Developmental exposure to lead (Pb) alters the expression of the human tau gene and its products in a transgenic animal model.

Tauopathies are a class of neurodegenerative diseases associated with the pathological aggregation of the tau protein in the human brain. The best known of these illnesses is Alzheimer's disease (AD); a disease where the microtubule associated protein tau (MAPT) becomes hyperphosphorylated (lowering its binding affinity to microtubules) and aggregates within neurons in the form of neurofibrillary tangles (NFTs). In this paper we examine whether environmental factors play a significant role in tau pathogenesis. Our studies were conducted in a double mutant mouse model that expressed the human tau gene and lacked the gene for murine tau. The human tau mouse model was tested for the transgene's ability to respond to an environmental toxicant. Pups were developmentally exposed to lead (Pb) from postnatal day (PND) 1-20 with 0.2% Pb acetate. Mice were then sacrificed at PND 20, 30, 40 and 60. Protein and mRNA levels for tau and CDK5 as well as tau phosphorylation at Ser396 were determined. In addition, the potential role of miRNA in tau expression was investigated by measuring levels of miR-34c, a miRNA that targets the mRNA for human tau, at PND20 and 50. The expression of the human tau transgene was altered by developmental exposure to Pb. This exposure also altered the expression of miR-34c. Our findings are the first of their kind to test the responsiveness of the human tau gene to an environmental toxicant and to examine an epigenetic mechanism that may be involved in the regulation of this gene's expression.

Effects of diabetes insipidus mutations on neurophysin folding and function.

Mechanisms underlying the pathogenicity of diabetes insipidus mutations were probed by studying their effects on the properties of bovine oxytocin-related neurophysin. The mutations G17V, DeltaE47, G57S, G57R, and C67STOP were each shown to have structural consequences that would diminish the conformational stability and folding efficiency of the precursors in which they were incorporated, and factors contributing to the origins of these property changes were identified. Effects of the mutations on dimerization of the folded proteins were similarly analyzed. The projected relative impact of the above mutations on precursor folding properties qualitatively parallels the reported relative severity of their effects on the biological handling of the human vasopressin precursor, but quantitative differences between thermodynamic effects and biological impact are noted and explored. The sole mutation for which no clear thermodynamic basis was found for its pathogenicity was 87STOP, suggesting that the region of the precursor deleted by this mutation plays a role in targeting independent from effects on folding, or participates in stabilizing interactions unique to the human vasopressin precursor.

Substrate interactions in BTEX and MTBE mixtures by an MTBE-degrading isolate.

Groundwater contaminant plumes from recent accidental gasoline releases often contain the fuel oxygenate MTBE (methyl tert-butyl ether) together with BTEX (benzene, toluene, ethylbenzene, o-xylene, m-xylene and p-xylene) compounds. This study evaluates substrate interactions during the aerobic biotransformation of MTBE and BTEX mixtures by a pure culture, PM1, capable of utilizing MTBE for growth. PM1 was unable to degrade ethylbenzene and two of the xylene isomers at concentrations of 20 mg/L following culture growth on MTBE. In addition, the presence of 20 mg/L of ethylbenzene or the xylenes in mixtures with MTBE completely inhibited MTBE degradation. When MTBE-grown cells of PM1 were exposed to MTBE/benzene and MTBE/toluene mixtures, MTBE degradation proceeded, while the degradation of benzene and toluene was delayed for several hours. Following this initial lag, benzene and toluene were degraded rapidly, while the rate of MTBE degradation slowed significantly. MTBE degradation did not increase to previous rates until benzene and toluene were almost entirely degraded. The lag in benzene and toluene degradation was presumably due to the induction of the enzymes necessary for BTEX degradation. Once these enzymes were induced, sequential additions of benzene or toluene were degraded rapidly, and growth on benzene and toluene was observed. The results of this study suggest that BTEX and MTBE degradation occurs primarily via two independent and inducible pathways. If subsurface microbial communities behave similarly to the culture used in this study, the observed severe inhibition of MTBE degradation by ethylbenzene and the xylenes and the partial inhibition by benzene and toluene suggest thatthe biodegradation of MTBE in subsurface environments would most likely be delayed until MTBE has migrated beyond the BTEX plume.

