BACKGROUND

The perceptions of patients and physicians regarding the symptoms and impact of allergic rhinitis (AR) were assessed in a prospective, cross-sectional, international survey. This paper presents the combined survey results from five European countries (Germany, France, Italy, Spain and the UK).

METHODS

Data were recorded by 1,482 patients and matched with records from 415 primary care physicians and specialists. Diagnostic tests to confirm AR had been performed on 1,279 (86.3%) patients. Both physicians and patients recorded the presence, severity and impact of symptoms at the time of consultation in addition to those symptoms frequently, but not currently, present. Health-related quality of life (HRQoL) was assessed using the Mini Rhinoconjunctivitis Quality of Life Questionnaire.

RESULTS

A large proportion of patients had moderate-severe disease (67.2%; n = 996), persistent disease (42.5%; n = 630) and comorbidities such as asthma (31.5%; n = 467). Overall, patients rated their disease as more severe than did physicians (P < 0.001). At the time of the consultation, one-third of all patients reported that their current nasal and ocular symptoms were moderate or severe in nature. According to the physicians' assessment, good control of nasal and ocular symptoms was achieved in 45.4% (n = 673) and 51.3% (n = 760) of patients, respectively, and poor symptom control in 18.0% (n = 267) and 12.1% (n = 179). Overall, 43.3% (n = 641) of those surveyed were using two or more medicines for their AR. Health-related quality of life was correlated with disease severity and with the number of days without symptoms in the previous 4 weeks. Allergic rhinitis had a significantly greater impact in patients with more persistent disease than in those with intermittent disease (2.3 +/- 1.3 vs 1.9 +/- 1.2; P < 0.001). Nonetheless, 81.8% (n = 601) of patients with intermittent disease reported some impairment of their daily life as a result of their AR.

CONCLUSIONS

Allergic rhinitis remains a significant health problem because of the high burden of symptoms and its impact on general well being and HRQoL among patients consulting for this condition. Overall, there was a poor correlation between patients and physicians in the reporting of disease severity.

BACKGROUND

The androgen receptor (AR) is expressed frequently in breast cancer, but its prognostic significance is unclear. Preclinical data suggest that expression of AR may modify clinical outcomes in early breast cancer with improved prognosis in estrogen receptor (ER)-positive disease and poorer prognosis in ER-negative disease.

METHODS

A systematic review of electronic databases was conducted to identify studies published between 1946 and July 2012 and to explore the association between AR expression and overall survival (OS) and disease-free survival (DFS) in women diagnosed with early breast cancer. The odds ratios (OR) for OS and DFS at 3 and 5 years were calculated and then weighted and pooled in a meta-analysis with Mantel-Haenszel random-effect modeling. All statistical tests were two-sided.

RESULTS

Nineteen studies with a total of 7693 women were included. AR expression was documented in 60.5% of patients. ER-positive tumors were more likely to express AR- than ER-negative tumors (74.8% vs 31.8%, χ(2) P < .001). Compared with tumors without AR expression, those expressing AR were associated with improved OS at both 3 and 5 years (OR = 0.47, 95% confidence interval [CI] = 0.39 to 0.58, P < .001; and OR = 0.40, 95% CI = 0.29 to 0.56, P < .001). The absolute differences in the probability of OS at 3 and 5 years were 6.7% (95% CI = 3.5% to 9.8%) and 13.5% (95% CI = 7.5% to 19.6%), respectively. Results for 3- and 5-year DFS were similar. Coexpression of the ER did not influence OS at 3 or at 5 years.

CONCLUSIONS

Expression of AR in women with breast cancer is associated with better OS and DFS irrespective of coexpression of ER.

