Presence Of Alae Research Articles

Acquiring of photosensitivity by Mycobacterium tuberculosis in vitro and inside infected macrophages is associated with accumulation of endogenous Zn–porphyrins

Mycobacterium tuberculosis (Mtb) is able to transition into a dormant state, causing the latent state of tuberculosis. Dormant mycobacteria acquire resistance to all known antibacterial drugs and can survive in the human body for decades before becoming active. In the dormant forms of M. tuberculosis, the synthesis of porphyrins and its Zn-complexes significantly increased when 5-aminolevulinic acid (ALA) was added to the growth medium. Transcriptome analysis revealed an activation of 8 genes involved in the metabolism of tetrapyrroles during the Mtb transition into a dormant state, which may lead to the observed accumulation of free porphyrins. Dormant Mtb viability was reduced by more than 99.99% under illumination for 30 min (300 J/cm2) with 565 nm light that correspond for Zn–porphyrin and coproporphyrin absorptions. We did not observe any PDI effect in vitro using active bacteria grown without ALA. However, after accumulation of active cells in lung macrophages and their persistence within macrophages for several days in the presence of ALA, a significant sensitivity of active Mtb cells (ca. 99.99%) to light exposure was developed. These findings create a perspective for the treatment of latent and multidrug-resistant tuberculosis by the eradication of the pathogen in order to prevent recurrence of this disease.

Optimization of Copper Stain Removal from Marble through the Formation of Cu(II) Complexes in Agar Gels.

Copper complexes with different ligands (ethylenediaminetetraacetic acid, EDTA, ammonium citrate tribasic, TAC, and alanine, ALA) were studied in aqueous solutions and hydrogels with the aim of setting the optimal conditions for copper stain removal from marble by agar gels, with damage minimization. The stoichiometry and stability of copper complexes were monitored by ultraviolet-visible (UV-Vis) spectroscopy and the symmetry of Cu(II) centers in the different gel formulations was studied by electron paramagnetic resonance (EPR) spectroscopy. Cleaning effectiveness in optimized conditions was verified on marble laboratory specimens through color variations and by determining copper on gels by inductively coupled plasma-mass spectrometry (ICP-MS). Two copper complexes with TAC were identified, one having the known stoichiometry 1:1, and the other 1:2, Cu(TAC)2, never observed before. The stability of all the complexes at different pH was observed to increase with pH. At pH 10.0, the gel’s effectiveness in removing copper salts from marble was the highest in the presence of ALA, followed by EDTA, TAC, and pure agar gel. Limited damage to the marble surface was observed when gels with added EDTA and TAC were employed, whereas agar gel with ALA was determined to be the most efficient and safe cleaning material.

α– Linolenic acid modulates phagocytosis and endosomal pathways of extracellular Tau in microglia

ABSTRACT Microglia, the resident immune cells, were found to be activated to inflammatory phenotype in Alzheimer’s disease (AD). The extracellular burden of amyloid-β plaques and Tau seed fabricate the activation of microglia. The seeding effect of extracellular Tau species is an emerging aspect to study about Tauopathies in AD. Tau seeds enhance the propagation of disease along with its contribution to microglia-mediated inflammation. The excessive neuroinflammation cumulatively hampers phagocytic function of microglia reducing the clearance of extracellular protein aggregates. Omega-3 fatty acids, especially docosahexaenoic acid and eicosapentaenoic acid, are recognized to induce anti-inflammatory phenotype of microglia. In addition to increased cytokine production, omega-3 fatty acids enhance phagocytic receptors expression in microglia. In this study, we have observed the phagocytosis of extracellular Tau in the presence of α-linolenic acid (ALA). The increased phagocytosis of extracellular Tau monomer and aggregates have been observed upon ALA exposure to microglia cells. After internalization, the degradation status of Tau has been studied with early and late endosomal markers Rab5 and Rab7. Further, the lysosome-mediated degradation of internalized Tau was studied with LAMP-2A, a lysosome marker. The enhanced migratory ability in the presence of ALA could be beneficial for microglia to access the target and clear it. The increased migration of microglia was found to induce the microtubule-organizing center repolarization. The data indicate that the dietary fatty acids ALA could significantly enhance phagocytosis and intracellular degradation of internalized Tau. Our results suggest that microglia could be influenced to reduce extracellular Tau seed with dietary fatty acids.

