Adenocarcinoma of colon differentiating as dome epithelium of gut-associated lymphoid tissue

Aims: An early adenocarcinoma of the ascending colon was confined to a mass of gut-associated lymphoid tissue (GALT). The first description of an adenocarcinoma of colon differentiating as dome epithelium is presented. Methods and results: A plaque-like carcinoma was identified opposite the ileocaecal valve in an asymptomatic 56-year-old man with a family history of colorectal cancer. Malignant epithelium was confined to a mass of GALT filling but limited to the submucosa, Characterization of the neoplasm was undertaken by means of mucin histochemistry, immunohistochemistry, electron microscopy and assessment of DNA microsatellite instability status. The malignant epithelium comprised well differentiated columnar cells with a microvillous brush border and expressing MUC1, but no goblet cells or expression of MUC2. The demonstration of focal clusters of intraepithelial B-lymphocytes supported the presence of functioning M-cells within the malignant neoplasm. The cancer was DNA microsatellite stable despite the finding of tumour infiltrating lymphocytes. Conclusions: There is evidence for the origin of colorectal neoplasia from dome epithelium in both experimental models and microreconstruction studies of early adenomas in nonpolypotic human colorectal mucose, It is suggested that the lymphocyte-rich subset of colorectal cancer that expresses MUC1 but not MUC2 may be differentiating as dome epithelium of gut-associated lymphoid tissue.

Neoplastic progression occurs through mutator pathways in hyperplastic polyposis of the colorectum

Aim-Colorectal cancer has been described in association with hyperplastic polyposis but the mechanism underlying this observation is unknown. The aim of this study was to characterise foci of dysplasia developing in the polyps of subjects with hyperplastic polyposis on the basis of DNA microsatellite status and expression of the DNA mismatch repair proteins hMLH1, hMSH2, and hMSH6. Materials and methods-The material was derived from four patients with hyperplastic polyposis and between one and six synchronous colorectal cancers. Normal (four), hyperplastic (13), dysplastic (13), and malignant (11) samples were microdissected and a PCR based approach was used to identify mutations at 10 microsatellite loci, TGF beta IIR, IGF2R, BAX, MSH3, and MSH6. Microsatellite instability-high (MSI-H) was diagnosed when 40% or more of the microsatellite loci showed mutational bandshifts. Serial sections were stained for hMLH1, hMSH2, and hMSH6. Result-DNA microsatellite instability was found in 1/13 (8%) hyperplastic samples, in 7/13 (54%) dysplastic foci, and in 8/11 (73%) cancers. None of the MSI-low (MSI-L) samples (one hyperplastic, three dysplastic, two cancers) showed loss of hMLH1 expression. All four MSI-H dysplastic foci and six MSI-H cancers showed loss of hMLH1 expression. Loss of hMLH1 in MSI-H but not in MSI-L lesions showing dysplasia or cancer was significant (p< 0.001, Fisher's exact test). Loss of hMSH6 occurred in one MSI-H cancer and one MSS focus of dysplasia which also showed loss of hMLH1 staining. Conclusion-Neoplastic changes in hyperplastic polyposis may occur within a hyperplastic polyp. Neoplasia may be driven by DNA instability that is present to a low (MSI-L) or high (MSI-H) degree. MSI-H but not MSI-L dysplastic foci are associated with loss of hMLH1 expression. At least two mutator pathways drive neoplasia in hyperplastic polyposis. The role of the hyperplastic polyp in the histogenesis of sporadic DNA microsatellite unstable colorectal cancer should be examined.

