proteasome inhibition enhances the killing effect of BikDD gene therapy  

PubMed Central

BikDD, a phosphorylation-mimic mutant of pro-apoptotic protein Bik, elicits strong apoptosis in cancer cells when introduced via an expression platform termed VP16-GAL4-WPRE integrated systemic amplifier (VISA) under the control of a cancer-specific promoter both in vitro and in vivo. C-VISA-BikDD expression plasmid encapsulated in liposomes is currently in the process to initiate a phase I clinical trial for pancreatic cancer. In this study, we report a potential combination approach of BikDD with proteasome inhibitors on the basis of our findings that exogenously expressed BikDD protein undergoes proteasome-mediated degradation via both ubiquitin-dependent and -independent pathways. Inhibition of proteasome increases the protein stability of BikDD, enhancing the apoptotic effect of BikDD. Hence, high proteasome activity may be a mechanism by which intrinsic and acquired resistance occurs in BikDD gene therapy, and a combination therapy with current clinically approved proteasome inhibitor may overcome resistance.

Sun, Ye; Ponz-Sarvise, Mariano; Chang, Shih-Shin; Chang, Wei-Chao; Chen, Chung-Hsuan; Hsu, Jennifer L; Hung, Mien-Chie

2015-01-01

119

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

120

Anti-gene peptide nucleic acid specifically inhibits MYCN expression in human neuroblastoma cells leading to cell growth inhibition and apoptosis.  

PubMed

We developed an anti-gene peptide nucleic acid (PNA) for selective inhibition of MYCN transcription in neuroblastoma cells, targeted against a unique sequence in the antisense DNA strand of exon 2 of MYCN and linked at its NH(2) terminus to a nuclear localization signal peptide. Fluorescence microscopy showed specific nuclear delivery of the PNA in six human neuroblastoma cell lines: GI-LI-N and IMR-32 (MYCN-amplified/overexpressed); SJ-N-KP and NB-100 (MYCN-unamplified/low-expressed); and GI-CA-N and GI-ME-N (MYCN-unamplified/unexpressed). Antiproliferative effects were observable at 24 hours (GI-LI-N, 60%; IMR-32, 70%) and peaked at 72 hours (GI-LI-N, 80%; IMR-32, 90%; SK-N-KP, 60%; NB-100, 50%); no reduction was recorded for GI-CA-N and GI-ME-N (controls). In MYCN-amplified/overexpressed IMR-32 cells and MYCN-unamplified/low-expressed SJ-N-KP cells, inhibition was recorded of MYCN mRNA (by real-time PCR) and N-Myc (Western blotting); these inhibitory effects increased over 3 days after single treatment in IMR-32. Anti-gene PNA induced G(1)-phase accumulation (39-53%) in IMR-32 and apoptosis (56% annexin V-positive cells at 24 hours in IMR-32 and 22% annexin V-positive cells at 48 hours in SJ-N-KP). Selective activity of the PNA was shown by altering three point mutations, and by the observation that an anti-gene PNA targeted against the noncoding DNA strand did not exert any effect. These findings could encourage research into development of an anti-gene PNA-based tumor-specific agent for neuroblastoma (and other neoplasms) with MYCN expression. PMID:15897242

Tonelli, Roberto; Purgato, Stefania; Camerin, Consuelo; Fronza, Raffaele; Bologna, Fabrizio; Alboresi, Simone; Franzoni, Monica; Corradini, Roberto; Sforza, Stefano; Faccini, Andrea; Shohet, Jason M; Marchelli, Rosangela; Pession, Andrea

2005-05-01

121

HPV-16 E2 contributes to induction of HPV-16 late gene expression by inhibiting early polyadenylation  

PubMed Central

We provide evidence that the human papillomavirus (HPV) E2 protein regulates HPV late gene expression. High levels of E2 caused a read-through at the early polyadenylation signal pAE into the late region of the HPV genome, thereby inducing expression of L1 and L2 mRNAs. This is a conserved property of E2 of both mucosal and cutaneous HPV types. Induction could be reversed by high levels of HPV-16 E1 protein, or by the polyadenylation factor CPSF30. HPV-16 E2 inhibited polyadenylation in vitro by preventing the assembly of the CPSF complex. Both the N-terminal and hinge domains of E2 were required for induction of HPV late gene expression in transfected cells as well as for inhibition of polyadenylation in vitro. Finally, overexpression of HPV-16 E2 induced late gene expression from a full-length genomic clone of HPV-16. We speculate that the accumulation of high levels of E2 during the viral life cycle, not only turns off the expression of the pro-mitotic viral E6 and E7 genes, but also induces the expression of the late HPV genes L1 and L2. PMID:22617423

Johansson, Cecilia; Somberg, Monika; Li, Xiaoze; Backström Winquist, Ellenor; Fay, Joanna; Ryan, Fergus; Pim, David; Banks, Lawrence; Schwartz, Stefan

2012-01-01

122

Discovery of Inhibitors of Aberrant Gene Transcription from Libraries of DNA Binding Molecules: Inhibition of LEF-1 Mediated Gene Transcription and Oncogenic Transformation  

PubMed Central

The screening of a >9000 compound library of synthetic DNA binding molecules for selective binding to the consensus sequence of the transcription factor LEF-1 followed by assessment of the candidate compounds in a series of assays that characterized functional activity (disruption of DNA–LEF-1 binding) at the intended target and site (inhibition of intracellular LEF-1 mediated gene transcription) resulting in a desired phenotypic cellular change (inhibit LEF-1 driven cell transformation) provided two lead compounds: lefmycin-1 and lefmycin-2. The sequence of screens defining the approach assures that activity in the final functional assay may be directly related to the inhibition of gene transcription and DNA binding properties of the identified molecules. Central to the implementation of this generalized approach to the discovery of DNA binding small molecule inhibitors of gene transcription was: (1) the use of a technically non-demanding fluorescent intercalator displacement (FID) assay for initial assessment of the DNA binding affinity and selectivity of a library of compounds for any sequence of interest, and (2) the technology used to prepare a sufficiently large library of DNA binding compounds. PMID:19216569

Stover, James S.; Shi, Jin; Jin, Wei; Vogt, Peter K.; Boger, Dale L.

2009-01-01

123

Vitamin D Growth Inhibition of Breast Cancer Cells: Gene Expression Patterns Assessed by cDNA Microarray  

Microsoft Academic Search

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3], the active metabolite of vitamin D, is a potent inhibitor of breast cancer cell growth. Although it is evident that 1,25(OH)2D3 inhibits growth of both estrogen receptor alpha-positive [ERa(+)] and -negative [ERa(-)] breast cancer cells, the cellular pathways contributing to these effects remain unclear. We studied the gene expression patterns in ERa(+) MCF-7 and ERa(-) MDA MB

Srilatha Swami; Nalini Raghavachari; Uwe R. Muller; Yijia P. Bao; David Feldman

2003-01-01

124

Inhibition of core gene of HCV 3a genotype using synthetic and vector derived siRNAs  

PubMed Central

Background Hepatitis C virus (HCV) is a major causative agent of liver associated diseases throughout the world, with genotype 3a responsible for most of the cases in Pakistan. Due to the limited efficiency of current therapy, RNA interference (RNAi) a novel regulatory and powerful silencing approach for molecular therapeutics through a sequence-specific RNA degradation process represents an alternative option. Results The current study was purposed to assess and explore the possibility of RNAi to silence the HCV-3a Core gene expression, which play complex role in regulation of cell growth and host genes expression essential for infectivity and disease progression. To identify the potent siRNA target sites, 5 small interfering RNAs (siRNAs) against Core gene were designed and in vitro transcribed after consensus sequence analysis of different HCV-3a isolates. Antiviral effects of siRNAs showed upto 80% inhibition of Core gene expression by different siRNAs into Huh-7 cells as compared with Mock transfected and control siRNAs treated cells. For long lasting effect of siRNAs, vector based short hairpin siRNAs (shRNAs) were designed and tested against HCV-3a Core which resulted in a similar pattern of inhibition on RNA and protein expression of HCV Core as synthetic siRNAs. Furthermore, the efficacy of cell culture tested siRNA and shRNA, were evaluated for inhibition of HCV replication in HCV infected serum inoculated Huh-7 cells and a significant decrease in HCV viral copy number was observed. Conclusions Our results support the possibility of using consensus siRNA and shRNA-based molecular therapy as a promising strategy in effective inhibition of HCV-3a genotype. PMID:21073745

2010-01-01

125

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

126

Retinoid X receptor and peroxisome proliferator-activated receptor-gamma agonists cooperate to inhibit matrix metalloproteinase gene expression  

Microsoft Academic Search

INTRODUCTION: We recently described the ability of retinoid X receptor (RXR) ligand LG100268 (LG268) to inhibit interleukin-1-beta (IL-1-?)-driven matrix metalloproteinase-1 (MMP-1) and MMP-13 gene expression in SW-1353 chondrosarcoma cells. Other investigators have demonstrated similar effects in chondrocytes treated with rosiglitazone, a ligand for peroxisome proliferator-activated receptor-gamma (PPAR?), for which RXR is an obligate dimerization partner. The goals of this study

Peter S Burrage; Adam C Schmucker; Yanqing Ren; Michael B Sporn; Constance E Brinckerhoff

2008-01-01

127

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

PubMed

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

128

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

129

Novel oligoamine analogues inhibit lysine-specific demethylase 1 (LSD1) and induce re-expression of epigenetically silenced genes  

PubMed Central

Purpose Abnormal DNA CpG island hypermethylation and transcriptionally repressive histone modifications are associated with the aberrant silencing of tumor suppressor genes. Lysine methylation is a dynamic, enzymatically-controlled process. Lysine-specific demethylase 1 (LSD1) has recently been identified as a histone lysine demethylase. LSD1 specifically catalyzes demethylation of mono- and dimethyl-lysine 4 of histone 3, key positive chromatin marks associated with transcriptional activation. We hypothesized that a novel class of oligoamine analogues would effectively inhibit LSD1 and thus cause the re-expression of aberrantly silenced genes. Experimental Design Human colorectal cancer cells were treated with the oligoamines and changes in mono- and dimethyl-lysine 4 of histone 3 (H3K4) and other chromatin marks were monitored. In addition, treated cells were evaluated for the re-expression of the aberrantly silenced secreted frizzled-related proteins (SFRPs) Wnt signaling pathway antagonist genes. Finally, the effects of the LSD1 inhibitors were evaluated in an in vivo xenograft model. Results Treatment of HCT116 human colon adenocarcinoma cells in vitro resulted in increased H3K4 methylation and re-expression of silenced SFRP genes. This re-expression is also accompanied by a decrease in H3K9me2 repressive mark. Importantly, co-treatment with low doses of oligoamines and a DNA methyltransferase (DNMT) inhibitor highly induces the re-expression of the aberrantly silenced SFRP2 gene and results in significant inhibition of the growth of established tumors in a human colon tumor model in vivo. Conclusions The use of LSD1-inhibiting oligoamine analogues in combination with DNMT inhibitors represents a highly promising and novel approach for epigenetic therapy of cancer. PMID:19934284

Huang, Yi; Stewart, Tracy Murray; Wu, Yu; Baylin, Stephen B.; Marton, Laurence J.; Perkins, Brandy; Jones, Richard J.; Woster, Patrick M.; Casero, Robert A.

2009-01-01

130

Replication-incompetent herpesvirus vector delivery of an interferon alpha gene inhibits human immunodeficiency virus replication in human monocytes.  

PubMed Central

Human monocytes and macrophages are nondividing cells that serve as a major reservoir for human immunodeficiency virus (HIV) at all stages of infection. To investigate viral-mediated gene delivery as a means of inhibiting HIV replication in human monocytes, a replication-incompetent herpes simplex virus vector was developed that expressed human interferon alpha. Monocytes infected with this herpes simplex virus vector and then challenged with HIV showed dramatically reduced cytopathic effects and HIV replication compared to control treated monocytes. Similar effects on HIV replication were observed if monocytes were first infected with HIV and then treated with the recombinant vectors. These results demonstrate that replication-incompetent herpes simplex virus gene delivery of interferon alpha directly to human monocytes can greatly decrease HIV replication and suggest that such a vector might deliver therapeutically important genes directly to sites of HIV infection. Images Fig. 1 Fig. 2 Fig. 3 PMID:8415668

Weir, J P; Elkins, K L

1993-01-01

131

Inhibition and genetic deficiency of p38 MAPK up-regulates heme oxygenase-1 gene expression via Nrf2.  

PubMed

Heme oxygenase (HO)-1 is the inducible isoform of the first and rate-limiting enzyme of heme degradation. The HO products carbon monoxide and bilirubin not only provide antioxidant cytoprotection, but also have potent anti-inflammatory and immunomodulatory functions. Although HO-1 has previously been shown to be induced by various stimuli via activation of the p38 MAPK signaling pathway, the role of this protein kinase for HO-1 gene regulation is largely unknown. In the present study, it is demonstrated that pharmacological inhibitors of p38 induced HO-1 expression in monocytic cells. Moreover, basal HO-1 gene expression levels were markedly higher in untreated murine embryonic fibroblasts (MEF) from p38alpha(-/-) mice compared with those from wild-type mice. Transfection studies with luciferase reporter gene constructs indicate that increased HO-1 gene expression via inhibition of p38 was mediated by the transcription factor Nrf2, which is a central regulator of the cellular oxidative stress response. Accordingly, inhibitors of p38 induced binding of nuclear proteins to a Nrf2 target sequence of the HO-1 promoter, but did not affect HO-1 protein expression and promoter activity in Nrf2(-/-) MEF. Genetic deficiency of p38 led to enhanced phosphorylation of ERK and increased cellular accumulation of reactive oxygen species. In addition, pharmacological blockage of ERK and scavenging of reactive oxygen species with N-acetylcysteine reduced HO-1 gene expression in p38(-/-) MEF, respectively. Taken together, it is demonstrated that pharmacological inhibition and genetic deficiency of p38 induce HO-1 gene expression via a Nrf2-dependent mechanism in monocytic cells and MEF. PMID:19454702

Naidu, Srivatsava; Vijayan, Vijith; Santoso, Sentot; Kietzmann, Thomas; Immenschuh, Stephan

2009-06-01

132

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

PubMed Central

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

2005-01-01

133

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. PMID:20957031

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

2010-01-01

134

Gene silencing of 4-1BB by RNA interference inhibits acute rejection in rats with liver transplantation.  

PubMed

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. PMID:23484089

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

2013-01-01

135

Cartilage oligomeric matrix protein gene multilayers inhibit osteogenic differentiation and promote chondrogenic differentiation of mesenchymal stem cells.  

PubMed

There are still many challenges to acquire the optimal integration of biomedical materials with the surrounding tissues. Gene coatings on the surface of biomaterials may offer an effective approach to solve the problem. In order to investigate the gene multilayers mediated differentiation of mesenchymal stem cells (MSCs), gene functionalized films of hyaluronic acid (HA) and lipid-DNA complex (LDc) encoding cartilage oligomeric matrix protein (COMP) were constructed in this study via the layer-by-layer self-assembly technique. Characterizations of the HA/DNA multilayered films indicated the successful build-up process. Cells could be directly transfected by gene films and a higher expression could be obtained with the increasing bilayer number. The multilayered films were stable for a long period and DNA could be easily released in an enzymatic condition. Real-time polymerase chain reaction (RT-PCR) assay presented significantly higher (p<0.01) COMP expression of MSCs cultured with HA/COMP multilayered films. Compared with control groups, the osteogenic gene expression levels of MSCs with HA/COMP multilayered films were down-regulated while the chondrogenic gene expression levels were up-regulated. Similarly, the alkaline phosphatase (ALP) staining and Alizarin red S staining of MSCs with HA/COMP films were weakened while the alcian blue staining was enhanced. These results demonstrated that HA/COMP multilayered films could inhibit osteogenic differentiation and promote chondrogenic differentiation of MSCs, which might provide new insight for physiological ligament-bone healing. PMID:25380520

Guo, Peng; Shi, Zhong-Li; Liu, An; Lin, Tiao; Bi, Fang-Gang; Shi, Ming-Min; Yan, Shi-Gui

2014-01-01

136

Cartilage Oligomeric Matrix Protein Gene Multilayers Inhibit Osteogenic Differentiation and Promote Chondrogenic Differentiation of Mesenchymal Stem Cells  

PubMed Central

There are still many challenges to acquire the optimal integration of biomedical materials with the surrounding tissues. Gene coatings on the surface of biomaterials may offer an effective approach to solve the problem. In order to investigate the gene multilayers mediated differentiation of mesenchymal stem cells (MSCs), gene functionalized films of hyaluronic acid (HA) and lipid-DNA complex (LDc) encoding cartilage oligomeric matrix protein (COMP) were constructed in this study via the layer-by-layer self-assembly technique. Characterizations of the HA/DNA multilayered films indicated the successful build-up process. Cells could be directly transfected by gene films and a higher expression could be obtained with the increasing bilayer number. The multilayered films were stable for a long period and DNA could be easily released in an enzymatic condition. Real-time polymerase chain reaction (RT-PCR) assay presented significantly higher (p < 0.01) COMP expression of MSCs cultured with HA/COMP multilayered films. Compared with control groups, the osteogenic gene expression levels of MSCs with HA/COMP multilayered films were down-regulated while the chondrogenic gene expression levels were up-regulated. Similarly, the alkaline phosphatase (ALP) staining and Alizarin red S staining of MSCs with HA/COMP films were weakened while the alcian blue staining was enhanced. These results demonstrated that HA/COMP multilayered films could inhibit osteogenic differentiation and promote chondrogenic differentiation of MSCs, which might provide new insight for physiological ligament-bone healing. PMID:25380520

Guo, Peng; Shi, Zhong-Li; Liu, An; Lin, Tiao; Bi, Fang-Gang; Shi, Ming-Min; Yan, Shi-Gui

2014-01-01

137

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

PubMed

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

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

2010-12-01

138

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

139

Species-Specific Dibutyl Phthalate Fetal Testis Endocrine Disruption Correlates with Inhibition of SREBP2-Dependent Gene Expression Pathways  

PubMed Central

Fetal rat phthalate exposure produces a spectrum of male reproductive tract malformations downstream of reduced Leydig cell testosterone production, but the molecular mechanism of phthalate perturbation of Leydig cell function is not well understood. By bioinformatically examining fetal testis expression microarray data sets from susceptible (rat) and resistant (mouse) species after dibutyl phthalate (DBP) exposure, we identified decreased expression of several metabolic pathways in both species. However, lipid metabolism pathways transcriptionally regulated by sterol regulatory element–binding protein (SREBP) were inhibited in the rat but induced in the mouse, and this differential species response corresponded with repression of the steroidogenic pathway. In rats exposed to 100 or 500 mg/kg DBP from gestational days (GD) 16 to 20, a correlation was observed between GD20 testis steroidogenic inhibition and reductions of testis cholesterol synthesis endpoints including testis total cholesterol levels, Srebf2 gene expression, and cholesterol synthesis pathway gene expression. SREBP2 expression was detected in all fetal rat testis cells but was highest in Leydig cells. Quantification of SREBP2 immunostaining showed that 500 mg/kg DBP exposure significantly reduced SREBP2 expression in rat fetal Leydig cells but not in seminiferous cords. By Western analysis, total rat testis SREBP2 levels were not altered by DBP exposure. Together, these data suggest that phthalate-induced inhibition of fetal testis steroidogenesis is closely associated with reduced activity of several lipid metabolism pathways and SREBP2-dependent cholesterologenesis in Leydig cells. PMID:21266533

Johnson, Kamin J.; McDowell, Erin N.; Viereck, Megan P.; Xia, Jessie Q.

2011-01-01

140

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. PMID:23805945

2013-01-01

141

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

142

Reovirus inhibition of cellular DNA synthesis: role of the S1 gene.  

PubMed

Type 3 reovirus inhibits L cell DNA synthesis, whereas type 1 reovirus exerts little or no effect on L cell DNA synthesis. By using recombinant viruses containing both type 1 and type 3 double-standard RNA segments, we determined that one double-stranded RNA segment, the reovirus type 3 S1 double-stranded RNA segment which encodes the viral hemagglutinin, segregates with and is responsible for the capacity of reovirus type 3 to inhibit L cell DNA synthesis. PMID:7241660

Sharpe, A H; Fields, B N

1981-04-01

143

Human DNA methyltransferase gene-transformed yeasts display an inducible flocculation inhibited by 5-aza-2'-deoxycytidine.  

PubMed

Mammalian DNA methyltransferases (DNMTs) play an important role in establishing and maintaining the proper regulation of epigenetic information. However, it remains unclear whether mammalian DNMTs can be functionally expressed in yeasts, which probably lack endogenous DNMTs. We cotransformed the budding yeast Saccharomyces cerevisiae with the human DNMT1 gene, which encodes a methylation maintenance enzyme, and the DNMT3A/3B genes, which encode de novo methylation enzymes, in an expression vector also containing the GAL1 promoter, which is induced by galactose, and examined the effects of the DNMT inhibitor 5-aza-2'-deoxycytidine (5AZ) on cell growth. Transformed yeast strains grown in galactose- and glucose-containing media showed growth inhibition, and their growth rate was unaffected by 5AZ. Conversely, 5AZ, but not 2'-deoxycytidine, dose-dependently interfered with the flocculation exhibited by DNMT-gene transformants grown in glucose-containing medium. Further investigation of the properties of this flocculation indicated that it may be dependent on the expression of a Flocculin-encoding gene, FLO1. Taken together, these findings suggest that DNMT-gene transformed yeast strains functionally express these enzymes and represent a useful tool for in vivo screening for DNMT inhibitors. PMID:25511699

Sugiyama, Kei-Ichi; Takamune, Makiko; Furusawa, Hiroko; Honma, Masamitsu

2015-01-01

144

Specific inhibition of histone deacetylase 8 reduces gene expression and production of proinflammatory cytokines in vitro and in vivo.  

PubMed

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

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

2015-01-23

145

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

PubMed Central

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

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

2014-01-01

146

Identification of functional toxin/immunity genes linked to contact-dependent growth inhibition (CDI) and rearrangement hotspot (Rhs) systems.  

PubMed

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. PMID:21829394

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

2011-08-01

147

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

148

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

149

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

150

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

151

The Krüppel-like factor KLF15 inhibits transcription of the adrenomedullin gene in adipocytes  

Microsoft Academic Search

KLF15 (Krüppel-like factor 15) plays a key role in adipocyte differentiation and glucose transport in adipocytes through activation of its target genes. We have now identified six target genes regulated directly by KLF15 in 3T3-L1 mouse adipocytes with the use of a combination of microarray-based chromatin immunoprecipitation and gene expression analyses. We confirmed the direct regulation by KLF15 of one

Tomoki Nagare; Hiroshi Sakaue; Mototsugu Takashima; Kazuhiro Takahashi; Hideyuki Gomi; Yasushi Matsuki; Eijiro Watanabe; Ryuji Hiramatsu; Wataru Ogawa; Masato Kasuga

2009-01-01

152

Ebola Virus Inhibits Induction of Genes by Double-Stranded RNA in Endothelial Cells  

Microsoft Academic Search

Fatal cases of filoviral infection are accompanied by a marked immunosuppression. Endothelial cells play a vital role in the host immune response through the expression of several immunomodulatory genes in addition to the expression of the antiviral genes, 2?,5?-oligoadenylate synthetase [2?-5?(A)N], and the double-stranded RNA (dsRNA)-activated protein kinase (PKR). dsRNA, an intermediate generated during viral replication and gene transcription of

Brian H. Harcourt; Anthony Sanchez; Margaret K. Offermann

1998-01-01

153

Oncogenic Forms of p53 Inhibit p53-Regulated Gene Expression  

Microsoft Academic Search

Mutant forms of the gene encoding the tumor suppressor p53 are found in numerous human malignancies, but the physiologic function of p53 and the effects of mutations on this function are unknown. The p53 protein binds DNA in a sequence-specific manner and thus may regulate gene transcription. Cotransfection experiments showed that wild-type p53 activated the expression of genes adjacent to

Scott E. Kern; Jennifer A. Pietenpol; Sam Thiagalingam; Albert Seymour; Kenneth W. Kinzler; Bert Vogelstein

1992-01-01

154

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

PubMed

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

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

2015-05-01

155

Interleukin-32? modulates promyelocytic leukemia zinc finger gene activity by inhibiting protein kinase C?-dependent sumoylation.  

PubMed

Interleukin-32 (IL-32) is a proinflammatory cytokine. However, there is growing evidence that IL-32 also plays a mediatory role intracellularly. In this study, we present evidence that IL-32? modifies and inhibits promyelocytic leukemia zinc finger (PLZF), a sequence-specific transcriptional regulator that regulates the expression of a subset of interferon (IFN)-stimulated genes (ISGs). We screened IL-32?-interacting proteins in a human spleen cDNA library using the yeast two-hybrid assay, and investigated the functional relevance of the interaction between IL-32? and PLZF. We demonstrated that IL-32? interacts with protein kinase C (PKC)? and PKC? in a phorbol 12-myristate 13-acetate (PMA) dependent way, and that PKC? regulates the interaction of IL-32? with PLZF. We verified the involvement of PKC? in the interaction between these proteins by using various PKC inhibitors. PLZF is known to be modified by small ubiquitin-like modifier (SUMO)-1, but it is unclear whether SUMO-2 conjugation of PLZF occurs. We showed that IL-32? inhibited SUMO-2-conjugation of PLZF. Further, we demonstrated that sumoylated PLZF decreased when IL-32? was co-expressed. PKC? affected the sumoylation of PLZF only in the presence of IL-32? because PKC inhibitor treatment did not reduce PLZF sumoylation in the absence of IL-32?. We finally investigated whether IL-32?-mediated inhibition of PLZF sumoylation affected the transcriptional activity of PLZF, and demonstrated that the inhibition of sumoylation of PLZF by IL-32? down-regulated ISGs induced by PLZF. Together, our data suggest that IL-32? associates with PLZF and PKC?, and then inhibits PLZF sumoylation, resulting in suppression of the transcriptional activity of PLZF. PMID:25178676

Park, Yun Sun; Kang, Jeong-Woo; Lee, Dong Hun; Kim, Man Sub; Bak, Yesol; Yang, Young; Lee, Hee-Gu; Hong, Jintae; Yoon, Do-Young

2014-10-01

156

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

PubMed

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

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

157

Inhibition of the Proprotein Convertases Represses the Invasiveness of Human Primary Melanoma Cells with Altered p53, CDKN2A and N-Ras Genes  

Microsoft Academic Search

BackgroundAltered tumor suppressor p53 and\\/or CDKN2A as well as Ras genes are frequently found in primary and metastatic melanomas. These alterations were found to be responsible for acquisition of invasive and metastatic potential through their defective regulatory control of metalloproteinases and urokinase genes.Methodology\\/Principal FindingsUsing primary human melanoma M10 cells with altered p53, CDKN2A and N-Ras genes, we found that inhibition

Claude Lalou; Nathalie Scamuffa; Samia Mourah; Francois Plassa; Marie-Pierre Podgorniak; Nadem Soufir; Nicolas Dumaz; Fabien Calvo; Nicole Basset-Seguin; Abdel-Majid Khatib

2010-01-01

158

Inhibiting AP-1 activity alters cocaine induced gene expression and potentiates sensitization  

PubMed Central

We have expressed A-FOS, an inhibitor of AP-1 DNA binding, in adult mouse striatal neurons. We observe normal behavior including locomotion and exploratory activities. Following a single injection of cocaine, locomotion increased similarly in both the A-FOS expressing and littermate controls. However, following repeated injections of cocaine, the A-FOS expressing mice showed increased locomotion relative to littermate controls, an increase that persisted following a week of withdrawal and subsequent cocaine administration. These results indicate that AP-1 suppresses this behavioral responses to cocaine. We analyzed mRNA from the striatum before and 4 and 24 hours after a single cocaine injection in both A-FOS and control striata using Affymetrix microarrays (430 2.0 Array) to identify genes mis-regulated by A-FOS that may mediate the increased locomotor sensitization to cocaine. A-FOS expression did not change gene expression in the basal state or 4 hours following cocaine treatment relative to controls. However, 24 hours after an acute cocaine treatment, 84 genes were identified that were differentially expressed between the A-FOS and control mice. 56 gene are down regulated while 28 genes are up regulated including previously identified candidates for addiction including BDNF and Per1. Using a random sample of identified genes, quantitative PCR was used to verify the microarray studies. The chromosomal location of these 84 genes was compared to human genome scans of addiction to identify potential genes in humans that are involved in addiction. PMID:18355967

Paletzki, Ronald F.; Myakishev, Max V.; Polesskaya, Oksana; Orosz, Andras; Hyman, Steven E.; Vinson, Charles

2008-01-01

159

Cycloheximide inhibition of delayed early gene expression in baculovirus-infected cells  

E-print Network

and their replication in host cells are intimately associated with host gene expression, they have served as model systems for the study of gene expression in both eukaryotic and prokaryotic cells. Aurograp)ta californica nuclear polyhedrosis virus (AcNPV) serves...

Ross, Larry Dale

1998-01-01

160

Antisense Oligodeoxynucleotide to the Cystic Fibrosis Gene Inhibits Anion Transport in Normal Cultured Sweat Duct Cells  

Microsoft Academic Search

We have tested the hypothesis that the cystic fibrosis (CF) gene product, called the CF transmembrane conductance regulator (CFTR), mediates anion transport in normal human sweat duct cells. Sweat duct cells in primary culture were treated with oligodeoxynucleotides that were antisense to the CFTR gene transcript in order to block the expression of the wild-type CFTR. Anion transport in CFTR

Eric J. Sorscher; Kevin L. Kirk; Mary L. Weaver; Tamas Jilling; J. Edwin Blalock; Robert D. Leboeuf

1991-01-01

161

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

Microsoft Academic Search

The authors have tested the hypothesis that the cystic fibrosis (CF) gene product, called the CF transmembrane conductance regulator (CFTR), mediates anion transport in normal human sweat duct cells. Sweat duct cells in primary culture were treated with oligodeoxynucleotides that were antisense to the CFTR gene transcript in order to block the expression of the wild-type CFTR. Anion transport in

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

1991-01-01

162

RNAi Silencing of the HaHMG-CoA Reductase Gene Inhibits Oviposition in the Helicoverpa armigera Cotton Bollworm  

PubMed Central

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

163

Inhibition of tumor necrosis factor alpha gene transcription by pentoxifylline reduces normothermic liver ischemia-reperfusion injury in rats.  

PubMed

Pentoxifylline (PTX) has been shown to protect the liver against normothermic ischemia-reperfusion (I-R) injury. The aims of this study were to investigate the action of PTX on tumor necrosis factor alpha (TNFalpha) gene transcription following normothermic liver I-R as well as to evaluate the resulting effects on liver function and survival. A segmental normothermic liver ischemia was induced for 90 minutes. Rats were divided into three groups: group 1, control, Ringer lactate administration; group 2, PTX treatment; group 3, sham-operated control rats. PTX (50 mg/kg) was injected intravenously 30 minutes before induction of ischemia and 30 minutes before reperfusion. The nonischemic liver lobes were resected at the end of ischemia. Survival rates were compared and serum activities of TNFalpha, aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase were measured. Liver histology was assessed 6 hours after reperfusion. Liver TNFalpha mRNA was assessed by polymerase chain reaction amplification at different times after reperfusion. PTX treatment significantly decreased serum activities of TNFalpha and inhibited liver expression of TNFalpha mRNA. The extent of liver necrosis and serum levels of liver enzymes were significantly decreased by PTX treatment, resulting in a significant increase in 7-day survival compared with nontreated control rats. In conclusion, PTX inhibits liver TNFalpha gene transcription, decreases serum TNFalpha levels, and reduces liver injury following normothermic I-R. PMID:17692605

El-Ghoneimi, A; Cursio, R; Schmid-Alliana, A; Tovey, M; Lasfar, A; Michiels, J-F; Rossi, B; Gugenheim, J

2007-01-01

164

Modulation of endogenous GATA-4 activity reveals its dual contribution to Müllerian inhibiting substance gene transcription in Sertoli cells.  

PubMed

Secretion of Müllerian inhibiting substance by fetal Sertoli cells is essential for normal male sex differentiation since it induces regression of the Müllerian ducts in the developing male embryo. Proper spatiotemporal expression of the MIS gene requires a specific combination of transcription factors, including the zinc finger factor GATA-4 and the nuclear receptor steroidogenic factor-1, which both colocalize with Müllerian inhibiting substance in Sertoli cells. To establish the molecular mechanisms through which GATA-4 contributes to MIS transcription, we have generated and characterized novel GATA-4 dominant negative competitors. The first one, which consisted solely of the GATA-4 zinc finger DNA-binding domain, was an efficient competitor of GATA transcription mediated both by direct GATA binding to DNA and protein-protein interactions involving GATA factors. The second type of competitor consisted of the same GATA-4 zinc finger DNA-binding domain but harboring mutations that prevented DNA binding. This second class of competitors repressed GATA-dependent transactivation by specifically competing for GATA protein-protein interactions without affecting the DNA-binding activity of endogenous GATA factors. These competitors, along with the GATA-4 cofactor FOG-2 (friend of GATA-2), were used to specifically modulate endogenous GATA-4 activity in Sertoli cells. Our results indicate that GATA-4 contributes to MIS promoter activity through two distinct mechanisms. Moreover, the GATA competitors described here should provide invaluable in vitro and in vivo tools for the study of GATA- dependent transcription and the identification of new target genes. PMID:11518812

Tremblay, J J; Robert, N M; Viger, R S

2001-09-01

165

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. PMID:23555746

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

166

Herpes Simplex Virus Gene Products Required for Viral Inhibition of Expression of G1-Phase Functions  

E-print Network

. These results provide evidence that HSV-1 ICP27 protein is essential for viral inhibition of G1-phase functions, these results show that HSV-1 ICP27 and vhs act jointly to reduce host mRNA levels in infected cells. © 2001

Knipe, David M.

167

G-patch domain containing 2, a gene highly expressed in testes, inhibits nuclear factor-?B and cell proliferation.  

PubMed

G-patch domain containing 2 (GPATC2), a human gene that is highly expressed in the testes, was implicated as a novel cancer/testis antigen. The present study investigated GPATC2 expression in a number of human cell lines and rat tissues, and its potential biological function in 293T cells. Semi?quantitative reverse transcription-polymerase chain reaction analysis showed that GPATC2 was widely expressed in 15 human cell lines (representing different lineages) and in 11 different rat tissues, and that the GPATC2 mRNA relative expression level was significantly higher in the testis than it was in other tissues. 293T cells were transiently transfected with GPATC2-p enhanced green fluorescent protein (EGFP)?N1 or GPATC2-pEGFP-C3 and the nuclei were stained with 4',6'?diamidino?2?phenylindole. The results showed that GPATC2 is predominantly expressed in the nucleus of 293T cells. Overexpression of GPATC2 may inhibit transcription of the NF-?B reporter gene. The role of GPATC2 in proliferation was analyzed with cell counting kit-8, colony-forming efficiency and flow cytometry assays. The results indicated that over?expression of GPATC2 in 293T cells significantly inhibited cell proliferation by decreasing the number of cells in S phase. By contrast, GPATC2 knockdown by RNA interference exhibited the opposite effect, suggesting that GPATC2 may be involved in inhibiting G1-S phase transition in 293T cells. In conclusion, these results provide novel insight into the breadth of expression of GPATC2 and its role in cell proliferation. PMID:25376275

Hu, Fen; Gou, Lixia; Liu, Qing; Zhang, Wendian; Luo, Mengmeng; Zhang, Xiujun

2015-02-01

168

Inhibition of p16 tumor suppressor gene expression via promoter hypermethylation in canine lymphoid tumor cells.  

