Kennedy disease, a degenerative disorder characterized by androgen-dependent neuromuscular weakness, is caused by a CAG/glutamine tract expansion in the androgen receptor (<em>Ar</em>) gene. We developed a mouse model of Kennedy disease, using gene targeting to convert mouse androgen receptor (<em>AR</em>) to human sequence while introducing 113 glutamines. <em>AR</em>113Q mice developed hormone and glutamine length-dependent neuromuscular weakness characterized by the early occurrence of myopathic and neurogenic skeletal muscle pathology and by the late development of neuronal intranuclear inclusions in spinal neurons. <em>AR</em>113Q males unexpectedly died at 2-4 months. We show that this androgen-dependent death reflects decreased expression of skeletal muscle chloride channel 1 (CLCN1) and the skeletal muscle sodium channel alpha-subunit, resulting in myotonic discharges in skeletal muscle of the lower urinary tract. <em>AR</em>113Q limb muscles show similar myopathic features and express decreased levels of mRNAs encoding neurotrophin-4 and glial cell line-derived neurotrophic factor. These data define an important myopathic contribution to the Kennedy disease phenotype and suggest a role for muscle in non-cell autonomous toxicity of lower motor neurons.

The rat A3 adenosine receptor (AR) is a recently characterized AR subtype cloned from testis and brain cDNA libraries. N6-2-(4-Amino-3-[125I]iodophenyl)ethyladenosine, a high affinity A1AR agonist, has served as the only radioligand available for study of the A3AR. The relatively low affinity of N6-2-(4-amino-3-[125I] iodophenyl)ethyladenosine for the A3AR and its greater A1AR selectivity necessitate the development of more appropriate radioligands for A3AR analysis. This report characterizes 125I-4-aminobenzyl-5'-N-methylcarboxamidoadenosine (125I-AB-MECA), a high affinity radioligand for the A3AR, in two cell lines that express this AR subtype. Membranes from Chinese hamster ovary (CHO) cells expressing the rat A3AR and from the rat mast cell line RBL-2H3 bound 125I-AB-MECA with Kd values of 1.48 +/- 0.33 nM and 3.61 +/- 0.30 nM, respectively. As determined by 125I-AB-MECA binding, levels of A3AR expression in the A3AR-CHO cell line and RBL-2H3 cells were 3.06 +/- 0.21 pmol/mg and 1.02 +/- 0.13 pmol/mg, respectively. Binding of 125I-AB-MECA was characterized in competition assays. In the A3AR-CHO cell line a potency order of cyclohexyl-5'-N-ethylcarboxamidoadenosine (cyclohexyl-NECA) = benzyl-NECA>> (-)-N6-[(R)-phenylisopropyl]adenosine = NECA was observed, and in RBL-2H3 cells (-)-N6-[(R)-phenylisopropyl]adenosine and NECA were equipotent. Xanthine amine congener (XAC) and 8-cyclopentyl-1,3-dipropylxanthine did not significantly inhibit 125I-AB-MECA binding. The parent compound, AB-MECA, dose-dependently inhibited forskolin-stimulated adenylyl cyclase activity in A3AR-CHO cell membranes. 125I-AB-MECA bound to the rat A1AR and canine A2aAR expressed in COS-7 cells with Kd values of 3.42 +/- 0.43 nM and 25.1 +/- 12.6 nM, respectively. This binding was significantly reduced in the presence of 1 microM XAC. In RBL-2H3 cells, XAC had no effect on 125I-AB-MECA affinity and reduced the level of radioligand binding by approximately 5%.

Genetic loss of function analysis is a powerful method for the study of protein function. However, some cell biological questions are difficult to address using traditional genetic strategies often due to the lack of appropriate genetic model systems. Here, we present a general strategy for the design and syntheses of molecules capable of inducing the degradation of selected proteins in vivo via the ubiquitin-proteasome pathway. Western blot and fluorometric analyses indicated the loss of two different targets: green fluorescent protein (GFP) fused with FK506 binding protein (FKBP12) and GFP fused with the androgen receptor (AR), after treatment with PROteolysis TArgeting Chimeric moleculeS (PROTACS) incorporating a FKBP12 ligand and dihydrotestosterone, respectively. These are the first in vivo examples of direct small molecule-induced recruitment of target proteins to the proteasome for degradation upon addition to cultured cells. Moreover, PROTAC-mediated protein degradation offers a general strategy to create "chemical knockouts," thus opening new possibilities for the control of protein function.

