Evidence for the role of calcineurin in morphogenesis and calcium homeostasis during mycelium-to-yeast dimorphism of Paracoccidioides brasiliensis

Paracoccidioides brasiliensis is a dimorphic fungus that causes paracoccidioidomycosis, the most prevalent human deep mycosis in Latin America. The dimorphic transition from mycelium to yeast (M-Y) is triggered by a temperature shift from 25°C to 37°C and is critical for pathogenicity. Intracellular Ca 2+ levels increased in hyphae immediately after temperature-induced dimorphism. The chelation of Ca 2+ with extracellular (EGTA) or intracellular (BAPTA) calcium chelators inhibited temperature-induced dimorphism, whereas the addition of extracellular Ca 2+ accelerated dimorphism. The calcineurin inhibitor cyclosporine A (CsA), but not tacrolimus (FK506), effectively decreased cell growth, halted the M-Y transition that is associated with virulence, and caused aberrant growth morphologies for all forms of P. brasiliensis. The difference between CsA and FK506 was ascribed by the higher levels of cyclophilins contrasted to FKBPs, the intracellular drug targets required for calcineurin suppression. Chronic exposure to CsA abolished intracellular Ca 2+ homeostasis and decreased mRNA transcription of the CCH1 gene for the plasma membrane Ca 2+ channel in yeast-form cells. CsA had no detectable effect on multidrug resistance efflux pumps, while the effect of FK506 on rhodamine excretion was not correlated with the transition to yeast form. In this study, we present evidence that Ca 2+/calmodulin-dependent phosphatase calcineurin controls hyphal and yeast morphology, M-Y dimorphism, growth, and Ca 2+ homeostasis in P. brasiliensis and that CsA is an effective chemical block for thermodimorphism in this organism. The effects of calcineurin inhibitors on P. brasiliensis reinforce the therapeutic potential of these drugs in a combinatory approach with antifungal drugs to treat endemic paracoccidioidomycosis. Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Plasma membrane calcium-ATPase 2 and 4 in human breast cancer cell lines

There is evidence to suggest that plasma membrane Ca2+-ATPase (PMCA) isoforms are important mediators of mammary gland physiology. PMCA2 in particular is upregulated extensively during lactation. Expression of other isoforms such as PMCA4 may influence mammary gland epithelial cell proliferation and aberrant regulation of PMCA isoform expression may lead or contribute to mammary gland pathophysiology in the form of breast cancers. To explore whether PMCA2 and PMCA4 expression may be deregulated in breast cancer, we compared mRNA expression of these PMCA isoforms in tumorigenic and non-tumorigenic human breast epithelial cell lines using real time RT-PCR. PMCA2 mRNA has a higher level of expression in some breast cancer cell lines and is overexpressed more than 100-fold in ZR-75-1 cells, compared to non-tumorigenic 184135 cells. Although differences in PMCA4 mRNA levels were observed between breast cell lines, they were not of the magnitude observed for PMCA2. We conclude that PMCA2 mRNA can be highly overexpressed in some breast cancer cells. The significance of PMCA2 overexpression on tumorigenicity and its possible correlation with other properties such as invasiveness requires further study. (c) 2005 Elsevier Inc. All rights reserved.

Interaction of calcineurin with a domain of the transcription factor NFAT1 that controls nuclear import.

The nuclear import of the nuclear factor of activated T cells (NFAT)-family transcription factors is initiated by the protein phosphatase calcineurin. Here we identify a regulatory region of NFAT1, N terminal to the DNA-binding domain, that controls nuclear import of NFAT1. The regulatory region of NFAT1 binds directly to calcineurin, is a substrate for calcineurin in vitro, and shows regulated subcellular localization identical to that of full-length NFAT1. The corresponding region of NFATc likewise binds calcineurin, suggesting that the efficient activation of NFAT1 and NFATc by calcineurin reflects a specific targeting of the phosphatase to these proteins. The presence in other NFAT-family transcription factors of several sequence motifs from the regulatory region of NFAT1, including its probable nuclear localization sequence, indicates that a conserved protein domain may control nuclear import of all NFAT proteins.

Head and eye movements and lane keeping in drivers with hemianopia and quadrantanopia compared to controls

Purpose: To compare the eye and head movements and lane-keeping of drivers with hemianopia and quadrantanopia with that of age-matched controls when driving under real world conditions. Methods: Participants included 22 hemianopes and 8 quadrantanopes (M age 53 yrs) and 30 persons with normal visual fields (M age 52 yrs) who were ≥ 6 months from the brain injury date and either a current driver or aiming to resume driving. All participants drove an instrumented dual-brake vehicle along a 14-mile route in traffic that included non-interstate city driving and interstate driving. Driving performance was scored using a standardised assessment system by two “backseat” raters and the Vigil Vanguard system which provides objective measures of speed, braking and acceleration, cornering, and video-based footage from which eye and head movements and lane-keeping can be derived. Results: As compared to drivers with normal visual fields, drivers with hemianopia or quadrantanopia on average were significantly more likely to drive slower, to exhibit less excessive cornering forces or acceleration, and to execute more shoulder movements off the seat. Those hemianopic and quadrantanopic drivers rated as safe to drive by the backseat evaluator made significantly more excursive eye movements, exhibited more stable lane positioning, less sudden braking events and drove at higher speeds than those rated as unsafe, while there was no difference between safe and unsafe drivers in head movements. Conclusions: Persons with hemianopic and quadrantanopic field defects rated as safe to drive have different driving characteristics compared to those rated as unsafe when assessed using objective measures of driving performance.

