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.     

Neurological Damage in MSUD: The Role of Oxidative Stress

Maple syrup urine disease (MSUD) is a metabolic disease caused by a deficiency in the branched-chain α-keto acid dehydrogenase complex, leading to the accumulation of branched-chain keto acids and their corresponding branched-chain amino acids (BCAA) in patients. Treatment involves protein-restricted diet and the supplementation with a specific formula containing essential amino acids (except BCAA) and micronutrients, in order to avoid the appearance of neurological symptoms. Although the accumulation of toxic metabolites is associated to appearance of symptoms, the mechanisms underlying the brain damage in MSUD remain unclear, and new evidence has emerged indicating that oxidative stress contributes to this damage. In this context, this review addresses some of the recent findings obtained from cells lines, animal studies, and from patients indicating that oxidative stress is an important determinant of the pathophysiology of MSUD. Recent works have shown that the metabolites accumulated in the disease induce morphological alterations in C6 glioma cells through nitrogen reactive species generation. In addition, several works demonstrated that the levels of important antioxidants decrease in animal models and also in MSUD patients (what have been attributed to protein-restricted diets). Also, markers of lipid, protein, and DNA oxidative damage have been reported in MSUD, probably secondary to the high production of free radicals. Considering these findings, it is well-established that oxidative stress contributes to brain damage in MSUD, and this review offers new perspectives for the prevention of the neurological damage in MSUD, which may include the use of appropriate antioxidants as a novel adjuvant therapy for patients.     

The rs225017 Polymorphism in the 3′UTR of the Human DIO2 Gene Is Associated with Increased Insulin Resistance

The Thr92Ala (rs225014) polymorphism in the type 2 deiodinase (DIO2) gene has been associated with insulin resistance (IR) and decreased enzyme activity in human tissues but kinetic studies failed to detect changes in the mutant enzyme, suggesting that this variant might be a marker of abnormal DIO2 expression. Thus, we aimed to investigate whether other DIO2 polymorphisms, individually or in combination with the Thr92Ala, may contribute to IR. The entire coding-region of DIO2 gene was sequenced in 12 patients with type 2 diabetes mellitus (T2DM). Potentially informative variants were evaluated in 1077 T2DM patients and 516 nondiabetic subjects. IR was evaluated using the homeostasis model assessment (HOMA-IR) index. DIO2 gene sequencing revealed no new mutation but 5 previously described single nucleotide polymorphisms (SNPs). We observed that all T2DM patients displaying high HOMA-IR index (n = 6) were homozygous for the rs225017 (T/A) polymorphism. Further analysis showed that the median fasting plasma insulin and HOMA-IR of T2DM patients carrying the T/T genotype were higher than in patients carrying the A allele (P = 0.013 and P = 0.002, respectively). These associations were magnified in the presence of the Ala92Ala genotype of the Thr92Ala polymorphism. Moreover, the rs225017 and the Thr92Ala polymorphisms were in partial linkage disequilibrium (|D′| = 0.811; r ² = 0.365). In conclusion, the rs225017 polymorphism is associated with greater IR in T2DM and it seems to interact with the Thr92Ala polymorphism in the modulation of IR.     

Prolonged use of letrozole causes morphological changes on gonads in Galea spixii

Letrozole is used as a therapeutic agent in reproductive disorders caused by high estrogen levels. Letrozole inhibits cytochrome P450 aromatase and reduces estrogen levels. However, the effects of long-term use on reproductive traits are unknown. The aim of this study was to evaluate the prolonged use of letrozole in the gonads of rodents (Spix''s yellow-toothed cavy; Galea spixii). Forty-eight rodents (24 males and 24 females) were randomly divided into the treated and control groups. Letrozole administration started at 15 days of age and continued weekly until 30, 45, 90, and 120 days of age. The body, testis, and ovary weights were analyzed, as well as the morphological progression of spermatogenesis and folliculogenesis. Macroscopically, body weight gain and gonads weight were increased in the letrozole group. Microscopically, the ovaries of treated females showed stratified epithelium and a cellular disorder of the tunica albuginea. In the testes of treated males, the development of seminiferous tubules was delayed and sperm was absent. The collective findings indicate that the prolonged use of letrozole alters secondary sexual characteristics, and causes weight gain, reproductive changes, and male infertility.     

Behavioral and metabolic effects of central injections of orexins/hypocretins in pigeons (Columba livia)

In the present study, the acute behavioral and ingestive effects of ICV injections of mammalian orexin-A (ORXA; vehicle, 0.2, 0.6 or 2 nmol) and of orexin-B (ORXB; vehicle, 0.2, 0.6 or 2 nmol), as well as possible long-term effects (through 24 h of continuous intake monitoring after 0.6 nmol of ORXA or ORXB) of these treatments in food/water intake and in blood levels of metabolic fuels (free fatty acids and glucose, after 0.2 or 0.6 nmol of ORXA) were examined in adult male pigeons. Both ORXA and ORXB treatments failed to produce acute (1–3 h) or long-term effects on feeding and drinking behaviors, and did not change blood free fatty acids and glucose 15 and 30 min after treatments, as compared to vehicle-treated animals. However, ORXA (but not ORXB) treatments evoked a dose-related, intense increase in exploratory behaviors, associated to reduced time spent in alert immobility and sleep-typical postures. These data substantiate the lack of orexigenic effects of ORXs in avian species, and suggest that an important role in vigilance control may represent a conserved functional attribute of orexinergic circuits in vertebrates.     

