Population genetic analysis of a medicinally significant Australian rainforest tree, Fontainea picrosperma C.T. White (Euphorbiaceae): biogeographic patterns and implications for species domestication and plantation establishment.

BACKGROUND: Fontainea picrosperma, a subcanopy tree endemic to the rainforests of northeastern Australia, is of medicinal significance following the discovery of the novel anti-cancer natural product, EBC-46. Laboratory synthesis of EBC-46 is unlikely to be commercially feasible and consequently production of the molecule is via isolation from F. picrosperma grown in plantations. Successful domestication and plantation production requires an intimate knowledge of a taxon's life-history attributes and genetic architecture, not only to ensure the maximum capture of genetic diversity from wild source populations, but also to minimise the risk of a detrimental loss in genetic diversity via founder effects during subsequent breeding programs designed to enhance commercially significant agronomic traits. RESULTS: Here we report the use of eleven microsatellite loci (PIC = 0.429; P ID = 1.72 × 10(-6)) to investigate the partitioning of genetic diversity within and among seven natural populations of F. picrosperma. Genetic variation among individuals and within populations was found to be relatively low (A = 2.831; H E = 0.407), although there was marked differentiation among populations (PhiPT = 0.248). Bayesian, UPGMA and principal coordinates analyses detected three main genotypic clusters (K = 3), which were present at all seven populations. Despite low levels of historical gene flow (N m = 1.382), inbreeding was negligible (F = -0.003); presumably due to the taxon's dioecious breeding system. CONCLUSION: The data suggests that F. picrosperma was previously more continuously distributed, but that rainforest contraction and expansion in response to glacial-interglacial cycles, together with significant anthropogenic effects have resulted in significant fragmentation. This research provides important tools to support plantation establishment, selection and genetic improvement of this medicinally significant Australian rainforest species.

The sap from Euphorbia peplus is effective against human nonmelanoma skin cancers.

BACKGROUND: The sap from Euphorbia peplus, commonly known as petty spurge in the U.K. or radium weed in Australia, has been used as a traditional treatment for a number of cancers. OBJECTIVE: To determine the effectiveness of E. peplus sap in a phase I/II clinical study for the topical treatment of basal cell carcinomas (BCC), squamous cell carcinomas (SCC) and intraepidermal carcinomas (IEC). METHODS: Thirty-six patients, who had refused, failed or were unsuitable for conventional treatment, were enrolled in a phase I/II clinical study. A total of 48 skin cancer lesions were treated topically with 100-300 µL of E. peplus sap once daily for 3 days. RESULTS: The complete clinical response rates at 1 month were 82% (n = 28) for BCC, 94% (n = 16) for IEC and 75% (n = 4) for SCC. After a mean follow-up of 15 months these rates were 57%, 75% and 50%, respectively. For superficial lesions < 16 mm, the response rates after follow-up were 100% for IEC (n = 10) and 78% for BCC (n = 9). CONCLUSIONS: The clinical responses for these relatively unfavourable lesions (43% had failed previous treatments, 35% were situated in the head and neck region and 30% were > 2 cm in diameter), are comparable with existing nonsurgical treatments. An active ingredient of E. peplus sap has been identified as ingenol mebutate (PEP005). This clinical study affirms community experience with E. peplus sap, and supports further clinical development of PEP005 for the treatment of BCC, SCC and IEC.

Conservation concerns associated with low genetic diversity for K'gari-Fraser Island dingoes.

The dingo population on world heritage-listed K'gari-Fraser Island (K'gari) is amongst the most well-known in Australia. However, an absence of population genetic data limits capacity for informed conservation management. We used 9 microsatellite loci to compare the levels of genetic diversity and genetic structure of 175 K'gari dingo tissue samples with 264 samples from adjacent mainland regions. Our results demonstrated that the K'gari population has significantly lower genetic diversity than mainland dingoes (AR, HE, PAR; p < 0.05) with a fourfold reduction in effective population size (Ne = 25.7 vs 103.8). There is also strong evidence of genetic differentiation between the island and mainland populations. These results are in accordance with genetic theory for small, isolated, island populations, and most likely the result of low initial diversity and founder effects such as bottlenecks leading to decreased diversity and drift. As the first study to incorporate a large sample set of K'gari dingoes, this provides invaluable baseline data for future research, which should incorporate genetic and demographic monitoring to ensure long-term persistence. Given that human-associated activities will continue to result in dingo mortality, it is critical that genetic factors are considered in conservation management decisions to avoid deleterious consequences for this iconic dingo population.

Antiproliferative activity of PEP005, a novel ingenol angelate that modulates PKC functions, alone and in combination with cytotoxic agents in human colon cancer cells.