PubMed

To investigate the epigenetic regulation of the p16 gene in canine lymphoid tumor cells, its methylation status was examined in four canine lymphoid tumor cell lines. In three canine lymphoid tumor cell lines (CLBL-1, GL-1, and UL-1) with low-level p16 mRNA expression, 20 CpG sites in the promoter region of p16 gene were consistently methylated although all of the CpG sites were not methylated in another cell line (CL-1) and normal lymph node cells. The expression level of p16 mRNA in these three cell lines was restored after cultivation in the presence of a methylation inhibitor, 5-Aza-2'-deoxycitidine, indicating inactivation of p16 gene via hypermethylation. This study revealed the inactivation of p16 gene through hypermethylation of its CpG island in a fraction of canine lymphoid tumor cells. PMID:24815345

Fujiwara-Igarashi, Aki; Goto-Koshino, Yuko; Mochizuki, Hiroyuki; Sato, Masahiko; Fujino, Yasuhito; Ohno, Koichi; Tsujimoto, Hajime

2014-08-01

169

Breast Cancer Metastasis Suppressor 1 Inhibits Gene Expression by Targeting Nuclear Factor-KB Activity  

Microsoft Academic Search

Breast cancer metastasis suppressor 1 (BRMS1) functions as a metastasis suppressor gene in breast cancer and melanoma cell lines, but the mechanism of BRMS1 suppression remains unclear. We determined that BRMS1 expression was inversely correlated with that of urokinase-type plasminogen activator (uPA), a prometastatic gene that is regulated at least in part by nuclear factor-KB (NF-KB). To further investigate the

Muzaffer Cicek; Ryuichi Fukuyama; Danny R. Welch; Graham Casey

2005-01-01

170

Specific inhibition of gene expression using a stably integrated, inducible small-interfering-RNA vector  

Microsoft Academic Search

We have designed a doxycycline-regulated form of the H1 promoter of RNA polymerase III that allows the inducible knockdown of gene expression by small interfering RNAs (siRNAs). As a proof-of-principle, we have targeted ?-catenin in colorectal cancer (CRC) cells. T-cell factor (TCF) target-gene expression is induced by accumulated ?-catenin, and is the main transforming event in these cells. We have

Marc van de Wetering; Irma Oving; Vanesa Muncan; Menno Tjon Pon Fong; Helen Brantjes; Dik van Leenen; Frank C. P. Holstege; Thijn R. Brummelkamp; Reuven Agami; Hans Clevers

2003-01-01

171

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. PMID:23544081

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

2013-01-01

172

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

PubMed Central

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

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

2015-01-01

173

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

PubMed Central

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

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

2011-01-01

174

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

PubMed

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

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

2015-03-01

175

Inhibition of Human UGT2B7 Gene Expression in Transgenic Mice by the Constitutive Androstane Receptor  

PubMed Central

The xenobiotic receptors, constitutive androstane receptor (CAR), and pregnane X receptor (PXR) regulate and alter the metabolism of xenobiotic substrates. Among the 19 functional UDP-glucuronosyltransferases (UGTs) in humans, UGT2B7 is involved in the metabolism of many structurally diverse xenobiotics and plays an important role in the clearance and detoxification of many therapeutic drugs. To examine whether this gene is regulated by CAR and PXR in vivo, transgenic mice expressing the entire UGT2B7 gene (TgUGT2B7) were created. Gene expression profiles revealed that UGT2B7 is differentially expressed in liver, kidney, adipocytes, brain, and estrogen-sensitive tissues, such as ovary and uterus. Liver UGT2B7 expression levels were decreased when TgUGT2B7 mice were treated with the CAR ligand 1,4-b-s-[2-(3,5,-dichloropyridyloxy)] (TCPOBOP) but not the PXR ligand pregnenolone 16?-carbonitrile. Although TCPOBOP decreased the levels of UGT2B7 mRNA in TgUGT2B7 mice, it had no affect on Tg(UGT2B7)Car(?/?) mice, adding support for a CAR-dependent mechanism contributing toward UGT2B7 gene suppression. Expression of promoter constructs in HepG2 cells showed the CAR-dependent inhibition was linked to hepatocyte nuclear factor-4? (HNF4?)-mediated transactivation of the UGT2B7 promoter. The inhibitory effect of CAR on UGT2B7 gene expression was validated in chromatin immunoprecipitation assays in which TCPOBOP treatment blocked HNF4? binding to the UGT2B7 promoter. These results suggest that HNF4? plays an important role in the constitutive expression of hepatic UGT2B7, and CAR acts as a negative regulator by interfering with HNF4? binding activity. PMID:21415305

Yueh, M. F.; Mellon, P. L.

2011-01-01

176

Inhibition of SMP30 gene expression influences the biological characteristics of human Hep G2 cells.  

PubMed

Senescence marker protein 30 (SMP30), a hepatocellular carcinoma (HCC) associated antigen had been identified by our research group. To study its mechanisms of regulation and associations with the occurrence and development of HCC, we inhibited expression by RNAi technique, and observed effects on the biological characteristics of Hep G2 cells. In cell viability assays, cell growth in the experimental group (with siRNA transfection) was elevated. In Transwell invasion assays, compared with blank and control groups, numbers of invading cells in the experimental group were significantly increased, whereas in apoptosis assays, the percentage apoptosis demonstrated no differences, but after UV irradiation, that in the experimental group was higher than the other two groups. In a word, SMP30 can inhibit the proliferation and invasion of human hepatoma cells and thus can be regarded as a cancer suppressive factor. PMID:24606440

Zhang, Sheng-Chang; Liang, Ming-Kang; Huang, Guang-Lin; Jiang, Kui; Zhou, Su-Fang; Zhao, Shuang

2014-01-01

177

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. PMID:20843837

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

178

Dynamic Telomerase Gene Suppression via Network Effects of GSK3 Inhibition  

Microsoft Academic Search

Background: Telomerase controls telomere homeostasis and cell immortality and is a promising anti-cancer target, but few small molecule telomerase inhibitors have been developed. Reactivated transcription of the catalytic subunit hTERT in cancer cells controls telomerase expression. Better understanding of upstream pathways is critical for effective anti- telomerase therapeutics and may reveal new targets to inhibit hTERT expression. Methodology\\/Principal Findings: In

Alan E. Bilsland; Stacey Hoare; Katrina Stevenson; Jane Plumb; Natividad Gomez-Roman; Claire Cairney; Sharon Burns; Kyle Lafferty-Whyte; Jon Roffey; Tim Hammonds; W. Nicol Keith

2009-01-01

179

Effective inhibition of Japanese encephalitis virus replication by small interfering RNAs targeting the NS5 gene  

Microsoft Academic Search

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, causes an acute infection of the central nervous system resulting in encephalitis of humans and many kinds of animals. NS5, the largest and most conserved flavivirus protein, is homologous to methyltransferase and RNA-dependent RNA polymerase. RNA interference is an effective anti-viral strategy to inhibit viral replication in vitro. In this study, four short

Wen-Bao Qi; Rong-Hong Hua; Li-Ping Yan; Guang-Zhi Tong; Gui-Hong Zhang; Tao Ren; Dong-Lai Wu; Ming Liao

2008-01-01

180

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. PMID:23565249

Lim, Chiat Koo; Kelley, Richard L.

2013-01-01

181

Cancer Targeted Gene Therapy of BikDD Inhibits Orthotopic Lung Cancer Growth and Improves Long-term Survival  

PubMed Central

Lung cancer is a leading cause of cancer death due to the high incidence of metastasis; therefore novel and effective treatments are urgently needed. A current strategy is cancer specific targeted gene therapy. While many identified cancer specific promoters are highly specific, they tend to have low activity compared to the ubiquitous CMV promoter, limiting their application. We developed a targeted gene therapy expression system for lung cancer that is highly specific with strong activity. Our expression vector uses the survivin promoter, highly expressed in many cancers but not normal adult tissues. We enhanced the survivin promoter activity comparable to the CMV promoter in lung cancer cell lines using an established platform technology, while the survivin promoter remained weak in normal cells. In mouse models, the transgene was specifically expressed in the lung tumor tissue, compared with the CMV promoter that was expressed in both normal and tumor tissues. Additionally, the therapeutic gene BikDD, a mutant form of pro-apoptotic Bik, induced cell killing in vitro, and inhibited cell growth and prolonged mouse survival in vivo. Importantly, there was virtually no toxicity when BikDD was expressed with our expression system. Thus, the current report provides a therapeutic efficacy and safe strategy worthy of development in clinical trials treating lung cancer. PMID:19597463

Sher, Yuh-Pyng; Tzeng, Tz-Fei; Kan, Shu-Fen; Hsu, Jennifer; Xie, Xiaoming; Han, Zhenbo; Lin, Wen-Chuan; Li, Long-Yuan; Hung, Mien-Chie

2009-01-01

182

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. PMID:24632777

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

2014-01-01

183

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

184

Vascular inflammation inhibits gene transfer to the pulmonary circulation in vivo.  

PubMed

We report gene transfer to the normal and injured murine pulmonary circulation via systemic (intravascular) and airway (intratracheal) delivery of novel polycationic liposomes (imidazolium chloride, imidazolinium chloride-cholesterol, and ethyl phosphocholine). With use of the reporter genes chloramphenicol acetyltransferase (CAT) or human placental alkaline phosphatase (hpAP), intravascular injection of lipid-DNA complexes resulted in gene expression primarily in the lung, with lesser expression in the heart (11% of lung, P < 0.05) and spleen (8% of lung, P < 0.05). Histochemical staining for the hpAP reporter gene showed localized transgene expression in the microvascular endothelium. Monocrotaline (80 mg/kg body wt sc) treatment produced endovascular inflammation and reduced lung CAT activity (2 days postintravascular transfection) by 75 +/- 8 and 86 +/- 6% at 7 and 21 days, respectively, after monocrotaline (P < 0. 05). Despite the apparent decrease in functional CAT protein, Southern blot analysis suggested maintained plasmid delivery, whereas quantitative PCR (TaqMan) showed decreased CAT mRNA levels in monocrotaline mice. In contrast, intratracheal delivery of lipid-DNA complexes showed enhanced CAT expression in monocrotaline mice. Transfection in alternate pulmonary vascular disorders was studied by inducing hypoxic pulmonary hypertension (4 wk at barometric pressure of 410 mmHg). Efficiency and duration of gene transfer, assessed by CAT activity, were similar in pulmonary hypertensive and normal lungs. We conclude that imidazolinium-derived polycationic liposomes provide a means of relatively selective and efficient gene transfer to the normal and injured murine microvascular circulation, although translation of transgene mRNA may be reduced by preexisting endothelial injury. PMID:10600891

Tyler, R C; Fagan, K A; Unfer, R C; Gorman, C; McClarrion, M; Bullock, C; Rodman, D M

1999-12-01

185

[Dose-effect research using nanopatch to deliver siRNA in the inhibition of HPV gene expression].  

PubMed

Hela is the cell line of adenocarcinoma of the uterine cervix, and human papillomavirus (HPV) 18 shows positive. We delivered siRNA with target specifically to HPV18 E7 mRNA into nude mice Hela tumor xenografts by nanopatch to inhibit the HPV gene expression, and further to study the superiority, the best action time and concentration of siRNA of using nanopatch to transfer siRNA in vivo. We designed siRNA that target specifically to HPV18 E7 mRNA (siE7) and checked the effect of siE7 in vitro. Tumor xenografts were transfected with siE7 and GenEscort III by nanopatch. Expression of HPV18 E7 mRNA and protein were detected 0 hours, 24 hours, 48 hours, 72 hours after transfection with PT-PCR and Western blot, and the best action time was analyzed using nanopatch to thansfect siRNA in vivo. We transfected GenEscort III and siE7 of Different concentration into tumor xenografts respectively by nanopatch and intraperitoneal injection. Expression of HPV18 E7 mRNA and protein was detected 72 hours after transfection by PT-PCR and Western blot, to analyze the best action concentration of siRNA and the superiority of using nanopatch to thansfect siRNA in vivo. The results proved that SiE7was efficient to inhibit expression of HPV18 E7 mRNA and to advance Hela apoptosis in vitro. SiE7 transfected by nanopatch into xenografts could inhibit effectively expression of HPV18 E7 mRNA and protein. The best action time and concentration of siRNA of using nanopatch to thansfect siRNA in vivo are 72 hour post-transfection and 2 micromol/L siE7. To compare intraperitoneal injection in delivering siRNA in vivo, the effect of nanopatch is very predominant. It can be well concluded that Nanopatch can effectively transfer siRNA in vivo, which can effectively inhibit the HPV gene expression. PMID:24645613

Xiong, Zhuo; Dong, Xiaojing; Sun, Peiwen; Zhang, Ying

2013-12-01

186

Basal and acidic fibroblast growth factor-induced atrial natriuretic peptide gene expression and secretion is inhibited by staurosporine.  

PubMed

We examined the mechanisms involved in the activation of atrial natriuretic peptide (ANP) gene expression and secretion in response to acidic fibroblast growth factor (aFGF) by studying the effects of staurosporine, a protein kinase C inhibitor, and 12-O-tetradecanoyl phorbol 13-acetate (TPA), an activator of protein kinase C, on basal and AFGF-induced ANP messenger RNA (mRNA) and immunoreactive ANP (IR-ANP) levels in cultured neonatal rat cardiac myocytes. Acidic FGF caused a dose- and time-dependent increase in IR-ANP and immunoreactive N-terminal fragment of proANP (IR-NT-proANP) release into the culture medium from ventricular but not from atrial myocytes. In ventricular cells, 50 ng/ml aFGF for 24 or 48 h resulted in a 70% or 181% increase, respectively, in the accumulation of IR-ANP into the culture medium. Acidic FGF also stimulated ANP gene expression significantly; after 48 h of incubation, the ANP mRNA levels of aFGF-treated ventricular myocytes were 205% (P < 0.001) higher than those of control cells. Staurosporine alone at concentration of 10 nM significantly decreased the basal IR-ANP and IR-NT-proANP secretion, and inhibited the aFGF-induced increase in ANP mRNA and IR-ANP levels in ventricular myocytes. TPA (100 nM) alone significantly stimulated ANP gene expression and secretion but these effects were not augmented by combining aFGF with TPA. High performance liquid chromatographical analysis showed that atrial and ventricular myocytes maintained in serum-free medium were capable of secreting processed, ANP99-126 sized material, and that aFGF did not alter the processing of ANP in ventricular cultures. These results demonstrate that aFGF is a potent stimulator of ANP gene expression and secretion in cultured neonatal rat ventricular but not in atrial cells. The observations that (a) staurosporine completely abolished the effects of aFGF on ANP gene expression and release and (b) ANP secretory and gene expression inducing effects of phorbol ester were not augmented by aFGF, suggest an important role of protein kinase C in mediating aFGF-induced ANP gene expression and secretion. PMID:7519562

Tokola, H; Salo, K; Vuolteenaho, O; Ruskoaho, H

1994-04-15

187

Doxorubicin Selectivity Inhibits Muscle Gene Expression in Cardiac Muscle Cells in vivo and in vitro  

Microsoft Academic Search

The anthracycline antibiotic doxorubicin produces a characteristic myopathy in cardiac muscle that limits its use in cancer therapy. We have shown in cultured neonatal rat cardiac muscle cells that doxorubicin treatment resulted in a rapid, selective decrease in the expression of muscle-specific genes, which preceded other changes characteristic of doxorubicin cardiomyopathy. Doxorubicin selectively and dramatically decreased the levels of mRNA

Hiroshi Ito; Steven C. Miller; Margaret E. Billingham; Hajime Akimoto; Suzy V. Torti; Robert Wade; Reinhold Gahlmann; Gary Lyons; Larry Kedes; Frank M. Torti

1990-01-01

188

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

Technology Transfer Automated Retrieval System (TEKTRAN)

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

189

Overexpression of PYL5 in rice enhances drought tolerance, inhibits growth, and modulates gene expression.  

PubMed

Abscisic acid (ABA) is a phytohormone that plays important roles in the regulation of seed dormancy and adaptation to abiotic stresses. Previous work identified OsPYL/RCARs as functional ABA receptors regulating ABA-dependent gene expression in Oryza sativa. OsPYL/RCARs thus are considered to be good candidate genes for improvement of abiotic stress tolerance in crops. This work demonstrates that the cytosolic ABA receptor OsPYL/RCAR5 in O. sativa functions as a positive regulator of abiotic stress-responsive gene expression. The constitutive expression of OsPYL/RCAR5 in rice driven by the Zea mays ubiquitin promoter induced the expression of many stress-responsive genes even under normal growth conditions and resulted in improved drought and salt stress tolerance in rice. However, it slightly reduced plant height under paddy field conditions and severely reduced total seed yield. This suggests that, although exogenous expression of OsPYL/RCAR5 is able to improve abiotic stress tolerance in rice, fine regulation of its expression will be required to avoid deleterious effects on agricultural traits. PMID:24474809

Kim, Hyunmi; Lee, Kyeyoon; Hwang, Hyunsik; Bhatnagar, Nikita; Kim, Dool-Yi; Yoon, In Sun; Byun, Myung-Ok; Kim, Sun Tae; Jung, Ki-Hong; Kim, Beom-Gi

2014-02-01

190

Construction of Expression Vector for Anti-Alpha-Fetoprotein Gene and Its Inhibition Effects on Alpha-Fetoprotein Positive Hepg2 Cells  

NASA Astrophysics Data System (ADS)

As research previously demonstrated, suppression of AFP expression or its biological activities might inhibit the proliferation of AFP positive human hepatocellular carcinoma cells. In this study, we constructed an anti-AFP gene vector and transfected it to HepG2 cells. RT-PCR showed AFP gene expression in the transfected cells was reduced. MTT assay suggested the proliferation of the transfected cells was also inhibited comparing with the untransfected cells. This result provides a new insight into AFP as the target for preventing and treating hepatocellular carcinoma.

Wang, Ze; Zhang, Hui

191

Alleviating transcriptional inhibition of the norepinephrine slc6a2 transporter gene in depolarized neurons.  

PubMed

Recent studies have brought to light additional experimental information, namely, that the MeCP2 protein complex is not only capable of associating with members of the ATPase-dependent bromodomain family, but also found on nonmethylated genomic sequences. These unexpected results are indicative of a multifunctional role for MeCP2, more importantly; our view of the molecular mechanisms that regulate gene activity may not be necessarily distinguishable. Depolarized mouse neuronal cortical cells were examined for increased Slc6a2 mRNA synthesis, changes in CpG methylation status using bisulfite sequencing, and binding of MeCP2 and Smarca2 on the Slc6a2 promoter sequence by chromatin immunopurification strategies. Increased Slc6a2 gene expression in response to membrane depolarization was strongly correlated with the dissociation of MeCP2 and Smarca2 complex on the unmethylated gene. We identified that gene expression in neuronal cortical cells involves increased histone hyperacetylation on the Slc6a2 promoter, which is commensurate with the recruitment of SP1 and RNA Polymerase II and is inversely correlated with H3K9 trimethylation. We hypothesize that the MeCP2 corepressor is capable of associating with multiple forms of SWI/SNF to remodel chromatin for important regulatory roles. The results of our experiments indicate that these proteins are asymmetrically bound to chromatin independent of DNA methylation and not inevitably diametrically opposed. These results now begin to offer a new perspective on the mechanism of Slc6a2 gene regulation. PMID:20107077

Harikrishnan, K N; Bayles, Richard; Ciccotosto, Giuseppe D; Maxwell, Scott; Cappai, Roberto; Pelka, Gregory J; Tam, Patrick P L; Christodoulou, John; El-Osta, Assam

2010-01-27

192

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

PubMed Central

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

1995-01-01

193

ZEB1 Imposes a Temporary Stage-Dependent Inhibition of Muscle Gene Expression and Differentiation via CtBP-Mediated Transcriptional Repression  

PubMed Central

Skeletal muscle development is orchestrated by the myogenic regulatory factor MyoD, whose activity is blocked in myoblasts by proteins preventing its nuclear translocation and/or binding to G/C-centered E-boxes in target genes. Recent evidence indicates that muscle gene expression is also regulated at the cis level by differential affinity for DNA between MyoD and other E-box binding proteins during myogenesis. MyoD binds to G/C-centered E-boxes, enriched in muscle differentiation genes, in myotubes but not in myoblasts. Here, we used cell-based and in vivo Drosophila, Xenopus laevis, and mouse models to show that ZEB1, a G/C-centered E-box binding transcriptional repressor, imposes a temporary stage-dependent inhibition of muscle gene expression and differentiation via CtBP-mediated transcriptional repression. We found that, contrary to MyoD, ZEB1 binds to G/C-centered E-boxes in muscle differentiation genes at the myoblast stage but not in myotubes. Its knockdown results in precocious expression of muscle differentiation genes and acceleration of myotube formation. Inhibition of muscle genes by ZEB1 occurs via transcriptional repression and involves recruitment of the CtBP corepressor. Lastly, we show that the pattern of gene expression associated with muscle differentiation is accelerated in ZEB1?/? mouse embryos. These results set ZEB1 as an important regulator of the temporal pattern of gene expression controlling muscle differentiation. PMID:23339872

Siles, Laura; Sánchez-Tilló, Ester; Lim, Jong-Won; Darling, Douglas S.; Kroll, Kristen L.

2013-01-01

194

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

PubMed Central

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

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

2013-01-01

195

Albizia lebbeck suppresses histamine signaling by the inhibition of histamine H1 receptor and histidine decarboxylase gene transcriptions.  

PubMed

Histamine plays major roles in allergic diseases and its action is mediated mainly by histamine H(1) receptor (H1R). We have demonstrated that histamine signaling-related H1R and histidine decarboxylase (HDC) genes are allergic diseases sensitive genes and their expression level affects severity of the allergic symptoms. Therefore, compounds that suppress histamine signaling should be promising candidates as anti-allergic drugs. Here, we investigated the effect of the extract from the bark of Albizia lebbeck (AL), one of the ingredients of Ayruvedic medicines, on H1R and HDC gene expression using toluene-2,4-diisocyanate (TDI) sensitized allergy model rats and HeLa cells expressing endogenous H1R. Administration of the AL extract significantly decreased the numbers of sneezing and nasal rubbing. Pretreatment with the AL extract suppressed TDI-induced H1R and HDC mRNA elevations as well as [(3)H]mepyramine binding, HDC activity, and histamine content in the nasal mucosa. AL extract also suppressed TDI-induced up-regulation of IL-4, IL-5, and IL-13 mRNA. In HeLa cells, AL extract suppressed phorbol-12-myristate-13-acetate- or histamine-induced up-regulation of H1R mRNA. Our data suggest that AL alleviated nasal symptoms by inhibiting histamine signaling in TDI-sensitized rats through suppression of H1R and HDC gene transcriptions. Suppression of Th2-cytokine signaling by AL also suggests that it could affect the histamine-cytokine network. PMID:21782040

Nurul, Islam Mohammed; Mizuguchi, Hiroyuki; Shahriar, Masum; Venkatesh, Pichairajan; Maeyama, Kazutaka; Mukherjee, Pulok K; Hattori, Masashi; Choudhuri, Mohamed Sahabuddin Kabir; Takeda, Noriaki; Fukui, Hiroyuki

2011-11-01

196

Inhibition of protein synthesis stimulates the transcription of human beta-interferon genes in Chinese hamster ovary cells.  

PubMed Central

Using Chinese hamster ovary (CHO) cells transfected with a plasmid carrying the human beta-interferon gene, we find that inhibitors of protein synthesis, in the absence of any other inducer, stimulate the production of interferon RNA; this effect is maintained in cells in which the plasmid sequences have been amplified 25- to 50-fold. Nuclear transcription assays show that a major effect of cycloheximide is to increase the rate of transcription of the interferon gene. This contradicts the generally accepted explanation that inhibitors of protein synthesis augment interferon production by stabilizing interferon mRNA. In addition, we have studied the effects of double stranded RNA [poly(rI) X poly(rC)] on the induction of interferon RNA in the presence and absence of cycloheximide. Our results indicate that poly(rI) X poly(rC) by itself causes a transient increase in interferon RNA; however, in the presence of cycloheximide this effect is prolonged. We do not, however, find an increase in transcription of the interferon gene(s) as an early response to poly(rI) X poly(rC). Finally, we have found that cells treated with cycloheximide or infected with Newcastle disease virus induce large amounts of a secreted 11-kDa protein. This cellular protein is not inducible by poly(rI) X poly(rC). We propose that both interferon and this 11-kDa protein belong to a family of proteins in which production is regulated in a coordinate fashion during viral inhibition of cellular protein synthesis. Images PMID:6330726

Ringold, G M; Dieckmann, B; Vannice, J L; Trahey, M; McCormick, F

1984-01-01

197

Celiprolol inhibits mitogen-activated protein kinase and endothelin-1 and transforming growth factor-beta(1) gene in rats.  

PubMed

We evaluated the cardioprotective effects of long-term treatment with celiprolol (for 5 weeks), a specific beta(1)-adrenoceptor antagonist with a weak beta(2)-adrenoceptor agonist action, on endothelin-1 and transforming growth factor (TGF)-beta(1) expression and cardiovascular remodeling in deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Upregulated preproendothelin-1, endothelin ET(A) receptor, TGF-beta(1), c-fos, and type I collagen expression and extracellular signal-regulated kinase activities were suppressed by celiprolol. Celiprolol effectively inhibited vascular lesion formation such as medial thickness and perivascular fibrosis. These observations suggested that extracellular signal-regulated kinase and c-fos gene pathway may contribute to the cardiovascular remodeling of DOCA rats, and that cardioprotective effects of celiprolol on cardiovascular remodeling may be mediated, at least in part, by suppressed expression of endothelin-1 and TGF-beta(1). PMID:12464353

Tsubokou, Yusuke; Kobayashi, Naohiko; Mita, Shin-ichiro; Yoshida, Kohtaro; Matsuoka, Hiroaki

2002-12-20

198

Selection on Glycine ?-1,3-Endoglucanase Genes Differentially Inhibited by a Phytophthora Glucanase Inhibitor Protein  

PubMed Central

Plant endo-?-1,3-glucanases (EGases) degrade the cell wall polysaccharides of attacking pathogens and release elicitors of additional plant defenses. Isozymes EGaseA and EGaseB of soybean differ in susceptibility to a glucanase inhibitor protein (GIP1) produced by Phytophthora sojae, a major soybean pathogen. EGaseA, the major elicitor-releasing isozyme, is a high-affinity ligand for GIP1, which completely inhibits it, whereas EGaseB is unaffected by GIP1. We tested for departures from neutral evolution on the basis of partial sequences of EGaseA and EGaseB from 20 widespread accessions of Glycine soja (the wild progenitor of soybean), from 4 other Glycine species, and across dicotyledonous plants. G. soja exhibited little intraspecific variation at either locus. Phylogeny-based codon evolution models detected strong evidence of positive selection on Glycine EGaseA and weaker evidence for selection on dicot EGases and Glycine EGaseB. Positively selected peptide sites were identified and located on a structural model of EGase bound to GIP1. Positively selected sites and highly variable sites were found disproportionately within 4.5 Ĺ of bound GIP1. Low variation within G. soja EGases, coupled with positive selection in both Glycine and dicot lineages and the proximity of rapidly evolving sites to GIP1, suggests an arms race involving repeated adaptation to pathogen attack and inhibition. PMID:15545660

Bishop, J. G.; Ripoll, D. R.; Bashir, S.; Damasceno, C. M. B.; Seeds, J. D.; Rose, J. K. C.

2005-01-01

199

Reciprocal regulation of gene transcription by insulin. Inhibition of the phosphoenolpyruvate carboxykinase gene and stimulation of gene 33 in a single cell type.  

PubMed

Two H4IIE hepatoma cell genes, phosphoenolpyruvate carboxykinase (PEPCK) and gene 33 (g33), are reciprocally regulated by insulin. Quantitation of mRNAPEPCK and mRNAg33 in total RNA isolated from cells treated with insulin showed a 7-fold increase in mRNAg33 amount and a 3-fold decrease of mRNAPEPCK. The cAMP analog 8-(4-chlorophenylthio)-cAMP induced mRNAPEPCK but had no effect on mRNAg33. The responses to various insulins and related molecules showed that the insulin receptor mediates the effects of physiologic concentrations of insulin on each of these genes. This inverse pattern of regulation by insulin was further characterized by determining the transcription rates of both genes in nuclei isolated at various times after the addition of insulin and 8-(4-chlorophenylthio)-cAMP to H4IIE cells. Insulin increased the rate of synthesis of mRNAg33 from 35 to 354 ppm and decreased the synthesis of mRNAPEPCK from 1175 to 109 ppm. These effects of insulin occurred rapidly and reached their maxima by 60 min. In both cases, greater effects were observed as insulin concentrations were increased from 10(-12) to 10(-8) M. Although the effects of insulin were concentration-dependent for both genes, the PEPCK gene was significantly more sensitive to low concentrations of insulin than was gene 33. The reciprocal effects of insulin on the synthesis of mRNAPEPCK and mRNAg33 in H4IIE cells provide a means of investigating how a hormone can exert opposing effects on two genes in the same cell. PMID:2843505

Chu, D T; Davis, C M; Chrapkiewicz, N B; Granner, D K

1988-09-15

200

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

PubMed Central

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

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

2015-01-01

201

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

PubMed Central

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

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

2014-01-01

202

Liposomal melatonin rescues methamphetamine-elicited mitochondrial burdens, pro-apoptosis, and dopaminergic degeneration through the inhibition PKC? gene.  

PubMed

We have demonstrated that mitochondrial oxidative damage and PKC? overexpression contribute to methamphetamine-induced dopaminergic degeneration. Although it is recognized that antioxidant melatonin is effective in preventing neurotoxicity induced by methamphetamine, its precise mechanism remains elusive. C57BL/6J wild-type mice exhibited a similar degree of dopaminergic deficit when methamphetamine was administered during light and dark phases. Furthermore, dopaminergic neuroprotection by genetic inhibition of PKC? during the light phase was comparable to that during the dark phase. Thus, we have focused on the light phase to examine whether melatonin modulates PKC?-mediated neurotoxic signaling after multiple high doses of methamphetamine. To enhance the bioavailability of melatonin, we applied liposomal melatonin. Treatment with methamphetamine resulted in hyperthermia, mitochondrial translocation of PKC?, oxidative damage (mitochondria > cytosol), mitochondrial dysfunction, pro-apoptotic changes, ultrastructural mitochondrial degeneration, dopaminergic degeneration, and behavioral impairment in wild-type mice. Treatment with liposomal melatonin resulted in a dose-dependent attenuation against degenerative changes induced by methamphetamine in wild-type mice. Attenuation by liposomal melatonin might be comparable to that by genetic inhibition (using PKC?((-/-)) mice or PKC? antisense oligonucleotide). However, liposomal melatonin did not show any additional protective effects on the attenuation by genetic inhibition of PKC?. Our results suggest that the circadian cycle cannot be a key factor in modulating methamphetamine toxicity under the current experimental condition and that PKC? is one of the critical target genes for melatonin-mediated protective effects against mitochondrial burdens (dysfunction), oxidative stress, pro-apoptosis, and dopaminergic degeneration induced by methamphetamine. PMID:25407782

Nguyen, Xuan-Khanh Thi; Lee, Jaehwi; Shin, Eun-Joo; Dang, Duy-Khanh; Jeong, Ji Hoon; Nguyen, Thuy-Ty Lan; Nam, Yunsung; Cho, Hyun-Jong; Lee, Jae-Chul; Park, Dae Hun; Jang, Choon-Gon; Hong, Jau-Shyong; Nabeshima, Toshitaka; Kim, Hyoung-Chun

2015-01-01

203

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. PMID:24810746

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

2014-01-01

204

Blocking signaling at the level of GLI regulates downstream gene expression and inhibits proliferation of canine osteosarcoma cells.  

PubMed

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. PMID:24810746

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

2014-01-01

205

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. PMID:25009749

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

2014-01-01

206

PIAS1 selectively inhibits interferon-inducible genes and is important in innate immunity  

Microsoft Academic Search

Interferon (IFN) activates the signal transducer and activator of transcription (STAT) pathway to regulate immune responses. The protein inhibitor of activated STAT (PIAS) family has been suggested to negatively regulate STAT signaling. To understand the physiological function of PIAS1, we generated Pias1?\\/? mice. Using PIAS1-deficient cells, we show that PIAS1 selectively regulates a subset of IFN-?- or IFN-?-inducible genes by

Bin Liu; Sheldon Mink; Kelly A Wong; Natalie Stein; Crescent Getman; Paul W Dempsey; Hong Wu; Ke Shuai

2004-01-01

207

Inhibition by insulin of resistin gene expression in 3T3-L1 adipocytes  

Microsoft Academic Search

Expression of the gene encoding resistin, a low molecular weight protein secreted from adipose tissue postulated to link obesity and type II diabetes, was examined in 3T3-L1 adipocytes. Resistin mRNA was detected in 3T3-L1 cells by day 3 following induction of differentiation into adipocytes; by day 4 the level of resistin mRNA peaked and remained high. The PPAR? activators, rosiglitazone

Fred Haugen; Aud Jřrgensen; Christian A. Drevon; Paul Trayhurn

2001-01-01

208

Knockdown of DNA methyltransferase-1 inhibits proliferation and derepresses tumor suppressor genes in myeloma cells  

PubMed Central

DNA methyltransferases (including DNMT1, DNMT3A and DNMT3B), catalyze the transfer of methyl groups from S-adenosyl-l-methionine to cytosine position 5; this methylation in promoter regions silences gene expression. In addition, DNMT1 plays a critical role in the maintenance of genomic DNA methylation during DNA replication. In the present study, silencing of DNMT1 with siRNA was performed in RPMI-8226 human multiple myeloma (MM) cells, and the impact on gene methylation status and proliferation of the cells was analyzed. Upon DNMT1 downregulation, proliferation decreased significantly compared with that in the control, non-transfected cells. The expression of B-cell lymphoma 2 and nuclear factor ?B proteins was also significantly reduced. Furthermore, nested methylation-specific polymerase chain reaction revealed that methylation of the tumor suppressor genes, suppressor of cytokine signaling 1 and p16, was significantly reduced upon DNMT1 knockdown. Our results suggest that DNMT1 silencing may be a promising strategy to consider during development of novel MM treatment strategies. PMID:25289094

ZHOU, WENWEN; CHEN, HUYING; HONG, XIULI; NIU, XIAOQING; LU, QUANYI

2014-01-01

209

The strawberry gene FaGAST affects plant growth through inhibition of cell elongation.  

