Characterization of the androgen receptor transcription unit

ln this study the androgen receptor (AR) transcription unit is presented. Chapter II describes the isolation and characterization of one genomic clone, from which the amino acid sequence of the N-terminai domain of the hAR was deduced. This amino acid sequence was characterized by the presence of several homopolymeric stretches, of which a long a-stretch {20 residues) and a long G-stretch (16 residues) are most conspicuous. In combination with a previously described eDNA clone, which encoded the hAR DBD and LSD, an open reading frame of 2730 bp was deduced, encoding a protein of 910 amino acids. Next, the complete coding region of the hAR gene was isolated from a genomic library, as described in Chapter Ill. The information for the hAR was found to be separated over eight exons. The total lenght of the single copy hAR gene, which is located on the X-chromosome, exceeds 90 kb. As described in Chapter ll, the N-terminal domain is present in one single exon. The DBD is encoded in the exons 2 and 3, with each exon containing the information for one "zinc-finger". The LBD is encoded by the exons 4 to 8. Interestingly, the positions of the introns were found to be conserved between the hAR gene and the cPR and hER genes. The experiments described in Chapter IV extend the work of the Chapters II and Ill and deal with the characterization of the complete hAR eDNA and gene. Northern blot analysis of hAR mRNA identified two hAR mRNA species of 11 and 8 kb, respectively, in the human prostatic tumor cell line LNCaP. A full-length hAR eDNA was constructed from eDNA and genomic clones. Structurally, the 11 kb eDNA consists of a long 5'-UTR (1.1 kb), a 2.7 kb ORF, and a very long 3'·UTR (6.8 kb). The complete 5'-UTR and 3'-UTR were found to be encoded in the exons 1 and 8 of the hAR gene, respectively, fixing the number of exons in this gene at 8. The promoter of the hAR gene, located 1.1 kb upstream from the ATG translation initiation codon in exon 1, was structurally and functionally characterized. Two major sites of transcription initiation in a 13 bp region were identified by 51-nuclease protection experiments. DNA fragments, spanning these sites of initiation, conferred promoter activity upon a promoterless reporter gene construct. 51-nuclease protection experiments with RNA from COS cells, transiently transfected with these constructs, showed usage of the correct initiation sites. Structurally, the promoter lacks TATA/CCAAT boxes and potential regulatory elements consist of a short GC-box (-59/-321. which includes an Sp1 binding sequence (-46/-37), and a long homopurine stretch (-60/-117). The 3'-UTR contains two equally effective polyadenylation signals at a mutual distance of 221 bp. In addition, it was shown that the 8 kb hAR mRNA results from an alternative splice in the 3'-UTR, which does not affect the hAR OR

Effect of Triangularity on Ion-Temperature-Gradient-Driven Turbulence

The linear and nonlinear properties of ion-temperature-gradient-driven (ITG) turbulence with adiabatic electrons are modeled for axisymmetric configurations for a broad range of triangularities δ, both negative and positive. Peak linear growth rates decrease with negative δ but increase and shift toward a finite radial wavenumber kx with positive δ. The growth-rate spectrum broadens as a function of kx with negative δ and significantly narrows with positive δ. The effect of triangularity on linear instability properties can be explained through its impact on magnetic polarization and curvature. Nonlinear heat flux is weakly dependent on triangularity for |δ| ≤ 0.5, decreasing significantly with extreme δ, regardless of sign. Zonal modes play an important role in nonlinear saturation in the configurations studied, and artificially suppressing zonal modes increased nonlinear heat flux by a factor of about four for negative δ, increasing with positive δ by almost a factor of 20. Proxies for zonal-flow damping and drive suggest that zonal flows are enhanced with increasing positive δ.</p

Wilson loop distributions, higher representations and centre dominance in SU(2)

