Genetic map construction and QTL mapping of resistance to blackleg (Leptosphaeria maculans) disease in Australian canola (Brassica napus L.) cultivars.

Genetic map construction and identification of quantitative trait loci (QTLs) for blackleg resistance were performed for four mapping populations derived from five different canola source cultivars. Three of the populations were generated from crosses between single genotypes from the blackleg-resistant cultivars Caiman, Camberra and (AV)Sapphire and the blackleg-susceptible cultivar Westar(10). The fourth population was derived from a cross between genotypes from two blackleg resistant varieties (Rainbow and (AV)Sapphire). Different types of DNA-based markers were designed and characterised from a collection of 20,000 EST sequences generated from multiple Brassica species, including a new set of 445 EST-SSR markers of high value to the international community. Multiple molecular genetic marker systems were used to construct linkage maps with locus numbers varying between 219 and 468, and coverage ranging from 1173 to 1800 cM. The proportion of polymorphic markers assigned to map locations varied from 70 to 89% across the four populations. Publicly available simple sequence repeat markers were used to assign linkage groups to reference nomenclature, and a sub-set of mapped markers were also screened on the Tapidor x Ningyou (T x N) reference population to assist this process. QTL analysis was performed based on percentage survival at low and high disease pressure sites. Multiple QTLs were identified across the four mapping populations, accounting for 13-33% of phenotypic variance (V (p)). QTL-linked marker data are suitable for implementation in breeding for disease resistance in Australian canola cultivars. However, the likelihood of shifts in pathogen race structure across different geographical locations may have implications for the long-term durability of such associations.

Triassic amphibian from Antarctica.

A fossil bone fragment-the first record of tetrapod life from Antarctica-was found near Graphite Peak in the upper Beardmore Glacier area (85 degrees 3.3'S; 172 degrees 19'E). The fragment was embedded in a pebbly quartzose sandstone, probably of fluvial origin, in the lower part of the Triassic Fremouw Formation (as yet undefined), which contains Dicroidium in the upper part. The fossil horizon is only 76 meters, stratigraphically, above the Glossopteris-bearing Buckley Formation, a coal-bearing sequence of Permian age. The bone fragment is the back portion of a left mandibular ramus of a labyrinthodont amphibian. This identification is based on the characteristic labyrinthodont external surface sculpturing, with indications of "mucous grooves," as well as on other osteological features.

Functional annotation of genomic data with metabolic inference.

Metabolomics is an appealing new approach in systems biology aimed at enabling an improved understanding of the dynamic biochemical composition of living systems. Biological systems are remarkably complex. Importantly, metabolites are the end products of cellular regulatory processes, and their concentrations reflect the ultimate response of a biological system to genetic or environmental changes. In this article, we describe the components of lipid metabolomics and then use them to investigate the metabolic basis for increased abdominal adiposity in 2 strains of divergently selected chickens. Lipid metabolomics were chosen due to the availability of well-developed analytical platforms and the pervasive physiological importance of lipids in metabolism. The analysis suggests that metabolic shifts that result in increased abdominal adiposity are not universal and vary with genetic background. Metabolomics can be used to reverse engineer selection programs through superior metabolic descriptions that can then be associated with specific gene networks and transcriptional profiles.

Nutritional and hormonal regulation of expression of the gene for malic enzyme.

We have provided a historical and personal description of the analysis of physiological and molecular mechanisms by which diet and hormones regulate the activity of hepatic malic enzyme. For the most part, our analyses have been reductionist in approach, striving for increasingly simpler systems in which we can ask more direct questions about the molecular nature of the signaling pathways that regulate the activity of malic enzyme. The reductionist approaches that were so successful at analyzing molecular mechanisms in cells in culture may now provide the means to analyze more definitively questions about the physiological mechanisms involved in nutritional regulation of gene expression. In addition to physiological questions, however, there are still many aspects of the molecular mechanisms that have not been elucidated. Despite considerable effort from many laboratories, the molecular mechanisms by which T3 regulates transcription are not clear. Similarly, the molecular details for the mechanisms by which glucagon, insulin, glucocorticoids, and fatty acids regulate gene expression remain to be determined. The role of fatty acids is particularly interesting because it may provide a model for mechanisms by which genes are regulated by metabolic intermediates; this is a form of transcriptional regulation widely used by prokaryotic organisms and extensively analyzed in prokaryotic systems, but poorly understood in higher eukaryotes. At any specific time, there is, of course, only one rate of transcription for each copy of the malic-enzyme gene in a cell. Our long-term objective is to understand how signals from all of the relevant regulatory pathways are integrated to bring about that rate.

