Behaviour of cyclic nucleotides and Ca2+ levels in liver tissue of rats poisoned by white phosphorus and trichlorobromomethane

The content of hepatic cyclic AMP was increased soon after intoxication by white phosphorus. Its level reached a maximum 4 h after poisoning, but in subsequent phases tended to return to normal. In contrast, the cyclic GMP concentration was altered only 24 and 36 h after treatment with the same hepatotoxin. Similar modifications of cAMP and cGMP content were also detected after poisoning by trichlorobromomethane (CBrCl3). As a consequence, an altered cGMP/cAMP ratio was found in both experimental conditions. Further, the modification of cAMP content after white phosphorus was detected prior to liver damage (steatosis and necrosis), while the highest concentration of the cyclic nucleotide in CBrCl3-poisoned rats was found when fatty liver was already evident. In addition, in phosphorus-poisoned rats, the hepatic content of Ca2+ was found to be unmodified during all phases of the intoxication, while after CBrCl3 a phasic increase of the Ca2+ level was observed at 4, 24 and 36 h.     

Release of ethane and pentane from rat tissue slices: effect of vitamin E, halogenated hydrocarbons, and iron overload

The effects of in vitro addition of halogenated hydrocarbons on the susceptibility of various rat tissues to lipid peroxidation, and of iron overload and dietary vitamin E in the intact rat on subsequent lipid peroxidation in rat tissue slices were examined. The ease and speed of tissue slice preparation allowed testing of multiple tissues from the same animals. Total ethane and pentane (TEP) released from the slices was as reliable as and more sensitive than thiobarbituric acid-reactive substances as an index of lipid peroxidation. TEP was released by tissues from vitamin E-deficient rats in the following order of magnitude:intestine = brain = kidney greater than liver = lung greater than heart greater than testes = diaphragm greater than skeletal muscle. The potency of halogenated hydrocarbons for causing increased TEP release from vitamin E-deficient rat liver slices was CBrCl3 greater than CCl4 = 1,1,2,2-tetrabromoethane = 1,1,2,2-tetrachloroethane greater than perchloroethylene. CBrCl3 also stimulated TEP release from kidney, intestine, and heart slices, thus identifying these as potential target organs for CBrCl3 toxicity. Dietary vitamin E decreased TEP release from liver and, to a lesser extent, from kidney. Iron overload in the rat increased TEP release by slices from all tissues tested except the brain.     

Heme peptide as a model substance for halogenomethane activation and heme modification.

Octa-heme peptide (CHP) obtained from Candida krusei cytochrome c was tested for suicidal activation of halogenomethanes. Under anaerobic conditions, CHP was kept in the reduced state in the presence of NADPH and NADPH-cytochrome P-450 reductase. Addition of CBrCl3 to the reduced CHP caused spectral changes such as rapid disappearance of alpha and beta bands and gradual decrease in the gamma-peak height, accompanied by oxidation of NADPH. Heme content of the reaction mixture, determined as pyridine hemochrome, also decreased NADPH dependently. CCl4 was less effective than CBrCl3, while CHCl3 had almost no effect. N-tert-butyl-alpha-phenylnitrone (PBN) suppressed the CBrCl3-induced heme damage, and resulted in the formation of radical adduct .PBN-CCl3 as evidenced by ESR spectroscopy. Radical formation was also observed with CCl4. The CHP damage induced by CBrCl3 was also accompanied by the release of Br- about 11-12-times molar excess of CHP, whereas the release of CHCl3 was about 20% that of Br-.FD-MS assay of the product of CHP reaction suggested that 10 trichloromethyl radicals bonded with CHP. Thus, CBrCl3 undergoes single-electron reduction in the presence of reduced CHP to trichloromethyl radicals, which covalently bind to CHP molecules. Heme peptide may be a useful tool in the study of mechanisms involved in the destruction of cytochrome P-450 by halogenomethanes.     

