Proline-Rich Homeodomain protein (PRH/HHEX) is a suppressor of breast tumour growth.

Breast tumours progress from hyperplasia to ductal carcinoma in situ (DCIS) and invasive breast carcinoma (IBC). PRH/HHEX (proline-rich homeodomain/haematopoietically expressed homeobox) is a transcription factor that displays both tumour suppressor and oncogenic activity in different disease contexts; however, the role of PRH in breast cancer is poorly understood. Here we show that nuclear localization of the PRH protein is decreased in DCIS and IBC compared with normal breast. Our previous work has shown that PRH phosphorylation by protein kinase CK2 prevents PRH from binding to DNA and regulating the transcription of multiple genes encoding growth factors and growth factor receptors. Here we show that transcriptionally inactive phosphorylated PRH is elevated in DCIS and IBC compared with normal breast. To determine the consequences of PRH loss of function in breast cancer cells, we generated inducible PRH depletion in MCF-7 cells. We show that PRH depletion results in increased MCF-7 cell proliferation in part at least due to increased vascular endothelial growth factor signalling. Moreover, we demonstrate that PRH depletion increases the formation of breast cancer cells with cancer stem cell-like properties. Finally, and in keeping with these findings, we show that PRH overexpression inhibits the growth of mammary tumours in mice. Collectively, these data indicate that PRH plays a tumour suppressive role in the breast and they provide an explanation for the finding that low PRH mRNA levels are associated with a poor prognosis in breast cancer.

In silico analysis of the 5' region of the Vitamin D receptor gene: functional implications of evolutionary conservation.

In recent years, the complexity of the 5' region of the Vitamin D receptor (VDR) gene has become apparent. Six exons, 1a-1f, lie upstream of the translation start codon in exon 2. Transcription has been reported beginning in exons 1f, 1a, 1d and 1c and alternative splicing can produce a large number of alternative mRNA transcripts. Exon 1d transcripts can code for two alternative proteins. This pattern of transcription produces several potential promoter regions. We have used a number of in silico tools to study the evolutionary conservation of the exon and promoter sequences of the 5' region. Those exons involved in the alternative VDR proteins, exons 1d and 1c, were well conserved from mouse and rat, unlike exons 1f, 1e and 1b which showed little homology. Exon 1a was also well conserved. Furthermore, 1a was shown to be within a strong CpG island and TraFac revealed a Sp1-MazR-Sp1-MazR cluster of transcription factor binding sites immediately upstream of exon 1a, a common motif in strong promoters. The promoter region upstream of 1f showed a highly conserved pattern of transcription factor binding sites and was also shown to be within a CpG island. No significant clusters of conserved sites were observed in the 1c promoter region, despite reports of 1c promoter activity.

Vitamin D receptor gene polymorphisms, particularly the novel A-1012G promoter polymorphism, are associated with vitamin D3 responsiveness and non-familial susceptibility in psoriasis.

Psoriasis is a genetically determined disease characterized by hyperproliferation and disordered maturation of the epidermis. Th1 lymphocytes are implicated in its pathogenesis. The vitamin D receptor (VDR) is a candidate modifying gene, having immunosuppressive effects and being involved in anti-proliferative and pro-differentiation pathways in keratinocytes. There is suggestive evidence that the A allele of the A-1012G polymorphism is associated with down-regulation of the Th1 response, via GATA-3. The F and T alleles of Fok1 and Taq1 have been associated with increased VDR activity. The present study aimed to test the hypothesis that the A allele of A-1012G is protective for occurrence and severity of psoriasis and enhances therapeutic response to vitamin D analogues and that these effects would be additive to those of Fok1 and Taq1. The study group comprised 206 psoriasis patients who had received topical calcipotriol treatment and 80 controls. There was no significant linkage disequilibrium between any pair of the three polymorphic sites (P=0.3-0.8). The A, F and T alleles were positively associated with calcipotriol response: AA genotype (compared to AG/GG), odds ratio (OR)=2.18 (P=0.04); TT, OR=1.97 (P=0.03); AAFF genotype combination, OR=4.11 (P=0.03); AATT, OR=5.64 (P=0.005); and FFTT, OR=3.22 (P=0.01). Comparing patients without, to patients with, a family history of psoriasis, the A allele was under represented (P=0.01) and the AAFF genotype combination even more so (compared to residual genotypes) (OR=0.24; P=0.005). AAFF was also under-represented in patients without a family history compared to controls (OR=0.31; P=0.04). There were no associations of family history with Fok1 and Taq1. There were no associations of severity of psoriasis with any polymorphism. In conclusion, the A-1012G, Fok1 and Taq1 VDR polymorphisms were associated with response to calcipotriol. A-1012G and Fok1 were associated with susceptibility to non-familial psoriasis.