Dinitrogen (N2) was reduced to ammonia at room temperature and 1 atmosphere with molybdenum catalysts that contain tetradentate [HIPTN3N]3- triamidoamine ligands (such as [HIPTN3N]Mo(N2), where [HIPTN3N]3- is [(3,5-(2,4,6-i-Pr3C6H2)2C6H3NCH2CH2)3N]3-) in heptane. Slow addition of the proton source [(2,6-lutidinium)(BAr'4), where Ar' is 3,5-(CF3)2C6H3]and reductant (decamethyl chromocene) was critical for achieving high efficiency ( approximately 66% in four turnovers). Numerous x-ray studies, along with isolation and characterization of six proposed intermediates in the catalytic reaction under noncatalytic conditions, suggest that N2 was reduced at a sterically protected, single molybdenum center that cycled from Mo(III) through Mo(VI) states.

The precise molecular mechanisms by which prostate cancer cells progress from androgen-sensitive to androgen-insensitive status still remain largely unclear. The hepatocyte growth factor/scatter factor (HGF/SF) plays a critical role in the regulation of cell growth, cell motility, morphogenesis, and angiogenesis. The aberrant expression of HGF/SF and its receptor, c-Met, often correlates with poor prognosis in a variety of human malignancies, including prostate cancer. Here, we investigate a potential link between androgen signaling and c-Met expression in prostate cancer cells. First, we showed that the androgen receptor (AR) represses the expression of c-Met in a ligand-dependent manner. Using different c-Met promoter/reporter constructs, we identified that Sp1 induces the transcription of c-Met and that AR can repress the Sp1-induced transcription in prostate cancer cells. Moreover, the data from electrophoretic mobility shift assay showed that AR interferes with the interaction between Sp1 and the functional Sp1 binding site within the c-Met promoter. Furthermore, we tested the effect of AR on c-Met expression in an androgen-insensitive prostate cancer cell line, CWR22Rv1. Finally, the repressive role of androgen signaling on c-Met expression was confirmed in prostate cancer xenografts. The above data indicate a dual role of AR in transcriptional regulation. Although the current androgen ablation therapy can repress the expression of growth-promoting genes that are activated by the AR, it may also attenuate the repressive role of AR on c-Met expression. Therefore, the therapeutic strategies to inhibit the activation of the HGF/c-Met pathway may be of benefit when combined with current androgen ablation treatment.

Castration-resistant prostate cancer continues to rely on androgen receptor (AR) expression. AR plays a central role in the development of prostate cancer and progression to castration resistance during and after androgen deprivation therapy. Here, we identified miR-let-7c as a key regulator of expression of AR. miR-let-7c suppresses AR expression and activity in human prostate cancer cells by targeting its transcription via c-Myc. Suppression of AR by let-7c leads to decreased cell proliferation of human prostate cancer cells. Down-regulation of Let-7c in prostate cancer specimens is inversely correlated with AR expression, whereas the expression of Lin28 (a repressor of let-7) is correlated positively with AR expression. Our study demonstrates that the miRNA let-7c plays an important role in the regulation of androgen signaling in prostate cancer by down-regulating AR expression. These results suggest that reconstitution of miR-let-7c may aid in targeting enhanced and hypersensitive AR in advanced prostate cancer.

The glucocorticoid receptor (GR) is a ubiquitously expressed transcription factor present in most cell types. Upon ligand binding, the GR is activated and translocates into the nucleus where it transmits the anti-inflammatory actions of glucocorticoids. Here, we describe the ligand-independent activation of GR by the beta2-adrenergic receptor (beta2-AR) agonists, salbutamol and salmeterol, in primary human lung fibroblasts and vascular smooth muscle cells. Immunohistochemistry demonstrated expression of GR and the beta2-AR by fibroblasts and vascular smooth muscle cells. Treatment of the cells with the beta2-AR agonists, salbutamol or salmeterol, resulted in translocation of GR into the nucleus beginning at 30 min, as shown by immunohistochemistry and Western blotting of cytosolic and nuclear cell extracts. In comparison, activation of GR induced by the corticosteroids dexamethasone and fluticasone occurred at the same time after treatment (30 min) but resulted in a more complete depletion of GR from the cytosolic compartment. Electrophoretic mobility shift assays confirmed that nuclear GR, activated by both beta2-AR agonists and glucocorticoids, actively bound to the GR consensus sequence (GR element). Functional activation of the GR was confirmed by a Luciferase reporter gene assay, using a GR driven promoter fragment from the p21((WAF1/CIP1)) gene. The effects of the beta2-AR agonists, salbutamol and salmeterol, were dependent upon binding to the beta2-AR, because blocking of beta2-AR with propranolol abrogated GR activation. GR activation appeared to involve cAMP. In summary, these data show that beta2-AR agonists are potent activators of GR. Ligand-independent activation of GR by beta2-AR agonists may substantially mediate the anti-inflammatory actions of these drugs observed in vitro and in vivo.