VIP and PACAP potentiation of nicotinic ACh-evoked currents in rat parasympathetic neurons is mediated by G-protein activation

The effects of vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP27 and PACAP38) on isolated parasympathetic neurons of rat intracardiac and submandibular ganglia were examined under voltage clamp using whole-cell patch-clamp recording techniques. VIP and PACAP (less than or equal to 10 nm) selectively and reversibly increased the affinity of nicotinic acetylcholine receptor channels (nAChRs) for their agonists resulting in a potentiation of acetylcholine (ACh)-evoked whole-cell currents at low agonist concentrations. VIP-induced potentiation was observed with either ACh or nicotine as the cholinergic agonist. The VIP- but not the PACAP-induced potentiation of ACh-evoked currents was inhibited by [Ac-Tyr(1), D-Phe(2)]-GRF 1-29, amide (100 nm), a selective antagonist of VPAC(1) and VPAC(2) receptors; whereas the PACAP38- but not the VIP-induced potentiation was inhibited by 100 nm PACAP6-38, a PAC(1) and VPAC(2) receptor antagonist. The signal transduction pathway mediating VIP- and PACAP-induced potentiation of nicotinic ACh-evoked currents involves a pertussis toxin (PTX)-sensitive G-protein. Intracellular application of 200 mu m GTP gamma S or GDP beta S inhibited VIP-induced potentiation of ACh-evoked whole-cell currents. GTP gamma S alone potentiated ACh- and nicotine-evoked currents and the magnitude of these currents was not further increased by VIP or PACAP. The G-protein subtype modulating the neuronal nAChRs was examined by intracellular dialysis with antibodies directed against alpha(o), alpha(i-1,2), alpha(i-3) or beta G-protein subunits. Only the anti-G alpha(o) and anti-G beta antibodies significantly inhibited the effect of VIP and PACAP on ACh-evoked currents. The potentiation of ACh-evoked currents by VIP and PACAP may be mediated by a membrane-delimited signal transduction cascade involving the PTX-sensitive G(o) protein.

Further evidence of linkage at 7p22 with familial hyperaldosteronism type II and exclusion of genetic defects in the RBAK coding regions

Once thought rare, primary aldosteronism (PAL) is now reported to be responsible for 5–10% of hypertension. Unlike familial hyperaldosteronism type I (FH-I), FH-II is not glucocorticoidremediable and not associated with the hybrid CYP11B1/CYP11B2 gene mutation. At least five times more common than FH-I, FH-II is clinically indistinguishable from apparently sporadic PAL, suggesting an even higher incidence. Studies performed in collaboration with C Stratakis (NIH, Bethesda) on our largest Australian family (eight affected members) demonstrated linkage at chromosome 7p22. Linkage at this region was also found in a South American family (DNA provided by MI New, Mount Sinai School of Medicine, New York) and in a second Australian family. The combined multipoint LOD score for these 3 families is 4.61 (q = 0) with markers D7S462 and D7S517, providing strong support for this locus harbouring mutations responsible for FH-II. A newly identified recombination event in our largest Australian family has narrowed the region of linkage by 1.8 Mb, permitting exclusion of approximately half the genes residing in the originally reported 5 Mb linked locus. Candidate genes that are involved in cell cycle control are of interest as adrenal hyperplasia and adrenal adenomas are common in FH-II patients. A novel candidate gene in this linked region produces the retinoblastoma-associated Kruppel-associated box protein (RBaK) which interacts with the retinoblastoma gene product to repress the expression of genes activated by members of the E2F family of transcription factors.

A polymorphism in the growth hormone-releasing hormone receptor gene: clinical significance?

Two forms of the growth hormone-releasing hormone (GHRH) receptor (GHRH-R) exist in terms of a polymorphism at codon 57. The most common allele possesses GCG, coding for Ala. This codon can also be ACG, replacing the Ala with Thr. The present study demonstrates that the latter occurs in about 20% of pituitary somatotrophinomas, removed from patients with acromegaly. Somatotrophinomas possessing the alternative allele respond, on average, more strongly to GHRH in terms of GH secretion in vitro than tumors which are homozygous for the more common allele. The distribution of the two allelic forms of the GHRH-R did not significantly differ between acromegalic and non-acromegalic subjects. Thus, while the alternative allelic forms may, at least partially, contribute to the variable response of serum GH levels to i.v. GHRH observed in acromegalic and normal subjects, it is unlikely that subjects possessing the rarer form containing Thr in place of Ala at residue 57 are at increased risk of developing acromegaly.

