Genetic rescue of Leishmania deficiency in porphyrin biosynthesis creates mutants suitable for analysis of cellular events in uroporphyria and for photodynamic therapy

Leishmania was found deficient in at least five and most likely seven of the eight enzymes in the heme biosynthesis pathway, accounting for their growth requirement for heme compounds. The xenotransfection of this trypanosomatid protozoan led to their expression of the mammalian genes encoding delta-aminolevulinate (ALA) dehydratase and porphobilinogen deaminase, the second and the third enzymes of the pathway, respectively. These transfectants still require hemin or protoporphyrin IX for growth but produce porphyrin when ALA was supplied exogenously. Leishmania is thus deficient in all first three enzymes of the pathway. Uroporphyrin I was produced as the sole intermediate by these transfectants, further indicating that they are also deficient in at least two porphyrinogen-metabolizing enzymes downstream of porphobilinogen deaminase, i.e. uroporphyrinogen III co-synthase and uroporphyrinogen decarboxylase. Pulsing the transfectants with ALA induced their transition from aporphyria to uroporphyria. Uroporphyrin I emerged in these cells initially as diffused throughout the cytosol, rendering them sensitive to UV irradiation. The porphyrin was subsequently sequestered in cytoplasmic vacuoles followed by its release and accumulation in the extracellular milieu, concomitant with a reduced photosensitivity of the cells. These events may represent cellular mechanisms for disposing soluble toxic waste from the cytosol. Monocytic tumor cells were rendered photosensitive by infection with uroporphyric Leishmania, suggestive of their potential application for photodynamic therapy.     

Transgenic Leishmania model for delta-aminolevulinate-inducible monospecific uroporphyria: cytolytic phototoxicity initiated by singlet oxygen-mediated inactivation of proteins and its ablation by endosomal mobilization of cytosolic uroporphyrin

Inherent deficiencies of Leishmania in heme biosynthesis were genetically complemented for delta-aminolevulinate-inducible biosynthesis and accumulation of light-excitable uroporphyrin. The phototoxic flagellar immobilization and cytolysis phenotypes and porphyrin mobilization noted previously were further analyzed biochemically and cytologically to delineate the mechanism of phototoxicity and detoxification in this monoporphyric model. Under optimal conditions of induction for approximately 3 days, cells remained viable but became increasingly uroporphyric, peaking at > or =90% of the population by approximately day 2; thereafter, a small population of less porphyric or aporphyric cells emerged. On exposure to light, the flagella of porphyric cells were immobilized in milliseconds, and singlet oxygen became detectable in their lysates. Both photosensitive phenotypes increased proportionally with the cellular uroporphyric levels and were susceptible to inhibition by azide, but not by D-mannitol. Brief irradiation of the uroporphyric cells produced no appreciable protein degradation but inactivated cytosolic neomycin phosphotransferase and significantly bleached cytosolic green fluorescent protein, which was azide reversible. These cells were irreparably photodamaged, as indicated by their subsequent loss of membrane permeability and viability. This is the first in situ demonstration that early inactivation of functional proteins by singlet oxygen initiates the cytolytic phototoxicity in uroporphyria. Detoxification appears to involve endocytic/exocytic mobilization of uroporphyrin from cytosol to "porphyrinosomes" for its eventual extracellular expulsion. This is proposed as the sole mechanism of detoxification, since it is attributable to the reversion of porphyric to aporphyric cells during uroporphyrinogenesis and repeated cycles of this event plus photolysis selected no resistant mutants, only aporphyric clones of the parental phenotypes. Further characterization of the transport system for uroporphyrin in this model is expected to benefit not only our understanding of the cellular mechanism for disposal of toxic soluble wastes but also potentially the effective management of human uroporphyria and the use of uroporphyric Leishmania for vaccine/drug delivery.     