Previous studies showed that epidermal growth factor (EGF) and TGFalpha mimic the action of LH on the resumption of oocyte maturation. We tested whether EGF-like agents, such as amphiregulin (AR), epiregulin (ER), and betacellulin (BTC), also mediate the LH stimulation of the ovulatory response in the rat. LH induced transient follicular expression of AR, ER, and BTC mRNA, reaching a maximum after 3-h incubation. Furthermore, the addition of ER, AR, and BTC to the culture medium could mimic some of LH actions. AR and ER fully simulated LH-induced resumption of meiosis in vitro, whereas BTC was less effective. To study the putative involvement of EGF-like factors in mediation of LH signal, the effect of the EGF receptor kinase inhibitor AG1478 was tested. When added with LH, AG1478, but not its inactive analog AG43, reduced EGF receptor phosphorylation and oocyte maturation compared with follicles treated with LH only. In addition to the inhibition of resumption of meiosis, AG1478 administration into the bursa (3 microg/bursa) resulted in 51% (P < 0.0005) inhibition of ovulation in the treated ovaries, compared with the untreated contralateral ones, as well as to the vehicle-treated ovaries (P < 0.02). LH, as well as ER, induced the expression of genes associated with the ovulatory response like rat hyaluronan synthase-2, cyclooxygenase-2, and TNFalpha-stimulated gene 6 mRNA, whereas AG1478 inhibited this effect of LH. Release of EGF-like factors from the membrane is dependent on activated metalloproteases. Indeed, Galardin, a broad-spectrum metalloprotease inhibitor, but not a specific matrix metalloprotease 2 and 9 inhibitor, suppressed meiotic maturation induced by LH. Conversely, meiotic maturation induced by ER was not affected by Galardin, thus, supporting the notion that LH releases follicular membrane-bound EGF-like agents. In summary, EGF-like factors such as ER, AR, and BTC seem to mediate, at least partially, the LH stimulation of oocyte maturation, ovulatory enzyme expression, and ovulation.

An autosomal recessive form of juvenile Parkinsonism (AR-JP) (MIM 600116) is a levodopa-responsive Parkinsonism whose pathological finding is a highly selective degeneration of dopaminergic neurons in the zona compacta of the substantia nigra. By linkage analysis of diallelic polymorphism of the Mn-superoxide dismutase gene (SOD2), we found a family with AR-JP showing perfect segregation of the disease with the SOD2 locus. By extending the linkage analysis to 13 families with AR-JP, we discovered strong evidence for the localization of the AR-JP gene at chromosome 6q25.2-27, including the SOD2 locus, with the maximal cumulative pairwise LOD scores of 7.26 and 7.71 at D6S305 (theta = .03) and D6S253 (theta = .02), respectively. Observation of obligate recombination events, as well as multipoint linkage analysis, placed the AR-JP gene in a 17-cM interval between D6S437 and D6S264. Delineation of the AR-JP gene will be an important step toward our understanding of the molecular mechanism underlying selective degeneration of the nigral neurons.

Fat intake has long been associated with the development of obesity. The studies described herein show that fat adversely affects adipocyte adrenergic receptor (AR) expression and function. As beta 3AR agonists have been shown to acutely reduce adipose tissue mass and improve thermogenesis in genetically obese rodents, we examined whether chronic supplementation of a high fat diet with a highly selective beta 3AR agonist, CL316,243 could prevent diet-induced obesity, and whether the effect could be sustained over prolonged treatment. C57BL/6J and A/J mice were weaned onto one of three diets: low fat (10.5% calories from fat), high fat (58% calories from fat), or high fat supplemented with 0.001% CL316,243. B/6J mice gained more weight on the high fat diet than A/J mice (at 16 weeks: B/6J, 36.6 +/- 1.4 g; A/J, 32.9 +/- 0.8 g; P < 0.002; n = 10), whereas weights on the low fat diets were similar (B/6J, 29.5 +/- 0.5 g; A/J, 28.8 +/- 0.6 g; P>> 0.05; n = 10). CL316,243 prevented the development of diet-induced obesity in A/J animals, but not in B/6J animals. A/J mice weighed 26.0 +/- 0.5 g at 16 weeks, whereas B/6J animals on the same diet weighed 34.1 +/- 0.8 g (P < 0.00001; n = 10), but food intake was not different between the strains throughout the study. beta-Adrenergic stimulation of adenylyl cyclase in obese B/6J mice was decreased by more than 75% in white adipose tissue and by more than 90% in brown adipose tissue (BAT). In contrast, in fat-fed A/J mice, beta-agonist-stimulated adenylyl cyclase was decreased in white adipose tissue by about 10%, whereas the activity in interscapular BAT was decreased by 50%, indicating significant retention of beta AR-stimulated activity in A/J mice compared to B/6J mice. High fat feeding was associated with decreased expression of beta 3AR and beta 1AR in white adipose tissue of both strains. However, chronic CL316,243 treatment prevented both the obesity and the decline in beta 3AR and beta 1AR messenger RNA levels in all adipose depots from A/J mice, but not B/6J mice. As CL316,243-treated A/J mice, but not B/6J mice, also showed marked uncoupling protein expression in white adipose depots, the ability of chronic CL316,243 treatment to prevent diet-induced obesity is dependent upon the elaboration of functional BAT in these regions.

