Noninvasive Evaluation of Heavy Metal Uptake and Storage in Micoralgae Using a Fluorescence Resonance Energy Transfer-Based Heavy Metal Biosensor

We have developed a fluorescence resonance energy transfer (FRET)-based heavy metal biosensor for the quantification of bioavailable free heavy metals in the cytoplasm of the microalga Chlamydomonas reinhardtii. The biosensor is composed of an end-to-end fusion of cyan fluorescent protein (CFP), chicken metallothionein II (MT-II), and yellow fluorescent protein (YFP). In vitro measurements of YFP/CFP fluorescence emission ratios indicated that the addition of metals to the purified biosensor enhanced FRET between CFP and YFP, consistent with heavy metal-induced folding of MT-II. A maximum YFP/CFP FRET ratio of 2.8 was observed in the presence of saturating concentrations of heavy metals. The sensitivity of the biosensor was greatest for Hg²⁺ followed by Cd²⁺ ≈ Pb²⁺ > Zn²⁺ > Cu²⁺. The heavy metal biosensor was unresponsive to metals that do not bind to MT-II (Na⁺ and Mg²⁺). When expressed in C. reinhardtii, we observed a differential metal-dependent response to saturating external concentrations (1.6 mm) of heavy metals (Pb²⁺ > Cd²⁺) that was unlike that observed for the isolated biosensor (in vitro). Significantly, analysis of metal uptake kinetics indicated that equilibration of the cytoplasm with externally applied heavy metals occurred within seconds. Our results also indicated that algae have substantial buffering capacity for free heavy metals in their cytosol, even at high external metal concentrations.Many proteins utilize metals to stabilize their structures or as cofactors to catalyze redox and other chemical reactions. Metals such as zinc, copper, iron, magnesium, cobalt, and manganese are required by most living organisms for their normal cellular functions. Essential metals are often present at low concentrations in the environment, however, and must be imported into cells, often at the expense of energy (Hanikenne et al., 2005; Merchant et al., 2006). In contrast to essential metals, toxic metals such as cadmium, lead, and mercury can disrupt cellular functions by competing with essential metals for their metal-binding sites and/or by altering the redox state of cells. Exposure of organisms to high concentrations of toxic metals can impair their cellular functions, growth, and reproduction. To prevent metal-induced cellular anomalies, organisms have evolved a variety of strategies to reduce the toxicity of heavy metals. One such strategy involves the selective binding of toxic metals in the cytoplasm by metal-binding proteins and other small molecules. As discussed below, both enzymatically and ribosomally synthesized Cys-rich peptides, including phytochelatins and metallothioneins (MTs), are utilized by a variety of organisms to sequester toxic heavy metals, including cadmium, mercury, lead, silver, and gold. The peptides may also serve as storage reserves for essential metals such as copper and zinc (Cobbett and Goldsbrough, 2002).Phytochelatins are enzymatically synthesized polypeptides containing repeating units of (γ-Glu-Cys)_n-Gly, where n = 2 to 11 (Rauser, 1990), whereas MTs are genetically encoded, ribosomally synthesized polypeptides (Cobbett and Goldsbrough, 2002). MTs have molecular mass values ranging from 6 to 7 kD and contain approximately 20 conserved Cys residues (Cobbett and Goldsbrough, 2002; Romero-Isart and Vasák, 2002). Metals are characteristically bound to MT via the thiolate sulfur ligands of Cys residues (Kägi, 1991). It is estimated that the metal-saturated MT contains about 10% thiolate sulfur and bound metals by mass (Romero-Isart and Vasák, 2002). Structural analyses of metal-free and metal-complexed MTs demonstrated that MTs undergo a structural transition from a metal-free random-coil structure to a metal-bound compact dumbbell-shaped structure having metal saturated α- and β-domains (Pearce et al., 2000; Romero-Isart and Vasak, 2002; Hong and Maret, 2003). The N-terminal β-domain binds three metal ion equivalents, and the C-terminal α-domain binds four metal ion equivalents (Romero-Isart and Vasák, 2002; Vasák, 2005). Furthermore, several decades of work on MTs have provided a great deal of information regarding their metal-binding affinity, specificity, and domain selectivity for select metals (Cobbett and Goldsbrough, 2002; Romero-Isart and Vasák, 2002; Vasák, 2005).Fluorescence resonance energy transfer (FRET) involves the nonradioactive transfer of energy between the excited state of a luminescent or fluorescent donor molecule and a nearby acceptor molecule that has overlapping excited state transitions. Proteins that are modified to have efficient energy donor and acceptor domains and that undergo structural changes upon binding a specific ligand are good candidates for FRET-based sensors. For ligand-specific FRET-based biosensors, the distance and/or the orientation between the energy donor and acceptor molecules is changed upon ligand binding in a concentration-dependent manner (Selvin, 1995; Weiss, 2000; Hong and Maret, 2003; Looger et al., 2005). Relevant to this discussion, a FRET-based biosensor with GFP variants fused to MT was previously shown to be an effective means to monitor metal release during nitric oxide-induced signaling in endothelial cells (Pearce et al., 2000).Unicellular algae such as Chlamydomonas species are often found in areas that might be contaminated by toxic heavy metals (Merchant et al., 2006). Chlamydomonas species have also been shown to sequester toxic metals (e.g. cadmium and mercury) and have potential use for bioremediation of these metals (Cai et al., 1999; Adhiya et al., 2002; Siripornadulsil et al., 2002; He et al., 2011; Priyadarshani et al., 2011). To determine the kinetics and selectivity of exogenous heavy metal uptake as well as free heavy metal concentration in the cytoplasm of Chlamydomonas species, we developed an MT, FRET-based metal-binding sensor and expressed this in the cytoplasm of the unicellular green alga Chlamydomonas reinhardtii. We demonstrate that heavy metal uptake is rapid in C. reinhardtii and that cytoplasmic free heavy metal concentrations are substantially lower than exogenous free heavy metal concentrations, implying that heavy metals are rapidly sequestered by various biological molecules in the cell.     

