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B Peeters

Publications and source records attributed to B Peeters.

At least 19 recordsLinked to original sources
PubMed1999

Differential DNA binding by the androgen and glucocorticoid receptors involves the second Zn-finger and a C-terminal extension of the DNA-binding domains.

The androgen and glucocorticoid hormones evoke specific in vivo responses by activating different sets of responsive genes. Although the consensus sequences of the glucocorticoid and androgen response elements are very similar, this in vivo specificity can in some cases be explained by differences in DNA recognition between both receptors. This has clearly been demonstrated for the androgen response element PB-ARE-2 described in the promoter of the rat probasin gene. Swapping of different fragments between the androgen- and glucocorticoid-receptor DNA-binding domains demonstrates that (i) the first Zn-finger module is not involved in this sequence selectivity and (ii) that residues in the second Zn-finger as well as a C-terminal extension of the DNA-binding domain from the androgen receptor are required. For specific and high-affinity binding to response elements, the DNA-binding domains of the androgen and glucocorticoid receptors need a different C-terminal extension. The glucocorticoid receptor requires 12 C-terminal amino acids for high affinity DNA binding, while the androgen receptor only involves four residues. However, for specific recognition of the PB-ARE-2, the androgen receptor also requires 12 C-terminal residues. Our data demonstrate that the mechanism by which the androgen receptor binds selectively to the PB-ARE-2 is different from that used by the glucocorticoid receptor to bind a consensus response element. We would like to suggest that the androgen receptor recognizes response elements as a direct repeat rather than the classical inverted repeat.

Amino Acid Sequence↗
PubMed1999

The first exon of the human sc gene contains an androgen responsive unit and an interferon regulatory factor element.

Secretory component (SC) plays a key role in the transport of IgA and IgM to the lumina of many glands. The gene is constitutively expressed, but can be modulated by hormonal and immunological stimuli. Recently, the promoter and the first exon of the human sc gene have been cloned. The first exon contains a putative androgen/glucocorticoid response element (ARE/GRE) and an Interferon Regulatory Factor Element (IRF-E). Here we show that the ARE/GRE can bind the DNA-binding domain (DBD) of both the androgen (AR) and glucocorticoid receptor (GR) with a preference for the AR-DBD. In transient transfection experiments, this element confers higher responsiveness to androgens than to glucocorticoids. The IRF-E can function as an IRF-2, but surprisingly not as an IRF-I responsive element. We postulate that these two regulatory elements play a key role in the complex regulation of the sc gene in vivo.

Androgens↗
PubMed1999

Complete nucleotide sequence of Newcastle disease virus: evidence for the existence of a new genus within the subfamily Paramyxovirinae.

We have completely sequenced the genome of Newcastle disease virus (NDV) vaccine strain LaSota. The sequences of the 3'- and 5'-terminal ends of the RNA genome were determined by sequencing cDNA fragments generated by rapid amplification of cDNA ends. The entire genomic sequence, which was established by sequencing cDNA fragments generated by high-fidelity RT-PCR, consists of 15186 nt. Comparison of the 5'-terminal sequence of NDV LaSota with the 5'-terminal sequences of ten members of the Paramyxovirinae showed that NDV LaSota has an unusually long 5' untranscribed region. Comparison of the entire genomic sequences showed that NDV is only distantly related to the other members of the genus Rubulavirus, to which NDV has been assigned. In this paper we present data which suggest that NDV should not be classified in the genus Rubulavirus, but instead should be considered as a member of a new genus within the subfamily Paramyxovirinae.

Base Sequence↗
PubMed1999

The androgen receptor amino-terminal domain plays a key role in p160 coactivator-stimulated gene transcription.