Antioxidant Effect of Cysteamine in Brain Cortex of Young Rats

Cysteamine is a cystine-depleting drug used in the treatment of cystinosis, a metabolic disorder caused by deficiency of the lysosomal cystine carrier. As a result, cystine accumulates within lysosomes in many tissues and organs, including the nervous system. Studies with cystine dimethyl ester loaded cells suggest that cystine might induce apoptosis through oxidative stress. Our objective was to investigate the effects of co-administration of cysteamine with the oxidant cystine dimethyl ester on several parameters of oxidative stress in the brain cortex of rats. Animals were injected with 1.6 μmol/g cystine dimethyl ester and/or 0.26 μmol/g body weight cysteamine. Cystine dimethyl ester induced lipoperoxidation, protein carbonylation, and stimulated superoxide dismutase, glutathione peroxidase and catalase activities, probably through the formation of free radicals. Cysteamine prevented those effects, possibly increasing cellular thiol pool and acting as a scavenger of free radicals. These results suggest that the antioxidant effect of cysteamine may be important in the treatment of cystinosis.     

Inhibitory effect of extracts from Brazilian medicinal plants on the adhesion of <Emphasis Type="Italic">Candida albicans</Emphasis> to buccal epithelial cells

Plants are known to produce a plethora of secondary metabolites which are recognized as a useful source of new drugs or drug leads. Extracts and fractions of Schinus terebinthifolius Raddi (Anacardiaceae), Piper regnellii C.D.C. (Piperaceae), Rumex acetosa L. (Polygonaceae), and Punica granatum L. (Punicaceae) were assessed for their antifungal activity against eight clinical isolates of C. albicans. They were also evaluated for their effect on the adhesion of these C. albicans isolates to buccal epithelial cells (BECs). The ethyl acetate fraction from the leaves of S. terebinthifolius showed promising activity, inhibiting the growth of three C. albicans isolates at 7.8 μg ml⁻¹ and significantly inhibiting their adhesion to BEC at 15 μg ml⁻¹ . In addition, this fraction did not show cytotoxic activity against murine macrophages. The results show the potential of the plant extracts studied as a source of new antifungal compounds. Further studies are necessary for isolation and characterization of the active compounds of these plants.     

Comparison of the mass and energy exchange of a pasture and a mature transitional tropical forest of the southern Amazon Basin during a seasonal transition

This research utilized tower‐based eddy covariance to quantify the trends in net ecosystem mass (CO₂ and H₂O vapor) and energy exchange of important land‐cover types of NW Mato Grosso during the March–December 2002 seasonal transition. Measurements were made in a mature transitional (ecotonal) tropical forest near Sinop, Mato Grosso, and a cattle pasture near Cotriguaçú, Mato Grosso, located 500 km WNW of Sinop. Pasture net ecosystem CO₂ exchange (NEE) was considerably more variable than the forest NEE over the seasonal transition, and the pasture had significantly higher rates of maximum gross primary production in every season except the dry–wet season transition (September–October). The pasture also had significantly higher rates of whole‐ecosystem dark respiration than the forest during the wetter times of the year. Average (±95% CI) rates of total daily NEE during the March–December 2002 measurement period were 26±15 mmol m^?2 day^?1 for the forest (positive values indicate net CO₂ loss by the ecosystem) and ?38±26 mmol m^?2 day^?1 for the pasture. While both ecosystems partitioned more net radiation (R_n) into latent heat flux (L_e), the forest had significantly higher rates of L_e and lower rates of sensible heat flux (H) than the pasture; a trend that became more extreme during the onset of the dry season. Large differences in pasture and forest mass and energy exchange occurred even though seasonal variations in micrometeorology (air temperature, humidity, and radiation) were relatively similar for both ecosystems. While the short measurement period and lack of spatial replication limit the ability to generalize these results to pasture and forest regions of the Amazon Basin, these results suggest important differences in the magnitude and seasonal variation of NEE and energy partitioning for pasture and transitional tropical forest.     

Propionic and methylmalonic acids increase cAMP levels in slices of cerebral cortex of young rats via adrenergic and glutamatergic mechanisms

We have previously described that propionic (PA) and methylmalonic (MMA) acids increased the in vitro phosphorylation of cytoskeletal proteins through cAMP-dependent protein kinase and glutamate. In the present study we investigated the in vitro effects of 1 mM glutamate, 2.5 mM MMA and 2.5 mM PA on cAMP levels in the slices of cerebral cortex of young rats. Results showed that PA, MMA and glutamate increased cAMP levels after 30 min of incubation, while the beta-adrenergic agonist epinephrine elicited a similar effect only at a shorter incubation time. Then effects were prevented by the beta-adrenergic antagonist propranolol, rather than by glutamate antagonists (AP5, CNQX and MCPG), suggesting that they were mediated by beta-adrenergic receptors. In addition, glutamate antagonists per se induced increased cAMP levels; however propranolol prevented only the effect elicited by the metabotropic glutamate antagonist MCPG. Taken together, it is feasible that PA and MMA increase cAMP synthesis via a beta-adrenergic/G protein coupled pathway, in a glutamate-dependent manner. Although additional studies will be necessary to evaluate the importance of these observations for the neuropathology of propionic and methylmalonic acidemias, it is possible that high brain cAMP levels may contribute to a certain extent to the neurological dysfunction of the affected individuals.