Conventionally Tilled and Permanent Raised Beds with Different Crop Residue Management: Effects on Soil C and N Dynamics

Conservation tillage in its version of permanent bed planting under zero-tillage with crop residue retention has been proposed as an alternative wheat production system for northwest Mexico. However, little is known about the dynamics of C and N in soils under wheat/maize on permanent beds (PB) where straw was burned, removed, partly removed or retained, as opposed to conventionally tilled beds (CTB) where straw was incorporated. We investigated the dynamics of soil C and N and normalized difference vegetative index (NDVI) crop values in zero-tilled PB and CTB after 26 successive maize and wheat crops. Organic C and total N were respectively, 1.15 and 1.17 times greater in PB with straw partly removed and with straw retained on the surface, than in CTB with straw incorporated. Organic C and total N were 1.10 times greater in soils with 300 kg N ha⁻¹ added than in unfertilized soil. Cumulative production of CO₂ was lower under CTB with straw incorporated than under PB treatments, and CO₂ production increased with increments in inorganic fertilizer. The N-mineralization rate was 1.18 times greater than in unamended soils when 150 kg inorganic N ha⁻¹ was applied, and 1.48 times greater when 300 kg inorganic N ha⁻¹ was added. The N-mineralization rate was significantly (1.66 times) greater in PB where the straw was burned or retained on the surface than in CTB where the straw was incorporated, but significantly (1.25 times) lower than in PB with straw partly removed. The NDVI values reached a maximum 56 days after planting and decreased thereafter. The NDVI for unfertilized soil were similar for CTB with straw incorporated, PB with straw partly removed, and PB with straw retained on the surface, but significantly lower for PB with straw burned and PB with straw removed. In soils to which 150 or 300 kg N ha⁻¹ was added, NDVI was significantly lower for PB with straw burned than for other treatments. Among other things, this suggests the utility of rotating maize or wheat with crops whose residues have lower C–N ratios, thus avoiding immobilization of large amounts of N for extended periods. PB with residue burning, however, is an unsustainable practice leading to low crop performance and soil and environmental degradation.     

Lifestyle and patterns of physical activity in Hadza foragers

Objectives

Physically active lifestyles are associated with several health benefits. Physical activity (PA) levels are low in post-industrial populations, but generally high throughout life in subsistence populations. The Hadza are a subsistence-oriented foraging population in Tanzania known for being physically active, but it is unknown how recent increases in market integration may have altered their PA patterns. In this study, we examine PA patterns for Hadza women and men who engage in different amounts of traditional foraging.

Materials and Methods

One hundred and seventy seven Hadza participants (51% female, 19–87 years) wore an Axivity accelerometer (dominant wrist) for ~6 days during dry season months. We evaluated the effects of age, sex, and lifestyle measures on four PA measures that capture different aspects of the PA profile.

Results

Participants engaged in high levels of both moderate-intensity PA and inactivity. Although PA levels were negatively associated with age, older participants were still highly active. We found no differences in PA between participants living in more traditional “bush” camps and those living in more settled “village” camps. Mobility was positively associated with step counts for female participants, and schooling was positively associated with inactive time for male participants.

