No description available.

No description available.

No description available.

This study reveals that bovine chromaffin cells express PKC-alpha, -beta, -epsilon and -iota and that they were differentially activated by nicotine receptors and a range of Gaq/11-coupled GPCRs. Thus, nicotinic receptor stimulation recruited PKC-alpha, -beta and -epsilon from the cytoplasm to the plasma membrane, indicative of activation. In contrast, activation of Gaq/11-coupled receptors with histamine activated all the expressed PKC isoforms and angiotensin II only activated PKC-alpha and -epsilon.;Inhibition of PKC using general or isoform-selective inhibitors potentiated catecholamine release in response to activation of Gaq/11-coupled receptors, most likely as consequence of potentiated phospholipase C-mediated signalling. However, inhibition of PKC, particularly PKCalpha, markedly inhibited nicotinic receptor-mediated catecholamine release. PKCalpha is a classical isoform of PKC, activated by Ca2+ and diacylglycerol (DAG).;The current study suggests that Ca2+ influx across the plasma membrane in response to nicotinic receptor activation is largely through the nicotinic receptors themselves. Furthermore, this Ca2+ entry activates PLC, generating both Ins(1,4,5)P3 and DAG. This activation of PLC contributes significantly to the activation of PKC. The mechanism through which PKC facilitates the release of catecholamines requires the PKC-dependent phosphorylation of myristoylated alanine-rich C protein kinase substrate (MARCKS), the subsequent disassembly of the cortical F-actin cytoskeleton and probably therefore, increased access of a reserve exocytotic vesicle pool to release sites at the plasma membrane. Moreover, this study also suggests that PLC-dependent generation of DAG recruits Munc13-1, a known vesicle priming agent. Thus, activation of Munc13-1 may also contribute to nicotinic receptor-mediated catecholamine release in a manner dependent on the activation of PLC.

Binding of the AR to selective and non-selective DNA response elements was investigated to determine how this specificity is achieved. The influence of other receptor domains on protein-DNA interactions was examined and structural studies were performed to study conformational changes in the receptor in the presence and absence of specific DNA response elements. It was confirmed that the affinity of the isolated AR DNA binding domain (DBD) was greater for non-selective response elements compared with selective response elements. Interestingly, a decrease in binding affinity for both non-selective and selective sequences was observed in the presence of the N terminal domain (NTD). Furthermore this inhibitory effect required the NTD to be attached to the DBD. There were no differences detected in protein/DNA contacts of the DBD in the absence or presence of the NTD. However modest differences detected in protein/DNA contacts of the DBD in the absence or presence of the NTD. However modest differences in sensitivity to DNA methylation were observed in the 3' flanking sequences of the non-selective C3(1) element. In addition, structural studies detected a change in conformation of the NTD on binding to DNA response elements suggesting a structural role for the NTD in recognition of androgen binding sites. However no dramatic differences in conformation were observed when selective and non-selective response elements were studied. Taken together these studies indicate that there are reciprocal intradomain communications between the AR DBD and the NTD, leading to modulation of receptor DNA-binding activity, and changes in receptor conformation. It is speculated that the latter may modify specific protein-protein interactions and receptor-dependent gene regulation. Therefore, it is clear from these studies that DNA binding is likely to lay a more active role in receptor activity than simply tethering of the protein to promoter or enhancer sequences and that the nature of the DNA response elements needs to be taken into consideration when studying receptor-dependent transactivation.