Extraction of RNA from archival tissues and measurement of thrombospondin-1 mRNA in normal, dysplastic, and malignant oral tissues.

Thrombospondin-1 (TSP-1) is an extracellular matrix glycoprotein implicated in the regulation of angiogenesis and tumour development. Our objectives were to ascertain the quantity and quality of RNA extracted from archival, formalin-fixed, paraffin embedded, oral tissues and their application in measuring the concentrations of TSP-1 mRNA in these tissues. We compared three techniques of isolation of RNA as well as related experimental variables. TSP-1 mRNA was measured in specimens of normal, dysplastic, and malignant oral tissues by real-time reverse transcriptase polymerase chain reaction (RT-PCR). RNA suitable for analysis by real-time RT-PCR was obtained by the three techniques tested, although the yield varied depending on the protocol used (range 0.2-3.6 microg/mm(3)). The mean (S.D.) concentrations of TSP-1 mRNA relative to 18S were 21.1 (7.2) in normal oral tissues (n=9), 11.0 (8.2) in dysplastic tissue (n=8) and 7.3 (5.3) in carcinomatous tissue (n=17). The difference between normal and carcinomatous specimens was significant (p=0.01). This reduction in expression of TSP-1 mRNA from normal to dysplasia to carcinoma may favour the angiogenic drive that accompanies the development of oral tumours.

Localization of basic fibroblast growth factor and transforming growth factor beta 1 in the human mammary gland.

The presence and distribution of basic fibroblast growth factor (bFGF) and transforming growth factor beta 1 in benign and malignant human breast tissue were determined by immunohistochemistry and immunoblotting. Peroxidase staining of biopsy specimens using a polyclonal antibody to amino acids 1-24 of bFGF and a monoclonal antibody to whole recombinant bFGF showed this growth factor to be localized in the myoepithelial cells of the benign breast. Epithelial cells and stroma were negative. In hyperplasia and intraductal carcinoma in situ staining was still seen around the perimeter of enlarged ducts. In malignant biopsies, however, staining was seen only when benign elements were present or residual myoepithelial cells and basement membrane remained. Antigen absorption and immunoblotting confirmed the antibody staining to be specific for bFGF. Transforming growth factor beta 1 was shown, using the same techniques, to be located in the periductal and intraductal stroma, closely associated with epithelial or myoepithelial cells in the benign and malignant breast. The relative localization of these two growth factors in the mammary gland may be significant in the control of breast development and/or tumor formation and progression.

Serum levels of renin, angiotensin-converting enzyme and angiotensin II in patients treated by surgical excision, propranolol and captopril for problematic proliferating infantile haemangioma.

The role of the renin-angiotensin system (RAS) in the biology of infantile haemangioma (IH) and its accelerated involution induced by ß-blockers was first proposed in 2010. This led to the first clinical trial in 2012 using low-dose captopril, an angiotensin-converting enzyme (ACE) inhibitor, demonstrating a similar response in these tumours. This study aimed to compare serial serum levels of the components of the RAS in patients before and after surgical excision, propranolol or captopril treatment for problematic proliferating IH. Patients with problematic proliferating IH underwent measurements of serum levels of plasma renin activity (PRA), ACE and angiotensin II (ATII) before, and 1-2 and 6 months following surgical excision, propranolol or captopril treatment. This study included 27 patients undergoing surgical excision (n = 8), propranolol (n = 11) and captopril (n = 8) treatment. Treatment with either surgical excision or propranolol resulted in significant decrease in the mean levels of PRA. Surgical excision or captopril treatment led to significant decline in the mean levels of ATII. All three treatment modalities had no significant effect on the mean levels of ACE. This study demonstrates the effect of surgical excision, propranolol and captopril treatment in lowering the levels of PRA and ATII, but not ACE, supporting a mechanistic role for the RAS in the biology of IH.