A new cognitive therapy (CT) program was compared with an established behavioral treatment. Sixty-two patients meeting Diagnostic and Statistical Manual of Mental Disorders (4th ed.; American Psychiatric Association, 1994) criteria for social phobia were randomly assigned to CT, exposure plus applied relaxation (EXP = AR), or wait-list (WAIT). CT and EXP = AR were superior to WAIT on all measures. On measures of social phobia, CT led to greater improvement than did EXP = AR. Percentages of patients who no longer met diagnostic criteria for social phobia at posttreatment-wait were as follows: 84% in CT, 42% in EXP = AR, and 0% in WAIT. At the 1-year follow-up, differences in outcome persisted. In addition, patients in EXP = AR were more likely to have sought additional treatment. Therapist effects were small and nonsignificant. CT appears to be superior to EXP = AR in the treatment of social phobia.

It has been demonstrated previously that both the cystic fibrosis transmembrane conductance regulator (CFTR) and beta(2) adrenergic receptor (beta(2)AR) can bind ezrinradixinmoesin-binding phosphoprotein 50 (EBP50, also referred to as NHERF) through their PDZ motifs. Here, we show that beta(2) is the major adrenergic receptor isoform expressed in airway epithelia and that it colocalizes with CFTR at the apical membrane. beta(2)AR stimulation increases CFTR activity, in airway epithelial cells, that is glybenclamide sensitive. Deletion of the PDZ motif from CFTR uncouples the channel from the receptor both physically and functionally. This uncoupling is specific to the beta(2)AR receptor and does not affect CFTR coupling to other receptors (e.g., adenosine receptor pathway). Biochemical studies demonstrate the existence of a macromolecular complex involving CFTR-EBP50-beta(2)AR through PDZ-based interactions. Assembly of the complex is regulated by PKA-dependent phosphorylation. Deleting the regulatory domain of CFTR abolishes PKA regulation of complex assembly. This report summarizes a macromolecular signaling complex involving CFTR, the implications of which may be relevant to CFTR-dysfunction diseases.

Advanced prostate cancer can progress to systemic metastatic tumors, which are generally androgen insensitive and ultimately lethal. Here, we report a comprehensive genomic survey for somatic events in systemic metastatic prostate tumors using both high-resolution copy number analysis and targeted mutational survey of 3508 exons from 577 cancer-related genes using next generation sequencing. Focal homozygous deletions were detected at 8p22, 10q23.31, 13q13.1, 13q14.11, and 13q14.12. Key genes mapping within these deleted regions include PTEN, BRCA2, C13ORF15, and SIAH3. Focal high-level amplifications were detected at 5p13.2-p12, 14q21.1, 7q22.1, and Xq12. Key amplified genes mapping within these regions include SKP2, FOXA1, and AR. Furthermore, targeted mutational analysis of normal-tumor pairs has identified somatic mutations in genes known to be associated with prostate cancer including AR and TP53, but has also revealed novel somatic point mutations in genes including MTOR, BRCA2, ARHGEF12, and CHD5. Finally, in one patient where multiple independent metastatic tumors were available, we show common and divergent somatic alterations that occur at both the copy number and point mutation level, supporting a model for a common clonal progenitor with metastatic tumor-specific divergence. Our study represents a deep genomic analysis of advanced metastatic prostate tumors and has revealed candidate somatic alterations, possibly contributing to lethal prostate cancer.