5-aminolevulinic acid regulates Krebs cycle, antioxidative system and gene expression in Brassica juncea L. to confer tolerance against lead toxicity

Heavy metal pollution seriously impairs crop production and poses serious concerns for human health. Exogenous application of biomolecules has been efficiently tested for enhancing plant resistance to metal toxicity. Current study evaluates the possible effect of 5-aminolevulinic acid (ALA) in Brassica juncea L. seedlings subjected to lead (Pb) stress. Our results showed that shoot length, root length and chlorophyll contents were significantly recovered in Pb stressed seedlings after ALA application, accompanied by reduction in the Pb accumulation. Significant reduction in the contents of reactive oxygen species (ROS) like superoxide anion, hydrogen peroxide and malondialdehyde were also observed in ALA treated seedlings under Pb stress. Furthermore, we also noticed enhancement in the activities of antioxidative enzymes like superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (POD), glutathione reductase (GR), glutathione-S-transferase (GST) and dehydroascorbate reductase (DHAR). We further noticed that ALA upregulated the expression of SOD (7.30 folds), POD (6.11 folds), CAT (3.52 folds), DHAR (6.42 folds), GR (6.04 folds), and GST (5.58 folds) under the Pb stress. However, RBOH1 (gene involved in ROS generation) and CHLASE (chlorophyllase) expressions were reduced in ALA treated seedlings grown under Pb stress (RBOH1 expression decreased to 3.44 from 6.50 fold and CHLASE expression decreased to 2.97 from 5.58 fold). Phenolic contents were increased in the presence of ALA and expression of genes like CHS (chalcone synthase; 7.50 fold) and PAL (phenylalanine ammonia lyase; 4.77 fold) was also stimulated by ALA under Pb stress. Furthermore, contents of the Krebs cycle metabolites (fumarate, succinate, malate and citrate) were also enhanced accompanied by upregulated expression of genes like CS (citrate Synthase; 8.13 fold), SUCLG1 (succinyl CoA ligase 1; 7.40 fold), SDH (succinate dehydrogenase; 5.10 fold) and FH (fumarate hydratase; 5.65 fold). In conclusion, current investigation revealed that ALA attenuated Pb toxicity by modulating the transcription patterns of key enzymes involved in plant defense system.

Chronic Treatment with α-Lipoic Acid Improves Endothelium-Dependent Vasorelaxation of Aortas in High-Fat Diet-Fed Mice

α-Lipoic acid (ALA) is used as a dietary supplement and known as an anti-oxidant. The present study aimed to examine whether ALA improves endothelial dysfunction in high-fat diet-fed obese mice. After feeding a high-fat diet to Institute of Cancer Research (ICR) mice for 4 weeks, the mice were maintained with a high-fat diet (group HF) or a high-fat diet containing ALA (25 mg/d, group HF + ALA) for an additional 20 weeks. Age-matched normal diet-fed mice were also used (group Normal). Chronic oral treatment with ALA did not affect various plasma parameters or body weights. As compared with the aortas of Normal mice, those from HF mice showed impaired endothelium-dependent relaxation in response to clonidine. However, such an impairment was not observed in the aortas from HF + ALA mice. The plasma levels of thiobarbituric acid reactive substances, an indicator of oxidative stress, were significantly decreased in HF + ALA mice compared with HF mice, confirming the anti-oxidative effects of ALA. In addition, when the impaired clonidine-induced vasorelaxation of aortas from normal mice under high glucose conditions was used as a model of acute oxidative stress, the vasorelaxation responses were improved in the presence of ALA at 100 µM. Our results suggested that the chronic oral administration of ALA improves endothelial dysfunction in high-fat diet-fed obese mice possibly through the reduction in oxidative stress in vivo.