Steroid receptors are conditional transcription factors that, upon binding to their response elements, regulate the expression of target genes via direct protein interactions with transcriptional coactivators. We have analyzed the functional interactions between the androgen receptor (AR) and 160-kDa nuclear receptor coactivators. Upon overexpression in mammalian cells, these coactivators enhance the transcriptional activity of both the amino-terminal domain (NTD) and the ligand-binding domain (LBD) of the AR. The coactivator activity for the LBD is strictly ligand-controlled and depends on the nature of the DNA-binding domain to which it is fused. We demonstrate that the NTD physically interacts with coactivators and with the LBD and that this interaction, like the functional interaction between the LBD and p160 coactivators, relies on the activation function 2 (AF2) core domain. The mutation of a highly conserved lysine residue in the predicted helix 3 of the LBD (K720A), however, blunts the functional interaction with coactivators but not with the NTD. Moreover, this mutation does not affect the transcriptional activity of the full-size AR. A mutation in the NTD of activation function AF1a (I182A/L183A), which dramatically impairs the activity of the AR, has no effect on the intrinsic transcriptional activity of the NTD but interferes with the cooperation between the NTD and the LBD. Finally, p160 proteins in which the three LXXLL motifs are mutated retain most of their coactivator activity for the full-size AR, although they are no longer functional for the isolated LBD. Together, these data suggest that in the native AR the efficient recruitment of coactivators requires a functional association of the NTD with the LBD and that the binding of coactivators occurs primarily through the NTD.

Amino Acid Sequence↗
PubMed1999

Interaction of the putative androgen receptor-specific coactivator ARA70/ELE1alpha with multiple steroid receptors and identification of an internally deleted ELE1beta isoform.

Steroid-regulated gene transcription requires the coordinate physical and functional interaction of hormone receptors, basal transcription factors, and transcriptional coactivators. In this context ARA70, previously called RFG and ELE1, has been described as a putative coactivator that specifically enhances the activity of the androgen receptor (AR) but not that of the glucocorticoid receptor (GR), the progesterone receptor, or the estrogen receptor (ER). Here we describe the cloning of the cDNA for ELE1/ARA70 by RT-PCR from RNA derived from different cell lines (HeLa, DU-145, and LNCaP). In accordance with the previously described sequence, we obtained a 1845-bp PCR product for the HeLa and the LNCaP RNA. Starting from T-47D RNA, however, an 860-bp PCR product was obtained. This shorter variant results from an internal 985-bp deletion and is called ELE1beta; accordingly, the longer isoform is referred to as ELE1alpha. The deduced amino acid sequence of ELE1alpha, but not that of ELE1beta, differs at specific positions from the one previously published by others, suggesting that these two proteins are encoded by different nonallelic genes. ELE1alpha is expressed in the three prostate-derived cell lines examined (PC-3, DU-145, and LNCaP), and this expression is not altered by androgen treatment. Of all rat tissues examined, ELE1alpha expression is highest in the testis. This is also the only tissue in which we could demonstrate ELE1beta expression. Both ELE1alpha and ELE1beta interact in vitro with the AR, but also with the GR and the ER, in a ligand-independent way. Overexpression of either ELE1 isoform in DU-145, HeLa, or COS cells had only minor effects on the transcriptional activity of the human AR. ELE1alpha has no intrinsic transcription activation domain or histone acetyltransferase activity, but it does interact with another histone acetyltransferase, p/CAF, and the basal transcription factor TFIIB. The interaction with the AR occurs through the ligand-binding domain and involves the region corresponding to the predicted helix 3. Mutation in this domain of leucine 712 to arginine greatly reduces the affinity of the AR for ELE1alpha but has only moderate effects on its transcriptional activity. Taken together, we have identified two isoforms of the putative coactivator ARA70/ELE1 that may act as a bridging factor between steroid receptors and components of the transcription initiation complex but which lack some fundamental properties of a classic nuclear receptor coactivator. Further experiments will be required to highlight the in vivo role of ELE1 in nuclear receptor functioning.

Acetyltransferases↗
PubMed1999

Androgen specificity of a response unit upstream of the human secretory component gene is mediated by differential receptor binding to an essential androgen response element.

