DL-2-Haloacid dehalogenase from Pseudomonas sp. 113 is a new class of dehalogenase catalyzing hydrolytic dehalogenation not involving enzyme-substrate ester intermediate.
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DL-2-Haloacid dehalogenase from Pseudomonas sp. 113 (DL-DEX 113) catalyzes the hydrolytic dehalogenation of D- and L-2-haloalkanoic acids, producing the corresponding L- and D-2-hydroxyalkanoic acids, respectively. Every halidohydrolase studied so far (L-2-haloacid dehalogenase, haloalkane dehalogenase, and 4-chlorobenzoyl-CoA dehalogenase) has an active site carboxylate group that attacks the substrate carbon atom bound to the halogen atom, leading to the formation of an ester intermediate. This is subsequently hydrolyzed, resulting in the incorporation of an oxygen atom of the solvent water molecule into the carboxylate group of the enzyme. In the present study, we analyzed the reaction mechanism of DL-DEX 113. When a single turnover reaction of DL-DEX 113 was carried out with a large excess of the enzyme in H(2)(18)O with a 10 times smaller amount of the substrate, either D- or L-2-chloropropionate, the major product was found to be (18)O-labeled lactate by ionspray mass spectrometry. After a multiple turnover reaction in H(2)(18)O, the enzyme was digested with trypsin or lysyl endopeptidase, and the molecular masses of the peptide fragments were measured with an ionspray mass spectrometer. No peptide fragments contained (18)O. These results indicate that the H(2)(18)O of the solvent directly attacks the alpha-carbon of 2-haloalkanoic acid to displace the halogen atom. This is the first example of an enzymatic hydrolytic dehalogenation that proceeds without producing an ester intermediate. (+info)
Enhanced apoptotic response to photodynamic therapy after bcl-2 transfection.
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Apoptosis is a cellular death process involving the sequential activation of a series of caspases, endonucleases, and other enzymes. The initiation of apoptosis can be inhibited by overexpression of bcl-2 and certain other members of a related family of proteins. We examined the effects of bcl-2 overexpression on the apoptotic response to photodynamic therapy (PDT), using aluminum phthalocyanine as the photosensitizing agent. In this study, we compared the immortalized human breast epithelial cell line MCF10A with a subline (MCF10A/bcl-2) transfected with the human bcl-2 gene. The latter was approximately 2-fold more sensitive to the phototoxic effects of PDT. At a 50 mJ/cm2 light dose, photodamage to MCF-10A/bcl-2 resulted in a greater loss of the mitochondrial membrane potential (delta(psi)m), enhanced release of mitochondrial cytochrome c, a more rapid and greater activation of caspase-3, and a greater apoptotic response. Western blot analysis revealed that the transfected cell line showed overexpression of both bcl-2 and bax, and that PDT caused selective destruction of bcl-2, leaving bax unaffected. The greater apoptotic response by the transfected line is, therefore, attributed to the higher bax:bcl-2 ratio after photodamage. (+info)
Cadmium(II), unlike nickel(II), inhibits 8-oxo-dGTPase activity and increases 8-oxo-dG level in DNA of the rat testis, a target organ for cadmium(II) carcinogenesis.
