Comparative population genetics of a mimicry locus among hybridizing Heliconius butterfly species.

The comimetic Heliconius butterfly species pair, H. erato and H. melpomene, appear to use a conserved Mendelian switch locus to generate their matching red wing patterns. Here we investigate whether H. cydno and H. pachinus, species closely related to H. melpomene, use this same switch locus to generate their highly divergent red and brown color pattern elements. Using an F2 intercross between H. cydno and H. pachinus, we first map the genomic positions of two novel red/brown wing pattern elements; the G locus, which controls the presence of red vs brown at the base of the ventral wings, and the Br locus, which controls the presence vs absence of a brown oval pattern on the ventral hind wing. The results reveal that the G locus is tightly linked to markers in the genomic interval that controls red wing pattern elements of H. erato and H. melpomene. Br is on the same linkage group but approximately 26 cM away. Next, we analyze fine-scale patterns of genetic differentiation and linkage disequilibrium throughout the G locus candidate interval in H. cydno, H. pachinus and H. melpomene, and find evidence for elevated differentiation between H. cydno and H. pachinus, but no localized signature of association. Overall, these results indicate that the G locus maps to the same interval as the locus controlling red patterning in H. melpomene and H. erato. This, in turn, suggests that the genes controlling red pattern elements may be homologous across Heliconius, supporting the hypothesis that Heliconius butterflies use a limited suite of conserved genetic switch loci to generate both convergent and divergent wing patterns.

Clinicopathological significance of erbB-2 expression in colorectal carcinoma.

The level of erbB-2 expression in both the tumour tissue and serum of individuals with colorectal carcinoma is unclear. This study aims to clarify expression levels and to relate these to a range of clinicopathological parameters. Overexpression of erbB-2 was detected in 25% of patient tumour tissue and an elevation in serum concentration of the extracellular domain (ECD) of erbB-2 was detected in 10% of patients. Surprisingly, an elevated serum ECD concentration did not correlate with erbB-2 overexpression within the primary tumour, and neither tumour or serum levels of the protein correlated with any clinicopathological parameters examined. These results indicate that erbB-2 overexpression in tissue and serum are not uncommon in colorectal carcinoma, but may not be useful as predictors of disease outcome.

Suggestions for HER-2/neu testing in breast carcinoma, based on a comparison of immunohistochemistry and fluorescence in situ hybridisation.

The arrival of Herceptin (Trastuzumab), an antibody against the HER-2 oncogene found in a proportion of breast carcinomas and other carcinomas, has emphasised the need for a standardised technique for demonstrating overexpression of HER-2. We compared the Dako A485 antibody and Dako HercepTest kit (HT) on a series of 122 breast carcinomas. Fluorescence in situ hybridisation (FISH) (Vysis) was performed on all cases with positive or equivocal immunohistochemical results. The Dako A485 showed HER-2 overexpression in 53% of carcinomas, while the HT showed 21% positive (HT 2+ 8%, HT 3+ 13%) and 79% negative (HT 0 67%, HT 1+ 12%). FISH for HER-2 gene amplification on all the HT 1+ and HT 2+ cases was negative, whereas FISH analysis of all HT 3+ cases was positive, with the exception of one case which could not be analysed for technical reasons. When histological subtype was analysed, only grade 3 infiltrating duct carcinomas were FISH-positive, suggesting that histological grading and subtyping may be able to triage carcinomas suitable for HER-2 testing. We suggest that the HT or a similar standardised immunohistochemical study for HER-2 can be used to screen breast carcinomas. We then recommend FISH where the carcinoma is HT 2+. FISH may also be appropriate in high grade, HT 1+ carcinomas where there are doubts regarding optimal tissue fixation or block storage conditions.

Delineation of two distinct type 1 activation functions in the androgen receptor amino-terminal domain.

