The ghrelin receptor isoforms (GHS-R1a and GHS-R1b) and GPR39 : an investigation into receptor dimerisation

Prostate cancer is the second most common cause of cancer-related deaths in Western males. Current diagnostic, prognostic and treatment approaches are not ideal and advanced metastatic prostate cancer is incurable. There is an urgent need for improved adjunctive therapies and markers for this disease. GPCRs are likely to play a significant role in the initiation and progression of prostate cancer. Over the last decade, it has emerged that G protein coupled receptors (GPCRs) are likely to function as homodimers and heterodimers. Heterodimerisation between GPCRs can result in the formation of novel pharmacological receptors with altered functional outcomes, and a number of GPCR heterodimers have been implicated in the pathogenesis of human disease. Importantly, novel GPCR heterodimers represent potential new targets for the development of more specific therapeutic drugs. Ghrelin is a 28 amino acid peptide hormone which has a unique n-octanoic acid post-translational modification. Ghrelin has a number of important physiological roles, including roles in appetite regulation and the stimulation of growth hormone release. The ghrelin receptor is the growth hormone secretagogue receptor type 1a, GHS-R1a, a seven transmembrane domain GPCR, and GHS-R1b is a C-terminally truncated isoform of the ghrelin receptor, consisting of five transmembrane domains. Growing evidence suggests that ghrelin and the ghrelin receptor isoforms, GHS-R1a and GHS-R1b, may have a role in the progression of a number of cancers, including prostate cancer. Previous studies by our research group have shown that the truncated ghrelin receptor isoform, GHS-R1b, is not expressed in normal prostate, however, it is expressed in prostate cancer. The altered expression of this truncated isoform may reflect a difference between a normal and cancerous state. A number of mutant GPCRs have been shown to regulate the function of their corresponding wild-type receptors. Therefore, we investigated the potential role of interactions between GHS-R1a and GHS-R1b, which are co-expressed in prostate cancer and aimed to investigate the function of this potentially new pharmacological receptor. In 2005, obestatin, a 23 amino acid C-terminally amidated peptide derived from preproghrelin was identified and was described as opposing the stimulating effects of ghrelin on appetite and food intake. GPR39, an orphan GPCR which is closely related to the ghrelin receptor, was identified as the endogenous receptor for obestatin. Recently, however, the ability of obestatin to oppose the effects of ghrelin on appetite and food intake has been questioned, and furthermore, it appears that GPR39 may in fact not be the obestatin receptor. The role of GPR39 in the prostate is of interest, however, as it is a zinc receptor. Zinc has a unique role in the biology of the prostate, where it is normally accumulated at high levels, and zinc accumulation is altered in the development of prostate malignancy. Ghrelin and zinc have important roles in prostate cancer and dimerisation of their receptors may have novel roles in malignant prostate cells. The aim of the current study, therefore, was to demonstrate the formation of GHS-R1a/GHS-R1b and GHS-R1a/GPR39 heterodimers and to investigate potential functions of these heterodimers in prostate cancer cell lines. To demonstrate dimerisation we first employed a classical co-immunoprecipitation technique. Using cells co-overexpressing FLAG- and Myc- tagged GHS-R1a, GHS-R1b and GPR39, we were able to co-immunoprecipitate these receptors. Significantly, however, the receptors formed high molecular weight aggregates. A number of questions have been raised over the propensity of GPCRs to aggregate during co-immunoprecipitation as a result of their hydrophobic nature and this may be misinterpreted as receptor dimerisation. As we observed significant receptor aggregation in this study, we used additional methods to confirm the specificity of these putative GPCR interactions. We used two different resonance energy transfer (RET) methods; bioluminescence