A 1.45 kb circular plasmid derived from yeast chromosome IV contains the autonomous replication element called ARSARSARSARSARSARSARSARS elements are origins of chromosome replication, then the experiment reveals times of activation for two origins.

Recent evidence indicates that testosterone is neuroprotective, however, the underlying mechanism(s) remains to be elucidated. In this study, we investigated the hypothesis that androgens induce mitogen-activated protein kinase (MAPK) signaling in neurons, which subsequently drives neuroprotection. We observed that testosterone and its non-aromatizable metabolite dihydrotestosterone (DHT) rapidly and transiently activate MAPK in cultured hippocampal neurons, as evidenced by phosphorylation of extracellular signal-regulated kinase (ERK)-1 and ERK-2. Importantly, pharmacological suppression of MAPK/ERK signaling blocked androgen-mediated neuroprotection against beta-amyloid toxicity. Androgen activation of MAPK/ERK and neuroprotection also was observed in PC12 cells stably transfected with androgen receptor (AR), but in neither wild-type nor empty vector-transfected PC12 cells. Downstream of ERK phosphorylation, we observed that DHT sequentially increases p90 kDa ribosomal S6 kinase (Rsk) phosphorylation and phosphorylation-dependent inactivation of Bcl-2-associated death protein (Bad). Prevention of androgen-induced phosphorylation of Rsk and Bad blocked androgen neuroprotection. These findings demonstrate AR-dependent androgen activation of MAPK/ERK signaling in neurons, and specifically identify a neuroprotective pathway involving downstream activation of Rsk and inactivation of Bad. Elucidation of androgen-mediated neural signaling cascades will provide important insights into the mechanisms of androgen action in brain, and may present a framework for therapeutic intervention of age-related neurodegenerative disorders.

To elucidate microscopic mechanisms underlying the modulation of cardiac excitation-contraction (EC) coupling by beta-adrenergic receptor (beta-AR) stimulation, we examined local Ca(2+) release function, ie, Ca(2+) spikes at individual transverse tubule-sarcoplasmic reticulum (T-SR) junctions, using confocal microscopy and our recently developed technique for release flux measurement. beta-AR stimulation by norepinephrine plus an alpha(1)-adrenergic blocker, prazosin, increased the amplitude of SR Ca(2+) release flux (J(SR)), its running integral (integralJ(SR)), and L-type Ca(2+) channel current (I(Ca)), and it shifted their bell-shaped voltage dependence leftward by approximately 10 mV, with the relative effects ranking I(Ca>> J(SR>>integralJ(SR). Confocal imaging revealed that the bell-shaped voltage dependence of SR Ca(2+) release is attributable to a graded recruitment of T-SR junctions as well as to changes in Ca(2+) spike amplitudes. beta-AR stimulation increased the fractional T-SR junctions that fired Ca(2+) spikes and augmented Ca(2+) spike amplitudes, without altering the SR Ca(2+) load, suggesting that more release units were activated synchronously among and within T-SR junctions. Moreover, beta-AR stimulation decreased the latency and temporal dispersion of Ca(2+) spike occurrence at a given voltage, delivering most of the Ca(2+) at the onset of depolarization rather than spreading it out throughout depolarization. Because the synchrony of Ca(2+) spikes affects Ca(2+) delivery per unit of time to contractile myofilaments, and because the myofilaments display a steep Ca(2+) dependence, our data suggest that synchronization of SR Ca(2+) release represents a heretofore unappreciated mechanism of beta-AR modulation of cardiac inotropy.

In the present study, we report that compound C, an inhibitor of a key intracellular energy sensor AMP-activated protein kinase (AMPK), can induce autophagy in cancer cells. The induction of autophagy in U251 human glioma cell line was demonstrated by acridine orange staining of intracellular acidic vesicles, Beclin 1 induction, p62 decrease and conversion of LC3-I to autophagosome-associated LC3-II in the presence of proteolysis inhibitors. The presence of autophagosome-like vesicles was confirmed by transmission electron microscopy. Compound C-mediated inhibition of AMPK and raptor in U251 cells was associated with paradoxical decrease in phosphorylation of AMPK/raptor-repressed mTOR, a major negative regulator of autophagy, and its downstream target p70S6K. The phosphorylation of an mTOR activator Akt and the PI3K-activating kinase Src was also impaired in compound C-treated cells. The siRNA-mediated AMPK silencing did not reduce the activity of the Akt/mTOR/p70S6K pathway and AMPK activators metformin and AIC AR failed to block compound C-induced autophagy. Autophagy inhibitors bafilomycin and chloroquine significantly increased the cytotoxicity of compound C towards U251 cells, as confirmed by increase in lactate dehydrogenase release, DNA fragmentation and caspase-3 activation. Similar effects of compound C were also observed in C6 rat glioma, L929 mouse fibrosarcoma and B16 mouse melanoma cell lines. Since compound C has previously been reported to suppress AMPK-dependent autophagy in different cell types, our findings suggest that the effects of compound C on autophagy might be dose-, cell type- and/or context-dependent. By demonstrating the ability of compound C to induce autophagic response in cancer cells via AMPK inhibition-independent downregulation of Akt/mTOR pathway, our results warrant caution when using compound C to inhibit AMPK-dependent cellular responses, but also support further exploration of compound C and related molecules as potential anticancer agents.