Effect of Catalase or Alpha Lipoic Acid Supplementation in the Vitrification Solution of Ovine Ovarian Tissue.

The present study evaluates the effect of different concentrations of antioxidants (catalase - CAT and alpha lipoic acid - ALA) on the follicular activation and morphology, DNA damage, ROS production, and mitochondrial activity in vitrified sheep ovarian tissue. This experiment was divided into two steps. First, ovarian fragments were distributed into the following treatments: fresh tissue or control (CTR), incubation (INC), vitrification without antioxidant (VWA), with CAT (10, 20, or 40 IU mL-1) or ALA (25, 50, or 100 μM mL-1). After vitrification/warming, the fragments were additionally incubated for 24 hours and evaluated for morphology and follicular activation, as well as reactive oxygen species (ROS) levels in the culture medium. For the second step, other ovarian fragments were submitted to CTR, VWA, CAT40, and ALA100. After vitrification/warming, the fragments were incubated for 24 hours and evaluated by cell density of ovarian stroma, DNA damage, and mitochondrial and intracellular ROS levels. The percentage of morphologically normal follicles in vitrified ovarian tissue in the presence of ALA in all concentrations did not differ (p > 0.05) from fresh tissue or CTRs. The percentage of activated follicles was higher in ALA100 μM mL-1 than those observed for the treatments INC, CAT (40 IU mL-1), or ALA (25 or 50 μM mL-1). The use of CAT affected (p < 0.05) the density of stromal cells (40 IU mL-1), ROS levels (10 and 20 IU mL-1), as well as DNA damage revealed by ©H2AX (40 IU mL-1). Although 100 μM/mL of ALA did not alter intracellular ROS, this concentration reduced the levels of ROS in the culture medium, preserved both the follicular morphology, as well as the mitochondrial activity, promoted follicle activation, and protected the follicles from DNA damage.

5‐Aminolevulinic acid promotes callus growth and paclitaxel production in light‐grown Taxus cuspidata suspension cultures

Cultured plant cells generally produce low levels of secondary metabolites, and elicitors of secondary metabolites usually inhibit callus growth. The aim of this study was to determine the effect of 5‐aminolevulinic acid (ALA), a chlorophyll precursor that promotes plant growth, on callus induction from leaves of Taxus cuspidata, and on callus growth on solid medium. ALA at 0.76, 7.6, and 76 μM had similar effects on callus induction and growth, while ALA at 760 μM had negative effects. Next, the effects of ALA concentrations on callus growth and paclitaxel production in suspension cultures in the dark were evaluated. The results showed that 0.76 and 7.6 μM ALA stimulated growth and paclitaxel production, while 76 μM ALA had negative effects. ALA is thought to promote cellular activity under light conditions. Therefore, the effects of light intensity on callus growth and paclitaxel production in the presence of ALA were evaluated. Our results showed that the best conditions for callus growth and paclitaxel production were 7.6 μM ALA under photosynthetically active radiation of 12 μmol photons m−2 s−1. Callus growth and paclitaxel production were inhibited under stronger light (24 μmol photons m−2 s−1). Together, these results show that ALA promoted callus growth and the production of paclitaxel by light‐grown cultured T. cuspidata cells.

Real-time detection of intracellular reactive oxygen species and mitochondrial membrane potential in THP-1 macrophages during ultrasonic irradiation for optimal sonodynamic therapy