resonance energy transfer (BRET) and fluorescence resonance energy transfer (FRET), to investigate interactions between the ghrelin receptor isoforms and GPR39. RET is the transfer of energy from a donor fluorophore to an acceptor fluorophore when they are in close proximity, and RET methods are, therefore, applicable to the observation of specific protein-protein interactions. Extensive studies using the second generation bioluminescence resonance energy transfer (BRET2) technology were performed, however, a number of technical limitations were observed. The substrate used during BRET2 studies, coelenterazine 400a, has a low quantum yield and rapid signal decay. This study highlighted the requirement for the expression of donor and acceptor tagged receptors at high levels so that a BRET ratio can be determined. After performing a number of BRET2 experimental controls, our BRET2 data did not fit the predicted results for a specific interaction between these receptors. The interactions that we observed may in fact represent ‘bystander BRET’ resulting from high levels of expression, forcing the donor and acceptor into close proximity. Our FRET studies employed two different FRET techniques, acceptor photobleaching FRET and sensitised emission FRET measured by flow cytometry. We were unable to observe any significant FRET, or FRET values that were likely to result from specific receptor dimerisation between GHS-R1a, GHS-R1b and GPR39. While we were unable to conclusively demonstrate direct dimerisation between GHS-R1a, GHS-R1b and GPR39 using several methods, our findings do not exclude the possibility that these receptors interact. We aimed to investigate if co-expression of combinations of these receptors had functional effects in prostate cancers cells. It has previously been demonstrated that ghrelin stimulates cell proliferation in prostate cancer cell lines, through ERK1/2 activation, and GPR39 can stimulate ERK1/2 signalling in response to zinc treatments. Additionally, both GHS-R1a and GPR39 display a high level of constitutive signalling and these constitutively active receptors can attenuate apoptosis when overexpressed individually in some cell types. We, therefore, investigated ERK1/2 and AKT signalling and cell survival in prostate cancer the potential modulation of these functions by dimerisation between GHS-R1a, GHS-R1b and GPR39. Expression of these receptors in the PC-3 prostate cancer cell line, either alone or in combination, did not alter constitutive ERK1/2 or AKT signalling, basal apoptosis or tunicamycin-stimulated apoptosis, compared to controls. In summary, the potential interactions between the ghrelin receptor isoforms, GHS-R1a and GHS-R1b, and the related zinc receptor, GPR39, and the potential for functional outcomes in prostate cancer were investigated using a number of independent methods. We did not definitively demonstrate the formation of these dimers using a number of state of the art methods to directly demonstrate receptor-receptor interactions. We investigated a number of potential functions of GPR39 and GHS-R1a in the prostate and did not observe altered function in response to co-expression of these receptors. The technical questions raised by this study highlight the requirement for the application of extensive controls when using current methods for the demonstration of GPCR dimerisation. Similar findings in this field reflect the current controversy surrounding the investigation of GPCR dimerisation. Although GHS-R1a/GHS-R1b or GHS-R1a/GPR39 heterodimerisation was not clearly demonstrated, this study provides a basis for future investigations of these receptors in prostate cancer. Additionally, the results presented in this study and growing evidence in the literature highlight the requirement for an extensive understanding of the experimental method and the performance of a range of controls to avoid the spurious interpretation of data gained from artificial expression systems. The future development of more robust techniques for investigating GPCR dimerisation is clearly required and will enable us to elucidate whether GHS-R1a, GHS-R1b and GPR39 form physiologically relevant dimers.