Conclusions

The similarity in PA patterns between Hadza participants in different camp types suggests that high PA levels characterize subsistence lifestyles generally. The sex-based difference in the effects of mobility and schooling on PA could be a reflection of the Hadza's gender-based division of labor, or indicate that changes to subsistence-oriented lifestyles impact women and men in different ways.

     

Influence of permanent raised bed planting and residue management on physical and chemical soil quality in rain fed maize/wheat systems

Densely populated, intensively cropped highland areas in the tropics and subtropics are prone to erosion and declining soil fertility, making agriculture unsustainable. Conservation agriculture in its version of permanent raised bed planting with crop residue retention has been proposed as an alternative wheat production system for this agro-ecological zone. A five years field experiment comparing permanent and tilled raised beds with different residue management under rainfed conditions was started at El Batán (Mexico) (2,240 m asl; 19.31°N, 98.50°W; Cumulic Phaeozem) in 1999. The objective of this study was to determine the soil quality status after five years of different management practices. The K concentration was 1.65 times and 1.43 times larger in the 0–5 cm and 5–20 cm profiles, respectively, for permanent raised beds compared to conventionally tilled raised beds. The Na concentration showed the opposite trend. Sodicity was highest for conventionally tilled raised beds and for permanent raised beds it increased with decreasing amounts of residue retained on the surface. Permanent raised beds with full residue retention increased soil organic matter content 1.4 times in the 0–5 cm layer compared to conventionally tilled raised beds with straw incorporated and it increased significantly with increasing amounts of residue retained on the soil surface for permanent raised beds. Soil from permanent raised beds with full residue retention had significantly higher mean weight diameter for wet and dry sieving compared to conventionally tilled raised beds. Permanent raised beds with full residue retention had significantly higher aggregate stability compared to those with residue removal. A lower aggregation resulted in a reduction of infiltration. Principal component analysis (PCA) was performed using these soil physicochemical variables that were significantly influenced by tillage or residue management. The PC1 and PC2 separated the conventionally tilled raised beds from the permanent raised beds and PC3 separated permanent raised beds with at least partial residue retention from permanent raised beds with no residue retention. These clear separations suggest that tillage and residue management have an effect on soil processes. The research indicates that permanent raised bed planting increases the soil quality and can be a sustainable production alternative for the (sub)tropical highlands. Extensive tillage with its associated high costs can be reduced by the use of permanent raised beds while at least partial surface residue retention is needed to insure production sustainability.     

Hydroxynonenal-generated crosslinking fluorophore accumulation in Alzheimer disease reveals a dichotomy of protein turnover

Lipid peroxidation generates reactive aldehydes, most notably hydroxynonenal (HNE), which covalently bind amino acid residue side chains leading to protein inactivation and insolubility. Specific adducts of lipid peroxidation have been demonstrated in intimate association with the pathological lesions of Alzheimer disease (AD), suggesting that oxidative stress is a major component of AD pathogenesis. Some HNE-protein products result in protein crosslinking through a fluorescent compound similar to lipofuscin, linking lipid peroxidation and the lipofuscin accumulation that commonly occurs in post-mitotic cells such as neurons. In this study, brain tissue from AD and control patients was examined by immunocytochemistry and immunoelectron microscopy for evidence of HNE-crosslinking modifications of the type that should accumulate in the lipofuscin pathway. Strong labeling of granulovacuolar degeneration (GVD) and Hirano bodies was noted but lipofuscin did not contain this specific HNE-fluorophore. These findings directly implicate lipid crosslinking peroxidation products as accumulating not in the lesions or the lipofuscin pathways, but instead in a distinct pathway, GVD, that accumulates cytosolic proteins.     

Macrophage receptors responsible for distinct recognition of low density lipoprotein containing pyrrole or pyridinium adducts: models of oxidized low density lipoprotein

