Mitochondrial function in the yeast form of the pathogenic fungus Paracoccidioides brasiliensis

Differences between the respiratory chain of the fungus Paracoccidioides brasiliensis and its mammalian host are reported. Respiration, membrane potential, and oxidative phosphorylation in mitochondria from P. brasiliensis spheroplasts were evaluated in situ, and the presence of a complete (Complex I-V) functional respiratory chain was demonstrated. In succinate-energized mitochondria, ADP induced a transition from resting to phosphorylating respiration. The presence of an alternative NADH-ubiquinone oxidoreductase was indicated by: (i) the ability to oxidize exogenous NADH and (ii) the lack of sensitivity to rotenone and presence of sensitivity to flavone. Malate/NAD(+)-supported respiration suggested the presence of either a mitochondrial pyridine transporter or a glyoxylate pathway contributing to NADH and/or succinate production. Partial sensitivity of NADH/succinate-supported respiration to antimycin A and cyanide, as well as sensitivity to benzohydroxamic acids, suggested the presence of an alternative oxidase in the yeast form of the fungus. An increase in activity and gene expression of the alternative NADH dehydrogenase throughout the yeast`s exponential growth phase was observed. This increase was coupled with a decrease in Complex I activity and gene expression of its subunit 6. These results support the existence of alternative respiratory chain pathways in addition to Complex I, as well as the utilization of NADH-linked substrates by P. brasiliensis. These specific components of the respiratory chain could be useful for further research and development of pharmacological agents against the fungus.

Characterization of Rubus fruticosus mitochondria and salicylic acid inhibition of reactive oxygen species generation at Complex III/Q cycle: potential implications for hypersensitive response in plants

In addition to adenosine triphosphate (ATP) production, mitochondria have been implicated in the regulation of several physiological responses in plants, such as programmed cell death (PCD) activation. Salicylic acid (SA) and reactive oxygen species (ROS) are essential signaling molecules involved in such physiological responses; however, the mechanisms by which they act remain unknown. In non-photosynthesizing tissues, mitochondria appear to serve as the main source of ROS generation. Evidence suggests that SA and ROS could regulate plant PCD through a synergistic mechanism that involves mitochondria. Herein, we isolate and characterize the mitochondria from non-photosynthesizing cell suspension cultures of Rubus fruticosus. Furthermore, we assess the primary site of ROS generation and the effects of SA on isolated organelles. Mitochondrial Complex III was found to be the major source of ROS generation in this model. In addition, we discovered that SA inhibits the electron transport chain by inactivating the semiquinone radical during the Q cycle. Computational analyses confirmed the experimental data, and a mechanism for this action is proposed.

Classical and alternative components of the mitochondrial respiratory chain in pathogenic fungi as potential therapeutic targets

The frequency of opportunistic fungal infection has increased drastically, mainly in patients who are immunocompromised due to organ transplant, leukemia or HIV infection. In spite of this, only a few classes of drugs with a limited array of targets, are available for antifungal therapy. Therefore, more specific and less toxic drugs with new molecular targets is desirable for the treatment of fungal infections. In this context, searching for differences between mitochondrial mammalian hosts and fungi in the classical and alternative components of the mitochondrial respiratory chain may provide new potential therapeutic targets for this purpose.

Dehydromonocrotaline induces cyclosporine A-insensitive mitochondrial permeability transition/cytochrome c release

Monocrotaline (MCT) is a pyrrolizidine alkaloid present in plants of the genus Crotalaria that causes cytotoxicity and genotoxicity in animals and humans. It is well established that the toxicity of MCT results from its hepatic bioactivation to dehydromonocrotaline (DHM), an alkylating agent, but the exact mechanism of action remains unknown. In a previous study, we demonstrated DHM`s inhibition of mitochondrial NADH-dehydrogenase activity at micromolar concentrations, which is an effect associated with a significant reduction in ATP synthesis. As a follow-up study, we have evaluated the ability of DHM to induce mitochondrial permeability transition (MPT) and its associated processes in isolated rat liver mitochondria. In the presence of 10 mu M Ca(2+), DHM (50-250 mu M) elicited MPT in a concentration-dependent, but cyclosporine A-independent manner, as assessed by mitochondrial swelling, which is associated with mitochondrial Ca(2+) efflux and cytochrome c release. DHM (50-250 mu M) did not cause hydrogen peroxide accumulation but did deplete endogenous glutathione and NAD(P)H, while oxidizing protein thiol groups. These results potentially indicate the involvement of mitochondria, via apoptosis, in the well-documented cytotoxicity of monocrotaline. (C) 2009 Elsevier Ltd. All rights reserved.