Reactive oxygen species (ROS) elevation and mitochondrial membrane potential (MMP) loss have been proven recently to be involved in sonodynamic therapy (SDT)-induced macrophage apoptosis and necrosis. This study aims to develop an experimental system to monitor intracellular ROS and MMP in real-time during ultrasonic irradiation in order to achieve optimal effect in SDT. Cultured THP-1 derived macrophages were incubated with 5-aminolevulinic acid (ALA), and then sonicated at different intensities. Intracellular ROS elevation and MMP loss were detected in real-time by fluorospectrophotometer using fluorescence probe DCFH-DA and jc-1, respectively. Ultrasound at low intensities (less than 0.48W/cm2) had no influence on ROS and MMP in macrophages, whereas at an intensity of 0.48W/cm2, ROS elevation and MMP loss were observed during ultrasonic irradiation. These effects were strongly enhanced in the presence of ALA. Quantitative analysis showed that ROS elevation and MMP loss monotonically increased with the rise of ultrasonic intensity between 0.48 and 1.16W/cm2. SDT at 0.48 and 0.84W/cm2 induced mainly apoptosis in THP-1 macrophages while SDT at 1.16W/cm2 mainly cell necrosis. This study supports the validity and potential utility of real-time ROS and MMP detection as a dosimetric tool for the determination of optimal SDT.

Identification of a Pair of Phospholipid:Diacylglycerol Acyltransferases from Developing Flax (Linum usitatissimum L.) Seed Catalyzing the Selective Production of Trilinolenin

The oil from flax (Linum usitatissimum L.) has high amounts of α-linolenic acid (ALA; 18:3(cis)(Δ9,12,15)) and is one of the richest sources of omega-3 polyunsaturated fatty acids (ω-3-PUFAs). To produce ∼57% ALA in triacylglycerol (TAG), it is likely that flax contains enzymes that can efficiently transfer ALA to TAG. To test this hypothesis, we conducted a systematic characterization of TAG-synthesizing enzymes from flax. We identified several genes encoding acyl-CoA:diacylglycerol acyltransferases (DGATs) and phospholipid:diacylglycerol acyltransferases (PDATs) from the flax genome database. Due to recent genome duplication, duplicated gene pairs have been identified for all genes except DGAT2-2. Analysis of gene expression indicated that two DGAT1, two DGAT2, and four PDAT genes were preferentially expressed in flax embryos. Yeast functional analysis showed that DGAT1, DGAT2, and two PDAT enzymes restored TAG synthesis when produced recombinantly in yeast H1246 strain. The activity of particular PDAT enzymes (LuPDAT1 and LuPDAT2) was stimulated by the presence of ALA. Further seed-specific expression of flax genes in Arabidopsis thaliana indicated that DGAT1, PDAT1, and PDAT2 had significant effects on seed oil phenotype. Overall, this study indicated the existence of unique PDAT enzymes from flax that are able to preferentially catalyze the synthesis of TAG containing ALA acyl moieties. The identified LuPDATs may have practical applications for increasing the accumulation of ALA and other polyunsaturated fatty acids in oilseeds for food and industrial applications.

GW24-e0405 Real-time detection of intracellular reactive oxygen species generation and mitochondrial membrane potential loss induced by sonodynamic therapy in THP-1 macrophages

ObjectivesReactive oxygen species (ROS) generation and mitochondrial membrane potential (MMP) loss have recently been shown to be involved in sonodynamic therapy (SDT)-induced macrophage death. This study aims to develop an...

QUANTIFICATION OF 5-AMINOLEVULINIC ACID IN SWINE MANURE EXTRACT BY HPLC-FLUORESCENCE

A modified high performance liquid chromatography (HPLC)-fluorescence method for determination of the 5-aminolevulinic acid (ALA) concentration in swine waste was studied. The method included digestion of the sample by heating in aqueous HCl and derivatization by acetyl acetone/formaldehyde reagent. The derivative was isolated and detected by fluorescence with excitation at 370 nm and emission at 460 nm. Chromatographic separation of ALA was carried out using the optimum mobile phase of a methanol/water/glacial acetic acid (49.5:49.5:1, v/v/v). The method produced a limit of detection of the ALA concentration of 0.10 µg/mL and a limit of quantification of the ALA concentration of 0.31 µg/mL. The method was used for determination of the ALA concentration in concentrated swine manure extract (CSME) fermented for 0, 7, 14, 21, and 28 d. The ALA concentrations in the CSME fermented for 0, 7, and 14 d were low and ranged from 0.216 to 0.399 µg/mL, but increased to a level ranging between 1.240 and 1.295 µg/mL at 21 and 28 d of fermentation. The results of the study confirmed the presence of ALA in the swine waste. The ability to quantify the ALA concentration in swine waste developed by the study was confirmed.

