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Leandro Nieto, Mariana Fuertes, Josefina Rosmino, Sergio Senin and Eduardo Arzt

inhibition. Other genes, under the control of RA and BMP-4 might be under the regulation of similar RA receptors/SMAD crosstalk mechanisms. It has been observed that COUP-TFI expression in the normal pituitary is the reason why RA does not act on these

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Gregory S Y Ong and Morag J Young

overlap in GR- and MR-regulated genes in the heart ( Latouche et al . 2010 ). Novel genes identified in these experiments require further investigation to establish the mechanism and functional outcomes of their regulation by MR. Rapid signalling

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S Baron, M Manin, C Aigueperse, M Berger, C Jean, G Veyssiere and L Morel

The akr1b7 gene encodes an aldose reductase-like protein that is responsible for detoxifying isocaproaldehyde generated by the conversion of cholesterol to pregnenolone. The regulation of gene expression by human chorionic gonadotropin (hCG) was first investigated in the MA-10 Leydig tumor cell line. The akr1b7 gene was constitutively expressed and accumulation of its mRNA was increased in a dose- and time-dependent manner by treatment with hCG. akr1b7 mRNA accumulation was sharply increased in the presence of 0.25 nM hCG and it reached a fivefold increase within 2 h. AKR1B7 protein accumulation was delayed compared with that of the corresponding mRNA. In agreement, hCG significantly increased the levels of mRNA and protein of akr1b7 in primary cultures of adult mouse Leydig cells, thus suggesting that LH potentially regulates akr1b7 gene expression in vivo. Expression of akr1b7 was developmentally regulated in the testis. Unexpectedly, levels of akr1b7 mRNA increased from embryonic day 15 to the day of birth and declined until adulthood while AKR1B7 protein levels followed an inverse pattern, suggesting an important role for translational mechanisms.

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YL Hsieh, A Chatterjee, JT Chien and JY Yu

The cDNAs encoding pituitary glycoprotein hormone alpha subunits (PGHalphas) of two species of duck (Muscovy duck, Cairina moschata and Pekin duck, Anas platyrhynchos domesticus) were cloned and sequenced to better understand the phylogenic diversity and evolution of PGHalpha molecules in vertebrates. Oligonucleotide primers were designed and used for reverse transcription PCR (RT-PCR) amplification of PGHalpha cDNA fragments from total cellular RNA of pituitary glands. The remaining sequences were completed by rapid amplification of the cDNA ends. The nucleotide sequence of isolated PGHalpha cDNA of both ducks are identical, including 81 bp of 5' untranslated region (UTR), 360 bp of coding region, and 272 bp of 3'-UTR followed by a 13 bp poly(A)(+) tract. The total number of amino acids deduced from the cDNA of the duck PGHalpha is 120 with a signal peptide of 24 amino acids and a mature protein of 96 amino acids. PGHalphas of the ducks (order Anseriformes) share 96% homology of amino acid sequence in signal peptide, and 100% homology in mature proteins with chicken, quail and turkey (order Galliformes). Our data thus demonstrate identical inter-order homology of PGHalpha mature protein in birds. Ten cysteine residues, presumably forming five disulfide bonds within the alpha subunit, and four proline residues, presumably responsible for folding of the molecule, are conserved in the alpha subunit of ducks. Northern blot analysis revealed that PGHalpha mRNA is expressed only in the pituitary. In order to study factors regulating the gene expression of PGHalpha mRNA, duck pituitary fragments were incubated with GnRH, TRH, testosterone, or triiodothyronine (T(3)). GnRH and TRH increased, while testosterone and T(3) decreased, PGHalpha mRNA levels. This is the first report in birds of TRH up-regulation and down-regulation by testosterone and T(3) under in vitro conditions. The present study demonstrates both ducks have the same cDNA nucleotide and deduced amino acid sequences in the PGHalpha subunit, exhibiting identical inter-genus homology within the family of Anatidae. The findings from mRNA expression work suggest that hypothalamic GnRH and TRH up-regulate, while testosterone and T(3) down-regulate, PGHalpha gene expression in ducks.

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Kay E Garnett, Philip Chapman, Julie A Chambers, Ian D Waddell and David S W Boam

on β-cell function. We performed further studies on the immediate-early gene, Egr-1, which was down-regulated in ZDF islets, because other studies suggest it plays a role in the regulation of cell proliferation, differentiation and survival ( Perez

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C Oury, E Alsat, P Jacquemin, D Evain-Brion, J A Martial and M Muller

ABSTRACT

Human chorionic somatomammotropin (hCS) is encoded by two highly related genes, hCS-A and hCS-B, located in the hGH/hCS gene locus. Both genes are expressed in the syncytiotrophoblast layer of the placenta and hCS release from trophoblast cells is known to be increased by cAMP and phorbol esters. However, it remains unclear whether this regulation acts at the level of hCS gene expression or secretion and whether both genes are affected. We examined the effects of cAMP and phorbol 12-myristate 13-acetate (PMA) on the transcription of the hCS-A and hCS-B genes. Transient expression experiments revealed a 7 bp cAMP- and PMA-responsive element (CRElhCS-A) spanning nucleotides −1102 to −1096 upstream of the hCS-A gene. In contrast, the homologous sequence upstream of hCS-B (CRElhCS-B), differing from CRElhCS-A by a single substitution, shows little or no response to cAMP. In band-shift assays, the CRElhCS-A oligonucleotide was shown to bind two factors related to CREBP and AP-1, whereas CRElhCS-B only competes for one of these complexes. Finally, Southwestern analysis revealed that the CRElhCS-A element binds two ubiquitous proteins of 100 kDa and 47 kDa respectively, whereas CRElhCS-B interacts only with the 47 kDa protein. Taken together, these results suggest that a 47 kDa protein binding to the CRElhCS-A and CRElhCS-B elements is involved in the PMA response of the hCS-A and hCS-B genes, and a 100 kDa protein plays a crucial role in cAMP regulation of the hCS-A gene.

