Estrogen inhibits the vascular injury response in estrogen receptor alpha-deficient mice.

The atheroprotective effects of estrogen in women are well recognized, but the underlying mechanisms responsible are not well understood. Blood vessel cells express the classic estrogen receptor, ER alpha (ref. 2-6), and are directly affected by estrogen, which inhibits the development of atherosclerotic and injury-induced vascular lesions. We have generated mice in which the ER alpha gene is disrupted and have used a mouse model of carotid arterial injury to compare the effects of estrogen on wild-type and estrogen receptor-deficient mice. Increases in vascular medial area and smooth muscle cell proliferation were quantified following vascular injury in ovariectomized mice treated with vehicle or with physiologic levels of 17 beta-estradiol. Surprisingly, in both wild-type and estrogen receptor-deficient mice, 17 beta-estradiol markedly inhibited to the same degree all measures of vascular injury. These data demonstrate that estrogen inhibits vascular by a novel mechanism that is independent of the classic estrogen receptor, ER alpha.

Cloning of human androgen receptor complementary DNA and localization to the X chromosome.

The androgen receptor (AR) mediates the actions of male sex steroids. Human AR genomic DNA was cloned from a flow-sorted human X chromosome library by using a consensus nucleotide sequence from the DNA-binding domain of the family of nuclear receptors. The AR gene was localized on the human X chromosome between the centromere and q13. Cloned complementary DNA, selected with an AR-specific oligonucleotide probe, was expressed in monkey kidney (COS) cells and yielded a high-affinity androgen-binding protein with steroid-binding specificity corresponding to that of native AR. A predominant messenger RNA species of 9.6 kilobases was identified in human, rat, and mouse tissues known to contain AR and was undetectable in tissues lacking AR androgen-binding activity, including kidney and liver from androgen-insensitive mice. The deduced amino acid sequence of AR within the DNA-binding domain has highest sequence identity with the progesterone receptor.

Estrogen receptor-alpha deficiency attenuates autoimmune disease in (NZB x NZW)F1 mice.

Estrogens promote lupus in humans and some mouse models of this disease. Nonetheless, little is known about the role of estrogen receptors in lupus pathogenesis. Here, we report that in females on the lupus-prone (NZB x NZW)F(1) background, disruption of estrogen receptor-alpha (ER alpha or Esr1) attenuated glomerulonephritis and increased survival. ER alpha deficiency also retarded development of anti-histone/DNA antibodies, suggesting that ER alpha promotes loss of immunologic tolerance. Furthermore, ER alpha deficiency in (NZB x NZW)F(1) females attenuated the subsequent development of anti-double-stranded DNA (dsDNA) IgG antibodies, which are associated with glomerulonephritis in this model. We provide evidence that ER alpha may promote lupus, at least in part, by inducing interferon-gamma, an estrogen-regulated cytokine that impacts this disease. ER alpha deficiency in (NZB x NZW)F(1) males increased survival and reduced anti-dsDNA antibodies, suggesting that ER alpha also modulates lupus in males. These studies demonstrate that ER alpha, rather than ER beta, plays a major role in regulating autoimmunity in (NZB x NZW)F(1) mice. Furthermore, our results suggest for the first time that ER alpha promotes lupus, at least in part, by impacting the initial loss of tolerance. These data suggest that targeted therapy disrupting ER alpha, most likely within the immune system, may be effective in the prevention and/or treatment of lupus.

Vascular estrogen receptors and endothelium-derived nitric oxide production in the mouse aorta. Gender difference and effect of estrogen receptor gene disruption.