The environmental control of reproduction in the female dace, Leuciscus leuciscus

An investigation was made into the nature and control of the annual reproductive cycle of the dace, Leuciscus leuciscus. It includes 1) a study of the natural reproductive cycle, 2) the use of Carp Pituitary Extract (CPE) to induce final maturation and ovulation in captive fish, 3) the effect of artificial light treatments on ovarian development and 4) the measurement of serum melatonin levels under different photoperiod regimes. Ovarian development was monitored by endocrinological data, notably serum cycles of 17-oestradiol (E2), testosterone (T), and calcium (as an index of vitellogenin), oocyte diameter, the gonadosomatic index and histological studies of the ovary. Under natural conditions, ovarian development can broadly be divided into 4 stages: 1) oogenesis which occurs immediately after spawning; 2) a primary growth phase (previtellogenic growth) prevalent between spawning and June; 3) a secondary growth phase (yolk vesicle plus vitellogenic growth) occurring between June and December and 4) final maturation and ovulation which occurs in mid-March. During the annual ovarian cycle, the sex steroids E2 and T showed two clear elevations. The first occurred initially in April followed by a rise in serum calcium levels. This subsequently initiated the appearance of yolk granules in the oocytes in June. The second rise occurred in September and levels were maintained until December, after which there was a decline in serum E2 levels. It is proposed that in the dace, high serum E2 levels between September and December were required to maintain vitellogenin production and therefore its uptake into the developing oocytes which occurred during this time, albeit at a slower rate than in the summer months. After December, prior to final maturation, whereas serum E2 and calcium levels declined, serum T levels remained elevated. In captivity, final maturation beyond the germinal vesicle migration stage failed to occur suggesting that the stimuli required for these events were absent. However ovulation could be induced by a single injection of CPE, which induced ovulation between 6 and 14 hours after treatment. Endocrine events associated with the artificial induction of spawning included a rise in serum levels of E2, T and the maturation inducing steroid 1720-dihydroxy progesterone. Photoperiodic manipulation demonstrated that whereas short or increasing daylengths were stimulatory to ovarian development, long days delayed development. Changes from long to short and constant short daylengths early in the reproductive cycle advanced maturation (up to 5 months), suggesting that the stimulus for ovarian development and maturation was a short day. However, experiments conducted later in the reproductive cycle demonstrated that only a simulated ambient photoperiod could induce final maturation. It is proposed therefore that under natural conditions the environmental stimulus for ovarian development and final maturation are short and increasing daylengths respectively. Further support that photoperiod is the dominant timing cue in this species was provided by the pattern of serum melatonin levels. Under different photoperiod treatments, serum melatonin, which is believed to be the chemical transducer of photoperiodic information (similar to other photoperiodic species) was elevated for the duration of the dark phase, indicating that the dace at least has the ability to `measure' changes in daylength.

Somatic cell gene therapy for diabetes mellitus: engineering a surrogate B-cell

Improved methods of insulin delivery are required for the treatment of insulin-dependent diabetes mellitus (IDDM) to achieve a more physiological profile of glucose homeostasis. Somatic cell gene therapy offers the prospect that insulin could be delivered by an autologous cell implant, engineered to secrete insulin in response to glucose. This study explores the feasibility of manipulating somatic cells to behave as a surrogate insulin-secreting β-cells. Initial studies were conducted using mouse pituitary AtT20 cells as a model, since these cells possess an endogenous complement of enzymes capable of processing proinsulin to mature insulin. Glucose sensitive insulin secretion was conferred to these cells by transfection with plasmids containing the human preproinsulin gene (hppI-1) and the GLUT2 gene for the glucose transporter isoform 2. Insulin secretion was responsive to changes in the glucose concentration up to about 50μM. Further studies to up-rate this glucose sensitivity into the mM range will require manipulation of the hexokinase and glucokinase enzymes. Intraperitoneal implantation of the manipulated AtT20 cells into athymic nude mice with streptozotocin-induced diabetes resulted in decreased plasma glucose concentrations. The cells formed vascularised tumours in vivo which were shown to contain insulin-secreting cells. To achieve proinsulin processing in non-endocrine cells, co-transfection with a suitable enzyme, or mutagenesis of the proinsulin itself are necessary. The mutation of the human preproinsulin gene to the consensus sequence for cleavage by the subtilisin-like serine protease, furin, was carried out. Co-transfection of human fibroblasts with wild-type proinsulin and furin resulted in 58% conversion to mature insulin by these cells. Intraperitoneal implantation of the mature-insulin secreting human fibroblasts into the diabetic nude mouse animal model gave less encouraging results than the AtT20 cells, apparently due to poor vascularisation. Cell aggregations removed from the mice at autopsy were shown to contain insulin secreting cells only at the periphery. This thesis provides evidence that it is possible to construct, by cellular engineering, a glucose-sensitive insulin-secreting surrogate β-cell. Therefore, somatic cell gene therapy offers a feasible alternative for insulin delivery in IDDM patients.