Delta-aminolevulinate-induced host-parasite porphyric disparity for selective photolysis of transgenic Leishmania in the phagolysosomes of mononuclear phagocytes: a potential novel platform for vaccine delivery

Leishmania double transfectants (DTs) expressing the 2nd and 3rd enzymes in the heme biosynthetic pathway were previously reported to show neogenesis of uroporphyrin I (URO) when induced with delta-aminolevulinate (ALA), the product of the 1st enzyme in the pathway. The ensuing accumulation of URO in DT promastigotes rendered them light excitable to produce reactive oxygen species (ROS), resulting in their cytolysis. Evidence is presented showing that the DTs retained wild-type infectivity to their host cells and that the intraphagolysosomal/parasitophorous vacuolar (PV) DTs remained ALA inducible for uroporphyrinogenesis/photolysis. Exposure of DT-infected cells to ALA was noted by fluorescence microscopy to result in host-parasite differential porphyrinogenesis: porphyrin fluorescence emerged first in the host cells and then in the intra-PV amastigotes. DT-infected and control cells differed qualitatively and quantitatively in their porphyrin species, consistent with the expected multi- and monoporphyrinogenic specificities of the host cells and the DTs, respectively. After ALA removal, the neogenic porphyrins were rapidly lost from the host cells but persisted as URO in the intra-PV DTs. These DTs were thus extremely light sensitive and were lysed selectively by illumination under nonstringent conditions in the relatively ROS-resistant phagolysosomes. Photolysis of the intra-PV DTs returned the distribution of major histocompatibility complex (MHC) class II molecules and the global gene expression profiles of host cells to their preinfection patterns and, when transfected with ovalbumin, released this antigen for copresentation with MHC class I molecules. These Leishmania mutants thus have considerable potential as a novel model of a universal vaccine carrier for photodynamic immunotherapy/immunoprophylaxis.     

Inhibition of the biosynthesis of uroporphyrinogen and heme in rat liver during obstructive jaundice produced by bile duct ligation

Altered hepatic microsomal drug metabolism has been reported to occur in afflicted with hyperbilirubinemia. Similarities of the chemical structures of hydroxymethylbilane, an intermediate in the biosynthesis of uroporphyrinogen, to bilirubin prompted investigations of the effect of bilirubin on the activity of uroporphyrinogen I synthase (porphobilinogen deaminase, EC 4.3.1.8) and the biosynthesis of heme. Bilirubin was found to be a reversible, noncompetitive inhibitor of uroporphyrinogen I synthase. The inhibition constant (Ki) for bilirubin was 1.5 microM. Bile acids had no effect on rat hepatic uroporphyrinogen I synthase activity. Hyperbilirubinemia was achieved in rats by biliary ligation in order to investigate whether elevated levels of bilirubin impair the biosynthesis of hepatic heme in vivo. The relative rate of heme biosynthesis, as measured by the rate of incorporation of delta-[4-14C]aminolevulinic acid into heme, was decreased 59% 24 h after biliary obstruction. The levels of hepatic microsomal heme and cytochrome P-450 were decreased by 43 and 40%, respectively, 72 h after biliary obstruction. The activities of hepatic delta-aminolevulinic acid synthase and uroporphyrinogen I synthase were increased by 39 and 46%, respectively, 72 h after biliary obstruction. During the 48- to 72-h period following biliary obstruction, the urinary excretion of porphobilinogen and uroporphyrin was increased 3.0- and 3.5-fold, respectively, whereas, the urinary excretion of delta-aminolevulinic acid was not altered. During this 48-to 72-h time interval following biliary obstruction, 100% of the uroporphyrin was excreted as isomer I. These results indicate that bilirubin is capable of depressing the biosynthesis of rat hepatic heme and thus cytochrome P-450-mediated drug metabolism by inhibition of the formation of uroporphyrinogen. These findings are a plausible mechanism for reports of impaired clearance of various drugs in patients afflicted with hyperbilirubinemic disease states.     

Yeast mutants deficient in heme biosynthesis and a heme mutant additionally blocked in cyclization of 2,3-oxidosqualene.

