Natural Background and the Anthropogenic Enrichment of Mercury in the Southern Florida Environment: A Review with a Discussion on Public Health.

Mercury (Hg) is a toxic metal that is easily released into the atmosphere as a gas or a particulate. Since Hg has serious health impacts based on human exposure, it is a major concern where it accumulates. Southern Florida is a region of high Hg deposition in the United States. It has entered the southern Florida environment for over 56 MY. For the past 3000 to 8000 years, Hg has accumulated in the Everglades peatlands, where approximately 42.3 metric tons of Hg was deposited. The pre-industrial source of mercury that was deposited into the Everglades was from the atmosphere, consisting of combined Saharan dust and marine evasion. Drainage and the development of the Everglades for agriculture, and other mixed land uses have caused a 65.7% reduction in the quantity of peat, therefore releasing approximately 28 metric tons of Hg into the southern Florida environment over a period of approximately 133 years. Both natural and man-made fires have facilitated the Hg release. The current range in mercury release into the southern Florida environment lies between 994.9 and 1249 kg/yr. The largest source of Hg currently entering the Florida environment is from combined atmospheric sources, including Saharan dust, aerosols, sea spray, and ocean flux/evasion at 257.1-514.2 kg/yr. The remobilization of Hg from the Everglades peatlands and fires is approximately 215 kg/yr. Other large contributors include waste to energy incinerators (204.1 kg/yr), medical waste and crematory incinerators (159.7+ kg/yr), and cement plant stack discharge (150.6 kg/yr). Minor emissions include fuel emissions from motorized vehicles, gas emissions from landfills, asphalt plants, and possible others. No data are available on controlled fires in the Everglades in sugar farming, which is lumped with the overall peatland loss of Hg to the environment. Hg has impacted wildlife in southern Florida with recorded excess concentrations in fish, birds, and apex predators. This bioaccumulation of Hg in animals led to the adoption of regulations (total maximum loads) to reduce the impacts on wildlife and warnings were given to consumers to avoid the consumption of fish that are considered to be contaminated. The deposition of atmospheric Hg in southern Florida has not been studied sufficiently to ascertain where it has had the greatest impacts. Hg has been found to accumulate on willow tree leaves in a natural environment in one recent study. No significant studies of the potential impacts on human health have been conducted in southern Florida, which should be started based on the high rates of Hg fallout in rainfall and known recycling for organic sediments containing high concentrations of Hg.

Differences in the photoacclimation and photoprotection exhibited by two species of the ciguatera causing dinoflagellate genus, Gambierdiscus.

In culture, Gambierdiscus spp. have been shown to prefer irradiances that are relatively low (≤250µmol photonsm-2s-1) versus those to which they are frequently exposed to in their natural environment (>500µmol photonsm-2s-1). Although several behavioral strategies for coping with such irradiances have been suggested, it is unclear as to how these dinoflagellates do so on a physiological level. More specifically, how do long term exposures (30days) affect cell size and cellular chlorophyll content, and what is the photosynthetic response to short term, high irradiance exposures (up to 1464µmol photonsm-2s-1)? The results of this study reveal that cell size and chlorophyll content exhibited by G. carolinianus increased with acclimation to increasing photon flux density. Additionally, both G. carolinianus and G. silvae exhibited reduced photosynthetic efficiency when acclimated to increased photon flux density. Photosynthetic yield exhibited by G. silvae was greater than that for G. carolinianus across all acclimation irradiances. Although such differences were evident, both G. carolinianus and G. silvae appear to have adequate biochemical mechanisms to withstand exposure to irradiances exceeding 250µmol photonsm-2s-1 for at least short periods of time following acclimation to irradiances of up to 150µmol photonsm-2s-1.

Factors influencing acceptability of voluntary HIV testing among pregnant women in Gamboma, Republic of Congo.

BACKGROUND: This study was carried out to identify factors affecting the acceptability of voluntary HIV testing among pregnant women in a semi-rural city, Gamboma, Republic of Congo. METHODS: A cross-sectional study was conducted between January and September 2012. Pregnant women attending antenatal heath care at an integrated health center were enrolled after informed consent and followed through voluntary HIV testing. RESULTS: Among 136 participants, 98 women (72 %) accepted voluntary HIV testing after pre-test counseling. Women with basic education, those who cited blood transfusion as a mode of transmission and prevention of mother-to-child transmission (MTCT) were more likely to accept testing as well those informed about free HIV testing. Interestingly, pregnant women who had heard about HIV/AIDS from hospital setting were less likely to accept testing. CONCLUSIONS: Our data indicate that increasing general education on HIV transmission/prevention modes is crucial for increasing acceptability of screening. Furthermore, HIV/AIDS knowledge disseminated to patients in hospital settings should be carefully monitored. Lastly, scaling-up MTCT services along with a better and larger community information, may address accessibility barriers observed in the present study.