Molecular biology and biochemistry of brain tumours

ßElucidating some molecular mechanisms and biochemistry of brain tumours is an important step towards the development of adjuvant medical therapies. The present study concentrates on cholecystokinin (CCK), a gut-brain peptide that has been described to be able to induce mitosis of rat gliomas as well as hormone secretion by the anterior pituitary, via the CCK-B receptor. The significance of a polymorphism in the growth hormone releasing hormone (GHRH) receptor (GHRH-R) gene was also determined. Finally, defects in the ß-catenin gene, an important component of the developmental pathway, in a sub-set of craniopharyngiomas were investigated. Reverse transcription-polymerase chain reaction (RT-PCR), restriction digestion analysis and direct sequencing demonstrated expression of CCK peptide itself and its A and B receptors by human gliomas, meningiomas and pituitary tumours. CCK peptides stimulated growth of cultured gliomas and meningiomas as well as in vitro hormone secretion [growth hormone (GH), luteinizing hormone (LH) and follicle stimulating hormone (FSH)] by human pituitary tumours. These biological effects were reduced or abolished by CCK antagonists. In addition, an antibody to CCK reduced mitosis by gliomas and meningiomas, and the same antibody inhibited hormone secretion by cultured human pituitary tumours. CCK peptides stimulated phosphatidylinositol (PI) hydrolysis, indicating coupling of the CCK receptors to phopsholipase C. Cyclic AMP was unaffected. In addition, caspase-3 activity was significantly and markedly increased, whilst proteasome activity was decreased. Taken together, these results may indicate an autocrine/paracrine role of CCK in the control of growth and/or functioning of gliomas, meningiomas and pituitary tumours. Primer induced restriction analysis (PIRA) of a rarer and alternative polymorphism in the GHRH-R receptor, in which Thr replaces Ala at codon 57, in human GH-secreting pituitary tumours was investigated. Whilst the rarer form correlated with an increased response of the pituitary cells to GHRH in vitro, allele distribution studies revealed that it is unlikely that the polymorphism contributes to increased risk of developing GH-secreting tumours and therefore acromegaly. Further findings of this study, using PCR and direct sequencing, were the demonstration of an association between b-catenin gene alterations and craniopharyngiomas of the adamantinomatous type. Since this gene product is involved with development, these results suggest that p-catenin mutations may contribute to the initiation and subsequent growth of congenital adamantinomatous craniopharyngiomas.

Down-regulation of central glucocorticoid receptor expression using antisense oligonucleotide technology