Oxidation of low density lipoproteins (LDL) induced by incubation with Cu(2+) ions results in the formation of a heterogeneous group of aldehydic adducts on lysyl residues (Lys) of apolipoprotein B (apoB) that are thought to be responsible for the uptake of oxidized LDL (oxLDL) by macrophages. To define the structural and chemical criteria governing such cell recognition, we induced two modifications of lysines in LDL that mimic prototypic adducts present in oxLDL; namely, epsilon-amino charge-neutralizing pyrrolation by treatment with 2,5-hexanedione (hdLDL), and epsilon-amino charge-retaining pyridinium formation via treatment with 2,4,6-trimethylpyrylium (tmpLDL). Both modifications led to recognition by receptors on mouse peritoneal macrophages (MPM). To assess whether the murine scavenger receptor class A-I (mSR-A) was responsible for recognition of hdLDL or tmpLDL in MPM, we measured binding at 4 degrees C and degradation at 37 degrees C of these modified forms of (125)I-labeled LDL by mSR-A-transfected CHO cells. Although uptake and degradation of hdLDL by mSR-A-transfected CHO cells was quantitatively similar to that of the positive control, acLDL, tmpLDL was not recognized by these cells. However, both tmpLDL and hdLDL were recognized by 293 cells that had been transfected with CD36. In the human monocytic cell line THP-1 that had been activated with PMA, uptake of tmpLDL was significantly inhibited by blocking monoclonal antibodies to CD36, further suggesting recognition of tmpLDL by this receptor. Macrophage uptake and degradation of LDL oxidized by brief exposure to Cu(2+) was inhibited more effectively by excess tmpLDL and hdLDL than was more extensively oxidized LDL, consistent with the recognition of the former by CD36 and the latter primarily by SR-A.Collectively, these studies suggest that formation of specific pyrrole adducts on LDL leads to recognition by both the mSR-A and mouse homolog of CD36 expressed on MPM, while formation of specific pyridinium adducts on LDL leads to recognition by the mouse homolog of CD 36 but not by mSR-A. As such, these two modifications of LDL may represent useful models for dissecting the relative contributions of specific modifications on LDL produced during oxidation, to the cellular uptake of this heterogeneous ligand.     

Differential inhibition of six copper amine oxidases by a family of 4-(aryloxy)-2-butynamines: evidence for a new mode of inactivation

A series of compounds derived from a previously identified substrate analogue of copper amine oxidases (CuAOs) (Shepard et al. (2002) Eur. J. Biochem. 269, 3645-3658) has been screened against six different CuAOs with a view to designing potent and selective inhibitors. The substrate analogues investigated were 4-(1-naphthyloxy)-2-butyn-1-amine, 4-(2-methylphenoxy)-2-butyn-1-amine, 4-(3-methylphenoxy)-2-butyn-1-amine, 4-(4-methylphenoxy)-2-butyn-1-amine, and 4-phenoxy-2-butyn-1-amine. These compounds were screened against equine plasma amine oxidase (EPAO), Pisum sativum amine oxidase (PSAO), Pichia pastoris lysyl oxidase (PPLO), bovine plasma amine oxidase (BPAO), human kidney diamine oxidase (KDAO), and Arthrobacter globiformis amine oxidase (AGAO) to examine the effect of different substituent groups on potency. Despite the similar structures of the 4-aryloxy analogues evaluated, striking differences in potency were observed. In addition, crystal structures of AGAO derivitized with 4-(2-naphthyloxy)-2-butyn-1-amine and 4-(4-methylphenoxy)-2-butyn-1-amine were obtained at a resolution of 1.7 A. The structures reveal a novel and unprecedented reaction mechanism involving covalent attachment of the alpha,beta-unsaturated aldehyde turnover product to the amino group of the reduced 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor. Collectively, the structural and inhibition results support the feasibility of designing selective mechanism-based inhibitors of copper amine oxidases.     

Ovine pedomics: the first study of the ovine foot 16S rRNA-based microbiome

We report the first study of the bacterial microbiome of ovine interdigital skin based on 16S rRNA by pyrosequencing and conventional cloning with Sanger-sequencing. Three flocks were selected, one a flock with no signs of footrot or interdigital dermatitis, a second flock with interdigital dermatitis alone and a third flock with both interdigital dermatitis and footrot. The sheep were classified as having either healthy interdigital skin (H) and interdigital dermatitis (ID) or virulent footrot (VFR). The ovine interdigital skin bacterial community varied significantly by flock and clinical condition. The diversity and richness of operational taxonomic units was greater in tissue from sheep with ID than H or VFR-affected sheep. Actinobacteria, Bacteriodetes, Firmicutes and Proteobacteria were the most abundant phyla comprising 25 genera. Peptostreptococcus, Corynebacterium and Staphylococcus were associated with H, ID and VFR, respectively. Sequences of Dichelobacter nodosus, the causal agent of ovine footrot, were not amplified because of mismatches in the 16S rRNA universal forward primer (27F). A specific real-time PCR assay was used to demonstrate the presence of D. nodosus, which was detected in all samples including the flock with no signs of ID or VFR. Sheep with ID had significantly higher numbers of D. nodosus (10⁴–10⁹ cells per g tissue) than those with H or VFR feet.