196 research outputs found
Selection effects in forensic science
In this report we consider the following question: does a forensic expert need to know exactly how the evidential material was selected? We set up a few simple models of situations in which the way evidence is selected may influence its value in court. Although reality is far from a probabilistic model, and one should be very careful when applying theoretical results to real life situations, we believe that the results in our models indicate how the selection of evidence affects its value. We conclude that selection effects in forensic science can be quite important, and that from a statistical point of view, improvements can be made to court room practice
Functional interactions of the AF-2 activation domain core region of the human androgen receptor with the amino-terminal domain and with the transcirptional coactivator TIF2 (Transcriptional Intermediary Factor 2)
Antiandrogens prevent stable DNA-binding of the androgen receptor
The androgen receptor (AR) is essential for development of the male gender
and in the growth of the majority of prostate cancers. Agonists as well as
most antagonists induce translocation of the receptor to the nucleus,
whereas only agonists can activate AR function. Antagonists are therefore
used in the therapy of metastasized prostate cancer. To obtain insight
into the mechanism by which antagonists block AR function in living cells,
we studied nuclear mobility and localization of green fluorescent protein
(GFP)-tagged AR in the presence of either the agonist R1881 or the
antagonists bicalutamide and hydroxyflutamide. As controls we investigated
a non-DNA-binding AR mutant (A573D) and two mutants (W741C and T877A) with
broadened ligand specificity. We demonstrate that in the presence of
R1881, AR localizes in numerous intranuclear foci and, using complementary
fluorescence recovery after photobleaching (FRAP) approaches and computer
modelling, that a fraction of AR ( approximately 10-15%) is transiently
immobilized in a DNA-binding-dependent manner (individual ARs being
immobile for approximately 45 seconds). By contrast, antagonist-bound
GFP-AR showed no detectable immobile fraction and the mobility was similar
to that of the R1881-liganded non-DNA-binding mutant (A573D), indicating
that antagonists do not induce the relatively stable DNA-binding-dependent
immobilization observed with agonist-bound AR. Moreover, in the presence
of bicalutamide and hydroxyflutamide GFP-AR was homogeneously distributed
in the nucleus. Binding of bicalutamide and hydroxyflutamide to
GFP-AR(W741C) and GFP-AR(T877A), respectively, resulted in similar
mobility and heterogeneous nuclear distribution as observed for
R1881-liganded GFP-AR. The live cell studies indicate that the
investigated antagonists interfere with events early in the
transactivation function of the AR
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
Accumulating Progenitor Cells in the Luminal Epithelial Cell Layer Are Candidate Tumor Initiating Cells in a Pten Knockout Mouse Prostate Cancer Model
The PSA-Cre;Pten-loxP/loxP mouse prostate cancer model displays clearly defined stages of hyperplasia and cancer. Here, the initial stages of hyperplasia development are studied. Immunohistochemical staining showed that accumulated pAkt+ hyperplastic cells overexpress luminal epithelial cell marker CK8, and progenitor cell markers CK19 and Sca-1, but not basal epithelial cell markers. By expression profiling we identified novel hyperplastic cell markers, including Tacstd2 and Clu. Further we showed that at young age prostates of targeted Pten knockout mice contained in the luminal epithelial cell layer single pAkt+ cells, which overexpressed CK8, Sca-1, Tacstd2 and Clu; basal epithelial cells were always pAkt−. Importantly, in the luminal epithelial cell layer of normal prostates we detected rare Clu+Tacstd2+Sca-1+ progenitor cells. These novel cells are candidate tumor initiating cells in Pten knockout mice. Remarkably, all luminal epithelial cells in the proximal region of normal prostates were Clu+Tacstd2+Sca-1+. However, in PSA-Cre;Pten-loxP/loxP mice, the proximal prostate does not contain hyperplastic foci. Small hyperplastic foci in prostates of PSA-Cre;Pten-loxP/+ mice found at old age, showed complete Pten inactivation and a progenitor marker profile. Finally, we present a novel model of prostate development and renewal, including lineage-specific luminal epithelial progenitor cells. It is proposed that Pten deficiency induces a shift in the balance of differentiation to proliferation in these cells
Functional interactions of the AF-2 activation domain core region of the human androgen receptor with the amino-terminal domain and with the transcriptional coactivator TIF2 (transcriptional intermediary factor2)
Previous studies in yeast and mammalian cells showed a functional
interaction between the amino-terminal domain and the carboxy-terminal,
ligand-binding domain (LBD) of the human androgen receptor (AR). In the
present study, the AR subdomains involved in this in vivo interaction were
determined in more detail. Cotransfection experiments in Chinese hamster
ovary (CHO) cells and two-hybrid experiments in yeast revealed that two
regions in the NH2-terminal domain are involved in the functional
interaction with the LBD: an interacting domain at the very NH2 terminus,
located between amino acid residues 3 and 36, and a second domain,
essential for transactivation, located between residues 370 and 494.
