Covalent inhibition of P. falciparum ferredoxin-NADP+ reductase: Exploring alternative strategies for the development of new antimalarial drugs.

The protozoan Plasmodium falciparum is the main aetiological agent of tropical malaria. Characteristic of the phylum is the presence of a plastid-like organelle which hosts several homologs of plant proteins, including a ferredoxin (PfFd) and its NADPH-dependent reductase (PfFNR). The PfFNR/PfFd redox system is essential for the parasite, while mammals share no homologous proteins, making the enzyme an attractive target for novel and much needed antimalarial drugs. Based on previous findings, three chemically reactive residues important for PfFNR activity were identified: namely, the active-site Cys99, responsible for hydride transfer; Cys284, whose oxidation leads to an inactive dimeric form of the protein; and His286, which is involved in NADPH binding. These amino acid residues were probed by several residue-specific reagents and the two cysteines were shown to be promising targets for covalent inhibition. The quantitative and qualitative description of the reactivity of few compounds, including a repurposed drug, set the bases for the development of more potent and specific antimalarial leads.

Ion-induced PCR strategy for mercury detection.

Mercury contamination is one of the most serious environmental problems. It can cause serious effects on the human health, such as case damage in the brain, nervous system, immune system, and kidney failure. Therefore, development of an accurate, sensitive, and simple operational detection method for mercury is very necessary. Herein, we report a new strategy for mercury ion detection based on commonly used PCR technique. High selectivity and sensitivity were achieved by the formation of the thymine-Hg-thymine (T-Hg-T) unnatural base pair at the 3'-end of PCR primers. The detection results of PCR amplification in presence of mercury ion could be reported either by using agarose gel analysis or through real-time fluorometric dye tracing for different detection purposes. To our knowledge, this study represents the first application of PCR based technique to the detection of metal ions.

Assessment of mobility and bioavailability of mercury compounds in sewage sludge and composts.

Content of heavy metals, including mercury, determines the method of management and disposal of sewage sludge. Excessive concentration of mercury in composts used as organic fertilizer may lead to accumulation of this element in soil and plant material. Fractionation of mercury in sewage sludge and composts provides a better understanding of the extent of mobility and bioavailability of the different mercury species and helps in more informed decision making on the application of sludge for agricultural purposes. The experimental setup comprises the composing process of the sewage sludge containing 13.1mgkg-1 of the total mercury, performed in static reactors with forced aeration. In order to evaluate the bioavailability of mercury, its fractionation was performed in sewage sludge and composts during the process. An analytical procedure based on four-stage sequential extraction was applied to determine the mercury content in the ion exchange (water soluble and exchangeable Hg), base soluble (Hg bound to humic and fulvic acid), acid soluble (Hg bound to Fe/Mn oxides and carbonates) and oxidizable (Hg bound to organic matter and sulphide) fractions. The results showed that from 50.09% to 64.55% of the total mercury was strongly bound to organo-sulphur and inorganic sulphide; that during composting, increase of concentrations of mercury compounds strongly bound with organic matter and sulphides; and that mercury content in the base soluble and oxidizable fractions was strongly correlated with concentration of dissolved organic carbon in those fractions.

Mechanisms involved in the transport of mercuric ions in target tissues.

Mercury exists in the environment in various forms, all of which pose a risk to human health. Despite guidelines regulating the industrial release of mercury into the environment, humans continue to be exposed regularly to various forms of this metal via inhalation or ingestion. Following exposure, mercuric ions are taken up by and accumulate in numerous organs, including brain, intestine, kidney, liver, and placenta. In order to understand the toxicological effects of exposure to mercury, a thorough understanding of the mechanisms that facilitate entry of mercuric ions into target cells must first be obtained. A number of mechanisms for the transport of mercuric ions into target cells and organs have been proposed in recent years. However, the ability of these mechanisms to transport mercuric ions and the regulatory features of these carriers have not been characterized completely. The purpose of this review is to summarize the current findings related to the mechanisms that may be involved in the transport of inorganic and organic forms of mercury in target tissues and organs. This review will describe mechanisms known to be involved in the transport of mercury and will also propose additional mechanisms that may potentially be involved in the transport of mercuric ions into target cells.