Development of the preterm infant gut microbiota is emerging as a critical research priority(1). Since preterm infants almost universally receive early and often extended antibiotic therapy(2), it is important to understand how these interventions alter gut microbiota development(3-6). Analysis of 401 stools from 84 longitudinally sampled preterm infants demonstrates that meropenem, cefotaxime and ticarcillin-clavulanate are associated with significantly reduced species richness. In contrast, vancomycin and gentamicin, the antibiotics most commonly administered to preterm infants, have non-uniform effects on species richness, but these can be predicted with 85% accuracy based on the relative abundance of only two bacterial species and two antibiotic resistance (AR) genes at treatment initiation. To investigate resistome development, we functionally selected resistance to 16 antibiotics from 21 faecal metagenomic expression libraries. Of the 794 AR genes identified, 79% had not previously been classified as AR genes. Combined with deep shotgun sequencing of all stools, we find that multidrug-resistant members of the genera Escherichia, Klebsiella and Enterobacter, genera commonly associated with nosocomial infections, dominate the preterm infant gut microbiota. AR genes that are enriched following specific antibiotic treatments are generally unique to the specific treatment and are highly correlated with the abundance of a single species. The most notable exceptions include ticarcillin-clavulanate and ampicillin, both of which enrich for a large number of overlapping AR genes, and are correlated with Klebsiella pneumoniae. We find that all antibiotic treatments are associated with widespread collateral microbiome impact by enrichment of AR genes that have no known activity against the specific antibiotic driver.

Fractional Gaussian noise (fGn) provides a parsimonious model for stationary increments of a self-similar process parameterised by the Hurst exponent, H, and variance, sigma2. Fractional Gaussian noise with H < 0.5 demonstrates negatively autocorrelated or antipersistent behaviour; fGn with H>> 0.5 demonstrates 1/f, long memory or persistent behaviour; and the special case of fGn with H = 0.5 corresponds to classical Gaussian white noise. We comparatively evaluate four possible estimators of fGn parameters, one method implemented in the time domain and three in the wavelet domain. We show that a wavelet-based maximum likelihood (ML) estimator yields the most efficient estimates of H and sigma2 in simulated fGn with 0 < H < 1. Applying this estimator to fMRI data acquired in the "resting" state from healthy young and older volunteers, we show empirically that fGn provides an accommodating model for diverse species of fMRI noise, assuming adequate preprocessing to correct effects of head movement, and that voxels with H>> 0.5 tend to be concentrated in cortex whereas voxels with H < 0.5 are more frequently located in ventricles and sulcal CSF. The wavelet-ML estimator can be generalised to estimate the parameter vector beta for general linear modelling (GLM) of a physiological response to experimental stimulation and we demonstrate nominal type I error control in multiple testing of beta, divided by its standard error, in simulated and biological data under the null hypothesis beta = 0. We illustrate these methods principally by showing that there are significant differences between patients with early Alzheimer's disease (AD) and age-matched comparison subjects in the persistence of fGn in the medial and lateral temporal lobes, insula, dorsal cingulate/medial premotor cortex, and left pre- and postcentral gyrus: patients with AD had greater persistence of resting fMRI noise (larger H) in these regions. Comparable abnormalities in the AD patients were also identified by a permutation test of local differences in the first-order autoregression AR(1) coefficient, which was significantly more positive in patients. However, we found that the Hurst exponent provided a more sensitive metric than the AR(1) coefficient to detect these differences, perhaps because neurophysiological changes in early AD are naturally better described in terms of abnormal salience of long memory dynamics than a change in the strength of association between immediately consecutive time points. We conclude that parsimonious mapping of fMRI noise properties in terms of fGn parameters efficiently estimated in the wavelet domain is feasible and can enhance insight into the pathophysiology of Alzheimer's disease.

