Danuta Chamot

Regulation of Cold Shock-Induced RNA Helicase Gene Expression in the Cyanobacterium Anabaena sp. Strain PCC 7120

Expression of the Anabaena sp. strain PCC 7120 RNA helicase gene crhC is induced by cold shock. crhC transcripts are not detectable at 30 degrees C but accumulate at 20 degrees C, and levels remain elevated for the duration of the cold stress. Light-derived metabolic capability, and not light per se, is required for crhC transcript accumulation. Enhanced crhC mRNA stability contributes significantly to the accumulation of crhC transcripts, with the crhC half-life increasing sixfold at 20 degrees C. The accumulation is reversible, with the cells responding more rapidly to temperature downshifts than to upshifts, as a result of the lack of active mRNA destabilization and the continuation of crhC transcription, at least transiently, after a temperature upshift. Translational inhibitors do not induce crhC expression to cold shock levels, indicating that inhibition of translation is only one of the signals required to activate the cold shock response in Anabaena. Limited amounts of protein synthesis are required for the cold shock-induced accumulation of crhC transcripts, as normal levels of accumulation occur in the presence of tetracycline but are abolished by chloramphenicol. Regulation of crhC expression may also extend to the translational level, as CrhC protein levels do not correlate completely with the pattern of mRNA transcript accumulation. Our experiments indicate that the regulation of crhC transcript accumulation is tightly controlled by both temperature and metabolic activity at the levels of transcription, mRNA stabilization, and translation.

Aquatic toxicity of manufactured nanomaterials: challenges and recommendations for future toxicity testing

Environmental context The increased use of nanomaterials in industrial and consumer products requires robust strategies to identify risks when they are released into the environment. Aquatic toxicologists are beginning to possess a clearer understanding of the chemical and physical properties of nanomaterials in solution, and which of the properties potentially affect the health of aquatic organisms. This review highlights the main challenges encountered in aquatic nanotoxicity testing, provides recommendations for overcoming these challenges, and discusses recent studies that have advanced our understanding of the toxicity of three important OECD nanomaterials, titanium dioxide, zinc oxide and silver nanomaterials. Abstract Aquatic nanotoxicologists and ecotoxicologists have begun to identify the unique properties of the nanomaterials (NMs) that potentially affect the health of wildlife. In this review the scientific aims are to discuss the main challenges nanotoxicologists currently face in aquatic toxicity testing, including the transformations of NMs in aquatic test media (dissolution, aggregation and small molecule interactions), and modes of NM interference (optical interference, adsorption to assay components and generation of reactive oxygen species) on common toxicity assays. Three of the major OECD (Organisation for Economic Co-operation and Development) priority materials, titanium dioxide (TiO2), zinc oxide (ZnO) and silver (Ag) NMs, studied recently by the Natural Sciences and Engineering Research Council of Canada (NSERC), National Research Council of Canada (NRC) and the Business Development Bank of Canada (BDC) Nanotechnology Initiative (NNBNI), a Canadian consortium, have been identified to cause both bulk effect, dissolution-based (i.e. free metal), or NM-specific toxicity in aquatic organisms. TiO2 NMs are most toxic to algae, with toxicity being NM size-dependent and principally associated with binding of the materials to the organism. Conversely, dissolution of Zn and Ag NMs and the subsequent release of their ionic metal counterparts appear to represent the primary mode of toxicity to aquatic organisms for these NMs. In recent years, our understanding of the toxicological properties of these specific OECD relevant materials has increased significantly. Specifically, researchers have begun to alter their experimental design to identify the different behaviour of these materials as colloids and, by introducing appropriate controls and NM characterisation, aquatic nanotoxicologists are now beginning to possess a clearer understanding of the chemical and physical properties of these materials in solution, and how these materials may interact with organisms. Arming nanotoxicologists with this understanding, combined with knowledge of the physics, chemistry and biology of these materials is essential for maintaining the accuracy of all future toxicological assessments.