PEP005 is a novel ingenol angelate that modulates protein kinases C (PKC) functions by activating PKC delta and inhibiting PKC alpha. This study assessed the antiproliferative effects of PEP005 alone and in combination with several other anticancer agents in a panel of 10 human cancer cell lines characterised for expression of several PKC isoforms. PEP005 displayed antiproliferative effects at clinically relevant concentrations with a unique cytotoxicity profile that differs from that of most other investigated cytotoxic agents, including staurosporine. In a subset of colon cancer cells, the IC(50) of PEP005 ranged from 0.01-140 microM. The antiproliferative effects of PEP005 were shown to be concentration- and time-dependent. In Colo205 cells, apoptosis induction was observed at concentrations ranging from 0.03 to 3 microM. Exposure to PEP005 also induced accumulation of cells in the G1 phase of the cell cycle. In addition, PEP005 increased the phosphorylation of PKC delta and p38. In Colo205 cells, combinations of PEP005 with several cytotoxic agents including oxaliplatin, SN38, 5FU, gemcitabine, doxorubicin, vinorelbine, and docetaxel yielded sequence-dependent antiproliferative effects. Cell cycle blockage induced by PEP005 in late G1 lasted for up to 24 h and therefore a 24 h lag-time between PEP005 and subsequent exposure to cytotoxics was required to optimise PEP005 combinations with several anticancer agents. These data support further evaluation of PEP005 as an anticancer agent and may help to optimise clinical trials with PEP005-based combinations in patients with solid tumours.

Characterisation of PAUSE-1, a powerful silencer in the human plasminogen activator inhibitor type 2 gene promoter.

Plasminogen activator inhibitor type 2 (PAI-2) is a serine protease inhibitor traditionally regarded as a regulator of fibrinolysis and extracellular matrix degradation. More recently, PAI-2 has been implicated in diverse processes such as keratinocyte differentiation, cell death and viral pathogenesis. The PAI-2 promoter tightly regulates PAI-2 gene expression in a cell-specific manner and this control is mediated, in part, by the upstream silencer element, PAUSE-1. Here we have defined PAUSE-1 and investigated its activity as a silencer. A series of mutations were generated within the PAUSE-1 element and analysed for transcription factor binding and transcriptional silencing activity. These studies have defined the minimal functional PAUSE-1 element as TCTN(x)AGAN(3)T(4), where x = 0, 2 or 4. Examination of related elements present in other promoters, such as the human IFNbeta promoter, suggests that PAUSE-1 is a member of a family of universal silencers with the consensus sequence TCTN(x)AGA. UV crosslinking analyses determined that the PAUSE-1 binding protein was approximately 67 kDa. Insertion of PAUSE-1 into the heterologous (SV40) or the minimal PAI-2 promoters silenced transcription by 2.5-fold. These data show that PAUSE-1 acts as a powerful silencer of PAI-2 gene transcription and is likely to be important in the silencing of other genes as well.

Localization, expression and genomic structure of the gene encoding the human serine protease testisin.

Testisin is a recently identified human serine protease expressed by premeiotic testicular germ cells and is a candidate tumor suppressor for testicular cancer. Here, we report the characterization of the gene encoding testisin, designated PRSS21, and its localization on the short arm of human chromosome 16 (16p13.3) between the microsatellite marker D16S246 and the radiation hybrid breakpoint CY23HA. We have further refined the localization to cosmid 406D6 in this interval and have established that the gene is approximately 4. 5 kb in length, and contains six exons and five intervening introns. The structure of PRSS21 is very similar to the human prostasin gene (PRSS8) which maps nearby on 16p11.2, suggesting that these genes may have evolved through gene duplication. Sequence analysis showed that the two known isoforms of testisin are generated by alternative pre-mRNA splicing. A major transcription initiation site was identified 97 nucleotides upstream of the testisin translation start and conforms to a consensus initiator element. The region surrounding the transcription initiation site lacks a TATA consensus sequence, but contains a CCAAT sequence and includes a CpG island. The 5'-flanking region contains several consensus response elements including Sp1, AP1 and several testis-specific elements. Analysis of testisin gene expression in tumor cell lines shows that testisin is not expressed in testicular tumor cells but is aberrantly expressed in some tumor cell lines of non-testis origin. These data provide the basis for identifying potential genetic alterations of PRSS21 that may underlie both testicular abnormalities and tumorigenesis.

Regioselective acylation of 3-O-angeloylingenol by Candida antarctica Lipase B.