Current approaches to inhibit nuclear receptor (NR) activity target the hormone binding pocket but face limitations. We have proposed that inhibitors, which bind to nuclear receptor surfaces that mediate assembly of the receptor's binding partners, might overcome some of these limitations. The androgen receptor (AR) plays a central role in prostate cancer, but conventional inhibitors lose effectiveness as cancer treatments because anti-androgen resistance usually develops. We conducted functional and x-ray screens to identify compounds that bind the AR surface and block binding of coactivators for AR activation function 2 (AF-2). Four compounds that block coactivator binding in solution with IC(50) approximately 50 microM and inhibit AF-2 activity in cells were detected: three nonsteroidal antiinflammatory drugs and the thyroid hormone 3,3',5-triiodothyroacetic acid. Although visualization of compounds at the AR surface reveals weak binding at AF-2, the most potent inhibitors bind preferentially to a previously unknown regulatory surface cleft termed binding function (BF)-3, which is a known target for mutations in prostate cancer and androgen insensitivity syndrome. X-ray structural analysis reveals that 3,3',5-triiodothyroacetic acid binding to BF-3 remodels the adjacent interaction site AF-2 to weaken coactivator binding. Mutation of residues that form BF-3 inhibits AR function and AR AF-2 activity. We propose that BF-3 is a previously unrecognized allosteric regulatory site needed for AR activity in vivo and a possible pharmaceutical target.

MCM2 and MCM3 are essential genes believed to play important roles in the initiation of DNA replication in Saccharomyces cerevisiae. Mutants defective in Mcm2 or Mcm3 are remarkably similar in phenotype. They both show an autonomously replicating sequence (ARS)-specific minichromosome maintenance defect, although their ARS specificities are not identical. In addition, these mutants exhibit a premitotic cell cycle arrest and an increase in chromosome loss and recombination. Genetic studies suggest that the two MCM gene products play interacting or complementary roles in DNA replication. Double mutants of mcm2-1 and mcm3-1 are inviable at the permissive growth temperature (23 degrees C) for each of the single mutants. Furthermore, overproduction of Mcm3 accentuates the deleterious effect of the mcm2-1 mutation, whereas overproduction of Mcm2 partially complements the mcm3-1 mutation. MCM2 encodes a protein of 890 amino acids containing a putative zinc-finger domain that is essential for Mcm2 function. Mcm2 shows striking homology to Mcm3 and three other proteins, Cdc46 of S. cerevisiae, and Nda4 and Cdc21 of Schizosaccharomyces pombe. The phenotypes of mutants defective in these proteins suggest that they belong to a protein family involved in the early steps of DNA replication.

Alternative splicing has critical roles in normal development and can promote growth and survival in cancer. Aberrant splicing, the production of noncanonical and cancer-specific mRNA transcripts, can lead to loss-of-function in tumor suppressors or activation of oncogenes and cancer pathways. Emerging data suggest that aberrant splicing products and loss of canonically spliced variants correlate with stage and progression in malignancy. Here, we review the splicing landscape of TP53, BARD1 and AR to illuminate roles for alternative splicing in cancer. We also examine the intersection between alternative splicing pathways and novel therapeutic approaches.

BACKGROUND

Activation of the NF-kappaB transcription factor has been previously demonstrated in two androgen receptor negative prostate cancer cell lines. We wished to extend this work to additional prostate cancer cells and to characterize the mechanisms responsible for constitutive NF-kappaB activation.

