Methylated Arsenic Species in Rice:Geographical Variation, Origin, and Uptake Mechanisms

Rice is a major source of inorganic arsenic (iAs) in the human diet because paddy rice. efficient at accumulating As Rice As speciation is dominated by iAs and dimethylarsinic acid (DMA). Here we review the global pattern in rice As speciation and the factors causing the variation. Rice produced in Asia shows a strong linear relationship between iAs and total As concentration with a slope of 0.78. Rice produced in Europe and the United States shows a more variable, but generally hyperbolic relationship with DMA being predominant in U.S. rice. Although there is significant genotypic variation in grain As speciation, the regional Variations are primarily attributed to environmental factors. Emerging evidence also indicates that methylated. As species in rice are derived from the soil, while rice plants lack the As methylation ability. Soil flooding and additions of organic matter increase microbial methylation of As, although the microbial community responsible for methylafion is poorly understood. Compared with iAs, methylated As species are taken up by rice roots less efficiently but are transported to the grain much, more efficiently, which may be an important factor responsible for the spikelet sterility disorder (straight head disease) in rice. DMA is a weak carcinogen, but the level of ingestion from rice consumption is much lower than that of concern. Questions that require further investigations are identified.

Characterization of the structure and iron uptake mechanisms of the Staphylococcus aureus ferric hydroxamate-binding lipoprotein FhuD2

The Reverse Vaccinology (RV) approach allows using genomic information for the delineation of new protein-based vaccines starting from an in silico analysis. The first powerful example of the application of the RV approach is given by the development of a protein-based vaccine against serogroup B Meningococcus. A similar approach was also used to identify new Staphylococcus aureus vaccine candidates, including the ferric hydroxamate-binding lipoprotein FhuD2. S. aureus is a widespread human pathogen, which employs various different strategies for iron uptake, including: (i) siderophore-mediated iron acquisition using the endogenous siderophores staphyloferrin A and B, (ii) siderophore-mediated iron acquisition using xeno-siderophores (the pathway exploited by FhuD2) and (iii) heme-mediated iron acquisition. In this work the high resolution crystal structure of FhuD2 in the iron (III)-siderophore-bound form was determined. FhuD2 belongs to the Periplasmic Binding Protein family (PBP ) class III, and is principally formed by two globular domains, at the N- and C-termini of the protein, that make up a cleft where ferrichrome-iron (III) is bound. The N- and C-terminal domains, connected by a single long α-helix, present Rossmann-like folds, showing a β-stranded core and an α-helical periphery, which do not undergo extensive structural rearrangement when they interact with the ligand, typical of class III PBP members. The structure shows that ferrichrome-bound iron does not come directly into contact with the protein; rather, the metal ion is fully coordinated by six oxygen donors of the hydroxamate groups of three ornithine residues, which, with the three glycine residues, make up the peptide backbone of ferrichrome. Furthermore, it was found that iron-free ferrichrome is able to subtract iron from transferrin. This study shows for the first time the structure of FhuD2, which was found to bind to siderophores ,and that the protein plays an important role in S. aureus colonization and infection phases.

Different endocytotic uptake mechanisms for nanoparticles in epithelial cells and macrophages.

Precise knowledge regarding cellular uptake of nanoparticles is of great importance for future biomedical applications. Four different endocytotic uptake mechanisms, that is, phagocytosis, macropinocytosis, clathrin- and caveolin-mediated endocytosis, were investigated using a mouse macrophage (J774A.1) and a human alveolar epithelial type II cell line (A549). In order to deduce the involved pathway in nanoparticle uptake, selected inhibitors specific for one of the endocytotic pathways were optimized regarding concentration and incubation time in combination with fluorescently tagged marker proteins. Qualitative immunolocalization showed that J774A.1 cells highly expressed the lipid raft-related protein flotillin-1 and clathrin heavy chain, however, no caveolin-1. A549 cells expressed clathrin heavy chain and caveolin-1, but no flotillin-1 uptake-related proteins. Our data revealed an impeded uptake of 40 nm polystyrene nanoparticles by J774A.1 macrophages when actin polymerization and clathrin-coated pit formation was blocked. From this result, it is suggested that macropinocytosis and phagocytosis, as well as clathrin-mediated endocytosis, play a crucial role. The uptake of 40 nm nanoparticles in alveolar epithelial A549 cells was inhibited after depletion of cholesterol in the plasma membrane (preventing caveolin-mediated endocytosis) and inhibition of clathrin-coated vesicles (preventing clathrin-mediated endocytosis). Our data showed that a combination of several distinguishable endocytotic uptake mechanisms are involved in the uptake of 40 nm polystyrene nanoparticles in both the macrophage and epithelial cell line.