Brevetoxin, the Dinoflagellate Neurotoxin, Localizes to Thylakoid Membranes and Interacts with the Light-Harvesting Complex II (LHCII) of Photosystem II.

The brevetoxins are neurotoxins that are produced by the "Florida red tide" dinoflagellate Karenia brevis. They bind to and activate the voltage-gated sodium channels in higher organisms, specifically the Nav 1.4 and Nav 1.5 channel subtypes. However, the native physiological function that the brevetoxins perform for K. brevis is unknown. By using fluorescent and photoactivatable derivatives, brevetoxin was shown to localize to the chloroplast of K. brevis where it binds to the light-harvesting complex II (LHCII) and thioredoxin. The LHCII is essential to non-photochemical quenching (NPQ), whereas thioredoxins are critical to the maintenance of redox homeostasis within the chloroplast and contribute to the scavenging of reactive oxygen. A culture of K. brevis producing low levels of toxin was shown to be deficient in NPQ and produced reactive oxygen species at twice the rate of the toxic culture, implicating a role in NPQ for the brevetoxins.

Transmembrane potential of red blood cells under low ionic strength conditions.

BACKGROUND/AIMS: In a variety of investigations described in the literature it was not clear to what extent the transmembrane potential red blood cells (RBCs) was changed after the cells have been transferred into low ionic strength (LIS) solutions. Another open question was to find out how fast the transmembrane potential of RBCs in LIS solution will change and which final new equilibrium value will be reached. METHODS: The transmembrane potential of human and bovine RBCs was investigated using the potential-sensitive fluorescent dye DIBAC4(3) (bis(1,3-dibutylbarbituric acid) trimethine oxonol) as well as the CCCP (carbonylcyanide-m-chlorophenylhydrazone) method. RESULTS: Under physiological conditions the transmembrane potential was about -10 mV in agreement with literature data. However, when the RBCs were transferred into an isosmotic low ionic strength medium containing sucrose the transmembrane potential increased to +73 mV and +81 mV for human and bovine RBCs, respectively. In case of human RBCs it continuously decreased reaching finally an equilibrium state of -10 mV again after 30 - 60 min. For bovine RBCs the transmembrane potential declined more slowly reaching a value of +72 mV after 30 min. CONCLUSIONS: Investigations of parameters of RBCs depending on transmembrane potential cannot be performed with human RBCs in LIS media.

Red cell investigations: art and artefacts.

Red blood cell research is important for both, the clinical haematology, such as transfusion medicine or anaemia investigations, and the basic research fields like exploring general membrane physiology or rheology. Investigations of red blood cells include a wide spectrum of methodologies ranging from population measurements with a billion cells evaluated simultaneously to single-cell approaches. All methods have a potential for pitfalls, and the comparison of data achieved by different technical approaches requires a consistent set of standards. Here, we give an overview of common mistakes using the most popular methodologies in red blood cell research and how to avoid them. Additionally, we propose a number of standards that we believe will allow for data comparison between the different techniques and different labs. We consider biochemical analysis, flux measurements, flow cytometry, patch-clamp measurements and dynamic fluorescence imaging as well as emerging single-cell techniques, such as the use of optical tweezers and atomic force microscopy.

Erythrocyte peripheral type benzodiazepine receptor/voltage-dependent anion channels are upregulated by Plasmodium falciparum.

Plasmodium falciparum relies on anion channels activated in the erythrocyte membrane to ensure the transport of nutrients and waste products necessary for its replication and survival after invasion. The molecular identity of these anion channels, termed "new permeability pathways" is unknown, but their currents correspond to up-regulation of endogenous channels displaying complex gating and kinetics similar to those of ligand-gated channels. This report demonstrates that a peripheral-type benzodiazepine receptor, including the voltage dependent anion channel, is present in the human erythrocyte membrane. This receptor mediates the maxi-anion currents previously described in the erythrocyte membrane. Ligands that block this peripheral-type benzodiazepine receptor reduce membrane transport and conductance in P falciparum-infected erythrocytes. These ligands also inhibit in vitro intraerythrocytic growth of P falciparum. These data support the hypothesis that dormant peripheral-type benzodiazepine receptors become the "new permeability pathways" in infected erythrocytes after up-regulation by P falciparum. These channels are obvious targets for selective inhibition in anti-malarial therapies, as well as potential routes for drug delivery in pharmacologic applications.

