Carbon turnover in the water-soluble protein of the adult human lens.

PURPOSE: Human eye lenses contain cells that persist from embryonic development. These unique, highly specialized fiber cells located at the core (nucleus) of the lens undergo pseudo-apoptosis to become devoid of cell nuclei and most organelles. Ostensibly lacking in protein transcriptional capabilities, it is currently believed that these nuclear fiber cells owe their extreme longevity to the perseverance of highly stable and densely packed crystallin proteins. Maintaining the structural and functional integrity of lenticular proteins is necessary to sustain cellular transparency and proper vision, yet the means by which the lens actually copes with a lifetime of oxidative stress, seemingly without any capacity for protein turnover and repair, is not completely understood. Although many years of research have been predicated upon the assumption that there is no protein turnover or renewal in nuclear fiber cells, we investigated whether or not different protein fractions possess protein of different ages by using the (14)C bomb pulse. METHODS: Adult human lenses were concentrically dissected by gently removing the cell layers in water or shaving to the nucleus with a curved micrometer-controlled blade. The cells were lysed, and the proteins were separated into water-soluble and water-insoluble fractions. The small molecules were removed using 3 kDa spin filters. The (14)C/C was measured in paired protein fractions by accelerator mass spectrometry, and an average age for the material within the sample was assigned using the (14)C bomb pulse. RESULTS: The water-insoluble fractions possessed (14)C/C ratios consistent with the age of the cells. In all cases, the water-soluble fractions contained carbon that was younger than the paired water-insoluble fraction. CONCLUSIONS: As the first direct evidence of carbon turnover in protein from adult human nuclear fiber cells, this discovery supports the emerging view of the lens nucleus as a dynamic system capable of maintaining homeostasis in part due to intricate protein transport mechanisms and possibly protein repair. This finding implies that the lens plays an active role in the aversion of age-related nuclear (ARN) cataract.

Development of fluorescent substrates for microsomal epoxide hydrolase and application to inhibition studies.

The microsomal epoxide hydrolase (mEH) plays a significant role in the metabolism of numerous xenobiotics. In addition, it has a potential role in sexual development and bile acid transport, and it is associated with a number of diseases such as emphysema, spontaneous abortion, eclampsia, and several forms of cancer. Toward developing chemical tools to study the biological role of mEH, we designed and synthesized a series of absorbent and fluorescent substrates. The highest activity for both rat and human mEH was obtained with the fluorescent substrate cyano(6-methoxy-naphthalen-2-yl)methyl glycidyl carbonate (11). An in vitro inhibition assay using this substrate ranked a series of known inhibitors similarly to the assay that used radioactive cis-stilbene oxide but with a greater discrimination between inhibitors. These results demonstrate that the new fluorescence-based assay is a useful tool for the discovery of structure-activity relationships among mEH inhibitors. Furthermore, this substrate could also be used for the screening chemical library with high accuracy and with a Z' value of approximately 0.7. This new assay permits a significant decrease in labor and cost and also offers the advantage of a continuous readout. However, it should not be used with crude enzyme preparations due to interfering reactions.

One pathway can incorporate either adenine or dimethylbenzimidazole as an alpha-axial ligand of B12 cofactors in Salmonella enterica.

Corrinoid (vitamin B12-like) cofactors contain various alpha-axial ligands, including 5,6-dimethylbenzimidazole (DMB) or adenine. The bacterium Salmonella enterica produces the corrin ring only under anaerobic conditions, but it can form "complete" corrinoids aerobically by importing an "incomplete" corrinoid, such as cobinamide (Cbi), and adding appropriate alpha- and beta-axial ligands. Under aerobic conditions, S. enterica performs the corrinoid-dependent degradation of ethanolamine if given vitamin B12, but it can make B12 from exogenous Cbi only if DMB is also provided. Mutants isolated for their ability to degrade ethanolamine without added DMB converted Cbi to pseudo-B12 cofactors (having adenine as an alpha-axial ligand). The mutations cause an increase in the level of free adenine and install adenine (instead of DMB) as an alpha-ligand. When DMB is provided to these mutants, synthesis of pseudo-B12 cofactors ceases and B12 cofactors are produced, suggesting that DMB regulates production or incorporation of free adenine as an alpha-ligand. Wild-type cells make pseudo-B12 cofactors during aerobic growth on propanediol plus Cbi and can use pseudo-vitamin B12 for all of their corrinoid-dependent enzymes. Synthesis of coenzyme pseudo-B12 cofactors requires the same enzymes (CobT, CobU, CobS, and CobC) that install DMB in the formation of coenzyme B12. Models are described for the mechanism and control of alpha-axial ligand installation.

The neutralizing effect of a polyclonal antibody raised against the N-terminal eighteen-aminoacid residues of birtoxin towards the whole venom of Parabuthus transvaalicus.