Aerobic MTBE biodegradation: an examination of past studies, current challenges and future research directions.

With the current practice of amending gasoline with up to 15% by volume MTBE, the contamination of groundwater by MTBE has become widespread. As a result, the bioremediation of MTBE-impacted aquifers has become an active area of research. A review of the current literature on the aerobic biodegradation of MTBE reveals that a number of cultures from diverse environments can either partially degrade or completely mineralize MTBE. MTBE is either utilized as a sole carbon and energy source or is degraded cometabolically by cultures grown on alkanes. Reported degradation rates range from 0.3 to 50 mg MTBE/g cells/h while growth rates (0.01-0.05 g MTBE/g cells/d) and cellular yields (0.1-0.2 g cells/g MTBE) are generally low. Studies on the mechanisms of MTBE degradation indicate that a monooxygenase enzyme cleaves the ether bond yielding tert-butyl alcohol (TBA) and formaldehyde as the dominant detectable intermediates. TBA is further degraded to 2-methyl-2-hydroxy-1-propanol, 2-hydroxyisobutyric acid, 2-propanol, acetone, hydroxyacteone and eventually, carbon dioxide. The majority of these intermediates are also common to mammalian MTBE metabolism. Laboratory studies on the degradation of MTBE in the presence of gasoline aromatics reveal that while degradation rates of other gasoline components are generally not inhibited by MTBE, MTBE degradation could be inhibited in the presence of more easily biodegradable compounds. Controlled field studies are clearly needed to elucidate MTBE degradation potential in co-contaminant plumes. Based on the reviewed studies, it is likely that a bioremediation strategy involving direct metabolism, cometabolism, bioaugmentation, or some combination thereof, could be applied as a feasible and cost-effective treatment method for MTBE contamination.

Bioremediation of MTBE: a review from a practical perspective.

The addition of methyl tert-butyl ether (MTBE) to gasoline has resulted in public uncertainty regarding the continued reliance on biological processes for gasoline remediation. Despite this concern, researchers have shown that MTBE can be effectively degraded in the laboratory under aerobic conditions using pure and mixed cultures with half-lives ranging from 0.04 to 29 days. Ex-situ aerobic fixed-film and aerobic suspended growth bioreactor studies have demonstrated decreases in MTBE concentrations of 83% and 96% with hydraulic residence times of 0.3 hrs and 3 days, respectively. In microcosm and field studies, aerobic biodegradation half-lives range from 2 to 693 days. These half-lives have been shown to decrease with increasing dissolved oxygen concentrations and, in some cases, with the addition of exogenous MTBE-degraders. MTBE concentrations have also been observed to decrease under anaerobic conditions; however, these rates are not as well defined. Several detailed field case studies describing the use of ex-situ reactors, natural attenuation, and bioaugmentation are presented in this paper and demonstrate the potential for successful remediation of MTBE-contaminated aquifers. In conclusion, a substantial amount of literature is available which demonstrates that the in-situ biodegradation of MTBE is contingent on achieving aerobic conditions in the contaminated aquifer.

Regulation of prostaglandin H2 synthase activity by nitrogen oxides.

Nitric oxide and its derivatives have been shown to both activate and inhibit prostaglandin H(2) synthase 1 (PGHS-1). We set out to determine the mechanisms by which different nitrogen oxide derivatives modulate PGHS-1 activity. To this end, we show that 3-morpholinosydnonimine hydrochloride (SIN-1), a compound capable of generating peroxynitrite, activates purified PGHS-1 and also stimulates PGE(2) production in arterial smooth muscle cells in the presence of exogenous arachidonic acid. The effect of SIN-1 in smooth muscle cells was abrogated by superoxide and peroxynitrite inhibitors, which supports the hypothesis that peroxynitrite is an activating species of PGHS-1. Indeed, authentic peroxynitrite also induced PGE(2) production in arachidonic acid-stimulated cells. In contrast, when cells were exposed to the nitric oxide-releasing compound 1-hydroxy-2-oxo-3-[(methylamino)propyl]-3-methyl-1-triazene (NOC-7), PGHS-1 enzyme activity was inhibited in the presence of exogenous arachidonic acid. Finally, in lipid-loaded smooth muscle cells, we demonstrate that SIN-1 stimulates arachidonic acid-induced PGE(2) production; albeit, the extent of activation is reduced compared to that under normal conditions. These results indicate that formation of peroxynitrite is a key intermediary step in PGHS-1 activation. However, other forms of NO(x)() inhibit PGHS-1. These results may have implications in the regulation of vascular function and tone in normal and atherosclerotic arteries.