Structural and Biochemical Characterization of a Copper-Binding Mutant of the Organomercurial Lyase MerB: Insight into the Key Role of the Active Site Aspartic Acid in Hg-Carbon Bond Cleavage and Metal Binding Specificity.

In bacterial resistance to mercury, the organomercurial lyase (MerB) plays a key role in the detoxification pathway through its ability to cleave Hg-carbon bonds. Two cysteines (C96 and C159; Escherichia coli MerB numbering) and an aspartic acid (D99) have been identified as the key catalytic residues, and these three residues are conserved in all but four known MerB variants, where the aspartic acid is replaced with a serine. To understand the role of the active site serine, we characterized the structure and metal binding properties of an E. coli MerB mutant with a serine substituted for D99 (MerB D99S) as well as one of the native MerB variants containing a serine residue in the active site (Bacillus megaterium MerB2). Surprisingly, the MerB D99S protein copurified with a bound metal that was determined to be Cu(II) from UV-vis absorption, inductively coupled plasma mass spectrometry, nuclear magnetic resonance, and electron paramagnetic resonance studies. X-ray structural studies revealed that the Cu(II) is bound to the active site cysteine residues of MerB D99S, but that it is displaced following the addition of either an organomercurial substrate or an ionic mercury product. In contrast, the B. megaterium MerB2 protein does not copurify with copper, but the structure of the B. megaterium MerB2-Hg complex is highly similar to the structure of the MerB D99S-Hg complexes. These results demonstrate that the active site aspartic acid is crucial for both the enzymatic activity and metal binding specificity of MerB proteins and suggest a possible functional relationship between MerB and its only known structural homologue, the copper-binding protein NosL.

Mercury binding to proteins disclosed by ESI MS experiments: The case of three organomercurials.

Solution interactions of three organomercury compounds, i.e., methylmercury chloride, thimerosal and phenylmercury acetate, with a group of biochemically relevant proteins, namely cytochrome c (Cyt c), ribonuclease A (RNase A), carbonic anhydrase I (hCA I), superoxide dismutase (SOD), and serum albumin (HSA), were investigated using an established ESI MS approach. Temporal analysis of sample aliquots provided insight into the binding kinetics, while comparative analysis of the obtained mass spectra disclosed adduct formation of each mercurial with the tested proteins and the relative abundance of the species. The three organomercurials bind, exclusively and tightly, to free cysteine residues as no binding was observed in the case of proteins lacking such groups. hCA I, SOD and HSA formed distinct mercury adducts, preserving the Hg bound alkyl/aryl ligands; yet, the three organomercurials displayed significant differences in reactivity in relation to their chemical structure. The investigation was then extended to analyze the reactions with the C-terminal dodecapeptide of the enzyme human thioredoxin reductase, which contains a characteristic selenol-thiol moiety: tight Hg binding was observed. Notably, this peptide was able to remove effectively and completely the alkyl/aryl ligands of the three tested organomercurials; this behavior may be relevant to the detoxification mechanism of organomercurials in mammals. Finally, a competition experiment was carried out to establish whether protein bound mercury centers may be displaced by other competing metals. Interestingly, and quite unexpectedly, we observed that a protein bound mercury fragment may be partially displaced from its coordination site in hCA I by the medicinal gold compound auranofin.

New insights into the metabolism of organomercury compounds: mercury-containing cysteine S-conjugates are substrates of human glutamine transaminase K and potent inactivators of cystathionine γ-lyase.