METHODS

Electrophoretic mobility shift assays were performed to measure NF-kappaB DNA-binding activity in prostate cancer cell lines, and immunohistochemistry was performed to detect nuclear localization of NF-kappaB in prostate cancer tissues. Western blot analysis was used to study the status of IkappaBalpha. Transient transfection assays were employed to characterize the contributions of IkappaB kinase (IKK), MAPK kinase kinases (MAPKKKs), androgen receptor (AR), and tyrosine phosphorylation to the constitutive activation of NF-kappaB in the prostate cancer cell lines.

RESULTS

Constitutive NF-kappaB activity was observed in AR-negative cell lines as well as in the prostate cancer patient samples, but was not present in AR positive cells. A "super-repressor" IkappaBalpha, as well as dominant negative forms of IKKbeta and NF-kappaB-inducing kinase (NIK), and tyrosine kinase inhibition were able to suppress NF-kappaB activity in the cells with constitutive activation.

CONCLUSIONS

The constitutive activation of NF-kappaB observed in prostate cancer cells is likely due to a signal transduction pathway involving tyrosine kinases, NIK, and IKK activation.

It has been found that 4-estren-3alpha,17beta-diol, a synthetic ligand for the estrogen receptor (ER) or androgen receptor (AR), which does not affect classical transcription, reverses bone loss in ovariectomized females or orchidectomized males without affecting the uterus or seminal vesicles, demonstrating that the classical genotropic actions of sex steroid receptors are dispensable for their bone-protective effects, but indispensable for their effects on reproductive organs. We have now investigated the mechanism of action of this compound. We report that, identically to 17beta-estradiol or dihydrotestosterone, but differently from raloxifene, estren alters the activity of Elk-1, CCAAT enhancer binding protein-beta (C/EBPbeta), and cyclic adenosine monophosphate-response element binding protein (CREB), or c-Jun/c-Fos by an extranuclear action of the ER or AR, resulting in activation of the Src/Shc/ERK pathway or downregulation of JNK, respectively. All of these effects are non-sex specific, require only the ligand-binding domain of the receptor, and are indispensable for the antiapoptotic action of these ligands on osteoblastic and HeLa cells. Moreover, administration of 17beta-estradiol or 4-estren-3alpha,17beta-diol to ovariectomized mice induces phosphorylation of ERKs, Elk-1, and C/EBPbeta, downregulates c-Jun, and upregulates the expression of egr-1, an ERK/SRE target gene. Kinase-initiated regulation of commonly used transcription factors offers a molecular explanation for the profound skeletal effects of sex steroid receptor ligands, including synthetic ones that are devoid of classical transcriptional activity.

BACKGROUND

The body of knowledge regarding rhinosinusitis(RS) continues to expand, with rapid growth in number of publications, yet substantial variability in the quality of those presentations. In an effort to both consolidate and critically appraise this information, rhinologic experts from around the world have produced the International Consensus Statement on Allergy and Rhinology: Rhinosinusitis (ICAR:RS).

METHODS

Evidence-based reviews with recommendations(EBRRs) were developed for scores of topics, using previously reported methodology. Where existing evidence was insufficient for an EBRR, an evidence-based review (EBR)was produced. The sections were then synthesized and the entire manuscript was then reviewed by all authors for consensus.

RESULTS

The resulting ICAR:RS document addresses multiple topics in RS, including acute RS (ARS), chronic RS (CRS)with and without nasal polyps (CRSwNP and CRSsNP), recurrent acute RS (RARS), acute exacerbation of CRS (AECRS), and pediatric RS.

CONCLUSIONS

As a critical review of the RS literature, ICAR:RS provides a thorough review of pathophysiology and evidence-based recommendations for medical and surgical treatment. It also demonstrates the significant gaps in our understanding of the pathophysiology and optimal management of RS. Too often the foundation upon which these recommendations are based is comprised of lower level evidence. It is our hope that this summary of the evidence in RS will point out where additional research efforts may be directed.