Structural basis of neurophysin hormone specificity: Geometry, polarity, and polarizability in aromatic ring interactions.

The structural origins of the specificity of the neurophysin hormone-binding site for an aromatic residue in peptide position 2 were explored by analyzing the binding of a series of peptides in the context of the crystal structure of liganded neurophysin. A new modeling method for describing the van der Waals surface of binding sites assisted in the analysis. Particular attention was paid to the unusually large (5 kcal/mol) difference in binding free energy between Phe and Leu in position 2, a value representing more than three times the maximum expected based on hydrophobicity alone, and additionally remarkable since modeling indicated that the Leu side chain was readily accommodated by the binding pocket. Although evidence was obtained of a weak thermodynamic linkage between the binding interactions of the residue 2 side chain and of the peptide alpha-amino group, two factors are considered central. (1) The bound Leu side chain can establish only one-third of the van der Waals contacts available to a Phe side chain. (2) The bound Phe side chain appears to be additionally stabilized relative to Leu by more favorable dipole and induced dipole interactions with nonaromatic polar and sulfur ligands in the binding pocket, as evidenced by examination of its interactions in the pocket, analysis of the detailed energetics of transfer of Phe and Leu side chains from water to other phases, and comparison with thermodynamic and structural data for the binding of residue 1 side chains in this system. While such polar interactions of aromatic rings have been previously observed, the present results suggest their potential for significant thermodynamic contributions to protein structure and ligand recognition.

Temperature effects and substrate interactions during the aerobic biotransformation of BTEX mixtures by toluene-enriched consortia and Rhodococcus rhodochrous.

A microbial consortium derived from a gasoline-contaminated aquifer was enriched on toluene (T) in a chemostat at 20 degrees C and was found to degrade benzene (B), ethylbenzene (E), and xylenes (X). Studies conducted to determine the optimal temperature for microbial activity revealed that cell growth and toluene degradation were maximized at 35 degrees C. A consortium enriched at 35 degrees C exhibited increased degradation rates of benzene, toluene, ethylbenzene, and xylenes in single-substrate experiments; in BTEX mixtures, enhanced benzene, toluene, and xylene degradation rates were observed, but ethylbenzene degradation rates decreased. Substrate degradation patterns over a range of BTEX concentrations (0 to 80 mg/L) for individual aromatics were found to differ significantly from patterns for aromatics in mixtures. Individually, toluene was degraded fastest, followed by benzene, ethylbenzene, and the xylenes. In BTEX mixtures, degradation followed the order of ethylbenzene, toluene, and benzene, with the xylenes degraded last. A pure culture isolated from the 35 degrees C-enriched consortium was identified as Rhodococcus rhodochrous. This culture was shown to degrade each of the BTEX compounds, individually and in mixtures, following the same degradation patterns as the mixed cultures. Additionally, R. rhodochrous was shown to utilize benzene, toluene, and ethylbenzene as primary carbon and energy sources. Studies conducted with the 35 degrees C-enriched consortium and R. rhodochrous to evaluate potential substrate interactions caused by the concurrent presence of multiple BTEX compounds revealed a range of substrate interaction patterns including no interaction, stimulation, competitive inhibition, noncompetitive inhibition, and cometabolism. In the case of the consortium, benzene and toluene degradation rates were slightly enhanced by the presence of o-xylene, whereas the presence of toluene, benzene, or ethylbenzene had a negative effect on xylene degradation rates. Ethylbenzene was shown to be the most potent inhibitor of BTEX degradation by both the mixed and pure cultures. Attempted quantification of these inhibition effects in the case of the consortium suggested a mixture of competitive and noncompetitive inhibition kinetics. Benzene, toluene, and the xylenes had a negligible effect on the biodegradation of ethylbenzene by both cultures. Cometabolism of o-, m-, and p-xylene was shown to be a positive substrate interaction.