To help understand the centre dominance picture of confinement, we look at Wilson loop distributions in pure SU(2) lattice gauge theory. A strong coupling approximation for the distribution is developed to use for comparisons. We perform a Fourier expansion of the distribution: centre dominance here corresponds to suppression of odd terms beyond the first. The Fourier terms correspond to SU(2) representations; hence Casimir scaling behaviour leads to centre dominance. We examine the positive plaquette model, where only thick vortices are present. We show that a simple picture of random, non-interacting centre vortices gives a string tension about 3/4 of the measured value. Finally, we attempt to limit confusion about the adjoint representation.Comment: 18 pages, 8 figures, LaTeX 2e with epsfig and amstex; discussion following eq. 24 modified; figure 8 replotted; various references added; substance unchange

Tissue specific and androgen-regulated expression of human prostate-specific transglutaminase

Transglutaminases (TGases) are calcium-dependent enzymes catalysing the post-translational cross-linking of proteins. In the prostate at least two TGases are present, the ubiquitously expressed tissue-type TGase (TGC), and a prostate-restricted TGase (TGP). This paper deals with the molecular cloning and characterization of the cDNA encoding the human prostate TGase (hTGP). For this purpose we have screened a human prostate cDNA library with a probe from the active-site region of TGC. The largest isolated cDNA contained an open reading frame encoding a protein of 684 amino acids with a predicted molecular mass of 77 kDa as confirmed by in vitro transcription-translation and subsequent SDS/PAGE. The hTGP gene was tissue-specifically expressed in the prostate, yielding an mRNA of approx. 3.5 kb. Furthermore, a 3-fold androgen-induced upregulation of hTGP mRNA expression has been demonstrated in the recently developed human prostate cancer cell line, PC346C. Other well established human prostate cancer cell lines, LNCaP and PC-3, showed no detectable hTGP mRNA expression on a Northern bolt. The gene coding for prostate TGase was assigned to chromosome 3

An androgen response element in a far upstream enhancer region is essential for high, androgen-regulated activity of the prostate-specific antigen promoter

Prostate-specific antigen (PSA) is expressed at a high level in the luminal epithelial cells of the prostate and is absent or expressed at very low levels in other tissues. PSA expression can be regulated by androgens. Previously, two functional androgenresponse elements were identified in the proximal promoter of the PSA gene. To detect additional, more distal control elements, DNaseI-hypersensitive sites (DHSs) upstream of the PSA gene were mapped in chromatin from the prostate-derived cell line LNCaP grown in the presence and absence of the synthetic androgen R1881. In a region 4.8 to 3.8 kb upstream of the transcription start site of the PSA gene, a cluster of three DHSs was detected. The middle DNAseI-hypersensitive site (DHSII, at ;24.2 kb) showed strong androgen responsiveness in LNCaP cells and was absent in chromatin from HeLa cells. Further analysis of the region encompassing DHSII provided evidence for the presence of a complex, androgen-responsive and cellspecific enhancer. In transient transfected LNCaP cells, PSA promoter constructs containing this upstream enhancer region showed approximately 3000-fold higher activity in the presence than in the absence of R1881. The core region of the enhancer could be mapped within a 440-bp fragment. The enhancer showed synergistic cooperation with the proximal PSA promoter and was found to be composed of at least three separate regulatory regions. In the center, a functionally active, high-affinity androgen receptor binding site (GGAACATATTGTATC) could be identified. Mutation of this element almost completely abolished PSA promoter activity. Transfection experiments in prostate and nonprostate cell lines showed largely LNCaP cell specificity of the upstream enhancer region, although some activity was found in the T47D mammary tumor cell line

The rat androgen receptor gene promoter

The androgen receptor (AR) is activated upon binding of testosterone or dihydrotestosterone and exerts regulatory effects on gene expression in androgen target cells. To study transcriptional regulation of the rat AR gene itself, the 5' genomic region of this gene was cloned from a genomic library and the promoter was identified. S1-nuclease protection analysis showed two major transcription start sites, located between 1010 and 1023 bp upstream from the translation initiation codon. The area surrounding these start sites was cloned in both orientations in a CAT reporter plasmid. Upon transfection of the constructs into COS cells, part of the promoter stimulated transcription in an orientation-independent manner, but the full promoter showed a higher and unidirectional activity. In the promoter/reporter gene constructs, transcription initiated from the same positions as in the native gene. Sequence analysis showed that the promoter of the rat AR gene lacks typical TATA and CCAAT box elements, but one SP1 site is located at about 60 bp upstream from the major start site of transcription. Other possible promoter elements are TGTYCT sequences at positions -174 to -179, -434 to -439., -466 to -471, and -500 to -505, resembling half-sites of the glucocorticoid-responsive element (GRE). Furthermore, a homopurine stretch containing a total of 8 GGGGA elements and similar to sequences that are present in several other GC-rich promoters, is located between -89 and -146 bp upstream from the major start site of transcriptio