Mutants of Saccharomyces cerevisiae were isolated which were blocked in heme biosynthesis and required heme for growth on a nonfermentable carbon source. They were rho+, and grew fermentatively on ergosterol or cholesterol and Tween 80, as a source of oleic acid. Cells grown on ergosterol and Tween 80 lacked cytochromes and catalase which were restored by growth on heme. The mutants comprised five nonoverlapping complementation groups. Tetrad analysis showed that the pleiotropic properties of each of the mutants resulted from a single mutation in one of five unlinked loci (hem1 to hem5) affecting heme biosynthesis. Biochemical studies confirmed that each mutation resulted in loss of a single enzyme activity. hem1 mutants grew on delta-aminolevulinate and lacked delta-aminolevulinate synthase activity, hem2 mutants lacked delta-aminolevulinate dehydratase, and hem3 mutants uroporphyrin I synthase. Mutants in hem1, hem2, and hem3 had an additional requirement for methionine on synthetic medium supplemented with either heme or ergosterol and Tween 80, owing to a lack of sulfite reductase which contains siroheme, a modified uroporphyrin III. Since hem4 and hem5 mutants have sulfite reductase activity under all growth conditions, they are blocked after uroporphyrin III. Cell extracts of a hem4 mutant incubated with delta-aminolevulinate accumulated coproporphyrin III suggesting a block in coproporphyrinogenase, the enzyme which converts coproporphyrinogen III to protoporphyrinogen. Cells and extracts of a hem5 mutant accumulated protoporphyrin IX. Since it was the only mutant that grew on heme but not on protoporphyrin IX, a block in ferrochelatase was suggested for this strain. Mutant strains grown on heme had the sterol composition of wild type cells, whereas without heme only squalene, small amounts of lanosterol, and added sterol was observed. A heme product therefore participates in the transformation of lanosterol to ergosterol. A hem3 mutant was isolated which was also blocked between 2,3-oxidosqualene and lanosterol (erg12). When grown on lanosterol or ergosterol (with Tween 80) it accumulated a compound which was identified as 2,3-oxidosqualene by comparison with the synthetic compound in thin layer and gas-liquid chromatography, and by proton magnetic resonance and mass spectroscopy. Supplementation with heme did not remove the requirement for sterol, but it enabled the mutant to convert lanosterol to ergosterol.     

Extracellular heme peroxidases in actinomycetes: a case of mistaken identity

Actinomycetes secrete into their surroundings a suite of enzymes involved in the biodegradation of plant lignocellulose; these have been reported to include both hydrolytic and oxidative enzymes, including peroxidases. Reports of secreted peroxidases have been based upon observations of peroxidase-like activity associated with fractions that exhibit optical spectra reminiscent of heme peroxidases, such as the lignin peroxidases of wood-rotting fungi. Here we show that the appearance of the secreted pseudoperoxidase of the thermophilic actinomycete Thermomonospora fusca BD25 is also associated with the appearance of a heme-like spectrum. The species responsible for this spectrum is a metalloporphyrin; however, we show that this metalloporphyrin is not heme but zinc coproporphyrin. The same porphyrin was found in the growth medium of the actinomycete Streptomyces viridosporus T7A. We therefore propose that earlier reports of heme peroxidases secreted by actinomycetes were due to the incorrect assignment of optical spectra to heme groups rather than to non-iron-containing porphyrins and that lignin-degrading heme peroxidases are not secreted by actinomycetes. The porphyrin, an excretory product, is degraded during peroxidase assays. The low levels of secreted peroxidase activity are associated with a nonheme protein fraction previously shown to contain copper. We suggest that the role of the secreted copper-containing protein may be to bind and detoxify metals that can cause inhibition of heme biosynthesis and thus stimulate porphyrin excretion.     

宽叶吊兰叶绿素生物合成的昼夜节律变化

在被子植物中,从谷氨酰-tRNA到叶绿素的生物合成是由许多酶催化的级联反应,其中间代谢产物具有较强的光反应活性和细胞毒性,因此这一过程在细胞内受到严格的调控。本研究通过检测宽叶吊兰叶片叶绿素生物合成途径的14种中间产物含量随昼夜节律的变化,探讨昼夜节律对宽叶吊兰叶绿素生物合成的影响。结果表明,中间产物ALA(δ-氨基乙酰丙酸)、PBG(胆色素原)、ProtoⅨ(原卟啉Ⅸ)、Heme(血红素)、Mg-ProtoⅨ(镁原卟啉Ⅸ)、Chlide a(叶绿素酸酯a)、Chlide b(叶绿素酸酯b)、Chl a(叶绿素a)、Chl b(叶绿素b)受光诱导,而UrogenⅢ(尿卟啉Ⅲ)、CoprogenⅢ(粪卟啉Ⅲ)和Pchlide(原叶绿素酸脂)受黑暗诱导,尤其是Pchlide在黑暗中的积累量显著增加;Mpe(镁原卟啉Ⅸ单甲酯)和Mpde(镁原卟啉Ⅸ二酯)具有2个积累峰值,分别出现在中午12∶00和夜间24∶00。说明叶绿素生物合成受昼夜节律的调控,但其中间代谢产物含量的变化规律与昼夜节律并不完全一致。     