Ion channels in human red blood cell membrane: actors or relics?

During the past three decades, electrophysiological studies revealed that human red blood cell membrane is endowed with a large variety of ion channels. The physiological role of these channels, if any, remains unclear; they do not participate in red cell homeostasis which is rather based on the almost total absence of cationic permeability and minute anionic conductance. They seem to be inactive in the "resting cell." However, when activated experimentally, ion channels can lead to a very high single cell conductance and potentially induce disorders, with the major risks of fast dehydration and dissipation of gradients. Could there be physiological conditions under which the red cell needs to activate these high conductances, or are ion channels relics of a function lost in anucleated cells? It has been demonstrated that they play a key role in diseases such as sickle cell anemia or malaria. This short overview of ion channels identified to-date in the human red cell membrane is an attempt to propose a dynamic role for these channels in circulating cells in health and disease.

Anion conductance of the human red cell is carried by a maxi-anion channel.

Historically, the anion transport through the human red cell membrane has been perceived to be mediated by Band 3, in the two-component concept with the large electroneutral anion exchange accompanied by the conductance proper, which dominated the total membrane conductance. The status of anion channels proper has never been clarified, and the informations obtained by different groups of electrophysiologists are rather badly matched. This study, using the cell-attached configuration of the patch-clamp technique, rationalizes and explains earlier confusing results by demonstrating that the diversity of anionic channel activities recorded in human erythrocytes corresponds to different kinetic modalities of a unique type of maxi-anion channel with multiple conductance levels and probably multiple gating properties and pharmacology, depending on conditions. It demonstrates the role of activator played by serum in the recruitment of multiple new conductance levels showing very complex kinetics and gating properties upon serum addition. These channels, which seem to be dormant under normal physiological conditions, are potentially activable and could confer a far higher anion conductance to the red cell than the ground leak mediated by Band 3.

Local membrane deformations activate Ca2+-dependent K+ and anionic currents in intact human red blood cells.

BACKGROUND: The mechanical, rheological and shape properties of red blood cells are determined by their cortical cytoskeleton, evolutionarily optimized to provide the dynamic deformability required for flow through capillaries much narrower than the cell's diameter. The shear stress induced by such flow, as well as the local membrane deformations generated in certain pathological conditions, such as sickle cell anemia, have been shown to increase membrane permeability, based largely on experimentation with red cell suspensions. We attempted here the first measurements of membrane currents activated by a local and controlled membrane deformation in single red blood cells under on-cell patch clamp to define the nature of the stretch-activated currents. METHODOLOGY/PRINCIPAL FINDINGS: The cell-attached configuration of the patch-clamp technique was used to allow recordings of single channel activity in intact red blood cells. Gigaohm seal formation was obtained with and without membrane deformation. Deformation was induced by the application of a negative pressure pulse of 10 mmHg for less than 5 s. Currents were only detected when the membrane was seen domed under negative pressure within the patch-pipette. K(+) and Cl(-) currents were strictly dependent on the presence of Ca(2+). The Ca(2+)-dependent currents were transient, with typical decay half-times of about 5-10 min, suggesting the spontaneous inactivation of a stretch-activated Ca(2+) permeability (PCa). These results indicate that local membrane deformations can transiently activate a Ca(2+) permeability pathway leading to increased [Ca(2+)](i), secondary activation of Ca(2+)-sensitive K(+) channels (Gardos channel, IK1, KCa3.1), and hyperpolarization-induced anion currents. CONCLUSIONS/SIGNIFICANCE: The stretch-activated transient PCa observed here under local membrane deformation is a likely contributor to the Ca(2+)-mediated effects observed during the normal aging process of red blood cells, and to the increased Ca(2+) content of red cells in certain hereditary anemias such as thalassemia and sickle cell anemia.

Effects of elevated intracellular calcium on the osmotic fragility of human red blood cells.

High throughput methodologies that measure the distribution of osmotic fragilities in red blood cell populations have enabled the investigation of dynamic changes in red cell homeostasis and membrane permeability in health and disease. The common assumption in the interpretation of dynamic changes in osmotic fragility curves is that left or right shifts reflect a decreased or increased hydration state of the cells, respectively, allowing direct inferences on membrane transport from osmotic fragility measurements. However, the assumed correlation between shifts in osmotic fragility and hydration state has never been directly explored, and may prove invalid in certain conditions. We investigated here whether this correlation holds for red cells exposed to elevated intracellular calcium. The results showed that elevated cell calcium causes a progressive increase in osmotic fragility with minimal contribution from cell hydration (<8%). Loss of membrane area by the release of 160+/-40nm diameter (mean+/-SD) vesicles is shown to be a major contributor, but may not account for the full non-hydration component. The rest must reflect a specific calcium-induced lytic vulnerability of the membrane causing rupture before the cells attain their maximal spherical volumes. The implications of these findings are discussed.