PubMed

The strawberry (Fragaria x ananassa) FaGAST gene encodes a small protein with 12 cysteine residues conserved in the C-terminal region similar to a group of proteins identified in other species with diverse assigned functions such as cell division, elongation, or elongation arrest. This gene is expressed in the fruit receptacle, with two peaks during ripening at the white and the red-ripe stages, both coincident with an arrest in the growth pattern. Expression is also high in the roots but confined to the cells at the end of the elongation zone. Exogenous application of gibberellin increased the transcript level of the FaGAST gene in strawberry fruits. Ectopic expression of FaGAST in transgenic Fragaria vesca under the control of the CaMV-35S promoter caused both delayed growth of the plant and fruits with reduced size. The same growth defect was observed in Arabidopsis thaliana plants overexpressing FaGAST. In addition, the transgenic plants exhibited late flowering and low sensitivity to exogenous gibberellin. Taken together, the expression pattern, the regulation by gibberellin, and the transgenic phenotypes point to a role for FaGAST in arresting cell elongation during strawberry fruit ripening. PMID:16804055

de la Fuente, José I; Amaya, Iraida; Castillejo, Cristina; Sánchez-Sevilla, José F; Quesada, Miguel A; Botella, Miguel A; Valpuesta, Victoriano

2006-01-01

210

Evolution of the Retroviral Restriction Gene Fv1: Inhibition of Non-MLV Retroviruses  

PubMed Central

Fv1 is the prototypic restriction factor that protects against infection by the murine leukemia virus (MLV). It was first identified in cells that were derived from laboratory mice and was found to be homologous to the gag gene of an endogenous retrovirus (ERV). To understand the evolution of the host restriction gene from its retroviral origins, Fv1s from wild mice were isolated and characterized. Most of these possess intact open reading frames but not all restricted N-, B-, NR-or NB-tropic MLVs, suggesting that other viruses could have played a role in the selection of the gene. The Fv1s from Mus spretus and Mus caroli were found to restrict equine infectious anemia virus (EIAV) and feline foamy virus (FFV) respectively, indicating that Fv1 could have a broader target range than previously thought, including activity against lentiviruses and spumaviruses. Analyses of the Fv1 sequences revealed a number of residues in the C-terminal region that had evolved under positive selection. Four of these selected residues were found to be involved in the novel restriction by mapping studies. These results strengthen the similarities between the two capsid binding restriction factors, Fv1 and TRIM5?, which support the hypothesis that Fv1 defended mice against waves of retroviral infection possibly including non-MLVs as well as MLVs. PMID:24603659

Yap, Melvyn W.; Colbeck, Emily; Ellis, Scott A.; Stoye, Jonathan P.

2014-01-01

211

Acanthoic acid inhibits melanogenesis through tyrosinase downregulation and melanogenic gene expression in B16 melanoma cells.  

PubMed

The aim of this study was to investigate the in vitro inhibitory effects of acanthoic acid (ACAN), isolated from Acanthopanax koreanum, on melanogenesis and its related enzymes such as tyrosinase, tyrosinase-related protein (TRP)-1, and TRP-2 in B16 melanoma cells. We found that ACAN significantly attenuates melanin synthesis and reduces the activity of intracellular tyrosinase, the rate-limiting melanogenic enzyme. Western blot analysis showed that ACAN also decreases tyrosinase, TRP-1, and TRP-2 protein expression. In addition, ACAN significantly decreased the expression of microphthalmia-associated transcription factor (MITF), a key regulator of melanogenesis. These results indicate that ACAN effectively inhibits melanin biosynthesis through down-regulation of MITF and thus could be useful as a new skin-whitening agent. PMID:24354173

Yoon, Weon-Jong; Ham, Young-Min; Yoon, Hun Seok; Lee, Wook-Jae; Lee, Nam Ho; Hyun, Chang-Gu

2013-10-01

212

Inhibition of protein deacetylation augments herpes simplex virus type 1-activated transcription of host fucosyltransferase genes associated with virus-induced sLex expression.  

PubMed

Herpes simplex virus type 1 induces expression of the selectin ligand sialyl Lewis X in infected cells by activating transcription of three normally silent host glycosyltransferase genes, FUT3, FUT5, and FUT6, a process that is initiated by binding of viral RNA to cellular protein kinase R. We investigated the involvement of protein deacetylation and promoter methylation in viral activation of host FUT genes by analysing the effects of appropriate inhibitors on the transcription rates of the FUT genes in virus-infected cells. The histone deacetylase inhibitor trichostatin A augmented the viral activation of FUT transcription, whereas inhibition of DNA methylation did not affect transcription of these genes. The trichostatin A enhancement did not involve interference with expression of viral late genes or viral DNA replication. Thus, the virus-activated FUT genes are at least partially suppressed by deacetylation of histones or other regulatory proteins in uninfected HEL cells, whereas promoter methylation is a less important factor. PMID:20039087

Nordén, Rickard; Nyström, Kristina; Olofsson, Sigvard

2010-03-01

213

Characterization of a lily anther-specific gene encoding cytoskeleton-binding glycoproteins and overexpression of the gene causes severe inhibition of pollen tube growth.  

PubMed

This work characterizes an anther/pollen-specific gene that encodes potential intermediate filament (IF)-binding glycoproteins in lily (Lilium longiflorum Thunb. cv. Snow Queen) anthers during the development and pollen germination. LLP13 is a single gene that encodes a polypeptide of 807 amino acids, and a calculated molecular mass of 91 kDa. The protein contains a predicted transmembrane domain at the N-terminus and a conserved domain of unknown function (DUF)593 at the C-terminal half of the polypeptide. Sequence analysis revealed that LLP13 shares significant identity (37-41 %) with two intermediate filament antigen-binding proteins, representing a unique subgroup of DUF593 domain proteins from known rice and Arabidopsis species. The expression of LLP13 gene is anther-specific, and the transcript accumulates only at the stage of pollen maturation. Both premature drying and abscisic acid (ABA) treatment of developing pollen indicated that LLP13 was not induced by desiccation and ABA, but by other developmental cues. Antiserum was raised against the overexpressed LLP13C fragment of the protein in Escherichia coli and affinity-purified antibodies were prepared. Immunoblot analyses revealed that the LLP13 protein was a heterogeneous, anther-specific glycoprotein that accumulated only at the stage of pollen maturation. The protein is not heat-soluble. The level of LLP13 protein remained for 24 h during germination in vitro. Overexpression of LLP13-GFP or GFP-LLP13 in lily pollen tubes caused severe inhibition of tube elongation. The LLP13 protein codistributed with mTalin in growing tubes, suggesting that it apparently decorates actin cytoskeleton and is likely a cytoskeleton-binding protein that binds with IFs that potentially exist in pollen tubes. PMID:24944111

Wang, Bing-Jyun; Hsu, Yi-Feng; Chen, Yun-Chu; Wang, Co-Shine

2014-09-01

214

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

PubMed

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

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

2014-01-01

215

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

216

Acute inhibition of casein kinase 1?/? rapidly delays peripheral clock gene rhythms.  

PubMed

Circadian rhythms are generated through a transcription-translation feedback loop involving clock genes and the casein kinases CSNK1D and CSNK1E. In this study, we investigated the effects of the casein kinase inhibitor PF-670462 (50 mg/kg) on rhythmic expression of clock genes in the liver, pancreas and suprachiasmatic nucleus (SCN) as well as plasma corticosterone, melatonin and running behaviour in rats and compared them to the responses to a 4 h extension of the light phase. PF-670462 acutely phase delayed the rhythmic transcription of Bmal1, Per1, Per2 and Nr1d1 in both liver and pancreas by 4.5 ± 1.3 and 4.5 ± 1.2 h, respectively, 1 day after administration. In the SCN, the rhythm of Nr1d1 and Dbp mRNA expression was delayed by 4.2 and 4 h, respectively. Despite these changes, the time of peak plasma melatonin secretion was not delayed, although the plasma corticosterone rhythm and onset of wheel-running activity were delayed by 2.1 and 1.1 h, respectively. These changes are in contrast to the effects of the 4 h light extension, which resulted in delays in peak expression of the clock genes of less than 1 h and no change in the melatonin or corticosterone rhythms. The ability of the casein kinase inhibitor to bring about large phase shifts in the rhythms of major metabolic target tissues may lead to new drugs being developed to rapidly phase adjust circadian rhythms to alleviate the metabolic impact of shift work. PMID:25245819

Kennaway, D J; Varcoe, T J; Voultsios, A; Salkeld, M D; Rattanatray, L; Boden, M J

2015-01-01

217

Inhibition of HCV 3a core gene through Silymarin and its fractions  

Microsoft Academic Search

Hepatitis C is a major health problem affecting 270 million individuals in world including Pakistan. Current treatment regimen,\\u000a interferon alpha and ribavirin only cure half of patients due to side effects and high cost.\\u000a \\u000a \\u000a \\u000a \\u000a Results  In the present study Silybum marianum (Milk thistle) seeds were collected, extracted and analyzed against HCV 3a core gene by transiently transfecting the liver\\u000a cells with

Ali Usman Ashfaq; Tariq Javed; Sidra Rehman; Zafar Nawaz; Sheikh Riazuddin

2011-01-01

218

Inhibition of proliferation of human Hela cells by small interference RNA against Pokemon gene  

Microsoft Academic Search

Objective  The transcriptional repressor Pokemon (encoded by the Zbtb7 gene) is a critical factor in oncogenesis. Pokemon overexpression\\u000a leads to overt oncogenic transformation both in vitro and in vivo in transgenic mice. The objective of this study was to investigate the effect of retrovirus expressing the siRNA targeting\\u000a Pokemon in human cervical cancer cells.\\u000a \\u000a \\u000a \\u000a Methods  We constructed and identified the recombinant retrovirus

Yi-jing Deng; Bing Ni; Man Jiang; Di Yang; Fan Li; Yu-zhang Wu

2008-01-01

219

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

PubMed

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

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

2004-06-25

220

Apigenin Inhibits Tumor Necrosis Factor-?-Induced Production and Gene Expression of Mucin through Regulating Nuclear Factor-Kappa B Signaling Pathway in Airway Epithelial Cells  

PubMed Central

In the present study, we investigated whether apigenin significantly affects tumor necrosis factor-? (TNF-?)-induced production and gene expression of MUC5AC mucin in airway epithelial cells. Confluent NCI-H292 cells were pretreated with apigenin for 30 min and then stimulated with TNF-? for 24 h or the indicated periods. The MUC5AC mucin gene expression and mucin protein production were measured by reverse transcription - polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Apigenin significantly inhibited MUC5AC mucin production and down-regulated MUC5AC gene expression induced by TNF-? in NCI-H292 cells. To elucidate the action mechanism of apigenin, effect of apigenin on TNF-?-induced nuclear factor kappa B (NF-?B) signaling pathway was also investigated by western blot analysis. Apigenin inhibited NF-?B activation induced by TNF-?. Inhibition of inhibitory kappa B kinase (IKK) by apigenin led to the suppression of inhibitory kappa B alpha (I?B?) phosphorylation and degradation, p65 nuclear translocation. This, in turn, led to the down-regulation of MUC5AC protein production in NCI-H292 cells. Apigenin also has an influence on upstream signaling of IKK because it inhibited the expression of adaptor protein, receptor interacting protein 1 (RIP1). These results suggest that apigenin can regulate the production and gene expression of mucin through regulating NF-?B signaling pathway in airway epithelial cells. PMID:25489420

Seo, Hyo-Seok; Sikder, Mohamed Asaduzzaman; Lee, Hyun Jae; Ryu, Jiho; Lee, Choong Jae

2014-01-01

221

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

PubMed

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

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

2014-10-01

222

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

Technology Transfer Automated Retrieval System (TEKTRAN)

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

223

Inhibition of Human Immunodeficiency Virus Type 1 Replication by Regulated Expression of a Polymeric Tat Activation Response RNA Decoy as a Strategy for Gene Therapy in AIDS  

Microsoft Academic Search

We are investigating a strategy for somatic gene therapy to treat human immunodeficiency virus type 1 (HIV-1) infection by intracellular expression of an RNA decoy and a ribozyme. The RNA decoy, consisting of polymeric Tat activation response elements (TARs), is designed to compete for Tat binding in an equilibrium with viral TAR RNA, thereby inhibiting viral replication. The expression of

Julianna Lisziewicz; Daisy Sun; Jason Smythe; Paolo Lusso; Franco Lori; Andrey Louie; Phillip Markham; John Rossi; Marvin Reitz; Robert C. Gallo

1993-01-01

224

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

PubMed Central

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

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

2014-01-01

225

Lipolytic inhibitor G0/G1 switch gene 2 inhibits reactive oxygen species production and apoptosis in endothelial cells.  

PubMed

G0/G1 switch gene 2 (G0S2), a novel target gene of peroxisome proliferator-activated receptor, is highly expressed in fat tissues. G0S2 acts as proapoptotic factor toward human cancer cells. Endothelial cell (EC) apoptosis may be an initiating event in the development of atherosclerosis. However, the expression and function of G0S2 in vascular ECs remain unknown. Here, we reported for the first time that G0S2 is expressed in arterial ECs. Ectopic expression of G0S2 increased neutral lipid accumulation in cultured ECs. However, G0S2 prevented ECs from serum-free starvation stress- and hydrogen peroxide (H2O2)-induced apoptosis. G0S2 blocked the H2O2-induced dissipation of mitochondrial membrane potential. G0S2 decreased the release of cytochrome c from mitochondria into the cytosol, followed by activation of caspase-9 and caspase-3. The anti-apoptotic effect of G0S2 was Bcl-2 and adipose triglyceride lipase independent. In contrast, gene silence of G0S2 increased serum-free starvation stress-induced EC apoptosis and decreased the formation of capillary-like structures. We further found that G0S2 couples with the F0F1-ATP synthase in ECs. Levels of ATP were elevated, whereas reactive oxygen species levels were reduced in G0S2-expressing ECs. G0S2 can inhibit endothelial denudation secondary to H2O2-induced injury to ECs in vivo. These results indicate that G0S2 acts as a prosurvival molecule in ECs. Taken together, our results indicate that G0S2 has a protective function in ECs and may be a potential target for the treatment of cardiovascular diseases associated with reactive oxygen species-induced EC injury, such as atherosclerosis and restenosis. PMID:25588877

Wang, Yinfang; Zhang, Yahui; Zhu, Yichun; Zhang, Peng

2015-03-15

226

?-Amylase inhibitor-1 gene from Phaseolus vulgaris expressed in Coffea arabica plants inhibits ?-amylases from the coffee berry borer pest  

PubMed Central

Background Coffee is an important crop and is crucial to the economy of many developing countries, generating around US$70 billion per year. There are 115 species in the Coffea genus, but only two, C. arabica and C. canephora, are commercially cultivated. Coffee plants are attacked by many pathogens and insect-pests, which affect not only the production of coffee but also its grain quality, reducing the commercial value of the product. The main insect-pest, the coffee berry borer (Hypotheneumus hampei), is responsible for worldwide annual losses of around US$500 million. The coffee berry borer exclusively damages the coffee berries, and it is mainly controlled by organochlorine insecticides that are both toxic and carcinogenic. Unfortunately, natural resistance in the genus Coffea to H. hampei has not been documented. To overcome these problems, biotechnological strategies can be used to introduce an ?-amylase inhibitor gene (?-AI1), which confers resistance against the coffee berry borer insect-pest, into C. arabica plants. Results We transformed C. arabica with the ?-amylase inhibitor-1 gene (?-AI1) from the common bean, Phaseolus vulgaris, under control of the seed-specific phytohemagglutinin promoter (PHA-L). The presence of the ?-AI1 gene in six regenerated transgenic T1 coffee plants was identified by PCR and Southern blotting. Immunoblotting and ELISA experiments using antibodies against ?-AI1 inhibitor showed a maximum ?-AI1 concentration of 0.29% in crude seed extracts. Inhibitory in vitro assays of the ?-AI1 protein against H. hampei ?-amylases in transgenic seed extracts showed up to 88% inhibition of enzyme activity. Conclusions This is the first report showing the production of transgenic coffee plants with the biotechnological potential to control the coffee berry borer, the most important insect-pest of crop coffee. PMID:20565807

2010-01-01

227

Genistein downregulates SREBP-1 regulated gene expression by inhibiting site-1 protease expression in HepG2 cells.  

PubMed

Genistein is one of the most abundant isoflavones in soy. The effects of genistein on cholesterol synthesis and fatty acid oxidation have been well documented, but the effect of genistein on fatty acid synthesis remains unclear. Thus, we investigated the effect of genistein on fatty acid synthase (FAS) expressions in HepG2 cells. In HepG2 cells treated with 10 micromol/L genistein, mRNA and protein expressions of FAS, as well as FAS activity, were significantly decreased. The promoter region of FAS contains binding sites for the transcription factor called sterol regulated element binding protein 1 (SREBP-1); SREBP-1 must be processed by site-1 (S1P) and site-2 proteases to be activated. We also investigated the effects of genistein on S1P, SREBP-1 expression, and subsequent SREBP-1 processing by S1P in HepG2 cells. Genistein reduced the expression of S1P and the processing of SREBP-1 but did not change the expression of SREBP-1 mRNA. SREBP-1 is also a transcription factor for lipogenic genes, such as stearoyl coenzyme-A desaturase1 (SCD1), glycerol-3-phosphate acyltransferase (GPAT), and acetyl-CoA carboxylase (ACC)1, and ACC2. Genistein also significantly inhibited the expression of these lipogenic genes. Thus, genistein treatment of HepG2 cells decreased the expression of lipogenic genes such as FAS, SCD1, GPAT, and ACC, which is, at least in part, mediated through the downregulation of S1P expression and subsequent SREBP-1 proteolytic cleavage. PMID:17449569

Shin, Eui Seok; Lee, Hyoung Ho; Cho, Si Young; Park, Hyun Woo; Lee, Sang Jun; Lee, Tae Ryong

2007-05-01

228

Molecular characterization and inhibition analysis of the acetylcholinesterase gene from the silkworm maggot, Exorista sorbillans.  

PubMed

Several organophosphorus (OP) insecticides can selectively kill the silkworm maggot, Exorista sorbillans (Es) (Diptera: Tachinidae), while not obviously affecting the host (Bombyx mori) larvae, but the mechanism is not yet clear. In this study, the cDNA encoding an acetylcholinesterase (AChE) from the field Es was isolated. One point mutation (Gly353Ala) was identified. The Es-353G AChE and Es-353A AChE were expressed in baculovirus- insect cell system, respectively. The inhibition results showed that for eserine and Chlorpyrifos, Es-353A AChE was significantly less sensitive than Es-353G AChE. Meanwhile, comparison of the I(50) values of eserine, dichlorvos, Chlorpyrifos and omethoate of recombinant Es AChEs with its host (Bombyx mori) AChEs indicated that, both Es AChEs are more sensitive than B. mori AChEs. The results give an insight of the mechanism that some OP insecticides can selectively kills Es while without distinct effect on its host, B. mori. PMID:20797321

Lang, Guo-Jun; Zhang, Ming-Yan; Li, Bao-Ling; Yu, Lin-Lin; Lu, Xing-Meng; Zhang, Chuan-Xi

2010-08-01

229

Post-translational modifications in activation and inhibition of Oct-1-DNA binding complex in H2B and other diverse gene regulation: prediction of interplay sites.  

PubMed

Octamer DNA binding transcription factors play important roles in housekeeping and specific gene regulations. Octamer DNA binding transcription factor-1 (Oct-1), expressed ubiquitously, is a multifunctional molecule. The binding sites of Oct-1 are the promoters of H2B gene and the genes of snRNA, U2, U6, and 7SK, yet Oct-1 has been described as constitutively expressed transcription factor regulating the expression of housekeeping genes. Diverse tissue-specific genes regulations by Oct-1 include genes for interleukins (IL) 2, 3, 5; the granulocyte-macrophagal colony-stimulating factor, immunoglobulins ?, ?, Ly9; the endocrine-associated Pit-1 gene; the genes for gonadoliberin, prolactin, the thyroid transcription factor, and thyrotropin. The most interesting aspect of the gene regulations of Oct-1 includes both activation and inhibition of transcription. These opposite regulations of Oct-1 have been described through presence/absence of a post-translational modification (PTM) in its different domains. We propose a mechanism of interplay of different PTMs or presence/absence of PTMs in the different domains of Oct-1. We also suggest that the absence of phosphorylation and acetylation in G1 and S phases of the cell cycle is associated with interplay of methylation and O-GlcNAc modification. This interplay of O-GlcNAc modification with the phosphorylation and methylation with acetylation in POU sub-domain of Oct-1 may facilitate the formation of Oct-1-DNA complex, consequently activating H2B gene transcription. Whereas, in G2 and M phases these sites are occupied by phosphate resulting in inhibition of Oct-1-DNA complex formation leading to the suppression of H2B gene transcription. PMID:22961715

Shakoori, Abdul Rauf; Hoessli, Daniel C; Nasir-ud-Din

2013-02-01

230

Effect of Nuclear Factor ?B Inhibition on Serotype 9 Adeno-Associated Viral (AAV9) Minidystrophin Gene Transfer to the mdx Mouse  

PubMed Central

Gene therapy studies for Duchenne muscular dystrophy (DMD) have focused on viral vector-mediated gene transfer to provide therapeutic protein expression or treatment with drugs to limit dystrophic changes in muscle. The pathological activation of the nuclear factor (NF)-?B signaling pathway has emerged as an important cause of dystrophic muscle changes in muscular dystrophy. Furthermore, activation of NF-?B may inhibit gene transfer by promoting inflammation in response to the transgene or vector. Therefore, we hypothesized that inhibition of pathological NF-?B activation in muscle would complement the therapeutic benefits of dystrophin gene transfer in the mdx mouse model of DMD. Systemic gene transfer using serotype 9 adeno-associated viral (AAV9) vectors is promising for treatment of preclinical models of DMD because of vector tropism to cardiac and skeletal muscle. In quadriceps of C57BL/10ScSn-Dmdmdx/J (mdx) mice, the addition of octalysine (8K)–NF-?B essential modulator (NEMO)-binding domain (8K-NBD) peptide treatment to AAV9 minidystrophin gene delivery resulted in increased levels of recombinant dystrophin expression suggesting that 8K-NBD treatment promoted an environment in muscle tissue conducive to higher levels of expression. Indices of necrosis and regeneration were diminished with AAV9 gene delivery alone and to a greater degree with the addition of 8K-NBD treatment. In diaphragm muscle, high-level transgene expression was achieved with AAV9 minidystoophin gene delivery alone; therefore, improvements in histological and physiological indices were comparable in the two treatment groups. The data support benefit from 8K-NBD treatment to complement gene transfer therapy for DMD in muscle tissue that receives incomplete levels of transduction by gene transfer, which may be highly significant for clinical applications of muscle gene delivery. PMID:22231732

Reay, Daniel P; Niizawa, Gabriela A; Watchko, Jon F; Daood, Molly; Reay, Ja’Nean C; Raggi, Eugene; Clemens, Paula R

2012-01-01

231

Inhibition of Experimental Liver Cirrhosis in Mice by Telomerase Gene Delivery  

NASA Astrophysics Data System (ADS)

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

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

2000-02-01

232

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. PMID:25009705

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

2014-01-01

233

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

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. PMID:25009705

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

2014-05-01

234

A novel function of interferon regulatory factor-1: inhibition of Th2 cells by down-regulating the Il4 gene during Listeria infection.  

PubMed

Infection with certain pathogens induces a shift of the Th subset balance to a Th1 dominant state. This, in turn, results in the suppression of Th2 responses. We focused on the involvement of interferon regulatory factor-1 (IRF-1) in the suppression of Th2 cells during Listeria infection. We found that the inhibition of IL-4 production by Th2 cells is mediated by a soluble factor (LmSN) produced by Listeria-infected antigen-presenting cells. The inhibition is not observed with T cells from Irf1 gene-targeted mice. IRF-1 suppresses transcription of the Il4 gene in Th2 cells. Under the influence of the LmSN, IRF-1 binds to the 3' untranslated region (UTR) region of the Il4 gene and down-regulates Il4 gene transcription. Finally, we identified IL-1? and IL-1? as the mediator of the LmSN activity. Signaling through IL-1R induces the stabilization and/or nuclear translocation of IRF-1. We propose that IRF-1 functions to induce the T-cell subset shift via a novel mechanism. Under the influence of IL-1, IRF-1 translocates into the nucleus and acts on the 3'UTR region of the Il4 gene, thus inhibiting its transcription in Th2 cells. As a result, the immune system shifts predominantly to a Th1 response during Listeria infection, resulting in effective protection of the host. PMID:25280793

Maruyama, Saho; Kanoh, Makoto; Matsumoto, Akira; Kuwahara, Makoto; Yamashita, Masakatsu; Asano, Yoshihiro

2015-03-01

235

Tandemly Duplicated Arabidopsis Genes That Encode Polygalacturonase-Inhibiting Proteins Are Regulated Coordinately by Different Signal Transduction Pathways in Response to Fungal Infection  

E-print Network

Polygalacturonase-inhibiting proteins (PGIPs) are plant proteins that counteract fungal polygalacturonases, which are important virulence factors. Like many other plant defense proteins, PGIPs are encoded by gene families, but the roles of individual genes in these families are poorly understood. Here, we show that in Arabidopsis, two tandemly duplicated PGIP genes are upregulated coordinately in response to Botrytis cinerea infection, but through separate signal transduction pathways. AtPGIP2 expression is mediated by jasmonate and requires COI1 and JAR1, whereas AtPGIP1 expression is upregulated strongly by oligogalacturonides but is unaffected by salicylic acid, jasmonate, or ethylene. Both AtPGIP1 and AtPGIP2 encode functional inhibitors of polygalacturonase from Botrytis, and their overexpression in Arabidopsis significantly reduces Botrytis disease symptoms. Therefore, gene duplication followed by the divergence of promoter regions may result in different modes of regulation of similar defensive proteins, thereby enhancing the likelihood of defense gene activation during pathogen infection.

Simone Ferrari; A Donatella Vairo; B Frederick M. Ausubel; A Felice Cervone; Giulia De Lorenzo B

236

CK2 Phosphorylates and Inhibits TAp73 Tumor Suppressor Function to Promote Expression of Cancer Stem Cell Genes and Phenotype in Head and Neck Cancer12  

PubMed Central

Cancer stem cells (CSC) and genes have been linked to cancer development and therapeutic resistance, but the signaling mechanisms regulating CSC genes and phenotype are incompletely understood. CK2 has emerged as a key signal serine/threonine kinase that modulates diverse signal cascades regulating cell fate and growth. We previously showed that CK2 is often aberrantly expressed and activated in head and neck squamous cell carcinomas (HNSCC), concomitantly with mutant (mt) tumor suppressor TP53, and inactivation of its family member, TAp73. Unexpectedly, we observed that classical stem cell genes Nanog, Sox2, and Oct4, are overexpressed in HNSCC with inactivated TAp73 and mtTP53. However, the potential relationship between CK2, TAp73 inactivation, and CSC phenotype is unknown. We reveal that inhibition of CK2 by pharmacologic inhibitors or siRNA inhibits the expression of CSC genes and side population (SP), while enhancing TAp73 mRNA and protein expression. Conversely, CK2 inhibitor attenuation of CSC protein expression and the SP by was abrogated by TAp73 siRNA. Bioinformatic analysis uncovered a single predicted CK2 threonine phosphorylation site (T27) within the N-terminal transactivation domain of TAp73. Nuclear CK2 and TAp73 interaction, confirmed by co-immunoprecipitation, was attenuated by CK2 inhibitor, or a T27A point-mutation of this predicted CK2 threonine phospho-acceptor site of TAp73. Further, T27A mutation attenuated phosphorylation, while enhancing TAp73 function in repressing CSC gene expression and SP cells. A new CK2 inhibitor, CX-4945, inhibited CSC related SP cells, clonogenic survival, and spheroid formation. Our study unveils a novel regulatory mechanism whereby aberrant CK2 signaling inhibits TAp73 to promote the expression of CSC genes and phenotype. PMID:25379016

Lu, Hai; Yan, Carol; Quan, Xin Xin; Yang, Xinping; Zhang, Jialing; Bian, Yansong; Chen, Zhong; Van Waes, Carter

2014-01-01

237

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. PMID:24281383

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

238

Metformin inhibits food intake and neuropeptide Y gene expression in the hypothalamus  

PubMed Central

Metformin may reduce food intake and body weight, but the anorexigenic effects of metformin are still poorly understood. In this study, Sprague-Dawley rats were administered a single intracere-broventricular dose of metformin and compound C, in a broader attempt to investigate the regula-tory effects of metformin on food intake and to explore the possible mechanism. Results showed that central administration of metformin significantly reduced food intake and body weight gain, par-ticularly after 4 hours. A reduction of neuropeptide Y expression and induction of AMP-activated protein kinase phosphorylation in the hypothalamus were also observed 4 hours after metformin administration, which could be reversed by compound C, a commonly-used antagonist of AMP-activated protein kinase. Furthermore, metformin also improved lipid metabolism by reducing plasma low-density lipoprotein. Our findings suggest that under normal physiological conditions, central regulation of appetite by metformin is related to a decrease in neuropeptide Y gene expres-sion, and that the activation of AMP-activated protein kinase may simply be a response to the anorexigenic effect of metformin. PMID:25206548

Duan, Yale; Zhang, Rui; Zhang, Min; Sun, Lijuan; Dong, Suzhen; Wang, Gang; Zhang, Jun; Zhao, Zheng

2013-01-01

239

Plp1 gene duplication inhibits airway responsiveness and induces lung inflammation.  

PubMed

Mice with Plp1 gene duplication model the most common form of Pelizaeus-Merzbacher disease (PMD), a CNS disease in which patients may suffer respiratory complications. We hypothesized that affected mice would lack airway responsiveness compared to wild-type and carrier mice during methacholine challenge. Wild-type (n = 10), carrier female (n = 6) and affected male (n = 8) mice were anesthetized-paralyzed, tracheostomized and ventilated. Respiratory mechanics were recorded at baseline and during escalating doses of nebulized methacholine followed by albuterol. Lung resistance (RL) was the primary endpoint. Lung tissues were assayed for inflammatory and histological differences. At baseline, phase angles were higher in carrier and affected mice than wild-type. Dose-response RL curves in affected and carrier mice indicated a lack of methacholine response. Albuterol reduced RL in wild-type and carrier, but not affected mice. Affected mice exhibited lower interleukin (IL)-6 tissue levels and alveolar inflammatory infiltrates. Affected and carrier mice, compared to wild-type, lacked airway reactivity during methacholine challenge, but only affected mice exhibited decreased lung tissue levels of IL-6 and inflammation. PMID:25445931

Rodriguez, Elena; Sakowski, Lauren; Hobson, Grace M; Armani, Milena Hirata; Kreiger, Portia A; Zhu, Yan; Waldman, Scott A; Shaffer, Thomas H

2015-02-01

240

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

241

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

SciTech Connect

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

Cho, Mi Ae [Division of Endocrinology, Pituitary Tumor Clinic, Research Institute of Endocrinology, Yonsei University College of Medicine, Seoul (Korea, Republic of); Endocrinology, Dong Rae Bong Seng Hospital, Busan (Korea, Republic of); Yashar, Parham [Endocrinology, Metabolism, and Molecular Medicine, Northwestern University, Feinberg School of Medicine, Chicago, IL 60611 (United States); Department of Neurosurgery, University of Southern California - Keck School of Medicine, Los Angeles, CA (United States); Kim, Suk Kyoung; Noh, Taewoong [Division of Endocrinology, Pituitary Tumor Clinic, Research Institute of Endocrinology, Yonsei University College of Medicine, Seoul (Korea, Republic of); Gillam, Mary P. [Endocrinology, Metabolism, and Molecular Medicine, Northwestern University, Feinberg School of Medicine, Chicago, IL 60611 (United States); Lee, Eun Jig [Division of Endocrinology, Pituitary Tumor Clinic, Research Institute of Endocrinology, Yonsei University College of Medicine, Seoul (Korea, Republic of); Endocrinology, Metabolism, and Molecular Medicine, Northwestern University, Feinberg School of Medicine, Chicago, IL 60611 (United States)], E-mail: EJLEE423@yuhs.ac; Jameson, J. Larry [Endocrinology, Metabolism, and Molecular Medicine, Northwestern University, Feinberg School of Medicine, Chicago, IL 60611 (United States)

2008-07-04

242

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

243

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

PubMed

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

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

2009-02-25

244

Circadian gene hClock enhances proliferation and inhibits apoptosis of human colorectal carcinoma cells in vitro and in vivo.  

PubMed

Colorectal carcinoma (CRC) is one of the most prevalent types of malignancy?associated mortality worldwide. Previous studies have demonstrated that amplification and overexpression of the human circadian locomotor output cycles kaput gene (hClock) was closely associated with a high risk for CRC as well as poor prognosis in CRC patients. However, the underlying molecular mechanisms of CRC remain to be fully elucidated. In the present study, hClock was exogenously overexpressed in the CRC cell line SW480 via infection of a lentivirus vector expressing hClock; in addition, a lentivirus vector?based RNA interference approach, using short hairpin RNA, was performed in order to knockdown hClock in SW620 cells. The results showed that upregulation of hClock promoted proliferation and inhibited apoptosis in SW480 cells in vitro and in vivo, while downregulation of hClock inhibited SW620 cell proliferation and accelerated apoptosis in vitro. Upregulation of hClock enhanced the activity of the anti?apoptotic gene phosphorpylated (p?)AKT and inhibited the expression of the pro?apoptotic gene B cell lymphoma?2 (Bcl?2)?associated X protein and Bcl?2 homology 3 interacting domain death agonist. Furthermore, targeted inhibition of hClock activity reduced p?AKT expression. In conclusion, the results of the present study suggested that the circadian gene hClock promoted CRC progression and inhibit tumor cell apoptosis in vitro and in vivo, while silencing hClock was able to reverse this effect. PMID:25625359

Wang, Yaping; Qian, Ruizhe; Sun, Ning; Lu, Chao; Chen, Zongyou; Hua, Luchun

2015-06-01

245

RNA interference-mediated silencing of eukaryotic translation initiation factor 3, subunit B (EIF3B) gene expression inhibits proliferation of colon cancer cells  

PubMed Central

Background A key factor underlying the control of the cellular growth, size and proliferation involves the regulation of the total protein synthesis. Most often, the initial stages of mRNA translation are rate limiting, which involves a group of eukaryotic translation initiation factors (EIFs). Research advances focused on the inhibition of their expression and activity hold the key to the initiation and progression of tumor and tumor prognosis. Method We performed RNA interference (RNAi) with the lentivirus vector system to silence the EIF3B gene using the colon cancer cell strain SW1116. The negative control included the normal target cells infected with the negative control virus whereas the knockdown cells included the normal target cells transfected with the RNAi target virus. We tested the inhibition resulting from the decreased expression of EIF3B gene on the proliferation rate of SW1116 cells, including the cell cycle, apoptosis and clonability. Results Compared with the negative control, the impact of EIF3B gene expression in SW1116 cells on the levels of mRNA and protein in the knockdown group, was significantly inhibited (P <0.01). Furthermore, the cell proliferation rate and clonability were also significantly inhibited (P <0.01). The apoptosis rate increased significantly (P <0.05). A significant decrease in the number of cells in the G1 phase (P <0.01) and significant increases in S (P <0.01) and G2 phases (P <0.05) were observed. Conclusions The silencing of EIF3B gene expression inhibits the proliferation of colon cancer cells. PMID:22734884

2012-01-01

246

Expression of the DisA amino acid decarboxylase from Proteus mirabilis inhibits motility and class 2 flagellar gene expression in Escherichia coli.  