Endogenous glucocorticoids and serotonin have been implicated in the pathophysiology of depression, anxiety and schizophrenia. This thesis investigates the potential of downregulating expression of central Type II glucocorticoid receptors (GR) both in vitro and in vivo, with empirically-designed antisense oligodeoxynucleotides (ODN), to characterise GR modulation of 5-HT2A receptor expression using quantitative RT-PCR, Western blot analysis and radioligand binding. The functional consequence of GR downregulation is also determined by measuring 1-(2,5-dimethoxy 4-iodophenyl)-2-amino propane hydrochloride (DOI) mediated 5-HT2A receptor specific headshakes. Using a library of random antisense ODN probes, RNAse H accessibility mapping of T7-primed, in vitro transcribed GR mRNA revealed several potential cleavage sites and identified an optimally effect GR antisense ODN sequence of 21-mer length (GRAS5). In vitro efficacy studies using rat C6 glioma cells showed a 56% downregulation in GR mRNA levels and 80% downregulation in GR protein levels. In the same cells a 29% upregulation in 5-HT2A mRNA levels and 32% upregulation in 5-HT2A protein levels was revealed. This confirmed the optimal nature of the GRAS5 sequence to produce marked inhibition of GR gene expression, and also revealed GR modulation of the 50-HT2A receptor subtype in C6 glioma cells to be a tonic repression of receptor expression. The distribution of a fluorescently-labelled GRAS5 ODN was detected in diverse areas of the rat brain after single ICV administration, although this fluorescence signal was not sustained over a period of 5 days. However, fluorescently-labelled GRAS5 ODN, when formulated in polymer microspheres, showed diverse distribution in the brain which was maintained for 5 days following a single ICV administration. This produced no apparent neurotoxic effects on rat behaviour and hypothalamic-pituitary-adrenal (HPA) axis homeostasis. Furthermore, a single polymer microsphere injection ICV proved to be an effective means of delivering antisense ODNs and this was adopted for the in vivo efficacy studies. In vivo characterisation of GRAS5 revealed marked downregulation of GR mRNA in rat brain regions such as the frontal cortex (26%), hippocampus (35%), and hypothalamus (39%). Downregulation of GR protein was also revealed in frontal cortex (67%), hippocampus (76%), and hypothalamus (80%). In the same animals upregulation of 5-HT2A mRNA levels was shown in frontal cortex (13%), hippocampus (7%), and hypothalamus (5%) while upregulation in 5-HT2A protein levels was shown in frontal cortex (21 %). This upregulation in 5-HT2A receptor density as a result of antisense-mediated inhibition of GR was further confirmed by a 55% increase in DOl-mediated 5-HT2A receptor specific headshakes. These results demonstrate that GR is involved in tonic inhibitory regulation of 5-HT2A receptor expression and function in vivo, thus providing the potential to control 5-HT2A-linked disorders through corticosteroid manipulation. These experiments have therefore established an antisense approach which can be used to investigate pharmacological characteristics of receptors.

Tetrahydrobiopterin metabolism in depression and senile dementia of Alzheimer type

Excretion of biopterin and the related pteridines neopterin and pterin was measured in urine samples from a group of 76 male and female unipolar and bipolar depressed outpatients receiving lithium therapy, and compared to 61 male and female control subjects. The ratio of neopterin to biopterin excreted (N/B) was significantly higher in the patients than the controls. The significant positive correlation between urinary neopterin and biopterin shown by the controls was absent in the patients, indicating disrupted biosynthesis of tetrahydrobiopterin.Urinary cortisol excretion in depressed patients was similar to controls, implying normal hypothalmus-pituitary-adrenal axis function in these patients, Serum folate was shown to correlate with urinary total biopterin excretion in female unipolar patients. Two groups of elderly females with senile dementia of Alzheimer type (SDAT) were examined for urinary pteridine excretion. In the first study of 10 patients, the N/B ratio was significantly higher than in 24 controls and the ratio B/B+ N significantly lower. A second study of 30 SDAT patients and 21 controls confirmed these findings. However, neopterin correlated with biopterin in both patients and controls, indicating that the alteration in tetrahydrobiopterin metabolism may be different to that shown in depression. Lithium had no effect in vivo or in vitro on Wistar rat brain or liver biosynthesis of tetrahydrobiopterin at a range of concentrations and duration of dosing period, showing that lithium was not responsible for the lowered biopterin excretion by depressed patients. No significant effects on tetrahydrobiopterin metabolism in the rat were shown by the tricyclic antidepressant imipramine, the anticonvulsant sodium valproate, the vitamin folic acid, the anticatecholaminergic agent amethylparatyrosine, the synthetic corticosteroid dexamethasone, or stimulation of natural cortisol by immobilisation stress. Scopolamine, an ant ichol inergic drug, lowered rat brain pterin which may relate to the tetrahydrobiopterin deficits shown in SDAT.