Anoikis is the apoptotic response induced in normal cells by inadequate or inappropriate adhesion to substrate. It is postulated that resistance to anoikis facilitates tumorigenesis and metastasis. Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is an immunoglobulin superfamily member overexpressed in a number of human cancers and implicated in anoikis resistance. We tested the effect of CEACAM6 gene silencing on anoikis in pancreatic adenocarcinoma cell lines. Anoikis was induced in PANC1, Capan2, MiaPaCa2 and Mia(AR) (a MiaPaCa2-derived anoikis-resistant subline) by culture in poly-2-hydroxyethylmethacrylate-coated wells. Anoikis was quantified by YO-PRO-1/propidium iodide staining and flow cytometry. The role of caspase activation was determined using fluorometric profiling and the caspase inhibitor Z-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-fmk). CEACAM6 expression was suppressed by RNA interference. Using a nude mouse orthotopic xenograft model, we assessed the effect of this treatment on in vivo metastatic ability. Anoikis resistance was associated with increased CEACAM6 expression. CEACAM6-specific short interfering ribonucleic acid (siRNA), but not control siRNA, increased susceptibility to caspase-mediated anoikis, an effect abrogated by Z-VAD-fmk, and decreased Akt phosphorylation (Ser-473) under anchorage-independent conditions. CEACAM6 gene silencing reversed the acquired anoikis resistance of Mia(AR) and inhibited its in vivo metastatic ability. CEACAM6 warrants further investigation as a novel therapeutic target for the treatment of pancreatic adenocarcinoma.

BACKGROUND

The efficacy of antireflux surgery (ARS) and proton pump inhibitor therapy in the control of gastroesophageal reflux disease is well established. A direct comparison between these therapies is warranted to assess the benefits of respective therapies.

METHODS

There were 310 patients with erosive esophagitis enrolled in the trial. There were 155 patients randomized to continuous omeprazole therapy and 155 to open antireflux surgery, of whom 144 later had an operation. Because of various withdrawals during the study course, 122 patients originally having an antireflux operation completed the 5-year followup; the corresponding figure in the omeprazole group was 133. Symptoms, endoscopy, and quality-of-life questionnaires were used to document clinical outcomes. Treatment failure was defined to occur if at least one of the following criteria were fulfilled: Moderate or severe heartburn or acid regurgitation during the last 7 days before the respective visit; Esophagitis of at least grade 2; Moderate or severe dysphagia or odynophagia symptoms reported in combination with mild heartburn or regurgitation; If randomized to surgery and subsequently required omeprazole for more than 8 weeks to control symptoms, or having a reoperation; If randomized to omeprazole and considered by the responsible physician to require antireflux surgery to control symptoms; If randomized to omeprazole and the patient, for any reason, preferred antireflux surgery during the course of the study. Treatment failure was the primary outcomes variable.

RESULTS

When the time to treatment failure was analyzed by use of the intention to treat approach, applying the life table analysis technique, a highly significant difference between the two strategies was revealed (p < 0.001), with more treatment failures in patients who originally were randomized to omeprazole treatment. The protocol also allowed dose adjustment in patients allocated to omeprazole therapy to either 40 or 60 mg daily in case of symptom recurrence. The curves subsequently describing the failure rates still remained separated in favor of surgery, although the difference did not reach statistical significance (p = 0.088). Quality of life assessment revealed values within normal ranges in both therapy arms during the 5 years.

CONCLUSIONS

In this randomized multicenter trial with a 5-year followup, we found antireflux surgery to be more effective than omeprazole in controlling gastroesophageal reflux disease as measured by the treatment failure rates. But if the dose of omeprazole was adjusted in case of relapse, the two therapeutic strategies reached levels of efficacy that were not statistically different.

Androgen receptor (AR) signaling persists in castration-resistant prostate carcinomas (CRPC), because of several mechanisms that include increased AR expression and intratumoral androgen metabolism. We investigated the mechanisms underlying aberrant expression of transcripts involved in androgen metabolism in CRPC. We compared gene expression profiles and DNA copy number alteration (CNA) data from 29 normal prostate tissue samples, 127 primary prostate carcinomas (PCa), and 19 metastatic PCas. Steroidogenic enzyme transcripts were evaluated by quantitative reverse transcriptase PCR in PCa cell lines and circulating tumor cells (CTC) from CRPC patients. Metastatic PCas expressed higher transcript levels for AR and several steroidogenic enzymes, including SRD5A1, SRD5A3, and AKR1C3, whereas expression of SRD5A2, CYP3A4, CYP3A5, and CYP3A7 was decreased. This aberrant expression was rarely associated with CNAs. Instead, our data suggest distinct patterns of coordinated aberrant enzyme expression. Inhibition of AR activity by itself stimulated AKR1C3 expression. The aberrant expression of the steroidogenic enzyme transcripts was detected in CTCs from CRPC patients. In conclusion, our findings identify substantial interpatient heterogeneity and distinct patterns of dysregulated expression of enzymes involved in intratumoral androgen metabolism in PCa. These steroidogenic enzymes represent targets for complete suppression of systemic and intratumoral androgen levels, an objective that is supported by the clinical efficacy of the CYP17 inhibitor abiraterone. A comprehensive AR axis-targeting approach via simultaneous, frontline enzymatic blockade, and/or transcriptional repression of several steroidogenic enzymes, in combination with GnRH analogs and potent antiandrogens, would represent a powerful future strategy for PCa management.