Beta(1)- and beta(2)-adrenergic receptors (beta(1)AR and beta(2)AR) are co-expressed in numerous tissues where they play a central role in the responses of various organs to sympathetic stimulation. Although the two receptor subtypes share some signaling pathways, each has been shown to have specific signaling and regulatory properties. Given the recent recognition that many G protein-coupled receptors can form homo- and heterodimers, the present study was undertaken to determine whether the beta(1)AR and beta(2)AR can form dimers in cells and, if so, to investigate the potential functional consequences of such heterodimerization. Using co-immunoprecipitation and bioluminescence resonance energy transfer, we show that beta(1)AR and beta(2)AR can form heterodimers in HEK 293 cells co-expressing the two receptors. Functionally, beta-adrenergic stimulated adenylyl cyclase activity was found to be identical in cells expressing beta(1)AR, beta(2)AR, or both receptors at similar levels, indicating that heterodimerization did not affect this signaling pathway. When considering ERK1/2 MAPK activity, a significant agonist-promoted activation was detected in beta(2)AR- but not beta(1)AR-expressing cells. Similarly to what was observed in cells expressing the beta(1)AR alone, no beta-adrenergic stimulated ERK1/2 phosphorylation was observed in cells co-expressing the two receptors. A similar inhibition of agonist-promoted internalization of the beta(2)AR was observed upon co-expression of the beta(1)AR, which by itself internalized to a lesser extent. Taken together, our data suggest that heterodimerization between beta(1)AR and beta(2)AR inhibits the agonist-promoted internalization of the beta(2)AR and its ability to activate the ERK1/2 MAPK signaling pathway.

We previously reported that our novel compound 3beta-hydroxy-17-(1H-benzimidazole-1-yl)androsta-5,16-diene (VN/124-1) is a potent 17alpha-hydroxylase/17,20-lyase (CYP17) inhibitor/antiandrogen and strongly inhibits the formation and proliferation of human prostate cancer LAPC4 tumor xenografts in severe combined immunodeficient mice. In this study, we report that VN/124-1 and other novel CYP17 inhibitors also cause down-regulation of androgen receptor (AR) protein expression in vitro and in vivo. This mechanism of action seems to contribute to their antitumor efficacy. We compared the in vivo antitumor efficacy of VN/124-1 with that of castration and a clinically used antiandrogen, Casodex, and show that VN/124-1 is more potent than castration in the LAPC4 xenograft model. Treatment with VN/124-1 (0.13 mmol/kg twice daily) was also very effective in preventing the formation of LAPC4 tumors (6.94 versus 2410.28 mm(3) in control group). VN/124-1 (0.13 mmol/kg twice daily) and VN/124-1 (0.13 mmol/kg twice daily) + castration induced regression of LAPC4 tumor xenografts by 26.55% and 60.67%, respectively. Treatments with Casodex (0.13 mmol/kg twice daily) or castration caused significant tumor suppression compared with control. Furthermore, treatment with VN/124-1 caused marked down-regulation of AR protein expression, in contrast to treatments with Casodex or castration that caused significant up-regulation of AR protein expression. The results suggest that VN/124-1 acts by several mechanisms (CYP17 inhibition, competitive inhibition, and down-regulation of the AR). These actions contribute to inhibition of the formation of LAPC4 tumors and cause regression of growth of established tumors. VN/124-1 is more efficacious than castration in the LAPC4 xenograft model, suggesting that the compound has potential for the treatment of prostate cancer.