Anion channels in Plasmodium-falciparum-infected erythrocytes and protein kinase A.

By replicating within red blood cells, malaria parasites are largely hidden from immune recognition; however, in the cells, nutrients are limiting and hazardous metabolic end products can rapidly accumulate. Therefore, to survive within erythrocytes, parasites alter the permeability of the host plasma membrane, either by upregulating existing transporters or by creating new permeation pathways. Recent electrophysiological studies of Plasmodium-infected erythrocytes have demonstrated that membrane permeability is mediated by transmembrane transport through ion channels in the infected erythrocyte. This article discusses the evidence and controversies concerning the nature of these channels and surveys the potential role of phosphorylation in activating anion channels that could be important in developing novel strategies for future malarial chemotherapies.

Further characterization of cation channels present in the chicken red blood cell membrane.

In this paper, we provide an update on cation channels in nucleated chicken erythrocytes. Patch-clamp techniques were used to further characterize the two different types of cation channels present in the membrane of chicken red blood. In the whole-cell mode, with Ringer in the bath and internal K+ saline in the pipette solution, the membrane conductance was generated by cationic currents, since the reversal potential was shifted toward cations equilibrium when the impermeant cation NMDG was substituted to small cations. The membrane conductance could be increased by application of mechanical deformation or by the addition of agonists of the cAMP-dependent pathway. At the unitary level, two different types of cationic channels were revealed and could account for the cationic conductance observed in whole-cell configuration. One of them belongs to the family of stretch-activated cationic channel showing changes in activity under conditions of membrane deformation, whereas the second one belongs to the family of the cAMP activated cationic channels. These two channels could be distinguished according to their unitary conductances and drug sensitivities. The stretch-activated channel was sensitive to Gd(3+) and the cAMP-dependent channel was sensitive to flufenamic acid. Possible role of these channels in cell volume regulation process is discussed.

Allelopathic activity among Cyanobacteria and microalgae isolated from Florida freshwater habitats.

We evaluated allelopathic interactions between strains of Cyanobacteria and green algae isolated from south and central Florida. Allelopathy, including inhibition or stimulation of growth, was assessed by cocultivation of each of the isolated strains, as well as by evaluation of extracts prepared from the isolates. All of the strains of Cyanobacteria, and four of the six isolates of green algae, showed some allelopathic activity (i.e. inhibition or stimulation of the growth of other strains). Of these, the most pronounced activity was observed for the cyanobacterial isolate Fischerella sp. strain 52-1. In the cocultivation experiments this cyanobacterium inhibited the growth of all tested green algae and Cyanobacteria. The crude lipophilic extracts from Fischerella sp. strain 52-1 isolated from both the biomass and the culture liquid inhibited photosynthesis of the green alga Chlamydomonas sp. in a concentration- and time-dependent manner and caused extensive loss of ultrastructural cell organization. Preliminary chemical characterization of compounds extracted from Fischerella sp. strain 52-1 indicated the presence of indole alkaloids, and further characterization has confirmed that these compounds belong to the hapalindoles previously isolated from other species of Fischerella and related genera. Further chemical characterization of these compounds, and further investigation of their apparent role in allelopathy is ongoing.

Plasmodium falciparum regulatory subunit of cAMP-dependent PKA and anion channel conductance.

Malaria symptoms occur during Plasmodium falciparum development into red blood cells. During this process, the parasites make substantial modifications to the host cell in order to facilitate nutrient uptake and aid in parasite metabolism. One significant alteration that is required for parasite development is the establishment of an anion channel, as part of the establishment of New Permeation Pathways (NPPs) in the red blood cell plasma membrane, and we have shown previously that one channel can be activated in uninfected cells by exogenous protein kinase A. Here, we present evidence that in P. falciparum-infected red blood cells, a cAMP pathway modulates anion conductance of the erythrocyte membrane. In patch-clamp experiments on infected erythrocytes, addition of recombinant PfPKA-R to the pipette in vitro, or overexpression of PfPKA-R in transgenic parasites lead to down-regulation of anion conductance. Moreover, this overexpressing PfPKA-R strain has a growth defect that can be restored by increasing the levels of intracellular cAMP. Our data demonstrate that the anion channel is indeed regulated by a cAMP-dependent pathway in P. falciparum-infected red blood cells. The discovery of a parasite regulatory pathway responsible for modulating anion channel activity in the membranes of P. falciparum-infected red blood cells represents an important insight into how parasites modify host cell permeation pathways. These findings may also provide an avenue for the development of new intervention strategies targeting this important anion channel and its regulation.