Anthropogenic practices and recycling in the environment through natural processes result in release of potentially harmful levels of mercury into the biosphere. Mercury, especially organic forms, accumulates in the food chain. Mercury reacts readily with sulfur-containing compounds and often exists as a thiol S-conjugate, such as the l-cysteine (Cys)-S-conjugate of methylmercury (CH(3)Hg-S-Cys) or inorganic mercury (Cys-S-Hg-S-Cys). These S-conjugates are structurally similar to l-methionine and l-cystine/l-cystathionine, respectively. Bovine and rat glutamine transaminase K (GTK) catalyze transamination of sulfur-containing amino acids. Recombinant human GTK (rhGTK) has a relatively open catalytic active site, and we report here that this enzyme, like the rat and bovine enzymes, can also utilize sulfur-containing l-amino acids, including l-methionine, l-cystine, and l-cystathionine as substrates. The current study extends this list to include mercuric S-conjugates, and shows that CH(3)Hg-S-Cys and Cys-S-Hg-S-Cys are substrates and reversible inhibitors of rhGTK. The homocysteine S-conjugates, Hcy-S-Hg-S-Hcy and CH(3)Hg-S-Hcy, are also inhibitors. Finally, we show that HgCl(2), CH(3)Hg-S-Cys and Cys-S-Hg-S-Cys are potent irreversible inhibitors of rat cystathionine γ-lyase. The present study broadens our knowledge of the biochemistry of mercury compounds by showing that Cys S-conjugates of mercury interact with enzymes that catalyze transformations of biologically important sulfur-containing amino acids.

Localizing organomercury uptake and accumulation in zebrafish larvae at the tissue and cellular level.

Using synchrotron x-ray fluorescence mapping, we have examined the uptake and localization of organic mercury in zebrafish larvae. Strikingly, the greatest accumulation of methyl and ethyl mercury compounds was highly localized in the rapidly dividing lens epithelium, with lower levels going to brain, optic nerve, and various other organs. The data suggest that the reported impairment of visual processes by mercury may arise not only from previously reported neurological effects, but also from direct effects on the ocular tissue. This novel approach is a powerful tool for directly investigating the molecular toxicology of heavy metals, and should be equally applicable to the study of a wide range of elements in developing embryos.

Volatilization of metal mercury from Organomercurials by highly mercury-resistant Acidithiobacillus ferrooxidans MON-1.

The iron-oxidizing bacterium Acidithiobacillus ferrooxidans MON-1 is highly resistant not only to mercuric chloride (HgCl(2)) but also to organomercurials such as methylmercury chloride (MMC). We have found that cytochrome c oxidase, purified from strain MON-1, reduces Hg(2+) to volatilizable metal mercury (Hg(0)) with reduced mammalian cytochrome c or Fe(2+) as an electron donor. In this study we found that cytochrome c oxidase can volatilize Hg(0) from MMC as well as from Hg(2+) with reduced mammalian cytochrome c or c-type cytochrome purified from strain MON-1 as an electron donor. We also found that MMC-Hg(0) volatilization activity is present in the MON-1 plasma membrane but not in the cytosol. These activities were strongly inhibited by sodium cyanide (NaCN) and the antibody produced against purified MON-1 cytochrome c oxidase. This is the first report to indicate that cytochrome c oxidase is involved in the degradation of organomercurials in microorganisms.

Levels of organic and inorganic mercury in human blood predicted from measurements of total mercury.

The toxicologically relevant mercury species inorganic and organic Hg in blood are frequently determined by separate measurements of total Hg and of inorganic Hg, with their difference indicating organic Hg. It is shown that the different partition of inorganic and organic Hg between erythrocytes and plasma (e/p ratio) can be used to calculate the concentrations of either Hg species in either blood constituent from measurement of total Hg only. This was tested on the blood of different groups of volunteers. The calculated concentrations of inorganic and organic Hg in cells and plasma were then compared by linear regression with their previously measured counterparts. An accurate prediction has been found for individual levels of inorganic Hg in plasma and organic Hg in cells. These calculated levels were little affected by variations of the e/p ratios. The coincidence between calculated and measured levels of inorganic Hg in cells and organic Hg in plasma was more sensitive to alterations of the e/p ratios. In conclusion, the relevant concentrations of inorganic Hg in plasma and organic Hg in cells can reliably be calculated from measurements of total Hg and from assumed e/p ratios. This means a sizeable reduction of analytical work, and also provides specific information in cases of low-level co-exposure to both Hg species. Besides the possibility to introduce automated analyses of total Hg in mercury speciation in blood, the proposed calculation scheme has the potential to easily enlarge the data base in epidemiological and toxicological surveys of mercury exposure.

Visualization of polymercurimethane-labeled for bacteriophage in the scanning transmission electron microscope.