Artful determination : "state of the art" and PCME Ltd v. Goyen COntrols Co. UK Ltd

In a recent decision by Mr Justice Laddie, a patent was held anticipated by, inter alia, prior use of a device which fell within the claims of the patent in suit, even though its circuitry was enclosed in resin. The anticipating invention had been "made available to the public" within the terms of section 2 (2) of the Patents Act 1977 because its essential integers would have been revealed by an interesting character, the "skilled forensic engineer".

Controls on soil respiration in semiarid soils

Soil respiration in semiarid ecosystems responds positively to temperature, but temperature is just one of many factors controlling soil respiration. Soil moisture can have an overriding influence, particularly during the dry/warm portions of the year. The purpose of this project was to evaluate the influence of soil moisture on the relationship between temperature and soil respiration. Soil samples collected from a range of sites arrayed across a climatic gradient were incubated under varying temperature and moisture conditions. Additionally, we evaluated the impact of substrate quality on short-term soil respiration responses by carrying out substrate-induced respiration assessments for each soil at nine different temperatures. Within all soil moisture regimes, respiration rates always increased with increase in temperature. For a given temperature, soil respiration increased by half (on average) across moisture regimes; Q(10) values declined with soil moisture from 3.2 (at -0.03 MPa) to 2.1 (-1.5 MPa). In summary, soil respiration was generally directly related to temperature, but responses were ameliorated with decrease in soil moisture. (C) 2004 Elsevier Ltd. All rights reserved.

INTS3 controls the hSSB1-mediated DNA damage response

Human SSB1 (single-stranded binding protein 1 [hSSB1]) was recently identified as a part of the ataxia telangiectasia mutated (ATM) signaling pathway. To investigate hSSB1 function, we performed tandem affinity purifications of hSSB1 mutants mimicking the unphosphorylated and ATM-phosphorylated states. Both hSSB1 mutants copurified a subset of Integrator complex subunits and the uncharacterized protein LOC58493/c9orf80 (henceforth minute INTS3/hSSB-associated element [MISE]). The INTS3–MISE–hSSB1 complex plays a key role in ATM activation and RAD51 recruitment to DNA damage foci during the response to genotoxic stresses. These effects on the DNA damage response are caused by the control of hSSB1 transcription via INTS3, demonstrating a new network controlling hSSB1 function.

Homodimerization controls the fibroblast growth factor 9 subfamily's receptor binding and heparan sulfate-dependent diffusion in the extracellular matrix

Uncontrolled fibroblast growth factor (FGF) signaling can lead to human diseases, necessitating multiple layers of self-regulatory control mechanisms to keep its activity in check. Herein, we demonstrate that FGF9 and FGF20 ligands undergo a reversible homodimerization, occluding their key receptor binding sites. To test the role of dimerization in ligand autoinhibition, we introduced structure-based mutations into the dimer interfaces of FGF9 and FGF20. The mutations weakened the ability of the ligands to dimerize, effectively increasing the concentrations of monomeric ligands capable of binding and activating their cognate FGF receptor in vitro and in living cells. Interestingly, the monomeric ligands exhibit reduced heparin binding, resulting in their increased radii of heparan sulfate-dependent diffusion and biologic action, as evidenced by the wider dilation area of ex vivo lung cultures in response to implanted mutant FGF9-loaded beads. Hence, our data demonstrate that homodimerization autoregulates FGF9 and FGF20's receptor binding and concentration gradients in the extracellular matrix. Our study is the first to implicate ligand dimerization as an autoregulatory mechanism for growth factor bioactivity and sets the stage for engineering modified FGF9 subfamily ligands, with desired activity for use in both basic and translational research.