A novel human keratinocyte-derived autocrine factor (KAF) was purified from conditioned medium by using heparin affinity chromatography as the first step. Purified KAF stimulated the growth of normal human keratinocytes, mouse AKR-2B cells, and a mouse keratinocyte cell line (BALB/MK). Heparin sulfate inhibited KAF mitogenic activity on all cell types tested and inhibited the ability of KAF to compete with epidermal growth factor for cell surface binding. Interestingly, KAF stimulated the growth of BALB/MK cells at high cell density but failed to stimulate these cells at clonal density. Protein microsequencing of the first 20 NH2-terminal amino acid residues of purified KAF revealed identity to the NH2 terminus of human amphiregulin (AR). Northern (RNA) blot analysis with AR-specific cRNA demonstrated that human keratinocytes, as well as mammary epithelial cell cultures, expressed high levels of AR mRNA. In contrast, AR mRNA was not detected in normal human fibroblasts or melanocytes and was present at reduced levels in several mammary tumor cell lines. The mitogenic activity of purified AR was also shown to be inhibited by heparin sulfate, and an AR-specific enzyme-linked immunosorbent assay (ELISA) revealed that KAF and AR are antigenically related. We have previously shown that human keratinocytes can grow in an autocrine manner. Our present study demonstrates that one of the growth factors responsible for this autocrine growth (KAF) is similar or identical to AR and that KAF and AR bioactivity can be negatively regulated by heparin sulfate.

There is a need to identify genetic mediators of solid-tumor cancers, such as prostate cancer, where invasion and distant metastases determine the clinical outcome of the disease. Whole-genome expression profiling offers promise in this regard, but can be complicated by the challenge of identifying the genes affected by a condition from the hundreds to thousands of genes that exhibit changes in expression. Here, we show that reverse-engineered gene networks can be combined with expression profiles to compute the likelihood that genes and associated pathways are mediators of a disease. We apply our method to non-recurrent primary and metastatic prostate cancer data, and identify the androgen receptor gene (AR) among the top genetic mediators and the AR pathway as a highly enriched pathway for metastatic prostate cancer. These results were not obtained on the basis of expression change alone. We further demonstrate that the AR gene, in the context of the network, can be used as a marker to detect the aggressiveness of primary prostate cancers. This work shows that a network biology approach can be used advantageously to identify the genetic mediators and mediating pathways associated with a disease.

OBJECTIVE

The G protein-coupled receptor kinase-2 (GRK2 or beta-ARK1) regulates beta-adrenergic receptors (beta-ARs) in the heart, and its cardiac expression is elevated in human heart failure (HF). We sought to determine whether myocardial levels and activity of GRK2 could be monitored using white blood cells, which have been used to study cardiac beta-ARs. Moreover, we were interested in determining whether GRK2 levels in myocardium and lymphocytes may be associated with beta-AR dysfunction and HF severity.

RESULTS

In myocardial biopsies from explanted failing human hearts, GRK activity was inversely correlated with beta-AR-mediated cAMP production (R(2)=-0.215, P<0.05, n=24). Multiple regression analysis confirmed that GRK activity participates with beta-AR density to regulate catecholamine-sensitive cAMP responses. Importantly, there was a direct correlation between myocardial and lymphocytes GRK2 activity (R(2)=0.5686, P<0.05, n=10). Lymphocyte GRK activity was assessed in HF patients with various ejection fractions (EFs) (n=33), and kinase activity was significantly higher in patients with lower EFs and was higher with increasing NYHA class (P<0.001).

CONCLUSIONS

Myocardial GRK2 expression and activity are mirrored by lymphocyte levels of this kinase, and its elevation in HF is associated with the loss of beta-AR responsiveness and appears to increase with disease severity. Therefore, lymphocytes may provide a surrogate for monitoring cardiac GRK2 in human HF.

Androgen independence is responsible for most prostate cancer lethality, yet currently there are no effective clinical treatments. We have been investigating the mechanisms underlying androgen-independent prostate cancer in Nkx3.1;Pten mutant mice, which display salient features of the disease, including a requirement for wild-type androgen receptor (AR) signaling. We now demonstrate that the Akt and Erk MAP kinase signaling pathways are activated in androgen-independent lesions of these mice. Forced activation of either Akt or Erk signaling in an androgen-responsive prostate cancer cell line promotes hormone-independent but AR-dependent growth in culture. Although these pathways act additively in culture, they act synergistically in vivo to promote tumorigenicity and androgen independence in the context of the prostate microenvironment. We propose that androgen independence emerges by means of epithelial-stromal competition, in which activation of Akt and Erk promotes AR activity in the prostate epithelium while counteracting antagonistic effects of the stroma.