A novel polymorphism in the 1A promoter region of the vitamin D receptor is associated with altered susceptibilty and prognosis in malignant melanoma.

The association of Taq 1 and Fok 1 restriction fragment length polymorphisms of the vitamin D receptor with occurrence and outcome of malignant melanoma (MM), as predicted by tumour (Breslow) thickness, has been reported previously. We now report a novel adenine-guanine substitution -1012 bp relative to the exon 1a transcription start site (A-1012G), found following screening by single-stranded conformational polymorphism of this promoter region. There was a total of 191 MM cases, which were stratified according to conventional Breslow thickness groups, cases being randomly selected from each group to form a distribution corresponding to the known distribution of Breslow thickness in our area, and this population (n=176) was compared to 80 controls. The A allele was over-represented in MM patients and, with GG as reference, odds ratio (OR) for AG was 2.5, 95% confidence interval (CI) 1.1-5.7, (P=0.03) and AA 3.3, CI 1.4-8.1, (P=0.007). The outcome was known in 171 of 191 patients and the A allele was related to the development of metastasis, the Kaplan-Meier estimates of the probability of metastasis at 5 years being: GG 0%; AG 9%, CI 4-16%; AA 21%, CI 12-36%; (P=0.008), and to thicker Breslow thickness groups (P=0.04). The effect on metastasis was independent of tumour thickness and A-1012G may have predictive potential, additional to Breslow thickness. Neither the Fok 1 nor Taq 1 variants (f and t) were significantly related to the development of metastasis, although there was a strong relationship of fftt with the thickest Breslow thickness group (P=0.005). There was an interaction between the A-1012G and Fok 1 polymorphisms (P=0.025) and the Fok 1 variant enhanced the effect of the A allele of the A-1012G polymorphism on metastasis, the probability of metastasis for AAff at 5 years follow-up being 57%, CI 24-92%.

Three subfamilies of pheromone and receptor genes generate multiple B mating specificities in the mushroom Coprinus cinereus.

The B mating type locus of the basidiomycete Coprinus cinereus encodes a large family of lipopeptide pheromones and their seven transmembrane domain receptors. Here we show that the B42 locus, like the previously described B6 locus, derives its unique specificity from nine multiallelic genes that are organized into three subgroups each comprising a receptor and two pheromone genes. We show that the three genes within each group are kept together as a functional unit by being embedded in an allele-specific DNA sequence. Using a combination of sequence analysis, Southern blotting, and DNA-mediated transformation with cloned genes, we demonstrate that different B loci may share alleles of one or two groups of genes. This is consistent with the prediction that the three subgroups of genes are functionally redundant and that it is the different combinations of their alleles that generate the multiple B mating specificities found in nature. The B42 locus was found to contain an additional gene, mfs1, that encodes a putative multidrug transporter belonging to the major facilitator family. In strains with other B mating specificities, this gene, whose functional significance was not established, lies in a region of shared homology flanking the B locus.

A large pheromone and receptor gene complex determines multiple B mating type specificities in Coprinus cinereus.

Pheromone signaling plays an essential role in the mating and sexual development of mushroom fungi. Multiallelic genes encoding the peptide pheromones and their cognate 7-transmembrane helix (7-TM) receptors are sequestered in the B mating type locus. Here we describe the isolation of the B6 mating type locus of Coprinus cinereus. DNA sequencing and transformation analysis identified nine genes encoding three 7-TM receptors and six peptide pheromone precursors embedded within 17 kb of mating type-specific sequence. The arrangement of the nine genes suggests that there may be three functionally independent subfamilies of genes each comprising two pheromone genes and one receptor gene. None of the nine B6 genes showed detectable homology to corresponding B gene sequences in the genomic DNA from a B3 strain, and each of the B6 genes independently alter B mating specificity when introduced into a B3 host strain. However, only genes in two of the B6 groups were able to activate B-regulated development in a B42 host. Southern blot analysis showed that these genes failed to cross-hybridize to corresponding genes in the B42 host, whereas the three genes of the third subfamily, which could not activate development in the B42 host, did cross-hybridize. We conclude that cross-hybridization identifies the same alleles of a particular subfamily of genes in different B loci and that B6 and B42 share alleles of one subfamily. There are an estimated 79 B mating specificities: we suggest that it is the different allele combinations of gene subfamilies that generate these large numbers.