Interleukin-6 protects PC12 cells from 4-hydroxynonenal-induced cytotoxicity by increasing intracellular glutathione levels

Oxidative stress plays an important role in neuronal cell death associated with many different neurodegenerative conditions, and it is reported that 4-hydroxynonenal (HNE), an aldehydic product of membrane lipid peroxidation, is a key mediator of neuronal cell death induced by oxidative stress. Previously, we have demonstrated that interleukin-6 (IL-6) protects PC12 cells from serum deprivation and 6-hydroxydopamine-induced toxicity. Therefore, in the present study, we examined the effects of interleukins on HNE toxicity in PC12 cells. Exposure of PC12 cells to HNE resulted in a decrease in levels of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction, which was due to necrotic and apoptotic cell death. Addition of IL-6 24 h before HNE treatment provided a concentration-dependent protection against HNE toxicity, whereas neither IL-1β nor IL-2 had any effect. Addition of glutathione (GSH)-ethyl ester, but not superoxide dismutase or catalase, before HNE treatment to the culture medium protected PC12 cells from HNE toxicity. We found that IL-6 increases intracellular GSH levels and the activity of γ-glutamylcysteine synthetase (γ-GCS) in PC12 cells. Buthionine sulfoximine (BSO), an inhibitor of γ-GCS, reversed the protective effect of IL-6 against HNE toxicity. These results suggest that IL-6 protects PC12 cells from HNE-induced cytotoxicity by increasing intracellular levels of GSH.     

Carbon tetrachloride toxicity on Escherichia coli exacerbated by superoxide

The effects of carbon tetrachloride (CCl4) and paraquat on the growth of Escherichia coli were investigated. Paraquat at 10 mM caused some inhibition of growth of E. coli in trypticase-soy-yeast extract medium. CCl4 enhanced growth inhibition by paraquat in a concentration-dependent manner. In the absence of paraquat, CCl4 had no effect on growth rate or on surviving cell numbers at stationary phase. CCl4 did not prevent the induction of manganese-superoxide dismutase by paraquat. Under anaerobic conditions, CCl4 and paraquat exhibited no effect on E. coli. In the presence of Mn(II) and paraquat, intracellular superoxide dismutase was markedly induced and protected E. coli against the toxicity of CCl4 and paraquat. The reactive free radical CCl3OO-, which can be formed from the reaction of O2- with CCl4, may cause cell damage. The growth-inhibiting effects of polyhalides in the presence of paraquat followed the order CBrCl3 greater than CCl4 greater than CHCl3 greater than CH2Cl2, which is in accord with that of the reaction rates of these compounds with O2- and with their hepatotoxicities. These results suggest that O2- plays a role in the hepatotoxicity of polyhalides.     

Vitamin E, diethylmaleate and bromotrichloromethane interactions in oxidative damage in vivo

In vivo interactions of vitamin E with diethylmaleate (DEM) and bromotrichloromethane (CBrCl3) were examined in rats fed a diet either without vitamin E or supplemented with 30 IU dl-alpha-tocopheryl acetate/kg. Groups of rats within each dietary group were given two injections 30 min apart. One group received two injections of the mineral oil carrier. The other groups were injected with either DEM and mineral oil, mineral oil and CBrCl3, or DEM and CBrCl3. The rats were killed 10 min after the second injection. Measurements were made of hepatic GSH, thiobarbituric acid-reactive substances (TBARS) as a lipid peroxidation index, and 11 enzymes as potential markers of oxidant damage. Special focus was placed on reactive cysteine-containing aldehyde dehydrogenase (ALDH). Although dietary vitamin E protected ALDH, the enzyme was highly susceptible to oxidant damage. ALDH activity was correlated with GSH (r = 0.83, p less than 0.001) and there was an inverse relationship between the logarithmic values of ALDH activity and TBARS (r = 0.78, p less than 0.001). Similar results were observed for a number of other enzymes when GSH depletion preceded oxidant treatment. Two-way analysis of variance revealed significant effects of vitamin E and of injection treatments on hepatic GSH. There was a significant interaction between vitamin E and the injection treatments on the activities of five enzymes. The results suggested that vitamin E and GSH functioned together to protect sensitive enzymes against oxidant stress. The sensitive enzymes may be useful markers of hepatic damage in vivo.     

