Community Priority setting for Fetal Alcohol Spectrum Disorder Research in Australia.

INTRODUCTION: Fetal Alcohol Spectrum Disorder (FASD) is a neurodevelopmental disorder caused by prenatal alcohol exposure (PAE). FASD research is a rapidly growing field that crosses multiple disciplines. To ensure research is relevant and meaningful for people living with FASD, their families, and the broader public there is a need to engage community members in setting priorities for research. OBJECTIVES: Our primary objective was to formally identify the views of people living with FASD, their parents/caregivers, service providers, and the general community on the research priorities for FASD and alcohol use in pregnancy in Australia. Our secondary objective was to provide an overview of current research in the highest priority areas identified. METHODS: The approach for this study involved two community surveys and a consensus workshop, followed by a rapid literature review. Survey responses (n = 146) were collected and grouped using qualitative thematic analysis. The themes identified were then ranked in a second survey (n = 45). The 22 highest ranked themes were considered in a workshop with 21 community members, and consensus on the top ten priority areas was sought. The priority areas were grouped into conceptually similar topics and rapid literature reviews were undertaken on each. RESULTS: A diverse range of priorities was identified within key areas of prevention, diagnosis, and therapy. On request from participants, separate priority lists were developed by Aboriginal and non-Aboriginal participants. CONCLUSION: There is need for a national network of researchers to take forward the research commenced by the Centre of Research Excellence, FASD Research Australia, in addressing community priorities. KEY WORDS: Community, priorities, FASD, rapid review, Australia.

Guanine nucleotide exchange factor Dock7 mediates HGF-induced glioblastoma cell invasion via Rac activation.

BACKGROUND: Glioblastoma multiforme (GBM), a highly invasive primary brain tumour, remains an incurable disease. Rho GTPases and their activators, guanine nucleotide exchange factors (GEFs), have central roles in GBM invasion. Anti-angiogenic therapies may stimulate GBM invasion via HGF/c-Met signalling. We aim to identify mediators of HGF-induced GBM invasion that may represent targets in a combination anti-angiogenic/anti-invasion therapeutic paradigm. METHODS: Guanine nucleotide exchange factor expression was measured by microarray analysis and western blotting. Specific depletion of proteins was accomplished using siRNA. Cell invasion was determined using matrigel and brain slice assays. Cell proliferation and survival were monitored using sulforhodamine B and colony formation assays. Guanine nucleotide exchange factor and GTPase activities were determined using specific affinity precipitation assays. RESULTS: We found that expression of Dock7, a GEF, is elevated in human GBM tissue in comparison with non-neoplastic brain. We showed that Dock7 mediates serum- and HGF-induced glioblastoma cell invasion. We also showed that Dock7 co-immunoprecipitates with c-Met and that this interaction is enhanced upon HGF stimulation in a manner that is dependent on the adaptor protein Gab1. Dock7 and Gab1 also co-immunoprecipitate in an HGF-dependent manner. Furthermore, Gab1 is required for HGF-induced Dock7 and Rac1 activation and glioblastoma cell invasion. CONCLUSIONS: Dock7 mediates HGF-induced GBM invasion. Targeting Dock7 in GBM may inhibit c-MET-mediated invasion in tumours treated with anti-angiogenic regimens.

Examining the role of Rac1 in tumor angiogenesis and growth: a clinically relevant RNAi-mediated approach.

Angiogenesis, the sprouting of new blood vessels from the pre-existing vasculature, is a well established target in anti-cancer therapy. It is thought that the Rho GTPase Rac1 is required during vascular endothelial growth factor (VEGF)-mediated angiogenesis. In the present study, we have used a clinically relevant RNA interference approach to silence Rac1 expression. Human umbilical vein endothelial cells were transiently transfected with non-specific control siRNA (siNS) or Rac1 siRNA (siRac1) using electroporation or Lipofectamine 2000. Functional assays with transfected endothelial cells were performed to determine the effect of Rac1 knockdown on angiogenesis in vitro. Silencing of Rac1 inhibited VEGF-mediated tube formation, cell migration, invasion and proliferation. In addition, treatment with Rac1 siRNA inhibited angiogenesis in an in vivo Matrigel plug assay. Intratumoral injections of siRac1 almost completely inhibited the growth of grafted Neuro2a tumors and reduced tumor angiogenesis. Together, these data indicate that Rac1 is an important regulator of VEGF-mediated angiogenesis. Knockdown of Rac1 may represent an attractive approach to inhibit tumor angiogenesis and growth.