The aromatic residue content of the enzyme rhodanese.

The aromatic amino acid composition of the enzyme rhodanese has been redetermined. Previous reports have varied from 5 to 11 tryptophans per 26 alanine residues. The present work has quantitated the aromatic residues by a combination of amino acid analysis, solvent perturbation difference spectroscopy, specific residue modification and direct ultraviolet spectral analysis. These methods indicate that rhodanese contains 10 tyrosines, eight tryptophans and 16 phenylalanines per 26 alanine residues. The results for tyrosine and phenylalanine are in reasonable agreement with previous results.

A reexamination of the postulated charge transfer interactions at the active site of the enzyme rhodanese.

Spectral and kinetic studies of the interaction of N-methylnicotinamide chloride and nicotinamide with the enzyme thiosulphate sulphurtransferase (thiosulphate: cyanide sulfurtransferase, EC 2.8.1.1) (also known as rhodanese) have been performed and compared with previous inhibition data obtained with N-1-(4-pyridyl)pyridinium chloride (NPP). Like NPP both N-methylnicotinamide chloride and nicotinamide are competitive inhibitors of rhodanese with respect to the substrate thiosulfate. Rhodanese binding of N-methylnicotinamide chloride gives rise to no charge transfer absorbtion band. In addition, the free energy of interaction (deltaG0) of NPP with rhodanese is approximately equal to the sum of the individual deltaG0 values of MNA and NA. These compounds are analogous to the two halves of the NPP structure. We conclude that NPP and N-methylnicotinamide chloride are not bound via a charge transfer mechanism. The major stabilizing influence appears to be an ionic interaction with an anionic enzyme site with accessory apolar stabilization. It is postulated that the ionized active site sulfhydryl group in rhodanese could provide the ionic site.

Denaturation-induced disulfide formation in the enzyme rhodanese.

The effect of denaturants on the quantitation of free sulfhydryl groups in the enzyme rhodanese (thiosulfate sulfurtransferase, EC 2.8.1.1) has been reinvestigated in some detail. The sulfhydryl assay with the colorimetric reagent 5, 5'-dithio-bis (2-nitrobenzoic acid) Nbs2 shows four sulfhydryl groups per enzyme molecule (mol. wt. 32 300) when the colorimetric reagent is added to the assay mixture before the denaturant, sodium dodecyl sulfate. On the other hand, only two sulfhydryl groups per molecule are observed when Nbs2 is added after denaturation has been initiated. The time dependence observed in this latter procedure indicates that the loss of the two groups is rapid and permanent. The results depend on the denaturant used: urea acts like sodium dodecyl sulfate while guanidine reveals four sulfhydryl groups independent of reagent order. The assay also gives four sulfhydryl groups independent of reagent order. The assay also gives four sulfhydryl groups independent of reagent order with urea or sodium dodecyl sulfate under conditions which are expected to limit metal ion-catalyzed oxidation of sulfhydryl groups (e.g. oxygen exclusion or metal ion chelation). Recent studies have shown that rhodanese has a molecular weight of 32 600, no disulfides and four sulfhydryl groups per molecule. These results together with the observations reported here are taken to indicate that a disulfide can be formed during denaturation of rhodanese and that the pathway of denaturation determines the result obtained.

Spectral studies of the tryptophan exposure in the enzyme rhodanese.

Tryptophan exposure in the enzyme rhodanese (thiosulfate:cyanide sulfur-transferase, EC 2.8.1.1.) has been studied by the methods of solvent perturbation difference spectroscopy, fluorescence spectroscopy and fluorescence quenching. The data from all these techniques are consistent with two classes of tryptophan:surface and buried. The surface residues appear to be relatively shielded and in an anionic environment. The buried residues have spectral characteristics suggesting they are in a very apolar environment and have less than the expected contact with the peptide backbone. The solvent perturbation difference spectra indicate further that additional tyrosine as well as tryptophan residues are buried as rhodanese is converted from the free enzyme to the sulfur-substituted enzyme: forms which are obligatory intermediates in the catalytic cycle.

Peroxisome proliferator-activated receptors: a family of lipid-activated transcription factors.