Thermodynamic role of the pro region of the neurophysin precursor in neurophysin folding: evidence from the effects of ligand peptides on folding.

Attention has focused recently on the role of amino-terminal precursor pro regions in protein folding, with particular emphasis on their effects on folding kinetics. We examined the kinetic and thermodynamic effects of ligand peptides on the folding of neurophysin from the reduced state; these peptides serve as analogs of the pro regions of the common precursors of the neurophysins and the hormones oxytocin and vasopressin. Folding of reduced, mononitrated bovine neurophysin-II was monitored by circular dichroism in a glutathione redox buffer. The results confirmed the ability of neurophysin to fold to a limited extent (20-25% in this system) in the absence of ligand peptides. Ligand peptides increased the efficiency of folding to 100%, the exact efficiency being dependent on peptide identity and concentration. However, the rate of folding was peptide-independent. Analysis of the folding reaction demonstrated relatively rapid conversion of the reduced state to a disulfide-scrambled state, which slowly converted (half-life of 5 h at pH 7.3) to the folded state. Native unliganded neurophysin also equilibrated with the disulfide-scrambled state in the same redox buffers. For each peptide, an equilibrium constant for the folding reaction, representing the amount of peptide bound in the folding system as a function of peptide concentration, was calculated. Comparison of this constant with the intrinsic binding constants of the native protein allowed the derivation, under conditions at or approaching thermodynamic reversibility, of the relative stability of the native and disulfide-scrambled states. The results indicate that the scrambled state, which probably represents the presence of incorrect disulfide pairs in both protein domains, is more stable than the native unliganded state by approximately 1 kcal/mol in this system. The role of ligand peptide therefore is to stabilize the folded protein after it is formed, i.e., it provides a thermodynamic sink. The results contrast with the putative behavior of exogenous peptides representative of the pro regions of subtilisin and alpha-lytic protease, which are generally considered to facilitate folding by reaction with folding intermediates. A potential alternative view of the role of propeptides in protease folding is suggested.

NMR behavior of the aromatic protons of bovine neurophysin-I and its peptide complexes: implications for solution structure and for function.

The NMR behavior of the aromatic protons of bovine neurophysin-I and its complexes was interpreted with reference to the 2.8 A crystal structure of the dipeptide complex of bovine neurophysin-II and to mechanisms underlying the thermodynamic linkage between neurophysin dimerization and peptide binding. Large binding-induced shifts in the ring proton signals of Tyr-2 of ligand peptides (approximately 0.5 ppm upfield and approximately 0.35 ppm downfield at 25 degrees C for the 3,5- and 2,6-ring protons, respectively) were demonstrated. Consistent with the crystal structure, and in disagreement with conclusions by other investigators, evidence is presented indicating the absence of dipolar contact between Tyr-2 ring protons and protein Phe ring protons. The large binding-induced shifts are attributed to a strong influence of proximal neurophysin carbonyl and disulfide groups on the bound Tyr-2 ring, of potential importance in binding specificity. Resolution of the behavior of neurophysin Phe residues -22 and -35 and of their proton NOE contacts provided insights into the conformational changes associated with peptide binding and with dimerization. Within the amino domain of the protein, as evidenced by the behavior of interface residue Phe-35 and its NOE contacts, binding-induced changes in the subunit interface appeared to involve principally the junction between this interface region and the 3,10-helix that connects it to the binding site in the liganded state. By contrast, as judged by the NOE contacts of His-80, the corresponding interface participant of the carboxyl domain, peptide binding induced a marked decrease in side-chain mobility within the carboxyl domain segment of the interface. Interactions of Phe-22 with protons assigned to Ala-68, neither of which is an interface participant, were demonstrated to be markedly altered both by dimerization in the unliganded state and by peptide binding to the dimer. Since Phe-22 is adjacent to the peptide-binding site, the results collectively support a model in which conformational differences between unliganded monomer and dimer are important contributors to the preferential binding of peptide to the dimer and indicate that the amino and carboxyl domain segments of the interface, which are homologous, are affected differently by peptide binding.