Studies on Syntaxin 12 and alcohol preference involving C57BL/6J and DBA/2J strains of mice.

C57BL/6J and DBA/2J inbred mouse strains have been extensively studied for the genetic dissection of alcohol-related phenotypes. We have previously found Syntaxin 12 to be associated with alcohol preference in C57BL/6J and DBA/2J due to its strain-specific and ethanol responsive expression in the male brain. In the current study, we combined genetic and expression analyses to assess the segregation of Syntaxin 12 c.*1370G>A polymorphism with its strain-specific expression and alcohol preference in an F (2) population (N = 427) derived from C57BL/6J and DBA/2J strains. Syntaxin 12 c.*1370G>A polymorphism was found to segregate with alcohol preference in the B6D2F2 population and a correlation was identified between Syntaxin 12 expression and alcohol preference in the selected B6D2F2 males (r = -0.473, r (2) = 0.22). We followed up our analysis in the BXD RI lines using resources from WebQTL and the Mouse Phenome Database. Our study detected significant associations of Syntaxin 12 molecular variants with its level of expression and alcohol preference in B6D2F2 males. Overall, our findings support a role for Syntaxin 12 as a potential contributor to alcohol preference in mice.

The application of machine learning techniques as an adjunct to clinical decision making in alcohol dependence treatment.

With few exceptions, research in the addictive sciences has relied on linear statistics and methodologies. Addiction involves a complex array of nonlinear behaviors. This study applies two machine learning techniques, Bayesian and decision tree classifiers, in the assessment of outcome of an alcohol dependence treatment program. These nonlinear approaches are compared to a standard linear analysis. Seventy-three alcohol-dependent subjects undertaking a 12-week cognitive-behavioral therapy (CBT) program and 66 subjects undertaking an identical program but also prescribed the relapse prevention agent Acamprosate were employed in this study. Demographic, alcohol use, dependence severity, craving, health-related quality of life, and psychological measures at baseline were used to predict abstinence at 12 weeks. Decision trees had a 77% predictive accuracy across both data sets, Bayesian networks 73%, and discriminant analysis 42%. Combined with clinical experience, machine learning approaches offer promise in understanding the complex relationships that underlie treatment outcome for abstinence-based alcohol treatment programs.

Hazard rate ratio and prospective epidemiological studies.

Analysis of prospective follow-up data usually includes a Cox regression model. When a hazard rate ratio, obtained as the exponential of an estimated regression coefficient from the Cox model, is greater than 1.0, it consistently exceeds relative risk, and is exceeded by the odds ratio. The divergence of these distinct epidemiologic measures increases with the product of three factors: (1) the length of follow-up, (2) the average rate of the end point occurence over the follow-up period, and (3) the magnitude of risk, either above or below 1. Cornfield's rare disease assumption is basically the product of the first two of these factors. However, risks in excess of 2.5 have a powerful effect on the divergence of these measures, and this point has received less emphasis. Conversely, and as seen frequently in applications, relative risk, hazard rate ratio, and odds ratio numerically approximate one another with shorter follow-up, rarer end points, and risks closer to 1. Although the hazard rate ratio is not always distinguished from relative risk, it is commonly close to, and is always between, relative risk and the odds ratio. Consistent and accurate terminology would have us use hazard rate ratio with Cox regression and odds ratio with logistic regression. The term "relative risk" seems to be a default choice, regardless of the model being used. However, when relative risk is the object of the model chosen, as in a Poisson regression approximation of two binomial proportions or an equivalent weighted least squares, then for us, relative risk is the accurate terminology.

Ras GTPases: singing in tune.

A review of the meeting "The Ras Superfamily of Small GTP-Binding Proteins," FASEB Summer Research Conference, Snowmass, Colorado, 15 through 20 July 2000 The molecular cloning of the human proto-oncogene encoding Ras was reported nearly 20 years ago. Since then, Ras has become the prototypical member of a superfamily of small guanosine triphosphatase proteins. Despite the maturity of this field of research, the discovery of new functions and interactions between the superfamily members continues unabated. Symons and Takai have written a meeting report on the latest findings on the Ras superfamily.

The small GTPase Rac suppresses apoptosis caused by serum deprivation in fibroblasts.