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8-Oxo-2'-deoxyguanosine 5'-triphosphate pyrophosphohydrolase (8-oxo-dGTPase) is an enzyme which prevents incorporation into DNA of promutagenic 8-oxo-2'-deoxyguanosine (8-oxo-dG) from a deoxynucleotide pool damaged by endogenous oxidants. Its inhibition may thus be carcinogenic. We previously found that Cd(II) inhibited 8-oxo-dGTPase in both cell free systems and cultured cells. To verify this finding in a relevant animal model, we investigated the effects of Cd(II) on cellular 8-oxo-dGTPase activity and nuclear DNA 8-oxo-dG levels in the rat testis, a target organ for Cd(II) carcinogenesis. Ni(II), which does not induce testicular tumors in rats and is a weaker in vitro inhibitor of 8-oxo-dGTPase than Cd(II), was investigated as a comparison. Male F344/NCr rats were given a single s.c. dose of 20 micromol Cd(II) acetate, 90 micromol Ni(II) acetate or 180 micromol sodium acetate (controls) per kg body wt and killed 2, 8, 24 or 48 h later (three rats/time point). Cd(II) caused a gradual decrease in testicular 8-oxo-dGTPase activity with time. It became significant only after 8 h post-injection (P < 0.05) and resulted in a final 50% loss of the enzyme activity at 48 h (P < 0. 01). Although the results for Ni(II) at 8 h and later were apparently lower than the controls, the decrease did not reach statistical significance. Treatment of rats with Cd(II) led to an early and progressive increase (from 130% at 2 h to 200% at 48 h versus the controls) of the 8-oxo-dG level in testicular DNA (P < 0. 05 or better). Ni(II) acetate also tended to raise the testicular 8-oxo-dG level, but the increase was transient, with an apparent maximum at 8 h, and did not approach statistical significance (P < 0. 2). Thus, Cd(II), unlike Ni(II), is able to inhibit 8-oxo-dGTPase activity and to raise 8-oxo-dG levels in rat testicular DNA. However, the time course of both effects indicates that 8-oxo-dGTPase inhibition is most likely not the sole cause of the increase in 8-oxo-dG. (+info)
S-adenosylmethionine attenuates the lipopolysaccharide-induced expression of the gene for tumour necrosis factor alpha.
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Intracellular deficiency of S-adenosylmethionine (AdoMet) and elevated serum concentrations of tumour necrosis factor alpha (TNF) are hallmarks of toxin-induced liver injury. In these models, the administration of either exogenous AdoMet or antibody/soluble receptor for TNF attenuates the injury. We have demonstrated previously that the administration of exogenous AdoMet to AdoMet-deficient rats attenuated lipopolysaccharide (LPS)-induced liver injury and serum TNF concentrations. Here we report that AdoMet lowered the amount of TNF secreted by LPS-stimulated murine macrophage cells (RAW 264.7) in a dose-dependent manner. The inhibition of TNF release was correlated with changes in the steady-state TNF mRNA concentrations. Changes in TNF mRNA were not due to its altered stability and might have been due to an attenuation of the transcription rate of the TNF gene. The inhibition of TNF release in RAW cells was not mediated by GSH because treatment with AdoMet did not increase intracellular GSH. In addition, N-acetylcysteine, whereas it did increase GSH concentration, had no effect on LPS-stimulated TNF release in these cells. Exogenous AdoMet also attenuated LPS-induced serum TNF levels in normal rats sensitized with lead. Thus AdoMet administration might exert its hepatoprotective effects at least in part by its inhibitory effect on expression of the gene for TNF. (+info)
Kinetic and equilibrium studies of incorporation of di-sulfonated aluminum phthalocyanine into unilamellar vesicles.
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The interactions of cis-di-sulfonated aluminum phthalocyanine (PcS(2)Al) with dimyristoylphosphatidylcholine (DMPC) unilamellar vesicles have been investigated by fluorescence spectroscopy. At pH 7.0, PcS(2)Al incorporates into the vesicles with a high affinity constant (2.7x10(6) M(-1), in terms of phospholipid concentration). The fluorescence changes following rapid mixing of PcS(2)Al with vesicles are biphasic. The first phase is attributed to the entry of PcS(2)Al into the vesicles, as deduced from the linear dependence of the rate upon lipid concentration. More surprisingly, this rate is strongly pH dependent with a marked maximum around pH 7.3, a result interpreted in terms of the coordination state of the aluminum ion in aqueous solutions. At this pH, a hydroxide ion neutralizes the residual positive charge of the metal ion that remains unbalanced after coordination by the phthalocyanine cycle. A water molecule is likely to complete the metal coordination sphere. Only this form, PcAl(+)(OH(-))(OH(2)), with an uncharged core is quickly incorporated into the vesicles. The protonation of OH(-) or the deprotonation of the coordinated H(2)O leading to a positively or negatively charged core, respectively, account for the observed pH effect. Studies on the effect of cholesterol addition and exchange of PcS(2)Al between vesicles and albumin all indicate the absence of transfer of the phthalocyanine between the vesicle hemileaflets, a result expected from the presence of the two negatively charged sulfonated groups at the ring periphery. Instead, the slower kinetic phase is likely due to the movement of the phthalocyanine becoming more buried within the outer leaflet upon the loss of the water molecule coordinated to the aluminum ion. (+info)
Mitochondrial depolarization is not required for neuronal apoptosis.