Here we report the first characterization of replication timing and its regulation in the fission yeast Schizosaccharomyces pombe. We used three different synchronization methods: centrifugal elutriation, cdc10 temperature-shift and release, and starvation for deoxyribonucleoside triphosphates (dNTPs) by treatment with hydroxyurea (HU) followed by removal of HU, to study the times when specific autonomously replicating sequence elements (ARS elements; potential replication origins) replicate during S phase. We found that individual ARS elements replicate at characteristic times, some early and some late, independently of synchronization method. In wild-type cells treated with HU, early ARS elements replicated but late ones did not. However, in HU-treated mutant cells lacking the Rad3 (similar to human ATR and ATM) or Cds1 (similar to human CHK2) checkpoint kinase, both early and late ARS elements were able to replicate. Thus under conditions of dNTP starvation the Rad3 and Cds1 kinases are needed to suppress the replication of normally late-replicating regions.

Adenosine is elaborated in injured tissues where it suppresses inflammatory responses of essentially all immune cells, including production of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha). Most of the anti-inflammatory actions of adenosine have been attributed to signaling through the A(2A) adenosine receptor (A(2A)AR). Previously, however, it has been shown that the A(3)AR agonist N(6)-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide (IB-MECA) potently inhibited TNF-alpha release from macrophages obtained from A(2A)AR "knockout" (A(2A)KO) mice, suggesting that the A(3)AR may also regulate cytokine expression. Here, we confirmed that the A(2A)AR is the primary AR subtype that suppresses TNF-alpha release from thioglycollate-elicited mouse peritoneal macrophages induced by both Toll-like receptor-dependent (TLR) and TLR-independent stimuli, but we determined that the A(2B)AR rather than the A(3)AR mediates the non-A(2A)AR actions of adenosine since 1) the ability of IB-MECA to inhibit TNF-alpha release was not altered in macrophages isolated from A(3)KO mice, and 2) the A(2B)AR antagonist 1,3-dipropyl-8-[4-[((4-cyanophenyl)carbamoylmethyl)oxy]phenyl]xanthine (MRS 1754) blocked the ability of the nonselective AR agonist adenosine-5'-N-ethylcarboxamide (NECA) to inhibit TNF-alpha release from macrophages isolated from A(2A)KO mice. Although A(2B)ARs seem capable of inhibiting TNF-alpha release, the A(2A)AR plays a dominant suppressive role since MRS 1754 did not block the ability of NECA to inhibit TNF-alpha release from macrophages isolated from wild-type (WT) mice. Furthermore, the potency and efficacy of adenosine to inhibit TNF-alpha release from WT macrophages were not influenced by blocking A(2B)ARs with MRS 1754. The data indicate that adenosine suppresses TNF-alpha release from macrophages primarily via A(2A)ARs, although the A(2B)AR seems to play an underlying inhibitory role that may contribute to the anti-inflammatory actions of adenosine under select circumstances.

We investigated the effects of changes in luminal [Ca2+] on the gating of native and purified sheep cardiac sarcoplasmic reticulum (SR) Ca(2+)-release channels reconstituted into planar phospholipid bilayers. The open probability (Po) of channels activated solely by cytosolic Ca2+ was greater at positive than negative holding potentials. Channels activated solely by 10 microM cytosolic Ca2+ exhibited no change in steady-state Po or in the relationship between Po and voltage when the luminal [Ca2+] was increased from nanomolar to millimolar concentrations. In the absence of activating concentrations of cytosolic Ca2+, the channel can be activated by the phosphodiesterase inhibitor sulmazole (AR-L 115BS). However, cytosolic Ca(2+)-independent activation of the channel by sulmazole requires luminal Ca2+. In the presence of sulmazole, at picomolar luminal [Ca2+] the channel remains completely closed. Increasing the luminal [Ca2+] to millimolar levels markedly increases the Po via an increase in the duration of open events. The Po and duration of the sulmazole-activated, luminal Ca(2+)-dependent channel openings are voltage dependent. In the presence of micromolar luminal Ca2+, the Po and duration of sulmazole-activated openings are greater at negative voltages. However, at millimolar luminal [Ca2+], long openings are also observed at positive voltages and the Po appears to be similar at positive and negative voltages. Our findings indicate that the regulation of channel gating by luminal Ca2+ depends on the mechanism of channel activation.