OBJECTIVE

Mechanisms mediating androgen receptor (AR) reactivation in prostate cancer that progresses after castration (castration-resistant prostate cancer; CRPC) and subsequent treatment with abiraterone (CYP17A1 inhibitor that further suppresses androgen synthesis) remain unclear.

METHODS

Prostate cancer xenografts were examined to identify mechanism of progression after castration and abiraterone.

RESULTS

AR reactivation in abiraterone-resistant VCaP xenografts was not associated with restoration of intratumoral androgens or alterations in AR coregulators. In contrast, mRNA encoding full-length AR (AR-FL) and a constitutively active splice variant (AR-V7) were increased compared with xenografts before castration, with an increase in AR-V7 relative to AR-FL. This shift toward AR-V7 was due to a feedback mechanism whereby the androgen-liganded AR stimulates expression of proteins that suppress generation of AR-V7 relative to AR-FL transcripts. However, despite the increases in AR-V7 mRNA, it remained a minor transcript (<1%) relative to AR-FL in resistant VCaP xenografts and CRPC clinical samples. AR-V7 protein expression was similarly low relative to AR-FL in castration-resistant VCaP xenografts and androgen-deprived VCaP cells, but the weak basal AR activity in these latter cells was further repressed by AR-V7 siRNA.

CONCLUSIONS

AR-V7 at these low levels is not adequate to restore AR activity, but its rapid induction after androgen deprivation allows tumors to retain basal AR activity that may be needed for survival until more potent mechanisms emerge to activate AR. Agents targeting AR splice variants may be most effective when used very early in conjunction with therapies targeting the AR ligand-binding domain.

The European Position Paper on Rhinosinusitis and Nasal Polyps 2020 is the update of similar evidence based position papers published in 2005 and 2007 and 2012. The core objective of the EPOS2020 guideline is to provide revised, up-to-date and clear evidence-based recommendations and integrated care pathways in ARS and CRS. EPOS2020 provides an update on the literature published and studies undertaken in the eight years since the EPOS2012 position paper was published and addresses areas not extensively covered in EPOS2012 such as paediatric CRS and sinus surgery. EPOS2020 also involves new stakeholders, including pharmacists and patients, and addresses new target users who have become more involved in the management and treatment of rhinosinusitis since the publication of the last EPOS document, including pharmacists, nurses, specialised care givers and indeed patients themselves, who employ increasing self-management of their condition using over the counter treatments. The document provides suggestions for future research in this area and offers updated guidance for definitions and outcome measurements in research in different settings. EPOS2020 contains chapters on definitions and classification where we have defined a large number of terms and indicated preferred terms. A new classification of CRS into primary and secondary CRS and further division into localized and diffuse disease, based on anatomic distribution is proposed. There are extensive chapters on epidemiology and predisposing factors, inflammatory mechanisms, (differential) diagnosis of facial pain, allergic rhinitis, genetics, cystic fibrosis, aspirin exacerbated respiratory disease, immunodeficiencies, allergic fungal rhinosinusitis and the relationship between upper and lower airways. The chapters on paediatric acute and chronic rhinosinusitis are totally rewritten. All available evidence for the management of acute rhinosinusitis and chronic rhinosinusitis with or without nasal polyps in adults and children is systematically reviewed and integrated care pathways based on the evidence are proposed. Despite considerable increases in the amount of quality publications in recent years, a large number of practical clinical questions remain. It was agreed that the best way to address these was to conduct a Delphi exercise . The results have been integrated into the respective sections. Last but not least, advice for patients and pharmacists and a new list of research needs are included. The full document can be downloaded for free on the website of this journal: http://www.rhinologyjournal.com.

Using meta-analysis of eight independent transplant datasets (236 graft biopsy samples) from four organs, we identified a common rejection module (CRM) consisting of 11 genes that were significantly overexpressed in acute rejection (AR) across all transplanted organs. The CRM genes could diagnose AR with high specificity and sensitivity in three additional independent cohorts (794 samples). In another two independent cohorts (151 renal transplant biopsies), the CRM genes correlated with the extent of graft injury and predicted future injury to a graft using protocol biopsies. Inferred drug mechanisms from the literature suggested that two FDA-approved drugs (atorvastatin and dasatinib), approved for nontransplant indications, could regulate specific CRM genes and reduce the number of graft-infiltrating cells during AR. We treated mice with HLA-mismatched mouse cardiac transplant with atorvastatin and dasatinib and showed reduction of the CRM genes, significant reduction of graft-infiltrating cells, and extended graft survival. We further validated the beneficial effect of atorvastatin on graft survival by retrospective analysis of electronic medical records of a single-center cohort of 2,515 renal transplant patients followed for up to 22 yr. In conclusion, we identified a CRM in transplantation that provides new opportunities for diagnosis, drug repositioning, and rational drug design.