The androgen receptor: Functional structure and expression in transplanted human prostate tumors and prostate tumor cell lines

Abstract The growth of the majority of prostate tumors is androgen-dependent, for which the presence of a functional androgen receptor is a prerequisite. Tumor growth can be inhibited by blockade of androgen receptor action. However, this inhibition is transient. To study the role of the androgen receptor in androgen-dependent and androgen-independent prostate tumor cell growth, androgen receptor mRNA expression was monitored in six different human prostate tumor cell lines and tumors, which were grown either in vitro or by transplantation on (male) nude mice. Androgen receptor mRNA was clearly detectable in three androgen-dependent (sensitive) tumors and absent or low in three androgen-independent tumors. Growth of the LNCaP prostate tumor cell line can be stimulated both by androgens and by fetal calf serum. In the former situation androgen receptor mRNA expression is downregulated, whereas in the latter no effect on androgen receptor mRNA levels can be demonstrated. Sequence analysis showed that the androgen receptor gene from LNCaP cells contains a point mutation in the region encoding the steroid-binding domain, which confers an ACT coVon encoding a threonine residue to GCT, encoding alanine

Heavy menstrual bleeding on direct factor Xa inhibitors: Rationale and design of the MEDEA study

Background: In premenopausal women, treatment with direct oral factor Xa inhibitors is associated with an increased risk of heavy menstrual bleeding (HMB) compared with vitamin K antagonists (VKA). Treatment with the direct oral thrombin inhibitor dabigatran appears to be associated with a reduced risk of HMB compared with VKA. These findings come from small observational studies or post hoc analyses of trials in which HMB was not a primary outcome. Use of tranexamic acid during the menstrual period may be effective in patients with HMB, but prospective data regarding efficacy and safety in patients on anticoagulant treatment are lacking. Rationale and Design: A direct comparison of a factor Xa inhibitor and a thrombin inhibitor with HMB as primary outcome, as well as an evaluation of the effects of adding tranexamic acid in women with anticoagulant-associated HMB is highly relevant for clinical practice. The MEDEA study is a randomized, open-label, pragmatic clinical trial to evaluate management strategies in premenopausal women with HMB associated with factor Xa inhibitor therapy. Outcomes: Women using factor Xa inhibitors with proven HMB, as assessed by a pictorial blood loss assessment chart (PBAC) score of >150, will be randomized to one of three study arms: (i) switch to dabigatran; (ii) continue factor Xa inhibitor with addition of tranexamic acid during the menstrual period; or (iii) continue factor Xa inhibitor without intervention. The primary outcome is the difference in PBAC score before and after randomization. Here, we present the rationale and highlight several unique features in the design of the study

Effect of Triangularity on Ion-Temperature-Gradient-Driven Turbulence

The linear and nonlinear properties of ion-temperature-gradient-driven (ITG) turbulence with adiabatic electrons are modeled for axisymmetric configurations for a broad range of triangularities δ, both negative and positive. Peak linear growth rates decrease with negative δ but increase and shift toward a finite radial wavenumber kx with positive δ. The growth-rate spectrum broadens as a function of kx with negative δ and significantly narrows with positive δ. The effect of triangularity on linear instability properties can be explained through its impact on magnetic polarization and curvature. Nonlinear heat flux is weakly dependent on triangularity for |δ| ≤ 0.5, decreasing significantly with extreme δ, regardless of sign. Zonal modes play an important role in nonlinear saturation in the configurations studied, and artificially suppressing zonal modes increased nonlinear heat flux by a factor of about four for negative δ, increasing with positive δ by almost a factor of 20. Proxies for zonal-flow damping and drive suggest that zonal flows are enhanced with increasing positive δ