Insulin gene therapy for the treatment of diabetes mellitus

Currently available treatments for insulin-dependent diabetes mellitus are often inadequate in terms of both efficacy and patient compliance. Gene therapy offers the possibility of a novel and improved method by which exogenous insulin can be delivered to a patient. This was approached in the present study by constructing a novel insulin-secreting cell line. For the purposes of this work immortalized cell lines were used. Fibroblasts and pituitary cells were transfected with the human preproisinulin gene to create stable lines of proinsulin- and insulin-secreting cells. The effect of known β-cell secretagogues on these cells were investigated, and found mostly to have no stimulatory effect, although IBMX, arginine and ZnSO4 each increased the rate of secretion. Cyclosporin (CyA) is currently the immunosuppresant of choice for transplant recipients; the effect of this treatment on endogenous β-cell function was assessed both in vivo and in vitro. Therapeutic doses of CyA were found to reduce plasma insulin concentrations and to impair glucose tolerance. The effect of immunoisolation on insulin release by HIT T15 cells was also investigated. The presence of an alginate membrane was found to severely impair insulin release. For the first implantation of the insulin-secreting cells, the animal model selected was the athymic nude mouse. This animal is immunoincompetent, and hence the use of an immunosuppressive regimen is circumvented. Graft function was assessed by measurement of plasma human C peptide concentrations, using a highly specific assay. Intraperitoneal implantation of genetically manipulated insulin-secreting pituitary cells into nude mice subsequently treated with a large dose of streptozotocin (STZ) resulted in a significantly delayed onset of hyperglycaemia when compared to control animals. Consumption of a ZnSO4 solution was shown to increase human C peptide release by the implant. Ensuing studies in nude mice examined the efficacy of different implantation sites, and included histochemical examination of the tumours. Aldehyde fuchsin staining and immunocytochemical processing demonstrated the presence of insulin containing cells within the excised tissue. Following initial investigations in nude mice, implantation studies were performed in CyA-immunosuppressed normal and STZ-diabetic mice. Graft function was found to be less efficacious, possibly due to the subcutaneous implantation site, or to the immunosuppresive regimen. Histochemical and transmission electron microscopic analysis of the tumour-like cell clusters found at autopsy revealed necrosis of cells at the core, but essentially normal cell morphology, with dense secretory granules in peripheral cells. The thesis provides evidence that gene therapy offers a feasibly new approach to insulin delivery.

The relationship between alexithymia and salivary cortisol levels in somatoform disorders

The purpose of this study was to investigate cortisol levels as a function of the hypothalamic-pituitary-adrenal axis (HPA) in relation to alexithymia in patients with somatoform disorders (SFD). Diurnal salivary cortisol was sampled in 32 patients with SFD who also underwent a psychiatric examination and filled in questionnaires (Toronto Alexithymia Scale, TAS scale; Screening for Somatoform Symptoms, SOMS scale; Hamilton Depression Scale, HAMD). The mean TAS total score in the sample was 55.69.6, 32% of patients being classified as alexithymic on the basis of their TAS scores. Depression scores were moderate (HAMD=13.2, Beck Depression Inventory, BDI=16.5). The patients' alexithymia scores (TAS scale Difficulty identifying feelings) correlated significantly positively with their somatization scale scores (Symptom Checklist-90 Revised, SCL-90-R); r=0.3438 (P0.05) and their scores on the Global Severity Index (GSI) on the SCL-90-R; r=0.781 (P0.01). Regression analysis was performed with cortisol variables as the dependent variables. Cortisol levels [measured by the area under the curve-ground (AUC-G), area under the curve-increase (AUC-I) and morning cortisol (MCS)] were best predicted in a multiple linear regression model by lower depressive scores (HAMD) and more psychopathological symptoms (SCL-90-R). No significant correlations were found between the patients' alexithymia scores (TAS) and cortisol levels. The healthy control group (n=25) demonstrated significantly higher cortisol levels than did the patients with SFD; in both tests P0.001 for AUC-G and AUC-I. However, the two groups did not differ in terms of their mean morning cortisol levels (P0.05). The results suggest that pre-existing hypocortisolism might possibly be associated with SFD.