Agonist-independent properties of the human beta 2-adrenergic receptor (beta 2AR) were studied using the baculovirus expression system in Sf9 cells. In the absence of agonist but in the presence of GTP, membranes from cells expressing the beta 2AR exhibited higher levels of cAMP production than did membranes from uninfected cells or from cells infected with wild-type baculovirus. The increase in cAMP production was proportional to the number of beta 2AR expressed, up to 40 pmol/mg of membrane protein, and it could be inhibited in a dose-dependent manner by beta AR antagonists. The increase and its reversal both were independent of the possible presence of contaminating catecholamines in the culture medium and thus appear to reflect spontaneous beta 2AR activity and direct antagonist-receptor interactions, respectively. The maximal level of inhibition varied among the beta AR ligands tested, to yield the following rank order of "inverse efficacy"; timolol>> or = propranolol>> alprenolol>> or = pindolol>> labetalol>> dichloroisoproterenol. The same rank order was observed using membranes prepared from Chinese hamster fibroblasts expressing beta 2AR. The effect of timolol was partly blocked by labetalol and dichloroisoproterenol, in an apparently competitive manner. The intracellular cAMP content of Sf9 cells cultured in serum-free medium was also increased by the expression of beta 2AR, and that increase was reversed by timolol and propranolol, consistent with observations in membrane preparations. The properties revealed by the expression of the beta 2AR in Sf9 cells suggest two agonist-independent traits of G protein-linked receptors, i.e., 1) that unliganded receptors are able to activate G proteins both in membrane preparations and in whole cells and 2) that antagonists may mediate their effects not only by preventing the binding of agonists but also by decreasing the propensity of the receptor to assume an active state.

The proliferation and differentiation of normal prostate epithelial cells depends upon the action of androgens produced by the testis. Prostate cancers retain the ability to respond to androgens in the initial stages of cancer development, but progressively become independent of exogenous androgens in advanced stages of the disease while maintaining the expression of functional androgen receptor (AR). In the present study, we have determined the potential of prostate cancer cells to synthesize androgens from cholesterol which may be involved in intracrine regulation of AR in advanced stages of the disease. Established androgen-independent prostate cancer cell lines, PC3 and DU145 cells, expressed mRNA and proteins for scavenger receptor type B1 (SRB1), steroidogenic acute regulatory (StAR) protein, cytochrome P450 cholesterol side chain cleavage (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and other enzymes involved in androgen biosynthesis. Expression of all these proteins and enzymes was significantly higher in the androgen-independent derivative of LNCaP prostate cancer cells (C81) than in the androgen-dependent cell line (C33). In serum-free cultures, the androgen-independent C81 cells secreted approximately 5-fold higher testosterone than C33 cells as determined in the conditioned media by immunoassays. These cells could also directly convert radioactive cholesterol into testosterone which was identified by thin layer chromatography. These results for the first time show that prostate cancer cells in advanced stages of the disease could synthesize androgens from cholesterol and hence are not dependent upon testicular and/or adrenal androgens.

The replication of putative replication origins (ARS elements) was examined for 200 kilobases of chromosome III of Saccharomyces cerevisiae. By using synchronous cultures and transfers from dense to light isotope medium, the temporal pattern of mitotic DNA replication of eight fragments that contain ARSs was determined. ARS elements near the telomeres replicated late in S phase, while internal ARS elements replicated in the first half of S phase. The results suggest that some ARS elements in the chromosome may be inactive as replication origins. The actively expressed mating type locus, MAT, replicated early in S phase, while the silent cassettes, HML and HMR, replicated late. Unexpectedly, chromosome III sequences were found to replicate late in G1 at the arrest induced by the temperature-sensitive cdc7 allele.