Nitrate and phosphate uptake characteristics of three species of brown algae cultured at low salinity.

Nitrate and phosphate uptake mechanisms have been characterised under conditions of 100 and 50% seawater in 3 common brown algae of NW Europe: Fucus vesiculosus, F. serratus and Laminaria digitata. Under low salinity, the growth rate and internal nitrate accumulation of F. serratus significantly increased (20 and 48%, respectively), but no significant changes were observed for F. vesiculosus and L. digitata. However, nitrate uptake rates were reduced in L. digitata, so that this species was less adaptable to low salinity than the Fucus species. Both F. vesiculosus and F. serratus reached a steady-state uptake rate after acclimation regardless of the salinity treatment. All 3 species had a high capacity for storing inorganic N and P intracellularly. The results for F. serratus pointed to a dual mechanism of adaptation to the special characteristics of the intertidal environment where it grows. Non-saturating (low affinity) nitrate uptake and biphasic (double Michaelis-Menten curve) phosphate uptake are adaptations to high nutrient concentrations. Temporal partition of cellular energy for carbon metabolism and nutrient uptake is also suggested as an adaptation to the transient nutrient inputs occurring in these environments.

Suppression of the high affinity phosphate uptake system:A mechanism of arsenate tolerance in Holcus lanatus L.

In Holcus lanatus L. phosphate and arsenate are taken up by the same transport system. Short-term uptake kinetics of the high affinity arsenate transport system were determined in excised roots of arsenate-tolerant and non-tolerant genotypes. In tolerant plants the V_max of ion uptake in plants grown in phosphate-free media was decreased compared to non-tolerant plants, and the affinity of the uptake system was lower than in the non-tolerant plants. Both the reduction in V_max and the increase in K_m led to reduced arsenate influx into tolerant roots. When the two genotypes were grown in nutrient solution containing high levels of phosphate, there was little change in the uptake kinetics in tolerant plants. In non-tolerant plants, however, there was a marked decrease in the V_max to the level of the tolerant plants but with little change in the K_m. This suggests that the low rate of arsenate uptake over a wide range of differing root phosphate status is due to loss of induction of the synthesis of the arsenate (phosphate) carrier. © 1992 Oxford University Press.

Comparative analyses of cadmium and zinc uptake correlated with changes in natural resistance-associated macrophage protein (NRAMP) expression in Solanum nigrum L. and Brassica rapa

Environmental context Soils contaminated with metals can pose both environmental and human health risks. This study showed that a common crop vegetable grown in the presence of cadmium and zinc readily accumulated these metals, and thus could be a source of toxicity when eaten. The work highlights potential health risks from consuming crops grown on contaminated soils. Abstract Ingestion of plants grown in heavy metal contaminated soils can cause toxicity because of metal accumulation. We compared Cd and Zn levels in Brassica rapa, a widely grown crop vegetable, with that of the hyperaccumulator Solanum nigrum L. Solanum nigrum contained 4 times more Zn and 12 times more Cd than B. rapa, relative to dry mass. In S. nigrum Cd and Zn preferentially accumulated in the roots whereas in B. rapa Cd and Zn were concentrated more in the shoots than in the roots. The different distribution of Cd and Zn in B. rapa and S. nigrum suggests the presence of distinct metal uptake mechanisms. We correlated plant metal content with the expression of a conserved putative natural resistance-associated macrophage protein (NRAMP) metal transporter in both plants. Treatment of both plants with either Cd or Zn increased expression of the NRAMP, with expression levels being higher in the roots than in the shoots. These findings provide insights into the molecular mechanisms of heavy metal processing by S. nigrum L. and the crop vegetable B. rapa that could assist in application of these plants for phytoremediation. These investigations also highlight potential health risks associated with the consumption of crops grown on contaminated soils.

