Simon B. Guild

Developing research skills in medical students: AMEE Guide No. 69

This Guide has been written to provide guidance for individuals involved in curriculum design who wish to develop research skills and foster the attributes in medical undergraduates that help develop research. The Guide will provoke debate on an important subject, and although written specifically with undergraduate medical education in mind, we hope that it will be of interest to all those involved with other health professionals’ education. Initially, the Guide describes why research skills and its related attributes are important to those pursuing a medical career. It also explores the reasons why research skills and an ethos of research should be instilled into professionals of the future. The Guide also tries to define what these skills and attributes should be for medical students and lays out the case for providing opportunities to develop research expertise in the undergraduate curriculum. Potential methods to encourage the development of research-related attributes are explored as are some suggestions as to how research skills could be taught and assessed within already busy curricula. This publication also discusses the real and potential barriers to developing research skills in undergraduate students, and suggests strategies to overcome or circumvent these. Whilst we anticipate that this Guide will appeal to all levels of expertise in terms of student research, we hope that, through the use of case studies, we will provide practical advice to those currently developing this area within their curriculum.

A novel 4.1 ezrin radixin moesin (FERM)-containing protein, Willin

The 4.1 superfamily of proteins contain a 4.1 Ezrin Radixin Moesin (FERM) domain and are described as linking the cytoskeleton with the plasma membrane. Here, we describe a new FERM domain‐containing protein called Willin. Willin has a recognizable FERM domain within its N‐terminus and is capable of binding phospholipids. Its intra‐cellular distribution can be cytoplasmic or at the plasma membrane where it can co‐localize with actin. However, the plasma membrane location of Willin is not influenced by cytochalasin D induced actin disruption but it is induced by the addition of epidermal growth factor.

Cross-referencing the Scottish Doctor and Tomorrow's Doctors learning outcome frameworks

Learning outcomes, organised into systems or frameworks which describe and define the output of an educational programme, are being created and used in healthcare education with increasing frequency (Harden , 2002). Medical schools may be required to conform to more than one such outcome framework. For example, both the UK General Medical Council (GMC) and the Scottish Deans’ Medical Curriculum Group (SDMCG) have created and published a systematic learning outcome framework for medical graduates. Although both of these publications are concerned with undergraduate medical education, they differ in their aims, and structure. In order to use, evaluate and validate them, a cross-referencing system which relates each learning outcome statement, term or groups of terms is required.  This paper describes the cross-referencing exercise undertaken by the SDMCG, the philosophy behind it, the practical steps taken, the findings, the lessons learnt and reflections upon how this work may be taken forward. It will be of interest to all those who are involved in curriculum development using outcomes, and especially those who use the GMCs Tomorrows Doctors or the SDMCGs Scottish Doctor frameworks and those who are interested in education informatics in general.

Effects of protein kinase C activators upon the late stages of the ACTH secretory pathway of AtT-20 cells.