- / -

The hypothalamic-pituitary-adrenal (HPA) axis regulates circulating levels of vital glucocorticoid hormones, and is the major neuroendocrine system in mammals that provides a rapid response and defence against stress. The dynamics of this system is characterized by an ultradian pulsatile pattern of hormone secretion, which becomes disrupted in different physiological and pathological conditions. The biological basis for these hormone oscillations is not known, but a neuronal "pulse generator" within the hypothalamus has remained a popular hypothesis. Using a mathematical model, we show that the combination of delay with negative feedback in the pituitary-adrenal system provides a mechanism for generating systems-level ultradian hormone oscillations, independent of pulsatile stimulation from the hypothalamus. Our model also predicts that these oscillations become disrupted for higher levels of hypothalamic drive. Testing these predictions in vivo, we show that a constant infusion of CRH and/or AVP induces ultradian oscillations in ACTH and glucocorti- coids, and confirm that higher constant levels of hypothalamic drive disrupt the oscillatory dynamics of the system. We also use our mathematical model to investigate how stressful stimuli perturb the pulsatile dynamics of the system. Using the theory of phase re- sponse curves, we demonstrate that an acute stress acts as a phase-resetting mechanism of the ultradian glucocorticoid oscillation. Our results demonstrate that pulsatile hypothalamic activity is not re- quired for generating ultradian glucocorticoid oscillations, and suggest that these oscillations emerge from a sub-hypothalamic pituitary-adrenal system, which functions as a deterministic peripheral hormone oscillator with a char- acteristic ultradian frequency. This constitutes a novel mechanism by which the level, rather than the pattern, of hypothalamic stimulation determines the dynamics of glucocorticoid hormone secretion. These findings could be important for our understanding of glucocorticoid signalling dynamics in both health and disease.

No description available.

Mutants were created within the main activation function domain to investigate the structure and function of this region. Structural studies based on limited proteolysis and fluorescence spectroscopy experiments, indicate that though the N-terminus is not as specially structured as either the LBD or DBD, there are regions of specific folding within the activation domain.  Results from <i>in silco</i> investigation suggest several possible regions of <span style='font-family: Symbol'>a helix within the AF-1 domain, and mutants designed to disrupt these regions were less folded than the wild-type protein. Protein-protein interaction studies showed that the four mutants designed to potentially disrupt function rather than structure, had reduced binding to the large subunit of TFIIF - RAP74, but had no effect on binding to the transcriptional co-activator SRC-1a. In a functional assay performed in yeast cells, these four same mutants all showed reduced activity, showing the same trend as binding to RAP74. This could be an indication that the function of the AR is dependent upon binding to the general transcription factor TFIIF. Interestingly one of the mutants that was found to show increased structure over the wild-type protein was previously shown to have a reduced interaction with RAP74. This implies that structure is important for interaction with the transcription machinery. This was confirmed by FTIR experiments which can detect changes in the proportion of secondary structure present in a protein. These data show that the proportion of <span style='font-family:Symbol'>a helix present in the AF-1 domain increases when it is complexed with RAP74. GST pull-down assays then demonstrated that the complex of AF-1 and RAP74 enhanced the binding of the co-activator SRC-1a. This is the first time that this cooperativity has been demonstrated with nuclear receptors and interacting proteins. Additionally, specific phosphorylation of the AF-1 domain by glycogen synthase kinase 3 also increases the level of binding with SRC-1a. These data together suggest a possible mechanism of action for the androgen receptor and its involvement in regulating transcription.

Previous studies have established that average profiles of salivary progesterone and oestradiol, differ considerably among populations. Diet, age, and energetics appear responsible for acute inter-populational differences, but significant, unexplained differences in chronic levels of reproductive steroids remain. Based on developmental hypotheses advanced by reproductive ecologists, a migration study was initiated to assess whether environmental conditions experienced during development can influence patterns of adult ovarian hormones. Salivary steroid profiles of Bangladeshi women who migrated to the UK at different times (infancy, childhood, adulthood) were compared to those of women in Bangladesh, second-generation Bangladeshi migrants, and white women bom and resident in the UK. Data on socio- demographics, anthropometry, physical activity, diet and reproductive history were also collected. The following hypotheses and predictions were examined: A) Early life conditions influence adult set points of ovarian steroid hormones - women in Bangladesh and adult migrants will have lower ovarian steroids than child migrants, second generation and white women B) improved conditions during childhood can alter levels of ovarian steroids child migrants will have levels of ovarian steroids that are negatively correlated with age at migration and C) alterations in conditions after maturation do not modify set points established during early life - adult migrants will have steroid levels that are comparable to Bangladeshi sedentees. The predictions were upheld for progesterone but not for oestradiol. Results point to infancy and childhood as a sensitive period when changes in environmental conditions determine the tempo of growth and maturation, as well as later adult progesterone levels. In contrast, no evidence was found of a developmental effect on adult levels of oestradiol. The alterations in hormones levels among Bangladeshi migrants, together with a changing diet and reproductive behaviours, may put child migrants and second-generation women at increased risk for breast cancer in later life.