Quantification of gold nanoparticle cell uptake under controlled biological conditions and adequate resolution

Aim: We examined cellular uptake mechanisms of fluorescently labeled polymer-coated gold nanoparticles (NPs) under different biological conditions by two quantitative, microscopic approaches. Materials & methods: Uptake mechanisms were evaluated using endocytotic inhibitors that were tested for specificity and cytotoxicity. Cellular uptake of gold NPs was analyzed either by laser scanning microscopy or transmission electron microscopy, and quantified by means of stereology using cells from the same experiment. Results: Optimal inhibitor conditions were only achieved with chlorpromazine (clathrin-mediated endocytosis) and methyl-β-cyclodextrin (caveolin-mediated endocytosis). A significant methyl-β-cyclodextrin-mediated inhibition (63-69%) and chlorpromazine-mediated increase (43-98%) of intracellular NPs was demonstrated with both imaging techniques, suggesting a predominant uptake via caveolin-medicated endocytois. Transmission electron microscopy imaging revealed more than 95% of NPs localized in intracellular vesicles and approximately 150-times more NP events/cell were detected than by laser scanning microscopy. Conclusion: We emphasize the importance of studying NP-cell interactions under controlled experimental conditions and at adequate microscopic resolution in combination with stereology. Original submitted 10 July 2012; Revised submitted 23 January 2013.

Bicarbonate uptake via an anion exchange protein is the main mechanism of inorganic carbon acquisition by the giant kelp Macrocystis pyrifera (Laminariales, Phaeophyceae) under variable pH

Macrocystis pyrifera is a widely distributed, highly productive, seaweed. It is known to use bicarbonate (HCO3-) from seawater in photosynthesis and the main mechanism of utilization is attributed to the external catalyzed dehydration of HCO3- by the surface-bound enzyme carbonic anhydrase (CAext). Here, we examined other putative HCO3- uptake mechanisms in M. pyrifera under pHT 9.00 (HCO3-: CO2 = 940:1) and pHT 7.65 (HCO3-: CO2 = 51:1). Rates of photosynthesis, and internal CA (CAint) and CAext activity were measured following the application of AZ which inhibits CAext, and DIDS which inhibits a different HCO3- uptake system, via an anion exchange (AE) protein. We found that the main mechanism of HCO3- uptake by M. pyrifera is via an AE protein, regardless of the HCO3-: CO2 ratio, with CAext making little contribution. Inhibiting the AE protein led to a 55%-65% decrease in photosynthetic rates. Inhibiting both the AE protein and CAext at pHT 9.00 led to 80%-100% inhibition of photosynthesis, whereas at pHT 7.65, passive CO2 diffusion supported 33% of photosynthesis. CAint was active at pHT 7.65 and 9.00, and activity was always higher than CAext, because of its role in dehydrating HCO3- to supply CO2 to RuBisCO. Interestingly, the main mechanism of HCO3- uptake in M. pyrifera was different than that in other Laminariales studied (CAext-catalyzed reaction) and we suggest that species-specific knowledge of carbon uptake mechanisms is required in order to elucidate how seaweeds might respond to future changes in HCO3-:CO2 due to ocean acidification.