1 The mouse AtT‐20/D16–16 anterior pituitary tumour cell line was used as a model system for the study of phorbol 12‐myristate 13‐acetate (PMA)‐mediated enhancement of calcium‐evoked adrenocorticotrophin (ACTH) secretion. 2 PMA stimulated ACTH secretion from intact cells in a concentration‐dependent manner. Other phorbol esters; phorbol 12, 13‐dibutyrate (PDBu) and phorbol 12, 13‐didecanoate (PDD) and diacylglycerol analogues; 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG) and 1, 2‐dioctanoyl‐sn‐glycerol (DOG) also stimulated ACTH release from intact AtT‐20 cells. This would suggest that activation of protein kinase C (PKC) stimulates ACTH secretion from AtT‐20 cells. 3 Calcium stimulated ACTH secretion from electrically‐permeabilized cells over the concentration‐range of 10−7 m to 10−5 m. PMA (10−7 m) enhanced the amount of ACTH secreted at every concentration of calcium investigated. The PKC inhibitor, chelerythrine (10−5m) blocked the PMA (10−7 m)‐evoked enhancement of calcium (10−5 m)‐stimulated ACTH secretion but did not alter significantly the calcium (10−5 m)‐evoked secretion itself. This suggests that PKC modulates the secretory response to increases in intracellular calcium but does not mediate the effects of calcium. 4 Guanosine 5′‐O‐(3‐thiotriphosphate) (GTP‐γ‐S, 10−5m) stimulated ACTH secretion from permeabilized cells in the absence of calcium and was additive with calcium‐evoked ACTH secretion up to a maximum value which could be achieved by calcium acting alone. This suggests that a GTP‐binding protein mediates the secretory response to increases in the intracellular calcium. PMA (10−7 m) enhanced ACTH secretion stimulated by the combination of calcium and GTP‐γ‐S (10−5 m). 5 GTP‐γ‐S stimulated ACTH secretion from permeabilized cells in a concentration‐dependent manner with a threshold of 10−6 m. PMA (10−7 m) increased the amount of ACTH secretion evoked by every concentration of GTP‐γ‐S investigated. Chelerythrine (10−5m) blocked the PMA (10−7 m)‐evoked enhancement of GTP‐γ‐S (10−4 m)‐stimulated ACTH secretion but did not significantly alter GTP‐γ‐S (10−4 m)‐evoked secretion itself. This suggests that PKC modulates the secretory response to GTP‐γ‐S but does not mediate the effects of GTP‐γ‐S. 6 GTP‐γ‐S (10−8‐10−4 m) stimulated ACTH secretion from permeabilized cells either in the presence or absence of ATP (5 mM) indicating that its effects on secretion are ATP‐independent. 7 The results of the present study support the hypothesis that, in AtT‐20 cells, PMA is acting at some site distal to calcium entry which modulates the ability of an increase in cytosolic calcium concentration to stimulate ACTH secretion. This site of action is either at the level of or at some stage distal to a GTP‐binding protein which mediates the effects of calcium upon secretion. 8 PMA, unlike adenosine 3′:5′‐cyclic monophosphate (cyclic AMP) (Guild, 1991), can stimulate ACTH secretion from permeabilized cells in the absence of added calcium and guanine nucleotides which suggests that PMA and cyclic AMP are acting through distinct mechanisms at this post calcium site of action.

CHARACTERISATION OF THE EFFECTS OF NATRIURETIC PEPTIDES UPON ACTH SECRETION FROM THE MOUSE PITUITARY

The involvement of natriuretic peptides in the regulation of ACTH secretion in mice hemi-pituitary preparations was investigated. Atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) all inhibited CRF (10(-9) M)-evoked ACTH secretion over a concentration range of 10(-12)-10(-10) M and also stimulated cyclic GMP accumulation over a concentration range of 10 (-8)-10(-5) M. CNP was the most effective both in the inhibition of ACTH secretion and in the stimulation of cyclic GMP accumulation. Coincubation of hemi-pituitaries with 8bromo-cyclic GMP (10(-4) M) completely inhibited CRF (10(-9) M)-evoked ACTH secretion. Northern blot analysis revealed that all three major isoforms of the natriuretic peptide receptors are expressed in the mouse pituitary. These results demonstrate that natriuretic peptides do inhibit CRF-stimulated ACTH secretion from mouse pituitary preparations. A role for cGMP in mediating this effect on hormone secretion is indicated but the discrepancy between the efficacies of natriuretic peptides in inhibiting the secretory response and stimulating cyclic GMP accumulation suggest a more complicated stimulus-secretion coupling pathway is in operation.

Involvement of multiple protein kinase C isozymes in the ACTH secretory pathway of AtT‐20 cells