Scorpion venom is composed among other things of a large number of neurotoxic peptides affecting all major types of ion channels. The majority of the toxicity of the venom is attributed to the presence of these peptides. In our previous studies using a combination of HPLC and mass spectrometry, we showed that birtoxin like peptides are the major peptidic components of the venom of Parabuthus transvaalicus. These peptides are quite similar to each other differing by only few amino acid residues. In addition they all share a common N-terminus of eighteen amino acid residues. We hypothesize that neutralization of this domain will decrease the toxicity of the whole venom of P. transvaalicus. Polyclonal antibodies against the common N-terminal region of the peptides are generated. Here we show by bioassays that the polyclonal antibodies neutralize the venom of P. transvaalicus in a dose dependent manner and by mass spectrometry and western blotting that these peptides indeed react with the polyclonal antibodies. Previously antibodies generated against a single major toxic component of venom have proven to be an effective strategy for antivenin production. In the case of P. transvaalicus the generated antibody is against the majority of the peptidic fraction due to the presence of several highly similar and highly toxic components in this venom. We show that using the knowledge obtained through biochemical characterization studies it is possible to design very specific antibodies that will be useful for clinical applications against Parabuthus envenomation.

Three structurally related, highly potent, peptides from the venom of Parabuthus transvaalicus possess divergent biological activity.

The venom of South African scorpion Parabuthus transvaalicus contains a novel group of peptide toxins. These peptides resemble the long chain neurotoxins (LCN) of 60-70 residues with four disulfide bridges; however they are 58 residues long and have only three disulfide bridges constituting a new family of peptide toxins. Here we report the isolation and characterization of three new members of this mammal specific group of toxins. Dortoxin is a lethal peptide, bestoxin causes writhing in mice and altitoxin is a highly depressant peptide. Binding ability of these peptides to rat brain synaptosomes is tested. While the crude venom of P. transvaalicus enhances the binding of [(3)H] BTX to rat brain synaptosomes none of these individual toxins had a positive effect on binding. Although the primary structures of these toxins are very similar to birtoxin, their 3D models indicate significant differences. Dortoxin, bestoxin and altitoxin cumulatively constitute at least 20% of the peptide contained in the venom of P. transvaalicus and contribute very significantly to the toxicity of the venom of this medically important scorpion species. Therefore the amino acid sequences presented here can be used to make more specific and effective antivenins. Possible approaches to a systematic nomenclature of toxins are suggested.

In vitro and in vivo metabolism of N-adamantyl substituted urea-based soluble epoxide hydrolase inhibitors.

N,N'-disubstituted urea-based soluble epoxide hydrolase (sEH) inhibitors are promising therapeutics for hypertension, inflammation, and pain in multiple animal models. The drug absorption and pharmacological efficacy of these inhibitors have been reported extensively. However, the drug metabolism of these inhibitors is not well described. Here we reported the metabolic profile and associated biochemical studies of an N-adamantyl urea-based sEH inhibitor 1-adamantan-1-yl-3-(5-(2-(2-ethoxyethoxy)ethoxy)pentyl)urea (AEPU) in vitro and in vivo. The metabolites of AEPU were identified by interpretation of liquid chromatography-mass spectrometry (LC-MS), liquid chromatography-tandem mass spectrometry (LC-MS/MS) and/or NMR. In vitro, AEPU had three major positions for phase I metabolism including oxidations on the adamantyl moiety, urea nitrogen atoms, and cleavage of the polyethylene glycol chain. In a rodent model, the metabolites from the hydroxylation on the adamantyl group and nitrogen atom were existed in blood while the metabolites from cleavage of polyethylene glycol chain were not found in urine. The major metabolite found in rodent urine was 3-(3-adamantyl-ureido)-propanoic acid, a presumably from cleavage and oxidation of the polyethylene glycol moiety. All the metabolites found were active but less potent than AEPU at inhibiting human sEH. Furthermore, cytochrome P450 (CYP) 3A4 was found to be a major enzyme mediating AEPU metabolism. In conclusion, the metabolism of AEPU resulted from oxidation by CYP could be shared with other N-adamantyl-urea-based compounds. These findings suggest possible therapeutic roles for AEPU and new strategies for drug design in this series of possible drugs.

Maternal transfer of BDE-47 to offspring and neurobehavioral development in C57BL/6J mice.