Despite differences between dendritic cells and Langerhans cells in the mechanism of papillomavirus-like particle antigen uptake, both cells cross-prime T cells

As human papillomavirus-like particles (HPV-VLP) represent a promising vaccine delivery vehicle, delineation of the interaction of VLP with professional APC should improve vaccine development. Differences in the capacity of VLP to signal dendritic cells (DC) and Langerhans cells (LC) have been demonstrated, and evidence has been presented for both clathrin-coated pits and proteoglycans (PG) in the uptake pathway of VLP into epithelial cells. Therefore, we compared HPV-VLP uptake mechanisms in human monocyte-derived DC and LC, and their ability to cross-present HPV VLP-associated antigen in the MHC class I pathway. DC and LC each took up virus-like particles (VLP). DC uptake of and signalling by VLP was inhibited by amiloride or cytochalasin D (CCD), but not by filipin treatment, and was blocked by several sulfated and non-sulfated polysaccharides and anti-CD16. In contrast, LC uptake was inhibited only by filipin, and VLP in LC were associated with caveolin, langerin, and CD1a. These data suggest fundamentally different routes of VLP uptake by DC and LC. Despite these differences, VLP taken up by DC and LC were each able to prime naive CD8(+) T cells and induce cytolytic effector T cells in vitro. (C) 2004 Elsevier Inc. All rights reserved.

Cellular uptake and biological activity of synthetic hammerhead ribozymes

Glioblastoma Multiforme (GBM) is a highly malignant form of brain cancer for which there is no effective cure. The over-expression of a number of genes, including the epidermal growth factor receptor (EGFr), has been implicated as a causative factor of tumourigenesis. Ribozymes are a class of ribonucleic acid that possess enzymatic properties. They can inhibit gene-expression in a highly sequence specific manner by catalysing the trans-cleavage of target RNA. The potential use of synthetic hammerhead ribozymes as novel anti-brain tumour agents was investigated in this study. The successful use of synthetic, exogenously administered ribozymes for such applications will require chemical modifications that improve biological stability and a fundamental understanding of cellular uptake mechanisms. Chimeric 2'-O-methylated hammerhead ribozymes proved to be significantly more stable (>4000-fold) in serum than unmodified RNA ribozymes and exhibited high in vitro catalytic activity. The cellular association of an internally [32P]-labelled 2'-O-methylated chimeric ribozyme in U87-MG human glioma cells was temperature-, energy- and pH-dependent and involved an active process that could be competed with a variety of polyanions. Indications are that the predominant mechanism of uptake is by adsorptive and / or receptor mediated endocytosis. Twenty 2'-O-methylated chimeric ribozymes were designed to cleave various sites along the EGFr mRNA. In vitro, 18 ribozymes exhibited high activity in cleaving a complementary short substrate. Using LipofectAMINETM as a delivery agent, the efficacy of these ribozymes was evaluated in the A431 cell line, which expresses amplified levels of EGFr. Studies revealed that although the ribozymes were taken up by the cells and remained stable over a period of 4 days, no significant reduction in either EGFr expression or cell proliferation was evident. The presence of telomerase, a ribonucleoprotein responsible for telomere elongation, has been strongly associated with tumour progression. The biological activity of a 2'-O-methylated ribozyme targeted against the RNA component of telomerase was determined. The ribozyme exhibited specific dose-dependent inhibition of telomerase activity in U87-MG cell lysates with an IC50 of –4μM. When 4μM ribozyme was delivered to intact U87-MG cells, complexed to LipofectAMINETM, telomerase activity was significantly reduced to 74.5±4.17% of the untreated control. Free ribozyme showed no significant inhibitory effect demonstrating the importance of an appropriate delivery system for optimum delivery of exogenously administered ribozymes.

Gene expression evidence for off-target effects caused by RNA interference-mediated gene silencing of Ubiquitin-63E in the cattle tick Rhipicephalus microplus.