The present study was designed to test the hypothesis that estrogen receptors (ER) in the blood vessel wall play a role in the modulation of the release of endothelium-derived nitric oxide (EDNO). Both basal and stimulated release of EDNO were determined in aortic rings isolated from female and male wild-type and male homozygous estrogen receptor knock-out (ERKO) mice. 125I-17beta-estradiol binding in aortic tissue showed significantly more high affinity cytosolic- nuclear-binding sites in male compared with female wildtype mice. Estrogen receptor transcripts were present in the aorta of male wild-type mice, but they were absent in male ERKO animals. Basal release of EDNO (determined by endothelium-dependent contraction caused by NG-nitro-arginine) was significantly higher in aorta of wild-type male mice compared with wild-type female mice, and significantly lower in the aorta of male ERKO compared with male wild-type mice. Acetylcholine-induced endothelium-dependent relaxation was similar in all groups studied. No difference was observed in the activity of calcium-dependent nitric oxide synthase in homogenates of lungs and brain taken from male wild-type and ERKO mice. These studies show a significant association between the number of estrogen receptors and basal release of EDNO in the aorta of mice, and suggest that decreased vascular estrogen receptor number may represent a novel risk factor for cardiovascular diseases.

Identification of DNA methylation markers for human breast carcinomas using the methylation-sensitive restriction fingerprinting technique.

We have developed a PCR-based method, called methylation-sensitive restriction fingerprinting (MSRF), to screen changes in DNA methylation in breast carcinomas. Two hypermethylation-containing fragments, HBC-1 (for "hypermethylation in breast cancer") and HBC-2, were identified in the amplified breast tumor DNA relative to the amplified normal breast DNA of a patient. Nucleotide sequence analysis revealed no significant matches between the sequence of HBC-1 and the known sequences in the GenBank database, whereas the sequence of HBC-2 matched the upstream region of an antisense WT1 (Wilms' tumor suppressor gene) promoter. The methylation status in the breast tumor DNA from this patient was confirmed by Southern hybridization using HBC-1 and HBC-2 as probes, respectively. Further analysis showed that HBC-1 was methylated aberrantly in 90% (17 of 19 patients) of the primary breast carcinomas examined. This study demonstrates that MSRF provides a useful means for screening aberrant changes in DNA methylation during tumorigenesis. The commonly methylated fragments identified by MSRF could potentially supplement pathological markers currently used for cancers and additionally lead to the discovery of novel methylated tumor suppressor genes.

Expression and localization of androgen receptor in the R-3327 Dunning rat prostatic adenocarcinoma.

The Dunning R-3327 rat prostatic adenocarcinoma and its sublines have been developed as a model system to study prostate tumor progression. We have used this system to study the changes in androgen receptor (AR) and AR mRNA expression which occur during tumor progression from androgen dependent to androgen independent growth. Dorsal prostate and all tumor sublines contained a 10-kilobase AR mRNA on Northern blot analysis. The levels of AR mRNA in each subline compared to dorsal prostate (100%) were: H (75%) greater than G (48%) greater than HI (25%) greater than HI-F = AT-1 = AT-3 = MAT-Lu = MAT-Ly-Lu = less than 5%. Immunocytochemistry showed AR predominantly in acinar epithelial cells of dorsal prostate and the androgen sensitive H subline. In the H subline, both acinar epithelial cells and locally invasive adenocarcinoma cells within the stroma showed positive immunostaining. The androgen responsive, anaplastic G subline also showed strong positive immunostaining. The androgen resistant AT-1 and MAT-Lu sublines lacked immunostaining for the AR. Steroid autoradiography revealed a similar cellular distribution of AR. These data suggest that in the Dunning system the loss of androgen binding and responsiveness is primarily due to selective changes in gene expression and not to gene rearrangements or posttranscriptional or translational modification of the AR mRNA or protein.

Repression of cancer protective genes by 17beta-estradiol: ligand-dependent interaction between human Nrf2 and estrogen receptor alpha.

