PTEN/MMAC1 expression in melanoma resection specimens

PTEN/MMAC1, a tumour suppressor gene located on chromosome 10q23.3, has been found to be deleted in several types of human malignancies. As the chromosomal region 10q22-qter commonly is affected by losses in melanomas, we addressed this gene as tumour suppressor candidate in melanomas. Investigating PTEN/MMAC1 expression at mRNA level by semi-quantitative reverse transcription-polymerase chain reaction, we did not find a statistically significant down-regulation in melanoma resection specimens in comparison to acquired melanocytic nevi from which melanomas quite often are known to arise. Upon immunohistochemistry, PTEN/MMAC1 protein expression in melanomas was not lost. Sequencing the PTEN/MMAC1 cDNAs in 26 melanoma resection specimens (21 primary melanomas, five metastases), we detected three point mutations and two nucleotide deletions which did not represent genetic polymorphisms. With respect to the predicted protein sequences, all three point mutations were silent whereas the two frame shifts at the extreme C-terminus resulted in a loss of the putative PDZ-targeting consensus sequence. As loss of this motif possibly impairs localization and function of PTEN/MMAC1 in the two corresponding primary tumours, alterations of this tumour suppressor protein may participate in some melanomas

The Creation and Physiological Relevance of Divergent Hydroxylation Patterns in the Flavonoid Pathway

Flavonoids and biochemically-related chalcones are important secondary metabolites, which are ubiquitously present in plants and therefore also in human food. They fulfill a broad range of physiological functions in planta and there are numerous reports about their physiological relevance for humans. Flavonoids have in common a basic C6-C3-C6 skeleton structure consisting of two aromatic rings (A and B) and a heterocyclic ring (C) containing one oxygen atom, whereas chalcones, as the intermediates in the formation of flavonoids, have not yet established the heterocyclic C-ring. Flavonoids are grouped into eight different classes, according to the oxidative status of the C-ring. The large number of divergent chalcones and flavonoid structures is from the extensive modification of the basic molecules. The hydroxylation pattern influences physiological properties such as light absorption and antioxidative activity, which is the base for many beneficial health effects of flavonoids. In some cases antiinfective properties are also effected

Pollination ecology and molecular systematics of Diuris (Orchidaceae)