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Y-L Zhao, W-D Han, Q Li, Y-M Mu, X-C Lu, L Yu, H-J Song, X Li, J-M Lu and C-Y Pan

reported here is to illustrate the mechanism of transcriptional regulation of LRP16 gene expression by E2, and to determine cis -elements present in the 2.6 kb fragment of human LRP16 gene promoter that confers the E2 transactivation effect. A proximal

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JA Vendrell, F Magnino, E Danis, MJ Duchesne, S Pinloche, M Pons, D Birnbaum, C Nguyen, C Theillet and PA Cohen

We explored, by cDNA mini-arrays, gene expression measurements of MVLN, a human breast carcinoma cell line derived from MCF-7, after 4 days of exposure to 17beta-estradiol (E(2)) treatment, in order to extend our understanding of the mechanism of the pharmacological action of estrogens. We focused on 22 genes involved in estrogen metabolism, cell proliferation regulation and cell transformation. The specificity of the E(2) response was reinforced by comparison with 4-hydroxytamoxifen (OH-Tam), ICI 182,780 and E(2)+OH-Tam expression profiles. Real-time quantitative PCR (RTQ-PCR) confirmed the variation of expression of known (TFF1, AREG, IRS1, IGFBP4, PCNA, ERBB2, CTSD, MYC) as well as novel (DLEU2, CCNA2, UGT1A1, ABCC3, ABCC5, TACC1, EFNA1, NOV, CSTA, MMP15, ZNF217) genes. The temporal response of these gene expression regulations was then investigated after 6 and 18 h of E(2) treatment and this allowed the identification of different time-course patterns. Cycloheximide treatment studies indicated first that estrogen affected the transcript levels of ABCC3 and ABCC5 through dissimilar pathways, and secondly that protein synthesis was needed for modulation of the expression of the CCNA2 and TACC1 genes by estrogens. Western blot analysis performed on TFF1, IRS1, IGFBP4, amphiregulin, PCNA, cyclin A2, TACC1 and ABCC5 proteins confirmed the mini-array and RTQ-PCR data, even for genes harboring low variations of mRNA expression. Our findings should enhance the understanding of changes induced by E(2) on the transcriptional program of human E(2)-responsive cells and permit the identification of new potential diagnostic/prognostic tools for the monitoring of estrogen-related disease conditions such as breast cancer.

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Hseng-Kuang Hsu, Pei-Lin Shao, Ke-Li Tsai, Huei-Chuan Shih, Tzu-Ying Lee and Chin Hsu

, Sheriff et al. 2002 ). These results suggest that NMDA receptor activation is involved in transcriptional regulation of gene expression. Therefore, the present study was designed to identify a possible pathway leading to gene regulation and subsequent

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N Boulle, H Schneid, A Listrat, P Holthuizen, M Binoux and A Groyer

ABSTRACT

Initial observations have indicated similarities between bovine and human IGF-II production during development. The aim of the present study was to investigate whether cattle could provide an experimental model that would mimic the complex pattern of human IGF-II gene expression. Expression of bovine IGF-II gene during development was studied by RNA hybridization using various human IGF-II probes. In fetal tissues and in adult muscle, the bovine IGF-II gene was expressed as a family of eight transcripts ranging in size from 5·2 to 1·1 kb. In adult bovine liver, a major IGF-II transcript of 4·4 kb was expressed that could not be detected in any fetal or adult extra-hepatic tissue. During fetal life, quantitative IGF-II mRNA expression differed in liver and muscle, and the relative amounts of the different transcripts varied with the tissue of origin. These observations suggest that the regulation of bovine IGF-II gene expression is specific to the stage of development and the tissue concerned. Moreover its pattern is very similar to that in its human counterpart.

In order to identify a putative homology between human and bovine gene structures, bovine mRNAs were examined for cross-hybridization with various non-coding exons of the human gene. Cross-hybridization was detected with human untranslated exons 5 and 6, suggesting the presence of two distinct promoters similar to the human promoters P3 and P4. The 4·4 kb mRNA species expressed in adult bovine liver failed to hybridize to a probe for human exons 1 and 2, suggesting that the leader sequences of this transcript were different from those present in the human gene. Finally, results obtained with a probe containing the 3′ untranslated end of exon 9 suggested the presence of at least two polyadenylation sites in the bovine gene.

Although differences in IGF-II gene structures were found between cattle and man, the similarities in the pattern of gene expression between the two species suggest that cattle may be a useful model to investigate some developmental aspects of the expression of the human IGF-II gene.