Synergistic induction of delta-aminolevulinate synthase by glutethimide and iron: relationship to the synergistic induction of heme oxygenase

Relationships between activities of delta-aminolevulinate synthase and heme oxygenase, respectively the rate-limiting enzymes of heme biosynthesis and degradation, have been studied in chick embryo liver cell cultures following exposure of the cultures to glutethimide and iron, a combination known to produce a synergistic induction of both enzymes. In time-course experiments, synergistic induction of heme oxygenase activity by glutethimide and iron preceded that of delta-aminolevulinate synthase by 4 h. Effects of selective inhibitors of both heme synthesis and degradation have also been studied with respect to effects on delta-aminolevulinate synthase and heme oxygenase activities. The synergistic induction of heme oxygenase by glutethimide and iron appears to be dependent upon cellular heme synthesis because addition of inhibitors of heme biosynthesis, 4,6-dioxoheptanoic acid or N-methyl-mesoporphyrin abolishes this synergistic induction. Exposure of cultures to tin-mesoporphyrin, a potent inhibitor of heme oxygenase, prevented the synergistic induction of delta-aminolevulinate synthase produced by glutethimide and iron, or, when added after induction was already established, promptly halted any further induction. These results suggest that the level of activity of heme oxygenase can reciprocally modulate intracellular heme levels and thus activity of delta-aminolevulinate synthase.     

Up-regulation of heme biosynthesis during differentiation of Neuro2a cells

Heme is an iron-containing tetrapyrrole molecule that functions as a prosthetic group for proteins such as mitochondrial respiratory enzymes. Several studies have suggested that heme has essential functions in the construction and maintenance of the nervous system. In this study, the contents of three biologically important forms of heme (types a, b, and c) and the expression of heme biosynthetic enzymes were examined in differentiating Neuro2a cells. During neuronal differentiation, there were increases in the cellular heme levels and increases in the mRNA levels for the rate-limiting enzymes of heme biosynthesis, such as aminolevulinic acid synthase (ALAS; EC 2.3.1.37) and coproporphyrinogen oxidase (EC 1.3.3.3). With respect to heme contents, heme b increased in the late phase of differentiation, but no apparent increase in heme a or b was observed in the early phase. In contrast, heme c (cytochrome c) markedly increased during the early phase of differentiation. This change preceded the increase in heme b and the up-regulation of the mRNA levels for heme biosynthetic enzymes. This study suggests the up-regulation of heme biosynthesis and differential regulation of the heme a, b, and c levels during neuronal differentiation.     

Extracellular Heme Peroxidases in Actinomycetes: a Case of Mistaken Identity

Actinomycetes secrete into their surroundings a suite of enzymes involved in the biodegradation of plant lignocellulose; these have been reported to include both hydrolytic and oxidative enzymes, including peroxidases. Reports of secreted peroxidases have been based upon observations of peroxidase-like activity associated with fractions that exhibit optical spectra reminiscent of heme peroxidases, such as the lignin peroxidases of wood-rotting fungi. Here we show that the appearance of the secreted pseudoperoxidase of the thermophilic actinomycete Thermomonospora fusca BD25 is also associated with the appearance of a heme-like spectrum. The species responsible for this spectrum is a metalloporphyrin; however, we show that this metalloporphyrin is not heme but zinc coproporphyrin. The same porphyrin was found in the growth medium of the actinomycete Streptomyces viridosporus T7A. We therefore propose that earlier reports of heme peroxidases secreted by actinomycetes were due to the incorrect assignment of optical spectra to heme groups rather than to non-iron-containing porphyrins and that lignin-degrading heme peroxidases are not secreted by actinomycetes. The porphyrin, an excretory product, is degraded during peroxidase assays. The low levels of secreted peroxidase activity are associated with a nonheme protein fraction previously shown to contain copper. We suggest that the role of the secreted copper-containing protein may be to bind and detoxify metals that can cause inhibition of heme biosynthesis and thus stimulate porphyrin excretion.     