BACKGROUND: The small GTPase Rac1 is a key signaling protein that mediates a number of important physiologic functions including the organization of the actin cytoskeleton, lipid metabolism, and gene transcription. Rac1 has also been implicated in oncogenic transformation. Expression of constitutively active Rac1 in Rat1 fibroblasts elicits serum- and anchorage-independent growth and causes tumorigenicity in nude mice. The signaling pathways that mediate the role of Rac in cell transformation remain to be identified. Here, we study the role of Rac in cell survival in the absence of serum. MATERIALS AND METHODS: The cell lines used in this study are Ratl fibroblasts that express constitutively active or dominant negative mutants of Rac1. We used long-term video time-lapse microscopy to analyze the effects of these Rac1 mutants on mitogenicity and apoptosis. RESULTS: We show that the increase in viability, which is stimulated by Rac1 in the absence of serum, is predominantly caused by an inhibition of apoptosis, with a minor increase in cell division. We also show that Rac1-stimulated cell viability in serum-starved cells is inhibited by chemical inhibition of phosphatidylinositol 3-kinase. CONCLUSIONS: Our observations indicate a role for Rac1 in survival signaling, possibly via activation of phosphatidylinositol 3-kinase. We propose that Rac1-stimulated cell survival may contribute to the role of Rac1 in serum-independent growth and cell transformation.

Self-treatment for gout.

After describing some of the symptoms of gout and considering some causes, such as an excess of ethanol, the source of the pain in the infected joint is discussed. This is known to be from urate crystals formed in the synovial fluid inside the joint. It is suggested herein that the pain is due to grinding from the crystals through the surface film of the joint, and possibly into the bone itself, which is relatively soft. The pain then stems in part from the resulting inflammation. The key hypothesis is that these urate crystals dissolve on warming. Hence, by warming the joint concerned in hot water, and moving the joint around to encourage diffusion, the urate concentration is reduced and crystals no longer form, provided the treatment is continued.

Hydrogen peroxide: a potent cytotoxic agent effective in causing cellular damage and used in the possible treatment for certain tumours.

H2O2, a highly reactive agent, can react under certain conditions with a variety of cellular components. These reactions include the lipid peroxidation of membrane and hydroxylation of proteins and DNA. The reactions can take place in the presence of oxygen and are fairly rapid, the H2O2 being converted to water and oxygen. Experiments were carried out in vitro to assess the ability of this agent to destroy cancer cells without generating dangerous by-products. The direct administration of aqueous H2O2 into solid tumours has the potential to cause tumour cell death. The efficacy of the use of H2O2 for treating 'solid' cancers will necessitate its delivery to the tumour site, for example by direct special multiple injection of H2O2 into a detectable tumour mass. We anticipate that, if suggested mode of delivery can be obtained, H2O2 can act as an anti-cancer drug with two distinct advantages over conventional chemotherapeutic agents: to produce minimal short- and long-term side-effects and is relatively cheap and cost effective.

Mechanism of nitrite-stimulated catalysis by lactoperoxidase.

The reactions of lactoperoxidase (LPO) intermediates compound I, compound II and compound III, with nitrite (NO2(-)) were investigated. Reduction of compound I by NO2(-) was rapid (k2 = 2.3 x 10(7) M(-1) x s(-1); pH = 7.2) and compound II was not an intermediate, indicating that NO2* radicals are not produced when NO2(-) reacts with compound I. The second-order rate constant for the reaction of compound II with NO2(-) at pH = 7.2 was 3.5 x 10(5) M(-1) x s(-1). The reaction of compound III with NO2(-) exhibited saturation behaviour when the observed pseudo first-order rate constants were plotted against NO2(-) concentrations and could be quantitatively explained by the formation of a 1 : 1 ratio compound III/NO2(-) complex. The Km of compound III for NO2(-) was 1.7 x 10(-4) M and the first-order decay constant of the compound III/ NO2(-) complex was 12.5 +/- 0.6 s(-1). The second-order rate constant for the reaction of the complex with NO2(-) was 3.3 x 10(3) M(-1) x s(-1). Rate enhancement by NO2(-) does not require NO2* as a redox intermediate. NO2(-) accelerates the overall rate of catalysis by reducing compound II to the ferric state. With increasing levels of H2O2, there is an increased tendency for the catalytically dead-end intermediate compound III to form. Under these conditions, the 'rescue' reaction of NO2(-) with compound III to form compound II will maintain the peroxidatic cycle of the enzyme.

Commentary: possible mechanisms for strand-breaks in DNA induced by stirring.

When aqueous solutions of DNA are stirred at room temperature, strand breaks occur extensively. Using various spin-traps, coupled with epr spectroscopy, we have shown that this does not proceed via homolysis. It is suggested that breaks occur by hydrolysis at strongly bent regions, momentarily induced by the stirring.

