Arginine decarboxylase: A novel biological target of mercury compounds identified in PC12 cells.

Mercury compounds are well-known toxic environmental pollutants and potently induce severe neurotoxicological effects in human and experimental animals. Previous studies showed that one of the mechanisms of mercury compounds neurotoxicity arose from the over-activation of the N-methyl d-aspartate (NMDA)-type glutamate receptor induced by increased glutamate release. In this work, we aimed to investigate the molecular mechanisms of Hg compounds neurotoxicities by identifying their biological targets in cells. Firstly, the inhibitory effects of four Hg compounds, including three organic (methyl-, ethyl- and phenyl-mercury) and one inorganic (Hg2+) Hg compounds, on the activity of arginine decarboxylase (ADC), a key enzyme in the central agmatinergic system, were evaluated. They were found to inhibit the ADC activity significantly with methylmercury (MeHg) being the strongest (IC50=7.96nM). Furthermore, they showed remarkable inhibitory effects on ADC activity in PC12 cells (MeHg>EtHg>PhHg>HgCl2), and led to a marked loss in the level of agmatine, an endogenous neuromodulatory and neuroprotective agent that selectively blocks the activation of NMDA receptors. MeHg was detected in the immunoprecipitated ADC from the cells, providing unequivocal evidence for the direct binding of MeHg with ADC in the cell. Molecular dynamics simulation revealed that Hg compounds could form the coordination bond not only with cofactor PLP of ADC, but also with substrate arginine. Our finding indicated that MeHg could attenuate the neuroprotective effects of agmatine by the inhibition of ADC, a new cellular target of MeHg, which might be implicated in molecular mechanism of MeHg neurotoxicity.

Role of MerC, MerE, MerF, MerT, and/or MerP in resistance to mercurials and the transport of mercurials in Escherichia coli.

The characteristics of bacteria take up mercury into cells via membrane potential-dependent sequence-divergent members of the mercuric ion (Mer) superfamily, i.e., a periplasmic mercuric ion scavenging protein (MerP) and one or more inner membrane-spanning proteins (MerC, MerE, MerF, and MerT), which transport mercuric ions into the cytoplasm, have been applied in engineering of bioreactor used for mercurial bioremediation. We engineered bacteria to express MerC, MerE, MerF, or MerT with or without MerP to clarify their individual role and potential in transport of mercurial. By immunoblot analysis using specific polyclonal antibody, the proteins encoded by merC, merE, merF, merT or merP, were certainly expressed and identified in the membrane fraction. Bacteria expressing MerC, MerE, MerF or MerT in the absence of MerP transported significantly more C6H5Hg(I) and Hg(II) across bacterial membrane than their isogenic strain. In vivo expression of MerP in the presence of all the transporters did not cause apparent difference to the C6H5Hg(I) transport, but gives an apparently higher Hg(II) transport than that did by MerE, MerF or MerT but not by MerC. Among the four transporters studied, MerC showed more potential to transport Hg(II) across bacterial membrane than MerE, MerF and MerT. Together these findings, we demonstrated for the first time that in addition to MerE and MerT, MerF and MerC are broad-spectrum mercury transporters that mediate both Hg(II) and phenylmercury transport into cells. Our results suggested that MerC is the most efficient tool for designing mercurial bioremediation systems, because MerC is sufficient for mercurial transport into cells.

Mesenchymal stem cell population isolated from the subepithelial layer of umbilical cord tissue.

The therapeutic use of stem cells to treat diseases and injuries is a promising tool in regenerative medicine. The umbilical cord provides a rich source of stem cells; we have previously reported a population of stem cells isolated from Wharton's jelly. In this report, we aimed to isolate a novel cell population that was different than those found in Wharton's jelly. We isolated stem cells from the subepithelial layer of the umbilical cord; the cells could be expanded for greater than 90 population doubling and had mesenchymal stem cell characteristics, expressing CD9, SSEA4, CD44, CD90, CD166, CD73, and CD146 but were negative for STRO-1. The cells can be directionally differentiated and undergo osteo-, chondro-, adipo-, and cardiogenesis. In addition, we have identified for the first time that mesenchymal stem cells isolated from umbilical cord can produce microvesicles, termed exosomes. This is the first report describing a stem cell population isolated from the subepithelial layer of the umbilical cord. Given the growth capacity, multilineage potential, and most importantly the low levels of HLA-ABC, we propose that this novel cell isolated from the subepithelial layer of umbilical cord is an ideal candidate for allogeneic cell-based therapy.