Peroxisome proliferator-activated receptors (PPARs) are a family of nuclear transcription factors that belong to the steroid receptor superfamily. This family of PPARs includes PPARalpha, PPARdelta, PPARgamma1, and PPARgamma2. These PPARs are related to the T3 and vitamin D(3) receptors and bind to a hexameric direct repeat as a heterodimeric complex with retinoid receptor Xalpha. PPARs regulate the expression of a wide array of genes that encode proteins involved in lipid metabolism, energy balance, eicosanoid signaling, cell differentiation, and tumorigenesis. A unique feature of these steroid-like receptors is that the physiologic ligands for PPARs appear to be fatty acids from the n-6 and n-3 families of fatty acids and their respective eicosanoid products. This review describes the characteristics, regulation, and gene targets for PPARs and relates their effects on gene expression to physiologic outcomes that affect lipid and glucose metabolism, thermogenesis, atherosclerosis, and cell differentiation.

Multiple forms of TGF-beta 1 in breast tissues: a biologically active form of the small latent complex of TGF-beta 1.

The aim of this study was to investigate whether breast cancer growth in vivo could be due to a failure in the activation of TGF-beta 1, a growth factor which has been shown to affect the development of normal breast tissue. Tissue samples of 40 breast carcinomas and the normal adjacent tissue from 37 (henceforth referred to as 'adjacent tissue'), as well as 13 specimens of benign lesions, were included in this study. The specimens were used in vitro to produce conditioned medium (CM), and this was examined for TGF-beta 1 activity by measuring growth inhibition of the mink lung epithelial cell line CCL-64. Immunoblotting and electrophoresis were used to detect the presence of TGF-beta 1 in CM and homogenised tissue samples. We demonstrated that the majority of TGF-beta 1 in breast cancer conditioned medium was biologically active, in direct contrast to CM prepared from benign disease specimens. Furthermore, active TGF-beta 1 was also identified in CM prepared from adjacent tissue, suggesting an important early role for this growth factor in the spread of this disease. Three distinct breast cancer related (BCR) molecular weight species of TGF-beta 1 (12.5/25 kDa, 50 kDa and 95 kDa) were identified. Both the 50 kDa and 95 kDa bands immunoprecipitated by an anti-TGF-beta 1 antibody were also immunoreactive with anti-TGF-beta 1 binding protein antibodies suggesting that the 50 kDa band may comprise at least part of the previously described small latent complex of TGF-beta 1. However, using the CCL-64 cell assay, we were able to demonstrate that the 50 kDa TGF-beta 1 BCR protein was biologically active whereas the large (95 kDa) TGF-beta 1 BCR latent complex protein was not. Adjacent tissue was more likely to contain the 50 kDa form than the tumour tissues (P < 0.05). Similarly, the 50 kDa molecule was also more common in patients who had oestrogen receptor (ER) negative tumours (compared with ER positive patients; P < 0.05) and in those who had received tamoxifen treatment prior to surgery (P < 0.01). In all of these cases, the increase in the incidence of the small active complex form was accompanied by a decrease in the incidence of the high molecular weight complex (95 kDa). We confirmed that, in vitro, the 95 kDa TGF-beta 1 BCR can be proteolytically cleaved to yield a 50 kDa TGF-beta 1 BCR. Finally, we observed a correlation between the presence of the 50 kDa complex protein and reduced levels of plasminogen activator (PA), which was significant in ER negative patients (P < 0.05) and tamoxifen-pretreated patients (P < 0.01). This suggests that the secretion of this active TGF-beta 1 protein may provide breast tumours with a mechanism whereby they can escape oestrogen dependence, and may provide an explanation for the common problem of tamoxifen resistance.

Acidic and basic fibroblast growth factors in human breast tissue.

Previously we have reported changes in fibroblast growth factors (FGF) in conditioned medium (CM) derived from rat mammary tumours undergoing remission. We have used a similar approach to assay for the presence of FGFs in human breast tissue and cell lines. The majority of cancer tissues (35/50), benign tissues (8/9) and all cancer adjacent normal tissues (20/20) released heat labile, NR6 transforming activity which coeluted from heparin with acidic FGF (aFGF) at 0.9-1.1 M NaCl and was neutralised by antibodies to aFGF. The conclusion that the majority of breast cancers contain active aFGF was supported by immunoblotting. The CM of a minority (15/50) of cancers and one benign tissue had highly transforming activity for NR6 cells, and was mitogenic for a breast cancer cell line, was heat labile, and strongly heparin binding, eluting at 1.5-2.0 M salt. It was not immunoreactive with antibodies to aFGF, basic FGF (bFGF) or Kaposi's FGF (kFGF) and its activity was reduced by the presence of aFGF, suggesting competition for the same receptor. Very little aFGF was observed in the CM of these tumours, and neither aFGF nor other FGF activity was detected in CM of breast cell lines.