This study tests the hypothesis that G-protein-coupled receptor (GPCR) signaling components involved in the regulation of adenylyl cyclase (AC) localize with caveolin (Cav), a protein marker for caveolae, in both cell-surface and intracellular membrane regions. Using sucrose density fractionation of adult cardiac myocytes, we detected Cav-3 in both buoyant membrane fractions (BF) and heavy/non-buoyant fractions (HF); beta2-adrenergic receptors (AR) in BF; and AC5/6, beta1-AR, M4-muscarinic acetylcholine receptors (mAChR), mu-opioid receptors, and Galpha(s) in both BF and HF. In contrast, M2-mAChR, Galpha(i3), and Galpha(i2) were found only in HF. Immunofluorescence microscopy showed co-localization of Cav-3 with AC5/6, Galpha(s), beta2-AR, and mu-opioid receptors in both sarcolemmal and intracellular membranes, whereas M2-mAChR were detected only intracellularly. Immunofluorescence of adult heart revealed a distribution of Cav-3 identical to that in isolated adult cardiac myocytes. Upon immunoelectron microscopy, Cav-3 co-localized with AC5/6 and Galpha(s) in sarcolemmal and intracellular vesicles, the latter closely allied with T-tubules. Cav-3 immunoprecipitates possessed components that were necessary and sufficient for GPCR agonist-promoted stimulation and inhibition of cAMP formation. The distribution of GPCR, G-proteins, and AC with Cav-3 in both sarcolemmal and intracellular T-tubule-associated regions indicates the existence of multiple Cav-3-localized cellular microdomains for signaling by hormones and drugs in the heart.

Capacitation of bovine sperm was evaluated by determining the ability of sperm to fertilize bovine oocytes in vitro and to undergo an acrosome reaction upon exposure to lysophosphatidylcholine (LC). Incubation of sperm with heparin (10 micrograms/ml) increased the percentage of oocytes fertilized, but this required exposing sperm to heparin for at least 4 h before adding them to oocytes. There was no effect on the percentage of motile or acrosome-reacted sperm after exposure of noncapacitated sperm to 100 micrograms/ml LC for 15 min. When sperm were incubated for 4 h with heparin, exposure to 100 micrograms/ml LC for 15 min had no effect on the percentage of sperm that were motile, but the percentage of acrosome-reacted sperm increased from less than 10% to over 70%. The acrosome reactions (ARs) induced by LC were synchronous, reached maximal levels within 15 min, and differed (p less than 0.001) between sperm incubated under capacitating (with heparin) and noncapacitating conditions (without heparin). The time course required for heparin to capacitate sperm as judged by in vitro fertilization and to render sperm sensitive to LC induction of the AR were found to be similar. The percentage of ARs induced by LC and percentage of oocytes fertilized by sperm were found to be heparin-dose-dependent, with the maximum responses occurring at 5-10 micrograms/ml heparin. The correlation between the mean fertilization and LC-induced AR percentages was 0.997 (p less than 0.01). These studies demonstrate capacitation of bovine sperm by heparin requires at least a 4-h exposure of sperm to heparin and suggest that plasma membrane changes prior to an AR can be detected by exposure of bovine sperm to LC.