The Australian terrestrial orchid genus Diuris is currently recognised to contain at least 61 species, with numerous new taxa expected to be recognised in the near future. Species are restricted to Australia, with the exception of Diuris fryana, which is endemic to Timor. Species of Diuris are well represented in the southern parts of western and eastern Australia, separated by the Nullarbor Plain, with a few species found in tropical Queensland. The eastern and western species mostly fall into morphologically distinct groups suggestive of distinct phylogenetic lineages. Despite considerable variation between and even within species, Diuris species share certain important features. Most species occur in open forest and woodland and have flowers that bear a resemblance to Australian native ‘egg and bacon’ pea flowers of the tribes Bossiaeae and Mirbeliae, with which they are frequently sympatric. In some species, the resemblance is very close, in others it is more general. Most existing work on pollination in this taxon is of an anecdotal nature, with only one formal study prior to this project, of one species (Diuris maculata at Fern Tree Gully, near Melbourne, Victoria in 1986). The Beardsell et al. (1986) study proposed that this orchid was a non-rewarding floral mimic of pea flowers of the genera Daviesia, Pultenaea and Dillwynia. It was sympatric with the peas, with which it bore a visual resemblance, flowered at the same time and was visited by native bees, plus a wasp species that pollinated the pea flowers. The overall purpose of my project was to advance knowledge of the pollination biology of Diuris. In particular, I planned to (i) test the effectiveness of AFLP markers for identifying the source of pollinium remnants collected from the bodies of putative pollinators, (ii) conduct detailed pollination studies on two species in the Sydney region (D. maculata and D. alba) to test the generality of the conclusion of floral mimicry in Diuris, drawn from the 1986 study in Melbourne; (iii) survey pollinator interactions in a range of taxa (D. aurea, D. punctata and D. sp. aff. punctata (Mellong Swamp)), using pollination observations, imaging of ultraviolet visual cues, colorimetric analysis of putative model and mimic flowers, testing for nectar production and DNA-based identification of pollinaria removed from captured insects, to test for patterns in pollinator-plant associations, and (iv) place these observations in the context of a complete a phylogenetic analysis of the genus Diuris. These main experimental findings are summarised as follows: 1. Amplified Fragment Length Polymorphism (AFLP) was shown not only to be capable of distinguishing many species, but also to possess high sensitivity. This latter feature had not been exploited previously. (See Chapter 2) 2. The pollination mechanism of Diuris maculata in this population was shown to be similar to the original Beardsell et al. (1986) study, but there were some significant differences – the timing of orchid flowering early in the flowering season of putative model pea species (cf. synchronised flowering in the Victorian population) and the role of male bees (cf. various male and female bees) in pollination, which may be quite common in Diuris. (See Chapter 3) 3. Diuris alba has flowers that are similar in form, but not colour, to other, putative pea-mimicking Diuris species. Diuris alba at Munmorah, New South Wales, was found to occur in a variety of habitats including sites where pea flowers are absent or rare and the pollination success was found to be not dependent on pea flowers. This species was also found to produce a meagre nectar reward. (See Chapter 4) 4. Unpublished pollination data were obtained for a number of Diuris taxa (Diuris aurea (Castlereagh Nature Reserve), D. punctata (Castlereagh Nature Reserve), D. sp. aff. punctata (Mellong Swamp), D. arenaria (Tomaree National Park) and Diuris sulphurea form Stringy Bark Ridge, Pennant Hills. It was found that D. aurea, D. punctata and D. sp. aff. punctata showed pollination features consistent with Batesian-type floral mimicry of yellow ‘egg and bacon’ pea flowers, despite the latter two taxa having a white floral anthoxanthin base colour (with pink/purple suffusions). Additionally, preliminary data was obtained for D. aurea and D. sp. aff. punctata that showed higher pollination success for plants clustered some distance from yellow pea flowers than was obtained for plants scattered among yellow pea flowers. The taxon D. arenaria was shown to have higher reproductive success, when scattered amongst yellow ‘egg and bacon’ pea flowers than would be expected for a Batesian-type mimic, a result suggestive of a more generalised pollination strategy. Meagre nectar was found in one plant of Diuris sulphurea tested for nectar production, an interesting result that requires confirmation with further testing. 5. The molecular phylogenetic analysis of Diuris (Orchidaceae) based on AFLP and ITS (Internal Transcribed Spacers of Ribosomal DNA) revealed three major clades and a basal species. Diuris sulphurea (subg. Paradiuris) is shown to a monotypic sister group to the rest of Diuris. (See Chapter 5) The conclusions of the study can be summarised as follows: 1. Some species show close visual mimicry of specific model species (strict Batesian mimicry) e.g. Diuris aequalis is proposed to mimic Gompholobium spp. 2. Many species show a more generalised mimicry of peas (loose Batesian-type mimicry) e.g. Diuris maculata shows general similarity to ‘egg and bacon’ peas. Over its wide distribution (southern Victoria to north of Sydney, New South Wales) this orchid occurs at many sites and its pollination is likely to involve dozens of species of both peas and pollinators. 3. Some species show apparent dependence on mimicry while showing only aspects of similarity, with other different, perhaps flamboyant features suggestive of non-model mimicry e.g Diuris sp. aff. punctata (Mellong Swamp). This species shows pollination outcomes in the presence of the yellow pea Dillwynia glaberrima as expected for a yellow-flowered pea mimic. Its flowers show general pea-like form. However, the pink/purple floral colouration is quite different to the sympatric yellow peas. It is also noticeably fragrant. It is suggested that the flowers of this species are ‘exciting’ to a bee foraging on pea flowers because its flowers possess strong floral cues, which could be considered to transcend strict mimicry. 4. Generalised pollination e.g. Diuris alba from Munmorah, New South Wales. In this case flowers were shown to have pea-like floral form, but with different colour (white cf. yellow), fragrance and nectar. Plants were shown to have high pollination success both in the presence and absence of pea flowers. The pollination system was analysed using colorimetric analysis with a model of predicted colourbased foraging errors. This pollination system was proposed to have evolved from pea mimicry and not entirely disconnected from it. Aspects of Batesian-type mimicry, non-model mimicry, the ‘magnet effect’ and the presence of a meagre nectar reward may all contribute to high pollination success in varied environments. 5. Diuris sulphurea is the most widespread of all Diuris species in eastern Australia, forms a basal clade to the rest of the genus and may show ancestral pollination features. Such a widespread species is unlikely to mimic a single pea species and must show fairly generic pea mimicry. It has a colony-forming growth habit, produces nectar and has high reproductive success. An understanding of its pollination mechanism is likely to lead to insights of pollination evolution within the genus. The phylogenetic data allow the following interpretations of current patterns of Diuris pollination: 1. The finding that D. sulphurea forms basal clade to all other Diuris suggests that this species may possess ancestral features, including pollination mechanism, which on the basis on preliminary evidence would appear to be fairly generalised (guild) pea mimicry combined with nectar reward. Its pollination mechanism, combined with a colony-forming habit might be expected to promote significant selfpollination. 2. Knowledge of species groupings (clades) will aid in focusing pollination studies since clades can be expected to contain species sharing morphological and pollination features. 3. Yellow base colour can be inferred to be ancestral in Diuris, with pink/purple colouration being a synapomorphy in Diuris subg. Diuris sect. Purpureo-albae (plus some species within Diuris subg. Xanthodiuris, sect. Pedunculatae, e.g. D. venosa). Preliminary data suggest that despite the floral colour difference, species closely related to D. punctata appear to mimic yellow pea flowers and have a similar pollination mechanism, while another closely related species, D. alba has been shown to have generalised pollination. Therefore, a detailed understanding of pollination of the species D. aurea, D. punctata, and D. alba can be expected to provide considerable information about pollination within this large species group and also to provide important insights into the evolution of Batesian-type mimicry in this orchid group. I propose the following hypothesis about the role of Batesian-type mimicy (which includes strict Batesian mimicry as commonly understood and looser forms, such as mimicry of a guild of Mullerian mimics) in the pollination systems of Australian east coast Diuris species. Strict Batesian mimicry is highly specialised and inevitably leads to rarity, and there are consequently few examples. However, the looser type of Batesian mimicry (sometimes termed ‘guild mimicry’) exemplified by Diuris maculata permits the exploitation of many ecologically similar environments, in which basically similar, but distinct pea flower species may all serve as models for this orchid. Within Diuris, subg. Diuris, sect. Purpureo-albae there are numerous species with pink/purple, or white base colour. Many of these, paradoxically, appear to depend on association with yellow ‘egg and bacon’ peas for pollination. Phylogenetic evidence suggests that these species have undergone recent and rapid evolutionary radiation. This could be viewed as a shift toward even looser Batesian-type mimicry and could account for their evolutionary success. Diuris alba represents a group of species which have developed a sufficiently generalised pollination system to be independent of pea flowers, although high reproductive success in the presence of pea flowers would suggest that the link to pea mimicry is not completely broken. It thus may be reasonably termed a ‘non-model’ mimic and it likely benefits from the ‘magnet effect’ of being in the proximity of abundant rewarding species (which often happen to be pea flowers). As resemblance of this species to pea flowers is somewhat unclear, it may not be meaningful to view pea flowers as ‘model’ flowers, or indeed this orchid as a pea ‘mimic’