The roles played by Aspergillus nidulans apoptosis-inducing factor (AIF)-like mitochondrial oxidoreductase (AifA) and NADH-ubiquinone oxidoreductases (NdeA-B and NdiA) in farnesol resistance

Farnesol (FOH) is a nonsterol isoprenold produced by dephosphorylanon of farnesyl pyrophosphate a catabolite of the cholesterol biosynthetic pathway These isoprenoids inhibit proliferation and induce apoptosis Here we show that Aspergillus nidulans MA encoding the apoptosis-Inducing factor (AIF)-like mitochondrial oxidoreductase plays a role in the function of the mitochondrial Complex I Additionally we demonstrated that ndeA B and ndiA encode external and internal alternative NADH dehydrogenases respectively that have a function in FOH resistance When exposed to FOH the Delta aifA and Delta ndeA strains have increased ROS production while Delta ndeB Delta ndeA Delta ndeB and Andul mutant strains showed the same ROS accumulation than in the absence of FOH We observed several compensatory mechanisms affecting the differential survival of these mutants to FOH (C) 2010 Elsevier Inc All rights reserved

Enzymatic inhibitory activity and trypanocidal effects of extracts and compounds from Siphoneugena densiflora O. Berg and Vitex polygama Cham.

Hexanic, methanolic, and hydroalcoholic extracts, and 34 isolated compounds from Vitex polygama Cham. (Lamiaceae, formely Verbenaceae) and Siphoneugena densiflora O. Berg (Myrtaceae) were screened for their trypanocidal effects on bloodstream forms of Trypanosoma cruzi and T brucei, as well as for their enzymatic inhibitory activities on glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) and trypanothione reductase (TR) enzymes from T cruzi and adeninephosphoribosyl transferase (APRT) enzyme from Leishmania tarentolae. In general, polar extracts displayed strong effects and some of the tested compounds have shown good results in comparison to positive controls of the bioassays.

A proteomic approach to identifying proteins differentially expressed in conidia and mycelium of the entomopathogenic fungus Metarhizium acridum

Metarhizium spp. is an important worldwide group of entomopathogenic fungi used as an interesting alternative to chemical insecticides in programs of agricultural pest and disease vector control. Metarhizium conidia are important in fungal propagation and also are responsible for host infection. Despite their importance, several aspects of conidial biology, including their proteome, are still unknown. We have established conidial and mycelial proteome reference maps for Metarhizium acridum using two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF MS). In all, 1130 +/- 102 and 1200 +/- 97 protein spots were detected in ungerminated conidia and fast-growing mycelia, respectively. Comparison of the two protein-expression profiles reveled that only 35 % of the protein spots were common to both developmental stages. Out of 94 2-DE protein spots (65 from conidia, 25 from mycelia and two common to both) analyzed using mass spectrometry, seven proteins from conidia, 15 from mycelia and one common to both stages were identified. The identified protein spots exclusive to conidia contained sequences similar to known fungal stress-protector proteins (such as heat shock proteins (HSP) and 6-phosphogluconate dehydrogenase) plus the fungal allergen Alt a 7, actin and the enzyme cobalamin-independent methionine synthase. The identified protein spots exclusive to mycelia included proteins involved in several cell housekeeping biological processes. Three proteins (HSP 90, 6-phosphogluconate dehydrogenase and allergen Alt a 7) were present in spots in conidial and mycelial gels, but they differed in their locations on the two gels. (c) 2010 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Amino acid synthesis in the symbiotic sea anemone Aiptasia pulchella

Symbiotic Aiptasia pulchella and freshly isolated zooxanthellae were incubated in (NaHCO3)-C-14 and NH4Cl for 1 to 240 min, and samples were analysed by reverse-phase high-performance liquid chromatography (HPLC) and an online radiochemical detector. NH4+ was first assimilated into C-14-glutamate and C-14-glutamine in the zooxanthellae residing in A. pulchella. The specific activities (dpm nmol(-1)) of C-14-glutamate and C-14-glutamine in vivo, were far greater in the zooxanthellae than in the host tissue, indicating that NH4+ was principally incorporated into the glutamate and glutamine pools of the zooxanthellae. C-14-alpha-ketoglutarate was taken up from the medium by intact A. pulchella and assimilated into a small amount of C-14-glutamate in the host tissue, but no C-14-glutamine was detected in the host fraction. The C-14-glutamate that was synthesized was most likely produced from transamination reactions as opposed to the direct assimilation of NH4+. The free aminoacid composition of the host tissue and zooxanthellae of A. pulchella was also measured. The results presented here demonstrate that NH4+ was initially assimilated by the zooxanthellae of A. pulchella.