Each of the 2700 coat proteins of fd bacteriophage was labeled with tetrakis(acetoxymercuri)methane (TAMM) or aquoglycylmethionineplatinum(II). The TAMM-labeled specimens reveal striking bright spots in the scanning transmission electron microscope which arise from clustering. Measurements of mass show increases consistent with the addition of four mercury atoms or one platinum atom, respectively, to each coat protein.

Regulation of steroid hormone receptor activity.

PIP: Studies in receptorology since the early 1960s have helped to broaden current knowledge regarding the mechanism of action of steroid hormones. A detailed biochemical explanation from a literature survey focusses attention on the functional activity of the receptor as it exists within the living cell instead of simply in an isolated in vitro tissue preparation. The studies have shown the assortment of exogenous and endogenous factors which influence receptor activity, to varying degrees and often in conflicting directions. Different cell populations in the same tissue may respond to any hormone in different ways. Dissimilarities exist across interspecies lines. It is obvious from all the receptor studies that the responsiveness of a cell to steroid hormone stimulation is dependent on the presence of functionally competent receptor proteins which can be measured in the cytoplasmic compartment of the resting cell. A general hypothesis as to the way in which hormone receptor functions is advanced and explained. There seem to be multiple regions of specific acceptor-type binding sites. Cells respond to estradiol by the 3 separate mechanisms of synthesis, reutilization, and recycling. Progesterone, on the other hand, seems to have an overall negative regulatory control.^ieng

Revision of reciprocal action of mercury and selenium.

Diverse forms of mercury (Hg) have various effects on animals and humans because of a variety of routes of administration. Inorganic mercury (iHg) binds to thiol groups of proteins and enzymes in one's body or is methylated by microorganisms. Organic form of Hg, contrary to the iHg, is more stable but may be demethylated to Hg2+ in the tissue of intestinal flora. Selenium (Se) also occurs in a variety of chemical forms in one's body but both of these elements behave very differently from one another. Mercury binding to selenide or Se-containing ligands is a primary molecular mechanism that reduces toxicity of Hg. Complexes formed in such a way are irreversible, and thus, biologically inactive. Se deficiency in a human body may impair normal synthesis of selenoproteins and its expression because expression of mRNA may be potentially regulated by the Se status. This paper provides a comprehensive review concerning Hg-Se reciprocal action as a potential mechanism of protective action of Se against Hg toxicity as well as a potential detoxification mechanism. Although interactions between Hg-Se have been presented in numerous studies concerning animals and humans, we have focused mainly on animal models so as to understand molecular mechanisms responsible for antagonism better. The review also investigates what conclusions have been drawn by researchers with respect to the chemical species of Se and Hg (and their relationship) in biological systems as well as genetic variations and expression and/or activity of selenoproteins related to the thioredoxin (thioredoxin Trx/TrxR) system and glutathione metabolism. Int J Occup Med Environ Health 2018;31(5):575-592.

Coupling two mercury resistance genes in Eastern cottonwood enhances the processing of organomercury.

Eastern cottonwood (Populus deltoides Bartr. ex Marsh.) trees were engineered to express merA (mercuric ion reductase) and merB (organomercury lyase) transgenes in order to be used for the phytoremediation of mercury-contaminated soils. Earlier studies with Arabidopsis thaliana and Nicotiana tabacum showed that this gene combination resulted in more efficient detoxification of organomercurial compounds than did merB alone, but neither species is optimal for long-term field applications. Leaf discs from in vitro-grown merA, nptII (neomycin phosphotransferase) transgenic cottonwood plantlets were inoculated with Agrobacterium tumefaciens strain C58 carrying the merB and hygromycin resistance (hptII) genes. Polymerase chain reaction of shoots regenerated from the leaf discs under selection indicated an overall transformation frequency of 20%. Western blotting of leaves showed that MerA and MerB proteins were produced. In vitro-grown merA/merB plants were highly resistant to phenylmercuric acetate, and detoxified organic mercury compounds two to three times more rapidly than did controls, as shown by mercury volatilization assay. This indicates that these cottonwood trees are reasonable candidates for the remediation of organomercury-contaminated sites.

Mercury in human brain, blood, muscle and toenails in relation to exposure: an autopsy study.