Testosterone and estrogen are essential for male behaviors in vertebrates. How these two signaling pathways interact to control masculinization of the brain and behavior remains to be established. Circulating testosterone activates the androgen receptor (AR) and also serves as the source of estrogen in the brain. We have used a genetic strategy to delete AR specifically in the mouse nervous system. This approach permits us to determine the function of AR in sexually dimorphic behaviors in males while maintaining circulating testosterone levels within the normal range. We find that AR mutant males exhibit masculine sexual and territorial displays, but they have striking deficits in specific components of these behaviors. Taken together with the surprisingly limited expression of AR in the developing brain, our findings indicate that testosterone acts as a precursor to estrogen to masculinize the brain and behavior, and signals via AR to control the levels of male behavioral displays.

BACKGROUND

In alpha1-AR knockout (alpha1ABKO) mice that lacked cardiac myocyte alpha1-adrenergic receptor (alpha1-AR) binding, aortic constriction induced apoptosis, dilated cardiomyopathy, and death. However, it was unclear whether these effects were attributable to a lack of cardiac myocyte alpha1-ARs and whether the alpha1A, alpha1B, or both subtypes mediated protection. Therefore, we investigated alpha1A and alpha1B subtype-specific survival signaling in cultured cardiac myocytes to test for a direct protective effect of alpha1-ARs in cardiac myocytes.

RESULTS

We cultured alpha1ABKO myocytes and reconstituted alpha1-AR signaling with adenoviruses expressing alpha1-GFP fusion proteins. Myocyte death was induced by norepinephrine, doxorubicin, or H2O2 and was measured by annexin V/propidium iodide staining. In alpha1ABKO myocytes, all 3 stimuli significantly increased apoptosis and necrosis. Reconstitution of the alpha1A subtype, but not the alpha1B, rescued alpha1ABKO myocytes from cell death induced by each stimulus. To address the mechanism, we examined alpha1-AR activation of extracellular signal-regulated kinase (ERK). In alpha1ABKO hearts, aortic constriction failed to activate ERK, and in alpha1ABKO myocytes, expression of a constitutively active MEK1 rescued alpha1ABKO myocytes from norepinephrine-induced death. In addition, only the alpha1A-AR activated ERK in alpha1ABKO myocytes, and expression of a dominant-negative MEK1 completely blocked alpha1A survival signaling in alpha1ABKO myocytes.

CONCLUSIONS

Our results demonstrate a direct protective effect of the alpha1A subtype in cardiac myocytes and define an alpha1A-ERK signaling pathway that is required for myocyte survival. Absence of the alpha1A-ERK pathway can explain the failure to activate ERK after aortic constriction in alpha1ABKO mice and can contribute to the development of apoptosis, dilated cardiomyopathy, and death.

The technique of in situ hybridization with specific ribonucleotide probes was used to determine the distribution patterns of mRNA encoding the alpha 1a-, alpha 1b- and alpha 1d-adrenoceptor (AR) subtypes in rat brain and spinal cord. The expression pattern of alpha 1a-AR mRNA has not been reported previously, and was found to be widespread throughout the rat central nervous system. High levels were found in regions of the olfactory system, several hypothalamic nuclei, and regions of the brainstem and spinal cord, particularly in areas related to motor function. Regions expressing moderate levels of mRNA for this receptor were the septum, bed nucleus of the stria terminalis, cerebral cortex, amygdala, cerebellum and pineal gland. Low expression levels were detected in the hippocampal formation. Most nuclei in the basal ganglia and thalamus expressed extremely low or undetectable levels of alpha 1a-AR mRNA. The expression patterns of the alpha 1b- and alpha 1d-AR mRNAs were similar to those described using oligonucleotide probes in earlier studies. High expression of alpha 1b-AR mRNA was noted in the pineal gland, most thalamic nuclei, lateral nucleus of the amygdala and dorsal and median raphe nuclei. Moderate expression levels were noted throughout the cerebral cortex, and in some olfactory, septal, and brainstem regions. The distribution of alpha 1d-AR mRNA was the most discrete of the three receptors examined. Expression was strong in the olfactory bulb, cerebral cortex, hippocampus, reticular thalamic nucleus, regions of the amygdala, motor nuclei of the brainstem, inferior olivary complex and spinal cord. Comparison of the distributions of the alpha 1a-, alpha 1b- and alpha 1d-AR mRNA suggests unique functional roles for each of these receptors.

