The generally accepted role of iron-regulatory protein 1 (IRP1) in orchestrating the fate of iron-regulated mRNAs depends on the interconversion of its cytosolic aconitase and RNA-binding forms through assembly/disassembly of its Fe-S cluster, without altering protein abundance. Here, we show that IRP1 protein abundance can be ironregulated. Modulation of IRP1 abundance by iron did not require assembly of the Fe-S cluster, since a mutant with all cluster-ligating cysteines mutated to serine underwent iron-induced protein degradation. Phosphorylation of IRP1 at S138 favored the RNA-binding form and promoted iron-dependent degradation. However, phosphorylation at S138 was not required for degradation. Further, degradation of an S138 phosphomimetic mutant was not blocked by mutation of cluster-ligating cysteines. These findings were confirmed in mouse models with genetic defects in cytosolic Fe-S cluster assembly/disassembly. IRP1 RNAbinding activity was primarily regulated by IRP1 degradation in these animals. Our results reveal a mechanism for regulating IRP1 action relevant to the control of iron homeostasis during cell proliferation, inflammation, and in response to diseases altering cytosolic Fe-S cluster assembly or disassembly.

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The exchange processes between the Maryland Coastal Bays system (MCBs) and their adjacent coastal ocean were simulated using a three-dimensional unstructured-grid based hydrodynamic model, which was validated by observed data including water level, current velocity and salinity. Idealized experiments were then carried out to investigate the impact of wind forcing on water exchange and salt flux. Through these experiments, the exchanges between the MCBs and coastal ocean were investigated at two inlets (Ocean City Inlet and Chincoteague Inlet). Given that winds and tides are two key external forces known to impact estuarine dynamics, the effect of each individual force on the exchange processes was studied to evaluate the corresponding influence on the inlet dynamics. It was found that wind forcing significantly impacts the inlet dynamics: the effect of wind directions on exchange processes under strong wind speeds is substantial; for example, northwesterly winds push flux to the southern part of the bays, while southwesterly winds pile up flux towards northern Chincoteague Bay. The effect of wind forcing on the exchange dynamics becomes stronger with the augmentation of its speed. Meanwhile, tidal forcing is the major driver of exchange dynamics at weak wind speeds (e.g., 3 m/s), and its effect on exchange process gradually weakens with stronger wind speeds (e.g., 7 m/s, 15 m/s). In addition, sensitivity tests elucidated that closing either inlet results in a significant impact on the water elevation, current velocity and salinity nearby the relevant cutoff inlet areas.

Iron-regulatory protein 1 (IRP1) is a dual-function protein with mutually exclusive roles as a posttranscriptional regulator of animal-cell iron metabolism or as the cytosolic isoform of the ironsulfur enzyme aconitase (c-acon). Much effort has focused on the role of IRP1 in posttranscriptional gene regulation and in factors that influence its interconversion with c-acon, but little is known about the metabolic function and regulation of c-acon. The role of PKC-dependent phosphorylation of S711 on IRP1͞c-acon function was examined. Phosphorylation state-specific antibodies revealed that S711 is phosphorylated by PKC in vitro and in human embryonic kidney cells treated with a PKC activator. In aco1 yeast, the phosphomimetic mutants S711D and S711E exhibited severely impaired aconitase function, whereas S711A and S711T were unaffected relative to the WT protein. Aconitase activity in yeast extracts displayed a similar pattern when assayed for capacity to convert citrate to isocitrate: WT, S711A, and S711T were active, but S711D and S711E activity was undetectable. In contrast, when measured by the conversion of isocitrate to cis-aconitate, S711D and S711E displayed substantial activity, indicating that phosphorylation impairs the citrate but not isocitrate mode of aconitase function. This possibility was confirmed in vivo by demonstrating that S711D and S711E specifically antagonized the requirement for isocitrate in two metabolic scenarios. Iron-responsive element RNA-binding affinity was unaffected by S711 mutations. Our results show that S711 is a target of phosphorylation capable of conferring distinct effects on c-acon function potentially dictating changes in cytosolic citrate͞isocitrate metabolism. I t is critical that organisms balance the metabolic requirement for iron with its potential toxicity. Vertebrates possess an elegant system to sense cellular iron status, regulate the uptake and metabolic fate of iron, and thereby maintain iron homeostasis. Iron-regulatory protein 1 (IRP1) and IRP2 are sensory components of this critical regulatory network that promote increased iron uptake when cells are iron depleted and storage of iron when cells are iron overloaded (1, 2). IRPs control mRNA fate by binding to iron-responsive elements (IRE) in the untranslated regions of mRNA encoding proteins that control iron homeostasis. The RNA-binding activity of IRP can be regulated by multiple factors. Iron promotes the loss of IRP1 RNA-binding activity through insertion of a [4Fe-4S] cluster into the protein, converting it to the cytosolic isoform of the tricarboxylic acid (TCA) cycle enzyme aconitase (c-acon). Solvent accessibility of the Fe-S cluster in aconitases allows for perturbants to promote loss of the cluster from c-acon, converting it to IRP1 (1, 2). Consequently, reactive species such as NO and H 2 O 2 are able to promote removal of the Fe-S cluster, favoring generation of IRP1 from c-acon, although H 2 O 2 may do so through direct and indirect mechanisms (reviewed in refs. 1 and 2).Whereas much is known a...