The thrombin receptor, PAR-1, causes transformation by activation of Rho-mediated signaling pathways.

We utilized a cDNA expression library derived from the B6SutA(1) mouse myeloid progenitor cell line to search for novel oncogenes that promote growth transformation of NIH3T3 cells. A 2.2 kb transforming cDNA was recovered that encodes the wild type thrombin-stimulated G protein-coupled receptor PAR-1. In addition to its potent focus forming activity, constitutive overexpression of PAR-1 in NIH3T3 cells promoted the loss of anchorage- and serum-dependent growth. Although inhibitors of thrombin failed to block PAR-1 transforming activity, a PAR-1 mutant that cannot be cleaved by thrombin was nontransforming. Since the foci of transformed cells induced by PAR-1 bear a striking resemblance to those induced by activated RhoA, we determined if PAR-1 transformation was due to the aberrant activation of a specific Rho family member. Like RhoA, PAR-1 cooperated with activated Raf-1 and caused synergistic enhancement of transforming activity, induced stress fibers when microinjected into porcine aortic endothelial cells, stimulated the activity of the serum response factor and NF-kappaB transcription factors, and PAR-1 transformation was blocked by co-expression of dominant negative RhoA. Finally, PAR-1 transforming activity was blocked by pertussis toxin and by co-expression of the RGS domain of Lsc, implicating Galpha(i) and Galpha(12)/Galpha(13) subunits, respectively, as mediators of PAR-1 transformation. Taken together, these observations suggest that PAR-1 growth transformation is mediated, in part, by activation of RhoA.

Rac1 protects epithelial cells against anoikis.

Rho family members play a critical role in malignant transformation. Anchorage-independent growth and the ability to avoid apoptosis caused by loss of anchorage (anoikis) are important features of transformed cells. Here we show that constitutive activation of Rac1 inhibits anoikis in Madin-Darby canine kidney (MDCK) epithelial cells. Constitutively active Rac1-V12 decreases DNA fragmentation and caspase activity by 50% in MDCK cells kept in suspension. In addition, expression of Rac1-V12 in MDCK cells in suspension conditions causes an increase in the number of surviving cells. We also investigated the signaling pathways that are activated by Rac1 to stimulate cell survival. We show that expression of Rac1-V12 in MDCK cells in suspension stimulates a number of signaling cascades that have been implicated in the control of cell survival, including the p42/44 ERK, p38, protein kinase B, and nuclear factor kappaB pathways. Using specific chemical or protein inhibitors of these respective pathways, we show that Rac1-mediated cell survival strongly depends on phosphatidylinositol 3-kinase activity and that activation of ERK, p38, and NF-kappaB are largely dispensable for Rac1 survival signaling. In conclusion, these studies demonstrate that Rac1 can suppress apoptosis in epithelial cells in anchorage-independent conditions and suggest a potential role for Rac1-mediated survival signaling in cell transformation.

Characterization of the radical product formed from the reaction of nitric oxide with the spin trap 3,5-dibromo-4-nitrosobenzene sulfonate.

Previously, 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS) has been used in combination with electron paramagnetic resonance (EPR) spectrometry to trap nitric oxide (NO(*)). The reaction between DBNBS and NO(*) yields a radical product which gives rise to an EPR signal consisting of three lines with an A(N) = 0.96 mT, but the structure of this product is unknown. A two-stage high-performance liquid chromatography fractionation was performed to isolate the radical product from the other components in the DBNBS/NO(*) reaction mixture. The fractions containing the radical product were identified by the presence of the three-line EPR signal, and then these fractions were analyzed by negative ion fast atom bombardment-mass spectrometry (FAB-MS). Collectively, the FAB-MS data suggested that the radical product is the monosodium electrostatic complex with the dianion, bis(2,6-dibromo-4-sulfophenyl) nitroxyl. Analysis of the Gaussian and Lorentzian linewidths of the EPR signal suggested that bis(2,6-dibromo-4-sulfophenyl) nitroxyl molecules may group together to form micelles. Further studies also indicated that significant amounts of nitrogen and nitrate were produced during the reaction between DBNBS and NO(*). A reaction scheme consistent with these results is presented.

The EPR spectrum for CuB in cytochrome c oxidase.