Substrate specificity of haloalkane dehalogenases

An enzyme's substrate specificity is one of its most important characteristics. The quantitative comparison of broad-specificity enzymes requires the selection of a homogenous set of substrates for experimental testing, determination of substrate-specificity data and analysis using multivariate statistics. We describe a systematic analysis of the substrate specificities of nine wild-type and four engineered haloalkane dehalogenases. The enzymes were characterized experimentally using a set of 30 substrates selected using statistical experimental design from a set of nearly 200 halogenated compounds. Analysis of the activity data showed that the most universally useful substrates in the assessment of haloalkane dehalogenase activity are 1-bromobutane, 1-iodopropane, 1-iodobutane, 1,2-dibromoethane and 4-bromobutanenitrile. Functional relationships among the enzymes were explored using principal component analysis. Analysis of the untransformed specific activity data revealed that the overall activity of wild-type haloalkane dehalogenases decreases in the following order: LinB~DbjA>DhlA~DhaA~DbeA~DmbA>DatA~DmbC~DrbA. After transforming the data, we were able to classify haloalkane dehalogenases into four SSGs (substrate-specificity groups). These functional groups are clearly distinct from the evolutionary subfamilies, suggesting that phylogenetic analysis cannot be used to predict the substrate specificity of individual haloalkane dehalogenases. Structural and functional comparisons of wild-type and mutant enzymes revealed that the architecture of the active site and the main access tunnel significantly influences the substrate specificity of these enzymes, but is not its only determinant. The identification of other structural determinants of the substrate specificity remains a challenge for further research on haloalkane dehalogenases.     

Isolation and characterization of a haloalkane halidohydrolase from Rhodococcus erythropolis Y2

Rhodococcus erythropolis strain Y2, isolated from soil by enrichment culture using 1-chlorobutane, was able to utilize a range of halogenated aliphatic compounds as sole sources of carbon and energy. The ability to utilize 1-chlorobutane was conferred by a single halidohydrolase-type haloalkane dehalogenase. The presence of the single enzyme in cell-free extracts was demonstrated by activity strain polyacrylamide gel electrophoresis. The purified enzyme was a monomeric protein with a relative molecular mass of 34 kDa and demonstrated activity against a broad range of haloalkanes, haloalcohols and haloethers. The highest activity was found towards alpha, omega disubstituted chloro- and bromo- C2-C6 alkanes and 4-chlorobutanol. The Km value of the enzyme for 1-chlorobutane was 0.26 mM. A comparison of the R. erythropolis Y2 haloalkane halidohydrolase with other haloalkane dehalogenases is discussed on the basis of biochemical properties and N-terminal amino acid sequence data.     

Mutual dependence of growth modifying effects of 4-hydroxynonenal and fetal calf serum in vitro

Recently, the hypothesis has been put forward that 4-hydroxynonenal (HNE), an aldehydic product of lipid peroxidation, contributes to the mechanism of oxygen toxicity and to the selective pressure exerted by exposure to hyperoxia. Here it has been studied whether HNE itself is involved in mechanisms that convey increased resistance of the cells to the toxicity of HNE. The following four cell lines, different in their basic biological features, were used: nonmalignant Chinese hamster lung fibroblasts V79 (established cell line), human carcinoma HeLa (established cell line), pigmented murine melanoma B16f10 (primary culture), and amelanotic murine melanoma B16BL6 (primary culture). The cells were pretreated in vitro with a toxic dose of HNE (50 μM), and afterwards the effects of a second exposure to the same dose of HNE on ³H-thymidine incorporation was examined. Cells were cultured in the absence and in the presence of fetal calf serum (FCS), because it had been shown that a growth modifying effect of HNE depends on an unknown serum factor. The results showed that, regardless of the type of cells, preculturing them with 50 μM HNE in the presence of serum changed the reactivity of the cells to added serum as well as to additional HNE treatment. Thus, HNE precultured cells incorporated less ³H-thymidine in the presence of serum than if cultured under serum-free conditions. On the other hand, HNE precultured cells became less sensitive to further HNE treatment, but only if cultured in the presence of serum. It was concluded that a toxic dose of HNE renders surviving cells more resistent to oxidative stress, possibly by forming a bioactive conjugate with a serum peptide/protein. It is supposed that the same humoral factor might be responsible for the growth modifying effects of high doses of HNE as well as for the growth inhibition in the presence of serum observed for HNE precultured cells.     

Utilization of trihalogenated propanes by Agrobacterium radiobacter AD1 through heterologous expression of the haloalkane dehalogenase from Rhodococcus sp. strain M15-3.