Molecular cloning and genetic analysis of functional merB gene from indian isolates of Escherichia coli.

Studies were carried out to characterize organomercurial lyase genes from wild type mercury-resistant Escherichia coli isolates, previously collected from five geographically distinct regions of the Indian subcontinent. PCR amplification followed by DNA sequencing of amplified fragments showed three merB identical to the previously characterized mer B from E. coli pR831b that were thus considered as the same gene. The remaining two genes derived from E. coli isolates of an almost mercury-free site (Dal lake, Kashmir) and designated as pIAAD3 merB and pIAAD14 merB showed slight variation (2%) at base. However, this variation in pIAAD3 due to the absence of base "T" at 479 position results in complete frame shift and the predicted MerB-like polypeptide derived from it showed 21.53% divergent at its C terminal end from the previously characterized pR831b MerB. The expression profile of pIAAD3 merB in pQE30 and pUC18 vectors each demonstrated 22.2 kDa proteins. The induced DH5alpha E. coli cells possessing pIAAD3 merB cloned in pUC18 vector split phenyl mercuric acetate (PMA) into benzene and inorganic mercury efficiently, thus giving a clue that the expressed gene product is biologically active. The current study suggests that such genetic changes may take place in the continued absence of mercury pressure, and with such modifications, they finally break down to act as vestigial remnants. Further work is going on in our lab to exploit pIAAD3 merB for the bioremediation of mercury-polluted sites.

Isolation of MMP-2 from MMP-2/TIMP-2 complex: characterization of the complex and the free enzyme in pulmonary vascular smooth muscle plasma membrane.

We have previously indicated that bovine pulmonary artery smooth muscle plasma membrane possesses a complex of 72-kDa gelatinase and TIMP-2 (MMP-2/TIMP-2 complex) [Mol. Cell. Biochem. 258 (2004) 73]. In this paper, we described isolation of MMP-2 from the MMP-2/TIMP-2 complex, characterizations of the isolated MMP-2 and also the complex. MMP-2/TIMP-2 complex was purified from bovine pulmonary vascular smooth muscle plasma membrane using a combination of purification steps. Heparin-sepharose (100 mM NaCl eluate)-purified preparation contained the MMP-2/TIMP-2 complex. The MMP-2/TIMP-2 complex, which was electrophoresed under reducing condition on the SDS-PAGE and immunobloted with a mixture of polyclonal MMP-2 and TIMP-2 antibodies, revealed two separate immunoreactive bands at their respective electrophoretic migration. Continuous elution electrophoresis of the complex resulted to MMP-2 free of any detectable TIMP-2. The homogeneity of the isolated MMP-2 and the complex was demonstrated by SDS-PAGE under nonreducing condition and also by nondenaturing native-PAGE. The purified TIMP-2 free enzyme electrophoresed as a single band of 72-kDa, which could be activated rapidly and fully by aminophenylmercuric acetate (APMA) with the formation of 62-kDa and 45-kDa active species like native MMP-2 purified from the same source (bovine pulmonary artery smooth muscle). Identical treatment of the MMP-2/TIMP-2 complex with APMA resulted to significantly slower and partial conversion of the active species. Addition of pure TIMP-2 to the TIMP-2 free MMP-2 formed a complex with the progelatinase and prevented the rapid autolytic conversion induced by APMA. Immunoblot study with polyclonal MMP-2 antibody suggested that the isolated 72-kDa gelatinase is the MMP-2. We have also presented additional data indicating that the isolated preparation of 72-kDa gelatinase exhibited properties that are identical with MMP-2 obtained from different sources.