The effect of endocrine therapy on fibroblast growth factor-like activity in nitrosomethylurea-induced rat mammary tumours.

Tumour regression following ovariectomy of rats bearing nitrosomethylurea-induced mammary tumours has been well characterised as a model for oestrogen receptor (ER)-positive breast cancer. We have shown that a similar regression response can be induced in these rats by the cytotoxic drug doxorubicin. Conditioned medium (CM) from serum-free explant cultures of the mammary tumours of ovariectomised rats showed a striking increase in its ability to transform NR6 cells compared to that of control or doxorubicin-treated rats (P = 0.001, t-test). Activity was also present in CM derived from rat uteri but not in ER-negative tissues such as skin and liver. Activity was further defined as fibroblast growth factor (FGF)-like by its strong affinity to heparin, partial neutralisation by antibodies to acidic FGF (aFGF) and partial co-elution with aFGF on salt elution from heparin. Both aFGF protein and mRNA were detected in tissue preparations of rat tumours and uterus.

Prognostic value of vascularity and vascular endothelial growth factor expression in non-small cell lung cancer.

AIMS: High expression of the angiogenic factor vascular endothelial growth factor (VEGF) in tumours has been found to be associated with poor prognosis in some studies, but not in others. The aims of this study were to determine the prognostic value of VEGF in operable non-small cell lung cancer (NSCLC) and its possible association with vascularity. METHODS: Sections from 81 NSCLC archival specimens were stained with antibodies to von Willebrand factor (vWF) and VEGF. Vascularity was measured by the average density of vWF positive vessels. VEGF expression in tumour cells was assessed by consensus of two independent observers according to three indices, namely: (1) percentage of area stained, (2) intensity of staining, and (3) final score (product of area and intensity). RESULTS: VEGF immunoreactivity was present in all tumours and adjacent normal lung tissue. None of the three VEGF indices was associated with vascularity or the clinical parameters examined. Mean survival times were shorter in patients with high VEGF expression, but the difference was not significant. This applied to the full cohort of patients, or when analysed separately according to tumour type or stage. However, high VEGF expression was associated with poor survival in patients with high vascularity (p = 0.02). VEGF had no discriminant value among patients with low vascularity. Vascularity had no prognostic value, except for late stage patients (UICC stages II and IIIa combined; n = 36), where high vascularity was associated with longer survival (p = 0.01). CONCLUSIONS: VEGF on its own has no prognostic value in NSCLC, but may become a useful indicator when combined with vascularity. VEGF may play a physiological role in the normal lung.

Fatty acid regulation of gene expression. Its role in fuel partitioning and insulin resistance.

Dietary polyenoic (n-6) and (n-3) fatty acids uniquely regulate fatty acid biosynthesis and fatty acid oxidation. They exercise this effect by modulating the expression of genes coding for key metabolic enzymes and, in doing this, PUFA govern the intracellular as well as the interorgan metabolism of glucose and fatty acids. During the past 20 years, we have gradually elucidated the cellular and molecular mechanism by which dietary PUFA regulate lipid metabolism. Central to this mechanism has been our ability to determine that dietary PUFA regulate the transcription of genes. We have only begun to elucidate the nuclear mechanisms by which PUFA govern gene expression, but one point is clear and that is that it is unlikely that one mechanism will explain the variety of genes governed by PUFA. The difficulty in providing a unifying hypothesis at this time stems from (a) the many metabolic routes taken by PUFA upon entering a cell and (b) the lack of identity of a specific PUFA-regulated trans-acting factor. Nevertheless, our studies have revealed that PUFA are not only utilized as fuel and structural components of cells, but also serve as important mediators of gene expression, and that in this way they influence the metabolic directions of fuels and they modulate the development of nutritionally related pathophysiologies such as diabetes.