Previous studies from this laboratory have described that LNCaP prostate tumor cells contain an androgen receptor (AR) with a point mutation in the steroid-binding domain (codon 868, Thr to Ala). This defect leads to a change in specificity of the AR. Estrogens, progestagens, and some anti-androgens (e.g., cyproterone acetate, hydroxyflutamide, nilutamide) stimulate LNCaP cell growth rate through the AR. The present studies indicate that not all anti-androgens showed agonistic effects with the mutated receptor. The growth rate of LNCaP cells did not increase with the anti-androgen ICI 176334, nor could this compound increase transcription activation of the reporter gene construct via the mutant receptor in a cotransfection system [HeLa cell cotransfection system with an androgen-regulated reporter gene construct (pG29G-tk-CAT) and the mutant receptor as trans-vector]. Interaction of the AR of LNCaP cells with heat-shock proteins was studied by isolation of the receptor with a specific monoclonal antibody and characterization of associated proteins. Hsp90, hsp70, and hsp56 were found to coprecipitate with the AR. Incubation of the cells at 37 degrees C with androgen (R1881, 10 nM) or the anti-androgen hydroxyflutamide, prior to receptor isolation, resulted in dissociation of the AR-heat-shock protein complex. This dissociation is paralleled by the transformation to a tight nuclear-binding form of the AR. In contrast, ICI 176334 could not induce a release of heat-shock proteins and did not increase nuclear binding, but inhibited the transformation process induced by R1881.(ABSTRACT TRUNCATED AT 250 WORDS)

Nuclear receptors (NRs) are ligand-regulated transcription factors important in human physiology and disease. In certain NRs, including the androgen receptor (AR), ligand binding to the carboxy-terminal domain (LBD) regulates transcriptional activation functions in the LBD and amino-terminal domain (NTD). The basis for NTD-LBD communication is unknown but may involve NTD-LBD interactions either within a single receptor or between different members of an AR dimer. Here, measurement of FRET between fluorophores attached to the NTD and LBD of the AR established that agonist binding initiated an intramolecular NTD-LBD interaction in the nucleus and cytoplasm. This intramolecular folding was followed by AR self-association, which occurred preferentially in the nucleus. Rapid, ligand-induced intramolecular folding and delayed association also were observed for estrogen receptor-alpha but not for peroxisome proliferator activated receptor-gamma2. An antagonist ligand, hydroxyflutamide, blocked the NTD-LBD association within AR. NTD-LBD association also closely correlated with the transcriptional activation by heterologous ligands of AR mutants isolated from hormone-refractory prostate tumors. Intramolecular folding, but not AR-AR affinity, was disrupted by mutation of an alpha-helical ((23)FQNLF(27)) motif in the AR NTD previously described to interact with the AR LBD in vitro. This work establishes an intramolecular NTD-LBD conformational change as an initial component of ligand-regulated NR function.

OBJECTIVE

This study tested the hypothesis that brief cycles of iterative ischemia-reperfusion at onset of reperfusion (termed "postconditioning", post-con) delays washout of intravascular adenosine and thereby increases endogenous adenosine receptor (AR) activation during the early moments of reperfusion (R).

METHODS

Isolated mouse hearts were subjected to 20 min global ischemia (I) and 30 min R with or without post-con (3 or 6 cycles of 10 s R&I). Intravascular purines in coronary effluent were analyzed by HPLC. To assess the functional role of endogenous AR activation in post-con, an open-chest rat model of myocardial infarction was employed. Rats were randomly divided into 11 groups: control, no intervention at R; post-con, three cycles of 10 s R followed by 10 s LCA re-occlusion immediately upon R. In the following interventions, drugs (or vehicle) were administered 5 min before R in the absence or presence (+/-) of post-con. Vehicle (DMSO < 300 microl/kg); 8-SPT (non-selective AR antagonist, 10 mg/kg) +/- post-con; DPCPX (A(1A)R antagonist, 0.1 mg/kg) +/- post-con; ZM241385 (A(2A)AR antagonist, 0.2 mg/kg) +/- post-con; MRS1523 (A(3)AR antagonist, 2 mg/kg) +/- post-con.

RESULTS

In isolated mouse hearts, post-con reduced diastolic pressure during both early (26+/-3* vs. 37+/-3 mmHg at 5 min) and late (22+/-3* vs. 34+/-3 mmHg at 30 min) R. Post-con also hastened the early recovery of contractile function (developed pressure 39+/-6* vs. 16+/-2 mmHg at 5 min R), although differences did not persist at 30 min R. Importantly, post-con was associated with reduced adenosine washout (58+/-5* vs. 155+/-16 nM/min/g) at 2 min R suggesting greater retention time of intravascular adenosine. In rats, post-con significantly attenuated infarct size compared to control (40+/-3% vs. 53 +/- 2%* in control), an effect that was unaltered by DPCPX (42 +/- 2%) but was abrogated by 8-SPT (50 +/- 2%), ZM241385 (49 +/- 3%) or MRS1523 (52 +/- 1%) (P < 0.02).