Modelling bus lost time : an additional parameter influencing bus dwell time and station platform capacity at a BRT station platform

The common approach to estimate bus dwell time at a BRT station is to apply the traditional dwell time methodology derived for suburban bus stops. In spite of being sensitive to boarding and alighting passenger numbers and to some extent towards fare collection media, these traditional dwell time models do not account for the platform crowding. Moreover, they fall short in accounting for the effects of passenger/s walking along a relatively longer BRT platform. Using the experience from Brisbane busway (BRT) stations, a new variable, Bus Lost Time (LT), is introduced in traditional dwell time model. The bus lost time variable captures the impact of passenger walking and platform crowding on bus dwell time. These are two characteristics which differentiate a BRT station from a bus stop. This paper reports the development of a methodology to estimate bus lost time experienced by buses at a BRT platform. Results were compared with the Transit Capacity and Quality of Servce Manual (TCQSM) approach of dwell time and station capacity estimation. When the bus lost time was used in dwell time calculations it was found that the BRT station platform capacity reduced by 10.1%.

High-challenge teaching for senior English as an Additional Language (EAL) learners in times of change

This paper will present a brief overview of the recent shifts within English and EAL/D (English as an additional language/dialect) curriculum documents and their focus on critical literacy, using the Queensland context as a case in point. The English syllabus landscape in Queensland has continued to morph in recent years. From 2002 to 2009, teachers of senior English and English as an Additional Language (EAL/D) have witnessed no less than four separate syllabus documents that impact on their daily work. The Australian Curriculum, when finally implemented, will also require teachers to navigate and grapple with its particular obligations and affordances. The combined effect of the shifts and tensions between recent policy documents has led to confusion about exactly how to cater for EAL/D learners in mainstream English. We discuss the possible effects of this on teachers as the agents of policy implementation and argue that in spite of such contradictions, EAL/D teachers can productively use syllabus frameworks to craft pedagogy to cater for their EAL/D learners’ language and literacy needs. Following this, we present aspects of the teaching practice of four teachers of senior EAL/D, who provide intellectually-engaging, critical literacy pedagogy that takes into account the language proficiency level of their learners, within the required curriculum. Such practice provides teachers with valuable pedagogic possibilities to meet EAL/D learners’ needs within continually varying policy terrain.

Hysteresis modulation of multilevel inverters

The hysteresis modulation for power electronic converters is attractive in many different applications because of its unmatched dynamic response and wide command-tracking bandwidth. Its application and beneftis for two-level converters are well understood, but the extension of this strategy to multilevel converters is still under development. This paper summarizes and reviews the various hysteresis modulation approaches available in the literature for multilevel converters. The pros and cons of various techniques are described and compared for tracking the reference signal in order to attain an adequate switching optimization, excellent dynamic responses and high accuracy in steady-state operation. By using the recently developed multilevel hysteresis modulation approaches the advantages of using several accessible dc potentials in a multilevel inverter has been fully exploited. All of these hysteresis modulation approaches are testing for tracking a current reference when applied to a fivelevel inveter. The relevant simulation and experimental result are also presented. This study will provide a useful framweork and point of reference for the future development of hysteresis modulation for multilevel converters.

Modulation of depth-dependent properties in tissue-engineered cartilage with a semi-permeable membrane and perfusion : a continuum model of matrix metabolism and transport

The functional properties of cartilaginous tissues are determined predominantly by the content, distribution, and organization of proteoglycan and collagen in the extracellular matrix. Extracellular matrix accumulates in tissue-engineered cartilage constructs by metabolism and transport of matrix molecules, processes that are modulated by physical and chemical factors. Constructs incubated under free-swelling conditions with freely permeable or highly permeable membranes exhibit symmetric surface regions of soft tissue. The variation in tissue properties with depth from the surfaces suggests the hypothesis that the transport processes mediated by the boundary conditions govern the distribution of proteoglycan in such constructs. A continuum model (DiMicco and Sah in Transport Porus Med 50:57-73, 2003) was extended to test the effects of membrane permeability and perfusion on proteoglycan accumulation in tissue-engineered cartilage. The concentrations of soluble, bound, and degraded proteoglycan were analyzed as functions of time, space, and non-dimensional parameters for several experimental configurations. The results of the model suggest that the boundary condition at the membrane surface and the rate of perfusion, described by non-dimensional parameters, are important determinants of the pattern of proteoglycan accumulation. With perfusion, the proteoglycan profile is skewed, and decreases or increases in magnitude depending on the level of flow-based stimulation. Utilization of a semi-permeable membrane with or without unidirectional flow may lead to tissues with depth-increasing proteoglycan content, resembling native articular cartilage.