Trihalogenated propanes are toxic and recalcitrant organic compounds. Attempts to obtain pure bacterial cultures able to use these compounds as sole carbon and energy sources were unsuccessful. Both the haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 (DhlA) and that from Rhodococcus sp. strain m15-3 (DhaA) were found to dehalogenate trihalopropanes to 2,3-dihalogenated propanols, but the kinetic properties of the latter enzyme are much better. Broad-host-range dehalogenase expression plasmids, based on RSF1010 derivatives, were constructed with the haloalkane dehalogenase from Rhodococcus sp. strain m15-3 under the control of the heterologous promoters P(lac), P(dhlA), and P(trc). The resulting plasmids yielded functional expression in several gram-negative bacteria. A catabolic pathway for trihalopropanes was designed by introducing these broad-host-range dehalogenase expression plasmids into Agrobacterium radiobacter AD1, which has the ability to utilize dihalogenated propanols for growth. The recombinant strain AD1(pTB3), expressing the haloalkane dehalogenase gene under the control of the dhlA promoter, was able to utilize both 1,2,3-tribromopropane and 1,2-dibromo-3-chloropropane as sole carbon sources. Moreover, increased expression of the haloalkane dehalogenase resulted in elevated resistance to trihalopropanes.     

Biodegradation of 1,2,3-Trichloropropane through Directed Evolution and Heterologous Expression of a Haloalkane Dehalogenase Gene

Using a combined strategy of random mutagenesis of haloalkane dehalogenase and genetic engineering of a chloropropanol-utilizing bacterium, we constructed an organism that is capable of growth on 1,2,3-trichloropropane (TCP). This highly toxic and recalcitrant compound is a waste product generated from the manufacture of the industrial chemical epichlorohydrin. Attempts to select and enrich bacterial cultures that can degrade TCP from environmental samples have repeatedly been unsuccessful, prohibiting the development of a biological process for groundwater treatment. The critical step in the aerobic degradation of TCP is the initial dehalogenation to 2,3-dichloro-1-propanol. We used random mutagenesis and screening on eosin-methylene blue agar plates to improve the activity on TCP of the haloalkane dehalogenase from Rhodococcus sp. m15-3 (DhaA). A second-generation mutant containing two amino acid substitutions, Cys176Tyr and Tyr273Phe, was nearly eight times more efficient in dehalogenating TCP than wild-type dehalogenase. Molecular modeling of the mutant dehalogenase indicated that the Cys176Tyr mutation has a global effect on the active-site structure, allowing a more productive binding of TCP within the active site, which was further fine tuned by Tyr273Phe. The evolved haloalkane dehalogenase was expressed under control of a constitutive promoter in the 2,3-dichloro-1-propanol-utilizing bacterium Agrobacterium radiobacter AD1, and the resulting strain was able to utilize TCP as the sole carbon and energy source. These results demonstrated that directed evolution of a key catabolic enzyme and its subsequent recruitment by a suitable host organism can be used for the construction of bacteria for the degradation of a toxic and environmentally recalcitrant chemical.     

Resveratrol protects against 4-HNE induced oxidative stress and apoptosis in Swiss 3T3 fibroblasts

Here we report on the marked protective effect of resveratrol on 4-hydroxynonenal (4-HNE) induced oxidative stress and apoptotic death in Swiss 3T3 fibroblasts. 4-HNE, one of the major aldehydic products of the peroxidation of membrane w-6 polyunsaturated fatty acids, has been suggested to contribute to oxidant stress mediated cell injury. Indeed, in vitro treatment of 3T3 fibroblasts with 4-HNE induced a condition of oxidative stress as monitored by the oxidation of dichlorofluorescein diacetate; this reaction was prevented when cells were pretreated with resveratrol. Further, 4-HNE-treated fibroblasts eventually underwent apoptotic death as determined by differential staining and internucleosomal DNA fragmentation. Resveratrol pretreatment also prevented 4-HNE induced DNA fragmentation and apoptosis. These observations are consistent with a potential role of lipid peroxidation-derived products in programmed cell death and demonstrate that resveratrol can counteract this effect by quenching cell oxidative stress.     

Utilization of Trihalogenated Propanes by Agrobacterium radiobacter AD1 through Heterologous Expression of the Haloalkane Dehalogenase from Rhodococcus sp. Strain m15-3