CONCLUSIONS

These data suggest that post-con involves endogenous activation of A(2A) and A3 but not A1AR subtypes. This activation may be linked to the delay in the washout of intravascular adenosine during the early minutes of R during which post-con is applied.

BACKGROUND

Enzalutamide is a novel antiandrogen with proven efficacy in metastatic castration-resistant prostate cancer (mCRPC).

OBJECTIVE

To evaluate enzalutamide's effects on cancer and on androgens in blood and bone marrow, and associate these with clinical observations.

METHODS

In this prospective phase 2 study, 60 patients with bone mCRPC received enzalutamide 160mg orally daily and had transilial bone marrow biopsies before treatment and at 8 wk of treatment.

METHODS

Androgen signaling components (androgen receptor [AR], AR splice variant 7 (ARV7), v-ets avian erythroblastosis virus E26 oncogene homolog [ERG], cytochrome P450, family 17, subfamily A, polypeptide 1 [CYP17]) and molecules implicated in mCRPC progression (phospho-Met, phospho-Src, glucocorticoid receptor, Ki67) were assessed by immunohistochemistry; testosterone, cortisol, and androstenedione concentrations were assessed by liquid chromatography-tandem mass spectrometry; AR copy number was assessed by real-time polymerase chain reaction. Descriptive statistics were applied.

CONCLUSIONS

Median time to treatment discontinuation was 22 wk (95% confidence interval, 19.9-29.6). Twenty-two (37%) patients exhibited primary resistance to enzalutamide, discontinuing treatment within 4 mo. Maximal prostate-specific antigen (PSA) decline ≥ 50% and ≥ 90% occurred in 27 (45%) and 13 (22%) patients, respectively. Following 8 wk of treatment, bone marrow and circulating testosterone levels increased. Pretreatment tumor nuclear AR overexpression >> 75%) and CYP17 >> 10%) expression were associated with benefit (p = 0.018). AR subcellular localization shift from the nucleus was confirmed in eight paired samples (with PSA decline) of 23 evaluable paired samples. Presence of an ARV7 variant was associated with primary resistance to enzalutamide (p = 0.018). Limited patient numbers warrant further validation.

CONCLUSIONS

The observed subcellular shift of AR from the nucleus and increased testosterone concentration provide the first evidence in humans that enzalutamide suppresses AR signaling while inducing an adaptive feedback. Persistent androgen signaling in mCRPC was predictive of benefit and ARV7 was associated with primary resistance.

RESULTS

We report a first bone biopsy study in metastatic prostate cancer in humans that searched for predictors of outcome of enzalutamide therapy. Benefit is linked to a pretreatment androgen-signaling signature.

BACKGROUND

ClinicalTrials.gov identifier NCT01091103.

The C terminus of the beta(2)-adrenoceptor (AR) interacts with G protein-coupled receptor kinases and arrestins in an agonist-dependent manner, suggesting that conformational changes induced by ligands in the transmembrane domains are transmitted to the C terminus. We used fluorescence resonance energy transfer (FRET) to examine ligand-induced structural changes in the distance between two positions on the beta(2)-AR C terminus and cysteine 265 (Cys-265) at the cytoplasmic end of transmembrane domain 6. The donor fluorophore FlAsH (Fluorescein Arsenical Helix binder) was attached to a CCPGCC motif introduced at position 351-356 in the proximal C terminus or at the distal C terminus. An acceptor fluorophore, Alexa Fluor 568, was attached to Cys-265. FRET analyses revealed that the average distances between Cys-265 and the proximal and distal FlAsH sites were 57 and 62A(,) respectively. These relatively large distances suggest that the C terminus is in an extended, relatively unstructured conformation. Nevertheless, we observed ligand-specific changes in FRET. All ligands induced an increase in FRET between the proximal C-terminal FlAsH site and Cys-265. Ligands that have been shown to induce arrestin-dependent ERK activation, including the catecholamine agonists and the inverse agonist ICI118551, led to a decrease in FRET between the distal FlAsH site and Cys-265, whereas other ligands had no effect or induced a small increase in FRET. Taken together the results provide new insight into the structure of the C terminus of the beta(2)-AR as well as ligand-induced conformational changes that may be relevant to arrestin-dependent regulation and signaling.