1 The mouse AtT‐20/D16‐16 anterior pituitary tumour cell line was used as a model system for the study of protein kinase C (PKC)‐mediated enhancement of calcium‐ and guanine nucleotide‐evoked adrenocorticotrophin (ACTH) secretion. 2 A profile of the PKC isozymes present in AtT‐20 cells was obtained by Western blotting analysis and it was found that AtT‐20 cells express the α, β, ∍ and ζ isoforms of PKC. 3 PKC isozymes were activated by the use of substances reported to activate particular isoforms of the enzyme. The effects of these substances were investigated in both intact and electrically‐permeabilized cells. Phorbol 12‐myristate 13‐acetate (PMA, EC50 = 1 ± 0.05 nM, which activates all isozymes of PKC, except the ζ isozyme), thymeleatoxin (TMX, EC50 = 10 ± 0.5 nM, which activates the α, β and γ isozymes) and 12‐deoxyphorbol 13‐phenylacetate 20‐acetate (dPPA, EC50 = 3 ± 0.5 nm, α β1 selective isozyme activator) all stimulated ACTH secretion from intact cells in a concentration‐dependent manner. Maximal TMX stimulated ACTH secretion was of a similar degree to that obtained in response to PMA but maximal dPPA‐stimulated ACTH secretion was only 60–70% of that obtained in response to PMA or TMX. 4 Calcium stimulated ACTH secretion from electrically‐permeabilized cells over the concentration‐range of 100 nM to 10 μm. PMA (100 nM), TMX (100 nM) but not dPPA (100 nM) enhanced the amount of ACTH secreted at every concentration of calcium investigated. PMA (100 nM) and TMX (100 nM) significantly enhanced ACTH secretion in the effective absence of calcium (i.e. where the free calcium concentration is 1 nM). 5 GTP‐γ‐S stimulated ACTH secretion from permeabilized cells in a concentration‐dependent manner with a threshold of 1 μm. PMA (100 nM), TMX (100 nM) but not dPPA (100 nM) increased the amount of ACTH secretion evoked by every concentration of GTP‐γ‐S investigated. 6 The PKC inhibitor, chelerythrine chloride (10 μm), blocked the PMA (100nM)‐evoked enhancement of calcium‐ and GTP‐γ‐S‐stimulated ACTH secretion but did not significantly alter calcium‐ or GTP‐γ‐S‐evoked secretion itself. 7 The present paper indicates that AtT‐20 cells express multiple isoforms of PKC and that these act at different sites in the secretory pathway for ACTH secretion. The α and ∍ isozymes of PKC can act distal to calcium entry to modulate the ability of increased cytosolic calcium concentrations to stimulate ACTH secretion. This site of action is either at the level of, or at some stage distal to, a GTP‐binding protein which mediates the effects of calcium upon ACTH secretion. The β isozyme of PKC may act at a stage early in the secretory pathway to regulate the cytosolic calcium concentration.

Effects of mastoparan upon the late stages of the ACTH secretory pathway of AtT‐20 cells

1 The mouse AtT‐20/D16‐16 anterior pituitary tumour cell line was used as a model system for the study of the effects of mastoparan upon the late stages of the adrenocorticotrophin (ACTH) secretory pathway. 2 Mastoparan (10−8‐10−5 m), an activator of heterotrimeric guanosine 5′‐triphosphate binding proteins (G‐proteins), stimulated ACTH secretion from electrically‐permeabilized AtT‐20 cells in a concentration‐dependent manner in the effective absence of calcium ions with a threshold of 10−6 m. Guanosine 5′‐0‐(3‐thiotriphosphate) (GTP‐γ‐S) (10−8‐10−4m) also stimulated ACTH secretion from electrically‐permeabilized AtT‐20 cells in a concentration‐dependent manner in the effective absence of calcium ions with a threshold of 10−6 m. This GTP‐γ‐S‐evoked secretion is consistent with previous studies which demonstrated that a G‐protein, termed GE, mediates calcium evoked ACTH secretion from AtT‐20 cells. GTP‐γ‐S‐evoked secretion however was not as great as that obtained in response to mastoparan. 3 Both mastoparan (10−5 m) and GTP‐γ‐S (10−4m) stimulated ACTH secretion from electrically‐permeabilized AtT20 cells in a time‐dependent manner. A time of 30 min was adopted as the standard incubation period for the study of both mastoparan and GTP‐γ‐S‐stimulated ACTH secretion from permeabilized AtT‐20 cells. 4 Mastoparan (10−8‐10−5 m) stimulated ACTH secretion from permeabilized AtT‐20 cells to the same extent in the presence and absence of the protein kinase C (PKC) inhibitor, chelerythrine chloride (10−5 m). 5 Mastoparan (10−8‐10−5 m)‐stimulated ACTH secretion from permeabilized AtT‐20 cells was significantly reduced in the presence of guanosine 5′‐0‐(2‐thiodiphosphate) (GDP‐β‐S, 10−4 m). 6 The mastoparan analogue, Mas‐7 (10−8‐10−5m) stimulated ACTH secretion from permeabilized AtT‐20 cells to a greater extent than mastoparan (10−8‐10−5m) however, the mastoparan analogue Mas‐17 (10−8‐10−5 m) had no effect upon ACTH secretion from permeabilized AtT‐20 cells. 7 Mastoparan (10−8‐10−5m) stimulated ACTH secretion from permeabilized AtT‐20 cells in the presence and absence of ATP, normally present in the standard permeabilization medium at a concentration of 5 mM. Mastoparan (10−8‐10−5 m)‐stimulated ACTH secretion as well as control secretion was reduced when ATP was omitted. 8 The results of the present study demonstrate that mastoparan stimulated ACTH secretion from permeabilized AtT‐20 cells and displayed characteristics consistent with calcium ion‐ and GTP‐γ‐S‐stimulated ACTH secretion from permeabilized AtT‐20 cells. This suggests that in permeabilized AtT‐20 cells, mastoparan directly activates GE and that this G‐protein may be a heterotrimeric G‐protein. This study also suggests mastoparan may be a useful alternative to GTP‐γ‐S as a means of directly activating GE.