Despite the initial androgen-dependent growth of most human prostate cancers, eventually all prostate cancers become androgen-independent at varying intervals after androgen ablation or anti-androgen therapy. In order to gain more insight into the role of the androgen receptor (AR) in this process, AR and prostate-specific antigen (PA) expression was evaluated immunohistochemically in prostatic tumour tissues from patients who developed urinary flow obstruction between 4 and 107 months after onset of treatment. AR expression was evaluated with a monoclonal antibody (MAb) specific for the N-terminal domain of the human AR. To substantiate the progressive tumour growth, proliferative activity was assessed immunohistochemically by staining with MAb Ki-67. Ki-67-defined tumour-growth fractions varied from 0.8-64.7%. In 13 of the 17 examined tumours over 80% of the tumour cells were AR-positive, 3 tumours showed a considerable heterogeneity in AR expression and in 1 tumour almost all tumour cells seemed to be AR-negative. Two-thirds of the examined tumours contained variable proportions of PA-positive tumour areas. These observations contrast with the view that androgen ablation induces a preferential outgrowth of receptor-negative tumour cells.

Toward development of a precision medicine framework for metastatic, castration-resistant prostate cancer (mCRPC), we established a multi-institutional clinical sequencing infrastructure to conduct prospective whole-exome and transcriptome sequencing of bone or soft tissue tumor biopsies from a cohort of 150 mCRPC affected individuals. Aberrations of AR, ETS genes, TP53, and PTEN were frequent (40%-60% of cases), with TP53 and AR alterations enriched in mCRPC compared to primary prostate cancer. We identified new genomic alterations in PIK3CA/B, R-spondin, BRAF/RAF1, APC, β-catenin, and ZBTB16/PLZF. Moreover, aberrations of BRCA2, BRCA1, and ATM were observed at substantially higher frequencies (19.3% overall) compared to those in primary prostate cancers. A total of 89% of affected individuals harbored a clinically actionable aberration, including 62.7% with aberrations in AR, 65% in other cancer-related genes, and 8% with actionable pathogenic germline alterations. This cohort study provides clinically actionable information that could affect treatment decisions for these affected individuals.

The A2A adenosine receptor (A2A-AR) transcript and radioligand binding sites have a distinct distribution in rat brain, restricted primarily to the striatum, nucleus accumbens and olfactory tubercles. We describe here the use of purified recombinant human A2A-ARs to generate a monoclonal antibody that has been used to better resolve the distribution of A2A-ARs in rat brain. The antibody can detect 1 ng of purified recombinant receptor by Western blotting and is potent (EC50 = 0.62 microg/ml) and highly selective for the A2A-AR subtype. By Western blotting, the apparent molecular mass of recombinant and rat striatal receptors shifts upon deglycosylation from 43-48 to 42 kilodaltons. Analyses of chimeric A1/A2A-ARs and synthesis of a blocking peptide pinpointed the epitope (SQPLPGER) of the antibody to the center of the third intracellular loop of the receptor. Incubation of rat striatal membranes with antibody reduces receptor coupling to G-proteins. In rat brain, dense A2A-AR-like immunoreactivity that is eliminated by the blocking peptide was found in the neuropil of the striatum, nucleus accumbens (rostral pole, core and shell), cell bridges of the striatum, olfactory tubercles, and areas of extended amygdala with somewhat lighter labeling in the globus pallidus and nucleus of the solitary tract. Light perikaryal labeling was found in other areas of the brain, including the cortex, hippocampus, thalamus, cerebellum, and portions of the hindbrain. The observed distribution of A2A-AR immunoreactivity throughout the neuraxis is consistent with the receptors' role in modulating dopaminergic neurotransmission and central control of cardiovascular function.

The androgen receptor (AR) plays a critical role in prostate cancer. We have identified a ubiquitin E3 ligase, RNF6, as an AR-associated protein in a proteomic screen. RNF6 induces AR ubiquitination and promotes AR transcriptional activity. Specific knockdown of RNF6 or mutation of RNF6-induced ubiquitination acceptor sites on AR selectively alters expression of a subset of AR target genes and diminishes recruitment of AR and its coactivators to androgen-responsive elements present in the regulatory region of these genes. Furthermore, RNF6 is overexpressed in hormone-refractory human prostate cancer tissues and required for prostate cancer cell growth under androgen-depleted conditions. Our data suggest that RNF6-induced ubiquitination may regulate AR transcriptional activity and specificity through modulating cofactor recruitment.