Hematodinium sp. is a parasitic dinoflagellate infecting the blue crab Callinectes sapidus and other crustaceans. PCR-based assays are currently being used to identify infections in crabs that would have been undetectable by traditional microscopic examination. We therefore sought to define the limits of quantitative PCR (qPCR) detection within the context of field collection protocols. We present a qPCR assay based on the Hematodinium sp. 18S rRNA gene that can detect 10 copies of the gene per reaction. Analysis of a cell dilution series vs. defined numbers of a cloned Hematodinium sp. 18S rRNA gene suggests a copy number of 10 000 per parasite and predicts a sensitivity of 0.001 cell equivalents. In practice, the assays are based on analysis of 1% of the DNA extracted from 200 µl of serum, yielding a theoretical detection limit of 5 cells ml -1 hemolymph, assuming that 1 cell is present per sample. When applied to a limited field survey of blue crabs collected in Maryland coastal bays from May to August 2005, 24 of 128 crabs (18.8%) were identified as positive for Hematodinium sp. infection using qPCR. In comparison, only 6 of 128 crabs (4.7%) were identified as positive using traditional hemolymph microscopic examination. The qPCR method also detected the parasite in gill, muscle, heart and hepatopancreas tissues, with 17.2% of the crabs showing infection in at least one of these tissues. Importantly, it is now possible to enumerate parasites within defined quantities of crab tissue, which permits collection of more detailed information on the epizootiology of the pathogen.

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BackgroundSignificant damage to crustacean fisheries worldwide has been associated with Hematodinium sp. It has been postulated that Hematodinium sp. requires passage through the water column and/or intermediate hosts to complete its life cycle. Thus, an understanding of the prevalence and seasonality of Hematodinium sp. within environmentally-derived samples should yield insight into potential modes of disease transmission, and how these relate to infection cycles in hosts.ResultsWe conducted a two year survey, from 2010–2011, in which 48 of 546 (8.8%) of environmental samples from the Maryland and Virginia coastal bays were positive for Hematodinium sp. between April and November, as based upon endpoint PCR analysis specific to blue crab isolates. Detection in both water and sediment was roughly equivalent, and there were no obvious seasonal patterns. However, there was a high detection in April water samples, which was unanticipated owing to the fact that crabs infected with Hematodinium sp. have not been observed in this early month of the seasonal disease cycle. Focusing on three sites of high prevalence (Sinnickson, VA; Tom’s Cove, VA; and Newport Bay, MD) Hematodinium sp. population diversity was analyzed using standard cloning methods. Of 131 clones, 109 (83.2%) were identical, 19 displayed a single nucleotide substitution, and 4 contain two nucleotide substitutions.ConclusionsOur data suggests a continuous presence of Hematodinium sp. in both water and sediment of a combined Maryland and Virginia coastal bay ecosystem. The detection of Hematodinium sp. in the water column in April is an earlier manifestation of the parasite than predicted, pointing to an as yet unknown stage in its development prior to infection. That the population is relatively homogenous ranging from April to November, at three distinct sites, supports a hypothesis that one species of Hematodinium is responsible for infections within the ecosystem.

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