Trihalogenated propanes are toxic and recalcitrant organic compounds. Attempts to obtain pure bacterial cultures able to use these compounds as sole carbon and energy sources were unsuccessful. Both the haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 (DhlA) and that from Rhodococcus sp. strain m15-3 (DhaA) were found to dehalogenate trihalopropanes to 2,3-dihalogenated propanols, but the kinetic properties of the latter enzyme are much better. Broad-host-range dehalogenase expression plasmids, based on RSF1010 derivatives, were constructed with the haloalkane dehalogenase from Rhodococcus sp. strain m15-3 under the control of the heterologous promoters P_lac, P_dhlA, and P_trc. The resulting plasmids yielded functional expression in several gram-negative bacteria. A catabolic pathway for trihalopropanes was designed by introducing these broad-host-range dehalogenase expression plasmids into Agrobacterium radiobacter AD1, which has the ability to utilize dihalogenated propanols for growth. The recombinant strain AD1(pTB3), expressing the haloalkane dehalogenase gene under the control of the dhlA promoter, was able to utilize both 1,2,3-tribromopropane and 1,2-dibromo-3-chloropropane as sole carbon sources. Moreover, increased expression of the haloalkane dehalogenase resulted in elevated resistance to trihalopropanes.     

Degradation of 1,3-Dichloropropene by Pseudomonas cichorii 170

The gram-negative bacterium Pseudomonas cichorii 170, isolated from soil that was repeatedly treated with the nematocide 1,3-dichloropropene, could utilize low concentrations of 1,3-dichloropropene as a sole carbon and energy source. Strain 170 was also able to grow on 3-chloroallyl alcohol, 3-chloroacrylic acid, and several 1-halo-n-alkanes. This organism produced at least three different dehalogenases: a hydrolytic haloalkane dehalogenase specific for haloalkanes and two 3-chloroacrylic acid dehalogenases, one specific for cis-3-chloroacrylic acid and the other specific for trans-3-chloroacrylic acid. The haloalkane dehalogenase and the trans-3-chloroacrylic acid dehalogenase were expressed constitutively, whereas the cis-3-chloroacrylic acid dehalogenase was inducible. The presence of these enzymes indicates that 1,3-dichloropropene is hydrolyzed to 3-chloroallyl alcohol, which is oxidized in two steps to 3-chloroacrylic acid. The latter compound is then dehalogenated, probably forming malonic acid semialdehyde. The haloalkane dehalogenase gene, which is involved in the conversion of 1,3-dichloropropene to 3-chloroallyl alcohol, was cloned and sequenced, and this gene turned out to be identical to the previously studied dhaA gene of the gram-positive bacterium Rhodococcus rhodochrous NCIMB13064. Mutants resistant to the suicide substrate 1,2-dibromoethane lacked haloalkane dehalogenase activity and therefore could not utilize haloalkanes for growth. PCR analysis showed that these mutants had lost at least part of the dhaA gene.     

Biodegradation of 1,2,3-trichloropropane through directed evolution and heterologous expression of a haloalkane dehalogenase gene

Using a combined strategy of random mutagenesis of haloalkane dehalogenase and genetic engineering of a chloropropanol-utilizing bacterium, we constructed an organism that is capable of growth on 1,2,3-trichloropropane (TCP). This highly toxic and recalcitrant compound is a waste product generated from the manufacture of the industrial chemical epichlorohydrin. Attempts to select and enrich bacterial cultures that can degrade TCP from environmental samples have repeatedly been unsuccessful, prohibiting the development of a biological process for groundwater treatment. The critical step in the aerobic degradation of TCP is the initial dehalogenation to 2,3-dichloro-1-propanol. We used random mutagenesis and screening on eosin-methylene blue agar plates to improve the activity on TCP of the haloalkane dehalogenase from Rhodococcus sp. m15-3 (DhaA). A second-generation mutant containing two amino acid substitutions, Cys176Tyr and Tyr273Phe, was nearly eight times more efficient in dehalogenating TCP than wild-type dehalogenase. Molecular modeling of the mutant dehalogenase indicated that the Cys176Tyr mutation has a global effect on the active-site structure, allowing a more productive binding of TCP within the active site, which was further fine tuned by Tyr273Phe. The evolved haloalkane dehalogenase was expressed under control of a constitutive promoter in the 2,3-dichloro-1-propanol-utilizing bacterium Agrobacterium radiobacter AD1, and the resulting strain was able to utilize TCP as the sole carbon and energy source. These results demonstrated that directed evolution of a key catabolic enzyme and its subsequent recruitment by a suitable host organism can be used for the construction of bacteria for the degradation of a toxic and environmentally recalcitrant chemical.     