Subcellular targeting of methylmercury lyase enhances its specific activity for organic mercury detoxification in plants.

Methylmercury is an environmental pollutant that biomagnifies in the aquatic food chain with severe consequences for humans and other animals. In an effort to remove this toxin in situ, we have been engineering plants that express the bacterial mercury resistance enzymes organomercurial lyase MerB and mercuric ion reductase MerA. In vivo kinetics experiments suggest that the diffusion of hydrophobic organic mercury to MerB limits the rate of the coupled reaction with MerA (Bizily et al., 2000). To optimize reaction kinetics for organic mercury compounds, the merB gene was engineered to target MerB for accumulation in the endoplasmic reticulum and for secretion to the cell wall. Plants expressing the targeted MerB proteins and cytoplasmic MerA are highly resistant to organic mercury and degrade organic mercury at 10 to 70 times higher specific activity than plants with the cytoplasmically distributed wild-type MerB enzyme. MerB protein in endoplasmic reticulum-targeted plants appears to accumulate in large vesicular structures that can be visualized in immunolabeled plant cells. These results suggest that the toxic effects of organic mercury are focused in microenvironments of the secretory pathway, that these hydrophobic compartments provide more favorable reaction conditions for MerB activity, and that moderate increases in targeted MerB expression will lead to significant gains in detoxification. In summary, to maximize phytoremediation efficiency of hydrophobic pollutants in plants, it may be beneficial to target enzymes to specific subcellular environments.

Simultaneous detection and removal of organomercurial compounds by using the genetic expression system of an organomercury lyase from the transposon Tn MERI1.

Using a newly identified organomercury lyase gene (merB3) expression system from Tn MERI1, the mercury resistance transposon first found in Gram-positive bacteria, a dual-purpose system to detect and remove organomercurial contamination was developed. A plasmid was constructed by fusing the promoterless luxAB genes as bioluminescence reporter genes downstream of the merB3 gene and its operator/promoter region. Another plasmid, encoding mer operon genes from merR1 to merA, was also constructed to generate an expression regulatory protein, MerR1, and a mercury reductase enzyme, MerA. These two plasmids were transformed into Escherichia coli cells to produce a biological system that can detect and remove environmental organomercury contamination. Organomercurial compounds, such as neurotoxic methylmercury at nanomolar levels, were detected using the biomonitoring system within a few minutes and were removed during the next few hours.

Role of MerT and MerP from Pseudomonas K-62 plasmid pMR26 in the transport of phenylmercury.

To investigate the individual role of MerT and MerP encoded by Pseudomonas K-62 pMR26 in the transport of phenylmercury, a series of mutants with a specific point mutation in merT and/or genetic deletion in merP were constructed and transformed into Escherichia coli XL1-Blue. Transport of phenylmercury across the cytoplasmic membrane of E. coli mediated by MerT and MerP was inhibited by NaCN and by cold temperatures. Deletion of merP reduced, but did not completely abolish the C6H5Hg+-hyperuptake and -hypersensitive phenotypes suggesting that transport of phenylmercury into the cytoplasm of E. coli is still occurring. Mutations of the vicinal cysteine residues (Cys24 and Cys25) in the first transmembrane region of MerT to serine caused complete loss of Hg2+-hyperuptake and -hypersensitivity, whereas the mutations did not affect the C6H5Hg+-hyperuptake and -hypersensitive phenotypes. In addition, no additive effect on the C6H5Hg+-hyperuptake and -hypersensitive phenotypes was found, when mutations of the two cysteines in MerT to serine were further introduced in the merP-deleted mutants. These results clearly demonstrated that the vicinal cysteine residues of MerT are not involved in the transport of C6H5Hg+, but indeed are involved in the transport of Hg2+ as previously reported.

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.

Zn2+ promotes the self-association of human immunodeficiency virus type-1 integrase in vitro.