Repression of cancer-protective phase II enzymes may help explain why estrogen exposure leads to the development of cancer. In an earlier report we described the ability of 17beta-estradiol (E(2)) to repress phase II enzyme activity in vivo. Phase II enzymes are coordinately regulated via the presence of the antioxidant response element (ARE) in their promoter. We wanted to determine if estrogen receptors (ER) repress ARE-dependent gene expression through a mechanism that requires interaction with Nrf2, the transcription factor that regulates ARE-mediated gene transcription. E(2)-bound ERalpha, but not ERbeta, represses ARE-regulated gene expression in the presence of exogenously expressed Nrf2 as well as when the transactivation domain of Nrf2 was fused to a heterologous DNA-binding domain. Deletion of the activation function-2 (AF-2) and the ligand-binding domain of ERalpha result in a constitutive repression of Nrf2-mediated transcription. Finally, E(2)-bound ERalpha co-immunoprecipitates with Nrf2. Repression of Nrf2-mediated transcription by E(2)-bound ERalpha expands our knowledge of E(2)-regulated genes and provides a potential drug-screening target for the development of selective estrogen receptor modulators with a lower risk of causing cancer.

Antibodies to steroid receptor deoxyribonucleic acid binding domains and their reactivity with the human glucocorticoid receptor.

Functional properties of the DNA-binding domain of the human glucocorticoid receptor were investigated using high titer polyclonal antibodies produced against single synthetic peptides or a mixture of peptides whose sequences were derived from the DNA-binding domain of steroid receptor proteins. Three of seven antisera recognized both native and denatured forms of the glucocorticoid receptor, although considerably lower antisera dilutions were required for antibody binding to native receptor. Activation of the glucocorticoid receptor to its DNA-binding form was required for antibody recognition of the native receptor. Antisera to the second finger region of the DNA-binding domain caused a portion of the activated 4S glucocorticoid receptor to sediment as 7 or 9S in sucrose gradients containing 0.4 M KCl, but did not alter the sedimentation of the nontransformed 8S receptor. Specificity of the glucocorticoid receptor-antibody interaction was demonstrated by loss of reactivity after preabsorption with peptide antigens. Antisera that interacted specifically with the glucocorticoid receptor inhibited DNA binding of the activated receptor by as much as 80%. Thus, antibody probes directed against DNA-binding domain sequences provide immunological evidence that glucocorticoid receptor activation exposes the DNA-binding region of the receptor.

Autologous down-regulation of androgen receptor messenger ribonucleic acid.

Autoregulation of androgen receptor (AR) mRNA was investigated using Northern blot analysis with AR cDNA fragments as probes. The amount of AR mRNA increased 2- to 10-fold with androgen withdrawal and decreased below control levels after androgen stimulation in rat ventral prostate, coagulating gland, epididymis, seminal vesicle, kidney, and brain, and in a human prostate cancer cell line, LNCaP. In rat ventral prostate, AR mRNA increased 2- to 3-fold within 24 h after castration and remained elevated for 4 days. Treatment with testosterone propionate beginning 24 h after castration reduced ventral prostate AR mRNA 4-fold within 8 h of androgen replacement. Administration of estradiol 24 h after castration had no significant effect on prostatic AR mRNA. Androgens, including testosterone and the synthetic androgen methyltrienolone (R1881), or the antiandrogen cyproterone acetate down-regulated AR mRNA in vitro in LNCaP cells, whereas estradiol was without effect. Administration of testosterone propionate to rats with androgen insensitivity did not decrease AR mRNA. Down-regulation of AR mRNA by androgen is therefore a receptor-mediated process which occurs in vivo in rat tissues that differ in androgen responsiveness and in cultured human prostate cells.

Novel antipeptide antibodies to the human glucocorticoid receptor: recognition of multiple receptor forms in vitro and distinct localization of cytoplasmic and nuclear receptors.