Redox regulation of chlorophyll biosynthesis

Chlorophyll captures and redirects light-energy and is thus essential for photosynthetic organisms. The demand for chlorophyll differs throughout the day and night and in response to changing light conditions. Moreover, the chlorophyll biosynthesis pathway is up to certain points shared between the different tetrapyrroles; chlorophyll, heme, siroheme and phytochromobilin, for which the cell has different requirements at different time points. Combined with the phototoxic properties of tetrapyrroles which, if not properly protected, can lead to formation of reactive oxygen species (ROS), the need for a strict regulation of the chlorophyll biosynthetic pathway is obvious. Here we describe the current knowledge on regulation of chlorophyll biosynthesis in plants by the chloroplast redox state with emphasis on the Mg-chelatase situated at the branch point between the heme and the chlorophyll pathway. We discuss the proposed role of the Mg-chelatase as a key regulator of the tetrapyrrole pathway by its effect on enzymes both up- and downstream in the pathway and we specifically describe how redox state might regulate the Mg-branch. Finally, we propose that a recently identified NADPH-dependent thioredoxin reductase (NTRC) could be involved in redox regulation or protection of chlorophyll biosynthetic enzymes and describe the possible modes of action by this enzyme.     

Novel inhibitors of glutamyl-tRNA(Glu) reductase identified through cell-based screening of the heme/chlorophyll biosynthetic pathway

The metabolite 5-aminolevulinic acid (ALA) is an early committed intermediate in the biosynthetic pathway of heme and chlorophyll formation. In plants, 5-aminolevulinic acid is synthesized via a two-step pathway in which glutamyl-tRNA(Glu) is reduced by glutamyl-tRNA(Glu) reductase (GluTR) to glutamate 1-semialdehyde, followed by transformation to 5-aminolevulinic acid catalyzed by glutamate 1-semialdehyde aminotransferase. Using an Escherichia coli cell-based high-throughput assay to screen small molecule libraries, we identified several chemical classes that specifically inhibit heme/chlorophyll biosynthesis at this point by demonstrating that the observed cell growth inhibition is reversed by supplementing the medium with 5-aminolevulinic acid. These compounds were further tested in vitro for inhibition of the purified enzymes GluTR and glutamate 1-semialdehyde aminotransferase as confirmation of the specificity and site of action. Several promising compounds were identified from the high-throughput screen that inhibit GluTR with an I(0.5) of less than 10 microM. Our results demonstrate the efficacy of cell-based high-throughput screening for identifying inhibitors of 5-aminolevulinic acid biosynthesis, thus representing the first report of exogenous inhibitors of this enzyme.     

Interaction of rabbit hemoplexin with copro- and uroporphyrins.

Rabbit hemopexin forms equimolar complexes in vitro with the I and III isomers of both coproporphyrin and uroporphyrin. The apparent dissociation constants (Kd) of these complexes are estimated to be 4-10(-7) M for coproporphyrin-hemopexin and 10(-6) M for uroporphyrin-hemopexin by equilibrium dialysis and quenching of protein fluorescence. Results of competitive binding experiments suggest that all four porphyrins bind at the heme-binding site of hemopexin, and that the relative affinity of rabbit hemopexin for these porphyrins is: deuteroheme greater than coproporphyrin I or III greater than uroporphyrin I or III. These findings provide further evidence that hemopexin may function as a transport protein for circulating coproporphyrins as well as for heme.     

Effects of iron deficiency on heme biosynthesis in Rhizobium japonicum.

The effects of iron deficiency on heme biosynthesis in Rhizobium japonicum were examined. Iron-deficient cells had a decreased maximum cell yield and a decreased cytochrome content and excreted protoporphyrin into the growth medium. The activities of the first two enzymes of heme biosynthesis, delta-aminolevulinic acid synthase (EC 2.3.1.37) and delta-aminolevulinic acid dehydrase (EC 4.2.1.24), were diminished in iron-deficient cells, but were returned to normal levels upon addition of iron to the cultures. The addition of iron salts, iron chelators, hemin, or protoporphyrin to cell-free extracts did not affect the activity of these enzymes. The addition of levulinic acid to iron-deficient cultures blocked protoporphyrin excretion and also resulted in high delta-aminolevulinic acid synthase and delta-aminolevulinic acid dehydrase activities. These results suggest the possibility that rhizobial heme biosynthesis in the legume root nodule may be affected by the release of iron from the host plant to the bacteroids.     