Varenicline, an alpha-4 beta-2 nicotinic acetylcholine receptor partial agonist, selectively decreases ethanol consumption and seeking

Abstract Alcohol dependence is a disease that impacts millions of individuals worldwide. There has been some progress with pharmacotherapy for alcohol-dependent individuals; however, there remains a critical need for the development of novel and additional therapeutic approaches. Alcohol and nicotine are commonly abused together, and there is evidence that neuronal nicotinic acetylcholine receptors (nAChRs) play a role in both alcohol and nicotine dependence. Varenicline, a partial agonist at the alpha4beta2 nAChRs, reduces nicotine intake and was recently approved as a smoking cessation aid. We have investigated the role of varenicline in the modulation of ethanol consumption and seeking using three different animal models of drinking. We show that acute administration of varenicline, in doses reported to reduce nicotine reward, selectively reduced ethanol but not sucrose seeking using an operant self-administration drinking paradigm and also decreased voluntary ethanol but not water consumption in animals chronically exposed to ethanol for 2 months before varenicline treatment. Furthermore, chronic varenicline administration decreased ethanol consumption, which did not result in a rebound increase in ethanol intake when the varenicline was no longer administered. The data suggest that the alpha4beta2 nAChRs may play a role in ethanol-seeking behaviors in animals chronically exposed to ethanol. The selectivity of varenicline in decreasing ethanol consumption combined with its reported safety profile and mild side effects in humans suggest that varenicline may prove to be a treatment for alcohol dependence.

Affect, attention, or anticipatory arousal? Human blink startle modulation in forward and backward affective conditioning

Affect modulates the blink startle reflex in the picture-viewing paradigm, however, the process responsible for reflex modulation during conditional stimuli (CSs) that have acquired valence through affective conditioning remains unclear. In Experiment 1, neutral shapes (CSs) and valenced or neutral pictures (USs) were paired in a forward (CS → US) manner. Pleasantness ratings supported affective learning of positive and negative valence. Post-acquisition, blink reflexes were larger during the pleasant and unpleasant CSs than during the neutral CS. Rather than affect, attention or anticipatory arousal were suggested as sources of startle modulation. Experiment 2 confirmed that affective learning in the picture–picture paradigm was not affected by whether the CS preceded the US. Pleasantness ratings and affective priming revealed similar extents of affective learning following forward, backward or simultaneous pairings of CSs and USs. Experiment 3 utilized a backward conditioning procedure (US → CS) to minimize effects of US anticipation. Again, blink reflexes were larger during CSs paired with valenced USs regardless of US valence implicating attention rather than anticipatory arousal or affect as the process modulating startle in this paradigm.

Modulation of the Chlamydia trachomatis In vitro transcriptome response by the sex hormones estradiol and progesterone

BACKGROUND:Chlamydia trachomatis is a major cause of sexually transmitted disease in humans. Previous studies in both humans and animal models of chlamydial genital tract infection have suggested that the hormonal status of the genital tract epithelium at the time of exposure can influence the outcome of the chlamydial infection. We performed a whole genome transcriptional profiling study of C. trachomatis infection in ECC-1 cells under progesterone or estradiol treatment.RESULTS:Both hormone treatments caused a significant shift in the sub-set of genes expressed (25% of the transcriptome altered by more than 2-fold). Overall, estradiol treatment resulted in the down-regulation of 151 genes, including those associated with lipid and nucleotide metabolism. Of particular interest was the up-regulation in estradiol-supplemented cultures of six genes (omcB, trpB, cydA, cydB, pyk and yggV), which suggest a stress response similar to that reported previously in other models of chlamydial persistence. We also observed morphological changes consistent with a persistence response. By comparison, progesterone supplementation resulted in a general up-regulation of an energy utilising response.CONCLUSION:Our data shows for the first time, that the treatment of chlamydial host cells with key reproductive hormones such as progesterone and estradiol, results in significantly altered chlamydial gene expression profiles. It is likely that these chlamydial expression patterns are survival responses, evolved by the pathogen to enable it to overcome the host's innate immune response. The induction of chlamydial persistence is probably a key component of this survival response.