We have synthesized two peptides that correspond to unique regions of the amino-terminus of the human glucocorticoid receptor (GR). Peptides representing amino acids 245-259 and 346-367 (designated 59 and 57, respectively) were chosen on the basis of hydrophobicity/hydrophilicity ratios as well as overall proline content. These peptides were then used as antigens to produce epitope-specific antibodies that recognize and interact with human GR in a variety of physical states. Antiserum directed against each peptide recognizes denatured, [3H] dexamethasone mesylate-labeled GR as well as unliganded receptor on Western blots. In contrast to other antipeptide GR antibodies, these antibodies recognize and form stable complexes with unactivated and molybdate-stabilized forms of the GR, indicating that neither epitope is occluded when the receptor exists in an oligomeric state. Activated, 4S DNA-binding forms of the receptor are also recognized by both antibodies. The interaction of antibodies 59 and 57 with human GR in various states is highly specific based on the observation that preincubation of either antiserum with the appropriate peptide completely precludes the recognition of receptor by antibody. Titration analysis of antisera reveals that an increase in the antibody concentration cause discrete increases in the sedimentation coefficient of GR on sucrose gradients. These shifts occur under high salt conditions and are consistent with the formation of multiple stable antibody-receptor complexes. Interestingly, neither antibody interferes with the ability of the GR to be activated into a DNA-binding form or with the ability of the activated GR to interact with DNA cellulose. Consistent with these observations, both antibodies recognize and form stable complexes with GR when the receptor is associated with DNA fragments that contain specific glucocorticoid-responsive elements. Thus, both antibodies appear to recognize all known forms of the human GR protein. Using immunohistochemical techniques to visualize GR in HeLa S3 cells as well as in Chinese hamster ovary cells that stably express transfected human GR, a cytoplasmic location for receptor is observed in the absence of ligand. In contrast, immunoreactive GR is predominantly nuclear after hormone treatment, further supporting a role for nuclear translocation in GR function.

The human androgen receptor: complementary deoxyribonucleic acid cloning, sequence analysis and gene expression in prostate.

Androgenic hormones mediate their effects on male sex differentiation and development through a high affinity receptor protein. We report here cloning of the complete coding sequence of the human androgen receptor (hAR). By sequence homology hAR is a member of the nuclear receptor family, with closest sequence identity to the progesterone, mineralocorticoid, and glucocorticoid receptors. Regions of highest homology include the DNA-binding domain and a small region within the hydrophobic ligand-binding domain. Comparison of the deduced 919 amino acid sequence of hAR (98,999 mol wt) to the 902 amino acid sequence of rat AR (98,227 mol wt) reveals identical sequences in the DNA- and hormone-binding domains, with an overall homology of 85%. In human prostate, the major androgen receptor mRNA species is 10 kilobases while a less abundant mRNA is approximately 7 kilobases. Rabbit polyclonal antibodies were raised against a synthetic peptide from the N-terminal region of hAR. Immunocytochemical analysis of human prostate tissue demonstrated that AR is localized predominantly in nuclei of glandular epithelial cells.

The rat androgen receptor: primary structure, autoregulation of its messenger ribonucleic acid, and immunocytochemical localization of the receptor protein.

A composite androgen receptor DNA sequence 4,181 base pairs in length was determined from three cDNA clones isolated from a rat epididymal bacteriophage lambda gt11 library. An open reading frame of 902 amino acids encodes a protein of 98,227 mol wt. Structural domains characteristic of the steroid receptor family include an amino-terminal region with five repeated amino acid motifs, a central DNA-binding domain homologous with other steroid receptors, and a carboxyl-terminal steroid-binding region. A receptor cDNA probe used in Northern blot analysis hybridized with a predominant 10-kilobase androgen receptor mRNA in male reproductive tissues of the rat. Autoregulation of androgen receptor mRNA was indicated in rat ventral prostate by an increase in the level of 10-kilobase mRNA after castration and suppression of receptor mRNA upon androgen restimulation. A 15 amino acid peptide with sequence derived from the deduced androgen receptor sequence was synthesized and used as immunogen in raising receptor antibodies in rabbits. Antisera reacted with high titer against the synthetic peptide by enzyme-linked immunosorbent assay and against the native [3H]dihydrotestosterone-labeled androgen receptor as evidenced by an increase in receptor sedimentation rate determined by sucrose gradient centrifugation. Immunocytochemical staining localized the androgen receptor to epithelial cell nuclei in rat ventral prostate.