PubMed

In Proteus mirabilis, a putative phenylalanine decarboxylase (DisA) acts in a regulatory pathway to inhibit class 2 flagellar gene expression and motility. In this study, we demonstrate that DisA expression in Escherichia coli blocked motility and resulted in a 50-fold decrease in the expression of class 2 (fliA) and class 3 (fliC) flagellar genes. However, the expression of flhDC encoding the class 1 activator of the flagellar cascade was unchanged by DisA expression at both the transcriptional and translational levels. Phenethylamine, a decarboxylation product derived from phenylalanine, was able to mimic DisA overexpression and decrease both motility and class 2/3 flagellar gene expression. In addition, both DisA overexpression and phenethylamine strongly inhibited biofilm formation in E. coli. DisA overexpression and exogenous phenethylamine could also reduce motility in other enteric bacteria, but had no effect on motility in non-enteric Gram-negative bacteria. It is hypothesized that phenethylamine or a closely related compound formed by the DisA decarboxylation reaction inhibits the formation or activity of the FlhD(4)C(2) complex required for activation of class 2 genes. PMID:22982608

Stevenson, Lindsay G; Szostek, Bree A; Clemmer, Katy M; Rather, Philip N

2013-01-01

247

Inhibition of CD44 Gene Expression in Human Skin Models, Using Self-Delivery Short Interfering RNA Administered by Dissolvable Microneedle Arrays  

PubMed Central

Abstract Treatment of skin disorders with short interfering RNA (siRNA)-based therapeutics requires the development of effective delivery methodologies that reach target cells in affected tissues. Successful delivery of functional siRNA to the epidermis requires (1) crossing the stratum corneum, (2) transfer across the keratinocyte membrane, followed by (3) incorporation into the RNA-induced silencing complex. We have previously demonstrated that treatment with microneedle arrays loaded with self-delivery siRNA (sd-siRNA) can achieve inhibition of reporter gene expression in a transgenic mouse model. Furthermore, treatment of human cultured epidermal equivalents with sd-siRNA resulted in inhibition of target gene expression. Here, we demonstrate inhibition of CD44, a gene that is uniformly expressed throughout the epidermis, by sd-siRNA both in vitro (cultured human epidermal skin equivalents) and in vivo (full-thickness human skin equivalents xenografted on immunocompromised mice). Treatment of human skin equivalents with CD44 sd-siRNA markedly decreased CD44 mRNA levels, which led to a reduction of the target protein as confirmed by immunodetection in epidermal equivalent sections with a CD44-specific antibody. Taken together, these results demonstrate that sd-siRNA, delivered by microneedle arrays, can reduce expression of a targeted endogenous gene in a human skin xenograft model. PMID:22480249

Lara, Maria Fernanda; González-González, Emilio; Speaker, Tycho J.; Hickerson, Robyn P.; Leake, Devin; Milstone, Leonard M.; Contag, Christopher H.

2012-01-01

248

Antisense inhibition of gene expression in cells by oligonucleotides incorporating locked nucleic acids: effect of mRNA target sequence and chimera design  

PubMed Central

Use of antisense oligonucleotides is a versatile strategy for achieving control of gene expression. Unfortunately, the interpretation of antisense-induced phenotypes is sometimes difficult, and chemical modifications that improve the potency and specificity of antisense action would be useful. The introduction of locked nucleic acid (LNA) bases into oligonucleotides confers exceptional improvement in binding affinity, up to 10°C per substitution, making LNAs an exciting option for the optimization of antisense efficacy. Here we examine the rules governing antisense gene inhibition within cells by oligonucleotides that contain LNA bases. LNA- containing oligomers were transfected into cells using cationic lipid and accumulated in the nucleus. We tested antisense gene inhibition by LNAs and LNA–DNA chimeras complementary to the 5?-untranslated region, the region surrounding the start codon and the coding region of mRNA, and identified effective antisense agents targeted to each of these locations. Our data suggest that LNA bases can be used to develop antisense oligonucleotides and that their use is a versatile approach for efficiently inhibiting gene expression inside cells. PMID:12466540

Braasch, Dwaine A.; Liu, Yinghui; Corey, David R.

2002-01-01

249

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. PMID:24460684

2014-01-01

250

Highly potent dUTPase inhibition by a bacterial repressor protein reveals a novel mechanism for gene expression control  

PubMed Central

Transfer of phage-related pathogenicity islands of Staphylococcus aureus (SaPI-s) was recently reported to be activated by helper phage dUTPases. This is a novel function for dUTPases otherwise involved in preservation of genomic integrity by sanitizing the dNTP pool. Here we investigated the molecular mechanism of the dUTPase-induced gene expression control using direct techniques. The expression of SaPI transfer initiating proteins is repressed by proteins called Stl. We found that ?11 helper phage dUTPase eliminates SaPIbov1 Stl binding to its cognate DNA by binding tightly to Stl protein. We also show that dUTPase enzymatic activity is strongly inhibited in the dUTPase:Stl complex and that the dUTPase:dUTP complex is inaccessible to the Stl repressor. Our results disprove the previously proposed G-protein-like mechanism of SaPI transfer activation. We propose that the transfer only occurs if dUTP is cleared from the nucleotide pool, a condition promoting genomic stability of the virulence elements. PMID:25274731

Szabó, Judit E.; Németh, Veronika; Papp-Kádár, Veronika; Nyíri, Kinga; Leveles, Ibolya; Bendes, Ábris Á.; Zagyva, Imre; Róna, Gergely; Pálinkás, Hajnalka L.; Besztercei, Balázs; Ozohanics, Olivér; Vékey, Károly; Liliom, Károly; Tóth, Judit; Vértessy, Beáta G.

2014-01-01

251

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

PubMed Central

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

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

2009-01-01

252

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. PMID:24849810

Spiegelberg, Linda; Swagemakers, Sigrid MA; van IJcken, Wilfred FJ; Oole, Edwin; Wolvius, Eppo B; Essers, Jeroen; Braks, Joanna AM

2014-01-01

253

Gene expression analysis reveals inhibition of radiation-induced TGF?-signaling by hyperbaric oxygen therapy in mouse salivary glands.  

PubMed

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. PMID:24849810

Spiegelberg, Linda; Swagemakers, Sigrid M A; Van Ijcken, Wilfred F J; Oole, Edwin; Wolvius, Eppo B; Essers, Jeroen; Braks, Joanna A M

2014-01-01

254

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

NASA Astrophysics Data System (ADS)

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

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

2010-11-01

255

Gonadotropin-inhibitory hormone inhibits GnRH-induced gonadotropin subunit gene transcriptions by inhibiting AC/cAMP/PKA-dependent ERK pathway in L?T2 cells.  

PubMed

A neuropeptide that directly inhibits gonadotropin secretion from the pituitary was discovered in quail and named gonadotropin-inhibitory hormone (GnIH). The presence and functional roles of GnIH orthologs, RF-amide-related peptides (RFRP), that possess a common C-terminal LPXRF-amide (X = L or Q) motif have also been demonstrated in mammals. GnIH orthologs inhibit gonadotropin synthesis and release by acting on pituitary gonadotropes and GnRH neurons in the hypothalamus via its receptor (GnIH receptor). It is becoming increasingly clear that GnIH is an important hypothalamic neuropeptide controlling reproduction, but the detailed signaling pathway mediating the inhibitory effect of GnIH on target cells is still unknown. In the present study, we investigated the pathway of GnIH cell signaling and its possible interaction with GnRH signaling using a mouse gonadotrope cell line, L?T2. First, we demonstrated the expression of GnIH receptor mRNA in L?T2 cells by RT-PCR. We then examined the inhibitory effects of mouse GnIH orthologs [mouse RFRP (mRFRP)] on GnRH-induced cell signaling events. We showed that mRFRP effectively inhibited GnRH-induced cAMP signaling by using a cAMP-sensitive reporter system and measuring cAMP levels, indicating that mRFRP function as an inhibitor of adenylate cyclase. We further showed that mRFRP inhibited GnRH-stimulated ERK phosphorylation, and this effect was mediated by the inhibition of the protein kinase A pathway. Finally, we demonstrated that mRFRP inhibited GnRH-stimulated gonadotropin subunit gene transcriptions and also LH release. Taken together, the results indicate that mRFRP function as GnIH to inhibit GnRH-induced gonadotropin subunit gene transcriptions by inhibiting adenylate cyclase/cAMP/protein kinase A-dependent ERK activation in L?T2 cells. PMID:22374973

Son, You Lee; Ubuka, Takayoshi; Millar, Robert P; Kanasaki, Haruhiko; Tsutsui, Kazuyoshi

2012-05-01

256

Butein, a tetrahydroxychalcone, inhibits nuclear factor (NF)-kappaB and NF-kappaB-regulated gene expression through direct inhibition of IkappaBalpha kinase beta on cysteine 179 residue.  

PubMed

Although butein (3,4,2',4'-tetrahydroxychalcone) is known to exhibit anti-inflammatory, anti-cancer, and anti-fibrogenic activities, very little is known about its mechanism of action. Because numerous effects modulated by butein can be linked to interference with the NF-kappaB pathway, we investigated in detail the effect of this chalcone on NF-kappaB activity. As examined by DNA binding, we found that butein suppressed tumor necrosis factor (TNF)-induced NF-kappaB activation in a dose- and time-dependent manner; suppressed the NF-kappaB activation induced by various inflammatory agents and carcinogens; and inhibited the NF-kappaB reporter activity induced by TNFR1, TRADD, TRAF2, NIK, TAK1/TAB1, and IKK-beta. We also found that butein blocked the phosphorylation and degradation of IkappaBalpha by inhibiting IkappaBalpha kinase (IKK) activation. We found the inactivation of IKK by butein was direct and involved cysteine residue 179. This correlated with the suppression of phosphorylation and the nuclear translocation of p65. In this study, butein also inhibited the expression of the NF-kappaB-regulated gene products involved in anti-apoptosis (IAP2, Bcl-2, and Bcl-xL), proliferation (cyclin D1 and c-Myc), and invasion (COX-2 and MMP-9). Suppression of these gene products correlated with enhancement of the apoptosis induced by TNF and chemotherapeutic agents; and inhibition of cytokine-induced cellular invasion. Overall, our results indicated that antitumor and anti-inflammatory activities previously assigned to butein may be mediated in part through the direct inhibition of IKK, leading to the suppression of the NF-kappaB activation pathway. PMID:17439942

Pandey, Manoj K; Sandur, Santosh K; Sung, Bokyung; Sethi, Gautam; Kunnumakkara, Ajaikumar B; Aggarwal, Bharat B

2007-06-15

257

SlrA/SlrR/SinR inhibits motility gene expression upstream of a hypersensitive and hysteric switch at the level of ?D in Bacillus subtilis  

PubMed Central

Exponentially growing Bacillus subtilis cultures are epigenetically differentiated into two subpopulations in which cells are either ON or OFF for ?D-dependent gene expression: a pattern suggestive of bistability. The gene encoding ?D, sigD, is part of the 31-gene fla/che operon where its location at the 3? end, 25 kb away from the strong Pfla/che promoter, determines its expression level relative to a threshold. Here we show that addition of a single extra copy of the slrA gene in the chromosome inhibited ?D-dependent gene expression. SlrA together with SinR and SlrR reduced sigD transcript by potentiating a distance-dependent decrease in fla/che operon transcript abundance that was not mediated by changes in expression from the Pfla/che promoter. Consistent with acting upstream of ?D, SlrA/SinR/SlrR could be bypassed by artificial ectopic expression of sigD that hysteretically maintained for 20 generations by engaging the sigD gene at the native locus. SlrA/SinR/SlrR was also bypassed by increasing fla/che transcription and resulted a hypersensitive output in flagellin expression. Thus, flagellin gene expression demonstrated hypersensitivity and hysteresis and we conclude that ?D-dependent gene expression is bistable. PMID:22329926

Cozy, Loralyn M.; Phillips, Andrew M.; Calvo, Rebecca A.; Bate, Ashley R.; Hsueh, Yi-Huang; Bonneau, Richard; Eichenberger, Patrick; Kearns, Daniel B.

2012-01-01

258

Superiority of combined phosphodiesterase PDE3/PDE4 inhibition over PDE4 inhibition alone on glucocorticoid- and long-acting ?2-adrenoceptor agonist-induced gene expression in human airway epithelial cells.  

PubMed

Glucocorticoids, also known as corticosteroids, induce effector gene transcription as a part of their anti-inflammatory mechanisms of action. Such genomic effects can be significantly enhanced by long-acting ?2-adrenoceptor agonists (LABAs) and may contribute to the clinical superiority of inhaled corticosteroid (ICS)/LABA combinations in asthma and chronic obstructive pulmonary disease (COPD) over ICSs alone. Using models of cAMP- and glucocorticoid-induced transcription in human bronchial epithelial BEAS-2B cells, we show that combining inhibitors of phosphodiesterase (PDE) 3 and PDE4 provides greater benefits compared with inhibiting either PDE alone. In respect to cAMP-dependent transcription, inhibitors of PDE3 (siguazodan, cilostazol) and PDE4 (rolipram, GSK256066, roflumilast N-oxide) each sensitized to the LABA, formoterol. This effect was magnified by dual PDE3 and PDE4 inhibition. Siguazodan plus rolipram was also more effective at inducing cAMP-dependent transcription than either inhibitor alone. Conversely, the concentration-response curve describing the enhancement of dexamethasone-induced, glucocorticoid response element-dependent transcription by formoterol was displaced to the left by PDE4, but not PDE3, inhibition. Overall, similar effects were described for bona fide genes, including RGS2, CD200, and CRISPLD2. Importantly, the combination of siguazodan plus rolipram prolonged the duration of gene expression induced by formoterol, dexamethasone, or dexamethasone plus formoterol. This was most apparent for RGS2, a bronchoprotective gene that may also reduce the proinflammatory effects of constrictor mediators. Collectively, these data provide a rationale for the use of PDE3 and PDE4 inhibitors in the treatment of COPD and asthma where they may enhance, sensitize, and prolong the effects of LABA/ICS combination therapies. PMID:25324049

BinMahfouz, Hawazen; Borthakur, Bibhusana; Yan, Dong; George, Tresa; Giembycz, Mark A; Newton, Robert

2015-01-01

259

Apple flavonoids inhibit growth of HT29 human colon cancer cells and modulate expression of genes involved in the biotransformation of xenobiotics.  

PubMed

Flavonoids from fruits and vegetables probably reduce risks of diseases associated with oxidative stress, including cancer. Apples contain significant amounts of flavonoids with antioxidative potential. The objectives of this study were to investigate such compounds for properties associated with reduction of cancer risks. We report herein that apple flavonoids from an apple extract (AE) inhibit colon cancer cell growth and significantly modulate expression of genes related to xenobiotic metabolism. HT29 cells were treated with AE at concentrations delivering 5-50 microM of one of the major ingredients, phloridzin ("phloridzin-equivalents," Ph.E), to the cell culture medium, with a synthetic flavonoid mixture mimicking the composition of the AE or with 5-100 microM individual flavonoids. HT29 cell growth was inhibited by the complex extract and by the mixture. HT29 cells were treated with nontoxic doses of the AE (30 microM, Ph.E) and after 24 h total RNA was isolated to elucidate patterns of gene expression using a human cDNA-microarray (SuperArray) spotted with 96 genes of drug metabolism. Treatment with AE resulted in an upregulation of several genes (GSTP1, GSSTT2, MGST2, CYCP4F3, CHST5, CHST6, and CHST7) and downregulation of EPHX1, in comparison to the medium controls. The enhanced transcriptional activity of GSTP1 and GSTT2 genes was confirmed with real-time qRT-PCR. On the basis of the pattern of differential gene expression found here, we conclude that apple flavonoids modulate toxicological defense against colon cancer risk factors. In addition to the inhibition of tumor cell proliferation, this could be a mechanism of cancer risk reduction. PMID:16369997

Veeriah, Selvaraju; Kautenburger, Tanja; Habermann, Nina; Sauer, Julia; Dietrich, Helmut; Will, Frank; Pool-Zobel, Beatrice Louise

2006-03-01

260

Silencing SlELP2L, a tomato Elongator complex protein 2-like gene, inhibits leaf growth, accelerates leaf, sepal senescence, and produces dark-green fruit  

PubMed Central

The multi-subunit complex Elongator interacts with elongating RNA polymerase II (RNAPII) and is thought to facilitate transcription through histone acetylation. Elongator is highly conserved in eukaryotes, yet has multiple kingdom-specific functions in diverse organisms. Recent genetic studies performed in Arabidopsis have demonstrated that Elongator functions in plant growth and development, and in response to biotic and abiotic stress. However, little is known about its roles in other plant species. Here, we study the function of an Elongator complex protein 2-like gene in tomato, here designated as SlELP2L, through RNAi-mediated gene silencing. Silencing SlELP2L in tomato inhibits leaf growth, accelerates leaf and sepal senescence, and produces dark-green fruit with reduced GA and IAA contents in leaves, and increased chlorophyll accumulation in pericarps. Gene expression analysis indicated that SlELP2L-silenced plants had reduced transcript levels of ethylene- and ripening-related genes during fruit ripening with slightly decreased carotenoid content in fruits, while the expression of DNA methyltransferase genes was up-regulated, indicating that SlELP2L may modulate DNA methylation in tomato. Besides, silencing SlELP2L increases ABA sensitivity in inhibiting seedling growth. These results suggest that SlELP2L plays important roles in regulating plant growth and development, as well as in response to ABA in tomato. PMID:25573793

Zhu, Mingku; Li, Yali; Chen, Guoping; Ren, Lijun; Xie, Qiaoli; Zhao, Zhiping; Hu, Zongli

2015-01-01

261

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. PMID:24403227

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

262

Mangostin Inhibits Inhibitor B Kinase Activity and Decreases Lipopolysaccharide-Induced Cyclooxygenase2 Gene Expression in C6 Rat Glioma Cells  

Microsoft Academic Search

We investigated the effect of -mangostin purified from the fruit hull of the medicinal plant Garcinia mangostana on spontane- ous prostaglandin E2 (PGE2) release and inducible cyclooxy- genase 2 (COX-2) gene expression in C6 rat glioma cells. An 18-h treatment with -mangostin potently inhibited spontane- ous PGE2 release in a concentration-dependent manner with the IC50 value of approximately 2 M,

Keigo Nakatani; Tohru Yamakuni; Nobuhiko Kondo; Tsutomu Arakawa; Kenji Oosawa; Susumu Shimura; Hiroyasu Inoue; Yasushi Ohizumi

2004-01-01

263

Cytomegalovirus-mediated induction of antisense mRNA expression to UL44 inhibits virus replication in an astrocytoma cell line: identification of an essential gene.  

PubMed Central

We have used an antisense RNA approach in the analysis of gene function in human cytomegalovirus (HCMV). An astrocytoma cell line (U373-MG) that is permissive for virus replication was permanently transfected with a construct bearing sequence from HCMV UL44 (coding for the major late DNA-binding protein, ppUL44, also known as pp52 or ICP36) in an antisense orientation and under the control of the immediate-early enhancer-promoter element. Upon HCMV infection at a high multiplicity, we found a marked reduction in UL44 protein products (the ICP36 family of proteins) in established cell transfectants and a strong inhibition of virus yield in infected-cell supernatants at two weeks postinfection, while herpes simplex virus replication was not affected. In infected cells, viral DNA replication was strongly inhibited. While gene products such as pUS22 and pUL32 were also inhibited, pUL123 and pUL82 accumulated in the infected cells over time. Our data suggest an essential role for the UL44 family of proteins in HCMV replication and represent a model of virus inhibition by virus-induced antisense RNA synthesis in genetically modified cells. PMID:7884850

Ripalti, A; Boccuni, M C; Campanini, F; Landini, M P

1995-01-01

264

Salt Stress Inhibits the Repair of Photodamaged Photosystem II by Suppressing the Transcription and Translation of psbA Genes in Synechocystis1  

PubMed Central

Light stress and salt stress are major environmental factors that limit the efficiency of photosynthesis. However, we have found that the effects of light and salt stress on photosystem II (PSII) in the cyanobacterium Synechocystis sp. PCC 6803 are completely different. Strong light induced photodamage to PSII, whereas salt stress inhibited the repair of the photodamaged PSII and did not accelerate damage to PSII directly. The combination of light and salt stress appeared to inactivate PSII very rapidly as a consequence of their synergistic effects. Radioactive labeling of cells revealed that salt stress inhibited the synthesis of proteins de novo and, in particular, the synthesis of the D1 protein. Northern- and western-blotting analyses demonstrated that salt stress inhibited the transcription and the translation of psbA genes, which encode D1 protein. DNA microarray analysis indicated that the light-induced expression of various genes was suppressed by salt stress. Thus, our results suggest that salt stress inhibits the repair of PSII via suppression of the activities of the transcriptional and translational machinery. PMID:12428009

Allakhverdiev, Suleyman I.; Nishiyama, Yoshitaka; Miyairi, Sachio; Yamamoto, Hiroshi; Inagaki, Noritoshi; Kanesaki, Yu; Murata, Norio

2002-01-01

265

Blockage by SP600125 of Fcepsilon receptor-induced degranulation and cytokine gene expression in mast cells is mediated through inhibition of phosphatidylinositol 3-kinase signalling pathway.  

PubMed

SP600125 is used as a specific inhibitor of c-Jun N-terminal kinase (JNK). We initially aimed to examine physiological roles of JNK in mast cells that play a central role in inflammatory and immediate allergic responses. We found that Fc receptor for IgE (FcepsilonRI)-induced degranulation (serotonin release) and cytokine gene expression [interleukin (IL)-6, tumour necrosis factor-alpha and IL-13] in bone marrow-derived mast cells, were almost completely inhibited by SP600125. However, the time course of FcepsilonRI-induced JNK activation did not correlate with that of serotonin release. Furthermore, FcepsilonRI-induced degranulation and cytokine gene expression were not impaired in a JNK activator, MKK7-deficient mast cells, in which JNK activation was lost. These results indicate that the inhibitory effects by SP600125 are not due to impaired JNK activation. Instead, we found that SP600125 markedly inhibited the FcepsilonRI-induced activation of phosphatidylinositol 3-kinase (PI3K) and Akt, the same as a PI3K inhibitor, wortmannin. Finally, we found that SP600125 specifically inhibits delta form of p110 catalytic subunit (p110delta) of PI3K. Thus, SP600125 exerts its influence on mast cell functions by inhibiting the kinase activity of PI3K, but not JNK. PMID:19106158

Tanemura, Shuhei; Momose, Haruka; Shimizu, Nao; Kitagawa, Daiju; Seo, Jungwon; Yamasaki, Tokiwa; Nakagawa, Kentaro; Kajiho, Hiroaki; Penninger, Josef M; Katada, Toshiaki; Nishina, Hiroshi

2009-03-01

266

Astragaloside IV ameliorates renal injury in streptozotocin-induced diabetic rats through inhibiting NF-?B-mediated inflammatory genes expression.  

PubMed

Accumulating evidence suggests that inflammatory processes are involved in the development of diabetic nephropathy (DN). However, there are no effective interventions for inflammation in the diabetic kidneys. Here, we tested the hypothesis that Astragaloside IV(AS-IV), a novel saponin purified from Astragalus membranaceus (Fisch) Bge, ameliorates DN in streptozotocin (STZ)-induced diabetic rats through anti-inflammatory mechanisms. Diabetes was induced with STZ (65 mg/kg) by intraperitoneal injection in rats. Two weeks after STZ injection, rats were divided into three groups (n=8/each group), namely, diabetic rats, diabetic rats treated with AS-IV at 5 and 10 mgkg(-1)d(-1), p.o., for 8 weeks. The normal rats were chosen as nondiabetic control group (n=8). The rats were sacrificed 10 weeks after induction of diabetes. AS-IV ameliorated albuminuria, renal histopathology and podocyte foot process effacement in diabetic rats. Renal NF-?B activity, as wells as protein and mRNA expression were increased in diabetic kidneys, accompanied by an increase in mRNA expression and protein content of TNF-?, MCP-1 and ICAM-1 in kidney tissues. The ?1-chain type IV collagen mRNA was elevated in the kidneys of diabetic rats. All of these abnormalities were partially restored by AS-IV. AS-IV also decreased the serum levels of TNF-?, MCP-1 and ICAM-1 in diabetic rats. These findings suggest that AS-IV, a novel anti-inflammatory agent, attenuated DN in rats through inhibiting NF-?B mediated inflammatory genes expression. PMID:23434274

Gui, Dingkun; Huang, Jianhua; Guo, Yongping; Chen, Jianguo; Chen, Yifang; Xiao, Wenzhen; Liu, Xusheng; Wang, Niansong

2013-03-01

267

Histone deacetylase inhibition rescues gene knockout levels achieved with integrase-defective lentiviral vectors encoding zinc-finger nucleases.  

PubMed

Zinc-finger nucleases (ZFNs) work as dimers to induce double-stranded DNA breaks (DSBs) at predefined chromosomal positions. In doing so, they constitute powerful triggers to edit and to interrogate the function of genomic sequences in higher eukaryotes. A preferred route to introduce ZFNs into somatic cells relies on their cotransduction with two integrase-defective lentiviral vectors (IDLVs) each encoding a monomer of a functional heterodimeric pair. The episomal nature of IDLVs diminishes the risk of genotoxicity and ensures the strict transient expression profile necessary to minimize deleterious effects associated with long-term ZFN activity. However, by deploying IDLVs and conventional lentiviral vectors encoding HPRT1- or eGFP-specific ZFNs, we report that DSB formation at target alleles is limited after IDLV-mediated ZFN transfer. This IDLV-specific underperformance stems, to a great extent, from the activity of chromatin-remodeling histone deacetylases (HDACs). Importantly, the prototypic and U.S. Food and Drug Administration-approved inhibitors of metal-dependent HDACs, trichostatin A and vorinostat, respectively, did not hinder illegitimate recombination-mediated repair of targeted chromosomal DSBs. This allowed rescuing IDLV-mediated site-directed mutagenesis to levels approaching those achieved by using their isogenic chromosomally integrating counterparts. Hence, HDAC inhibition constitutes an efficacious expedient to incorporate in genome-editing strategies based on transient IDLV-mediated ZFN expression. Finally, we compared two of the most commonly used readout systems to measure targeted gene knockout activities based on restriction and mismatch-sensitive endonucleases. These experiments indicate that these enzymatic assays display a similar performance. PMID:24059449

Pelascini, Laetitia P L; Maggio, Ignazio; Liu, Jin; Holkers, Maarten; Cathomen, Toni; Gonçalves, Manuel A F V

2013-12-01

268

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

269

Guava Leaf Extract Inhibits Quorum-Sensing and Chromobacterium violaceum Induced Lysis of Human Hepatoma Cells: Whole Transcriptome Analysis Reveals Differential Gene Expression  

PubMed Central

Quorum sensing (QS) is a process mediated via small molecules termed autoinducers (AI) that allow bacteria to respond and adjust according to the cell population density by altering the expression of multitudinous genes. Since QS governs numerous bioprocesses in bacteria, including virulence, its inhibition promises to be an ideal target for the development of novel therapeutics. We found that the aqueous leaf extract of Psidium guajava (GLE) exhibited anti-QS properties as evidenced by inhibition of violacein production in Chromobacterium violaceum and swarming motility of Pseudomonas aeruginosa. The gram-negative bacterium, C. violaceum is a rare pathogen with high mortality rate. In this study, perhaps for the first time, we identified the target genes of GLE in C. violaceum MTCC 2656 by whole transcriptome analysis on Ion Torrent. Our data revealed that GLE significantly down-regulated 816 genes at least three fold, with p value?0.01, which comprises 19% of the C. violaceum MTCC 2656 genome. These genes were distributed throughout the genome and were associated with virulence, motility and other cellular processes, many of which have been described as quorum regulated in C. violaceum and other gram negative bacteria. Interestingly, GLE did not affect the growth of the bacteria. However, consistent with the gene expression pattern, GLE treated C. violaceum cells were restrained from causing lysis of human hepatoma cell line, HepG2, indicating a positive relationship between the QS-regulated genes and pathogenicity. Overall, our study proposes GLE as a QS inhibitor (QSI) with the ability to attenuate virulence without affecting growth. To the best of our knowledge, this is the first report which provides with a plausible set of candidate genes regulated by the QS system in the neglected pathogen C. violaceum. PMID:25229331

Tiwary, Bipransh Kumar; Kumar, Anoop

2014-01-01

270

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. PMID:21805138

Zhu, Qingsong; Huang, Yi; Marton, Laurence J.; Woster, Patrick M.; Davidson, Nancy E.; Casero, Robert A.

2011-01-01

271

RNA interference-mediated silencing of the acetyl-CoA-carboxylase-alpha gene induces growth inhibition and apoptosis of prostate cancer cells.  

PubMed

Overexpression of lipogenic enzymes is a common characteristic of many cancers. Thus far, studies aimed at the exploration of lipogenic enzymes as targets for cancer intervention have focused on fatty acid synthase (FAS), the enzyme catalyzing the terminal steps in fatty acid synthesis. Chemical inhibition or RNA interference (RNAi)-mediated knockdown of FAS consistently inhibits the growth and induces death of cancer cells. Accumulation of the FAS substrate malonyl-CoA has been implicated in the mechanism of cytotoxicity of FAS inhibition. Here, using RNAi technology, we have knocked down the expression of acetyl-CoA carboxylase-alpha (ACC-alpha), the enzyme providing the malonyl-CoA substrate. Silencing of the ACC-alpha gene resulted in a similar inhibition of cell proliferation and induction of caspase-mediated apoptosis of highly lipogenic LNCaP prostate cancer cells as observed after FAS RNAi. In nonmalignant cells with low lipogenic activity, no cytotoxic effects of knockdown of ACC-alpha or FAS were observed. These findings indicate that accumulation of malonyl-CoA is not a prerequisite for cytotoxicity induced by inhibition of tumor-associated lipogenesis and suggest that in addition to FAS, ACC-alpha is a potential target for cancer intervention. PMID:16061653

Brusselmans, Koen; De Schrijver, Ellen; Verhoeven, Guido; Swinnen, Johannes V

2005-08-01

272

Selective inhibition of monocyte chemoattractant protein-1 gene expression in human embryonal kidney cells by specific triple helix-forming oligonucleotides.  

PubMed

Monocyte chemoattractant protein-1 (MCP-1) is a chemokine that is expressed by a variety of tissue cells in response to inflammatory stimuli, such as IL-1beta, TNF-alpha, and IFN-gamma. A major function of MCP-1 is the recruitment and activation of monocytes and T lymphocytes. Overexpression of MCP-1 has been implicated in a number of diseases, including glomerulonephritis and rheumatoid arthritis, indicating that the modulation of MCP-1 activity and/or expression is a desired therapeutic strategy. In the present study, our aim was to test whether the MCP-1 expression could be inhibited at the transcriptional level using triple helix-forming oligonucleotides (TFOs). We designed a TFO targeted to the SP-1 binding site in the human MCP-1 gene promoter. Gel mobility shift assays demonstrated that the phosphodiester TFO formed a sequence-specific triplex with its dsDNA target with an EC50 of approximately 1.9 x 10(-7) M. The corresponding phosphorothioated oligonucleotide was also effective in this assay with an 8-fold higher EC50 value. Binding of the TFO to the target DNA prevented the binding of rSP-1 and of nuclear proteins in vitro. The TFO could also partially inhibit endogenous MCP-1 gene expression in cultured human embryonic kidney cells. Treatment of TNF-alpha-stimulated human embryonic kidney 293 cells with the TFO inhibited the secretion of MCP-1 in a dose-dependent manner (up to 45% at 5 microM oligonucleotide). The inhibition of MCP secretion was caused at the level of gene transcription, because MCP-1 mRNA levels in oligonucleotide-treated cells were also decreased by approximately 40%. PMID:10657660

Marchand, P; Resch, K; Radeke, H H

2000-02-15

273

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

274

Calcitonin gene-related peptide stimulates stromal cell osteogenic differentiation and inhibits RANKL induced NF-?B activation, osteoclastogenesis and bone resorption  

PubMed Central

Previously we observed that capsaicin treatment in rats inhibited sensory neuropeptide signaling, with a concurrent reduction in trabecular bone formation and bone volume, and an increase in osteoclast numbers and bone resorption. Calcitonin gene-related peptide (CGRP) is a neuropeptide richly distributed in sensory neurons innervating the skeleton and we postulated that CGRP signaling regulates bone integrity. In this study we examined CGRP effects on stromal and bone cell differentiation and activity in vitro. CGRP receptors were detected by immunocytochemical staining and real time PCR assays in mouse bone marrow stromal cells (BMSCs) and bone marrow macrophages (BMMs). CGRP effects on BMSC proliferation and osteoblastic differentiation were studied using BrdU incorporation, PCR products, alkaline phosphatase (ALP) activity, and mineralization assays. CGRP effects on BMM osteoclastic differentiation and activity were determined by quantifying tartrate-resistant acid phosphatase positive (TRAP+) multinucleated cells, pit erosion area, mRNA levels of TRAP and cathepsin K, and nuclear factor-?B (NF-?B) nuclear localization. BMSCs, osteoblasts, BMMs, and osteoclasts all expressed CGRP receptors. CGRP (10-10-10-8M) stimulated BMSC proliferation, up-regulated the expression of osteoblastic genes, and increased ALP activity and mineralization in the BMSCs. In BMM cultures CGRP (10-8M) inhibited receptor activator of NF-?B ligand (RANKL) activation of NF-?B. CGRP also down-regulated osteoclastic genes like TRAP and cathepsin K, decreased the numbers of TRAP+ cells, and inhibited bone resorption activity in RANKL stimulated BMMs. These results suggest that CGRP signaling maintains bone mass both by directly stimulating stromal cell osteoblastic differentiation and by inhibiting RANKL induced NF-?B activation, osteoclastogenesis, and bone resorption. PMID:19962460

Wang, Liping; Shi, Xiaoyou; Zhao, Rong; Halloran, Bernard P.; Clark, David J.; Jacobs, Christopher R.; Kingery, Wade S.

2010-01-01

275

Wild-type p53 inhibits pro-invasive properties of TGF-?3 in breast cancer, in part through regulation of EPHB2, a new TGF-? target gene.  