A series of brief ischemia/reperfusion cycles (termed ischemic preconditioning, IPC) limits myocardial injury produced by a subsequent prolonged period of coronary artery occlusion and reperfusion. Over the last 2 decades our understanding of IPC's mechanism has increased exponentially. Hearts exposed to IPC have a better metabolic and ionic status during prolonged ischemia compared to naïve hearts. However, this difference is not thought to be the main mechanism by which IPC protects against infarction. Signaling pathways that are activated by IPC distinguish IPC hearts from naïve hearts. During the trigger phase of IPC, adenosine, bradykinin and opioid receptors are occupied. Although these three receptors trigger signaling through divergent pathways, the signaling converges on protein kinase C. We have proposed that at the end of the index ischemia the activated PKC sensitizes the low-affinity A(2b) adenosine receptor (A(2b)AR) through phosphorylation of either the receptor or its coupling proteins so that A(2b)AR can be activated by endogenous adenosine released by the previously ischemic cardiomyocytes. The sensitized A(2b)AR would then be responsible for activation of the survival kinases including PI3 kinase, Akt and ERK which then act to inhibit lethal mitochondrial permeability transition pore formation which normally uncouples mitochondria and destroys many myocytes in the first minutes of reperfusion. Herein we review the evidence for the above mechanisms and their functional details.

Y-27632, an inhibitor of the Rho-associated kinase ROCK, is a therapeutic lead for Huntington disease (HD). The downstream targets that mediate its inhibitory effects on huntingtin (Htt) aggregation and toxicity are unknown. We have identified profilin, a small actin-binding factor that also interacts with Htt, as being a direct target of the ROCK1 isoform. The overexpression of profilin reduces the aggregation of polyglutamine-expanded Htt and androgen receptor (AR) peptides. This requires profilin's G-actin binding activity and its direct interaction with Htt, which are both inhibited by the ROCK1-mediated phosphorylation of profilin at Ser-137. Y-27632 blocks the phosphorylation of profilin in HEK293 cells and primary neurons, which maintains profilin in an active state. The knockdown of profilin blocks the inhibitory effect of Y-27632 on both AR and Htt aggregation. A signaling pathway from ROCK1 to profilin thus controls polyglutamine protein aggregation and is targeted by a promising therapeutic lead for HD.

BACKGROUND

Dementia drug development aims to modulate pathological processes that cause clinical syndromes. Population data (epidemiological neuropathology) will help to model and predict the potential impact of such therapies on dementia burden in older people. Presently this can only be explored through post mortem findings. We report the attributable risks (ARs) for dementia at death for common age-related degenerative and vascular pathologies, and other factors, in the MRC Cognitive Function and Ageing Study (MRC CFAS).

RESULTS

A multicentre, prospective, longitudinal study of older people in the UK was linked to a brain donation programme. Neuropathology of 456 consecutive brain donations assessed degenerative and vascular pathologies. Logistic regression modelling, with bootstrapping and sensitivity analyses, was used to estimate AR at death for dementia for specific pathologies and other factors. The main contributors to AR at death for dementia in MRC CFAS were age (18%), small brain (12%), neocortical neuritic plaques (8%) and neurofibrillary tangles (11%), small vessel disease (12%), multiple vascular pathologies (9%), and hippocampal atrophy (10%). Other significant factors include cerebral amyloid angiopathy (7%) and Lewy bodies (3%).

CONCLUSIONS

Such AR estimates cannot be derived from the living population; rather they estimate the relative contribution of specific pathologies to dementia at death. We found that multiple pathologies determine the overall burden of dementia. The impact of therapy targeted to a specific pathology may be profound when the dementia is relatively "pure," but may be less impressive for the majority with mixed disease, and in terms of the population. These data justify a range of strategies, and combination therapies, to combat the degenerative and vascular determinants of cognitive decline and dementia. Please see later in the article for the Editors' Summary.