BACKGROUND: The main forms of mercury (Hg) exposure in the general population are methylmercury (MeHg) from seafood, inorganic mercury (I-Hg) from food, and mercury vapor (Hg0) from dental amalgam restorations. While the distribution of MeHg in the body is described by a one compartment model, the distribution of I-Hg after exposure to elemental mercury is more complex, and there is no biomarker for I-Hg in the brain. The aim of this study was to elucidate the relationships between on the one hand MeHg and I-Hg in human brain and other tissues, including blood, and on the other Hg exposure via dental amalgam in a fish-eating population. In addition, the use of blood and toenails as biological indicator media for inorganic and organic mercury (MeHg) in the tissues was evaluated. METHODS: Samples of blood, brain (occipital lobe cortex), pituitary, thyroid, abdominal muscle and toenails were collected at autopsy of 30 deceased individuals, age from 47 to 91 years of age. Concentrations of total-Hg and I-Hg in blood and brain cortex were determined by cold vapor atomic fluorescence spectrometry and total-Hg in other tissues by sector field inductively coupled plasma-mass spectrometry (ICP-SFMS). RESULTS: The median concentrations of MeHg (total-Hg minus I-Hg) and I-Hg in blood were 2.2 and 1.0 microg/L, and in occipital lobe cortex 4 and 5 microg/kg, respectively. There was a significant correlation between MeHg in blood and occipital cortex. Also, total-Hg in toenails correlated with MeHg in both blood and occipital lobe. I-Hg in both blood and occipital cortex, as well as total-Hg in pituitary and thyroid were strongly associated with the number of dental amalgam surfaces at the time of death. CONCLUSION: In a fish-eating population, intake of MeHg via the diet has a marked impact on the MeHg concentration in the brain, while exposure to dental amalgam restorations increases the I-Hg concentrations in the brain. Discrimination between mercury species is necessary to evaluate the impact on Hg in the brain of various sources of exposure, in particular, dental amalgam exposure.

Phytodetoxification of hazardous organomercurials by genetically engineered plants.

Methylmercury is a highly toxic, organic derivative found in mercury-polluted wetlands and coastal sediments worldwide. Though commonly present at low concentrations in the substrate, methylmercury can biomagnify to concentrations that poison predatory animals and humans. In the interest of developing an in situ detoxification strategy, a model plant system was transformed with bacterial genes (merA for mercuric reductase and merB for organomercurial lyase) for an organic mercury detoxification pathway. Arabidopsis thaliana plants expressing both genes grow on 50-fold higher methylmercury concentrations than wild-type plants and up to 10-fold higher concentrations than plants that express merB alone. An in vivo assay demonstrated that both transgenes are required for plants to detoxify organic mercury by converting it to volatile and much less toxic elemental mercury.

Structure of HIV-1 reverse transcriptase in a complex with the non-nucleoside inhibitor alpha-APA R 95845 at 2.8 A resolution.

BACKGROUND: HIV-1 reverse transcriptase (RT) is a multifunctional enzyme that copies the RNA genome of HIV-1 into DNA. It is a heterodimer composed of a 66 kDa (p66) and a 51 kDa (p51) subunit. HIV-1 RT is a crucial target for structure-based drug design, and potent inhibitors have been identified, whose efficacy, however, is limited by drug resistance. RESULTS: The crystal structure of HIV-1 RT in complex with the non-nucleoside inhibitor alpha-anilinophenyl-acetamide (alpha-APA) R95845 has been determined at 2.8 A resolution. The inhibitor binds in a hydrophobic pocket near the polymerase active site. The pocket contains five aromatic amino acid residues and the interactions of the side chains of these residues with the aromatic rings of non-nucleoside inhibitors appear to be important for inhibitor binding. Most of the amino acid residues where mutations have been correlated with high levels of resistance to non-nucleoside inhibitors of HIV-1 RT are located close to alpha-APA. The overall fold of HIV-1 RT in complex with alpha-APA is similar to that found when in complex with nevirapine, another non-nucleoside inhibitor, but there are significant conformational changes relative to an HIV-1 RT/DNA/Fab complex. CONCLUSIONS: The non-nucleoside inhibitor-binding pocket has a flexible structure whose mobility may be required for effective polymerization, and may be part of a hinge that permits relative movements of two subdomains of the p66 subunit denoted the 'palm' and 'thumb'. An understanding of the structure of the inhibitor-binding pocket, of the interactions between HIV-1 RT and alpha-APA, and of the locations of mutations that confer resistance to inhibitors provides a basis for structure-based design of chemotherapeutic agents for the treatment of AIDS.