An additional step towards modelling the mechanical behaviour of sugar cane bagasse

A better understanding of the behaviour of prepared cane and bagasse, especially the ability to model the mechanical behaviour of bagasse as it is squeezed in a milling unit to extract juice, would help identify how to improve the current milling process; for example to reduce final bagasse moisture. Previous investigations have proven with certainty that juice flow through bagasse obeys Darcy’s permeability law, that the grip of the rough surface of the grooves on the bagasse can be represented by the Mohr- Coulomb failure criterion for soils, and that the internal mechanical behaviour of the bagasse can be represented by critical state behaviour similar to that of sand and clay. Current Finite Element Models (FEM) available in commercial software have adequate permeability models. However, commercial software does not contain an adequate mechanical model for bagasse. Progress has been made in the last ten years towards implementing a mechanical model for bagasse in finite element software code. This paper builds on that progress and carries out a further step towards obtaining an adequate material model. In particular, the prediction of volume change during shearing of normally consolidated final bagasse is addressed.

Pharmacological consequence of the A118G μ opioid receptor polymorphism on morphine-and fentanyl-mediated modulation of Ca2+ channels in humanized mouse sensory neurons

Background: The most common functional single nucleotide polymorphism of the human OPRM1 gene, A118G, has been shown to be associated with interindividual differences in opioid analgesic requirements, particularly with morphine, in patients with acute postoperative pain. The purpose of this study was to examine whether this polymorphism would modulate the morphine and fentanyl pharmacological profile of sensory neurons isolated from a humanized mouse model homozygous for either the 118A or 118G allele. Methods: The coupling of wild-type and mutant μ opioid receptors to voltage-gated Ca channels after exposure to either ligand was examined by employing the whole cell variant of the patch-clamp technique in acutely dissociated trigeminal ganglion neurons. Morphine-mediated antinociception was measured in mice carrying either the 118AA or 118GG allele. RESULTS:: The biophysical parameters (cell size, current density, and peak current amplitude potential) measured from both groups of sensory neurons were not significantly different. In 118GG neurons, morphine was approximately fivefold less potent and 26% less efficacious than that observed in 118AA neurons. On the other hand, the potency and efficacy of fentanyl were similar for both groups of neurons. Morphine-mediated analgesia in 118GG mice was significantly reduced compared with the 118AA mice. Conclusions: This study provides evidence to suggest that the diminished clinical effect observed with morphine in 118G carriers results from an alteration of the receptor's pharmacology in sensory neurons. In addition, the impaired analgesic response with morphine may explain why carriers of this receptor variant have an increased susceptibility to become addicted to opioids. © 2011 the American Society of Anesthesiologists, Inc. Lippincott Williams & Wilkins. Anesthesiology.

The effect of emotional and attentional load on attentional startle modulation

The interactive effects of emotion and attention on attentional startle modulation were investigated in two experiments. Participants performed a discrimination and counting task with two visual stimuli during which acoustic eyeblink startle-eliciting probes were presented at long lead intervals. In Experiment 1, this task was combined with aversive Pavlovian conditioning. In Group Attend CS+, the attended stimulus was followed by an aversive unconditional stimulus (US) and the ignored stimulus was presented alone whereas the ignored stimulus was paired with the US in Group Attend CS−. In Experiment 2, a non-aversive reaction time task US replaced the aversive US. Regardless of the conditioning manipulation and consistent with a modality non-specific account of attentional startle modulation, startle magnitude was larger during attended than ignored stimuli in both experiments. Blink latency shortening was differentially affected by the conditioning manipulations suggesting additive effects of conditioning and discrimination and counting task on blink startle.

The relationship between self-reported animal fear and ERP modulation : Evidence for enhanced processing and fear of harmless invertebrates in snake- and spider-fearful individuals

The present study used ERPs to compare processing of fear-relevant (FR) animals (snakes and spiders) and non-fear-relevant (NFR) animals similar in appearance (worms and beetles). EEG was recorded from 18 undergraduate participants (10 females) as they completed two animal-viewing tasks that required simple categorization decisions. Participants were divided on a post hoc basis into low snake/spider fear and high snake/spider fear groups. Overall, FR animals were rated higher on fear and elicited a larger LPC. However, individual differences qualified these effects. Participants in the low fear group showed clear differentiation between FR and NFR animals on subjective ratings of fear and LPC modulation. In contrast, participants in the high fear group did not show such differentiation between FR and NFR animals. These findings suggest that the salience of feared-FR animals may generalize on both a behavioural and electro-cortical level to other animals of similar appearance but of a non-harmful nature.