The expression of secretory component (SC), the epithelial receptor for poly-immunoglobulins, is regulated in a highly tissue-specific manner. In several tissues, e.g. lacrimal gland and prostate, SC synthesis is enhanced by androgens at the transcriptional level. In this study, we describe the presence of an androgen response unit, located 3.3 kb upstream of the sc transcription initiation site and containing several 5'-TGTTCT-3'-like motifs. Although each of these elements is implicated in the enhancer function, one element, the ARE1.2 motif, is found to be the main interaction site for the androgen receptor as demonstrated in in vitro binding assays as well as in transient transfection assays. A high-affinity binding site for nuclear factor I, adjacent to this ARE, is also involved in the correct functioning of the sc upstream enhancer. The ARE1.2 motif consists of an imperfect direct repeat of two core binding elements with a three-nucleotide spacer and therefore constitutes a nonconventional ARE. We demonstrate that this element displays selectivity for the androgen receptor as opposed to glucocorticoid receptor both in in vitro binding assays and in transfection experiments. Mutational analysis suggests that the direct nature of the half-site repeat is responsible for this selectivity. We have thus determined a complex and androgen-specific response unit in the far upstream region of the human SC gene, which we believe to be involved in its androgen responsiveness in epithelial cells of different organs such as prostate and lacrimal gland. We were also able to demonstrate that the primary sequence of a single nonconventional ARE motif within the enhancer is responsible for its androgen specificity.

Androgens↗
PubMed1998

Primary rat lacrimal cells undergo acinar-like morphogenesis on reconstituted basement membrane and express secretory component under androgen stimulation.

Single cells or small cell clusters, isolated from the rat lacrimal gland, were incubated on reconstituted basement membrane (matrigel) in a well-defined serum-free medium. During the first days of culture, cells reassociated and reorganized in structures resembling acini. These multicellular structures, maintained in culture for 2 weeks, consisted of well-polarized cuboidal cells surrounding a central lumen and exhibiting apically located microvilli. Myoepithelial cells were observed at the periphery of the acinar structures. Both in the native lacrimal and in the cultured aggregates, epithelial cells displayed strong immunoreactivity for cytokeratin 8, while myoepithelial cells were immunoreactive for vimentin and alpha-smooth muscle isoactin. These data indicate that the cultured aggregates closely mimic the in vivo architecture of lacrimal glands both by morphology and immunohistochemistry. We further demonstrated the presence of an intact androgen receptor and the ability of the cultured aggregates to respond to androgens with increased secretion of the secretory component. Comparable androgen responses were observed in lacrimal gland cultures of 5-week-old male and female rats. In conclusion, we report a morphologically and functionally differentiated culture system of primary rat lacrimal cells, in which androgen-regulated gene expression was observed. This culture model provides a unique experimental paradigm for studying the effects of hormones, cytokines, and growth factors on the morphogenesis, growth, and functional differentiation of lacrimal glands.

Actins↗
PubMed1997

Characterization of the human secretory component gene promoter.

Secretory Component (SC) is a receptor molecule implicated in the transepithelial transport of polymeric immunoglobulins. We have cloned and characterized the first exon, part of the first intron and 3500 bp of the upstream region of the gene and determined the transcription initiation region. A GC rich region immediately upstream of the transcription start region is interrupted by a potential TATA-box (TTTAA) at position -28. Promoter activity was demonstrated in transient transfection experiments in HepG2 and HeLa cells. The smallest fragment still showing transcriptional activity contains 48 bp of SC promoter. A number of putative recognition sites for transcription factors possibly involved in the regulation of SC transcription by steroids, peptide hormones and cytokines were found in the upstream region.

Base Sequence↗
PubMed1997

Biologically safe, non-transmissible pseudorabies virus vector vaccine protects pigs against both Aujeszky's disease and classical swine fever.

Envelope glycoprotein D (gD) of pseudorabies virus (PRV) is essential for penetration but is not required for cell-to-cell spread. When animals are inoculated with a phenotypically complemented PRV gD mutant, the virus is able to spread locally by means of direct cell-to-cell transmission, but progeny virions released by infected cells are non-infectious because they lack gD. Therefore, the virus cannot be transmitted from inoculated animals to other animals. This property makes a PRV gD mutant an attractive candidate as a safe vaccine vector. To examine whether a self-restricted, non-transmissible PRV mutant can be used as a biologically safe vaccine vector, a gD/gE-negative PRV recombinant virus which expresses envelope glycoprotein E2 of classical swine fever virus was constructed. Vaccination of pigs showed that the recombinant virus was able to protect pigs against both Aujeszky's disease and classical swine fever.