Substitution of glutamic acid by glutamine at position 888 (E888Q) in the
AF-2 activation domain (AD) core region in the LBD, markedly decreased the
interaction with the NH2-terminal domain. This mutation neither influenced
hormone binding nor LBD homodimerization, suggesting a role of the AF-2 AD
core region in the functional interaction between the NH2-terminal domain
and the LBD. The AF-2 AD core region was also involved in the interaction
with the coactivator TIF2 (transcriptional intermediary factor 2), as the
E888Q mutation decreased the stimulatory effect of TIF2 on AR AF-2
activity. Cotransfection of TIF2 and the AR NH2-terminal domain expression
vectors did not result in synergy between both factors in the induction of
AR AF-2 activity. TIF2 highly induced AR AF-2 activity on a complex
promoter [mouse mammary tumor virus (MMTV)], but it was hardly active on a
minimal promoter (GRE-TATA). In contrast, the AR NH2-terminal domain
induced AR AF-2 activity on both promoter constructs. These data indicate
that both the AR NH2-terminal domain and the coactivator TIF2 functionally
interact, either directly or indirectly, with the AF-2 AD core region in
the AR-LBD, but the level of transcriptional response induced by TIF2
depends on the promoter context
Amino acids 3-13 and amino acids in and flanking the 23FxxLF27 motif modulate the interaction between the N-terminal and ligand-binding domain of the androgen receptor
The N-terminal domain (NTD) and the ligand-binding domain (LBD) of the
androgen receptor (AR) exhibit a ligand-dependent interaction (N/C
interaction). Amino acids 3-36 in the NTD (AR3-36) play a dominant role in
this interaction. Previously, it has been shown that a PhixxPhiPhi motif
in AR3-36, 23FxxLF27, is essential for LBD interaction. We demonstrate in
the current study that AR3-36 can be subdivided into two functionally
distinct fragments: AR3-13 and AR16-36. AR3-13 does not directly interact
with the AR LBD, but rather contributes to the transactivation function of
the AR.NTD-AR.LBD complex. AR16-36, encompassing the 23FxxLF27 motif, is
predicted to fold into a long amphipathic alpha-helix. A second
PhixxPhiPhi candidate protein interaction motif within the helical
structure, 30VREVI34, shows no affinity to the LBD. Within AR16-36, amino
acid residues in and flanking the 23FxxLF27 motif are demonstrated to
modulate N/C interaction. Substitution of Q24 and N25 by alanine residues
enhances N/C interaction. Substitution of amino acids flanking the
23FxxLF27 motif by alanines are inhibitory to LBD interaction
Substitution of Ala564 in the first zinc cluster of the deoxyribonucleic acid (DNA)-binding domain of the androgen receptor by Asp, Asn, or Leu exerts differential effects on DNA binding
In the androgen receptor of a patient with androgen insensitivity, the
alanine residue at position 564 in the first zinc cluster of the
DNA-binding domain was substituted by aspartic acid. In other members of
the steroid receptor family, either valine or alanine is present at the
corresponding position, suggesting the importance of a neutral amino acid
residue at this site. The mutant receptor was transcriptionally inactive,
which corresponded to the absence of specific DNA binding in gel
retardation assays, and its inactivity in a promoter interference assay.
Two other receptor mutants with a mutation at this same position were
created to study the role of position 564 in the human androgen receptor
on DNA binding in more detail. Introduction of asparagine at position 564
resulted in transcription activation of a mouse mammary tumor virus
promoter, although at a lower level compared with the wild-type receptor.
Transcription activation of an (ARE)2-TATA promoter was low, and binding
to different hormone response elements could not be visualized. The
receptor with a leucine residue at position 564 was as active as the
wild-type receptor on a mouse mammary tumor virus promoter and an
(ARE)2-TATA promoter, but interacted differentially with several hormone
response elements in a gel retardation assay. The results of the
transcription activation and DNA binding studies could partially be
predicted from three-dimensional modeling data. The phenotype of the
patient was explained by the negative charge, introduced at position 564
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