Neuroprotective effect of α-lipoic acid on cultured retinal ganglion cells under elevated pressure in vitro

Objective To observe the neuroprotective effect of α-lipoic acid (ALA) on cultured retinal ganglion cells (RGC-5) under elevated pressure in vitro.Methods Cultured RGC-5 cells were divided randomly into 4 groups,including normal control group (group A),negative control group (group B),elevated pressure group (group C) and elevated pressure + ALA group (group D).The cells of group A and C were not intervened with ALA.The cells of group B were treated with 200 μmol/L ALA.The cells of group D were treated with different concentrations of ALA (50,100,200 μmol/L) for one hour.Then cells of group C and D were exerted to 50 mm Hg (1 mm Hg=0.133 kPa) for 24 hours,while the cells of group A and B were exerted to normal pressures for 24 hours.The cell viability was measured using the methyl thiazolyl tetrazolium (MTT) assay and apoptosis was evaluated using 4',6 diamidino-2-phenylindole (DAPI) staining.Expression of MnSOD was determined by real-time polymerase chain reaction (RT-PCR)and Western blot,respectively.Results The cell viability of group B was (65.6 ± 3.4) ％,which lower than that in group D of three concentrations of ALA[ (75.1± 3.3)％,(81.8 ± 2.9 ) ％,(87.9 ± 3.1 ) ％ ],the differences were significantly (t =5.108,10.007,12.513; P＜0.05).DAPI staining revealed that characteristic apoptotic changes,such as chromatin condensation,convoluted nuclei with cavitations,fragmentation of the nucleus,and apoptotic bodies appeared in RGC-5 cells after 24 ours pressure.There was almost no evidence of apoptosis in group D.RT-PCR and Western blot analysis revealed that the expression of MnSOD mRNA and protein were weakly expressed in group C compared with control A (t=22.045,26.979; P＜0.01).Compared group C with group D,the level of MnSOD mRNA and protein in group D increased significantly (t=9.171,12.267,23.567,7.723,12.009,28.198;P＜0.05).In addition,the presence of ALA was found to inhibit hydrostatic pressure induced damage of RGC-5 cells in a dose-dependent manner (F=134.273,194.597;P＜0.01).Conclusion ALA can effectively improve the expression of MnSOD in RGC-5 cells under the condition of elevated pressure,enhance the ability of RGC-5 cells against oxidative damage. Key words: Retinal ganglion cells/ pathology; Thioctic acid/therapeutic use; Cells, cultured; Animal experimentation

Novel asparagine-derived lipid enhances distearoylphosphatidylcholine bilayer resistance to acidic conditions.

A novel asparagine-derived lipid analogue (ALA(11,17)) bearing a tetrahydropyrimidinone headgroup and two fatty chains (11 and 17 indicate the lengths of linear alkyl groups) was synthesized in high yield and purity. The thin film hydration of formulations containing 5 mol % or greater ALA(11,17) in distearoylphosphatidylcholine (DSPC) generated multilamellar vesicles (MLVs) that remained unaggregated according to optical microscopy, while those formed from DSPC only were highly clustered. The MLVs were processed into unilamellar liposomes via extrusion and were characterized by dynamic light scattering (DLS), zeta potential, turbidity, and scanning electron microscopy (SEM) analysis. Results show that the presence of ALA(11,17) in DSPC liposomes significantly alters the morphology, colloidal stability, and retention of encapsulated materials in both acidic and neutral conditions. The ability of ALA(11,17)-hybrid liposomes to encapsulate and retain inclusions under neutral and acidic conditions (pH < 2) was demonstrated by calcein dequenching experiments. DLS and SEM confirmed that ALA(11,17)/DSPC liposomes remained intact under these conditions. The bilayer integrity observed under neutral and acidic conditions and the likely biocompatibility of these fatty amino acid analogues suggest that ALA(11,17) is a promising additive for modulating phosphatidylcholine lipid bilayer properties.