Although the majority of primary human breast cancers express the androgen receptor (AR), the role of androgens in breast cancer growth and progression is poorly understood. We have investigated the effects of the naturally occurring androgen, dihydrotestosterone (DHT), and a synthetic non-metabolizable androgen, mibolerone, on the proliferation of six human breast cancer cell lines. The anti-proliferative and proliferative effects of androgens were only observed in cell lines that expressed the AR. Two of the AR-positive cell lines, T47-D and ZR-75-1 were growth inhibited in the presence of either DHT or mibolerone, while the proliferation of MCF-7 and MDA-MB-453 cells was increased by both androgens. Co-incubation of cultures with 1 nM DHT and a 100-fold excess of the androgen receptor antagonist, hydroxyflutamide, resulted in reversal of both inhibitory and stimulatory effects of DHT on T47-D, MCF-7 and MDA-MB-453 cell proliferation, indicating that DHT action is mediated by the AR in these lines. Hydroxyflutamide only partially reversed the DHT-induced growth inhibition of ZR-75-1 cultures, which suggests that growth inhibition of these cells may be mediated by non-AR pathways of DHT (or DHT metabolite) action. Mibolerone action on breast cancer cell growth was similar to that of DHT, with the exception that growth stimulation of MCF-7 and MDA-MB-453 cells was only partially reversed in the presence of a 100-fold excess of hydroxyflutamide. Anandron, another androgen receptor antagonist, was able to reverse all inhibitory and stimulatory actions of the androgens. AR antisense oligonucleotides reduced the level of immunoreactive AR expression in MDA-MB-453 and ZR-75-1 cells by more than 60%, but only reversed the growth inhibitory action of mibolerone in ZR-75-1 cultures. The results suggest that androgen action in breast cancer cell lines may not be solely mediated by binding of androgen to the AR. For example, metabolites of DHT with oestrogenic activity, or androgen binding to receptors other than the AR, may explain the divergent responses to androgens observed in different breast cancer cell lines.

BACKGROUND

Bacteria develop resistance to aminoglycosides by producing aminoglycoside-modifying enzymes such as acetyltransferase, phosphorylase, and adenyltransferase. These enzymes, however, cannot confer consistent resistance to various aminoglycosides because of their substrate specificity. Notwithstanding, a Pseudomonas aeruginosa strain AR-2 showing high-level resistance (minimum inhibitory concentration >1024 mg/L) to various aminoglycosides was isolated clinically. We aimed to clone and characterise the genetic determinant of this resistance.

METHODS

We used conventional methods for DNA manipulation, susceptibility testing, and gene analyses to clone and characterise the genetic determinant of the resistance seen. PCR detection of the gene was also done on a stock of P aeruginosa strains that were isolated clinically since 1997.

RESULTS

An aminoglycoside-resistance gene, designated rmtA, was identified in P aeruginosa AR-2. The Escherichia coli transformant and transconjugant harbouring the rmtA gene showed very high-level resistance to various aminoglycosides, including amikacin, tobramycin, isepamicin, arbekacin, kanamycin, and gentamicin. The 756-bp nucleotide rmtA gene encoded a protein, RmtA. This protein showed considerable similarity to the 16S rRNA methylases of aminoglycoside-producing actinomycetes, which protect bacterial 16S rRNA from intrinsic aminoglycosides by methylation. Incorporation of radiolabelled methyl groups into the 30S ribosome was detected in the presence of RmtA. Of 1113 clinically isolated P aeruginosa strains, nine carried the rmtA gene, as shown by PCR analyses.

CONCLUSIONS

Our findings strongly suggest intergeneric lateral gene transfer of 16S rRNA methylase gene from some aminoglycoside-producing microorganisms to P aeruginosa. Further dissemination of the rmtA gene in nosocomial bacteria could be a matter of concern in the future.

In Alzheimer's disease (AD), there is a significant loss of locus ceruleus (LC) noradrenergic neurons. However, functional and anatomical evidence indicates that the remaining noradrenergic neurons may be compensating for the loss. Because the noradrenergic system plays an important role in learning and memory, it is important to determine whether compensation occurs in noradrenergic neurons in the LC and hippocampus of subjects with AD or a related dementing disorder, dementia with Lewy bodies (DLB). We observed profound neuronal loss in the LC in AD and DLB subjects with three major changes in the noradrenergic system consistent with compensation: (1) an increase in tyrosine hydroxylase (TH) mRNA expression in the remaining neurons; (2) sprouting of dendrites into peri-LC dendritic zone, as determined by alpha2-adrenoreceptors (ARs) and norepinephrine transporter binding sites; and (3) sprouting of axonal projections to the hippocampus as determined by alpha2-ARs. In AD and DLB subjects, the postsynaptic alpha1-ARs were normal to elevated. Expression of alpha1A- and alpha2A-AR mRNA in the hippocampus of AD and DLB subjects were not altered, but expression of alpha1D- and alpha2C-AR mRNA was significantly reduced in the hippocampus of AD and DLB subjects. Therefore, in AD and DLB subjects, there is compensation occurring in the remaining noradrenergic neurons, but there does appear to be a loss of specific AR in the hippocampus. Because changes in these noradrenergic markers in AD versus DLB subjects were similar (except neuronal loss and the increase in TH mRNA were somewhat greater in DLB subjects), the presence of Lewy bodies in addition to plaques and tangles in DLB subjects does not appear to further affect the noradrenergic compensatory changes.