Linking measures of health gain to explicit priority setting by an area health service in Australia.

A demonstration project was undertaken to develop an integer programming model that could help a regional health authority to take into account data on service effectiveness when allocating resources to acute inpatient services. The model was designed to find the mix of services that would maximise health gain from the available resources, and so provide information that could be used to encourage hospitals to change their patient mix. It was developed in collaboration with an Area Health Service in New South Wales, Australia, with the aim of assessing its potential as a decision support tool. Acute inpatient services were categorised in the model using classes derived from the Australian National Diagnosis Related Groups (AN-DRG) classification and the classes developed by the Oregon Health Services Commission. Estimates for the effectiveness of each service was derived from the Oregon benefit data. Estimates of resource use were derived from AN-DRG data. The expected demand for each service was derived from local activity data. Various scenarios were developed to assess the potential of the model to support decision makers. These mimicked plausible policy options and tested the sensitivity of the results to changes in the data. The scenarios demonstrated the model could reveal the consequences of different policy options, but also suggested that the difference in the cost-effectiveness of services close to the margin would be small and so a rigid approach to priority setting is undesirable. Difficulties in developing the model also demonstrate that incorporating health gain data into resource allocation decisions will not be straight-forward for health planners.

Expression of two closely linked hydrophobin genes of Coprinus cinereus is monokaryon-specific and down-regulated by the oid-1 mutation.

A protein with characteristic properties of a fungal hydrophobin (CoH1) was isolated from the monokaryotic stage of the basidiomycete Coprinus cinereus. A cosmid clone containing the corresponding gene (coH1) was identified using a cDNA probe derived by RT-PCR. Hybridization and sequence analysis identified a second gene, coH2, just 4.1 kb downstream of coH1 encoding a hydrophobin (CoH2) with 64% sequence identity. Both coH1 and coH2 are subject to developmental regulation. They are expressed in vegetative monokaryotic cells but not in the asexual oidia produced on the surface of monokaryons. Transcripts of the genes were barely detected in dikaryotic mycelium and were absent from fruit bodies. Loss of aerial growth due to a mutation known as oid-1 was correlated with lack of both hydrophobins.

In vitro 31P-magnetic resonance spectroscopy of muscle extracts in malignant hyperthermia-susceptible patients.

BACKGROUND: It was recently suggested that malignant hyperthermia-susceptible (MHS) patients could have an elevated peak of phosphodiesters in leg muscles using in vivo phosphorus magnetic resonance spectroscopy. In the current study, analysis of the phosphodiesters of muscle extracts of MHS and malignant hyperthermia-negative patients was performed using in vitro phosphorus magnetic resonance spectroscopy to chemically identify and to compare the muscle concentrations of water-soluble compounds between the two groups with respect to the muscle fiber type composition. METHODS: Perchloric acid extracts of the vastus medialis muscle of seven MHS patients and ten malignant hyperthermia-negative patients on the basis of the European malignant hyperthermia contracture test were subjected to in vitro phosphorus magnetic resonance spectroscopy carried out at 9.4 T. In addition, chemical identification of the phosphodiester region and histologic examination of the muscle specimens were performed. RESULTS: The peak in the phosphodiester region was assigned to glycerophosphorylcholine. Muscle perchloric acid extracts of MHS patients had a significantly (P < 0.05) higher glycerophosphorylcholine to the sum of phosphocreatine and inorganic phosphate (glycerophosphorylcholine/ [phosphocreatine +inorganic phosphate]) value than those of malignant hyperthermia-negative patients. Neither a difference in the fiber type composition between the two groups nor any specific myopathy were found. CONCLUSIONS: In the absence of histologic differences between muscle specimens of MHS and malignant hyperthermia-negative patients, these results could suggest that glycerophosphorylcholine could be a marker of an impairment in the phospholipid metabolism in the skeletal muscle of MHS patients.

Information services in medical mycology.

The need for information services in medical mycology is discussed. The information service provided by the Review of Medical and Veterinary Mycology is described and compared with other information services.

Effect of halothane on the rate of acid production, lactate production and pyruvate dehydrogenase activity of malignant hyperpyrexia human muscle.

The rates of acid production were compared in thin strips of muscle biopsy samples isolated from malignant hyperpyrexia and control vastus internus human muscle. Halothane doubled the rate of acid production by malignant hyperpyrexia susceptible muscle but had no effect on control samples. This increased rate of release of acid from muscle was not from lactate. In addition, the pyruvate dehydrogenase activity of both control and malignant hyperpyrexia muscle samples was not stimulated by halothane.