Anti-leucocyte antibodies and embryonic mortality in embryo transferred cows

Cows carrying unrelated transferred embryos (ET) produced anti-leucocyte serum antibodies (aLA) more often than cows carrying their own embryos. Cows carrying the ET showed a higher frequency of cytotoxic reactions against leucocytes from 40-60 randomly chosen cows than individuals carrying their own embryos. The percentage of animals with aLA was higher in cows carrying their second or third transferred embryo than in those with their first transferred embryo. There was no change in the frequency of cytotoxic reactions with repeated pregnancies from transferred embryos. There was no difference in the toxicity of aLA in normal pregnant cows and those carrying transferred embryos. Embryonic mortality (EM) of 35, 73 and 88% was noted during pregnancies from the first, second and third successful ET, respectively. Mortality of 48% occurred in the first pregnancy following an unsuccessful ET. Embryonic mortality of 31% occurred in cows simultaneously carrying their own and a transferred embryo. A direct relationship between the presence of aLA and EM in recipients was not proved. Other fertility problems may lead to EM in cows subjected to repeated transfer of foreign embryos.

Modelling the photosensitization-based inactivation ofBacillus cereus

To study and to develop a model for the photo-destruction of the foodborne pathogen Bacillus cereus, initially treated with a precursor of endogenous photosensitizers (5-aminolevulinic acid, ALA). The cells were incubated in the presence of ALA (3 or 7.5 mmol l(-1)) for incubation times ranging from 2 to 60 min, inoculated onto the surface of LB Agar plates and submitted to light irradiation. The Weibull model was used to describe the survival curves of B. cereus. Quadratic equations were used to describe the effects of ALA concentration and incubation time on the Weibull model parameters. ALA-based photosensitization proved to be an effective tool for inactivation of B. cereus. The decrease in viable counts observed after 20 min of irradiation, ranged from 4 to 6 log CFU g(-1). The developed model proved to be a parsimonious and robust solution to describe the observed data. The study demonstrates the effectiveness of photosensitization on B. cereus on agar plates. The model developed may be useful to optimize inactivation treatments by photosensitization.

DNA pre-condensation with an amino acid-based cationic amphiphile. A viable approach for liposome-based gene delivery

A study related to the development and characterization of a new gene delivery system was performed. The approach consists in both the pre-condensation of plasmid DNA with an arginine-based cationic surfactant, arginine-N-lauroyl amide dihydrochloride (ALA), which was found not to be toxic, and the incorporation of the blood protein transferrin (Tf) into the formulations. Two cationic liposome formulations were used, one composed of a mixture of dioleoyl trimethylammoniopropane and cholesterol (DOTAP:Chol) and the other a pH sensitive formulation constituted of DOTAP, Chol, Dioleoyl phosphatidylethanolamine (DOPE) and cholesteryl hemisuccinate (CHEMS). Particles with different ALA/DNA and cationic lipid/DNA charge ratios were produced and a physicochemical characterization of the systems developed was performed. DNA conformational changes in the presence of ALA were studied by Circular Dichroism (CD) and the ALA binding to DNA was followed by surface tension measurements. Insight into the structure and morphology of the various ALA-complexes (complexes composed of ALA, DNA, Tf and liposomes) was obtained by cryogenic-Transmission Electron Microscopy (cryo-TEM) and the sizes of the ALA-complexes were determined through Photon Correlation Spectroscopy (PCS). We found that the transfection capacity of these systems is directly related with the presence of ALA and the lipidic composition. Complexes based on the pH sensitive liposome formulation present better transfection profiles. The correlation between the inner structure, density and size of the ALA-complexes and their biological activity is discussed. Overall, we demonstrate that the presence of ALA improves the transfection efficiency when conjugated with cationic liposome systems.

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.