Phospholipase C-η2 is activated by elevated intracellular Ca2+ levels

Phospholipase C-η2 (PLCη2) is a novel enzyme whose activity in a cellular context is largely uncharacterised. In this study the activity of PLCη2 was examined via [(3)H]inositol phosphate release in COS7 cells expressing the enzyme. PLCη2 activity increased approximately 5-fold in response to monensin, a Na(+)/H(+) antiporter. This was significantly inhibited by CGP-37157 which implies that the effect of monensin was due, at least in part, to mitochondrial Na(+)/Ca(2+)-exchange. Direct activation of PLCη2 by <1μM Ca(2+) was confirmed in permeabilised transfected cells. The roles of the PH and C2 domains in controlling PLCη2 activity via membrane association were also investigated. A PH domain-lacking mutant exhibited no detectable activity in response to monensin or Ca(2+) due to an inability to associate with the cell membrane. Within the C2 domain, mutation of D920 to alanine at the predicted Ca(2+)-binding site dramatically reduced enzyme activity highlighting an important regulatory role for this domain. Mutation of D861 to asparagine also influenced activity, most likely due to altered lipid selectivity. Of the C2 mutations investigated, none altered sensitivity to Ca(2+). This suggests that the C2 domain is not responsible for Ca(2+) activation. Collectively, this work highlights an important new component of the Ca(2+) signalling toolkit and given its sensitivity to Ca(2+), this enzyme is likely to facilitate the amplification of intracellular Ca(2+) transients and/or crosstalk between Ca(2+)-storing compartments in vivo.

Heterotrimeric G-protein candidates for Ge in the ACTH secretory pathway

The mouse AtT-20/D16-16 anterior pituitary tumour cell line was used to identify candidate heterotrimeric G-proteins for G-exocytosis (Ge) which mediates calcium ion-stimulated adrenocorticotrophin (ACTH) secretion in this cell line. AtT-20 cells express several heterotrimeric G-protein alpha subunits; Gs alpha, Gt alpha, Gq alpha, G11alpha, G12alpha, G13alpha, G14alpha, G15alpha, Gz alpha, Gi2alpha, Gi3alpha, and Go alpha and so heterotrimeric G-protein selective agents were used to differentiate between these candidates. Agents which stimulate ACTH secretion via Ge were not pertussis toxin (PTX)-sensitive nor was cholera toxin (CTX) able to stimulate ACTH secretion from permeabilised cells in the absence of calcium. G-protein antagonists which inhibit activation of Gs, Gi, and Gq subfamilies did not attenuate Ge-stimulated ACTH secretion from permeabilised AtT-20 cells. In AtT-20 cells the stimulatory G-protein involved in the late stages of the ACTH secretory pathway does not belong to the Gs, Gi (with the exception of Gz) or Gq subfamilies of heterotrimeric G-proteins leaving Gz, G12 or G13 as the strongest candidates for Ge.

Atrial natriuretic peptide effects in AtT-20 pituitary tumour cells

Whether atrial natriuretic peptide (ANP)-evoked inhibition of corticotrophin-releasing factor (CRF)-stimulated ACTH secretion was also manifest in ACTH secreting AtT-20 pituitary tumour cells was investigated. ANP stimulated increases in cGMP accumulation at concentrations of the peptide above 10(-8) M which indicates the presence of the ANP receptors on these cells. CRF stimulated a concentration-dependent increase in ACTH secretion from AtT-20 cells which was unaffected by ANP, 8-bromo-cGMP, or sodium nitroprusside (SNP). Calcium stimulated a concentration-dependent increase in ACTH secretion from electrically permeabilised cells which was unaffected by co-incubation with cGMP but potentiated by cAMP. These results reveal the presence of ANP receptors on AtT-20 cells but suggest that an incomplete expression of the stimulus-secretion coupling mechanisms for ANP, at some point after cGMP production, prevents the effects of natriuretic peptides upon ACTH secretion being manifest in these cells.