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Mitochondria are sites of cellular energy production but may also influence life and death decisions by initiating or inhibiting cell death. Mitochondrial depolarization and the subsequent release of pro-apoptotic factors have been suggested to be required for the activation of a cell death program in some forms of neuronal apoptosis. We induced apoptosis in cultured rat hippocampal neurons by exposure to the protein kinase inhibitor staurosporine (STS) (300 nM). The time course of mitochondrial membrane potential (DeltaPsi(m)) during apoptosis was examined using the probe tetramethylrhodamine ethyl ester (TMRE). Cells exhibited no decrease in TMRE fluorescence, indicative of mitochondrial depolarization, up to 8 hr after STS exposure. Rather, baseline TMRE fluorescence remained unchanged up to 2 hr and thereafter actually increased significantly. Throughout this time period, the mitochondria could also be depolarized with the protonophore carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP, 0.1 microM), exhibiting the same relative magnitude of fluorescence release (unquenching) as controls. Even after 16 hr of staurosporine treatment, neurons that showed signs of nuclear apoptosis maintained DeltaPsi(m) and could be depolarized with FCCP. In contrast, caspase-3-like activity had increased roughly sevenfold by 2 hr and >20-fold by 8 hr. Double-labeling of hippocampal neurons with the potential-sensitive probe Mitotracker Red Chloromethyl X-Rosamine and an antibody to cytochrome c demonstrated at the subcellular level that mitochondrial cytochrome c release also occurred in the absence of mitochondrial depolarization. These data suggest that mitochondrial depolarization is not a decisive event in neuronal apoptosis. (+info)
Structural effects of cobalt-amine compounds on DNA condensation.
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Light scattering and electron microscopy have been used to investigate the structural effects of the trivalent complexes hexaammine cobalt (III) chloride (Cohex), tris(ethylenediamine) cobalt(III) chloride (Coen), and cobalt(III) sepulchrate chloride (Cosep) on DNA condensation. These cobalt-amine compounds have similar ligand coordination geometries but differ slightly in size. Their hydrophobicity is in the order Cosep > Coen > Cohex, according to the numbers of methylene groups in these ligands. All of these compounds effectively precipitate DNA at high concentrations; but despite a lower surface charge density, Cosep condenses DNA twice as effectively as Coen or Cohex. UV and CD measurements of the supernatants of cobalt-amine/DNA solutions reveal a preferential binding of Delta-Coen over Lambda-Coen to the precipitated DNA, but there is no chiral selectivity for Cosep. Competition experiments show that the binding strengths of these three cobalt-amine compounds to aggregated DNA are comparable. A charge neutralization of 88-90% is required for DNA condensation. Our data indicate that 1) electrostatic interaction is the main driving force for binding of multivalent cations to DNA; 2) DNA condensation is dependent on the structure of the condensing agent; and 3) the hydration pattern or polarization of water molecules on the surface of condensing agents plays an important role in DNA condensation and chiral recognition. (+info)
Lead acetate exposure inhibits nitric oxide synthase activity in capillary and synaptosomal fractions of mouse brain.
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The toxicity of lead (Pb) is of concern to public health due to its persistence in the environment. Brain is one of the major target organs where severe neurologic alterations may be triggered after exposure. The primary effects of lead on brain functions are thought to be a damage to the nervous system microvasculature. However, the mechanism of this toxicity is poorly understood. Nitric oxide synthase (NOS) may be a target for lead and changes in its function can result in a cascade of pathophysiological effects that may be observed in isolated capillaries and synaptosomes. We have determined the concentration of lead in blood, capillaries and synaptosomes in brain from mice receiving 0, 250, 500, and 1000 ppm of lead for 14 days, through the drinking water. NOS activity was determined in the capillaries and synaptosomes by following the conversion of 3H-L-arginine to 3H-L-citrulline. The results show that blood lead levels were dose-dependent. Brain capillaries showed a preferential accumulation of lead as compared to synaptosomes. With all Pb treatments, synaptosomal constitutive NOS was inhibited (about 50% of control) while the inducible NOS activity in capillaries was enhanced. These data suggest that inhibition of cNOS activity and increase in iNOS may contribute to the Pb effects on the CNS. (+info)