It has been recently demonstrated that the Mg(2+)-dependent 3'-processing activity of purified human immunodeficiency virus type-1 (HIV-1) integrase is stimulated by the addition of exogenous Zn2+ [Lee, S. P., & Han, M. K. (1996) Biochemistry 35, 3837-3844]. This activation was hypothesized to result from integrase self-association. In this report, we examine the Zn2+ content of purified HIV-1 integrase by atomic absorption spectroscopy and by application of a thiol modification reagent, p-(hydroxymercuri)benzenesulfonate, with a metallochromic indicator, 4-(2-pyridylazo)resorcinol. We find that the Zn2+ content of HIV-1 integrase varies from 0.1 to 0.92 equiv of Zn2+ per monomer depending on the conditions of protein purification. In vitro activity assays, time-resolved fluorescence emission anisotropy, and gel filtration chromatographic analyses all indicate that EDTA yields an apoprotein which is predominantly monomeric and less active with Mg2+. Further, sedimentation equilibrium studies reveal that reconstitution of the apoprotein with Zn2+ results in a monomer-tetramer-octamer transition. These results suggest that Zn2+ promotes a conformation with enhanced oligomerization and thereby stimulates Mg(2+)-dependent 3'-processing. This may also imply that multimers larger than dimers (tetramers and possibly octamers) are required for in vitro activity of integrase in the presence of Zn2+ and Mg2+. It should be noted, however, that the content of Zn2+ did not significantly affect the 3'-processing and strand transfer reactions with Mn2+ in vitro.

Metal-binding site in a class I tRNA synthetase localized to a cysteine cluster inserted into nucleotide-binding fold.

The 10 class I aminoacyl-tRNA synthetases share a common N-terminal nucleotide-binding fold. Idiosyncratic polypeptide insertions into this fold introduce residues important for activity, including those that interact with the tRNA acceptor helix. The class I Escherichia coli methionyl-tRNA synthetase (L-methionine:tRNA(Met) ligase, EC 6.1.1.10), a 676-amino acid homodimer, was shown previously by others to contain zinc and to have an activity dependent on its presence. We show here by atomic absorption spectroscopy and zinc titrations the presence of 1 mol of zinc per polypeptide. Replacement of zinc with cobalt yields an active enzyme with a visible absorption spectrum characteristic of tetrahedral coordination to sulfur ligands and an intense metal-to-sulfur charge-transfer band at 340 nm. Mapping of the metal-binding site by zinc blotting of recombinant and proteolytic fragments localized the site to a polypeptide insertion between two strands and a beta-sheet in the N-terminal nucleotide-binding fold that contains the catalytic site. Beginning at Cys-145, this insertion contains a Cys-Xaa2-Cys-Xaa9-Cys-Xaa2-Cys motif. Site-directed substitution of these cysteines with serines yielded proteins that were stable but generally devoid of activity. With this result there is now at least one example of a class I and of a class II E. coli tRNA synthetase with a metal-binding domain important for activity inserted into the catalytic domain.

Phenylmercuric acetate biodegradation by environmental strains of Pseudomonas species.

Organomercurial pollution occurring in the Rhine river in 1986 led us to study the possibility of depollution by mercury-resistant environmental aquatic strains. Four species of Pseudomonas were investigated for their ability to biotransform phenylmercuric acetate (PMA). Such biological depollution was demonstrated to be due to an enzymatic activity in whole cells and in cell-free extracts from Pseudomonas fluorescens and other Pseudomonas species. PMA biotransformation was followed by high-performance liquid chromatography. Some of those bacteria growing between 4 and 41 degrees C probably represent a natural means of organomercurial depollution, which acts slowly in interaction with other organisms and non-organic porous surfaces.

The catalytic requirements for reduction and acetylation of protein X and the related regulation of various forms of resolved pyruvate dehydrogenase kinase.

