Cooperativity of Glucocorticoid Response Elements Located Far Upstream of the Tyrosine Aminotransferase Gene

Two glucocorticoid response elements (GREs) located 2.5 kb upstream of the transcription initiation site of the tyrosine aminotransferase gene were identified by gene transfer experiments and shown to bind to purified glucocorticoid receptor. Although the proximal GRE has no inherent capacity by itself to stimulate transcription, when present in conjunction with the distal GRE, this element synergistically enhances glucocorticoid induction of gene expression. Cooperativity of the two GREs is maintained when they are transposed upstream of a heterologous promoter. An oligonucleotide of 22 bp representing the distal GRE is sufficient to confer glucocorticoid inducibility. As evidenced by the mapping of DNAase I hypersensitive sites, local alterations in the structure of chromatin at the GREs take place as a consequence of hormonal treatment

Extinction of tyrosine aminotransferase gene activity in somatic cell hybrids involves modification and loss of several essential transcription factors

Extinction is defined as the loss of cell type-specific gene expression that occurs in somatic cell hybrids derived by fusion of cells with dissimilar phenotypes. To explore the basis of this dominant-negative regulation, we have studied the activities of the control elements of the liver-specific gene encoding tyrosine aminotransferase (TAT) in hepatoma/fibroblast hybrid crosses. We show that extinction in complete somatic cell hybrids is accompanied by the loss of activity of all known cell type-specific control elements of the TAT gene. This inactivity is the result of first, lack of expression of genes coding for the transcriptional activators HNF4 and HNF3[~ and HNF33,, which bind to essential elements of the enhancers; and second, loss of in vivo binding and activity of ubiquitous factors to these enhancers, including CREB, which is the target for repression by the tissue-specific extinguisher locus TSE1. Complete extinction of TAT gene activity is therefore a multifactorial process affecting all three enhancers controlling liver-specific and hormone-inducible expression. It results from lack of activation, rather than active repression, and involves both post-translational modification and loss of essential transcriptional activators

Delivery as nanoparticles reduces imatinib mesylate-induced cardiotoxicity and improves anticancer activity

Clinical effectiveness of imatinib mesylate in cancer treatment is compromised by its off-target cardiotoxicity. In the present study, we have developed physically stable imatinib mesylate-loaded poly(lactide-co-glycolide) nanoparticles (INPs) that could sustainably release the drug, and studied its efficacy by in vitro anticancer and in vivo cardiotoxicity assays. MTT (methylthiazolyldiphenyl-tetrazolium bromide) assay revealed that INPs are more cytotoxic to MCF-7 breast cancer cells compared to the equivalent concentration of free imatinib mesylate. Wistar rats orally administered with 50 mg/kg INPs for 28 days showed no significant cardiotoxicity or associated changes. Whereas, increased alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase levels, and reduced white blood cell, red blood cell, and hemoglobin content were observed in the animals administered with free drug. While the histological sections from hearts of animals that received INPs did not show any significant cardiotoxic symptoms, loss of normal architecture and increased cytoplasmic vacuolization were observed in the heart sections of animals administered with free imatinib mesylate. Based on these results, we conclude that nano-encapsulation of imatinib mesylate increases its efficacy against cancer cells, with almost no cardiotoxicity

Role of cyclic AMP in the control of cell-specific gene expression

Genes have to be expressed in specific cell types at appropriate times of development dependent on external signals. cAMP signaling occurs in all cells, thus raising the question of how this signal transduction pattern is integrated into mechanisms determining cell-specific gene expression. We have analyzed expression of the tyrosine aminotransferase gene as a model to study the basis of this cell type specificity of hormone induction. We found that cell-type-specific expression is generated by combined action of cAMP signal-dependent and liver cell-specific transcription factors. The interdependence of the cAMP response element and an element determining liver cell specificity enables a gene to respond to an ubiquitous signal in a cell-specific manner

Die Funktion des Responseregulators ARR2 in der Entwicklung von Arabidopsis thaliana

In dieser Arbeit konnte mittels physiologischer Experimente, Expressionsanalysen, Phosphorylierungsassays und Transaktivierungsanalysen eine komplexe Funktion von ARR2 in verschiedenen Signaltransduktionswegen von Arabidopsis aufgedeckt werden. Die Analyse von Keimlingen in der arr2-Nullmutante zeigt eine hyposensitive Reaktion auf Cytokinin. Mit transienten Transaktivierungsanalysen in Protoplasten konnte die Induktion Cytokinin-responsiver Gene durch ARR2 und ARR2D80E nachgewiesen werden. Zusammenfassend führen diese Ergebnisse zur Hypothese, dass ARR2 eine regulierende Funktion in der Cytokininantwort einnimmt. Die vergleichende physiologische Analyse von Dunkelrotlicht-bestrahlten Keimlingen sowie die Expression des Chalkonsynthasegens identifizieren ARR2 als einen positiven Regulator in der phyA-vermittelten "high irradiance response". Der Nachweis, dass ARR2 in die Ethylensignaltransduktion involviert ist, wurde in verschiedenen Ansätze erbracht. Durch physiologische Analysen unter Verwendung einer arr2-Nullmutante sowie ARR2D80E-überexprimierenden Linien wurde ARR2 als regulierende Komponente der Ethylensignaltransduktion identifiziert. Dies wurde mittels transienter Transaktivierungsanalysen in Arabidopsis-Protoplasten bestätigt. Der zellfreie Phosphorelayassay identifizierte die Ethylenrezeptorhybridkinase ETR1 als putative, stromaufwärtsliegende, phosphorylierende Komponente von ARR2. Die Untersuchungen unter Einsatz der pathogenen Pilze Botrytis cinerea und Peronospora parasitica sowie Trockenstress-Exyperimente lassen eine Rolle von ARR2 in der Pathogen- und dieser abiotischen Stressantwort vermuten. Auf Mikroarrays-basierende Expressionsanalysen der arr2-Nullmutante und ARR2D80E-Pflanzen deuten darauf hin, dass das Zwei-Komponentennetzwerk direkt oder indirekt verschiedene Signaltransduktionswege beeinflussen kann. Zusammenfassend zeigen diese Daten, dass das Zwei-Komponentensystem nicht nur einen primären Signalmechanismus darstellt, sondern zusätzlich das molekulare Grundgerüst für ein komplexes Signaltransduktionsnetzwerk bildet, welches die Feinabstimmung von Signalen übernimmt und die Kommunikation von verschiedenen Signalwegen untereinander ermöglicht