Coordinate induction of peroxisomal acyl-CoA oxidase and UCP-3 by dietary fish oil: a mechanism for decreased body fat deposition.

Rats fed dietary fats rich in 20- and 22-carbon polyenoic fatty acids deposit less fat and expend more energy at rest than rats fed other types of fats. We hypothesized that this decrease in energetic efficiency was the product of: (a) enhanced peroxisomal fatty acid oxidation and/or (b) the up-regulation of genes encoding proteins that were involved with enhanced heat production, i.e. mitochondrial uncoupling proteins (UCP-2, UCP-3) and peroxisomal fatty acid oxidation proteins. Two groups of male Fisher 344 rats 3-4 week old (n=5 per group) were pair fed for 6 weeks a diet containing 40% of its energy fat derived from either fish oil or corn oil. Epididymal fat pads from rats fed the fish oil diet weighed 25% (P < 0.05) less than those found in rats fed corn oil. The decrease in fat deposition associated with fish oil ingestion was accompanied by a significant increase in the abundance of skeletal muscle UCP-3 mRNA. The level of UCP-2 mRNA skeletal muscle was unaffected by the type of dietary oil, but the abundance of UCP-2 mRNA in the liver and heart were significantly lower (P < 0.05) in rats fed fish oil than in rats fed corn oil. In addition to inducing UCP-3 expression, dietary fish oil induced peroxisomal acyl-CoA oxidase gene expression 2-3 fold in liver, skeletal muscle and heart. These data support the hypothesis that dietary fish oil reduces fat deposition by increasing the expression of mitochondrial uncoupling proteins and increasing fatty acid oxidation by the less efficient peroxisomal pathway.

High levels of apoptosis are associated with improved survival in non-small cell lung cancer.

Tumour growth is accompanied by angiogenesis and reduced apoptosis in experimental animals. The aim of this study was to examine the prognostic value of apoptosis and the association between apoptosis and vascularity in non-small cell lung cancer (NSCLC). Following in-situ end-labelling of DNA, apoptotic cells were quantified by three different indices: as a percentage, either counting total cells (AI-tc) or point-counting (AI-pc), or as cells per area (AI-area). Blood vessels were stained with vWF antibody and vascularity was quantified by three methods. Median values for AI-tc, AI-pc and AI-area were 0.38, 0.32 and 10.7, respectively. High values were associated with improved survival, reaching statistical significance for AI-area (p < 0.05). All three apoptotic indices were significantly correlated with each other, but no correlation was found between indices of apoptosis and vascularity. As previously reported, vascularity had no prognostic value. These results indicate that, in NSCLC, vascularity is not informative, but apoptotic index may be a useful prognostic factor.

VEGF expression in skin warts. Relevance to angiogenesis and vasodilation.

Verrucae vulgaris (skin warts) are benign proliferative lesions which are generally associated with human papillomavirus type 2 (HPV-2) infection. Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen able to induce angiogenesis and vasodilation. Our previous findings indicate that these two processes take place during the formation of skin warts. The purpose of this study was to determine whether VEGF expression in these lesions was associated with HPV infection, angiogenesis or vasodilation. To this end, paraffin-embedded specimens of skin warts which were either negative for HPV-1, -2, -3 and -4 (HPV-; n = 18), or positive for HPV-2 (HPV+; n = 21) were compared with histologically normal perilesional skin (n = 13). Serial sections were stained with antibodies to von Willebrand Factor (vWF) and to VEGF. Vascularity was quantified by point counting vWF-positive blood vessels. Small and large vessels were quantified separately, using a cut-off value of 50 microm diameter. VEGF expression in the epidermis was estimated by consensus of two independent observers according to three indices: (1) percentage of cells stained, (2) intensity of the staining, and (3) product of area and intensity (final score). Results were analysed by nonparametric tests. Similar levels of VEGF were found in specimens of normal skin, HPV- and HPV+ warts, irrespective of the index used. There was no significant correlation between VEGF expression and vascularity values for either small or large vessels. These results indicate that, on its own, VEGF expression is not associated with angiogenesis, vasodilation or HPV infection in skin warts. The presence of VEGF in normal skin suggests that it may play a role in tissue homeostasis.