Controls on iron in soils and soil waters of a forested, coastal catchment in subtropical Australia

Soluble organic matter derived from exotic Pinus vegetation forms stronger complexes with iron (Fe) than the soluble organic matter derived from most native Australian species. This has lead to concern about the environmental impacts related to the establishment of extensive exotic Pinus plantations in coastal southeast Queensland, Australia. It has been suggested that the Pinus plantations may enhance the solubility of Fe in soils by increasing the amount of organically complexed Fe. While this remains inconclusive, the environmental impacts of an increased flux of dissolved, organically complexed Fe from soils to the fluvial system and then to sensitive coastal ecosystems are potentially damaging. Previous work investigated a small number of samples, was largely laboratory based and had limited application to field conditions. These assessments lacked field-based studies, including the comparison of the soil water chemistry of sites associated with Pinus vegetation and undisturbed native vegetation. In addition, the main controls on the distribution and mobilisation of Fe in soils of this subtropical coastal region have not been determined. This information is required in order to better understand the relative significance of any Pinus enhanced solubility of Fe. The main aim of this thesis is to determine the controls on Fe distribution and mobilisation in soils and soil waters of a representative coastal catchment in southeast Queensland (Poona Creek catchment, Fraser Coast) and to test the effect of Pinus vegetation on the solubility and speciation of Fe. The thesis is structured around three individual papers. The first paper identifies the main processes responsible for the distribution and mobilisation of labile Fe in the study area and takes a catchment scale approach. Physicochemical attributes of 120 soil samples distributed throughout the catchment are analysed, and a new multivariate data analysis approach (Kohonen’s self organising maps) is used to identify the conditions associated with high labile Fe. The second paper establishes whether Fe nodules play a major role as an iron source in the catchment, by determining the genetic mechanism responsible for their formation. The nodules are a major pool of Fe in much of the region and previous studies have implied that they may be involved in redox-controlled mobilisation and redistribution of Fe. This is achieved by combining a detailed study of a ferric soil profile (morphology, mineralogy and micromorphology) with the distribution of Fe nodules on a catchment scale. The third component of the thesis tests whether the concentration and speciation of Fe in soil solutions from Pinus plantations differs significantly from native vegetation soil solutions. Microlysimeters are employed to collect unaltered, in situ soil water samples. The redox speciation of Fe is determined spectrophotometrically and the interaction between Fe and dissolved organic matter (DOM) is modelled with the Stockholm Humic Model. The thesis provides a better understanding of the controls on the distribution, concentration and speciation of Fe in the soils and soil waters of southeast Queensland. Reductive dissolution is the main mechanism by which mobilisation of Fe occurs in the study area. Labile Fe concentrations are low overall, particularly in the sandy soils of the coastal plain. However, high labile Fe is common in seasonally waterlogged and clay-rich soils which are exposed to fluctuating redox conditions and in organic-rich soils adjacent to streams. Clay-rich soils are most common in the upper parts of the catchment. Fe nodules were shown to have a negligible role in the redistribution of dissolved iron in the catchment. They are formed by the erosion, colluvial transport and chemical weathering of iron-rich sandstones. The ferric horizons, in which nodules are commonly concentrated, subsequently form through differential biological mixing of the soil. Whereas dissolution/ reprecipitation of the Fe cements is an important component of nodule formation, mobilised Fe reprecipitates locally. Dissolved Fe in the soil waters is almost entirely in the ferrous form. Vegetation type does not affect the concentration and speciation of Fe in soil waters, although Pinus DOM has greater acidic functional group site densities than DOM from native vegetation. Iron concentrations are highest in the high DOM soil waters collected from sandy podosols, where they are controlled by redox potential. Iron concentrations are low in soil solutions from clay and iron oxide rich soils, in spite of similar redox potentials. This is related to stronger sorption to the reactive clay and iron oxide mineral surfaces in these soils, which reduces the amount of DOM available for microbial metabolisation and reductive dissolution of Fe. Modelling suggests that Pinus DOM can significantly increase the amount of truly dissolved ferric iron remaining in solution in oxidising conditions. Thus, inputs of ferrous iron together with Pinus DOM to surface waters may reduce precipitation of hydrous ferric oxides and increase the flux of dissolved iron out of the catchment. Such inputs are most likely from the lower catchment, where podosols planted with Pinus are most widely distributed. Significant outcomes other than the main aims were also achieved. It is shown that mobilisation of Fe in podosols can occur as dissolved Fe(II) rather than as Fe(III)-organic complexes. This has implications for the large body of work which assumes that Fe(II) plays a minor role. Also, the first paper demonstrates that a data analysis approach based on Kohonen’s self organising maps can facilitate the interpretation of complex datasets and can help identify geochemical processes operating on a catchment scale.

Serious firearm offending in New Zealand : issues for gun controls and public safety

Reducing or preventing the misuse of firearms (whether criminal, suicidal or accidental) is central to gun controls policy. In June 1997 the New Zealand Government received the recommendations of the Review of Firearms Control, an independent inquiry commissioned by the Minister of Police and chaired by Sir Thomas Thorp.The Review comprehensively examined several firearm-related issues, including the nature and extent of serious firearm offending in New Zealand. The research presented in this article was commissioned by the Review. Based on a population of 709 offences for the 12 months ending 30 June 1996, this study provides an analysis of serious firearm offending in New Zealand and discusses the implications of these findings for gun controls policy.