PubMed

The p53 tumor suppressor protein is primarily known for its important role in tumor suppression. In addition, p53 affects tumor cell migration, invasion, and epithelial-mesenchymal transition (EMT); processes also regulated by the transforming growth factor-? (TGF-?) signaling pathway. Here, we investigated the role of p53 in breast tumor cell invasion, migration, and EMT and examined the interplay of p53 with TGF-?3 in these processes. MCF-10A1 and MCF-10CA1a breast cancer cells were treated with Nutlin-3 and TGF-?3, and the effects on tumor cell migration and invasion were studied in transwell and 3D spheroid invasion assays. The effects of Nutlin-3 and TGF-?3 on EMT were examined in NMuMG cells. To identify genes involved in TGF-?-induced invasion that are modulated by p53, a Human Tumor Metastasis-specific RT-PCR array was performed. Verification of EPHB2 regulation by TGF-?3 and p53 was performed on breast cancer tumor cell lines. We demonstrate that p53 inhibits basal and TGF-?3-induced invasion, migration, and EMT in normal breast epithelial and breast cancer cells. Pharmacological activation of p53 inhibited induction of several TGF-?3 targets involved in TGF-?3-induced tumor cell invasion, i.e., matrix metallo proteinase (MMP)2, MMP9, and integrin ? 3 . The ephrin-type B receptor 2 (EPHB2) gene was identified as a new TGF-? target important for TGF-?3-mediated invasion and migration, whose transcriptional activation by TGF-?3 is also inhibited by p53. The results show an intricate interplay between p53 and TGF-?3 whereby p53 inhibits the TGF-?3-induced expression of genes, e.g., EPHB2, to impede tumor cell invasion and migration. PMID:25257729

Lam, Suzanne; Wiercinska, Eliza; Teunisse, Amina F A S; Lodder, Kirsten; ten Dijke, Peter; Jochemsen, Aart G

2014-11-01

276

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. PMID:23102993

Li, Bingbing X.; Yamanaka, Kinrin; Xiao, Xiangshu

2012-01-01

277

Enhancing microRNA transfection to inhibit survivin gene expression and induce apoptosis: could it be mediated by a novel combination of sonoporation and polyethylenimine?  

PubMed

Apoptosis is a physiologically essential mechanism of cell and plays an important role in reducing the development and progression of tumors. The appealing strategy for cancer therapy is to target the lesions that induce apoptosis in cancer cells. Survivin, the smallest member of the mammalian inhibitors of the apoptosis protein family, is upregulated in various malignancies to protect cells from apoptosis. Survivin knockdown could induce cancer cell apoptosis and inhibit tumor-angiogenesis. Survivin expression would be silenced by microRNA (miRNA)-mediated RNA interference. However, noninvasive and tissue-specific gene delivery techniques remain absent recently and the utilizations of miRNA expression vectors have been limited by inefficient delivery technique, especially in vivo. On the other hand, safe and promising technologies of gene transfection would be valuable in clinical gene therapy. Successful treatment of gene transfer method would lead to a new and readily available approach in the anticancer research. Sonoporation is an alternative technique of gene delivery that uses ultrasound targeted microbubble destruction to create pores in the cell membrane. Based on our previous studies, in this article, we postulated that the transfection of miRNA could be mediated by the combination of sonoporation and polyethylenimine (PEI) which was one of the most effective poly-cationic gene vectors and enhance the endocytosis of plasmids DNA and hypothesized that the gene silencing and apoptosis induction with miRNA targeting human Survivin would be improved by this novel technique. In our opinion, this novel combination of sonoporation and PEI could enhance targeted gene delivery effectively and might be a feasible, novel candidate for gene therapy. PMID:22340183

Chen, Zhi-Yi; Liang, Kun; Qiu, Ri-Xiang; Luo, Liang-Ping

2011-11-01

278

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. PMID:7868612

Fuqua, C; Burbea, M; Winans, S C

1995-01-01

279

Pattern Triggered Immunity (PTI) in Tobacco: Isolation of Activated Genes Suggests Role of the Phenylpropanoid Pathway in Inhibition of Bacterial Pathogens  

PubMed Central

Background Pattern Triggered Immunity (PTI) or Basal Resistance (BR) is a potent, symptomless form of plant resistance. Upon inoculation of a plant with non-pathogens or pathogenicity-mutant bacteria, the induced PTI will prevent bacterial proliferation. Developed PTI is also able to protect the plant from disease or HR (Hypersensitive Response) after a challenging infection with pathogenic bacteria. Our aim was to reveal those PTI-related genes of tobacco (Nicotiana tabacum) that could possibly play a role in the protection of the plant from disease. Methodology/Principal Findings Leaves were infiltrated with Pseudomonas syringae pv. syringae hrcC- mutant bacteria to induce PTI, and samples were taken 6 and 48 hours later. Subtraction Suppressive Hybridization (SSH) resulted in 156 PTI-activated genes. A cDNA microarray was generated from the SSH clone library. Analysis of hybridization data showed that in the early (6 hpi) phase of PTI, among others, genes of peroxidases, signalling elements, heat shock proteins and secondary metabolites were upregulated, while at the late phase (48 hpi) the group of proteolysis genes was newly activated. Microarray data were verified by real time RT-PCR analysis. Almost all members of the phenyl-propanoid pathway (PPP) possibly leading to lignin biosynthesis were activated. Specific inhibition of cinnamic-acid-4-hydroxylase (C4H), rate limiting enzyme of the PPP, decreased the strength of PTI - as shown by the HR-inhibition and electrolyte leakage tests. Quantification of cinnamate and p-coumarate by thin-layer chromatography (TLC)-densitometry supported specific changes in the levels of these metabolites upon elicitation of PTI. Conclusions/Significance We believe to provide first report on PTI-related changes in the levels of these PPP metabolites. Results implicated an actual role of the upregulation of the phenylpropanoid pathway in the inhibition of bacterial pathogenic activity during PTI. PMID:25101956

Szatmári, Ágnes; Zvara, Ágnes; Móricz, Ágnes M.; Besenyei, Eszter; Szabó, Erika; Ott, Péter G.; Puskás, László G.; Bozsó, Zoltán

2014-01-01

280

Combined inhibition of DNA methylation and histone acetylation enhances gene re-expression and drug sensitivity in vivo  

Microsoft Academic Search

Histone deacetylation and DNA methylation have a central role in the control of gene expression in tumours, including transcriptional repression of tumour suppressor genes and genes involved in sensitivity to chemotherapy. Treatment of cisplatin-resistant cell lines with an inhibitor of DNA methyltransferases, 2-deoxy-5?azacytidine (decitabine), results in partial reversal of DNA methylation, re-expression of epigenetically silenced genes including hMLH1 and sensitisation

N Steele; P Finn; R Brown; J A Plumb

2009-01-01

281

Immune interferon inhibits proliferation and induces 2'-5'-oligoadenylate synthetase gene expression in human vascular smooth muscle cells.  

PubMed Central

Proliferation of vascular smooth muscle cells (SMC) contributes to formation of the complicated human atherosclerotic plaque. These lesions also contain macrophages, known to secrete SMC mitogens, and T lymphocytes. Many of the SMC in the lesions express class II major histocompatibility antigens, an indication that activated T cells secrete immune IFN-gamma locally in the plaque. We therefore studied the effect of IFN-gamma on the proliferation of cultured SMC derived from adult human blood vessels. IFN-gamma (1,000 U/ml) reduced [3H]thymidine (TdR) incorporation into DNA by SMC stimulated with the well-defined mitogens IL 1 (from 15.3 +/- 0.7 to 6.2 +/- 0.7 dpm X 10(-3)/24 h) or platelet-derived growth factor (PDGF) (from 18.5 +/- 1.0 to 7.3 +/- 0.7 dpm X 10(-3)/24 h). Kinetic and nuclear labeling studies indicated that this effect of IFN-gamma was not due to altered thymidine transport or specific radioactivity of TdR in the cell. In longer term experiments (4-16 d) IFN-gamma prevented net DNA accumulation by SMC cultures stimulated by PDGF. IFN-gamma also delayed (from 30 to 60 min) the time to peak level of c-fos RNA in IL 1-treated SMC. It is unlikely that cytotoxicity caused these effects of IFN-gamma, as the inhibition of growth was reversible and we detected no cell death in SMC cultures exposed to this cytokine. Activation of 2'-5' oligoadenylate synthetase gene expression may mediate certain antiproliferative and antiviral effects of interferons. Both IFN-gamma and type I IFNs (IFN-alpha or IFN-beta) induced 2'-5' oligoadenylate synthetase mRNA and enzyme activity in SMC cultures, but with concentration dependence and time course that may not account for all of IFN-gamma's cytostatic effect on SMC. The accumulation of SMC in human atherosclerotic lesions is a long-term process that must involve altered balance between growth stimulatory and inhibitory factors. The cytostatic effect of IFN-gamma on human SMC demonstrated here may influence this balance during human atherogenesis, because T cells present in the complicated atherosclerotic plaque likely produce this cytokine. Images PMID:2495301

Warner, S J; Friedman, G B; Libby, P

1989-01-01

282

Canopy architectural and physiological characterization of near-isogenic wheat lines differing in the tiller inhibition gene tin  

PubMed Central

Tillering is a core constituent of plant architecture, and influences light interception to affect plant and crop performance. Near-isogenic lines (NILs) varying for a tiller inhibition (tin) gene and representing two genetic backgrounds were investigated for tillering dynamics, organ size distribution, leaf area, light interception, red: far-red ratio, and chlorophyll content. Tillering ceased earlier in the tin lines to reduce the frequencies of later primary and secondary tillers compared to the free-tillering NILs, and demonstrated the genetically lower tillering plasticity of tin-containing lines. The distribution of organ sizes along shoots varied between NILs contrasting for tin. Internode elongation commenced at a lower phytomer, and the peduncle was shorter in the tin lines. The flag leaves of tin lines were larger, and the longest leaf blades were observed at higher phytomers in the tin than in free-tillering lines. Total leaf area was reduced in tin lines, and non-tin lines invested more leaf area at mid-canopy height. The tiller economy (ratio of seed-bearing shoots to numbers of shoots produced) was 10% greater in the tin lines (0.73–0.76) compared to the free-tillering sisters (0.62–0.63). At maximum tiller number, the red: far-red ratio (light quality stimulus that is thought to induce the cessation of tillering) at the plant-base was 0.18–0.22 in tin lines and 0.09–0.11 in free-tillering lines at levels of photosynthetic active radiation of 49–53% and 30–33%, respectively. The tin lines intercepted less radiation compared to their free-tillering sisters once genotypic differences in tiller numbers had established, and maintained green leaf area in the lower canopy later into the season. Greater light extinction coefficients (k) in tin lines prior to, but reduced k after, spike emergence indicated that differences in light interception between NILs contrasting in tin cannot be explained by leaf area alone but that geometric and optical canopy properties contributed. The careful characterization of specifically-developed NILs is refining the development of a physiology-based model for tillering to improve understanding of the value of architectural traits for use in cereal improvement. PMID:25520724

Moeller, Carina; Evers, Jochem B.; Rebetzke, Greg

2014-01-01

283

Heterogeneous gene expression changes in colorectal cancer cells share the WNT pathway in response to growth suppression by APHS-mediated COX-2 inhibition  

PubMed Central

Cyclooxygenase-2 (COX-2), the prostaglandin (PG)-synthesizing enzyme overexpressed in colorectal cancer (CRC), has pleiotropic, cancer-promoting effects. COX-2 inhibitors (CIBs) interfere with many cancer-associated processes and show promising antineoplastic activity, however, a common mechanism of CIB action has not yet been established. We therefore investigated by microarray the global response towards the CIB APHS at a dose significantly inhibiting the growth of three COX-2-positive CRC but not of two COX-2-negative cell lines. None of the genes significantly (p = 0.005) affected by APHS were common to all three cell lines and 83% of the altered pathways were cell line-specific. Quantitative polymerase chain reaction (QPCR) on selected pathways confirmed cell line-specific expression alterations induced by APHS. A low stringency data analysis approach using BRB array tools coupled with QPCR, however, identified small expression changes shared by all COX-2-positive cell lines in genes related to the WNT pathway, the key driver of colonic carcinogenesis. Our data indicates a substantial cell line-specificity of APHS-induced expression alterations in CRC cells and helps to explain the divergent effects reported for CIBs. Further, the shared inhibition of the WNT pathway by APHS suggests one potential common mechanism behind the antineoplastic effects of COX-2 inhibition. PMID:19707365

Humar, Bostjan; McNoe, Les; Dunbier, Anita; Heathcott, Rosemary; Braithwaite, Antony W; Reeve, Anthony E

2008-01-01

284

Heat shock selectively inhibits ribosomal RNA gene transcription and down-regulates E1BF/Ku in mouse lymphosarcoma cells.  

PubMed Central

The effect of heat shock on RNA polymerase I (pol I)-directed transcription of the rRNA gene was studied in S-100 extract derived from mouse lymphosarcoma cells, and by in vivo labelling of rRNA. Exposure of cells to 42 degrees C for 2 h resulted in complete inhibition of rRNA synthesis in vivo. Pol I transcription was inhibited by 50% within 2 h of heat shock and was abolished after 3 h exposure at 42 degrees C. Under this condition, the core-promoter-binding activity of the factor (CPBF) that modulates pol I transcription was unaffected. In contrast, the promoter-binding activity of enhancer-1-binding factor, a protein related to the Ku autoantigen, which is involved in pol I transcription initiation, was reduced by 50 and 90% after 2 and 3 h of heat shock respectively. Western-blot analysis with antibodies specific for the two subunits of Ku protein showed the absence of p72 subunit after 3 h of heat shock. Under this condition, pol II transcription from the adenovirus major late promoter and pol III transcription of 5 S RNA gene remained unaffected. Mixing experiments ruled out the possibility that the inhibition of transcription was due to activation of nucleases or other inhibitors. This is the first report to show selective down-regulation of pol I transcription in vitro by heat shock and of the potential involvement of a pol I transcription factor in this process. PMID:8760351

Ghoshal, K; Jacob, S T

1996-01-01

285

Inhibition of cyclin D1 expression in human pancreatic cancer cells is associated with increased chemosensitivity and decreased expression of multiple chemoresistance genes.  

PubMed

Cyclin D1 belongs to a family of protein kinases that have been implicated in cell cycle regulation. Inhibition of cyclin D1 expression has been recently shown (M. Kornmann, et al., J. Clin. Invest, 101: 344-352, 1998) to suppress pancreatic cancer cell growth and increase cytotoxic actions of cisplatinum. The aim of the present study was to determine whether inhibition of cyclin D1 expression also modulates the effects of other antineoplastic drugs and whether it is associated with alterations in the level of expression of drug resistance genes. The suppression of cyclin D1 expression after the stable transfection of a cyclin D1 antisense construct in PANC-1 and COLO-357 human pancreatic cancer cells resulted in a significant increase in sensitivity to the fluoropyrimidines 5-fluorouracil and 5-fluoro-2'-deoxyuridine and to mitoxantrone. All of the antisense-expressing dones exhibited a decrease in thymidylate synthase and an increase in thymidine phosphorylase mRNA expression as determined by reverse transcription-PCR analysis and decreased levels of MDR-1 and MRP mRNA as determined by Northern blotting. These findings demonstrate that the inhibition of cyclin D1, in addition to suppressing the growth of pancreatic cancer cells, enhances their responsiveness to multiple chemotherapeutic agents and suggest that this effect may be due to the altered expression of several chemoresistance genes. PMID:10416617

Kornmann, M; Danenberg, K D; Arber, N; Beger, H G; Danenberg, P V; Korc, M

1999-07-15

286

Orostachys japonicus Inhibits Expression of the TLR4, NOD2, iNOS, and COX-2 Genes in LPS-Stimulated Human PMA-Differentiated THP-1 Cells by Inhibiting NF-?B and MAPK Activation  

PubMed Central

Orostachys japonicus is traditionally used as an inflammatory agent. In this report, we investigated the effects of O. japonicus extract on the expression of genes encoding pathogen-recognition receptors (TLR2, TLR4, NOD1, and NOD2) and proinflammatory factors (iNOS, COX-2, and cytokines) in LPS-stimulated PMA-differentiated THP-1 cells and the NF-?B and MAPK pathways. O. japonicus induced toxicity at high concentrations but had no effect at concentrations lower than 25??g/mL. O. japonicus inhibited LPS-induced TLR4 and NOD2 mRNA levels, suppressed LPS-induced iNOS and COX-2 transcription and translocation, and downregulated LPS-induced proinflammatory cytokine (IL-1?, IL-6, IL-8, and TNF-?) mRNA levels. In addition, O. japonicus inhibited LPS-induced NF-?B activation and I?B? degradation and suppressed LPS-induced JNK, p38 MAPK, and ERK phosphorylation. Overall, our results demonstrate that the anti-inflammatory effects of O. japonicus are mediated by suppression of NF-?B and MAPK signaling, resulting in reduced TLR4, NOD2, iNOS, and COX-2 expression and inhibition of inflammatory cytokine expression.

Woo, Hong-Jung; Kim, Youngchul

2015-01-01

287

Metformin inhibits epithelial-mesenchymal transition in prostate cancer cells: involvement of the tumor suppressor miR30a and its target gene SOX4.  

PubMed

Tumor metastasis is the leading cause of mortality and morbidity of prostate cancer (PCa) patients. Epithelial-mesenchymal transition (EMT) plays a critical role in cancer progression and metastasis. Recent evidence suggested that diabetic patients treated with metformin have lower PCa risk and better prognosis. This study was aimed to investigate the effects of metformin on EMT in PCa cells and the possible microRNA (miRNA)-based mechanisms. MiRNAs have been shown to regulate various processes of cancer metastasis. We herein showed that metformin significantly inhibits proliferation of Vcap and PC-3 cells, induces G0/G1 cell cycle arrest and inhibits invasiveness and motility capacity of Vcap cells. Metformin could inhibit TGF-?-induced EMT in Vcap cells, as manifested by inhibition of the increase of N-cadherin (p=0.013), Vimentin (p=0.002) and the decrease of E-cadherin (p=0.0023) and ?-catenin (p=0.034) at mRNA and protein levels. Notably, we demonstrated significant upregulation of miR30a levels by metformin (P<0.05) and further experiments indicated that miR30a significantly inhibits proliferation and EMT process of Vcap cells. Interestingly, we identified that SOX4, a previously reported oncogenic transcriptional factor and modulator of EMT, is a direct target gene of miR30a. Finally, we screened the expression of miR30a and SOX4 in 84 PCa cases with radical prostatectomy. Of note, SOX4 overexpression is significantly associated with decreased levels of miR30a in PCa cases. In all, our study suggested that inhibition of EMT by metformin in PCa cells may involve upregulation of miR30a and downregulation of SOX4. PMID:25201727

Zhang, Jing; Shen, Chengwu; Wang, Lin; Ma, Quanping; Xia, Pingtian; Qi, Mei; Yang, Muyi; Han, Bo

2014-09-26

288

The Mg-Chelatase H Subunit of Arabidopsis Antagonizes a Group of WRKY Transcription Repressors to Relieve ABA-Responsive Genes of Inhibition[W][OA  

PubMed Central

The phytohormone abscisic acid (ABA) plays a vital role in plant development and response to environmental challenges, but the complex networks of ABA signaling pathways are poorly understood. We previously reported that a chloroplast protein, the magnesium-protoporphyrin IX chelatase H subunit (CHLH/ABAR), functions as a receptor for ABA in Arabidopsis thaliana. Here, we report that ABAR spans the chloroplast envelope and that the cytosolic C terminus of ABAR interacts with a group of WRKY transcription factors (WRKY40, WRKY18, and WRKY60) that function as negative regulators of ABA signaling in seed germination and postgermination growth. WRKY40, a central negative regulator, inhibits expression of ABA-responsive genes, such as ABI5. In response to a high level of ABA signal that recruits WRKY40 from the nucleus to the cytosol and promotes ABAR–WRKY40 interaction, ABAR relieves the ABI5 gene of inhibition by repressing WRKY40 expression. These findings describe a unique ABA signaling pathway from the early signaling events to downstream gene expression. PMID:20543028

Shang, Yi; Yan, Lu; Liu, Zhi-Qiang; Cao, Zheng; Mei, Chao; Xin, Qi; Wu, Fu-Qing; Wang, Xiao-Fang; Du, Shu-Yuan; Jiang, Tao; Zhang, Xiao-Feng; Zhao, Rui; Sun, Hai-Li; Liu, Rui; Yu, Yong-Tao; Zhang, Da-Peng

2010-01-01

289

Cyclosporin A Inhibits T-Cell Growth Factor Gene Expression at the Level of mRNA Transcription  

Microsoft Academic Search

Cyclosporin A (CsA) is a potent immunosuppressive agent, now gaining wide application in human organ transplantation. The immunosuppressive activity of CsA is at least in part due to inhibition of lymphokine production by activated T lymphocytes. Specifically, inhibition of T-cell growth factor (TCGF; also designated interleukin 2) production appears to be an important pathway by which CsA impairs T-cell function.

Martin Kronke; Warren J. Leonard; Joel M. Depper; Suresh K. Arya; Flossie Wong-Staal; Robert C. Gallo; Thomas A. Waldmann; Warner C. Greene

1984-01-01

290

Nitric oxide inhibition induces early activation of type I collagen gene in renal resistance vessels and glomeruli in transgenic mice. Role of endothelin.  

PubMed Central

Hypertension is often associated with the development of nephroangio- and glomerulo-sclerosis. This pathophysiological process is due to increased extracellular matrix protein, particularly type I collagen, accumulation. This study investigated whether nitric oxide (NO) synthesis is involved in the mechanism(s) regulating activation of the collagen I gene in afferent arterioles and glomeruli. Experiments were performed on transgenic mice harboring the luciferase gene under the control of the collagen I-alpha2 chain promoter [procolalpha2(I)]. Measurements of luciferase activity provide highly sensitive estimates of collagen I gene activation. NO synthesis was inhibited by NG-nitro-L-arginine methyl ester (L-NAME) (20 mg/kg per day) for a period of up to 14 wk. Systolic blood pressure was increased after 6 wk of treatment (117+/-2 versus 129+/-2 mmHg, P < 0.01) and reached a plateau after 10 wk (around 160 mmHg). Luciferase activity was increased in freshly isolated afferent arterioles and glomeruli as early as week 4 of L-NAME treatment (150 and 200% of baseline, P < 0.01, respectively). The activation of procolalpha2(I) became more pronounced with time, and at 14 wk increased four- and tenfold compared with controls in afferent arterioles and glomeruli, respectively (P < 0.001). In contrast, luciferase activity remained unchanged in aorta and heart up to 8 wk and was increased thereafter. Increased histochemical staining for extracellular matrix deposition, and particularly of collagen I, was detected in afferent arterioles and glomeruli after 10 wk of L-NAME treatment. This fibrogenic process was accompanied by an increased urinary excretion rate of endothelin. In separate experiments, the stimulatory effect of L-NAME on collagen I gene activation was abolished when animals were treated with bosentan, an endothelin receptor antagonist. Similarly, bosentan reduced the increased extracellular matrix deposition in afferent arterioles and glomeruli during NO inhibition. Interestingly, bosentan had no effect on the L-NAME- induced increase of systolic pressure. These data indicate that NO inhibition induces an early activation of the collagen I gene in afferent arterioles and glomeruli. This activation in the kidney precedes the increase in blood pressure and the procolalpha2(I) activation in heart and aorta, suggesting a specific renal effect of NO blockade on collagen I gene expression that is independent of increased blood pressure and, at least partly, mediated through stimulation of the endothelin receptor. Use of procolalpha2(I) transgenic mice provides a novel and efficient model to study the pathophysiological mechanism(s) regulating renal fibrosis. PMID:9637712

Chatziantoniou, C; Boffa, J J; Ardaillou, R; Dussaule, J C

1998-01-01

291

Pinitol targets nuclear factor-kappaB activation pathway leading to inhibition of gene products associated with proliferation, apoptosis, invasion, and angiogenesis.  

PubMed

Pinitol (3-O-methyl-chiroinositol), a component of traditional Ayurvedic medicine (talisapatra), has been shown to exhibit anti-inflammatory and antidiabetic activities through undefined mechanisms. Because the transcription factor nuclear factor-kappaB (NF-kappaB) has been linked with inflammatory diseases, including insulin resistance, we hypothesized that pinitol must mediate its effects through modulation of NF-kappaB activation pathway. We found that pinitol suppressed NF-kappaB activation induced by inflammatory stimuli and carcinogens. This suppression was not specific to cell type. Besides inducible, pinitol also abrogated constitutive NF-kappaB activation noted in most tumor cells. The suppression of NF-kappaB activation by pinitol occurred through inhibition of the activation of IkappaBalpha kinase, leading to sequential suppression of IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 phosphorylation, p65 nuclear translocation, and NF-kappaB-dependent reporter gene expression. Pinitol also suppressed the NF-kappaB reporter activity induced by tumor necrosis factor receptor (TNFR)-1, TNFR-associated death domain, TNFR-associated factor-2, transforming growth factor-beta-activated kinase-1 (TAK-1)/TAK1-binding protein-1, and IkappaBalpha kinase but not that induced by p65. The inhibition of NF-kappaB activation thereby led to down-regulation of gene products involved in inflammation (cyclooxygenase-2), proliferation (cyclin D1 and c-myc), invasion (matrix metalloproteinase-9), angiogenesis (vascular endothelial growth factor), and cell survival (cIAP1, cIAP2, X-linked inhibitor apoptosis protein, Bcl-2, and Bcl-xL). Suppression of these gene products by pinitol enhanced the apoptosis induced by TNF and chemotherapeutic agents and suppressed TNF-induced cellular invasion. Our results show that pinitol inhibits the NF-kappaB activation pathway, which may explain its ability to suppress inflammatory cellular responses. PMID:18566231

Sethi, Gautam; Ahn, Kwang Seok; Sung, Bokyung; Aggarwal, Bharat B

2008-06-01

292

Survival motor neuron gene 2 silencing by DNA methylation correlates with spinal muscular atrophy disease severity and can be bypassed by histone deacetylase inhibition  

PubMed Central

Spinal muscular atrophy (SMA), a common neuromuscular disorder, is caused by homozygous absence of the survival motor neuron gene 1 (SMN1), while the disease severity is mainly influenced by the number of SMN2 gene copies. This correlation is not absolute, suggesting the existence of yet unknown factors modulating disease progression. We demonstrate that the SMN2 gene is subject to gene silencing by DNA methylation. SMN2 contains four CpG islands which present highly conserved methylation patterns and little interindividual variations in SMN1-deleted SMA patients. The comprehensive analysis of SMN2 methylation in patients suffering from severe versus mild SMA carrying identical SMN2 copy numbers revealed a correlation of CpG methylation at the positions ?290 and ?296 with the disease severity and the activity of the first transcriptional start site of SMN2 at position ?296. These results provide first evidence that SMN2 alleles are functionally not equivalent due to differences in DNA methylation. We demonstrate that the methyl-CpG-binding protein 2, a transcriptional repressor, binds to the critical SMN2 promoter region in a methylation-dependent manner. However, inhibition of SMN2 gene silencing conferred by DNA methylation might represent a promising strategy for pharmacologic SMA therapy. We identified histone deacetylase (HDAC) inhibitors including vorinostat and romidepsin which are able to bypass SMN2 gene silencing by DNA methylation, while others such as valproic acid and phenylbutyrate do not, due to HDAC isoenzyme specificities. These findings indicate that DNA methylation is functionally important regarding SMA disease progression and pharmacological SMN2 gene activation which might have implications for future SMA therapy regimens. PMID:18971205

Hauke, Jan; Riessland, Markus; Lunke, Sebastian; Eyüpoglu, Ilker Y.; Blümcke, Ingmar; El-Osta, Assam; Wirth, Brunhilde; Hahnen, Eric

2009-01-01

293

Astrocyte production of the chemokine macrophage inflammatory protein-2 is inhibited by the spice principle curcumin at the level of gene transcription  

PubMed Central

Background In neuropathological processes associated with neutrophilic infiltrates, such as experimental allergic encephalitis and traumatic injury of the brain, the CXC chemokine, macrophage inflammatory protein-2 (MIP-2) is thought to play a pivotal role in the induction and perpetuation of inflammation in the central nervous system (CNS). The origin of MIP-2 in inflammatory disorders of the brain has not been fully defined but astrocytes appear to be a dominant source of this chemokine. Curcumin is a spice principle in, and constitutes approximately 4 percent of, turmeric. Curcumin's immunomodulating and antioxidant activities suggest that it might be a useful adjunct in the treatment of neurodegenerative illnesses characterized by inflammation. Relatively unexplored, but relevant to its potential therapeutic efficacy in neuroinflammatory syndromes is the effect of curcumin on chemokine production. To examine the possibility that curcumin may influence CNS inflammation by mechanisms distinct from its known anti-oxidant activities, we studied the effect of this spice principle on the synthesis of MIP-2 by astrocytes. Methods Primary astrocytes were prepared from neonatal brains of CBA/CaJ mice. The cells were stimulated with lipopolysaccharide in the presence or absence of various amount of curcumin or epigallocatechin gallate. MIP-2 mRNA was analyzed using semi-quantitative PCR and MIP-2 protein production in the culture supernatants was quantified by ELISA. Astrocytes were transfected with a MIP-2 promoter construct, pGL3-MIP-2, and stimulated with lipopolysaccharide in the presence or absence of curcumin. Results The induction of MIP-2 gene expression and the production of MIP-2 protein were inhibited by curcumin. Curcumin also inhibited lipopolysaccharide-induced transcription of the MIP-2 promoter reporter gene construct in primary astrocytes. However MIP-2 gene induction by lipopolysaccharide was not inhibited by another anti-oxidant, epigallocatechin gallate. Conclusion Our results indicate that curcumin potently inhibits MIP-2 production at the level of gene transcription and offer further support for its potential use in the treatment of inflammatory conditions of the CNS. PMID:15733321

Tomita, Michiyo; Holman, Brita J; Santoro, Christopher P; Santoro, Thomas J

2005-01-01

294

Inhibition of interferon gene activation by death-effector domain-containing proteins from the molluscum contagiosum virus.  

PubMed

Apoptosis, NF-?B activation, and IRF3 activation are a triad of intrinsic immune responses that play crucial roles in the pathogenesis of infectious diseases, cancer, and autoimmunity. FLIPs are a family of viral and cellular proteins initially found to inhibit apoptosis and more recently to either up- or down-regulate NF-?B. As such, a broad role for FLIPs in disease regulation is postulated, but exactly how a FLIP performs such multifunctional roles remains to be established. Here we examine FLIPs (MC159 and MC160) encoded by the molluscum contagiosum virus, a dermatotropic poxvirus causing skin infections common in children and immunocompromised individuals, to better understand their roles in viral pathogenesis. While studying their molecular mechanisms responsible for NF-?B inhibition, we discovered that each protein inhibited IRF3-controlled luciferase activity, identifying a unique function for FLIPs. MC159 and MC160 each inhibited TBK1 phosphorylation, confirming this unique function. Surprisingly, MC159 coimmunoprecipitated with TBK1 and IKK? but MC160 did not, suggesting that these homologs use distinct molecular mechanisms to inhibit IRF3 activation. Equally surprising was the finding that the FLIP regions necessary for TBK1 inhibition were distinct from those MC159 or MC160 regions previously defined to inhibit NF-?B or apoptosis. These data reveal previously unappreciated complexities of FLIPs, and that subtle differences within the conserved regions of FLIPs possess distinct molecular and structural fingerprints that define crucial differences in biological activities. A future comparison of mechanistic differences between viral FLIP proteins can provide new means of precisely manipulating distinct aspects of intrinsic immune responses. PMID:24379396

Randall, Crystal M H; Biswas, Sunetra; Selen, Catherine V; Shisler, Joanna L

2014-01-14

295

Inhibition of interferon gene activation by death-effector domain-containing proteins from the molluscum contagiosum virus  

PubMed Central

Apoptosis, NF-?B activation, and IRF3 activation are a triad of intrinsic immune responses that play crucial roles in the pathogenesis of infectious diseases, cancer, and autoimmunity. FLIPs are a family of viral and cellular proteins initially found to inhibit apoptosis and more recently to either up- or down-regulate NF-?B. As such, a broad role for FLIPs in disease regulation is postulated, but exactly how a FLIP performs such multifunctional roles remains to be established. Here we examine FLIPs (MC159 and MC160) encoded by the molluscum contagiosum virus, a dermatotropic poxvirus causing skin infections common in children and immunocompromised individuals, to better understand their roles in viral pathogenesis. While studying their molecular mechanisms responsible for NF-?B inhibition, we discovered that each protein inhibited IRF3-controlled luciferase activity, identifying a unique function for FLIPs. MC159 and MC160 each inhibited TBK1 phosphorylation, confirming this unique function. Surprisingly, MC159 coimmunoprecipitated with TBK1 and IKK? but MC160 did not, suggesting that these homologs use distinct molecular mechanisms to inhibit IRF3 activation. Equally surprising was the finding that the FLIP regions necessary for TBK1 inhibition were distinct from those MC159 or MC160 regions previously defined to inhibit NF-?B or apoptosis. These data reveal previously unappreciated complexities of FLIPs, and that subtle differences within the conserved regions of FLIPs possess distinct molecular and structural fingerprints that define crucial differences in biological activities. A future comparison of mechanistic differences between viral FLIP proteins can provide new means of precisely manipulating distinct aspects of intrinsic immune responses. PMID:24379396

Randall, Crystal M. H.; Biswas, Sunetra; Selen, Catherine V.; Shisler, Joanna L.

2014-01-01

296

Inhibition of human immunodeficiency virus type 1 replication by a Tat-activated, transduced interferon gene: targeted expression to human immunodeficiency virus type 1-infected cells.  

PubMed Central

We have examined the feasibility of using interferon (IFN) gene transfer as a novel approach to anti-human immunodeficiency virus type 1 (HIV-1) therapy in this study. To limit expression of a transduced HIV-1 long terminal repeat (LTR)-IFNA2 (the new approved nomenclature for IFN genes is used throughout this article) hybrid gene to the HIV-1-infected cells, HIV-1 LTR was modified. Deletion of the NF-kappa B elements of the HIV-1 LTR significantly inhibited Tat-mediated transactivation in T-cell lines, as well as in a monocyte line, U937. Replacement of the NF-kappa B elements in the HIV-1 LTR by a DNA fragment derived from the 5'-flanking region of IFN-stimulated gene 15 (ISG15), containing the IFN-stimulated response element, partially restored Tat-mediated activation of LTR in T cells as well as in monocytes. Insertion of this chimeric promoter (ISG15 LTR) upstream of the human IFNA2 gene directed high levels of IFN synthesis in Tat-expressing cells, while this promoter was not responsive to tumor necrosis factor alpha-mediated activation. ISG15-LTR-IFN hybrid gene inserted into the retrovirus vector was transduced into Jurkat and U937 cells. Selected transfected clones produced low levels of IFN A (IFNA) constitutively, and their abilities to express interleukin-2 and interleukin-2 receptor upon stimulation with phytohemagglutinin and phorbol myristate acetate were retained. Enhancement of IFNA synthesis observed upon HIV-1 infection resulted in significant inhibition of HIV-1 replication for a period of at least 30 days. Virus isolated from IFNA-producing cells was able to replicate in the U937 cells but did not replicate efficiently in U937 cells transduced with the IFNA gene. These results suggest that targeting IFN synthesis to HIV-1-infected cells is an attainable goal and that autocrine IFN synthesis results in a long-lasting and permanent suppression of HIV-1 replication. PMID:7983701

Su, Y; Popik, W; Pitha, P M

1995-01-01

297

Inhibition of tumor growth through suppression of angiogenesis by brain-specific angiogenesis inhibitor 1 gene transfer in murine renal cell carcinoma.  