5-Aminolevulinic acid synthesis in epimastigotes of Trypanosoma cruzi

Background and aims: Trypanosoma cruzi is the causative agent of Chagas disease or American trypanosomiasis. The parasite manifests a nutritional requirement for heme compounds because of its biosynthesis deficiency. The aim of this study has been to investigate the presence of metabolites and enzymes of porphyrin pathway, as well as ALA formation in epimastigotes of T. cruzi, Tulahuén strain, Tul 2 stock. Methods: Succinyl CoA synthetase, 5-aminolevulinic acid (ALA) synthetase, 4,5-dioxovaleric (DOVA) transaminase, ALA dehydratase and porphobilinogenase activities, as well as ALA, porphobilinogen (PBG), free porphyrins and heme content were measured in a parasite cells-free extract. Extracellular content of these metabolites was also determined. Results: DOVA, PBG, porphyrins and heme were not detected in acellular extracts of T. cruzi. However ALA was detected both intra- and extracellularly This is the first time that the presence of ALA (98% of intracellularly formed ALA) is demonstrated in the extracellular medium of a parasite culture. Regarding the ALA synthesizing enzymes, DOVA transaminase levels found were low (7.13±0.49 EU/mg protein), whilst ALA synthetase (ALA-S) activity was undetectable. A compound of non-protein nature, low molecular weight, heat unstable, inhibiting bacterial ALA-S activity was detected in an acellular extract of T. cruzi. This inhibitor could not be identified with either ALA, DOVA or heme. Conclusions: ALA synthesis is functional in the parasite and it would be regulated by the heme levels, both directly and through the inhibitor factor detected. ALA formed can not be metabolized further, because the necessary enzymes are not active, therefore it should be excreted to avoid intracellular cytotoxicity.

Haem precursor delta-aminolaevulinic acid induces activation of the cytosolic iron regulatory protein 1.

Control of cellular iron homoeostasis is performed by iron regulatory protein 1 (IRP1) through post-transcriptional modifications. This protein is sensitive to intracellular iron availability, being activated at low iron levels and inactivated at high iron levels, conditions that signal the increased expression of the transferrin receptor or of ferritin respectively. IRP1 is known to be activated by some oxidants such as H2O2 and NO. delta-Aminolaevulinic acid (ALA), previously found to produce reactive oxygen species and a carbon-centred radical, to release iron from ferritin, and to increase rat liver and brain non-haem iron and ferritin, was investigated for its effects on IRP1 activity in cultured hamster pulmonary fibroblasts. We have found that 1-2 mM ALA produced a 2-3-fold activation of IRP. On incubation with 1-4 mM succinylacetone methyl ester, a potent ALA dehydratase inhibitor, a 3-4-fold activation of the protein was observed, accompanied by a 40% increase in the intracellular ALA concentration. When cells were incubated in the presence of ALA or succinylacetone methyl ester, N-acetylcysteine inhibited IRP1 activation, suggesting that the observed effect is mediated by an oxidative process. We surmise that ALA-induced IRP1 activation might act as a co-sensor of iron homoeostasis.

Evidence that 4-aminobutyric acid and 5-aminolevulinic acid share a common transport system into Saccharomyces cerevisiae

It has been previously reported that 5-aminolevulinic acid (ALA) and 4-aminobutyric acid (GABA) share a common permease in Saccharomyces cerevisiae (Bermúdez Moretti et al., 1993). The aim of the present work was to determine the relationship between the transport of these compounds in isolated cells. Assessment of amino acid incorporation was performed in S. cerevisiae using 14C-ALA or 3H-GABA. Initial rates of ALA incorporation in cells grown in the presence of 5 mM ALA and 5 mM GABA, were three to four times lower than in cells grown without supplements. Kinetic studies indicate that GABA competitively inhibits ALA transport. During the growth phase GABA uptake was also inhibited by 74% and 60% in the presence of ALA and GABA, respectively. These findings indicate that in S. cerevisiae the structurally related compounds, ALA and GABA, may be incorporated into the cells by a common carrier protein. Should this occur in other lukaryotic cells it may explain the neurotoxic effect attributed to ALA in the pathogenesis of acute porphyrias