Effect of insulin and glucagon on accumulation of uroporphyrin and coproporphyrin from 5-aminolevulinate in hepatocyte cultures

Primary cultures of chick embryo hepatocytes have been used to study the mechanisms by which various drugs and other chemicals cause accumulation of porphyrin intermediates of the heme pathway. When these cultures are incubated with the heme precursor, 5-aminolevulinic acid (ALA), there is a major accumulation of protoporphyrin. However, in the presence of ALA, addition of insulin caused a striking increase in accumulation of uroporphyrin I and coproporphyrin III, whereas addition of glucagon mainly caused an increase in uroporphyrin I. Treatment with both insulin and glucagon resulted in additive increases in uroporphyrin, but not coproporphyrin. Antioxidants abolished the uroporphyrin I accumulation and increased coproporphyrin III. Insulin caused an increase in uptake of ALA and an increase in porphobilinogen accumulation, suggesting that the accumulation of uroporphyrin I is due to increased flux through the heme pathway. Apparently, this increased flux could particularly affect the utilization of the intermediate hydroxymethylbilane, which would result in accumulation of uroporphyrin I.     

Identification and partial characterization of two eukaryotic UDP-galactopyranose mutases

Galactofuranose metabolism is valued as an important target for the development of new antituberculosis drugs. UDP-galactopyranose mutase, a central enzyme in galactofuranose biosynthesis, is essential for the growth and viability of mycobacteria. This enzyme catalyzes the conversion of UDP-galactopyranose into UDP-galactofuranose, the donor used by various galacto-furanosyltransferases. While D-galactofuranose residues are often found in important surface glycoconjugates of pathogenic bacteria, fungi and protozoan parasites, they are absent in the mammalian host, and thus their biosynthesis is an attractive target for the development of novel therapeutic strategies. In contrast to mycobacteria, the importance of galactofuranose for eukaryotic pathogens has not been ascertained because the enzymes involved in galactofuranose metabolism are unknown. Here, we report the identification and characterization of the first eukaryotic UDP-galactopyranose mutases. The genes encoding the enzymes were cloned from two different human pathogens: the parasite Leishmania major and the opportunistic fungus Aspergillus fumigatus. The newly identified eukaryotic enzymes exhibit 51% sequence identity, but are less than 20% identical to the prokaryotic counterparts. The sequence identity between pro- and eukaryotic enzymes is concentrated at amino acid residues that are involved in substrate and cofactor binding. Therefore, an inhibitor of UDP-galactopyranose mutase might be effective against a wide range of pathogenic organisms.     

Effects of diphenyl ether herbicides on porphyrin accumulation by cultured hepatocytes

Several diphenyl ether herbicides, such as acifluorfen methyl, have been previously shown to cause large accumulations of the heme and chlorophyll precursor, protoporphyrin, in plants. Lightinduced herbicidal damage is mediated by the photoactive porphyrin. Here we investigate whether diphenyl ether herbicides can affect porphyrin synthesis in rat and chick hepatocytes. In rat hepatocyte cultures, protoporphyrin, as well as coproporphyrin, accumulated after treatment with acifluorfen or acifluorfen methyl. Combination of acifluorfen methyl with an esterase inhibitor to prevent the conversion of acifluorfen methyl to acifluorfen resulted in a greater accumulation of porphyrins than caused by acifluorfen methyl or acifluorfen alone. In vitro enzyme studies of hepatic mitochondria isolated from rat and chick embryos demonstrated that protopor-phyrinogen oxidase, the penultimate enzyme of heme biosynthesis, was inhibited by low concentrations of acifluorfen, nitrofen, or acifluorfen methyl with the latter being the most potent inhibitor. These findings indicate that diphenyl ether treatment can cause protoporphyrin accumulation in rat hepatocyte cultures and suggest that this accumulation was associated with the inhibition of protoporphyrinogen oxidase. In cultured chick embryo hepatocytes, treatment with acifluorfen methyl plus an esterase inhibitor caused massive accumulation of uroporphyrin rather than protoporphyrin or coproporphyrin. Specific isozymes of cytochrome P450 were also induced in chick embryo hepatocytes. These effects were not observed in the absence of an esterase inhibitor. These results suggest that diphenyl ether herbicides can cause uroporphyrin accumulation similar to that induced by other cytochrome P450-inducing chemicals such as polyhalogenated aromatic hydrocarbons in the chick hepatocyte system.     