PubMed

This study was designed to elucidate the therapeutic effect of transfering the brain-specific angiogenesis inhibitor 1 (BAI1) gene to a mouse renal cell carcinoma cell line (Renca). Female BALB/c mice were inoculated subcutaneously with wild-type Renca (Renca/Wild) cells or Renca cells transfected with the BAI-1 (Renca/BAI-1) or LacZ (Renca/LacZ) gene. Tumor growth was observed every other day from 3 to 35 days after implantation. Moreover, the intratumoral injection of the adenovirus vector containing the gene encoding BAI1 was conducted at two-day intervals from 11 to 31 days after implantation of the Renca/Wild or Renca/BAI1 tumor. Tumor blood flow was measured by colorimetric angiogenesis assay (CAA). The concentration of the vascular endothelial growth factor (VEGF) in the cell culture supernatants was determined by enzyme-linked immunoassay. The size of the Renca/BAI1 tumor was significantly (p<0.01) suppressed compared to the Renca/Wild and Renca/LacZ tumors 21 days after tumor implantation. The injection of the BAI1 viral vector at 2-day intervals significantly inhibited the growth of both the Renca/Wild and Renca/BAI1 tumors. The blood volume measured by CAA and microvessel density was significantly lower in the Renca/BAI1 than in the Renca/Wild and Renca/LacZ tumors (p<0.01 and p<0.05, respectively). A significant (p<0.01) reduction in VEGF concentration in the supernatant was demonstrated in the Renca/BAI1 compared with the Renca/Wild and Renca/LacZ cell cultures. These observations suggest that the transfer of the BAI1 gene to Renca can suppress the tumor growth via the inhibition of angiogenesis. The down-regulation of VEGF production in tumor cells contributes to this anti-tumor effect. PMID:17786337

Kudo, Shigetaka; Konda, Ryuichiro; Obara, Wataru; Kudo, Daisuke; Tani, Kenzaburo; Nakamura, Yusuke; Fujioka, Tomoaki

2007-10-01

298

Low Concentrations of o,p’-DDT Inhibit Gene Expression and Prostaglandin Synthesis by Estrogen Receptor-Independent Mechanism in Rat Ovarian Cells  

PubMed Central

o,p’-DDT is an infamous xenoestrogen as well as a ubiquitous and persistent pollutant. Biomonitoring studies show that women have been internally exposed to o,p’-DDT at range of 0.3–500 ng/g (8.46×10?10 M?1.41×10?6 M) in blood and other tissues. However, very limited studies have investigated the biological effects and mechanism(s) of o,p’-DDT at levels equal to or lower than current exposure levels in human. In this study, using primary cultures of rat ovarian granulosa cells, we determined that very low doses of o,p’-DDT (10?12?10?8 M) suppressed the expression of ovarian genes and production of prostaglandin E2 (PGE2). In vivo experiments consistently demonstrated that o,p’-DDT at 0.5–1 mg/kg inhibited the gene expression and PGE2 levels in rat ovary. The surprising results from the receptor inhibitors studies showed that these inhibitory effects were exerted independently of either classical estrogen receptors (ERs) or G protein-coupled receptor 30 (GPR30). Instead, o,p’-DDT altered gene expression or hormone action via inhibiting the activation of protein kinase A (PKA), rather than protein kinase C (PKC). We further revealed that o,p’-DDT directly interfered with the PKA catalytic subunit. Our novel findings support the hypothesis that exposure to low concentrations of o,p’-DDT alters gene expression and hormone synthesis through signaling mediators beyond receptor binding, and imply that the current exposure levels of o,p’-DDT observed in the population likely poses a health risk to female reproduction. PMID:23209616

Liu, Jing; Zhao, Meirong; Zhuang, Shulin; Yang, Yan; Yang, Ye; Liu, Weiping

2012-01-01

299

1-Dehydro-[10]-gingerdione from ginger inhibits IKK? activity for NF-?B activation and suppresses NF-?B-regulated expression of inflammatory genes  

PubMed Central

BACKGROUND AND PURPOSE Pungent constituents of ginger (Zingiber officinale) have beneficial effects on inflammatory pain and arthritic swelling. However, the molecular basis for these pharmacological properties is only partially understood. Here, we investigated the molecular target of 1-dehydro-[10]-gingerdione (D10G), one of the pungent constituents of ginger, that mediates its suppression of NF-?B-regulated expression of inflammatory genes linked to toll-like receptor (TLR)-mediated innate immunity. EXPERIMENTAL APPROACH RAW 264.7 macrophages or primary macrophages-derived from bone marrows of C57BL/6 or C3H/HeJ mice were stimulated with the TLR4 agonist LPS in the presence of D10G. Catalytic activity of inhibitory ?B (I?B) kinase ? (IKK?) was determined by a kinase assay and immunoblot analysis, and the expression of inflammatory genes by RT-PCR analysis and a promoter-dependent reporter assay. KEY RESULTS D10G directly inhibited the catalytic activity of cell-free IKK?. Moreover, D10G irreversibly inhibited cytoplasmic IKK?-catalysed I?B? phosphorylation in macrophages activated by TLR agonists or TNF-?, and also IKK? vector-elicited NF-?B transcriptional activity in these cells. These effects of D10G were abolished by substitution of the Cys179 with Ala in the activation loop of IKK?, indicating a direct interacting site of D10G. This mechanism was shown to mediate D10G-induced disruption of NF-?B activation in LPS-stimulated macrophages and the suppression of NF-?B-regulated gene expression of inducible NOS, COX-2 and IL-6. CONCLUSION AND IMPLICATIONS This study demonstrates that IKK? is a molecular target of D10G involved in the suppression of NF-?B-regulated gene expression in LPS-activated macrophages; this suggests D10G has therapeutic potential in NF-?B-associated inflammation and autoimmune disorders. PMID:22489648

Lee, Hwa Young; Park, Sun Hong; Lee, Misoon; Kim, Hye-Jin; Ryu, Shi Yong; Kim, Nam Doo; Hwang, Bang Yeon; Hong, Jin Tae; Han, Sang-Bae; Kim, Youngsoo

2012-01-01

300

Inhibition of human cytomegalovirus IE gene expression by dihydro-beta-agarofuran sesquiterpenes isolated from Euonymus species.  

PubMed

The development of strategies intended to inhibit human cytomegalovirus (HCMV) immediate-early (IE) antigen expression is an important goal in research designed to prevent and treat certain forms of cancer. The aim of this study was to identify potent IE antigen-targeting natural compounds as antitumor promoters in an in vitro model of tumor promotion. Nineteen dihydro-beta-agarofuran sesquiterpenes isolated from Euonymus species were evaluated for their ability to inhibit HCMV IE antigen expression in human lung adenocarcinoma (A549) cells. Five esters of penta- and hexahydroxydihydro-beta-agarofuran proved to be active components in these Euonymus species, inhibiting the IE antigen expression of HCMV. The highest activity was displayed by 2beta,6alpha,15-triacetoxy1beta-benzoyloxy-9alpha-nicotinoyloxydihydro-beta-agarofuran. These effective compounds may be regarded as prototypes of antitumor promoters, as secondary chemopreventive agents which can modify or halt tumor promotion in general. PMID:19181007

Pusztai, Rozália; Hohmann, Judit; Rédei, Dora; Engi, Helga; Molnár, Joseph

2008-01-01

301

Cloning, characterization, expression analysis and inhibition studies of a novel gene encoding Bowman-Birk type protease inhibitor from rice bean.  

PubMed

This paper presents the first study describing the isolation, cloning and characterization of a full length gene encoding Bowman-Birk protease inhibitor (RbTI) from rice bean (Vigna umbellata). A full-length protease inhibitor gene with complete open reading frame of 327 bp encoding 109 amino acids was cloned from rice bean seeds using degenerate primer set. BlastP search revealed that the RbTI encoded amino acid of approx 13.0 kDa and shared 99% homology each with BBI from Phaseolus parvulus, Vigna trilobata and Vigna vexilata. Phylogenetic tree also showed close relationship of RbTI with BBI from other members of Leguminaceae family. RbTI gene was further confirmed as intronless (GenBank accession no. KJ159908). The secondary and 3D-structural models for the RbTI were predicted with homology modeling. qRT-PCR studies revealed the highest RbTI expression in the seeds nearing maturity, whereas the low expression of the gene was noticed in young leaves. The isolated RbTI was successfully expressed in Escherichiacoli and the highest expression was recorded after 5.5h of induction. Study on the inhibitory activity of expressed protein against the gut proteases of Hessian fly larvae revealed 87% inhibition. The novel RbTI gene will further broaden the pool of plant defense genes and could be an ideal choice for developing transgenic crops resistant to insect pests with high economic value. In addition, it has the potential to be used as a probe for selection of insect- and pathogen-resistant genotypes. PMID:24905651

Katoch, Rajan; Singh, Sunil Kumar; Thakur, Neelam; Dutt, Som; Yadav, Sudesh Kumar; Shukle, Rich

2014-08-10

302

Wilms’ Tumor Gene 1 (WT1) Silencing Inhibits Proliferation of Malignant Peripheral Nerve Sheath Tumor sNF96.2 Cell Line  

PubMed Central

Wilms’ tumor gene 1 (WT1) plays complex roles in tumorigenesis, acting as tumor suppressor gene or an oncogene depending on the cellular context. WT1 expression has been variably reported in both benign and malignant peripheral nerve sheath tumors (MPNSTs) by means of immunohistochemistry. The aim of the present study was to characterize its potential pathogenetic role in these relatively uncommon malignant tumors. Firstly, immunohistochemical analyses in MPNST sNF96.2 cell line showed strong WT1 staining in nuclear and perinuclear areas of neoplastic cells. Thus, we investigated the effects of silencing WT1 by RNA interference. Through Western Blot analysis and proliferation assay we found that WT1 knockdown leads to the reduction of cell growth in a time- and dose-dependent manner. siWT1 inhibited proliferation of sNF96.2 cell lines likely by influencing cell cycle progression through a decrease in the protein levels of cyclin D1 and inhibition of Akt phosphorylation compared to the control cells. These results indicate that WT1 knockdown attenuates the biological behavior of MPNST cells by decreasing Akt activity, demonstrating that WT1 is involved in the development and progression of MPNSTs. Thus, WT1 is suggested to serve as a potential therapeutic target for MPNSTs. PMID:25474318

Parenti, Rosalba; Cardile, Venera; Graziano, Adriana Carol Eleonora; Parenti, Carmela; Venuti, Assunta; Bertuccio, Maria Paola; Furno, Debora Lo; Magro, Gaetano

2014-01-01

303

(?)-Xanthatin up-regulation of the GADD45? tumor suppressor gene in MDA-MB-231 breast cancer cells: Role of topoisomerase II? inhibition and reactive oxygen species  

PubMed Central

Previously, we reported that (?)-xanthatin, a naturally occurring xanthanolide present in the Cocklebur plant, exhibits potent anti-proliferative effects on human breast cancer cells, accompanied by an induction of the growth arrest and DNA damage-inducible gene 45? (GADD45?), recognized recently as a novel tumor suppressor gene. However, the mechanisms mediating this activation were unknown. Topoisomerase II? (Topo II?) inhibition has been reported to produce a cell death response accompanied by an atypical DNA laddering fragmentation profile, similar to that noted previously for (–)-xanthatin. Therefore we hypothesized that (?)-xanthatin’s GADD45? activation was mediated through the Topo II? pathway. Here, we identify that (?)-xanthatin does function as a catalytic inhibitor of Topo II?, promoting DNA damage. In addition, reactive oxygen species (ROS) were elevated in cells treated with this agent. Mechanistically, it was determined that the induced levels of GADD45? mRNA resulting from (?)-xanthatin exposures were stabilized by coordinately produced ROS, and that the consequent induction of GADD45? mRNA, GADD45? protein and ROS generation were abrogated by co-treatment with N-acetyl-l-cysteine. Taken together, the data support the concept that Topo II? inhibition by (?)-xanthatin is a trigger that stimulates expression of DNA damage-inducible GADD45? mRNA and that concomitantly produced ROS act downstream to further enhance the GADD45? mRNA/GADD45? protein induction process, resulting in breast cancer cell death. PMID:23313378

Takeda, Shuso; Noguchi, Momoko; Matsuo, Kazumasa; Yamaguchi, Yasuhiro; Kudo, Taichi; Nishimura, Hajime; Okamoto, Yoshiko; Amamoto, Toshiaki; Shindo, Mitsuru; Omiecinski, Curtis J.; Aramaki, Hironori

2014-01-01

304

Delivery of inhibitor of growth 4 (ING4) gene significantly inhibits proliferation and invasion and promotes apoptosis of human osteosarcoma cells  

PubMed Central

Growing evidence has suggested that inhibitor of growth 4 (ING4), a novel member of ING family proteins, plays a critical role in the development and progression of different tumors via multiple pathways. However, the function of ING4 in human osteosarcoma remains unclear. To understand its potential roles and mechanisms in inhibiting osteosarcoma, we constructed an expression vector pEGFP-ING4 and transfected the human osteosarcoma cells using this vector. We then studied the effects of over-expressed ING4 in the transfected cells on the proliferation, apoptosis and invasion of the osteosarcoma cells. The up-regulation of ING4 in the osteosarcoma cells, arising from the stable pEGFP-ING4 gene transfection, was found to significantly inhibit the cell proliferation by the cell cycle alteration with S phase reduction and G0/G1 phase arrest, induce cell apoptosis via the activation of the mitochondria pathway, and suppress cell invasion through the down-regulation of the matrix metalloproteinase 2 (MMP-2) and MMP-9 expression. In addition, increased ING4 level evoked the blockade of NF-?B signaling pathway and down-regulation of its target proteins. Our work suggests that ING4 can suppress osteosarcoma progression through signaling pathways such as mitochondria pathway and NF-?B signaling pathway and ING4 gene therapy is a promising approach to treating osteosarcoma. PMID:25490312

Li, Mei; Zhu, Ye; Zhang, Hongbin; Li, Lihua; He, Peng; Xia, Hong; Zhang, Yu; Mao, Chuanbin

2014-01-01

305

The tumor-suppressor gene LZTS1 suppresses colorectal cancer proliferation through inhibition of the AKT-mTOR signaling pathway.  

PubMed

The Leucine zipper tumor suppressor gene 1 (LZTS1/FEZ1) gene was originally identified as a potential tumor suppressor. However, the expression pattern and the role of LZTS1 in the progression of colorectal cancer (CRC) have not been well characterized. Herein, we reported that LZTS1 was markedly reduced in CRC tissues compared with matched adjacent normal intestine epithelial tissues. In analysis of 160 CRC specimens, we revealed that decreased expression of LZTS1 was correlated to aggressive characteristics and poor survival of patients with CRC. Moreover, we found that expression of LZTS1 in CRC cells significantly inhibited cell proliferation in vitro and prohibited tumor growth in vitro. On the contrary, silence of LZTS1 promoted cell proliferation and tumor growth in CRC cells. Furthermore, we demonstrated that LZTS1 inhibited cell proliferation and tumor growth in CRC in part via suppression of AMT-mTOR, subsequently down-regulating p27Kip and up-regulating cyclin D1. These findings suggest that LZTS1 plays a potential tumor suppressor role in CRC progression and represents a valuable clinical prognostic marker of this disease. PMID:25667121

Zhou, Wei; He, Mei-Rong; Jiao, Hong-Li; He, Liu-Qing; Deng, Dan-Ling; Cai, Juan-Juan; Xiao, Zhi-Yuan; Ye, Ya-Ping; Ding, Yan-Qing; Liao, Wen-Ting; Liu, Si-De

2015-04-28

306

Flavocoxid Inhibits Phospholipase A2, Peroxidase Moieties of the Cyclooxygenases (COX), and 5-Lipoxygenase, Modifies COX-2 Gene Expression, and Acts as an Antioxidant  

PubMed Central

The multiple mechanisms of action for flavocoxid relating to arachidonic acid (AA) formation and metabolism were studied in vitro. Flavocoxid titrated into rat peritoneal macrophage cultures inhibited cellular phospholipase A2 (PLA2) (IC50 = 60??g/mL). In in vitro enzyme assays, flavocoxid showed little anti-cyclooxygenase (CO) activity on COX-1/-2 enzymes, but inhibited the COX-1 (IC50 = 12.3) and COX-2 (IC50 = 11.3??g/mL) peroxidase (PO) moieties as well as 5-lipoxygenase (5-LOX) (IC50 = 110??g/mL). No detectable 5-LOX inhibition was found for multiple traditional and COX-2 selective NSAIDs. Flavocoxid also exhibited strong and varied antioxidant capacities in vitro and decreased nitrite levels (IC50 = 38??g/mL) in rat peritoneal macrophages. Finally, in contrast to celecoxib and ibuprofen, which upregulated the cox-2 gene, flavocoxid strongly decreased expression. This work suggests that clinically favourable effects of flavocoxid for management of osteoarthritis (OA) are achieved by simultaneous modification of multiple molecular pathways relating to AA metabolism, oxidative induction of inflammation, and neutralization of reactive oxygen species (ROS). PMID:21765617

Burnett, Bruce P.; Bitto, Alessandra; Altavilla, Domenica; Squadrito, Francesco; Levy, Robert M.; Pillai, Lakshmi

2011-01-01

307

Pure enantiomers of benzoylamino-tranylcypromine: LSD1 inhibition, gene modulation in human leukemia cells and effects on clonogenic potential of murine promyelocytic blasts.  

PubMed

The pure enantiomers of the N-(2-, 3-, and 4-(2-aminocyclopropyl)phenyl)benzamides hydrochlorides 11a-j were prepared and tested against LSD1 and MAO enzymes. The evaluation of the regioisomers 11a-j highlighted a net increase of the anti-LSD1 potency by shifting the benzamide moiety from ortho to meta and mainly to para position of tranylcypromine phenyl ring, independently from their trans or cis stereochemistry. In particular, the para-substituted 11a,b (trans) and 11g,h (cis) compounds displayed LSD1 and MAO-A inhibition at low nanomolar levels, while were less potent against MAO-B. The meta analogs 11c,d (trans) and 11i,j (cis) were in general less potent, but more efficient against MAO-A than against LSD1. In cellular assays, all the para and meta enantiomers were able to inhibit LSD1 by inducing Gfi-1b and ITGAM gene expression, with 11b,c and 11g-i giving the highest effects. Moreover, 11b and 11g,h strongly inhibited the clonogenic potential of murine promyelocytic blasts. PMID:25768700

Valente, Sergio; Rodriguez, Veronica; Mercurio, Ciro; Vianello, Paola; Saponara, Bruna; Cirilli, Roberto; Ciossani, Giuseppe; Labella, Donatella; Marrocco, Biagina; Monaldi, Daria; Ruoppolo, Giovanni; Tilset, Mats; Botrugno, Oronza A; Dessanti, Paola; Minucci, Saverio; Mattevi, Andrea; Varasi, Mario; Mai, Antonello

2015-04-13

308

Short-hairpin RNA-mediated Heat shock protein 90 gene silencing inhibits human breast cancer cell growth in vitro and in vivo  

SciTech Connect

Highlights: Black-Right-Pointing-Pointer Hsp90 is over-expressed in human breast cancer. Black-Right-Pointing-Pointer The shRNA-mediated gene silencing of Hsp90 resulted in inhibition of cell growth. Black-Right-Pointing-Pointer Akt and NF-kB were down-regulation after transfection due to Hsp90 silencing. Black-Right-Pointing-Pointer The tumor growth ratio was decline due to Hsp90 silencing. Black-Right-Pointing-Pointer The PCNA expression was down-regulation due to Hsp90 silencing. -- Abstract: Hsp90 interacts with proteins that mediate signaling pathways involved in the regulation of essential processes such as proliferation, cell cycle control, angiogenesis and apoptosis. Hsp90 inhibition is therefore an attractive strategy for blocking abnormal pathways that are crucial for cancer cell growth. In the present study, the role of Hsp90 in human breast cancer MCF-7 cells was examined by stably silencing Hsp90 gene expression with an Hsp90-silencing vector (Hsp90-shRNA). RT-PCR and Western blot analyses showed that Hsp90-shRNA specifically and markedly down-regulated Hsp90 mRNA and protein expression. NF-kB and Akt protein levels were down-regulated in Hsp90-shRNA transfected cells, indicating that Hsp90 knockout caused a reduction of survival factors and induced apoptosis. Treatment with Hsp90-shRNA significantly increased apoptotic cell death and caused cell cycle arrest in the G1/S phase in MCF-7 cells, as shown by flow cytometry. Silencing of Hsp90 also reduced cell viability, as determined by MTT assay. In vivo experiments showed that MCF-7 cells stably transfected with Hsp90-shRNA grew slowly in nude mice as compared with control groups. In summary, the Hsp90-shRNA specifically silenced the Hsp90 gene, and inhibited MCF-7 cell growth in vitro and in vivo. Possible molecular mechanisms underlying the effects of Hsp90-shRNA include the degradation of Hsp90 breast cancer-related client proteins, the inhibition of survival signals and the upregulation of apoptotic pathways. shRNA-mediated interference may have potential therapeutic utility in human breast cancer.

Zuo, Keqiang [Department of General Surgery, Shanghai 10th People's Hospital, Tongji University School of Medicine, Shanghai 200072 (China)] [Department of General Surgery, Shanghai 10th People's Hospital, Tongji University School of Medicine, Shanghai 200072 (China); Li, Dan [Department of Nuclear Medicine, Shanghai 10th People's Hospital, Tongji University School of Medicine, Shanghai 200072 (China)] [Department of Nuclear Medicine, Shanghai 10th People's Hospital, Tongji University School of Medicine, Shanghai 200072 (China); Pulli, Benjamin [Center for Systems Biology, Massachusetts General Hospital, Harvard Medical School, 185 Cambridge Street, Boston, MA 02114 (United States)] [Center for Systems Biology, Massachusetts General Hospital, Harvard Medical School, 185 Cambridge Street, Boston, MA 02114 (United States); Yu, Fei; Cai, Haidong; Yuan, Xueyu [Department of Nuclear Medicine, Shanghai 10th People's Hospital, Tongji University School of Medicine, Shanghai 200072 (China)] [Department of Nuclear Medicine, Shanghai 10th People's Hospital, Tongji University School of Medicine, Shanghai 200072 (China); Zhang, Xiaoping, E-mail: zxpsibs@163.com [Department of Nuclear Medicine, Shanghai 10th People's Hospital, Tongji University School of Medicine, Shanghai 200072 (China)] [Department of Nuclear Medicine, Shanghai 10th People's Hospital, Tongji University School of Medicine, Shanghai 200072 (China); Lv, Zhongwei, E-mail: heyixue163@163.com [Department of Nuclear Medicine, Shanghai 10th People's Hospital, Tongji University School of Medicine, Shanghai 200072 (China)] [Department of Nuclear Medicine, Shanghai 10th People's Hospital, Tongji University School of Medicine, Shanghai 200072 (China)

2012-05-04

309

Mechanisms of hormonal regulation of CAD gene expression and inhibition by Aryl hydrocarbon receptor agonist in human breast cancer cells  

E-print Network

-mediated pathway. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and other aryl hydrocarbon receptor (AhR) ligands suppress several E2-induced responses in the rodent uterus and mammary tumors and in human breast cancer cells. TCDD inhibited hormone...

Khan, Shaheen Munawar Ali

2007-04-25

310

Transcriptional and Posttranscriptional Regulation of Cytokine Gene Expression in HIV-1 Antigen-Specific CD8+ T Cells That Mediate Virus Inhibition  

PubMed Central

ABSTRACT The ability of CD8+ T cells to effectively limit HIV-1 replication and block HIV-1 acquisition is determined by the capacity to rapidly respond to HIV-1 antigens. Understanding both the functional properties and regulation of an effective CD8+ response would enable better evaluation of T cell-directed vaccine strategies and may inform the design of new therapies. We assessed the antigen specificity, cytokine signature, and mechanisms that regulate antiviral gene expression in CD8+ T cells from a cohort of HIV-1-infected virus controllers (VCs) (<5,000 HIV-1 RNA copies/ml and CD4+ lymphocyte counts of >400 cells/?l) capable of soluble inhibition of HIV-1. Gag p24 and Nef CD8+ T cell-specific soluble virus inhibition was common among the VCs and correlated with substantial increases in the abundance of mRNAs encoding the antiviral cytokines macrophage inflammatory proteins MIP-1?, MIP-1?P (CCL3L1), and MIP-1?; granulocyte-macrophage colony-stimulating factor (GM-CSF); lymphotactin (XCL1); tumor necrosis factor receptor superfamily member 9 (TNFRSF9); and gamma interferon (IFN-?). The induction of several of these mRNAs was driven through a coordinated response of both increased transcription and stabilization of mRNA, which together accounted for the observed increase in mRNA abundance. This coordinated response allows rapid and robust induction of mRNA messages that can enhance the CD8+ T cells' ability to inhibit virus upon antigen encounter. IMPORTANCE We show that mRNA stability, in addition to transcription, is key in regulating the direct anti-HIV-1 function of antigen-specific memory CD8+ T cells. Regulation at the level of RNA helps enable rapid recall of memory CD8+ T cell effector functions for HIV-1 inhibition. By uncovering and understanding the mechanisms employed by CD8+ T cell subsets with antigen-specific anti-HIV-1 activity, we can identify new strategies for comprehensive identification of other important antiviral genes. This will, in turn, enhance our ability to inhibit virus replication by informing both cure strategies and HIV-1 vaccine designs that aim to reduce transmission and can aid in blocking HIV-1 acquisition. PMID:24899193

Payne, Tamika L.; Blackinton, Jeff; Frisbee, Alyse; Pickeral, Joy; Sawant, Sheetal; Vandergrift, Nathan A.; Freel, Stephanie A.; Ferrari, Guido; Keene, Jack D.

2014-01-01

311

The 3C Protein of Enterovirus 71 Inhibits Retinoid Acid-Inducible Gene I-Mediated Interferon Regulatory Factor 3 Activation and Type I Interferon Responses?  

PubMed Central

Enterovirus 71 (EV71) is a human pathogen that induces hand, foot, and mouth disease and fatal neurological diseases. Immature or impaired immunity is thought to associate with increased morbidity and mortality. In a murine model, EV71 does not facilitate the production of type I interferon (IFN) that plays a critical role in the first-line defense against viral infection. Administration of a neutralizing antibody to IFN-?/? exacerbates the virus-induced disease. However, the molecular events governing this process remain elusive. Here, we report that EV71 suppresses the induction of antiviral immunity by targeting the cytosolic receptor retinoid acid-inducible gene I (RIG-I). In infected cells, EV71 inhibits the expression of IFN-?, IFN-stimulated gene 54 (ISG54), ISG56, and tumor necrosis factor alpha. Among structural and nonstructural proteins encoded by EV71, the 3C protein is capable of inhibiting IFN-? activation by virus and RIG-I. Nevertheless, EV71 3C exhibits no inhibitory activity on MDA5. Remarkably, when expressed in mammalian cells, EV71 3C associates with RIG-I via the caspase recruitment domain. This precludes the recruitment of an adaptor IPS-1 by RIG-I and subsequent nuclear translocation of interferon regulatory factor 3. An R84Q or V154S substitution in the RNA binding motifs has no effect. An H40D substitution is detrimental, but the protease activity associated with 3C is dispensable. Together, these results suggest that inhibition of RIG-I-mediated type I IFN responses by the 3C protein may contribute to the pathogenesis of EV71 infection. PMID:20519382

Lei, Xiaobo; Liu, Xinlei; Ma, Yijie; Sun, Zhenmin; Yang, Yaowu; Jin, Qi; He, Bin; Wang, Jianwei

2010-01-01

312

HOS3, an ELO-Like Gene, Inhibits Effects of ABA and Implicates a S-1-P/Ceramide Control System for Abiotic Stress Responses in Arabidopsis thaliana  

PubMed Central

A hyper-osmotically sensitive mutant of Arabidopsis thaliana, designated hos3-1 (high expression of osmotically responsive genes), was identified based on its hyper-luminescence of RD29A:LUC promoter fusion plants upon treatment with NaCl and ABA. These responses implicate the disrupted gene as a direct or indirect negative regulator of the RD29A stress-responsive pathway. By sequencing the flanking regions of the T-DNA borders, it was determined that the disrupted gene is at locus At4g36830, annotated as encoding a putative protein with high homology to CIG30 (ELO2/FEN1). CIG30 has been implicated in synthesis of very long chain fatty acids (VLCFA), which are essential precursors for sphingolipids and ceramides. Altered stress responses characteristic of ABA-hypersensitivity, including reduced root growth inhibition and reduced germination with ABA treatment and reduced water loss from leaves, were exhibited by allelic hos3-1 and hos3-2 mutants. The hos3-2 mutant is partially suppressed in its transcript abundance and is inherited as a recessive trait. Further, the HOS3 ORF under the control of the 35SCaMV promoter restored wild-type NaCl- and ABA-root growth sensitivity as well as RD29A:LUC luminescence in mutant plants. We also show here that the HOS3 wild-type gene functionally complements the sensitivity of elo2 and elo3 yeast mutants to monensin. Furthermore, both hos3-1 and hos3-2 alleles shared increased sensitivity to the herbicide Metolachlor, which inhibits acyl chain elongation in synthesis of VLCFA, and HOS3 functionally complemented both elo2 and elo3 and restored levels of VLCFA. Together, these data establish that HOS3 inhibits ABA-mediated stress responses and implicate the VLCFA pathway and products as control points for several aspects of abiotic stress signaling and responses. The results also provide support for a role of ceramide in the control of stomatal behavior. PMID:19529829

Quist, Tanya M.; Sokolchik, Irina; Shi, Huazhong; Joly, Robert J.; Bressan, Ray A.; Maggio, Albino; Narsimhan, Meena; Li, Xia

2009-01-01

313

Inhibition of the binding of MSG-intermolt-specific complex, MIC, to the sericin-1 gene promoter and sericin-1 gene expression by POU-M1/SGF-3.  

PubMed

The sericin-1 gene encoding a glue protein is expressed in the middle silk gland (MSG) of the silkworm, Bombyx mori. A member of the class III POU domain transcription factors, POU-M1, was cloned as the factor bound to the SC site of the sericin-1 promoter and has been proposed to be a positive transcription factor. In this study, we analyzed the expression pattern of the POU-M1 gene in fourth and fifth instars in comparison with the pattern of the sericin-1 gene. The POU-M1 gene was expressed strongly in the region anterior to the sericin-1-expressing portion of the silk gland at both feeding stages. As the sericin-1-expressing region expands from the posterior to middle portions of the MSG in the fifth instar, the POU-M1-expressing region retreated from the middle to anterior portion. Introduction of the expression vector of POU-M1 into the silk glands by gene gun technology repressed promoter activity of the sericin-1 gene, suggesting that POU-M1 regulates the sericin-1 gene negatively. An in vitro binding assay showed that POU-M1 bound not only to the SC site but also to other promoter elements newly detected in vivo. Another spatiotemporal specific factor MIC binds to these elements, and POU-M1 competed with MIC to bind at the -70 site essential for promoter activity. These results suggest that POU-M1 is involved in restricting the anterior boundary of the sericin-1-expressing region in the silk gland by inhibiting the binding of the transcriptional activator to the promoter elements. PMID:23070540

Kimoto, Mai; Kitagawa, Tsuyuki; Kobayashi, Isao; Nakata, Tomohiro; Kuroiwa, Asato; Takiya, Shigeharu

2012-11-01

314

Inhibition of both HIV-1 reverse transcription and gene expression by a cyclic peptide that binds the Tat-transactivating response element (TAR) RNA.  

PubMed

The RNA response element TAR plays a critical role in HIV replication by providing a binding site for the recruitment of the viral transactivator protein Tat. Using a structure-guided approach, we have developed a series of conformationally-constrained cyclic peptides that act as structural mimics of the Tat RNA binding region and block Tat-TAR interactions at nanomolar concentrations in vitro. Here we show that these compounds block Tat-dependent transcription in cell-free systems and in cell-based reporter assays. The compounds are also cell permeable, have low toxicity, and inhibit replication of diverse HIV-1 strains, including both CXCR4-tropic and CCR5-tropic primary HIV-1 isolates of the divergent subtypes A, B, C, D and CRF01_AE. In human peripheral blood mononuclear cells, the cyclic peptidomimetic L50 exhibited an IC(50) ?250 nM. Surprisingly, inhibition of LTR-driven HIV-1 transcription could not account for the full antiviral activity. Timed drug-addition experiments revealed that L-50 has a bi-phasic inhibition curve with the first phase occurring after HIV-1 entry into the host cell and during the initiation of HIV-1 reverse transcription. The second phase coincides with inhibition of HIV-1 transcription. Reconstituted reverse transcription assays confirm that HIV-1 (-) strand strong stop DNA synthesis is blocked by L50-TAR RNA interactions in-vitro. These findings are consistent with genetic evidence that TAR plays critical roles both during reverse transcription and during HIV gene expression. Our results suggest that antiviral drugs targeting TAR RNA might be highly effective due to a dual inhibitory mechanism. PMID:21625572

Lalonde, Matthew S; Lobritz, Michael A; Ratcliff, Annette; Chamanian, Mastooreh; Athanassiou, Zafiria; Tyagi, Mudit; Wong, Julian; Robinson, John A; Karn, Jonathan; Varani, Gabriele; Arts, Eric J

2011-05-01

315

Inhibition of Both HIV-1 Reverse Transcription and Gene Expression by a Cyclic Peptide that Binds the Tat-Transactivating Response Element (TAR) RNA  

PubMed Central

The RNA response element TAR plays a critical role in HIV replication by providing a binding site for the recruitment of the viral transactivator protein Tat. Using a structure-guided approach, we have developed a series of conformationally-constrained cyclic peptides that act as structural mimics of the Tat RNA binding region and block Tat-TAR interactions at nanomolar concentrations in vitro. Here we show that these compounds block Tat-dependent transcription in cell-free systems and in cell-based reporter assays. The compounds are also cell permeable, have low toxicity, and inhibit replication of diverse HIV-1 strains, including both CXCR4-tropic and CCR5-tropic primary HIV-1 isolates of the divergent subtypes A, B, C, D and CRF01_AE. In human peripheral blood mononuclear cells, the cyclic peptidomimetic L50 exhibited an IC50 ?250 nM. Surprisingly, inhibition of LTR-driven HIV-1 transcription could not account for the full antiviral activity. Timed drug-addition experiments revealed that L-50 has a bi-phasic inhibition curve with the first phase occurring after HIV-1 entry into the host cell and during the initiation of HIV-1 reverse transcription. The second phase coincides with inhibition of HIV-1 transcription. Reconstituted reverse transcription assays confirm that HIV-1 (?) strand strong stop DNA synthesis is blocked by L50-TAR RNA interactions in-vitro. These findings are consistent with genetic evidence that TAR plays critical roles both during reverse transcription and during HIV gene expression. Our results suggest that antiviral drugs targeting TAR RNA might be highly effective due to a dual inhibitory mechanism. PMID:21625572

Lalonde, Matthew S.; Lobritz, Michael A.; Ratcliff, Annette; Chamanian, Mastooreh; Athanassiou, Zafiria; Tyagi, Mudit; Wong, Julian; Robinson, John A.; Karn, Jonathan; Varani, Gabriele; Arts, Eric J.

2011-01-01

316

MiR-429 inhibits cells growth and invasion and regulates EMT-related marker genes by targeting Onecut2 in colorectal carcinoma.  