Volatilization and recovery of mercury from mercury-polluted soils and wastewaters using mercury-resistant Acidithiobacillus ferrooxidans strains SUG 2-2 and MON-1.

Iron-oxidizing bacterium, Acidithiobacillus ferrooxidans, is one of the most important bacteria for the bioleaching of copper and gold ores. In order to use the mercury reducing activity of A. ferrooxidans for the bioremediation of mercury, mercury-resistant A. ferrooxidans strains SUG 2-2 and MON-1 were screened among 150 strains of iron-oxidizing bacteria isolated from natural environments. It was found that strains SUG 2-2 and MON-1 have a novel ferrous iron-dependent mercury volatilization activity as well as an NADPH-dependent mercury reductase activity. Strain MON-1 has an organomercurial lyase-like activity and grew most rapidly in an iron medium with 0.1 microM p-chloromercuribenzoic acid among 11 A. ferrooxidans strains tested. Nearly 100% of the total mercury in mercury-polluted soil or mercury wastewater was volatilized and recovered by incubating SUG 2-2 or MON-1 cells in 20 ml of an acidified water (pH 2.5) with ferrous iron, suggesting that these mercury-resistant strains can be used for the bioremediation of inorganic and organic mercurial compounds. We show for the first time that MON-1 cells immobilized in polyvinyl alcohol (PVA) resins could efficiently volatilize mercury from 2 L of a synthetic mercury-polluted wastewater (pH 2.5) containing 40 microM Hg(2+) and ferrous iron. The MON-1-immobilized PVA resins were used repeatedly.

Compensatory Renal Hypertrophy and the Uptake of Cysteine S-Conjugates of Hg2+ in Isolated S2 Proximal Tubular Segments.

Chronic kidney disease is characterized by a progressive and permanent loss of functioning nephrons. In order to compensate for this loss, the remaining functional nephrons undergo significant structural and functional changes. We hypothesize that luminal uptake of inorganic mercury (Hg2+), as a conjugate of cysteine (Cys; Cys-S-Hg-S-Cys), is enhanced in S2 segments of proximal tubules from the remnant kidney of uninephrectomized (NPX) rabbits. To test this hypothesis, we measured uptake and accumulation of Cys-S-Hg-S-Cys in isolated perfused S2 segments of proximal tubules from normal (control) and NPX rabbits. The remnant kidney in NPX rabbits undergoes significant hypertrophy during the initial 3 weeks following surgery. Tubules isolated from NPX rabbits were significantly larger in diameter and volume than those from control rabbits. Moreover, real-time PCR analyses of proximal tubules indicated that the expression of selected membrane transporters was greater in kidneys of NPX animals than in kidneys of control animals. When S2 segments from control and NPX rabbits were perfused with cystine or Cys-S-Hg-S-Cys, we found that the rates of luminal disappearance and tubular accumulation of Hg2+ were greater in tubules from NPX animals. These increases were inhibited by the addition of various amino acids to the perfusate. Taken together, our data suggest that hypertrophic changes in proximal tubules lead to an enhanced ability of these tubules to take up and accumulate Hg2.

Specific modification of Escherichia coli RNA polymerase with monomercury derivative of fluorescein acetate.

The method of specific modification of RNA polymerase with a monomercuric fluorescein derivative, fluorescein-monomercuriacetate (FMMA), is proposed. Under an appropriate condition of modification, FMMA is capable of mercaptid bonding with one of the alpha-subunits. It is shown that covalent modification with FMMA does not affect the kinetic parameters (KB and k2) of RNA synthesis nor does it lead to the inhibition of the overall RNA synthesis. The spectral characteristics of FMMA covalently bound to RNA polymerase were found to be sensitive to some temperature-induced conformational alterations of RNA polymerase, indicating that the labeled enzyme allows study of conformational behaviour of RNA polymerase during its functioning.