Cortisol alters gene expression during involution of the rat ventral prostate.

The ability of high doses of cortisol to retard the involution process in the rat ventral prostate was related to alterations in the pattern of gene expression. Poly(A)+ RNA preparations from the prostates of noncastrated, castrated, and castrated rats injected daily for 7 days with cortisol were compared by Northern blot hybridizations for the relative expression of genes associated with cell differentiation and maintenance (the C1 prostatic steroid binding protein gene and alpha-tubulin), with cell death (TRPM-2, hsp 70, and c-fos), and with hormone regulation (the androgen and glucocorticoid receptors). As anticipated, the concentration of C1 mRNA in the prostate fell to less than 4% of that in the noncastrated controls within 4 days after castration and was nearly undetectable after 7 days. This decline was retarded by cortisol treatment of 7-day castrated animals which sustained the level of C1 transcripts at approximately 50% of control. While the pattern of expression of alpha-tubulin indicated some minor fluctuations, with the highest level occurring 7 days after castration, the prostates of the cortisol-treated group had essentially the same concentration of this mRNA as the noncastrates. Cortisol also modified the expression of genes associated with prostatic cell death. The large increase in prostatic TRPM-2 mRNA, seen 7 days after castration, was reduced by over 80% after treatment with the glucocorticoid. Although not as abundantly expressed as TRPM-2, the castration-induced levels of transcripts for both hsp 70 and the protooncogene c-fos were substantially reduced by cortisol.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional characterization of naturally occurring mutant androgen receptors from subjects with complete androgen insensitivity.

Mutations in the androgen receptor (AR) are thought to cause complete androgen insensitivity (CAIS) in 46,XY human subjects who have a female phenotype despite normal adult male concentrations of plasma testosterone. Assays of AR binding in cultured skin fibroblasts from subjects with CAIS show either an apparent absence of AR (AR-) or normal levels of AR (AR+) binding. In several subjects with CAIS, AR-, no gross AR mutation was detected by Southern blot analyses of genomic DNA and normal sized 10 kilobase mRNA was present on Northern blots of poly(A+) RNA from cultured genital skin fibroblasts. We have used the polymerase chain reaction to amplify individual exons within the human AR gene of subjects with CAIS and have identified point mutations in three subjects. In one AR- subject (R774C), amino acid 774 was changed from arginine (CGC) to cysteine (TGC), in another AR- subject (R831Q), arginine (CGA) was changed to glutamine (CAA) at position 831, and in an AR+ subject (V866M) a methionine (ATG) was substituted for valine (GTG) at position 866. Transfection of wild type and mutant AR cDNA clones into COS cells results in detection of AR protein by immunoblotting. AR ligand binding activity is absent in cells transfected with AR mutants R774C and R831Q, but present with AR mutant V866M. Androgen binding in cells transfected with AR mutant V866M has a 6-fold lower apparent binding affinity than that of wild-type AR. Transcriptional activation of the MMTV-CAT reporter gene was androgen dependent and specific and nearly maximal at physiological concentrations (10(-10) M) of androgen when wild-type AR was transfected into cells, whereas neither AR mutants R774C nor R831Q were able to stimulate CAT activity even at 10(-8) M androgen. AR mutant V866M was able to stimulate CAT activity but the androgen dose dependency was shifted toward pharmacological concentrations of steroid that exceed in vivo levels. The molecular basis of CAIS in humans exhibits genetic heterogeneity. Our study shows that some cases of CAIS are explained by an inability to form a functional AR-steroid complex and hence, the AR is unable to activate transcription of genes essential for male sex differentiation during fetal development.

Role of estrogen receptor-alpha in the anterior pituitary gland.