4-Hydroxyhexenal: a lipid peroxidation product derived from oxidized docosahexaenoic acid

4-Hydroxynonenal and 4-hydroxyhexenal are cytotoxic aldehydic products of lipid peroxidation with high biological activity. Peroxidation of n - 6 fatty acids produces 4-hydroxynonenal, but the origin of 4-hydroxyhexenal has been uncertain. We now present evidence that 4-hydroxyhexenal is generated by oxidation of docosahexaenoic acid, the most abundant n-3 fatty acid in tissues.     

Influence of acetone on the severity of the liver injury induced by haloalkane mixtures.

Acetone potentiation of haloalkane-induced liver injury is a well-known phenomenon. Acetone-treated rats challenged with a trichloroethylene-CCl4 mixture exhibit a more sever liver injury than that predicted by the addition of the single potentiating effects of each. The purpose of the present study was to determine if acetone exerted similar interactions with other haloalkane mixtures. The testing protocol used was designed and performed to allow categorization of interactions occurring among two or three agents. Rats were treated (p.o.) with corn oil or acetone (10.2 mmol/kg) and were administered (i.p.) 18 h later 1,1-dichloroethylene (0.6 mmol/kg), trichloroethylene (5.6 mmol/kg), tetrachloroethylene (19.6 mmol/kg), 1,1,1-trichloroethane (10.0 mmol/kg), 1,1,2-trichloroethane (1.1 mmol/kg), 1,1,2,2-tetrachloroethane (1.0 mmol/kg), CHCl3 (6.2 mmol/kg), CCl4 (1.0 mmol/kg), or a mixture of two haloalkanes (all 28 combinations were tested). Liver injury was assessed 24 h later using plasma alanine aminotransferase activity and a quantitative histological evaluation. In corn oil pretreated rats, the hepatotoxic responses observed for the 28 mixtures were additive for 26 of 28 mixtures and supra-additive for 2 of 28, whereas in acetone-pretreated rats the responses observed were additive for 17 of 28, infra-additive for 10 of 28, and supra-additive for 1 of 28. Mixtures containing 1,1,1-trichloroethane or tetrachloroethylene resulted only in no change in toxicity or infra-additivity. Increased toxic responses (additivity and supra-additivity) were observed with certain binary mixtures containing CCl4, CHCl3, 1,1,2-trichloroethane, or 1,1-dichloroethylene.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vivo and in vitro evidence concerning the role of lipid peroxidation in the mechanism of hepatocyte death due to carbon tetrachloride

Isolated rat hepatocytes exposed to CCl₄ showed a stimulated formation of malonaldehyde after only 30–60 min incubation. Conversely, the onset of hepatocyte death was a relatively late event, being significant only after 2–3 h of treatment. A cause–effect relationship between the two phenomena has been demonstrated by using hepatocytes isolated from rats pretreated with alpha-tocopherol. Comparable results were obtained in vivo where supplementation with alpha-tocopherol 15 h before CCl₄ dosing induced a partial or complete protection against the drug's necrogenic effect, depending on the concentration of the haloalkane used. Moreover, the vitamin supplementation prevented the CCl₄-induced increase of liver total calcium content, probably by blocking alterations in the liver cell plasma membranes due to lipid peroxidation.     

Reconstruction of mycobacterial dehalogenase Rv2579 by cumulative mutagenesis of haloalkane dehalogenase LinB

The homology model of protein Rv2579 from Mycobacterium tuberculosis H37Rv was compared with the crystal structure of haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26, and this analysis revealed that 6 of 19 amino acid residues which form an active site and entrance tunnel are different in LinB and Rv2579. To characterize the effect of replacement of these six amino acid residues, mutations were introduced cumulatively into the six amino acid residues of LinB. The sixfold mutant, which was supposed to have the active site of Rv2579, exhibited haloalkane dehalogenase activity with the haloalkanes tested, confirming that Rv2579 is a member of the haloalkane dehalogenase protein family.     

Biological interactions of alpha,beta-unsaturated aldehydes

This article describes the chemical nature of alpha,beta-unsaturated aldehydes and some of their toxicological effects based on their ability to function as direct-acting alkylating agents. Selected compounds discussed include alpha,beta-unsaturated aldehydic environmental pollutants, metabolites of xenobiotics and natural products, and lipid peroxidation--and DNA oxidation products derived from cellular constituents. Briefly reviewed are sources and mechanisms of formation of the aldehydes, their reactivity with respect to glutathione and amino-groups, their toxicity based on interaction with sulfhydryl and amino targets in cells, and modulation of their toxicity by metabolism.