Polybrominated diphenyl ethers (PBDEs) are flame retardants used worldwide in a variety of commercial goods, and are now widely found in both environmental and biological samples. BDE-47 is one of the most pervasive of these PBDE congeners and therefore is of particular concern. In this study C57BL/6J mice were exposed perinatally to 0.03, 0.1 or 1mg/kg/day of BDE-47, a dose range chosen to encompass human exposure levels. Tissue levels of BDE-47 were measured in the blood, brain, fat and milk of dams and in whole fetal homogenate and blood and brain of pups on gestational day (GD) 15, and postnatal days (PNDs) 1, 10 and 21. From GD 15 to PND 1 levels of BDE-47 increased within dam tissues and then decreased from PNDs 1 to 21. Over the period of lactation levels in dam milk were comparatively high when compared to both brain and blood for all dose groups. Measurable levels of BDE-47 were found in the fetus on GD 15 confirming gestational exposure. From PNDs 1 to 21, levels of BDE-47 in pup tissue increased over the period of lactation due to the transfer of BDE-47 through milk. Behavioral tests of fine motor function and learning and memory were carried out between postnatal weeks 5-17 in order to evaluate the neurobehavioral toxicity of BDE-47. Behavioral deficits were only seen in the Barnes spatial maze where mice in the three exposure groups had longer latencies and traveled longer distances to find the escape hole when compared to vehicle control mice. These results support the conclusions that perinatal exposure to BDE-47 can have neurodevelopmental consequences, and that lactational exposure represents a significant exposure risk during development.

Mutation of bacterium Vibrio gazogenes for selective preparation of colorants.

A novel marine bacterium strain effectively produced prodiginine type pigments. These colorants could dye wool, silk and synthetic fabrics such as polyester and polyacrylic and also show antibacterial properties against Escherichia coli and Staphylococcus aureus bacteria on the dyed products. Methyl nitrosoguanidine was used as a mutation agent to increase the genetic diversity and the production yield of the bacteria of the family of Vibrio gazogenes. The analysis of the mutated samples showed that two new main colorants as well as three previously found ones were produced. Liquid chromatography electro spray ionization mass spectrometry (LC-ESI-MS) and nuclear magnetic resonance (NMR) spectroscopic techniques were used to elucidate the structures of the newly produced colorants. Mass measurements revealed that the colorants C1, C2, C3, C4 have molecular masses of 321, 323, 351, and 295 Da. One unstable colorant C5 with molecular mass of 309 Da was detected as well. The mutated bacteria strains increased the yield of pigment production by about 81% and produced prodigiosin in 97% purity. The antibiotic activities of pure colorants are discussed as well. Based on their bio-activity and excellent dyeing capabilities, these colorants could be employed in cosmetic and textile industries.

Antibacterial colorants: characterization of prodiginines and their applications on textile materials.

A strain of Vibrio sp. isolated from marine sediments produced large quantities of bright red pigments that could be used to dye many fibers including wool, nylon, acrylics, and silk. Characterization of the pigments by electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR) revealed three prodiginine-like structures with nonpolar characteristics and low molecular mass. UV-visible spectra of the major constituent in methanol solution showed absorbance at lambda max 530 nm wavelength. The accurate mass result showed that the main isolated product has a molecular mass of m/z 323.1997. Further analysis using mass fragmentation (MS/MS), 1H NMR, COSY, HMQC NMR and DEPT confirmed the detailed structure of the pigment with an elementary composition of C20H25N3O. Fabrics dyed with the microbial prodiginines demonstrated antibacterial activity.

Conformation-dependent stability of junctophilin 1 (JP1) and ryanodine receptor type 1 (RyR1) channel complex is mediated by their hyper-reactive thiols.

Junctophilin 1 (JP1), a 72-kDa protein localized at the skeletal muscle triad, is essential for stabilizing the close apposition of T-tubule and sarcoplasmic reticulum membranes to form junctions. In this study we report that rapid and selective labeling of hyper-reactive thiols found in both JP1 and ryanodine receptor type 1 (RyR1) with 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin, a fluorescent thiol-reactive probe, proceeded 12-fold faster under conditions that minimize RyR1 gating (e.g. 10 mM Mg2+) compared with conditions that promote high channel activity (e.g. 100 microM Ca2+, 10 mM caffeine, 5 mM ATP). The reactivity of these thiol groups was very sensitive to oxidation by naphthoquinone, H2O2, NO, or O2, all known modulators of the RyR1 channel complex. Using preparative SDS-PAGE, in-gel tryptic digestion, high pressure liquid chromatography, and mass spectrometry-based peptide sequencing, we identified 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin-thioether adducts on three cysteine residues of JP1 (101, 402, and 627); the remaining five cysteines of JP1 were unlabeled. Co-immunoprecipitation experiments demonstrated a physical interaction between JP1 and RyR1 that, like thiol reactivity, was sensitive to RyR1 conformation and chemical status of the hyper-reactive cysteines of JP1 and RyR1. These findings support a model in which JP1 interacts with the RyR1 channel complex in a conformationally sensitive manner and may contribute integral redox-sensing properties through reactive sulfhydryl chemistry.