Autoregulation of RNA Helicase Expression in Response to Temperature Stress in Synechocystis sp. PCC 6803

RNA helicases are ubiquitous enzymes whose modification of RNA secondary structure is known to regulate RNA function. The pathways controlling RNA helicase expression, however, have not been well characterized. Expression of the cyanobacterial RNA helicase, crhR, is regulated in response to environmental signals that alter the redox poise of the electron transport chain, including light and temperature. Here we analyze crhR expression in response to alteration of abiotic conditions in wild type and a crhR mutant, providing evidence that CrhR autoregulates its own expression through a combination of transcriptional and post-transcriptional mechanisms. Temperature regulates crhR expression through alteration of both transcript and protein half-life which are significantly extended at low temperature (20°C). CrhR-dependent mechanisms regulate both the transient accumulation of crhR transcript at 20°C and stability of the CrhR protein at all temperatures. CrhR-independent mechanisms regulate temperature sensing and induction of crhR transcript accumulation at 20°C and the temperature regulation of crhR transcript stability, suggesting CrhR is not directly associated with crhR mRNA turnover. Many of the processes are CrhR- and temperature-dependent and occur in the absence of a correlation between crhR transcript and protein abundance. The data provide important insights into not only how RNA helicase gene expression is regulated but also the role that rearrangement of RNA secondary structure performs in the molecular response to temperature stress. We propose that the crhR-regulatory pathway exhibits characteristics similar to the heat shock response rather than a cold stress-specific mechanism.

Inactivation of a Low Temperature-Induced RNA Helicase in Synechocystis sp. PCC 6803: Physiological and Morphological Consequences

Inactivation of the DEAD box RNA helicase, crhR, has dramatic effects on the physiology and morphology of the photosynthetic cyanobacterium, Synechocystis sp. PCC 6803. These effects are observed at both normal growth temperature (30°C) and under cold stress (20°C), indicating that CrhR performs crucial function(s) at all temperatures. A major physiological effect is the rapid cessation of photosynthesis upon temperature downshift from 30 to 20°C. This defect does not originate from an inability to transport or accumulate inorganic carbon or a deficiency in photosynthetic capacity as the mutant has sufficient electron transport and enzymatic capacity to sustain photosynthesis at 30°C and inorganic carbon (Ci) accumulation at 20°C. Oxygen consumption in the presence of methyl viologen indicated that while electron transport capacity is sufficient to accumulate Ci, the mutant does not possess sufficient activity to sustain carbon fixation at maximal rates. These defects are correlated with severely impaired cell growth and decreased viability, cell size and DNA content at low temperature. The ΔcrhR mutant also progressively accumulates structural abnormalities at low temperature that cannot be attributed solely to reactive oxygen species (ROS)-induced photooxidative damage, suggesting that they are manifestations of pre-existing defects that are amplified over time. The data indicate that the observed physiological and morphological effects are intimately related to crhR mutation, implying that the lack of CrhR RNA unwinding/annealing activity results in the inability to execute one or more vital steps in photosynthesis that are required at all temperatures but are crucial at low temperature.

Mechanisms of Cl− uptake in rainbow trout: Cloning and expression of slc26a6, a prospective Cl−/HCO3− exchanger

In fresh waters, fishes continuously acquire ions to offset diffusive losses to a more dilute ambient environment and to maintain acid-base status. The objectives of the present study were to clone slc26a6, a prospective Cl(-)/HCO3(-) exchanger from rainbow trout, investigate its expression patterns in various tissues, at different developmental stages and after differential salinity exposure, and probe the mechanisms of Cl(-) uptake in rainbow trout embryos during development using a pharmacological inhibitor approach combined with (36)Cl(-) unidirectional fluxes. Results showed that the cloned gene encoded a 783 amino acid protein with conserved domains characteristic of the SLC26a family of anion exchange proteins. Phylogenetic analysis of this sequence against all subfamilies of the SLC26a family demonstrated that this translated protein shared a common ancestor with other actinopterygii and mammalian SLC26a6 isoforms and thus confirmed the identity of the cloned gene. Expression of slc26a6 was detected in all tissues and developmental stages assayed but was highest in the gill of juvenile trout. In trout embryos, Cl(-) uptake increased significantly post-hatch and was demonstrated to be mediated via an anion exchanger specific (DIDS sensitive) pathway that was also sensitive to hypercapnia. This parallels well with the predicted function of slc26a6, and the detection of the transcript in embryos and tissues of trout. In conclusion, this study is the first report of slc26a6 in rainbow trout and functional and expression analyses indicate its likely involvement in Cl(-)/HCO3(-) exchange in two life stages of rainbow trout.

Characterization of developmental Na(+) uptake in rainbow trout larvae supports a significant role for Nhe3b.