PubMed

The 5-year survival rate for colorectal cancer is approximately 55 % because of its invasion and metastasis. The epithelial-mesenchymal transition (EMT) is one of the well-defined processes during the invasion and distant metastasis of primary epithelial tumors. miR-429, a member of the miR-200 family of microRNAs, was previously shown to inhibit the expression of transcriptional repressors ZEB1/delta EF1 and SIP1/ZEB2, and regulate EMT. In this study, we showed that miR-429 was significantly downregulated in colorectal carcinoma (CRC) tissues and cell lines. We found that miR-429 inhibited the proliferation and growth of CRC cells in vitro and in vivo, suggesting that miR-429 could play a role in CRC tumorigenesis. We also showed that downregulation of miR-429 may contribute to carcinogenesis and the initiation of EMT of CRC by targeting Onecut2. Further researches indicated that miR-429 inhibited the cells migration and invasion and reversed TGF-?-induced EMT changes in SW620 and SW480 cells. miR-429 could reverse the change of EMT-related markers genes induced by TGF-?1, such as E-cadherin, CTNNA1, CTNNB1, TFN, CD44, MMP2, Vimentin, Slug, Snail, and ZEB2 by targeting Onecut2. Taken together, our data showed that transcript factor Onecut2 is involved in the EMT, migration and invasion of CRC cells; miR-429 inhibits the initiation of EMT and regulated expression of EMT-related markers by targeting Onecut2; and miR-429 or Onecut2 is the important therapy target for CRC. PMID:24402783

Sun, Yingnan; Shen, Shourong; Liu, Xiaoping; Tang, Hailin; Wang, Zeyou; Yu, Zhibin; Li, Xiayu; Wu, Minghua

2014-05-01

317

125I seed irradiation induces up-regulation of the genes associated with apoptosis and cell cycle arrest and inhibits growth of gastric cancer xenografts  

PubMed Central

Background Iodine 125 (125I) seed irradiation can be used as an important supplementary treatment for unresectable advanced gastric cancer. Here, we aim to comprehensively elucidate the biological effects induced by 125I seed irradiation in human gastric cancer xenograft model by using global expression and DNA methylation analyses. Methods The 48 mice bearing NCI-N87 gastric cancer xenografts were randomly separated into 2 groups: sham seeds (O mCi) were implanted into the control group (n?=?24); 125?l seeds (0.9?mCi) were implanted into the treatment group (n?=?24). The mitotic index and apoptotic index were evaluated by quantitative morphometric analysis of the expression of proliferating cell nuclear antigen (PCNA) and in situ terminal transferase-mediated fluorescein deoxy- UTP nick end labeling (TUNEL), respectively. Global gene expression changes induced by 125I seed irradiation were analyzed by using Nimblegen Human gene expression array. DNA methylation profile in the tumors from control group was investigated with methylated DNA immunoprecipitation (MeDIP) and Nimblegen CpG promoter microarrays. The changes in the methylation status of selected genes were further investigated by using MeDIP-PCR. Results 125I seed irradiation suppresses the growth of gastric cancer xenografts in nude mice. PCNA staining and tissue TUNEL assays showed that both inhibition of cell proliferation and induction of apoptosis contribute to the 125I-induced tumor suppression in nude mouse model. Gene expression profiles revealed that the expression levels of several hundred genes, many of which are associated with apoptosis or cell cycle arrest, including BMF, MAPK8, BNIP3, RFWD3, CDKN2B and WNT9A, were upregulated following 125I seed irradiation. Furthermore, the up-regulation of some of these genes, such as BNIP3 and WNT9A, was found to be associated with irradiation-induced DNA demethylation. Conclusions This study revealed that 125I seed irradiation could significantly induce the up-regulation of apoptosis- and cell cycle-related genes in human gastric cancer xenografts. And some of the up-regulation might be attributed to 125I-irradiation induced demethylation in gene promoter regions. Collectively, these findings provided evidence for the efficacy of this modality for the treatment of gastric cancer. PMID:22827957

2012-01-01

318

Peroxisome Proliferator-activated Receptor ? Positively Regulates Complement C3 Expression but Inhibits Tumor Necrosis Factor ?-mediated Activation of C3 Gene in Mammalian Hepatic-derived Cells*  

PubMed Central

Complement C3 is a pivotal component of three cascades of complement activation. The liver is the main source of C3 in circulation and expression and secretion of C3 by hepatocytes is increased during acute inflammation. However, the mechanism of the regulation of the C3 gene in hepatocytes is not well elucidated. We showed that the C3 gene is the direct target for peroxisome proliferator-activated receptor ? (PPAR?) in human hepatoma HepG2 cells and mouse liver. Using PPAR? siRNA and synthetic PPAR? agonist WY-14643 and antagonist MK886 we showed that activation of PPAR? results in up-regulation of C3 gene expression and protein secretion by HepG2 cells. The PPAR response element (PPRE), which is able to bind PPAR? in vitro and in vivo, was found in the human C3 promoter. PPRE is conserved between human and mouse, and WY-14643 stimulates mouse C3 expression in the liver. TNF? increases C3 gene via NF-?B and, to a lesser extent, MEK1/2 signaling pathways, whereas TNF?-mediated stimulation of C3 protein secretion depends on activation of MEK1/2, p38, and JNK in HepG2 cells. Activation of PPAR? abolishes TNF?-mediated up-regulation of C3 gene expression and protein secretion due to interference with NF-?B via PPRE-dependent mechanism in HepG2 cells. TNF? decreases PPAR? protein content via NF-?B and MEK1/2 signaling pathways and inhibits PPAR? binding with the human C3 promoter in HepG2 cells. These results suggest novel mechanism controlling C3 expression in hepatocytes during acute phase inflammation and demonstrate a crosstalk between PPAR? and TNF? in the regulation of complement system. PMID:23168409

Mogilenko, Denis A.; Kudriavtsev, Igor V.; Shavva, Vladimir S.; Dizhe, Ella B.; Vilenskaya, Ekaterina G.; Efremov, Alexander M.; Perevozchikov, Andrej P.; Orlov, Sergey V.

2013-01-01

319

Peroxisome proliferator-activated receptor ? positively regulates complement C3 expression but inhibits tumor necrosis factor ?-mediated activation of C3 gene in mammalian hepatic-derived cells.  

PubMed

Complement C3 is a pivotal component of three cascades of complement activation. The liver is the main source of C3 in circulation and expression and secretion of C3 by hepatocytes is increased during acute inflammation. However, the mechanism of the regulation of the C3 gene in hepatocytes is not well elucidated. We showed that the C3 gene is the direct target for peroxisome proliferator-activated receptor ? (PPAR?) in human hepatoma HepG2 cells and mouse liver. Using PPAR? siRNA and synthetic PPAR? agonist WY-14643 and antagonist MK886 we showed that activation of PPAR? results in up-regulation of C3 gene expression and protein secretion by HepG2 cells. The PPAR response element (PPRE), which is able to bind PPAR? in vitro and in vivo, was found in the human C3 promoter. PPRE is conserved between human and mouse, and WY-14643 stimulates mouse C3 expression in the liver. TNF? increases C3 gene via NF-?B and, to a lesser extent, MEK1/2 signaling pathways, whereas TNF?-mediated stimulation of C3 protein secretion depends on activation of MEK1/2, p38, and JNK in HepG2 cells. Activation of PPAR? abolishes TNF?-mediated up-regulation of C3 gene expression and protein secretion due to interference with NF-?B via PPRE-dependent mechanism in HepG2 cells. TNF? decreases PPAR? protein content via NF-?B and MEK1/2 signaling pathways and inhibits PPAR? binding with the human C3 promoter in HepG2 cells. These results suggest novel mechanism controlling C3 expression in hepatocytes during acute phase inflammation and demonstrate a crosstalk between PPAR? and TNF? in the regulation of complement system. PMID:23168409

Mogilenko, Denis A; Kudriavtsev, Igor V; Shavva, Vladimir S; Dizhe, Ella B; Vilenskaya, Ekaterina G; Efremov, Alexander M; Perevozchikov, Andrej P; Orlov, Sergey V

2013-01-18

320

The effect of restraint stress on prepulse inhibition and on corticotropin-releasing factor (CRF) and CRF receptor gene expression in Wistar-Kyoto and Brown Norway rats  

PubMed Central

Stress plays a role in many psychiatric disorders that are characterized by deficits in prepulse inhibition (PPI), a form of sensorimotor gating. Corticotropin-releasing factor (CRF) is one of the most important neurotransmitters involved in behavioral components of the stress response, and central infusion of CRF decreases PPI in rodents. We recently demonstrated that restraint stress decreases PPI and attenuates the increase in PPI caused by repeated testing. To broaden our investigation into how restraint affects PPI, we subjected Wistar-Kyoto (WKY) and Brown Norway (BN) rats to 10 consecutive days of 2-hour restraint, or to brief handling, prior to assessing PPI. We next examined the effects of 1 or 10 days of 2-hour restraint on plasma corticosterone levels in order to determine whether the endocrine response to stress parallels the behavioral effect of stress. Finally, we examined the effects of 1 or 10 days of 2-hour restraint on CRF and CRF receptor gene expression in the amygdala, hippocampus, frontal cortex, and hypothalamus in order to determine whether a temporal pattern of gene expression parallels the change in the behavioral response to stress. The major findings of the present study are that 1) restraint stress attenuates the increase in PPI caused by repeated testing in both WKY and BN rats, and BN rats are more sensitive to the effects of restraint on PPI than WKY rats, 2) restraint-induced increases in corticosterone levels mirror the effect of restraint on PPI in WKY rats but not in BN rats, 3) laterality effects on gene expression were observed for the amygdala, whereby restraint increases CRF gene expression in the left, but not right, amygdala, and 4) some restraint-induced changes in CRF and CRF receptor gene expression precede changes in PPI while other changes coincide with altered PPI in a rat strain- and brain region-dependent manner. PMID:20709096

Sutherland, Jane E.; Burian, Linda C.; Covault, Jonathan; Conti, Lisa H.

2010-01-01

321

Angiostatin Gene Transfer: Inhibition of Tumor Growth in vivo by Blockage of Endothelial Cell Proliferation Associated with a Mitosis Arrest  

Microsoft Academic Search

The antitumoral effects that follow the local delivery of the N-terminal fragment of human plasminogen (angiostatin K3) have been studied in two xenograft murine models. Angiostatin delivery was achieved by a defective adenovirus expressing a secretable angiostatin K3 molecule from the cytomegalovirus promoter (AdK3). In in vitro studies, AdK3 selectively inhibited endothelial cell proliferation and disrupted the G2\\/M transition induced

Frank Griscelli; Hong Li; Annelise Bennaceur-Griscelli; Jeannette Soria; Paule Opolon; Claudine Soria; Michel Perricaudet; Patrice Yeh; He Lu

1998-01-01

322

Exploring signatures of positive selection in pigmentation candidate genes in populations of East Asian ancestry  

PubMed Central

Background Currently, there is very limited knowledge about the genes involved in normal pigmentation variation in East Asian populations. We carried out a genome-wide scan of signatures of positive selection using the 1000 Genomes Phase I dataset, in order to identify pigmentation genes showing putative signatures of selective sweeps in East Asia. We applied a broad range of methods to detect signatures of selection including: 1) Tests designed to identify deviations of the Site Frequency Spectrum (SFS) from neutral expectations (Tajima’s D, Fay and Wu’s H and Fu and Li’s D* and F*), 2) Tests focused on the identification of high-frequency haplotypes with extended linkage disequilibrium (iHS and Rsb) and 3) Tests based on genetic differentiation between populations (LSBL). Based on the results obtained from a genome wide analysis of 25 kb windows, we constructed an empirical distribution for each statistic across all windows, and identified pigmentation genes that are outliers in the distribution. Results Our tests identified twenty genes that are relevant for pigmentation biology. Of these, eight genes (ATRN, EDAR, KLHL7, MITF, OCA2, TH, TMEM33 and TRPM1,) were extreme outliers (top 0.1% of the empirical distribution) for at least one statistic, and twelve genes (ADAM17, BNC2, CTSD, DCT, EGFR, LYST, MC1R, MLPH, OPRM1, PDIA6, PMEL (SILV) and TYRP1) were in the top 1% of the empirical distribution for at least one statistic. Additionally, eight of these genes (BNC2, EGFR, LYST, MC1R, OCA2, OPRM1, PMEL (SILV) and TYRP1) have been associated with pigmentary traits in association studies. Conclusions We identified a number of putative pigmentation genes showing extremely unusual patterns of genetic variation in East Asia. Most of these genes are outliers for different tests and/or different populations, and have already been described in previous scans for positive selection, providing strong support to the hypothesis that recent selective sweeps left a signature in these regions. However, it will be necessary to carry out association and functional studies to demonstrate the implication of these genes in normal pigmentation variation. PMID:23848512

2013-01-01

323

The BEL1-type homeobox gene SH5 induces seed shattering by enhancing abscission-zone development and inhibiting lignin biosynthesis.  

PubMed

Seed shattering is an important trait that influences grain yield. A major controlling quantitative trait locus in rice is qSH1. Although the degree of shattering is correlated with the level of expression of qSH1, some qSH1-defective cultivars display moderate shattering while others show a non-shattering phenotype. Os05 g38120 (SH5) on chromosome 5 is highly homologous to qSH1. Although we detected SH5 transcripts in various organs, this gene was highly expressed at the abscission zone (AZ) in the pedicels. When expression of this gene was suppressed in easy-shattering 'Kasalath', development of the AZ was reduced and thereby so was seed loss. By contrast, the extent of shattering, as well as AZ development, was greatly enhanced in moderate-shattering 'Dongjin' rice when SH5 was overexpressed. Likewise, overexpression of SH5 in the non-shattering 'Ilpum' led to an increase in seed shattering because lignin levels were decreased in the basal region of spikelets in the absence of development of an AZ. We also determined that two shattering-related genes, SHAT1 and Sh4, which are necessary for proper formation of an AZ, were induced by SH5. Based on these observations, we propose that SH5 modulates seed shattering by enhancing AZ development and inhibiting lignin biosynthesis. PMID:24923192

Yoon, Jinmi; Cho, Lae-Hyeon; Kim, Song Lim; Choi, Heebak; Koh, Hee-Jong; An, Gynheung

2014-09-01

324

Inhibition of cell proliferation and induction of apoptosis by oleanane triterpenoid (CDDO-Me) in pancreatic cancer cells is associated with the suppression of hTERT gene expression and its telomerase activity  

SciTech Connect

Highlights: Black-Right-Pointing-Pointer CDDO-Me inhibits hTERT gene expression. Black-Right-Pointing-Pointer CDDO-Me inhibits hTERT protein expression. Black-Right-Pointing-Pointer CDDO-Me inhibits hTERT telomerase activity. Black-Right-Pointing-Pointer CDDO-Me inhibits hTERT regulatory proteins. -- Abstract: Methyl-2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oate (CDDO-Me) is a multifunctional oleanane synthetic triterpenoid with potent anti-inflammatory and antitumorigenic properties. The mechanisms of the antisurvival and apoptosis-inducing activities of CDDO-Me and related derivatives of oleanolic acid have been defined; however, to date, no study has been carried out on the effect of CDDOs on human telomerase reverse transcriptase (hTERT) gene or telomerase activity. Here we report for the first time that inhibition of cell proliferation and induction of apoptosis by CDDO-Me in pancreatic cancer cell lines is associated with the inhibition of hTERT gene expression, hTERT telomerase activity and a number of proteins that regulate hTERT expression and activity. Furthermore, abrogation or overexpression of hTERT protein altered the susceptibility of tumor cells to CDDO-Me. These findings suggest that telomerase (hTERT) is a relevant target of CDDO-Me in pancreatic cancer cells.

Deeb, Dorrah; Gao, Xiaohua; Liu, Yongbo [Department of Surgery, Henry Ford Health System, Detroit, MI (United States)] [Department of Surgery, Henry Ford Health System, Detroit, MI (United States); Kim, Sahn-Ho [Department of Urology, Henry Ford Health System, Detroit, MI (United States)] [Department of Urology, Henry Ford Health System, Detroit, MI (United States); Pindolia, Kirit R. [Department of Medical Genetics, Henry Ford Health System, Detroit, MI (United States)] [Department of Medical Genetics, Henry Ford Health System, Detroit, MI (United States); Arbab, Ali S. [Department of Radiology, Henry Ford Health System, Detroit, MI (United States)] [Department of Radiology, Henry Ford Health System, Detroit, MI (United States); Gautam, Subhash C., E-mail: sgautam1@hfhs.org [Department of Surgery, Henry Ford Health System, Detroit, MI (United States)

2012-06-15

325

Hydrodynamics-based transfection of rat interleukin-10 gene attenuates porcine serum-induced liver fibrosis in rats by inhibiting the activation of hepatic stellate cells  

PubMed Central

Liver fibrosis is the common pathological outcome for the majority of chronic liver diseases. Interleukin-10 (IL-10) is a cytokine that downregulates proinflammatory responses and has a modulatory effect on liver fibrogenesis. However, little is known regarding the effect of rat interleukin-10 (rIL-10) gene by hydrodynamics-based transfection (HBT) on liver fibrosis in rats. The aim of this study was to investigate the effect of the rIL-10 gene by HBT on the progression of liver fibrosis induced by porcine serum (PS) in rats and explore its possible mechanism. Plasmid-expressing rIL-10 was transferred into rats by HBT and immunohistochemistry and RT-PCR were used to detect the major organ expressing rIL-10. Liver fibrosis was induced in rats by intraperitoneal administration of PS for 8 weeks. Plasmid pcDNA3-rIL-10 solution was administered weekly by HBT starting at the 5th week. Liver function and hepatic histology were examined. The possible molecular mechanisms of rIL-10 gene therapy were assessed in liver tissue and hepatic stellate cells (HSCs) co-cultured with BRL cells (a hepatocyte line) in vitro. The results showed rIL-10 expression occurred mainly in the liver following rIL-10 gene transfer by HBT. Maintaining a stable expression of rIL-10 in serum was assessed by repeated administration. The rIL-10 gene treatment attenuated liver inflammation and fibrosis in PS-induced fibrotic rats, reduced the deposition of collagen and the expression of ?-smooth muscle actin (?-SMA) in fibrotic rats. The in vitro experiment showed that the expression of a-SMA and procollagen type I in HSCs co-cultured with the BRL-transfected rIL-10 gene were significantly decreased. These findings indicate that rIL-10 gene therapy by HBT attenuates PS-induced liver fibrosis in rats and that its mechanism is associated with rIL-10 inhibiting the activation of HSCs and promoting the degeneration of collagen. PMID:24993843

HUANG, YUE-HONG; CHEN, YUN-XIN; ZHANG, LI-JUAN; CHEN, ZHI-XIN; WANG, XIAO-ZHONG

2014-01-01

326

TET2 Inhibits Differentiation of Embryonic Stem Cells but Does Not Overcome Methylation-Induced Gene Silencing  

PubMed Central

TET2 is a methylcytosine dioxygenase that is frequently mutated in myeloid malignancies, notably myelodysplasia and acute myeloid leukemia. TET2 catalyses the conversion of 5?-methylcytosine to 5?-hydroxymethylcytosine within DNA and has been implicated in the process of genomic demethylation. However, the mechanism by which TET2 loss of function results in hematopoietic dysplasia and leukemogenesis is poorly understood. Here, we show that TET2 is expressed in undifferentiated embryonic stem cells and that its knockdown results in reduction of 5?-hydroxymethylcytosine in genomic DNA. We also present DNA methylation data from bone marrow samples obtained from patients with TET2-mutated myelodysplasia. Based on these findings, we sought to identify the role of TET2 in regulating pluripotency and differentiation. We show that overexpression of TET2 in a stably integrated transgene leads to increased alkaline phosphatase expression in differentiating ES cells and impaired differentiation in methylcellulose culture. We speculate that this effect is due to TET2-mediated expression of stem cell genes in ES cells via hydroxylation of 5?-methylcytosines at key promoter sequences within genomic DNA. This leads to relative hypomethylation of gene promoters as 5?-hydroxymethylcytosine is not a substrate for DNMT1-mediated maintenance methylation. We sought to test this hypothesis by cotransfecting the TET2 gene with methylated reporter genes. The results of these experiments are presented. PMID:25276435

2014-01-01

327

Root Development in Mice Lacking Functional Tissue Non-specific Alkaline Phosphatase Gene: Inhibition of Acellular Cementum Formation  

Microsoft Academic Search

Tissue non-specific alkaline phosphatase (TNAP) is richly present in developing teeth including the cells of the periodontal ligament. Here, we investigated tooth and root development in mice lacking the TNAP gene. Heterozygous mutants were obtained from The Jackson Laboratory, Animal Resources (Bar Harbor, ME, USA) and bred. TNAP-deficient mice and their littermates were killed from 6 to 25 days after

W. Beertsen; T. VandenBos; V. Everts

1999-01-01

328

Cell Growth Inhibition and Gene Expression Induced by the Histone Deacetylase Inhibitor, Trichostatin A, on Human Hepatoma Cells  

Microsoft Academic Search

Objective: Histone deacetylase (HDAC) inhibitors have been reported to induce cell growth arrest, apoptosis and differentiation in tumor cells. The effect of the HDAC inhibitor, trichostatin A (TSA), on hepatoma cells, however, has not been well studied. In this study, we examined cell viability and gene expression profile in hepatoma cell lines treated with TSA. Methods: To study cell growth

Tetsuhiro Chiba; Osamu Yokosuka; Kenichi Fukai; Hiroshige Kojima; Motohisa Tada; Makoto Arai; Fumio Imazeki; Hiromitsu Saisho

2004-01-01

329

Multidrug-resistance gene 1-type p-glycoprotein (MDR1 p-gp) inhibition by tariquidar impacts on neuroendocrine and behavioral processing of stress  

PubMed Central

SUMMARY The multidrug-resistance gene 1-type p-glycoprotein (MDR1 p-gp) is a major gate-keeper at the blood-brain barrier (BBB), protecting the central nervous system from accumulation of toxic xenobiotics and drugs. In addition, MDR1 p-gp has been found to control the intracerebral access of glucocorticoid hormones and thus to modulate the activity of the hypothalamic-pituitary-adrenocortical (HPA) system. In view of the implication of glucocorticoids in the control of behavior, we examined how acute pharmacological inhibition of MDR1 p-gp at the BBB by tariquidar (XR9576; 12 mg/kg, PO) impacts on the neuroendocrine and behavioral processing of stress in C57BL/6JIcoHim inbred mice. Inhibition of MDR1 p-gp at the BBB did not alter emotional behavior at baseline. However, mice that were sensitized by water-avoidance stress, a mild psychological stressor, displayed significantly reduced anxiety-related behavior in the elevated plus-maze test when treated with tariquidar. Tariquidar, however, had no effect on stress-coping performance assessed in the forced swim test. Investigating the impact of acute MDR1 p-gp inhibition on the glucocorticoid system, we observed a significant attenuation of the mild stress-induced increase of plasma corticosterone after tariquidar administration. In order to examine whether the anti-anxiety effect of tariquidar in sensitized animals is mediated by glucocorticoids, the animals were treated with corticosterone (1 mg/kg, SC immediately after exposure to water-avoidance stress. Corticosterone caused a significant anxiolytic-like effect in this stress-related anxiety protocol, whereas tariquidar could not further enhance corticosterone’s anti-anxiety effects. The current data show for the first time that pharmacological inhibition of MDR1 p-gp at the murine BBB by tariquidar alters emotional behavior and HPA axis activity. By facilitating the entry of corticosterone into the brain, tariquidar enhances feedback inhibition of the HPA system and in this way improves anxiety-related stress processing. These findings highlight a novel approach to the treatment of stress-related affective disorders in humans. PMID:17881135

Thoeringer, Christoph K.; Wultsch, Thomas; Shahbazian, Anaid; Painsipp, Evelin; Holzer, Peter

2015-01-01

330

Inhibition of hTERT Gene Expression by Silibinin-Loaded PLGA-PEG-Fe3O4 in T47D Breast Cancer Cell Line  

PubMed Central

Introduction: Nowadays, using drug delivery is an essential method to improve cancer therapy through decreasing drug toxicity and increasing efficiency of treatment. Silibinin (C25H22O10), a polyphenolic flavonoid which is isolated from the milk thistle plant, has various applications in cancer therapy but it has hydrophobic structure with low water solubility and bioavailability. To increase the effect of silibinin, silibinin-loaded PLGA-PEG-Fe3O4 was prepared to determine the inhibitory effect of this nanodrug on Telomerase gene expression. Methods: The rate of silibinin loaded into PLGA-PEG-Fe3O4 was measured. Then, the cytotoxic effect of silibinin-loaded PLGA-PEG-Fe3O4 was determined by Methyl Thiazol Tetrazolium (MTT) assay. After that, inhibition of Telomerase gene expression was indicated through Real-time PCR. Results: Data analysis from MTT assay showed that silibinin-loaded PLGA-PEG-Fe3O4 had dose dependent cytotoxic effect on T47D cell line. MTT assay showed no cytotoxic effect of free PLGA-PEG-Fe3O4 on T47D breast cancer cell line. Real Time PCR analysis showed that the level of telomerase gene expression more efficiently decreased with silibinin-loaded PLGA-PEG-Fe3O4 than with free silibinin alone. Conclusion: The present study indicates that this nanodrug causes down-regulation of Telomerase gene expression in cancer cells. Therefore, PLGA-PEG-Fe3O4 could be an appropriate carrier for hydrophobic agents such as silibinin to improve their action in cancer therapy. PMID:23878789

Ebrahimnezhad, Zohreh; Zarghami, Nosratollah; Keyhani, Manoutchehr; Amirsaadat, Soumaye; Akbarzadeh, Abolfazl; Rahmati, Mohammad; Mohammad Taheri, Zohreh; Nejati-Koshki, Kazem

2013-01-01

331

Spi-1/PU.1 Is a Positive Regulator of the Fli-1 Gene Involved in Inhibition of Erythroid Differentiation in Friend Erythroleukemic Cell Lines  

PubMed Central

Spi-1/PU.1 and Fli-1 are two members of the ETS family of transcription factors whose expression is deregulated by proviral insertion in most erythroleukemic cell lines induced by the spleen focus-forming virus (SFFV) and Friend murine leukemia virus (F-MuLV) components of the Friend viral complex, respectively. In this study, we present evidence that transcription of the Fli-1 gene is positively regulated by Spi-1/PU.1 in SFFV-transformed cell lines: (i) all SFFV-transformed cell lines expressing Spi-1/PU.1 are characterized by a specific pattern of Fli-1 gene transcripts initiated in the ?200 region instead of position ?400 as reported for F-MuLV-transformed cell lines; (ii) these Fli-1 transcripts initiated in the ?200 region are downregulated in parallel with that of Spi-1/PU.1 during hexamethylenebisacetamide (HMBA) induced differentiation; and (iii) Fli-1 transcription is upregulated in SFFV cells lines following stable transfection of a Spi-1/PU.1 expression vector. Furthermore, we found by transient transfection assays that the ?270/?41 region of the Fli-1 gene displays promoter activity which is transactivated by Spi-1/PU.1. This promoter is strictly dependent on the integrity of two highly conserved ETS DNA binding sites that bind the Spi-1/PU.1 protein in vitro. Finally, we show that transfection of constitutive or inducible Fli-1 expression vectors in SFFV-transformed cells inhibits their erythroid differentiation induced by HMBA. Overall, these data indicate that Fli-1 is a target gene of the Spi-1/PU.1 transcription factor in SFFV-transformed cell lines. We further suggest that deregulated synthesis of Fli-1 may trigger a common mechanism contributing to erythroleukemia induced by either SFFV or F-MuLV. PMID:9858537

Starck, Joëlle; Doubeikovski, Alexandre; Sarrazin, Sandrine; Gonnet, Colette; Rao, Govinda; Skoultchi, Arthur; Godet, Jacqueline; Dusanter-Fourt, Isabelle; Morle, François

1999-01-01

332

Overexpression of D-Xylose Reductase (xyl1) Gene and Antisense Inhibition of D-Xylulokinase (xyiH) Gene Increase Xylitol Production in Trichoderma reesei  

PubMed Central

T. reesei is an efficient cellulase producer and biomass degrader. To improve xylitol production in Trichoderma reesei strains by genetic engineering, two approaches were used in this study. First, the presumptive D-xylulokinase gene in T. reesei (xyiH), which has high homology to known fungi D-xylulokinase genes, was silenced by transformation of T. reesei QM9414 strain with an antisense construct to create strain S6-2-2. The expression of the xyiH gene in the transformed strain S6-2-2 decreased at the mRNA level, and D-xylulokinase activity decreased after 48?h of incubation. This led to an increase in xylitol production from undetectable levels in wild-type T. reesei QM9414 to 8.6?mM in S6-2-2. The T. reesei ?xdh is a xylose dehydrogenase knockout strain with increased xylitol production compared to the wild-type T. reesei QM9414 (22.8?mM versus undetectable). The copy number of the xylose reductase gene (xyl1) in T. reesei ?xdh strain was increased by genetic engineering to create a new strain ?9-5-1. The ?9-5-1 strain showed a higher xyl1 expression and a higher yield of xylose reductase, and xylitol production was increased from 22.8?mM to 24.8?mM. Two novel strains S6-2-2 and ?9-5-1 are capable of producing higher yields of xylitol. T. reesei has great potential in the industrial production of xylitol. PMID:25013760

Hong, Yuanyuan; Dashtban, Mehdi; Kepka, Greg; Chen, Sanfeng; Qin, Wensheng

2014-01-01

333

Onset of lactation in the bovine mammary gland: gene expression profiling indicates a strong inhibition of gene expression in cell proliferation  

Microsoft Academic Search

The mammary gland undergoes dramatic functional and metabolic changes during the transition from late pregnancy to lactation.\\u000a To better understand the molecular events underlying these changes, we analyzed expression profiles of approximately 23,000\\u000a gene transcripts in bovine mammary tissue about day 5 before parturition and day 10 after parturition. At the cutoff criteria\\u000a of the signed fold change ?2 or

Kiera A. Finucane; Thomas B. McFadden; Jeffrey P. Bond; John J. Kennelly; Feng-Qi Zhao

2008-01-01

334

Inhibition of Lipolysis in the Novel Transgenic Quail Model Overexpressing G0/G1 Switch Gene 2 in the Adipose Tissue during Feed Restriction  

PubMed Central

In addition to the issue of obesity in humans, the production of low-fat meat from domestic animals is important in the agricultural industry to satisfy consumer demand. Understanding the regulation of lipolysis in adipose tissue could advance our knowledge to potentially solve both issues. Although the G0/G1 switch gene 2 (G0S2) was recently identified as an inhibitor of adipose triglyceride lipase (ATGL) in vitro, its role in vivo has not been fully clarified. This study was conducted to investigate the role of G0S2 gene in vivo by using two independent transgenic quail lines during different energy conditions. Unexpectedly, G0S2 overexpression had a negligible effect on plasma NEFA concentration, fat cell size and fat pad weight under ad libitum feeding condition when adipose lipolytic activity is minimal. A two-week feed restriction in non-transgenic quail expectedly caused increased plasma NEFA concentration and dramatically reduced fat cell size and fat pad weight. Contrary, G0S2 overexpression under a feed restriction resulted in a significantly less elevation of plasma NEFA concentration and smaller reductions in fat pad weights and fat cell size compared to non-transgenic quail, demonstrating inhibition of lipolysis and resistance to loss of fat by G0S2. Excessive G0S2 inhibits lipolysis in vivo during active lipolytic conditions, such as food restriction and fasting, suggesting G0S2 as a potential target for treatment of obesity. In addition, transgenic quail are novel models for studying lipid metabolism and mechanisms of obesity. PMID:24964090

Shin, Sangsu; Choi, Young Min; Han, Jae Yong; Lee, Kichoon

2014-01-01

335

Expression of tolerance associated gene-1, a mitochondrial protein inhibiting T cell activation, can be used to predict response to immune modulating therapies.  

PubMed

Immune modulating therapies gain increasing importance in treatment of patients with autoimmune diseases such as psoriasis. None of the currently applied biologics achieves significant clinical improvement in all treated patients. Because the therapy with biologics is cost intensive and sometimes associated with side effects, noninvasive diagnostic tools for early prediction of responders are of major interest. We studied the effects of Alefacept (LFA3Ig), an approved drug for treatment of psoriasis, on leukocytes in vitro and in vivo to identify gene markers predictive for treatment response and to further investigate its molecular mechanisms of action. In an open-label study, 20 psoriasis patients were treated weekly with 15 mg Alefacept over 12 wk. We demonstrate that transcription of the tolerance-associated gene (TOAG-1) is significantly up-regulated whereas receptor for hyaluronic acid mediated migration (RHAMM) transcription is down-regulated in PBMCs of responding patients before clinical improvement. TOAG-1 is exclusively localized within mitochondria. Overexpression of TOAG-1 in murine T cells leads to increased susceptibility to apoptosis. Addition of Alefacept to stimulated human T cells in vitro resulted in reduced frequencies of activated CD137(+) cells, increased TOAG-1 but reduced RHAMM expression. This was accompanied by reduced proliferation and enhanced apoptosis. Inhibition of proliferation was dependent on enhanced PDL1 expression of APCs. Thus, peripheral changes of TOAG-1 and RHAMM expression can be used to predict clinical response to Alefacept treatment in psoriasis patients. In the presence of APCs Alefacept can inhibit T cell activation and survival by increasing expression of TOAG-1 on T cells and PDL1 on APCs. PMID:19684086

Keeren, Kathrin; Friedrich, Markus; Gebuhr, Inga; Philipp, Sandra; Sabat, Robert; Sterry, Wolfram; Brandt, Christine; Meisel, Christian; Grütz, Gerald; Volk, Hans-Dieter; Sawitzki, Birgit

2009-09-15

336

Co-Inoculation with Rhizobia and AMF Inhibited Soybean Red Crown Rot: From Field Study to Plant Defense-Related Gene Expression Analysis  

PubMed Central

Background Soybean red crown rot is a major soil-borne disease all over the world, which severely affects soybean production. Efficient and sustainable methods are strongly desired to control the soil-borne diseases. Principal Findings We firstly investigated the disease incidence and index of soybean red crown rot under different phosphorus (P) additions in field and found that the natural inoculation of rhizobia and arbuscular mycorrhizal fungi (AMF) could affect soybean red crown rot, particularly without P addition. Further studies in sand culture experiments showed that inoculation with rhizobia or AMF significantly decreased severity and incidence of soybean red crown rot, especially for co-inoculation with rhizobia and AMF at low P. The root colony forming unit (CFU) decreased over 50% when inoculated by rhizobia and/or AMF at low P. However, P addition only enhanced CFU when inoculated with AMF. Furthermore, root exudates of soybean inoculated with rhizobia and/or AMF significantly inhibited pathogen growth and reproduction. Quantitative RT-PCR results indicated that the transcripts of the most tested pathogen defense-related (PR) genes in roots were significantly increased by rhizobium and/or AMF inoculation. Among them, PR2, PR3, PR4 and PR10 reached the highest level with co-inoculation of rhizobium and AMF. Conclusions Our results indicated that inoculation with rhizobia and AMF could directly inhibit pathogen growth and reproduction, and activate the plant overall defense system through increasing PR gene expressions. Combined with optimal P fertilization, inoculation with rhizobia and AMF could be considered as an efficient method to control soybean red crown rot in acid soils. PMID:22442737

Gao, Xiang; Lu, Xing; Wu, Man; Zhang, Haiyan; Pan, Ruqian; Tian, Jiang; Li, Shuxian; Liao, Hong

2012-01-01

337

RNA Interference-Mediated Knockdown of Astrocyte Elevated Gene-1 Inhibits Growth, Induces Apoptosis, and Increases the Chemosensitivity to 5-Fluorouracil in Renal Cancer Caki-1 Cells  

PubMed Central

Astrocyte elevated gene-1 (AEG-1) is a recently discovered oncogene that has been reported to be highly expressed in various types of malignant tumors, including renal cell carcinoma. However, the precise role of AEG-1 in renal cancer cell proliferation and apoptosis has not been clarified. In this study, we transfected the renal cancer cell line Caki-1 with a plasmid expressing AEG-1 short hairpin RNA (shRNA) and obtained cell colonies with stable knockdown of AEG-1. We found that AEG-1 down-regulation inhibited cell proliferation and colony formation and arrested cell cycle progression at the sub-G1 and G0/G1 phase. Western blot analysis indicated that the expression of proliferating cell nuclear antigen (PCNA), cyclin D1 and cyclin E were significantly reduced following AEG-1 down-regulation. In addition, AEG-1 knockdown led to the appearance of apoptotic bodies in renal cancer cells, and the ratio of apoptotic cells significantly increased. Expression of the anti-apoptotic factor Bcl-2 was dramatically reduced, whereas the pro-apoptotic factors Bax, caspase-3 and poly (ADP-ribose) polymerase (PARP) were significantly activated. Finally, AEG-1 knockdown in Caki-1 cells remarkably suppressed cell proliferation and enhanced cell apoptosis in response to 5-fluorouracil (5-FU) treatment, suggesting that AEG-1 inhibition sensitizes Caki-1 cells to 5-FU. Taken together, our data suggest that AEG-1 plays an important role in renal cancer formation and development and may be a potential target for future gene therapy for renal cell carcinoma. PMID:25431427

Wang, Peng; Yin, Bo; Shan, Liping; Zhang, Hui; Cui, Jun; Zhang, Mo; Song, Yongsheng

2014-01-01

338

Genetic evidence that the yopA gene-encoded Yersinia outer membrane protein Yop1 mediates inhibition of the anti-invasive effect of interferon.  