Acylation of 3-O-angeloylingenol (1) with vinyl acetate, vinyl decanoate and vinyl cinnamate, catalyzed by Candida antarctica Lipase B, was investigated. In each case, compound 1 was quantitatively and regioselectively acylated to afford a single product, 3-O-angeloyl-20-O-acetylingenol (1a), 3-O-angeloyl-20-O-decanoylingenol (1b) and 3-O-angeloyl-20-O-cinnamoylingenol (1c), respectively. The structures of the novel compounds 1b-1c were determined by MS and NMR, and product 1a by comparison of RP-HPLC and TLC with a standard. Compounds 1b-1c induced a bipolar morphology of MM96L melanoma cells at a similar concentration as compound 1, as well as having activity in inhibiting the growth of MM96L melanoma cells.

Transcriptional control and the role of silencers in transcriptional regulation in eukaryotes.

Mechanisms controlling transcription and its regulation are fundamental to our understanding of molecular biology and, ultimately, cellular biology. Our knowledge of transcription initiation and integral factors such as RNA polymerase is considerable, and more recently our understanding of the involvement of enhancers and complexes such as holoenzyme and mediator has increased dramatically. However, an understanding of transcriptional repression is also essential for a complete understanding of promoter structure and the regulation of gene expression. Transcriptional repression in eukaryotes is achieved through 'silencers', of which there are two types, namely 'silencer elements' and 'negative regulatory elements' (NREs). Silencer elements are classical, position-independent elements that direct an active repression mechanism, and NREs are position-dependent elements that direct a passive repression mechanism. In addition, 'repressors' are DNA-binding trasncription factors that interact directly with silencers. A review of the recent literature reveals that it is the silencer itself and its context within a given promoter, rather than the interacting repressor, that determines the mechanism of repression. Silencers form an intrinsic part of many eukaryotic promoters and, consequently, knowledge of their interactive role with enchancers and other transcriptional elements is essential for our understanding of gene regulation in eukaryotes.

Regulation of the plasminogen activator inhibitor type-2 gene in monocytes: localization of an upstream transcriptional silencer.

Transcriptional regulation of the plasminogen activator inhibitor type-2 (PAI-2) gene appears to be an important factor in the response of mononuclear phagocytes to inflammation. We have investigated here the molecular basis for PAI-2 synthesis in monocytic cells by reporter gene deletion analysis. A DNA fragment containing 5.1 kb of 5' flanking region through to the start of the second exon was fused to a chloramphenicol acetyl transferase (CAT) reporter gene, transfected into macrophage and nonmacrophage cells and tested for PAI-2 promoter-directed CAT activity in the presence and absence of phorbol ester. Deletion analysis showed the existence of three major transcription regulatory regions. (1) A positive regulatory region contained in the proximal promoter mediates basal transcription and 12-phorbol 13-myristate acetate inducibility. (2) A negative regulatory region, or silencer, present between -1977 and -1675, was found to repress PAI-2 promoter activity in an orientation- and position-independent manner, but not in a cell-specific manner. (3) A second positive regulatory element, located upstream between approximately -5100 and -3300, appears to overcome inhibition mediated by the silencer in a cell-specific manner, suggesting a mechanism for the regulation of this gene. We have localized the motif responsible for silencer activity to a 28-bp DNA sequence containing a unique 12-bp palindrome centered at an Xba I restriction enzyme site, CTCTCTAGAGAG, which is designated the PAI-2-upstream silencer element-1 (PAUSE-1). This element binds a specific PAUSE-1 binding factor as determined by mobility shift analysis. We conclude that PAI-2 gene transcription is regulated by both positive and negative control mechanisms that may be important for the regulation of other genes as well.

Cloning of the cDNA and localization of the gene encoding human NRBP, a ubiquitously expressed, multidomain putative adapter protein.

Adapter proteins modulate multiple signaling pathways by regulating the aggregation of other factors into signaling complexes. Here we have identified a novel human cDNA encoding NRBP, a multidomain putative adapter protein containing (i) two putative nuclear receptor binding motifs (LXXLL), (ii) a putative binding domain for Src homology-2 (SH2) domain containing proteins, (iii) a kinase-like domain, (iv) a bipartite nuclear localization signal, and (v) three sequences rich in glutamic acid, serine, proline, and threonine (PEST) residues. The NRBP mRNA transcript, of approximately 2.4 kb, was ubiquitously expressed in a wide range of normal human tissues and 15 human tumor cell lines. The NRBP cDNA is predicted to encode a polypeptide of 535 amino acids with a molecular mass of 59.8 kDa. Translation of NRBP mRNA in vitro reveals three translation products of 60, 51, and 43 kDa, suggesting that translation of NRBP may initiate at multiple sites. The NRBP gene was localized to human chromosome 2p23, near the location of the NCOA1 gene encoding the nuclear receptor coactivator, steroid receptor coactivator-1 (SRC-1). The features of NRBP predict a function as an adapter protein potentially linking signaling pathways involving nuclear receptors and SH2 domain containing proteins.