The pyruvate dehydrogenase kinase consists of a catalytic subunit (Kc) and a basic subunit (Kb) which appear to be anchored to the dihydrolipoyl transacetylase core component (E2) by another subunit, referred to as protein X (Rahmatullah, M., Jilka, J. M., Radke, G. A., and Roche, T. E. (1986) J. Biol. Chem. 261, 6515-6523). We determined the catalytic requirements for reduction and acetylation of the lipoyl moiety in protein X and linked those changes in protein X to regulatory effects on kinase activity. Using fractions prepared by resolution and proteolytic treatments, we evaluated which subunits are required for regulatory effects on kinase activity. With X-KcKb fraction (treated to remove the mercurial agent used in its preparation), we found that the resolved pyruvate dehydrogenase component, the isolated inner domain of E2 (lacking the lipoyl-bearing region of E2), and the dihydrolipoyl dehydrogenase component directly utilize protein X as a substrate. The resulting reduction and acetylation of protein X occurs in association with enhancement of kinase activity. Following tryptic cleavage of E2 and protein X into subdomains, full acetylation of the lipoyl-bearing subdomains of these proteins is retained along with the capacity of acetylating substrates to stimulate kinase activity. All kinase-containing fractions, including those in which the Kb subunit was digested, were inhibited by pyruvate or ADP, alone, and synergistically by the combination suggesting that pyruvate and ADP bind to Kc. Our results suggest that the Kb subunit of the kinase does not contribute to the observed regulatory effects. A dynamic role of protein X in attenuating kinase activity based on changes in the mitochondrial redox and acetylating potentials is considered.

Target molecular weights for red cell band 3 stilbene and mercurial binding sites.

Radiation inactivation was used to measure the target sizes for binding of disulfonic stilbene anion transport inhibitor 4,4'-dibenzamido-2,2'-disulfonic stilbene (DBDS) and mercurial water transport inhibitor p-chloromercuribenzene sulfonate (pCMBS) to human erythrocytes. The measured target size for erythrocyte ghost acetylcholinesterase was 78 +/- 3 kDa. DBDS binding to ghost membranes was measured by a fluorescence enhancement technique. Radiation (0-26 Mrad) had no effect on total membrane protein and DBDS binding affinity, whereas DBDS binding stoichiometry decreased exponentially with radiation dose, giving a target size of 59 +/- 4 kDa. H2-4,4'-diisothiocyano-2,2'-disulfonic stilbene (H2-DIDS, 5 microM) blocked greater than 95% of DBDS binding at all radiation doses. pCMBS binding was measured from the time course of tryptophan fluorescence quenching in ghosts treated with the sulfhydryl reagent N-ethylmaleimide (NEM). Radiation did not affect the kinetics of tryptophan quenching, whereas the total amplitude of the fluorescence signal inactivated with radiation with a target size of 31 +/- 6 kDa. These results support the notion that DBDS and pCMBS bind to the transmembrane domain of erythrocyte band 3 in NEM-treated ghosts and demonstrate that radiation inactivation may probe a target significantly smaller than a covalently linked protein subunit. The small target size for the band 3 stilbene binding site may correspond to the intramembrane domain of the band 3 monomer (52 kDa), which is physically distinct from the cytoplasmic domain (42 kDa).

Prolinase and non-specific dipeptidase of human kidney.

Human kidney prolinase, assayed with Pro-Ala, and non-specific dipeptidase, assayed with Gly-Leu, were purified by using DEAE-cellulose, gel-filtration, metal-ion-chelate, hydrophobic and adsorption chromatography and chromatofocusing. Both enzymes gave single peaks of activity that were congruent and the ratio of their activities was constant throughout the purification. Gel filtration indicated an Mr of 100 000 and chromatofocusing a pI of 5.4. Ni2+-chelate chromatography demonstrated the presence of exposed histidine residues on the enzyme and was an effective separative procedure. Polyacrylamide-gel electrophoresis of the final preparation showed the two enzyme activities to be coincident. Both enzyme activities decayed at the same rate at 53 degrees C and were inhibited to the same extent by p-hydroxymercuribenzoate. Of six non-specific dipeptidase substrates tested Gly-Leu gave the highest activity, and of six prolinase substrates Pro-Leu had the highest activity. Gly-Leu was hydrolysed at double the rate of Pro-Leu. Pro-Ala was a competitive inhibitor of activity towards Gly-Leu, and Gly-Leu was a competitive inhibitor of activity towards Pro-Ala. Mixed-substrate studies strongly suggested that Gly-Leu and Pro-Ala were hydrolysed at a common active site. The data are consistent with prolinase and non-specific dipeptidase activity in human kidney being due to a single enzyme.