Incubation of cytochrome c oxidase (CcO) in its resting state in saturated ammonium sulfate, at room temperature overnight, gave EPR signals characteristic of a single Cu(II) center. From the g// and A// values it is concluded that this is a square-planar type 2 copper center, and superhyperfine splitting shows the presence of three nearly equivalent 14N nuclei in the plane. It is suggested that this center, also formed by incubating the enzyme in 10% methanol followed by direct irradiation, must be the CuB center. This type 2 copper EPR spectrum is identical to the EPR spectrum of CuB reported for the isolated cytochrome bo3 complex from Escherichia coli; and to the EPR spectrum reported for the sulfobetaine 12 heat-treated cytochrome c oxidase complex. It is argued that a small perturbation in the system causes decoupling of the magnetic coupling of the heme a3-CuB binuclear center and the appearance of the type 2 EPR signal.

Rho family GTPases regulate mammary epithelium cell growth and metastasis through distinguishable pathways.

BACKGROUND: Relatively few genes have been shown to directly affect the metastatic phenotype of breast cancer epithelial cells in vivo. The Rho family of proteins, incluing the Rho, Rac and Cdc42 subfamilies, are related to the small GTP binding protein Ras and regulated diverse biological processes including gene transcription, cytoskeletal organization, cell proliferation and transformation. The effects of Cdc42, Rac and Rho on the actin cytoskeleton suggested a possible role for Rho proteins in cellular motility and metastasis; however, a formal analysis of the role of Rho proteins in breast cancer cellular growth and metastasis in vivo had not previously been performed. MATERIALS AND METHODS: We generated a panel of MTLn3 rat mammary adenocarcinoma cells that expressed similar levels of dominant inhibitory mutants of Cdc42-, Rac- and Rho-dependent signaling, to examine the contribution of these GTPases to cell spreading, guided chemotaxis, and metastasis in vivo. The ability of Rho proteins to regulate intravasation into the peripheral blood was determined by implanting MTLn3 cell stable dominant negative lines in nude mice and measuring the formation of breast cancer cell colonies grown from the peripheral blood. Serial sectioning of the lungs was performed to determine the presence of metastasis in mice in which mammary tumors expressing the dominant negative Rho family proteins had grown to a similar size. RESULTS: Cell spreading of MTLn3 cells was selectively abrogated by N17Rac1. N19RhoA and N17Cdc42 reduced the number of focal contacts (FCs) and disrupted the co-localization of vinculin with phosphotyrosine at FCs. While N17Rac1 and N17Cdc42 preferentially inhibited colony formation in soft agar, all three GTPases affected cell growth in vivo. To distinguish effects on tumorigenicity from intravasation into the bloodstream, implanted tumors were grown to the same size in nude mice. Each dominant inhibitory Rho protein reduced intravasation into the peripheral blood. Lung metastasis of MTLn3 cells was also abrogated by the dominant inhibitory Rho proteins, despite the presence of residual CFU. CONCLUSIONS: These studies demonstrate for the first time a critical role for the Rho GTPases involving independent signaling pathways to limit mammary tumor cellular growth and metastasis in vivo.

Synaptojanin 2, a novel Rac1 effector that regulates clathrin-mediated endocytosis.

The small GTPase Rac has been implicated in a wide range of cellular processes, including the organization of the actin cytoskeleton, transcriptional control and endocytic vesicle trafficking [1-3]. The signaling components that mediate these functions downstream of Rac largely remain to be identified. In this study, we have identified synaptojanin 2, a polyphosphoinositide phosphatase as a novel Rac1 effector. Synaptojanin 2 directly and specifically interacts with Rac1 in a GTP-dependent manner. Expression of constitutively active Rac1 caused the translocation of synaptojanin 2 from the cytoplasm to the plasma membrane. Both activated Rac1 and a membrane-targeted version of synaptojanin 2 inhibited endocytosis of the epidermal growth factor (EGF) and transferrin receptors, a process that is known to be dependent on polyphosphoinositide lipids. Endocytosis of growth factor receptors is thought to play an important role in the regulation of cell proliferation. Thus, these results suggest that synaptojanin 2 may mediate the inhibitory effect of Rac1 on endocytosis and could contribute to Rac1-mediated control of cell growth.

A method for reducing the pain and danger of certain arthritic joints.

It has been found that by hanging upside-down from the feet, the pain arising from an arthritic hip joint is largely eliminated. If this method is not used, the pain returns rapidly. It is suggested that the direct cause of the pain and damage is the presence of small, hard crystals of apatite or related compounds present in the synovial fluid of the joint. Under the normal pressures involved in standing, walking or running, these damage the joint involved by a process of grinding. If this joint is opened by hanging and swivelling, the crystals are induced to move out of the joint region, thereby alleviating the problem.