Identical N-terminal deletions in the wild-type rat androgen receptor (rAR) and a constitutively active rAR (ARDelta641-902) devoid of the ligand-binding domain (LBD) resulted in dissimilar consequences in transcriptional activation: deletion of residues 149-295 abolished wild-type AR activity, but did not influence that of ARDelta641-902. The activity of the N-terminal transactivation domain is thus controlled by the hormone-occupied LBD, suggesting that the N- and C-terminal regions of rAR communicate. Consistent with this idea, a strong androgen-dependent interaction between the N-terminal region and LBD was demonstrated in a mammalian two-hybrid system using GAL4 and VP16 fusion proteins. This interaction can be direct or indirect. Several nuclear receptor coactivators (CBP, F-SRC-1, SRC-1, and RIP140) that interact with other steroid receptors were tested as potential mediators of the N- and C-terminal interaction of rAR using the mammalian two-hybrid system. CBP or F-SRC-1 not only enhanced AR-mediated transactivation, but also facilitated the androgen-dependent interaction between the N- and C-terminal domains, implying that part of the coactivator-dependent transcriptional activation occurs via this mechanism. In contrast, SRC-1, a coactivator for the progesterone receptor, inhibited both AR-mediated transactivation and interaction between the N and C termini. Recruitment of coregulators may involve AR domains other than the LBD, as F-SRC-1 and CBP enhanced, but SRC-1 repressed, the transcriptional activity of ARDelta641-902. Collectively, interplay between the N-terminal region and LBD of rAR results in the formation of a transactivation complex that includes coregulators and that is mandatory for optimal activation of androgen-induced promoters.

Leaf development in C4 plants such as maize involves the differentiation of two photosynthetic cell types [bundle sheath (BS) and mesophyll (M)] to form Kranz-type leaf anatomy. This cellular dimorphism partitions photosynthetic activities so that each enzyme of the C4 pathway accumulates only in the appropriate cell type. We have exploited this property to study BS and M cell interactions in developing maize leaves. Our previous studies showed that C4 proteins appear concurrently with the appearance of Kranz anatomy. To look at earlier events in BS and M cell development we have used three of the corresponding C4 mRNAs as cell-specific markers. We have followed, in situ, the accumulation of malic enzyme (ME), phosphoenolpyruvate carboxylase (PEPCase), and ribulose bisphosphate carboxylase (RuBPCase) mRNAs in developing leaves of both normal and mutant argentia (ar) maize. We have isolated a partial cDNA clone for maize ME to examine ME mRNA expression. We show that throughout the development of light-grown seedlings, all three mRNAs accumulate in a cell-specific fashion in both normal and ar leaves. The pattern of C4 mRNA accumulation longitudinally along the veins, laterally across the leaf, and locally around individual veins reveals the spatial and temporal sequence of BS and M cell development. BS cell-specific mRNAs accumulate around developing veins before Kranz anatomy is evident morphologically. Our analysis of the ar mutant, in which C4 mRNA appearance is delayed relative to the appearance of Kranz anatomy, demonstrates first that BS and M cells develop in clusters across the leaf blade and second that BS cells surrounding any individual vein are activated asynchronously. We discuss our results in relation to models and mechanisms of BS and M cell development.

The beta(2)-adrenoceptor (beta(2)AR) was one of the first Family A G protein-coupled receptors (GPCRs) shown to form oligomers in cellular membranes, yet we still know little about the number and arrangement of protomers in oligomers, the influence of ligands on the organization or stability of oligomers, or the requirement for other proteins to promote oligomerization. We used fluorescence resonance energy transfer (FRET) to characterize the oligomerization of purified beta(2)AR site-specifically labelled at three different positions with fluorophores and reconstituted into a model lipid bilayer. Our results suggest that the beta(2)AR is predominantly tetrameric following reconstitution into phospholipid vesicles. Agonists and antagonists have little effect on the relative orientation of protomers in oligomeric complexes. In contrast, binding of inverse agonists leads to significant increases in FRET efficiencies for most labelling pairs, suggesting that this class of ligand promotes tighter packing of protomers and/or the formation of more complex oligomers by reducing conformational fluctuations in individual protomers. The results provide new structural insights into beta(2)AR oligomerization and suggest a possible mechanism for the functional effects of inverse agonists.

Compartmentation of cAMP is thought to generate the specificity of Gs-coupled receptor action in cardiac myocytes, with phosphodiesterases (PDEs) playing a major role in this process by preventing cAMP diffusion. We tested this hypothesis in adult rat ventricular myocytes by characterizing PDEs involved in the regulation of cAMP signals and L-type Ca2+ current (I(Ca,L)) on stimulation with beta1-adrenergic receptors (beta1-ARs), beta2-ARs, glucagon receptors (Glu-Rs) and prostaglandin E1 receptors (PGE1-Rs). All receptors but PGE1-R increased total cAMP, and inhibition of PDEs with 3-isobutyl-1-methylxanthine strongly potentiated these responses. When monitored in single cells by high-affinity cyclic nucleotide-gated (CNG) channels, stimulation of beta1-AR and Glu-R increased cAMP, whereas beta2-AR and PGE1-R had no detectable effect. Selective inhibition of PDE3 by cilostamide and PDE4 by Ro 20-1724 potentiated beta1-AR cAMP signals, whereas Glu-R cAMP was augmented only by PD4 inhibition. PGE1-R and beta2-AR generated substantial cAMP increases only when PDE3 and PDE4 were blocked. For all receptors except PGE1-R, the measurements of I(Ca,L) closely matched the ones obtained with CNG channels. Indeed, PDE3 and PDE4 controlled beta1-AR and beta2-AR regulation of I(Ca,L), whereas only PDE4 controlled Glu-R regulation of I(Ca,L) thus demonstrating that receptor-PDE coupling has functional implications downstream of cAMP. PGE1 had no effect on I(Ca,L) even after blockade of PDE3 or PDE4, suggesting that other mechanisms prevent cAMP produced by PGE1 to diffuse to L-type Ca2+ channels. These results identify specific functional coupling of individual PDE families to Gs-coupled receptors as a major mechanism enabling cardiac cells to generate heterogeneous cAMP signals in response to different hormones.