Based on the finding that some transcription factors contain multiple transcriptional regulatory activities, we constructed a panel of rat androgen receptor (AR) mutants containing small internal deletions and point mutations within the amino-terminal region of the receptor. Trans-activation assays in CV-1 cells using AR-responsive reporter genes were performed and led to the identification of two noncontiguous trans-activation regions in the AR amino terminus. One of these regions, termed activator function 1a (AF-1a) is a highly-conserved 14-amino acid segment that is predicted to form a beta-turn followed by an acidic amphipathic alpha-helix. Point mutagenesis within AF-1a revealed that two adjacent hydrophobic residues were required for full AR trans-activation function, as arginine substitutions resulted in a 60% reduction in transcriptional activity. A second amino-terminal region was also identified and has been designated AF-1b. Deletion of the 65-amino acid AF-1b segment, which contains numerous glutamate and aspartate residues, caused a 55% decrease in trans-activation function. An AF-1a/AF-1b double mutant retains less than 10% trans-activation function compared with wild-type AR, suggesting that AF-1a and AF-1b may each contribute separately to maximal AR activity. To determine whether AF-1a and AF-1b play a role in AR-mediated trans-repression of AP-1 function, we tested single and double AF-1a/AF-1b mutants in a transient trans-repression assay. Our results showed that neither AF-1a nor AF-1b was required for AP-1 trans-repression, demonstrating that AR-mediated trans-repression and trans-activation are discrete functions.

The length and location of CAG trinucleotide repeats in the androgen receptor N-terminal domain affect transactivation function.

Some transcription factors contain stretches of polyglutamine encoded by repeats of the trinucleotide CAG. Expansion of the CAG repeat in the androgen receptor (AR) has been correlated with the incidence and severity of X-linked spinal and bulbar muscular atrophy (Kennedy's disease). In order to understand the relationship of this mutation to AR function, we constructed ARs that varied in the position and size of the polyglutamine tract, and assayed for the abilities of these mutant receptors to bind androgen and to activate transcription of several different AR-responsive reporter genes. Elimination of the tract in both human and rat AR resulted in elevated transcriptional activation activity, strongly suggesting that the presence of the polyglutamine tract is inhibitory to transactivation. Progressive expansion of the CAG repeat in human AR caused a linear decrease of transactivation function. Importantly, expansion of the tract did not completely eliminate AR activity. We postulate that this residual AR activity may be sufficient for development of male primary and secondary sex characteristics, but may fall below a threshold level of activity necessary for normal maintenance of motor neuron function. This functional abnormality may be representative of other genetic diseases that are associated with CAG expansion mutations in open reading frames, such as spinocerebellar ataxia type I and Huntington's disease.

Carols in the wind.

AIM: To compare mood and emotional responses to music played on pipe organs and pipeless (digital) organs. DESIGN: A two-organ (pipe v. digital) crossover study. SETTING: St Theodore's Anglican Church, Adelaide. SUBJECTS: 43 non-musician volunteers. MAIN OUTCOME MEASURES: Changes in mood and emotions measured by the Profile of Mood States. RESULTS: Both instruments produced significant reductions in the emotions commonly experienced during the Christmas season: tension-anxiety, depression-rejection, anger-hostility and fatigue. There were no significant changes for vigour or confusion-bewilderment, and no significant differences between the mood-altering effects of the two instruments. CONCLUSIONS: Pipeless organs are as effective as pipe organs in inducing beneficial mood changes.

Assessment of the therapeutic potential of cytokines, cytotoxic drugs and effector cell populations for the treatment of multiple myeloma using the 5T33 murine myeloma model.

The therapeutic potential of six cytokines, eight cytotoxic drugs and two effector cell populations for the treatment of multiple myeloma was assessed in vitro using the 5T33 murine myeloma model. The efficacy of combination IFN-alpha and melphalan therapy was also evaluated in vitro and in vivo. Of the cytokines tested in vitro using the MTT assay, only IFN-alpha demonstrated significant inhibition of myeloma cell growth at non-toxic concentrations (ED50 = 1508.3 +/- 181.3 U/mL and 2617.9 +/- 334.0 U/mL for murine IFN-alpha [mIFN-alpha] and human IFN-alpha hybrid B/D [hIFN-alpha B/D], respectively). The ED50 for the eight cytotoxic drugs tested ranged from 2.3 x 10(-9) to 4.3 x 10(-13) mol/L and all were within the therapeutic range for humans. Combination hIFN-alpha B/D and melphalan were found to be additive in their inhibitory effects on myeloma cell growth in vitro and this finding was confirmed in vivo in C57BL/KaLwRij mice bearing disseminated 5T33 myeloma. Control animals demonstrated a median survival duration of 25.3 days whereas hIFN-alpha B/D or melphalan treatment alone increased survival to 30.5 and 33.3 days, respectively (P < 0.001). Combination IFN-alpha/melphalan therapy increased median survival duration to 38.5 days (P < 0.001) which was also significantly greater than that obtained with single agent therapy (P < 0.01). The murine myeloma cells were found to be resistant to NK cell lysis but susceptible to lysis by LAK cells (49.3 +/- 6.3% lysis at an effector to target ratio of 100:1).(ABSTRACT TRUNCATED AT 250 WORDS)

A cell-specific and selective effect on transactivation by the androgen receptor.