Mode of action of fenarimol against Leishmania spp

This paper evaluates the effects of certain herbicides on Leishmania spp., their mechanism of action, and the evolutionary origin of the relevant susceptible leishmanial targets. We demonstrated that a relatively nontoxic herbicide, fenarimol, successfully interferes with a leishmanial target, which is probably a relic of an ancient ancestor. Fenarimol impairs the function of leishmanial 14alpha-sterol demethylase, a key enzyme in the sterol biosynthetic pathway. Therefore, fenarimol or its derivatives may be candidates for development of anti-leishmanial drugs. Of the herbicides that have the capability to act as potential inhibitors of the metabolism of Leishmania spp., fenarimol was found as the most active substance against both promastigotes and amastigotes in culture. In addition, it ameliorated lesions caused by Leishmania major in mice. Light microscopy demonstrated rounding of the parasite shape. Increase of osmophilic vacuoles and autophagosomal structures were observed by transmission electron microscopy. Biochemical studies demonstrated that fenarimol inhibited sterol biosynthesis. Docking of fenarimol to the modeled catalytic binding site of 14alpha-lanosterol demethylase of L. major showed a geometrical fit. Fenarimol is stabilized via hydrophobic interactions with the residues that surround it and interactions with the heme ring. These results provide support to the hypothesis that fenarimol inhibits leishmanial sterol biosynthesis. Overall, the findings suggest an additional source of substances for development of anti-leishmanial drugs.     

Reduction of Uroporphyrinogen Decarboxylase by Antisense RNA Expression Affects Activities of Other Enzymes Involved in Tetrapyrrole Biosynthesis and Leads to Light-Dependent Necrosis

We introduced a full-length cDNA sequence encoding tobacco (Nicotiana tabacum) uroporphyrinogen III decarboxylase (UROD; EC 4.1.1.37) in reverse orientation under the control of a cauliflower mosaic virus 35S promoter derivative into the tobacco genome to study the effects of deregulated UROD expression on tetrapyrrole biosynthesis. Transformants with reduced UROD activity were characterized by stunted plant growth and necrotic leaf lesions. Antisense RNA expression caused reduced UROD protein levels and reduced activity to 45% of wild type, which was correlated with the accumulation of uroporphyrin(ogen) and with the intensity of necrotic damage. Chlorophyll levels were only slightly reduced (up to 15%), indicating that the plants sustained cellular damage from accumulating photosensitive porphyrins rather than from chlorophyll deficiency. A 16-h light/8-h dark regime at high-light intensity stimulates the formation of leaf necrosis compared with a low-light or a 6-h high-light treatment. Transgenic plants grown at high light also showed inactivation of 5-aminolevulinate dehydratase and porphobilinogen deaminase, whereas the activity of coproporphyrinogen oxidase and the 5-aminolevulinate synthesizing capacity were not altered. We conclude that photooxidation of accumulating uroporphyrin(ogen) leads to the generation of oxygen species, which destabilizes other enzymes in the porphyrin metabolic pathway. This porphyrin-induced necrosis resembles the induction of cell death observed during pathogenesis and air pollution.     

Effect of endogenous heme generation on delta-aminolevulinic acid synthase activity in rat liver mitochondria.

This study examined the possibility that generation of heme within mitochondria may provide a local concentration sufficient to inhibit the activity of delta-aminolevulinic acid (ALA) synthase, the enzyme that catalyzes the rate-limiting step in hepatic heme biosynthesis. This was accomplished by simultaneously running ALA synthase and heme synthase activities in intact mitochondria isolated from rat liver. Radiochemical assays were used to measure the enzyme activities. ALA synthase activity did not decrease as the rate of heme formation was increased by varying the concentration of substrates for heme synthase. Even at a rate of heme generation estimated to be at least 75 times the rate occuring in vivo, ALA synthase activity was unchanged. We conclude that end product inhibition of ALA synthase activity by heme is not an important physiological mechanism for regulation of hepatic heme biosynthesis.