Proton NMR study of the heme complex of hemopexin.

Proton nuclear magnetic resonance spectroscopy of the complex of heme with hemopexin, a plasma protein with an exceptionally high affinity for heme, is reported. Characteristic spectra are shown for heme.hemopexin of cow, human, rabbit, and rat. Each of these spectra demonstrate that the iron of heme bound by hemopexin is paramagnetic and low-spin. Rabbit heme.hemopexin, which exhibits the best signal-to-noise ratio, is studied in detail. Deuterium isotope labeling experiments indicate that the methyls in heme positions 1-, 3-, and 8- are resolved downfield from the protein envelope of resonances; the 5-methyl may lie in the -5 to +12 ppm region. Two-dimensional nuclear Overhauser effect spectroscopy locates other protons of the heme periphery, including from the 2-vinyl. Strongly relaxed upfield resonances are identified and assigned to protons on the axial ligands. Cyanide interaction with heme.hemopexin produces an additional low-spin adduct.

Proton NMR study of the interaction of tin(IV) protoporphyrin IX monomers and dimers with apomyoglobin.

Events during the reconstitution of apomyoglobin to form the holoprotein were probed by porphyrin-metal substitution. Thus interactions between tin(IV) protoporphyrin IX (SnPP) and equine apomyoglobin (apoEqMb), and between tin(IV) protoporphyrin IX dimers [(SnPP)2] and apoEqMb, were observed by 1H NMR and optical absorbance spectroscopic techniques. The chief advantages of using SnPP are that products and intermediates can easily be related to SnPP.EqMb which has been studied [Deeb, R.S., & Peyton, D.H. (1991) J. Biol. Chem. 266, 3728-3733] and that at least one step during reconstitution is slowed considerably as compared to heme. Reactions of apoEqMb with SnPP and (SnPP)2 produce different intermediates, although the final product, SnPP.EqMb, is the same for each. An intermediate observed for reaction of SnPP with apoEqMb at pH 10 is in exchange with free SnPP, with the observed rate constant koff approximately 1 s-1. meso-Proton resonances were assigned for this intermediate by correlation to SnPP resonances via chemical exchange. The intermediate observed for reaction of (SnPP)2 with apoEqMb at pH 7.5 is heterogeneous. The reaction of either SnPP or (SnPP)2 with apoEqMb at neutral pH produces another species which may be the alternate porphyrin-insertion isomer arising from a 180 degree rotation about the alpha, gamma-meso axis of the porphyrin. Although optical absorbance spectroscopy of the Soret region shows evidence for each reaction, only in combination with 1H NMR are the various processes assigned.

1H and 119Sn magnetic resonance study of the SnIV protoporphyrin IX complex of equine myoglobin. Structure of the porphyrin-binding pocket.

Tin protoporphyrin IX (SnPP) is being used in the treatment of hyperbilirubinemia. We have studied the SnPP complex with equine myoglobin (EqMb) by 1H and 119Sn nuclear magnetic resonance spectroscopy (NMR) as a general model for SnPP interaction with hemoproteins. The complex formed from SnPP and EqMb, SnPP.EqMb, was found to have essentially the same porphyrin-binding pocket as EqMbCO, including the same porphyrin orientation in the major form of EqMbCO. 119Sn NMR spectroscopy has been used to demonstrate that the proximal His93F8-metal coordination is likely to be intact in SnPP.EqMb. Minor shifts in the side chain positions of some of the residues are indicated, possibly reflecting the presence of water in the sixth coordination site. SnPP.EqMb appears to be stable; it persists at room temperature for weeks and exhibits very slow exchange rates (2H for 1H) for a large number of amide protons in the pH range 7-9.