The androgen (AR) and glucocorticoid receptors (GR) are related ligand-activated transcriptional regulators which bind the same cis-acting element and are coexpressed in a variety of cell types. Despite a shared DNA binding site, these receptors mediate diverse cellular responses. To explain this paradox, the existence of cell-specific factors that interact with, and modulate the function of, distinct receptors has been proposed. Prostate epithelial cell growth is sensitive to androgens, but is not affected by glucocorticoids, even though both AR and GR are expressed in these cells. We have recently isolated a unique panel of prostate epithelial cell lines from normal rats and have used these cell lines to examine cell-specific steroid responses. In this study, we compared the abilities of AR and GR to enhance transcription of several different reporter genes regulated by simple (i.e., noncompsite) hormone response elements (HREs) in prostate and nonprostate cell lines. The cell-specific effect occurred independently of the AR hormone binding domain and could be observed with a GAL4 fusion protein containing only the AR N-terminal regulatory domain. Gel shift analyses showed that the relative DNA binding affinity of AR for a probe containing a simple HRE was similar in prostate and nonprostate cell extracts. Presently, the only factors known to mediate steroid receptor-specific gene regulation are cJun and cFos, but there were no cell-specific differences in the functional levels of these proteins which could account for a preferential effect on AR-dependent transcription. Taken together, these results suggest that cell-specific activities exist which can preferentially modulate transcriptional transactivation by AR.

The mitogen-activated protein kinase pathway contributes to vanadate toxicity in vascular smooth muscle cells.

Vanadate has been considered in the treatment of diabetes because of its insulin-like effects. However, it has severe toxic effects in both animal and man. In cultured cells, vanadate can either cause death or be growth stimulatory, depending on the cell type and growth conditions. Here, we report that in baboon aortic smooth muscle cells (SMCs), vanadate induced p42/p44 mitogen-activated protein kinase (MAPK) activity. This effect was abolished in the presence of the specific MAPK kinase (MAPKK) inhibitor PD098059. Although activation of p42/p44MAPK/MAPKK is generally thought to be necessary for proliferation, in SMCs, vanadate did not promote DNA synthesis and inhibited thymidine incorporation stimulated by platelet-derived growth factor (PDGF)-BB in a dose dependent fashion (IC50: 30 microM). Prolonged exposure to vanadate exerted cytotoxic effects. Cells retracted, rounded up and detached from the substratum. These vanadate-induced morphological changes were blocked in the presence of PD098059. The addition of PDGF-BB further activated p42/p44MAPK/MAPKK in the presence of vanadate and substantially increased vanadate toxicity. We conclude from these observations that activation of the p42/p44MAPK/MAPKK signalling module contributes to the cytotoxic effects induced by vanadate.

Associations between rural background and where South Australian general practitioners work.

OBJECTIVE: To determine the association between rural background on practice location of general practitioners (GPs) (rural or urban). DESIGN: Comparison of data from two postal surveys. SUBJECTS: 268 rural and 236 urban GPs practising in South Australia. MAIN OUTCOME MEASURES: Association between practice location (rural or urban) and demographic characteristics, training, qualifications, and rural background. RESULTS: Rural GPs were younger than urban GPs (mean age 47 versus 50 years, P < 0.01) and more likely to be male (81% versus 67%, P = 0.001), to be Australian-born (72% versus 61%, P = 0.01), to have a partner (95% versus 85%, P = 0.001), and to have children (94% versus 85%, P = 0.001). Similar proportions of rural and urban GPs were trained in Australia and were Fellows of the Royal Australian College of General Practitioners, but more rural GPs were vocationally registered (94% versus 84%, P = 0.001). Rural GPs were more likely to have grown up in the country (37% versus 27%, P = 0.02), to have received primary (33% versus 19%, P = 0.001) and secondary (25% versus 13%, P = 0.001) education there, and to have a partner who grew up in the country (49% versus 24%, P = 0.001). In multivariate analysis, only primary education in the country (odds ratio [OR], 2.43; 95% CI, 1.09-5.56) and partner of rural background (OR, 3.14; 95% CI, 1.96-5.10) were independently associated with rural practice. CONCLUSION: Our findings support the policy of promoting entry to medical school of students with a rural background and provide an argument for policies that address the needs of partners and maintain quality primary and secondary education in the country.