Knowledge of cattle tick (Rhipicephalus (Boophilus) microplus; Acari: Ixodidae) molecular and cellular pathways has been hampered by the lack of an annotated genome. In addition, most of the tick expressed sequence tags (ESTs) available to date consist of similar to 50% unassigned sequences without predicted functions. The most common approach to address this has been the application of RNA interference (RNAi) methods to investigate genes and their pathways. This approach has been widely adopted in tick research despite minimal knowledge of the tick RNAi pathway and double-stranded RNA (dsRNA) uptake mechanisms. A strong knockdown phenotype of adult female ticks had previously been observed using a 594 bp dsRNA targeting the cattle tick homologue for the Drosophila Ubiquitin-63E gene leading to nil or deformed eggs. A NimbleGen cattle tick custom microarray based on the BmiGI.V2 database of R. microplus ESTs was used to evaluate the expression of mRNAs harvested from ticks treated with the tick Ubiquitin-63E 594 bp dsRNA compared with controls. A total of 144 ESTs including TC6372 (Ubiquitin-63E) were down-regulated with 136 ESTs up-regulated following treatment. The results obtained substantiated the knockdown phenotype with ESTs identified as being associated with ubiquitin proteolysis as well as oogenesis, embryogenesis, fatty acid synthesis and stress responses. A bioinformatics analysis was undertaken to predict off-target effects (OTE) resulting from the in silico dicing of the 594 bp Ubiquitin-63E dsRNA which identified 10 down-regulated ESTs (including TC6372) within the list of differentially expressed probes on the microarrays. Subsequent knockdown experiments utilising 196 and 109 bp dsRNAs, and a cocktail of short hairpin RNAs (shRNA) targeting Ubiquitin-63E, demonstrated similar phenotypes for the dsRNAs but nil effect following shRNA treatment. Quantitative reverse transcriptase PCR analysis confirmed differential expression of TC6372 and selected ESTs. Our study demonstrated the minimisation of predicted OTEs in the shorter dsRNA treatments (similar to 100-200 bp) and the usefulness of microarrays to study knockdown phenotypes.

Biofunctionalized surface-modified silver nanoparticles for gene delivery

Silver nanoparticles (AgNPs) find use in different biomedical applications including wound healing and cancer. We propose that the efficacy of the nanoparticles can be further augmented by using these particles for gene delivery applications. The objective of this work was to engineer biofunctionalized stable AgNPs with good DNA binding ability for efficient transfection and minimal toxicity. Herein, we report on the one-pot facile green synthesis of polyethylene glycol (PEG) stabilized chitosan-g-polyacrylamide modified AgNPs. The size of the PEG stabilized AgNPs was 38 +/- 4 nm with a tighter size distribution compared to the unstabilized nanoparticles which showed bimodal distribution of particle sizes of 68 +/- 5 nm and 7 +/- 4 nm. To enhance the efficiency of gene transfection, the Arg-Gly-Asp-Ser (RGDS) peptide was immobilized on the silver nanoparticles. The transfection efficiency of AgNPs increased significantly after immobilization of the RGDS peptide reaching up to 42 +/- 4% and 30 +/- 3% in HeLa and A549 cells, respectively, and significantly higher than 34 +/- 3% and 23 +/- 2%, respectively, with the use of polyethyleneimine (25 kDa). These nanoparticles were found to induce minimal cellular toxicity. Differences in cellular uptake mechanisms with RGDS immobilization resulting in improved efficiency are elucidated. This study presents biofunctionalized AgNPs for potential use as efficient nonviral carriers for gene delivery with minimal cytotoxicity toward augmenting the therapeutic efficacy of AgNPs used in different biomedical products.

Contamination and distribution of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/F) in agriculture fields in Ya-Er Lake area, China