Developing freshwater fish must compensate for the loss of ions, including sodium (Na(+)), to the environment. In this study, we used a radiotracer flux approach and pharmacological inhibitors to investigate the role of sodium/hydrogen exchange proteins (Nhe) in Na(+) uptake in rainbow trout (Oncorhynchus mykiss) reared from fertilization in soft water (0.1mM Na(+)). For comparison, a second group of embryos/larvae reared in hard water (2.2mM Na(+), higher pH and [Ca(2+)]) were also included in the experiment but were fluxed in soft water, only. Unidirectional rates of Na(+) uptake increased throughout development and were significantly higher in embryos/larvae reared in soft water. However, the mechanisms of Na(+) uptake in both groups of larvae were not significantly different, either in larvae immediately post-hatch or later in development: the broad spectrum Na(+) channel blocker amiloride inhibited 85-90% of uptake and the Nhe-inhibitor EIPA also caused near maximal inhibitions of Na(+) uptake. These data indicated Na(+) uptake was Nhe-mediated in soft water. A role of Nhe3b (but not Nhe2 or Nhe3a) in Na(+) uptake in soft water was also supported through gene expression analyses: expression of nhe3b increased throughout development in whole embryos/larvae in both groups and was significantly higher in those reared in soft water. This pattern of expression correlated well with measurements of Na(+) uptake. Together these data indicate that in part, rainbow trout embryos/larvae reared in low Na(+) soft water maintained Na(+) homeostasis by an EIPA sensitive component of Na(+) uptake, and support a primary role for Nhe3b.

Conditional, temperature-induced proteolytic regulation of cyanobacterial RNA helicase expression

Conditional proteolysis is a crucial process regulating the abundance of key regulatory proteins associated with the cell cycle, differentiation pathways, or cellular response to abiotic stress in eukaryotic and prokaryotic organisms. We provide evidence that conditional proteolysis is involved in the rapid and dramatic reduction in abundance of the cyanobacterial RNA helicase, CrhR, in response to a temperature upshift from 20 to 30°C. The proteolytic activity is not a general protein degradation response, since proteolysis is only present and/or functional in cells grown at 30°C and is only transiently active at 30°C. Degradation is also autoregulatory, since the CrhR proteolytic target is required for activation of the degradation machinery. This suggests that an autoregulatory feedback loop exists in which the target of the proteolytic machinery, CrhR, is required for activation of the system. Inhibition of translation revealed that only elongation is required for induction of the temperature-regulated proteolysis, suggesting that translation of an activating factor was already initiated at 20°C. The results indicate that Synechocystis responds to a temperature shift via two independent pathways: a CrhR-independent sensing and signal transduction pathway that regulates induction of crhR expression at low temperature and a CrhR-dependent conditional proteolytic pathway at elevated temperature. The data link the potential for CrhR RNA helicase alteration of RNA secondary structure with the autoregulatory induction of conditional proteolysis in the response of Synechocystis to temperature upshift.

The role of the Human Metabolome Database in inborn errors of metabolism

Metabolomics holds considerable promise to advance our understanding of human disease, including our understanding of inborn errors of metabolism (IEM). The application of metabolomics in IEM research has already led to the discovery of several novel IEMs and the identification of novel IEM biomarkers. However, with hundreds of known IEMs and more than 700 associated IEM metabolites, it is becoming increasingly challenging for clinical researchers to keep track of IEMs, their associated metabolites, and their corresponding metabolic mechanisms. Furthermore, when using metabolomics to assist in IEM biomarker discovery or even in IEM diagnosis, it is becoming much more difficult to properly identify metabolites from the complex NMR and MS spectra collected from IEM patients. To that end, comprehensive, open access metabolite databases that provide up-to-date referential information about metabolites, metabolic pathways, normal/abnormal metabolite concentrations, and reference NMR or MS spectra for compound identification are essential. Over the last few years, a number of compound databases, including the Human Metabolome Database (HMDB), have been developed to address these challenges. First described in 2007, the HMDB is now the world’s largest and most comprehensive metabolomic resource for human metabolic studies. The latest release of the HMDB contains 114,100 metabolite entries (with 247 being relevant to IEMs), thousands of metabolite concentrations (with 600 being relevant to IEMs), and ~33,000 metabolic and disease-associated pathways (with 202 being relevant to IEMs). Here we provide a summary of the HMDB and offer some guidance on how it can be used in metabolomic studies of IEMs.