A method for photon beam dose calculations is described. The primary photon beam is raytraced through the patient, and the distribution of total radiant energy released into the patient is calculated. Polyenergetic energy deposition kernels are calculated from the spectrum of the beam, using a database of monoenergetic kernels. It is shown that the polyenergetic kernels can be analytically described with high precision by (A exp( -ar) + B exp( -br)/r2, where A, a, B, and b depend on the angle with respect to the impinging photons and the accelerating potential, and r is the radial distance. Numerical values of A, a, B, and b are derived and used to convolve energy deposition kernels with the total energy released per unit mass (TERMA) to yield dose distributions. The convolution is facilitated by the introduction of the collapsed cone approximation. In this approximation, all energy released into coaxial cones of equal solid angle, from volume elements on the cone axis, is rectilinearly transported, attenuated, and deposited in elements on the axis. Scaling of the kernels is implicitly done during the convolution procedure to fully account for inhomogeneities present in the irradiated volume. The number of computational operations needed to compute the dose with the method is proportional to the number of calculation points. The method is tested for five accelerating potentials; 4, 6, 10, 15, and 24 MV, and applied to two geometries; one is a stack of slabs of tissue media, and the other is a mediastinum-like phantom of cork and water. In these geometries, the EGS4 Monte Carlo system has been used to generate reference dose distributions with which the dose computed with the collapsed cone convolution method is compared. Generally, the agreement between the methods is excellent. Deviations are observed in situations of lateral charged particle disequilibrium in low density media, however, but the result is superior compared to that of the generalized Batho method.

Caffeine causes most of its biological effects via antagonizing all types of adenosine receptors (ARs): A1, A2A, A3, and A2B and, as does adenosine, exerts effects on neurons and glial cells of all brain areas. In consequence, caffeine, when acting as an AR antagonist, is doing the opposite of activation of adenosine receptors due to removal of endogenous adenosinergic tonus. Besides AR antagonism, xanthines, including caffeine, have other biological actions: they inhibit phosphodiesterases (PDEs) (e.g., PDE1, PDE4, PDE5), promote calcium release from intracellular stores, and interfere with GABA-A receptors. Caffeine, through antagonism of ARs, affects brain functions such as sleep, cognition, learning, and memory, and modifies brain dysfunctions and diseases: Alzheimer's disease, Parkinson's disease, Huntington's disease, Epilepsy, Pain/Migraine, Depression, Schizophrenia. In conclusion, targeting approaches that involve ARs will enhance the possibilities to correct brain dysfunctions, via the universally consumed substance that is caffeine.

Recent years have seen tremendous breakthroughs in structure determination of G-protein-coupled receptors (GPCRs). In 2011, two agonist-bound active-state structures of rhodopsin have been published. Together with structures of several rhodopsin activation intermediates and a wealth of biochemical and spectroscopic information, they provide a unique structural framework on which to understand GPCR activation. Here we use this framework to compare the recent crystal structures of the agonist-bound active states of the β(2) adrenergic receptor (β(2)AR) and the A(2A) adenosine receptor (A(2A)AR). While activation of these three GPCRs results in rearrangements of TM5 and TM6, the extent of this conformational change varies considerably. Displacements of the cytoplasmic side of TM6 ranges between 3 and 8Å depending on whether selective stabilizers of the active conformation are used (i.e. a G-protein peptide in the case of rhodopsin or a conformationally selective nanobody in the case of the β(2)AR) or not (A(2A)AR). The agonist-induced conformational changes in the ligand-binding pocket are largely receptor specific due to the different chemical nature of the agonists. However, several similarities can be observed, including a relocation of conserved residues W6.48 and F6.44 towards L5.51 and P5.50, and of I/L3.40 away from P5.50. This transmission switch links agonist binding to the movement of TM5 and TM6 through the rearrangement of the TM3-TM5-TM6 interface, and possibly constitutes a common theme of GPCR activation.