Evidence for the asymmetrical binding of p-chloromercuriphenyl sulphonate to the human erythrocyte nucleoside transporter.

Nucleosides cross the human erythrocyte membrane by a facilitated-diffusion process which is selectively inhibited by nanomolar concentrations of nitrobenzylthioinosine (NBMPR). The chemical asymmetry of the transporter was investigated by studying the effects of p-chloromercuriphenyl sulphonate (PCMBS) on uridine transport and high-affinity NBMPR binding in inside-out and right-side-out membrane vesicles, unsealed erythrocyte ghosts and intact cells. PCMBS was an effective inhibitor of the transporter (50% inhibition at 30 microM), but only when the organomercurial had access to the cytoplasmic membrane surface. PCMBS inhibition of NBMPR binding to ghosts was reversed by incubation with dithiothreitol. Both uridine and NBMPR were able to protect the transporter against PCMBS inhibition.

Octanol/water partition coefficients as a model system for assessing antidotes for methylmercury(II) poisoning, and for studying mercurials with medicinal applications.

1-Octanol/water partition coefficients, [HgII]octanol/[HgII]water, provide a simple but limited model system for aspects of the biological behavior of methylmercury(II) and commonly used organomercury(II) medicinal compounds. In an octanol/water system some widely studied antidotes for mercury poisoning at least partly displace the biological thiols L-cysteine and glutathione from binding to MeHgII at pH 6.9. Addition of the antidote meso-dimercaptosuccinic acid to MeHgII in the presence of glutathione results in formation of metallic mercury. For RHgII derivatives of L-cysteine and glutathione, octanol/water partition coefficients follow the order Ph greater than Et greater than Me. An exceptionally high value for diphenylmercury, compared with PhHgII derivatives of L-cysteine and glutathione, is consistent with reported results of the distribution of mercury compounds in rats. Ethylmercury(II) is partly displaced from thimerosal by L-cysteine and glutathione in the octanol/water system, indicating that the active form of thimerosal in vivo may involve binding of EtHgII to biological ligands.

Initial uptake and insulin releasing action of chloromercuribenzene-p-sulphonic acid (CMBS) in suspensions of pancreatic islet cells.

The effects of chloromercuribenzene-p-sulphonic acid on dispersed cells prepared from beta-cell-rich ob/ob-mouse islets were studied. 1) Chloromercuribenzene-p-sulphonic acid at concentrations of 0.1 mmol/l or higher diminished cell viability which was partially counteracted by increasing concentrations of bovine serum albumin. 2) The uptake of 203Hg-chloromercuribenzene-p-sulphonic acid after incubation for 4 seconds or longer showed that most of the non-toxic concentrations of chloromercuribenzene-p-sulphonic acid was bound to the cell within 40 seconds. Maximal uptake was achieved after 3 minutes of incubation. The uptake of radioactive chloromercuribenzene-p-sulphonic acid was inhibited by bovine serum albumin. 3) The dynamics of insulin release from perifused dispersed beta-cells embedded in fibrin showed a maximal 40--50-fold stimulation by 0.03 mmol/l chloromercuribenzene-p-sulphonic acid within 10 minutes of perifusion. 4) Scanning electron microscopy of beta-cells revealed no major changes in the cell surface under conditions of maximal binding and insulin releasing effects of chloromercuribenzene-p-sulphonic acid. These data support the concept that the ability of chloromercuribenzene-p-sulphonic acid to induce insulin release is related to its initial binding to the beta-cell surface. The binding of chloromercuribenzene-p-sulphonic acid and the subsequent release of insulin seem to occur without major changes in beta-cell surface morphology.