Animals↗
PubMed1997

Identification of a functional androgen-response element in the exon 1-coding sequence of the cystatin-related protein gene crp2.

Two hormone-responsive segments, one in the region of the promoter and one in intron 1, are identified in two homologous androgen-regulated and differentially expressed rat genes encoding the cystatin-related proteins (CRPs). Footprint analysis with the androgen receptor (AR) DNA-binding domain on the promoter-containing fragments reveals an AR-binding site downstream of the transcription start point in the crp2 gene (ARBSd/crp2, +40/+63). It displays an androgen response element-like sequence motif 5'-AGAAGAaaaTGTACA-3' and overlaps with the ATG translation start codon. A double-stranded oligonucleotide containing this sequence forms a DNA-protein complex with the full-length AR synthesized by vaccinia, as seen in band shift assays. Additional AR-binding sites, ARBSu/crp1 and ARBSu/crp2, occur 5' upstream of the transcription start point and are located at an identical position (-142/ -120) in crp1 and crp2. The AR affinity for these two slightly different sequence motifs is relatively weak. The biological function of all three AR-binding sites as transcription control elements has been studied. The ARBSd/crp2 element clearly shows androgen-response element characteristics. The contribution of the common upstream element to the androgen-dependent control of reporter gene transcription is less clear. The transcription of a reporter gene construct containing the crp2 footprint fragment crp2F (-273/+88) is hormonally regulated as determined by transfection into the human breast cancer cell line T-47D. Androgens, but also glucocorticoids, efficiently stimulate steroid-dependent transcription of the chloramphenicol acetyltransferase gene. Mutation of the 5'-TGTACA-3' sequence in ARBSd/crp2 destroys the AR binding and abolishes the androgen-dependent synthesis of chloramphenicol acetyltransferase. A large fragment derived from intron 1 of the crp1 and crp2 gene can also provide the androgen-dependent transcription of chimeric constructs in T-47D cells. However, the induction measured is less than the one observed with crp2F (-273/+88), and this activity seems to reside in several subfragments that each display a low but consistent androgen responsiveness.

Androgens↗
PubMed1996

Androgenic induction of cystatin-related protein and the C3 component of prostatic binding protein in primary cultures from the rat lacrimal gland.

Cystatin-related protein (CRP) and the C3 component of prostatic binding protein (C3) are synthesized in vivo under androgen control in the lacrimal gland and ventral prostate of adult male rats [1,2]. Androgen administration to female or 7-day castrated male rats, which do not express CRP, can induce its synthesis [3]. In this study, we show androgen-dependent expression of CRP1 and C3 in primary cultures of acinar cells of the lacrimal gland of female rats. Addition of androgens to the culture medium results in the synthesis and secretion of both proteins in a time- and dose-dependent way. Estradiol or progesterone are unable to induce their expression. Dexamethasone in low concentrations and present as a basal component of the serum free defined medium, is needed to sensitize the culture system for androgens. In high concentrations, this synthetic glucocorticoid seems to play a similar role as androgens in CRP1 and C3 regulation.

Androgen-Binding Protein↗
PubMed1996

The androgen-specific probasin response element 2 interacts differentially with androgen and glucocorticoid receptors.

The nuclear receptors constitute a large family of transcription factors characterized by a well conserved DNA-binding domain. The receptors for glucocorticoids, progestins, mineralocorticoids, and androgens constitute a subgroup because they bind in vitro with high affinity to DNA elements containing a partial palindrome of the core sequence 5'-TGTTCT-3'. In vivo, however, the corresponding steroids differentially regulate the expression of their target genes, even when more than one receptor type is present in a particular cell. The DNA-binding domains of the androgen and of the glucocorticoid receptors bind most androgen response elements with similar relative affinities. In contrast, one element (5'-GGTTCTTGGAGTACT-3') which was recently described in the promoter region of the probasin gene selectively interacts with the DNA-binding domain of the androgen receptor and not with that of the glucocorticoid receptor. From studies with chimeric elements, it can be deduced that it is the left subsequence 5'-GGTTCT-3' which excludes the glucocorticoid receptor domain from binding. In co-transfection experiments where the ARE of the C3(1) gene is responsive to both androgens and glucocorticoids, the probasin element is induced only by androgens and not by glucocorticoids. The existence of response elements which are recognized preferentially by the androgen receptor provides yet another possible mechanism to explain the differences of the in vivo effects between androgens and other steroids of the subgroup.