Targeted insertional disruption of the mouse estrogen receptor-alpha (ER alpha) gene has provided a genetic model in which to test hypotheses that estrogens exert important effects in development and homeostatic functions of the anterior pituitary gland, particularly in the lactotroph and gonadotroph cell types. Analysis of ER alpha gene-disrupted mice reveals a marked reduction in PRL mRNA and a decrease in lactotroph cell number, but normal specification of lactotroph cell phenotype. Gonadotropin mRNA levels in ER alpha gene-disrupted female mice are elevated, consistent with previously described transcriptional suppression of gonadotropin subunit gene expression in response to sustained administration of estrogen in wild type mice. These results provide genetic evidence that ER alpha plays a critical role in PRL and gonadotropin gene transcription and is involved in lactotroph cell growth, but is not required for specification of lactotroph cell phenotype.

Expression of recombinant androgen receptor in cultured mammalian cells.

Full-length rat and human androgen receptor (AR) cDNA clones were expressed in COS-7 and CV1 monkey kidney cells to analyze the AR protein using immunological and cotransfection techniques. The studies were aided by the development of two rabbit polyclonal antibodies, designated AR32 and AR52, directed against epitopes within the N-terminal region of AR. Each antibody recognizes native AR by sucrose gradient analysis and detects a 114-kilodalton protein in COS cells transfected with human or rat AR cDNA. Covalent binding of the synthetic androgen [3H]methyltrienolone (R1881) to the 114-kDa protein was saturable. The endogenous native AR was similarly 114 kDa on immunoblots of a human prostate adenocarcinoma cell line, LNCaP, and rat sex accessory gland extracts. AR was localized in nuclei of transfected COS cells and in LNCaP cells by immunocytochemical staining. Androgen induction of CAT activity was dose dependent in CV1 cells cotransfected with the AR expression vector and a reporter plasmid containing the mouse mammary tumor virus promoter linked to the chloramphenicol acetyltransferase gene. It is concluded that antipeptide antibodies are useful reagents in characterizing both native and denatured forms of the AR protein. The 114-kDa protein expressed transiently in cultured cells represents the full-length AR protein, has a molecular size equivalent to that of endogenous AR, and mediates androgen-dependent transcriptional activation in CV1 cells.

A frameshift mutation destabilizes androgen receptor messenger RNA in the Tfm mouse.

A composite mouse androgen receptor DNA sequence was obtained by amplifying genomic DNA or cDNA using the polymerase chain reaction. The open reading frame was 2,697 basepairs, encoding a polypeptide of 899 amino acids (98,204 mol wt). Amino acid sequence comparisons indicated that the mouse androgen receptor (AR) is 97% homologous with rat AR and 83% with human AR. The amino acid sequences of the three receptors are identical within the DNA- and steroid-binding domains. Northern blot analysis revealed the predominant mouse AR mRNA to be 10 kilobases (kb). A 1.7-kb mRNA species was detected in mouse kidney using a cDNA probe containing only 5' untranslated AR sequence. Lack of hybridization with AR-coding sequence probes suggested that the 1.7-kb mRNA was not a truncated form of AR mRNA. Sequencing of genomic DNA isolated from testicular feminized (Tfm) mice revealed a single base deletion in the N-terminal domain, resulting in a frameshift mutation. Cycloheximide treatment caused a dramatic increase in AR mRNA in kidneys of Tfm mice, but not wild-type mice, suggesting that the Tfm mutation results in an unstable AR mRNA.

Analysis of transcription and estrogen insensitivity in the female mouse after targeted disruption of the estrogen receptor gene.