PubMed Central

HEp-2 cell monolayers were challenged with genetic variants of Yersinia pseudotuberculosis YPIII(pIB1) and Yersinia enterocolitica W22708(pYL4). Both strains were represented by (i) variants harboring the 70-kilobase virulence plasmid, (ii) their isogenic plasmid-cured derivatives, and (iii) two transposon mutants constructed by insertional inactivation of the plasmid genes encoding outer membrane protein Yop1 and Ca2+ dependency in strains YPIII(pIB1) and W22708(pYL4). When the HEp-2 cells were pretreated with recombinant alpha interferon subtype A, all invasive variants of Y. enterocolitica and Y. pseudotuberculosis, except those variants which expressed Yop1, showed a significantly reduced ability to localize intracellularly. The anti-invasive effect of interferon was abolished when the gene was expressed or when a sterile filtered sonic extract of a Yop1-producing strain was added to the cell cultures. To obtain further evidence of a potential role of Yop1, a DNA fragment encoding Yop1 cloned into the vector pBR322 was used. After introduction of the resultant recombinant plasmid pYMS2 into the plasmid-cured variant YPIII and the Yop1-negative transposon mutant YPIII(pIB102) of Y. pseudotuberculosis, both transformants regained the ability to produce Yop1 and showed complete inhibition of the interferon effect. Moreover, the sterile sonic extract of an Escherichia coli strain, which carried pYMS2, neutralized the anti-invasive effect of interferon. The results provide direct genetic evidence that Yop1 mediates inhibition of the anti-invasive effect of interferon in HEp-2 cell cultures. The results also demonstrated that Yop1 itself reduces the ability of Yersinia spp. to localize intracellularly in HEp-2 cells. Images PMID:2194966

Bukholm, G; Kapperud, G; Skurnik, M

1990-01-01

339

Recombinant lentivirus targeting the pleotrophin gene reduces pleotrophin protein expression in pancreatic cancer cells and inhibits neurite outgrowth of dorsal root ganglion neurons.  

PubMed

The objectives of the present study were to construct the recombinant primate lentivirus?short hairpin RNA-pleiotrophin (pLV-shRNA-PTN) vector, to investigate the silencing effect of pLV-shRNA-PTN on PTN expression in MIA PaCa-2 cells and to observe the inhibition efficiency of pLV-shRNA?PTN on neurite outgrowth from dorsal root ganglion (DRG) neurons in vitro. The construction procedure for recombinant lentivirus pLV-shRNA-PTN has been described previously. In the present study, pLV-shRNA?PTN was used to infect MIA PaCa-2 pancreatic cancer cells and the efficiency of the knockdown of the PTN gene on day 7 following infection was analyzed using western blotting. The morphological changes in the cultured DRG neurons were observed by monoculture of DRG neurons and co-culture with MIA PaCa-2 cells in vitro. The recombinant lentivirus pLV-shRNA?PTN was successfully constructed. The western blot analysis showed that the inhibition rates of PTN expression were 46, 80, 20 and 21%, respectively, following pLV-shRNA?PTN-A, B, C and D infection. pLV-shRNA-PTN?B showed the highest knockdown efficiency. DRG neurons co-cultured with infected MIA PaCa-2 cells were decreased in size when compared with the control, and there was a significant decrease in the number and length of neurites. The results suggest that efficient and specific knockdown of PTN in MIA PaCa-2 pancreatic cancer cells and the subsequent reduction in PTN expression results in the inhibition of neurite outgrowth from DRG neurons. PMID:24469464

Yao, Jun; Li, Wen-Yao; Li, Shuo-Guo; Feng, Xiao-Shan; Gao, She-Gan

2014-03-01

340

Role of the nuclear receptors HNF4 alpha, PPAR alpha, and LXRs in the TNF alpha-mediated inhibition of human apolipoprotein A-I gene expression in HepG2 cells.  

PubMed

The expression of the apolipoprotein A-I gene (apoA-I) in hepatocytes is repressed by pro-inflammatory cytokines such as IL-1beta and TNFalpha. In this work, we have demonstrated that treatment of HepG2 human hepatoma cells with chemical inhibitors for JNK, p38 protein kinases, and NFkappaB transcription factor abolishes the TNFalpha-mediated inhibition of human apoA-I gene expression in HepG2 cells. In addition, we have shown that TNFalpha decreases also the rate of secretion of apoA-I protein by HepG2 cells, and this effect depends on JNK and p38, but not on NFkappaB and MEK1/2 signaling pathways. The inhibitory effect of TNFalpha has been found to be mediated by the hepatic enhancer of the apoA-I gene. The decrease in the level of human apoA-I gene expression under the impact of TNFalpha appears to be partly mediated by the inhibition of HNF4alpha and PPARalpha gene expression. Treatment of HepG2 cells with PPARalpha antagonist (MK886) or LXR agonist (TO901317) abolishes the TNFalpha-mediated decrease in the level of apoA-I gene expression. PPARalpha agonist (WY-14643) abolishes the negative effect of TNFalpha on apoA-I gene expression in the case of simultaneous inhibition of MEK1/2, although neither inhibition of MEK1/2 nor addition of WY-14643 leads to the blocking of the TNFalpha-mediated decrease in the level of apoA-I gene expression individually. The ligand-dependent regulation of apoA-I gene expression by PPARalpha appears to be affected by the TNFalpha-mediated activation of MEK1/2 kinases, probably through PPARalpha phosphorylation. Treatment of HepG2 cells with PPARalpha and LXR synthetic agonists also blocks the inhibition of apoA-I protein secretion in HepG2 cells under the impact of TNFalpha. A chromatin immunoprecipitation assay demonstrates that TNFalpha leads to a 2-fold decrease in the level of PPARalpha binding with the apoA-I gene hepatic enhancer. At the same time, the level of LXRbeta binding with the apoA-I gene hepatic enhancer is increased 3-fold under the impact of TNFalpha. These results suggest that nuclear receptors HNF4alpha, PPARalpha, and LXRs are involved in the TNFalpha-mediated downregulation of human apoA-I gene expression and apoA-I protein secretion in HepG2 cells. PMID:19883121

Mogilenko, Denis A; Dizhe, Ella B; Shavva, Vladimir S; Lapikov, Ivan A; Orlov, Sergey V; Perevozchikov, Andrey P

2009-12-22

341

Overexpression of N?myc downstream?regulated gene 1 inhibits human glioma proliferation and invasion via phosphoinositide 3?kinase/AKT pathways.  

PubMed

N?myc downstream?regulated gene 1 (NDRG1) was previously shown to exhibit low expression in glioma tissue as compared with that in normal brain tissue; however, the role of NDRG1 in human glioma cells has remained to be elucidated. The present study used the U87 MG and SHG?44 human glioma cell lines as well as the normal human astrocyte cell line 1800, which are known to have differential NDRG1 expression. Small interfering (si)RNA targeting NDRG1, and NDRG1 overexpression vectors were transfected into the SHG?44 and U87 MG glioma cells, respectively. Cell proliferation, invasion, apoptosis and cell cycle arrest were subsequently examined by MTT assay, transwell chamber assay, flow cytometry and western blot analysis, respectively. Furthermore, a subcutaneous tumor mouse model was used to investigate the effects of NDRG1 on the growth of glioma cells in vivo. Overexpression of NDRG1 was shown to inhibit cell proliferation and invasion, and induce apoptosis in the U87 MG glioma cells, whereas NDRG1 downregulation increased proliferation, suppressed apoptosis and promoted invasion of the SHG?44 glioma cells. In addition, in the subcutaneous tumor mouse model, overexpression of NDRG1 in U?87 MG cells suppressed tumorigenicity in vivo. The findings of the present study indicated that NDRG1 is required for the inhibition of gliomagenesis; therefore, targeting NDRG1 and its downstream targets may represent novel therapies for the treatment of glioma. PMID:25777142

Ma, Wei; Na, Meng; Tang, Chongyang; Wang, Haiyang; Lin, Zhiguo

2015-07-01

342

A potent inhibition of oxidative stress induced gene expression in neural cells by sustained ferulic acid release from chitosan based hydrogel.  

PubMed

Traumatic brain injury (TBI) is an extremely cataclysmic neurological disorder and the inhibition of oxidative stress following TBI could effectively protect the brain from further impairments. An injectable thermosensitive chitosan/gelatin/?-Glycerol phosphate (C/G/GP) hydrogel for the controlled release of the phenolic antioxidant ferulic acid (FA) to inhibit the neurological oxidative stress was demonstrated. The C/G/GP hydrogel ensures an excellent clinical expediency with a gelation temperature of 32.6°C and gelation time of 75.58s. In-vitro cytotoxicity assays of C/G/GP hydrogel and FA have revealed an excellent biocompatibility with the Neuro-2a cells. 500?M of FA was considered to be an effective concentration to reduce the oxidative stress in Neuro-2a cells. TUNEL staining images evidenced that the H2O2 induced DNA fragmentation was comprehensively controlled after FA treatment. The mRNA gene expression profiles markedly authenticate the neuroprotectivity of FA by down-regulating ROS, inflammatory and apoptosis related markers. The outcomes of this study suggest that, C/G/GP hydrogel carrying ferulic acid could effectively protect further secondary traumatic brain injury associated impairments. PMID:25686998

Dong, Guo-Chung; Kuan, Che-Yung; Subramaniam, Sadhasivam; Zhao, Jiong-Yao; Sivasubramaniam, Savitha; Chang, Hwan-You; Lin, Feng-Huei

2015-04-01

343

Inhibition of HCV 5?-NTR and Core Expression by a Small Hairpin RNA Delivered by a Histone Gene Carrier, HPhA  

PubMed Central

siRNA (small interfering RNA) interference represents an exciting new technology that could have therapeutic applications for the treatment of viral infections. However, a major challenge in the use of siRNA as a therapeutic agent is the development of a suitable delivery system. We demonstrated that a new non-viral transgene carrier, recombinant archaeal histone from the hyperthermophile Pyrococcus horikoshii OT3 (HPhA), can transfect short hairpin RNA (shRNA) expressing plasmids into HL-7702 cells to inhibit the expression of HCV 5'NTR and Core protein and mRNA. Plasmids Psilencirle transfected by HPhA inhibited the expression of HCV 5'-NTR and Core protein and mRNA in HL-7702 cells. The transfection efficiency of HPhA in HL-7702 cells was not affected by 10% fetal calf serum (FCS). HPhA exhibited effects of transfection without apparent toxicity, and with high affinity for DNA. This suggests that HPhA may be useful for RNAi-based gene therapy in vivo. PMID:23801881

Ding, Yanhua; Zhang, Hong; Li, Yuxiang; Wu, Di; He, Shumei; Wang, Yang; Li, Yuanyuan; Wang, Feng; Niu, Junqi

2013-01-01

344

Tea Polyphenol ()-Epigallocatechin-3Gallate Inhibits DNA Methyltransferase and Reactivates Methylation-Silenced Genes in Cancer Cell Lines  

Microsoft Academic Search

Hypermethylation of CpG islands in the promoter regions is an impor- tant mechanism to silence the expression of many important genes in cancer. The hypermethylation status is passed to the daughter cells through the methylation of the newly synthesized DNA strand by 5-cyto- sine DNA methyltransferase (DNMT). We report herein that ()-epigal- locatechin-3-gallate (EGCG), the major polyphenol from green tea,

Ming Zhu Fang; Yimin Wang; Ni Ai; Zhe Hou; Yi Sun; Hong Lu; William Welsh; Chung S. Yang

2003-01-01

345

Inhibition of Gene Expression and Growth by Antisense Peptide Nucleic Acids in a Multiresistant  -Lactamase-Producing Klebsiella pneumoniae Strain  

Microsoft Academic Search

Klebsiella pneumoniae causes common and severe hospital- and community-acquired infections with a high incidence of multidrug resistance. The emergence and spread of -lactamase-producing K. pneumoniae strains highlight the need to develop new therapeutic strategies. In this study, we developed antisense peptide nucleic acids (PNAs) conjugated to the (KFF)3K peptide and investigated whether they could mediate gene-specific antisense effects in K.

Prathiba Kurupati; K. S. W. Tan; G. Kumarasinghe; C. L. Poh

2007-01-01

346

Saccharomyces boulardii produces a soluble anti-inflammatory factor that inhibits NF-?B-mediated IL8 gene expression  

Microsoft Academic Search

Saccharomyces boulardii (Sb) is a non-pathogenic yeast that ameliorates intestinal injury and inflammation caused by a wide variety of enteric pathogens. We hypothesized that Sb may exert its probiotic effects by modulation of host cell signaling and pro-inflammatory gene expression. Human HT-29 colonocytes and THP-1 monocytes were stimulated with IL-1?, TNF? or LPS in the presence or absence of Sb

Stavros Sougioultzis; Simos Simeonidis; K. Ramakrishnan Bhaskar; Xinhua Chen; Pauline M. Anton; Sarah Keates; Charalabos Pothoulakis; Ciarán P. Kelly

2006-01-01

347

Progesterone Inhibits basal and gonadotropin-releasing hormone induction of luteinizing hormone beta-subunit gene expression.  

PubMed

LH and FSH play critical roles in mammalian reproduction by mediating steroidogenesis and gametogenesis in the gonad. Gonadal steroid hormone feedback to the hypothalamus and pituitary influences production of the gonadotropins. We previously demonstrated that progesterone differentially regulates the expression of the LH and FSH beta-subunits at the level of the gonadotrope: FSHbeta transcription is induced, whereas LHbeta is repressed. In this study, we investigated the mechanism of progesterone repression of LHbeta gene expression using immortalized gonadotrope-derived LbetaT2 cells. The progesterone suppression of both basal and GnRH-induced LHbeta gene expression occurs in a hormone- and receptor-dependent manner. Chromatin immunoprecipitation demonstrates that the hormone-bound progesterone receptor (PR) is recruited to the endogenous mouse LHbeta promoter. In addition, suppression requires both the amino-terminal and DNA-binding regions of PR. Furthermore, progesterone suppression does not require direct PR binding to the promoter, and, thus, PR is likely recruited to the promoter via indirect binding through other transcription factors. These data demonstrate that the molecular mechanism for progesterone action on the LHbeta promoter is distinct from FSHbeta, which involves direct PR binding to the promoter to produce activation. It also differs from androgen repression of LHbeta gene expression in that, rather than Sp1 or steroidogenic factor-1 elements, it requires elements within -300/-250 and -200/-150 that also contribute to basal expression of the LHbeta promoter. Altogether, our data indicate that progesterone feedback at the level of the pituitary gonadotrope is likely to play a key role in differential production of the gonadotropin genes. PMID:19106225

Thackray, Varykina G; Hunnicutt, Jennifer L; Memon, Aisha K; Ghochani, Yasmin; Mellon, Pamela L

2009-05-01

348

Isoproterenol inhibits resistin gene expression through a G S-protein-coupled pathway in 3T3-L1 adipocytes  

Microsoft Academic Search

Resistin was recently identified as a hormone secreted by adipocytes which leads to insulin resistance in vivo and in vitro and might therefore be an important link between obesity and diabetes. To clarify the regulation of resistin gene expression, 3T3-L1 adipocytes were treated with various agents known to modulate insulin sensitivity, and resistin mRNA was measured by quantitative real-time reverse

Mathias Fasshauer; Johannes Klein; Susanne Neumann; Markus Eszlinger; Ralf Paschke

2001-01-01

349

High Temperature Inhibits Ascorbate Recycling and Light Stimulation of the Ascorbate Pool in Tomato despite Increased Expression of Biosynthesis Genes  

PubMed Central

Understanding how the fruit microclimate affects ascorbate (AsA) biosynthesis, oxidation and recycling is a great challenge in improving fruit nutritional quality. For this purpose, tomatoes at breaker stage were harvested and placed in controlled environment conditions at different temperatures (12, 17, 23, 27 and 31°C) and irradiance regimes (darkness or 150 µmol m-2 s-1). Fruit pericarp tissue was used to assay ascorbate, glutathione, enzymes related to oxidative stress and the AsA/glutathione cycle and follow the expression of genes coding for 5 enzymes of the AsA biosynthesis pathway (GME, VTC2, GPP, L-GalDH, GLDH). The AsA pool size in pericarp tissue was significantly higher under light at temperatures below 27°C. In addition, light promoted glutathione accumulation at low and high temperatures. At 12°C, increased AsA content was correlated with the enhanced expression of all genes of the biosynthesis pathway studied, combined with higher DHAR and MDHAR activities and increased enzymatic activities related to oxidative stress (CAT and APX). In contrast, at 31°C, MDHAR and GR activities were significantly reduced under light indicating that enzymes of the AsA/glutathione cycle may limit AsA recycling and pool size in fruit pericarp, despite enhanced expression of genes coding for AsA biosynthesis enzymes. In conclusion, this study confirms the important role of fruit microclimate in the regulation of fruit pericarp AsA content, as under oxidative conditions (12°C, light) total fruit pericarp AsA content increased up to 71%. Moreover, it reveals that light and temperature interact to regulate both AsA biosynthesis gene expression in tomato fruits and AsA oxidation and recycling. PMID:24367665

Massot, Capucine; Bancel, Doriane; Lopez Lauri, Félicie; Truffault, Vincent; Baldet, Pierre; Stevens, Rebecca; Gautier, Hélčne

2013-01-01

350

Antisense Oligonucleotides Specific to Mutated K-ras Genes Inhibit Invasiveness of Human Pancreatic Cancer Cell Lines  

Microsoft Academic Search

Background\\/Aims: Point mutations of the K-ras gene are detected in >90% of human pancreatic cancers and may play an important role in tumorigenesis. However, correlations between mutant K-ras and the invasive activity of the tumor have remained unclarified. Methods: 17-mer phosphorothioate antisense oligonucleotides targeting K-ras point mutations were transfected into three kinds of human pancreatic cancer cell lines (MIAPaCa-2, PANC-1,

Yuji Nakada; Seiji Saito; Kouji Ohzawa; Cintia Yoko Morioka; Kei-ichiro Kita; Masami Minemura; Terumi Takahara; Akiharu Watanabe

2001-01-01

351

Inhibition of inflammatory gene expression in keratinocytes using a composition containing carnitine, thioctic Acid and saw palmetto extract.  

PubMed

Chronic inflammation of the hair follicle (HF) is considered a contributing factor in the pathogenesis of androgenetic alopecia (AGA). Previously, we clinically tested liposterolic extract of Serenoa repens (LSESr) and its glycoside, ?-sitosterol, in subjects with AGA and showed a highly positive response to treatment. In this study, we sought to determine whether blockade of inflammation using a composition containing LSESr as well as two anti-inflammatory agents (carnitine and thioctic acid) could alter the expression of molecular markers of inflammation in a well-established in vitro system. Using a well-validated assay representative of HF keratinocytes, specifically, stimulation of cultured human keratinocyte cells in vitro, we measured changes in gene expression of a spectrum of well-known inflammatory markers. Lipopolysaccharide (LPS) provided an inflammatory stimulus. In particular, we found that the composition effectively suppressed LPS-activated gene expression of chemokines, including CCL17, CXCL6 and LTB(4) associated with pathways involved in inflammation and apoptosis. Our data support the hypothesis that the test compound exhibits anti-inflammatory characteristics in a well-established in vitro assay representing HF keratinocyte gene expression. These findings suggest that 5-alpha reductase inhibitors combined with blockade of inflammatory processes could represent a novel two-pronged approach in the treatment of AGA with improved efficacy over current modalities. PMID:19692448

Chittur, Sridar; Parr, Brian; Marcovici, Geno

2011-01-01

352

Inhibition of Inflammatory Gene Expression in Keratinocytes Using a Composition Containing Carnitine, Thioctic Acid and Saw Palmetto Extract  

PubMed Central

Chronic inflammation of the hair follicle (HF) is considered a contributing factor in the pathogenesis of androgenetic alopecia (AGA). Previously, we clinically tested liposterolic extract of Serenoa repens (LSESr) and its glycoside, ?-sitosterol, in subjects with AGA and showed a highly positive response to treatment. In this study, we sought to determine whether blockade of inflammation using a composition containing LSESr as well as two anti-inflammatory agents (carnitine and thioctic acid) could alter the expression of molecular markers of inflammation in a well-established in vitro system. Using a well-validated assay representative of HF keratinocytes, specifically, stimulation of cultured human keratinocyte cells in vitro, we measured changes in gene expression of a spectrum of well-known inflammatory markers. Lipopolysaccharide (LPS) provided an inflammatory stimulus. In particular, we found that the composition effectively suppressed LPS-activated gene expression of chemokines, including CCL17, CXCL6 and LTB(4) associated with pathways involved in inflammation and apoptosis. Our data support the hypothesis that the test compound exhibits anti-inflammatory characteristics in a well-established in vitro assay representing HF keratinocyte gene expression. These findings suggest that 5-alpha reductase inhibitors combined with blockade of inflammatory processes could represent a novel two-pronged approach in the treatment of AGA with improved efficacy over current modalities. PMID:19692448

Chittur, Sridar; Parr, Brian; Marcovici, Geno

2011-01-01

353

Molecular cloning and functional analysis of three genes encoding polygalacturonase-inhibiting proteins from Capsicum annuum, and their relation to increased resistance to two fungal pathogens  

Technology Transfer Automated Retrieval System (TEKTRAN)

Polygalacturonase-inhibiting proteins (PGIPs) are plant cell wall glycoproteins that can inhibit fungal endopolygalacturonases (PGs). Inhibiting by PGIPs directly reduces potential PG activity in specific plant pathogenic fungi, reducing their aggressiveness. Here, we isolated and functionally chara...

354

New-generation taxoid SB-T-1214 inhibits stem cell-related gene expression in 3D cancer spheroids induced by purified colon tumor-initiating cells  

PubMed Central

Background Growing evidence suggests that the majority of tumors are organized hierarchically, comprising a population of tumor-initiating, or cancer stem cells (CSCs) responsible for tumor development, maintenance and resistance to drugs. Previously we have shown that the CD133high/CD44high fraction of colon cancer cells is different from their bulk counterparts at the functional, morphological and genomic levels. In contrast to the majority of colon cancer cells expressing moderate levels of CD133, CD44 and CD166, cells with a high combined expression of CD133 and CD44 possessed several characteristic stem cell features, including profound self-renewal capacity in vivo and in vitro, and the ability to give rise to different cell phenotypes. The present study was undertaken for two aims: a) to determine stem cell-related genomic characteristics of floating 3D multicellular spheroids induced by CD133high/CD44high colon cancer cells; and b) to evaluate CSC-specific alterations induced by new-generation taxoid SB-T-1214. Results Selected CSC phenotype was isolated from three independent invasive colon cancer cell lines, HCT116, HT29 and DLD-1. A stem cell-specific PCR array assay (SABiosciences) revealed that colonospheres induced by purified CD133high/CD44high expressing cells display profound up-regulation of stem cell-related genes in comparison with their bulk counterparts. The FACS analysis has shown that the 3D colonospheres contained some minority cell populations with high levels of expression of Oct4, Sox2, Nanog and c-Myc, which are essential for stem cell pluripotency and self-renewal. Single administration of the SB-T-1214 at concentration 100 nM-1 ?M for 48 hr not only induced growth inhibition and apoptotic cell death in these three types of colon cancer spheroids in 3D culture, but also mediated massive inhibition of the stem cell-related genes and significant down-regulation of the pluripotency gene expression. PCR array and FACS data were confirmed with western blotting. Importantly, viable cells that survived this treatment regimen were no longer able to induce secondary floating spheroids and exhibited significant morphological abnormalities. Conclusions We report here that a new-generation taxoid SB-T-1214 possesses significant activity against colon cancer spheroids induced by and enriched with drug resistant tumorigenic CD133high/CD44high cells and efficiently inhibited expression of the majority of stem cell-related genes. Our data indicates that the previously observed long-term efficacy of SB-T-1214 against drug resistant colon tumors in vivo may be explained by the down-regulation of multiple stem cell-related genes in the tumorigenic cell population, in addition to its known efficacy as a mitotic poison against proliferating cancer cells. PMID:20630067

2010-01-01

355

Genes  

NSDL National Science Digital Library

Illustration of the placement of genes in a chromosome. A gene can be defined as a region of DNA that controls a hereditary characteristic. It usually corresponds to a sequence used in the production of a specific protein or RNA. A gene carries biological information in a form that must be copied and transmitted from each cell to all its progeny. This includes the entire functional unit: coding DNA sequences, non-coding regulatory DNA sequences, and introns. Genes can be as short as 1000 base pairs or as long as several hundred thousand base pairs. It can even be carried by more than one chromosome. The estimate for the number of genes in humans has decreased as our knowledge has increased. As of 2001, humans are thought to have between 30,000 and 40,000 genes.

BEGIN:VCARD VERSION:2.1 FN:Access Excellence N:Excellence; Access REV:2005-03-12 END:VCARD

2005-03-12

356

IgG immune complexes inhibit IFN-gamma-induced transcription of the Fc gamma RI gene in human monocytes by preventing the tyrosine phosphorylation of the p91 (Stat1) transcription factor.  

PubMed

Immune complexes (IC) modulate Ag-driven immune responses in part by their ability to inhibit IFN-gamma-dependent MHC class II expression. Because many genes, including MHC class II Ags, transcriptionally activated by IFN-gamma require the tyrosine phosphorylation of the transcription factor p91 (Stat1), we examined whether IC could suppress IFN-gamma-induced expression of the Fc gamma receptor I gene (Fc gamma RI) in human monocytes and whether this occurred through inhibition of p91 phosphorylation. Preincubation of monocytes on gamma-globulin-coated dishes resulted in a 80% reduction in steady state levels of RNA for the Fc gamma RI gene. Nuclear run-on analysis confirmed that the inhibition was at the level of transcription. Treatment with IC resulted in no change in the IFN-gamma receptor number. In monocytes pretreated with IC, there was a 79% reduction in the formation of FcRF gamma, a p91-containing DNA binding protein complex that is rapidly activated by IFN-gamma, and which recognizes the gamma response region enhancer within the promoter of the Fc gamma RI gene. Furthermore, there was a marked reduction in the tyrosine phosphorylation of p91. Pretreatment with IC resulted in the inhibition of the tyrosine phosphorylation of the tyrosine kinases, Jak1 and Jak2, both of which are involved in IFN-gamma signal transduction. Therefore, culture of monocytes on IC inhibits IFN-gamma-induced expression of the Fc gamma RI gene by preventing tyrosine phosphorylation of p91, probably by the associated inhibition of the tyrosine kinases Jak1 and Jak2. PMID:7995951

Feldman, G M; Chuang, E J; Finbloom, D S

1995-01-01

357

Antisense inhibition of ATM gene enhances the radiosensitivity of head and neck squamous cell carcinoma in mice  

PubMed Central

Background Treatment failure after radiotherapy of head and neck squamous cell carcinoma (HNSCC) could be a significant problem. Our objective is to sensitize SCCVII cells to ionizing radiation in vitro and in vivo through inhibiting ATM expression using antisense oligodeoxynucleotides (AS-ODNs), and investigate the potential mechanism of radiosensitization. Methods We designed and synthesized AS-ODNs that target ATM mRNA to reduce the ATM expression. The influence on the expression of ATM mRNA and protein in SCCVII cells were analysed by real-time quantitative PCR and western blotting respectively. Clonogenic survival assay was performed to detect the survival ability of SCCVII cells after irradiation, while flow cytometry used to analyse the cell cycle and apoptosis. The volume of solid tumors generated with SCCVII cells was measured, and cell apoptosis was analysed by TUNEL assay after irradiation. Results The relative ATM mRNA and protein expression in SCCVII cells treated with ATM AS-ODNs were decreased to 25.7 ± 3.1% and 24.1 ± 2.8% of that in untreated cells respectively (P < 0.05). After irradiation, the survival fraction (SF) of cells treated with ATM AS-ODNs was lower than that of other groups at the same dose of radiation (P < 0.05), while the percentage of cells in G2/M phase decreased and apoptotic rate of cells increased(P < 0.05). The inhibition rate in SCCVII cells solid tumor exposed to X-ray alone was 23.2 ± 2.7%, while it was 56.1 ± 3.8% in the group which irradiated in combination with the treatment of ATM AS-ODNs (P < 0.05). The apoptotic index for the group irradiated in combination with ATM AS-ODNs injection was 19.6 ± 3.2, which was significantly higher than that of others (P < 0.05) Conclusion Inhibition of ATM expression sensitized SCCVII cells to ionizing radiation in vitro and in vivo. The potential mechanism should be the defective G2/M cell cycle checkpoint control and enhanced radiation-induced apoptosis. PMID:18950535

Zou, Jian; Qiao, Xiaoming; Ye, Huiping; Yang, Yuqiong; Zheng, Xuelian; Zhao, Houyu; Liu, Shixi

2008-01-01

358

Detection of the genetic variation of polygalacturonase-inhibiting protein gene 2 in autotetraploid alfalfa (Medicago sativa) using an improved SSCP technique.  

PubMed

In this study, 2 approaches were adopted to obtain good single-strand conformation polymorphism (SSCP) data for autotetraploid alfalfa; primers were added to PCR products, and fluorescent-labeled primers were utilized. PCR-SSCP conditions for a 331-bp fragment in the coding region of polygalacturonase-inhibiting protein gene 2 in alfalfa (MsPGIP2) were optimized, and the results showed that the best SSCP gel pattern could be obtained when the loading mixture was made by mixing 1 ?L PCR products, 0.2 to 0.8 ?L unlabeled primers (50 ?M) and 4 to 16 ?L loading buffer. Furthermore, the use of the fluorescent-labeled primers resulted in 2 separated electrophoresis images from 2 complementary single DNA strands, thus making the determination of alleles and idiotypes a relatively easy task. In addition, the results of sequencing prove that the determination of alleles and idiotypes were accurate based on SSCP analysis. Finally, a total of 9 alleles with 18 SNP sites were identified for MsPGIP2 in the alfalfa variety 'Algonquin'. In conclusion, MsPGIP2 possessed great genetic variation, and the addition of primers to the PCR products in combination with the fluorescent labeling of primers could significantly improve the sensitivity and resolution of SSCP analysis. This technique could be used for genetic diversity detection and marker-assisted breeding of useful genes in autopolyploid species such as alfalfa. PMID:25501230

Gui, Z; Liu, H Q; Wang, Y; Yuan, Q H; Xin, N; Zhang, X; Li, X L; Pi, Y S; Gao, J M

2014-01-01

359

Engineered external guide sequences are highly effective in inducing RNase P for inhibition of gene expression and replication of human cytomegalovirus  

PubMed Central

External guide sequences (EGSs), which are RNA molecules derived from natural tRNAs, bind to a target mRNA and render the mRNA susceptible to hydrolysis by RNase P, a tRNA processing enzyme. Using an in vitro selection procedure, we have previously generated EGS variants that efficiently direct human RNase P to cleave a target mRNA in vitro. In this study, a variant was used to target the overlapping region of the mRNAs encoding human cytomegalovirus (HCMV) essential transcription regulatory factors IE1 and IE2. The EGS variant was ?25-fold more active in inducing human RNase P to cleave the mRNA in vitro than the EGS derived from a natural tRNA. Moreover, a reduction of 93% in IE1/IE2 gene expression and a reduction of 3000-fold in viral growth were observed in HCMV-infected cells that expressed the variant, while cells expressing the tRNA-derived EGS exhibited a reduction of 80% in IE1/IE2 expression and an inhibition of 150-fold in viral growth. Our results provide the first direct evidence that EGS variant is highly effective in blocking HCMV gene expression and growth and furthermore, demonstrate the feasibility of developing effective EGS RNA variants for anti-HCMV applications by using in vitro selection procedures. PMID:16432261

Yang, Yong-Hua; Li, Hongjian; Zhou, Tianhong; Kim, Kihoon; Liu, Fenyong

2006-01-01

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Adenovirus-mediated gene transfer of endostatin in vivo results in high level of transgene expression and inhibition of tumor growth and metastases  

NASA Astrophysics Data System (ADS)

Inhibition of angiogenesis has been shown to be an effective strategy in cancer therapy in mice. However, its widespread application has been hampered by difficulties in the large-scale production of the antiangiogenic proteins. This limitation may be resolved by in vivo delivery and expression of the antiangiogenic genes. We have constructed a recombinant adenovirus that expresses murine endostatin that is biologically active both in vitro, as determined in endothelial cell proliferation assays, and in vivo, by suppression of angiogenesis induced by vascular endothelial growth factor 165. Persistent high serum levels of endostatin (605-1740 ng/ml; mean, 936 ng/ml) were achieved after systemic administration of the vector to nude mice, which resulted in significant reduction of the growth rates and the volumes of JC breast carcinoma and Lewis lung carcinoma (P < 0.001 and P < 0.05, respectively). In addition, the endostatin vector treatment completely prevented the formation of pulmonary micrometastases in Lewis lung carcinoma (P = 0.0001). Immunohistochemical staining of the tumors demonstrated a decreased number of blood vessels in the treatment group versus the controls. In conclusion, the present study clearly demonstrates the potential of vector-mediated antiangiogenic gene therapy as a component in cancer therapy.

Sauter, Bernhard V.; Martinet, Olivier; Zhang, Wei-Jian; Mandeli, John; Woo, Savio L. C.

2000-04-01