Frequent Loss of Heterozygosity Targeting the Inactive X Chromosome in Melanoma

After previous preliminary observations of paradoxical deletion events affecting the inactive X chromosome in melanoma, we have surveyed the X chromosome for deletions using 23 polymorphic microsatellite markers in 28 informative (female XX) metastatic melanomas. Ten tumors (36%) showed at least one loss of heterozygosity (LOH) event, and in two cases an entire chromosome showed LOH at all informative loci. Four distinct X chromosome smallest regions of overlap can be resolved. An 18.6-Mb region on the p arm involving 9 of 28 (32%) samples lies between the markers DXS1061 and DXS1068. An equally frequently deleted smallest region of overlap straddled the centromere, bounded by DX1204 on the p arm and DXS983 14.6 Mb away in Xq11-12. One tumor potentially defines this region more tightly to a 10.6-Mb smallest region of overlap bounded by DXS1190 and DXS981 that contains the androgen receptor (AR) gene. A 6.2-Mb deleted region can be defined between the markers DXS8051 and DXS9902 in 8 of 28 (28%) tumors. An additional, less frequently deleted region of 25.7 Mb was found on distal Xq between the markers DXS1212 and DXS1193 in 5 of 28 (18%) tumors. X inactivation analysis of five tumors with LOH, using the AR exon I CAG repeat, showed that in each case, the inactive, hypermethylated allele was the one deleted. Analysis of copy number in this region by quantitative PCR showed restoration to disomy and, in one case, trisomy at AR

Generalised pollination of Diuris alba (Orchidaceae) by small bees and wasps

Most Diuris species possess flowers of pea-like form and colour, and occur in association with flowering peas of the tribe Mirbeliae. Previous studies of the pollination of Diuris maculata sensu lato have found evidence for guild mimicry of pea flowers. The flowers of Diuris alba are also pea-like in form but not in colour, and this species is frequently found in habitats where peas are uncommon or absent. We investigated the pollination of Diuris alba, which we expected may have a distinct pollination system at Lake Munmorah, New SouthWales. Many Diuris species lack floral rewards, but D. alba produced a small amount of nectar. Flower visitors, and hence putative pollinators, were mainly female Exoneura bees, but also the wasps Eurys pulcher and a Paralastor species. Reproductive success of D. alba, both in woodland containing abundant Dillwynia retorta and in heathland where this pea was absent, was higher than in the previously studied D. maculata s.l.We suggest that the pollination of D. alba is more generalised than that found in the legume guild mimic D. maculata s.l. Although its flowers may display structural similarity to pea flowers, other characteristics suggest that its pollination system has diverged from a presumed pea-mimicry ancestral condition