Androgen-Binding Protein↗
PubMed1996

An indirect double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) using baculovirus-expressed antigen for the detection of antibodies to glycoprotein E of pseudorabies virus and comparison of the method with blocking ELISAs.

Antibodies in porcine sera against glycoprotein E (gE) of pseudorabies virus (PRV) are usually measured in blocking enzyme-linked immunosorbent assays (ELISAs) with one or two murine monoclonal antibodies (MAbs) directed against gE. Our aim was to develop a confirmation assay which is based on another principle and which is able to detect antibodies directed against most potential binding sites on gE with high specificity. Therefore, we developed an indirect double-antibody sandwich assay (IDAS) using recombinant gE expressed by baculovirus (BacgE960). A fragment of the gE gene consisting of nucleotide positions +60 to +1020 of gE, coding for the major antigenic sites of gE but not the transmembrane region, was cloned behind the signal sequence of PRV gG and the p10 promoter in a baculovirus vector. Immunoblot analysis showed that the expressed protein reacted with MAbs directed against five of the six antigenic sites on gE. Although the conformation of some antigenic sites, notably antigenic sites E and C, was not identical to their natural conformation, the expressed protein bound gE-specific antibodies in porcine sera in Western blots (immunoblots) and ELISAs. For the IDAS, a coating MAb directed against the nonimmunodominant antigenic site A on gE was chosen. A major obstacle in binding ELISAs, such as the IDAS, appeared to be the high nonspecific binding activity observed in porcine sera. As a result, sera could be tested only in relatively high dilutions in the BacgE960 IDAS, in contrast to the testing of sera in blocking ELISAs. The sensitivity and specificity of the newly developed BacgE960 IDAS were evaluated and compared with those of five commercially available blocking ELISAs by using several sets of sera of known PRV disease history. The BacgE960 IDAS assay had a high diagnostic specificity and a moderate sensitivity. The five blocking ELISAs differed remarkably in sensitivity and specificity, thereby illustrating the need for standardization and confirmation. We conclude that the BacgE960 IDAS is a useful and specific additional (confirmatory) test for the detection of antibodies to gE.

Animals↗
PubMed1996

Glycoprotein D-negative pseudorabies virus can spread transneuronally via direct neuron-to-neuron transmission in its natural host, the pig, but not after additional inactivation of gE or gI.

Envelope glycoprotein D (gD) is essential for entry of pseudorabies virus (PRV) into cells but is not required for the subsequent steps in virus replication. Phenotypically complemented gD mutants can infect cells and can spread, both in vitro and in mice, by direct cell-to-cell transmission. Progeny virions released by infected cells are noninfectious because they lack gD. The aim of this study was to determine the role of gD in the neuropathogenicity of PRV in its natural host, the pig. We investigated whether gD-negative PRV can spread transneuronally via synaptically linked neurons of the olfactory and trigeminal routes. High doses of a phenotypically complemented gD mutant and gD mutants that are unable to express either gI or gI plus gE were inoculated intranasally in 3- to 5-week-old pigs. Compared with the wild-type virus, the virulence of the gD mutant was reduced. However, pigs inoculated with the gD mutant still developed fever and respiratory signs. Additional inactivation of either gI or gI plus gE further decreased virulence for pigs. Immunohistochemical examination of infected pigs showed that a PRV gD mutant could replicate and spread transneuronally into the central nervous system (CNS). Compared with the wild-type virus, the gD mutant had infected fewer neurons of the CNS on day 2. Nevertheless, on day 3, the gD-negative PRV had infected more neurons and viral antigens were present in second- and third-order neurons in the olfactory bulb, brain stem, and medulla oblongata. In contrast, gD mutants which are unable to express either gI or gI plus gE infected a limited number of first-order neurons in the olfactory epithelium and in the trigeminal ganglion and did not spread transneuronally or infect the CNS. Thus, transsynaptic spread of PRV in pigs can occur independently of gD. Possible mechanisms of transsynaptic transport of PRV are discussed.