The contamination and distribution of polychlorinated dibeinizo-p-dioxins and dibenzofurans (PCDD/Fs) from two agricultural fields of a heavily polluted lake area in China (Ya-Er Lake) are presented. The vertical distribution pattern of total PCDD/Fs in soil cores reveals that the maximum concentration was in the layer of 20-30 cm. The concentrations in the top layer of soil at the two sites were similar (17.48 ng/kg at Site 1 and 18.10 ng/kg at Site 2), but the maximum concentration of Site 1 (120.8 ng/kg) was two times higher than that of Site 2 (64.39 ng/kg). The maximum concentration of PCDD/Fs in mud cores in rice fields (0-50 cm) at Sites 1 and 2 was in the layer of 0-10 cm. The maximum PCDD/F concentration in the top layer in mud at Site 1 (203.1 ng/kg) was higher than that at Site 2: (143.3 ng/kg). Significant correlations were found between the mind PCDD/Fs and the organic carbon content (R = 0.9743, P< 0,05 at Site 1; R = 0.9821, P< 0.05 at Site 2), the two variables being highly correlated (R = 0.9049, P< 0.05, at Site 1; R = 0.9916, P< 0.05 at Site 2). All correlation coefficients were significant at the 95% level. Concentrations were highly correlated with organic carbon, indicating that sorption to organic carbon was the dominant mechanism. Using principal component analysis, the homologue profiles of soil, mud, and plants (rice and radish) were compared. The PCDD/F patterns in plants were found not to be correlated to those in soil and mud. This suggests that atmospheric deposition may be the main source of PCDD/Fs in rice grain. However, mixed exposure involving uptake mechanisms and atmospheric deposition is considered main the source of PCDD/F pollution in radishes. (C) 2002 Elsevier Science (USA).

Particulate and dissolved aluminum and titanium in the upper water column of the Atlantic Ocean

This study investigates the oceanic behavior of the lithogenic trace elements Al and Ti in the upper 200 m of the Atlantic Ocean. The distribution of both metals in the dissolved and particulate phases was assessed along an E-W transect in the eastern tropical North Atlantic (December 2009) and along a meridional Atlantic transect (April-May 2010). The surface water concentrations of particulate and dissolved Al and Ti reflected the previously observed pattern of atmospheric inputs into the Atlantic Ocean. Subsurface minima at stations with pronounced fluorescence maxima were observed, suggesting a link between biological productivity and the removal of both dissolved and particulate Al and Ti. This may include uptake mechanisms, adsorption and aggregation processes on biogenic particle surfaces and the formation of large, fast sinking biogenic particles, e.g., fecal pellets. Residence times in the upper water column (100 m) of the tropical and subtropical North Atlantic were estimated to range in the order of days to weeks in the particulate phases (Al: 3-22 days, Ti: 4-37 days) and were 0.9-3.8 years for Al and 10-31 years for Ti in the dissolved phases. Longer residence times in both phases in the South Atlantic are consistent with lower biological productivity and decreased removal rates. In the upper water column, Al was predominantly present in the dissolved form, whereas Ti mostly occurred in the particulate form. Largest deviations in the partition coefficients between the particulate and dissolved phases were found in the surface waters, together with excess dissolved Al over Ti compared to the crustal source. This likely reflects elevated dissolution of Al compared to Ti from atmospheric mineral particles.

Arsenite transport into paddy rice (Oryza sativa) roots

Here the mechanism of arsenite transport into paddy rice (Oryza sativa) roots, uptake of which is described by Michaelis-Menten kinetics, is reported. A recent study on yeast (Saccharomyces cerevisiae) showed that undissociated arsenite (its pK_a is 9.2) was transported across the plasma membrane via a glycerol transporting channel. To investigate whether the same mechanism of transport was involved for rice, competitive studies with glycerol, which is transported into cells via aquaporins, were performed. Glycerol competed with arsenite for transport in a dose-dependent manner, indicating that arsenite and glycerol uptake mechanisms were the same. Arsenate transport was unaffected by glycerol, confirming that arsenate and arsenite are taken up into cells by different mechanisms. Antimonite, an arsenite analogue that is transported into S. cerevisiae cells by aquaporins, also competed with arsenite transport in a dose-dependent manner, providing further evidence that arsenite is transported into rice roots via glycerol transporting channels. Mercury (Hg²⁺) inhibited both arsenite and arsenate uptake, suggesting that inhibition of influx was due to general cellular stress rather than the specific action of Hg²⁺ on aquaporins. Arsenite uptake by pea (Pisum sativum) and wheat (Triticum aestivum) was also described by Michaelis-Menten kinetics.