We employed homologous recombination in mouse embryonic stem cells to disrupt the estrogen receptor (ER) gene. Subsequently generated mice that are homozygous for the gene disruption, termed ERKO, possess no demonstrable wild-type ER by Western blot analysis. However, the presence of residual high affinity binding, as detected by [3H]estradiol binding assays and sucrose gradients in uterine extracts from ERKO females prompted further investigation of transcription and translation products from the disrupted ER gene. Analysis of ERKO uterine messenger RNA (mRNA) by reverse transcriptase-polymerase chain reaction demonstrated that although no full-length wild-type ER mRNA was present, two smaller transcripts, labeled E1 and E2, were identified and partially sequenced. Both ERKO transcripts are splicing variants that result in the disrupting NEO sequence being partially or completely removed from the mRNA. In the ERKO-E2 variant, this results in a frame shift and the creation of at least two stop codons downstream. In the ERKO-E1 variant, the ER reading frame is preserved and encodes for a smaller mutant ER that could be the source of the residual estradiol binding. When this mutant form is overexpressed and characterized in vitro, it results in a smaller protein of the predicted size that possesses significantly reduced estrogen-dependent transcriptional activity compared with that of the wild-type ER. Despite residual amounts of an impaired ER variant, estrogen insensitivity in the female ERKOs was confirmed by the failure of estrogen treatment to induce known uterine markers of estrogen action, such as increased DNA synthesis, and transcription of the progesterone receptor, lactoferrin, and glucose-6-phosphate dehydrogenase genes. Furthermore, serum levels of estradiol in the ERKO female are more than 10-fold higher than those in the wild type, consistent with a syndrome of hormone insensitivity.

Complete androgen insensitivity due to deletion of exon C of the androgen receptor gene highlights the functional importance of the second zinc finger of the androgen receptor in vivo.

Androgen-dependent gene transcription is mediated by the androgen receptor (AR) through interaction of its central zinc finger region with specific DNA sequences on target genes. Failure of this receptor-mediated gene transcription results in end organ resistance to androgens-the androgen insensitivity syndromes. In a pair of siblings with complete androgen insensitivity who had supranormal levels of androgen binding in genital skin fibroblasts, polymerase chain reaction and Southern blot analysis of the androgen receptor gene confirmed by polymerase chain reaction and sequence analysis of AR cDNA, revealed an in-frame deletion of exon C encoding the second zinc finger of the receptor. The mutant receptor in cultured genital skin fibroblasts had normal androgen binding affinity and was localized in the nucleus but had markedly reduced DNA-binding affinity. When recreated in vitro and tested in a cotransfection assay system the mutant receptor failed to activate transcription of an androgen-responsive reporter gene. This naturally occurring mutation highlights the functional dependence of the AR upon its second zinc finger in vivo and explains the complete insensitivity to androgen manifest by the affected individuals despite increased androgen binding. The elevated AR levels in the subjects' genital skin fibroblasts further suggests a possible role for the second zinc finger in autoregulation of receptor levels in vivo.

Effects of Elderberry Juice from Different Genotypes on Oxidative and Inflammatory Responses in Microglial Cells.

Many species of berries are nutritious food and offer health benefits. However, among the different types of berries, information on health effects of American elderberries (Sambucus nigra subsp. canadensis) has been lacking and little is known about whether elderberry consumption can confer neuroprotective effects on the central nervous system. Microglial cells constitute a unique class of immune cells and exhibit characteristic properties to carry out multifunctional duties in the brain. Activation of microglial cells has been implicated in brain injury and in many types of neurodegenerative diseases. Our recent studies demonstrated the ability for endotoxin (lipopolysaccharide, LPS) and interferon gamma (IFNγ) to induce reactive oxygen species (ROS) and nitric oxide (NO) in murine microglial cells (BV-2) through activating NADPH oxidase and the MAPK pathways. In this study, BV-2 microglial cells were used to examine effects of elderberry juice obtained from different genotypes on oxidative and inflammatory responses induced by LPS and IFNγ. Results show that 'Wyldewood' extract demonstrated antioxidant properties by inhibiting IFNγ-induced ROS production and p-ERK1/2 expression. On the other hand, most juice extracts exerted small effects on LPS-induced NO production and some extracts showed an increase in NO production upon stimulation with IFNγ. The disparity of responses on ROS and NO production from different extracts suggests possible presence of unknown endogenous factor(s) in the extract in promoting the IFNγ-induced iNOS synthesis pathway.