Animals↗
PubMed1996

Androgens transcriptionally regulate the expression of cystatin-related protein and the C3 component of prostatic binding protein in rat ventral prostate and lacrimal gland.

In this report, it is demonstrated that the C3 component of prostatic binding protein (PBP) is also expressed and androgen regulated in the exorbital lacrimal gland, as shown previously for cystatin-related protein (CRP), another abundant secretory protein from the ventral prostate. The presence of C3 messenger RNA (mRNA) could be demonstrated by both Northern blot hybridization and PCR amplification and sequencing. The mRNAs encoding the C1 and C2 components of PBP, however, were undetectable. At the protein level, the C3 component in the lacrimal gland is glycosylated and linked by disulfide bridges to a new 10-kDa component not reacting with the PBP antiserum. As shown previously for CRP, the expression of C3 in the lacrimal gland requires the simultaneous presence of androgens and a functional androgen receptor. The effects of castration and androgen treatment on CRP and C3 mRNA concentrations were studied by Northern blot and dot blot hybridization; effects on transcription rates were determined by nuclear run-on assay. Two days after castration, the relative abundance of CRP mRNA had declined significantly (P < 0.01) to 10.5 +/- 1.5% (+/-SEM) of precastration levels in the prostate and to 14.5 +/- 8.0% in the lacrimal gland; the transcription rates declined to 14.3% and 10.0%, respectively. The C3 mRNA level and transcription rate in the prostate showed a more moderate decrease (P < 0.05) to 40.6 +/- 8.5% and 41.7%, but were hardly measurable in the lacrimal gland. Androgen administration resulted in a rapid increase in the transcription rates, which reached or exceeded control levels after 6-9 h of treatment and clearly preceded the increase in mRNA levels. It is concluded that the lacrimal gland, which can be studied conveniently in female and long term androgen-depleted animals offers a suitable model for the study of androgen-regulated gene expression.

Analysis of Variance↗
PubMed1995

The US3-encoded protein kinase from pseudorabies virus affects egress of virions from the nucleus.

We examined the influence of inactivation of various genes located in the unique short (U(S)) region of pseudorabies virus on virus replication and assembly in porcine nasal mucosa explant cultures. The following strains were used: the virulent wild-type strain NIA-3, and strains derived from NIA-3 containing a mutation inactivating the genes encoding either the US3-encoded protein kinase (PK), gG, gD, gI, gE, the 28 kDa ('28K') protein (single mutant), or the 28K and 11 kDa ('11K') proteins (double mutant). In addition a wild-type rescuant was used, which was generated by marker rescue from a PK- mutant. All virus strains infected nasal epithelium and had invaded the stroma after approximately 24 h. The morphogenesis in nasal epithelium cells of two PK- mutants showed the most striking differences compared to the parent NIA-3 strain and the other mutant strains. The changes could be ascribed to the US3-encoded PK because the rescue mutant showed a similar morphogenesis to wild-type NIA-3. The transmembrane transport of the PK- mutants was impaired at the outer nuclear membrane which resulted in an accumulation of virions in the perinuclear space. These results suggest that proteins, phosphorylated by the US3-encoded PK, are involved in debudding of virus particles at the outer nuclear membrane. This defect in the transport of the US3 mutant probably explains their reduced replication in vitro. The gG-, gD-, gI-, gE-, 28K- and 11K- mutant strains showed minor or no changes in viral assembly. Thus the reported decreased virulence of the gD-, gI- and gE- mutants was, in contrast to that of the PK- mutants, not associated with clear alterations in morphogenesis.

Animals↗