Toward a unifying model of malaria-induced channel activity.

Infection of RBC by the malaria parasite Plasmodium falciparum activates, at the trophozoite stage, a membrane current 100- to 150-fold larger than in uninfected RBC. This current is carried by small anion channels initially described in supraphysiological ion concentrations (1.115 M Cl(-)) and named plasmodial surface anion channels (PSAC), suggesting their plasmodial origin. Our results obtained with physiological ion concentrations (0.145 M Cl(-)) support the notion that the parasite-induced channels represent enhanced activity versions of anion channels already present in uninfected RBCs. Among them, an 18-pS inwardly rectifying anion channel (IRC) and a 4- to 5-pS small conductance anion channel (SCC) were present in most single-channel recordings of infected membranes. The aim of this study was to clarify disparities in the reported electrophysiological data and to investigate possible technical reasons why these discrepancies have arisen. We demonstrate that PSAC is the supraphysiological correlate of the SCC and is inhibited by Zn(2+), suggesting that it is a ClC-2 channel. We show that in physiological solutions 80% of the membrane conductance in infected cells can be accounted for by IRC and 20% can be accounted for by SCC whereas in supraphysiological conditions the membrane conductance is almost exclusively carried by SCC (PSAC) because the IRC is functionally turned off.

Chloride channels in normal and cystic fibrosis human erythrocyte membrane.

Electrophysiological studies on human RBCs have been difficult due to fragility and small size of cells, and little is known of ionic conductive pathways present in the RBC membrane in health and disease. We report on anionic channels in cells of healthy donors (control) and cystic fibrosis (CF) patients. Anion channel activity (8-12 pS, linear) was induced in cell-attached configuration by forskolin (50 microM) and in excised inside-out configuration by PKA (100 nM) and ATP (1 mM) but control and CF RBCs differed by their respective kinetics and gating properties. These channels were permeable to ATP (100 mM, symmetrical Tris-ATP). These data suggest either the existence of two different anionic channel types or regulation of a single channel type either by the CFTR (cystic fibrosis transmembrane regulator) protein or by different cytosolic factors. Another anionic channel type displaying outward rectification (approximately 80 pS, outward conductance) was present in 30% of CF cell patches but was not observed in normal cell patches. The frequently recorded activity of this channel in CF patches suggests a down-regulation in normal RBCs.

Electrophysiological studies of malaria parasite-infected erythrocytes: current status.

The altered permeability characteristics of erythrocytes infected with malaria parasites have been a source of interest for over 30 years. Recent electrophysiological studies have provided strong evidence that these changes reflect transmembrane transport through ion channels in the host erythrocyte plasma membrane. However, conflicting results and differing interpretations of the data have led to confusion in this field. In an effort to unravel these issues, the groups involved recently came together for a week of discussion and experimentation. In this article, the various models for altered transport are reviewed, together with the areas of consensus in the field and those that require a better understanding.

Three types of spontaneously active anionic channels in malaria-infected human red blood cells.

The electrophysiological study of red blood cells (RBCs), using the patch-clamp technique, has been going through a renaissance with the recent discovery of novel channel activity in the host plasma membrane of Plasmodium falciparum-infected human RBCs (S.A. Desai et al., Nature 406, 1001-1005, 2000; S.M. Huber et al., EMBO J. 21 (2002) 22-30; S. Egee et al., J. Physiol. 542 (2002) 795-801). This arose from the finding that malaria-infected RBCs have altered permeability characteristics due to the induction of new permeation pathways (NPPs) (H. Ginsburg, Novartis Foundation Symposium 226 (1999) 99-108; K. Kirk, Physiol. Rev. 81 (2001) 495-537), which are defined, using non-electrophysiological techniques, as having the general characteristics of anion channels (i.e. high anion permeability, linear concentration dependence, inability to distinguish between stereo-isomers of permeant solutes). Discovering potent and specific inhibitors of the NPPs is an important therapeutic challenge, but too many questions remain unanswered: do the NPPs correspond to a single path or multiple pathways? Are they parasite-derived proteins? Are they up-regulated or modified endogenous quiescent red blood cell proteins? This article concerns the identification of different types of anionic channels that are expressed in malaria-infected human RBCs. Implications regarding the presence of these different types of channels in infected RBCs and their functional significance are discussed.