Receptor Activity Modifying Proteins (RAMPs) interact with the VPAC2 Receptor and CRF1 receptors and modulate their function

Background and Purpose Although it is established that the receptor activity modifying proteins (RAMPs) can interact with a number of GPCRs, little is known about the consequences of these interactions. Here the interaction of RAMPs with the glucagon-like peptide 1 receptor (GLP-1 receptor), the human vasoactive intestinal polypeptide/pituitary AC-Activating peptide 2 receptor (VPAC) and the type 1 corticotrophin releasing factor receptor (CRF) has been examined. Experimental Approach GPCRs were co-transfected with RAMPs in HEK 293S and CHO-K1 cells. Cell surface expression of RAMPs and GPCRs was examined by elisa. Where there was evidence for interactions, agonist-stimulated cAMP production, Ca mobilization and GTPγS binding to G, G, G and G were examined. The ability of CRF to stimulate adrenal corticotrophic hormone release in Ramp2 mice was assessed. Key Results The GLP-1 receptor failed to enhance the cell surface expression of any RAMP. VPAC enhanced the cell surface expression of all three RAMPs. CRF enhanced the cell surface expression of RAMP2; the cell surface expression of CRF was also increased. There was no effect on agonist-stimulated cAMP production. However, there was enhanced G-protein coupling in a receptor and agonist-dependent manner. The CRF: RAMP2 complex resulted in enhanced elevation of intracellular calcium to CRF and urocortin 1 but not sauvagine. In Ramp2 mice, there was a loss of responsiveness to CRF. Conclusions and Implications The VPAC and CRF receptors interact with RAMPs. This modulates G-protein coupling in an agonist-specific manner. For CRF, coupling to RAMP2 may be of physiological significance. © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.

Nutritional influences on cognitive function:mechanisms of susceptibility

The impact of nutritional variation, within populations not overtly malnourished, on cognitive function and arousal is considered. The emphasis is on susceptibility to acute effects of meals and glucose loads, and chronic effects of dieting, on mental performance, and effects of cholesterol and vitamin levels on cognitive impairment. New developments in understanding dietary influences on neurohormonal systems, and their implications for cognition and affect, allow reinterpretation of both earlier and recent findings. Evidence for a detrimental effect of omitting a meal on cognitive performance remains equivocal: from the outset, idiosyncrasy has prevailed. Yet, for young and nutritionally vulnerable children, breakfast is more likely to benefit than hinder performance. For nutrient composition, despite inconsistencies, some cautious predictions can be made. Acutely, carbohydrate-rich–protein-poor meals can be sedating and anxiolytic; by comparison, protein-rich meals may be arousing, improving reaction time but also increasing unfocused vigilance. Fat-rich meals can lead to a decline in alertness, especially where they differ from habitual fat intake. These acute effects may vary with time of day and nutritional status. Chronically, protein-rich diets have been associated with decreased positive and increased negative affect relative to carbohydrate-rich diets. Probable mechanisms include diet-induced changes in monoamine, especially serotoninergic neurotransmitter activity, and functioning of the hypothalamic pituitary adrenal axis. Effects are interpreted in the context of individual traits and susceptibility to challenging, even stressful, tests of performance. Preoccupation with dieting may impair cognition by interfering with working memory capacity, independently of nutritional status. The change in cognitive performance after administration of glucose, and other foods, may depend on the level of sympathetic activation, glucocorticoid secretion, and pancreatic β-cell function, rather than simple fuelling of neural activity. Thus, outcomes can be predicted by vulnerability in coping with stressful challenges, interacting with nutritional history and neuroendocrine status. Functioning of such systems may be susceptible to dietary influences on neural membrane fluidity, and vitamin-dependent cerebrovascular health, with cognitive vulnerability increasing with age.