Increased expression of mitochondrial genes in human alcoholic brain revealed by differential display

Polymerase chain reaction (PCR)-based differential display was used to screen for alterations in gene expression in the mesolimbic system of the human alcoholic brain. Total RNA was extracted from the nucleus accumbens of five alcoholic and five control brains. A selected subpopulation of mRNA was reverse-transcribed to cDNA and amplified by PCR. A differentially expressed cDNA fragment was recovered, cloned, and sequenced. Full sequence analysis of this 467 bp fragment revealed 98.2% homology with the human mitochondrial 12S rRNA gene. Dot-blot analysis showed increased expression of this gem in nucleus accumbens and hippocampus, but not in the superior frontal cortex, primary motor cortex, caudate, and pallidus/putamen In a total of eight human alcoholic brains, compared with seven control brains. A similar increased expression was observed by dot-blot analysis, using RNA from the cerebral cortex of rats chronically treated with alcohol vapor. Hybridization of a 16S rRNA oligonucleotide probe indicated that the expression of both rRNAs genes was significantly increased in nucleus accumbens. These results indicate that chronic alcohol consumption induces alteration in expression of mitochondrial genes in selected brain regions. The altered gene expression may reflect mitochondrial dysfunction In the alcohol-affected brain.

Substrate-dependent regulation of human arylamine N-acetyltransferase-1 in cultured cells

Arylamine N-acetyltransferase-1 (NAT1) is a polymorphically expressed enzyme that is widely distributed throughout the body. In the present study, we provide evidence for substrate-dependent regulation of this enzyme. Human peripheral blood mononuclear cells cultured in medium supplemented with p-aminobenzoic acid (PABA; 6 mu M) for 24 h showed a significant decrease (50-80%) in NAT1 activity. The loss of activity was concentration-dependent (EC50 similar to 2 mu M) and selective because PABA had no effect on the activity of constitutively expressed lactate dehydrogenase or aspartate aminotransferase. PABA also induced down-regulation of NAT1 activity in several human cell lines grown at confluence. Substrate-dependent downregulation was not restricted to PABA. Addition of other NAT1 substrates, such as p-aminosalicylic acid, ethyl-p-aminobenzoate, or p-aminophenol to peripheral blood mononuclear cells in culture also resulted in significant (P < .05) decreases in NAT1 activity. However, addition of the NAT2-selective substrates sulfamethazine, dapsone, or procainamide did not alter NAT1 activity. Western blot analysis using a NAT1-specific antibody showed that the loss of NAT1 activity was associated with a parallel reduction in the amount of NAT1 protein (r(2) = 0.95). Arylamines that did not decrease NAT1 activity did not alter NAT1 protein levels. Semiquantitative reverse transcriptase polymerase chain reaction of mRNA isolated from treated and untreated cells revealed no effect of PABA on NAT1 mRNA levels. We conclude that NAT1 can be down-regulated by arylamines that are themselves NAT1 substrates. Because NAT1 is involved in the detoxification/activation of various drugs and carcinogens, substrate-dependent regulation may have important consequences with regard to drug toxicity and cancer risk.

Sulfite : Cytochrome c oxidoreductase from Thiobacillus novellus - Purification, characterization, and molecular biology of a heterodimeric member of the sulfite oxidase family

Direct oxidation of sulfite to sulfate occurs in various photo- and chemotrophic sulfur oxidizing microorganisms as the final step in the oxidation of reduced sulfur compounds and is catalyzed by sulfite:cytochrome c oxidoreductase (EC 1.8.2.1), Here we show that the enzyme from Thiobacillus novellus is a periplasmically located alpha beta heterodimer, consisting of a 40.6-kDa subunit containing a molybdenum cofactor and an 8.8-kDa monoheme cytochrome c(552) smbunit (midpoint redox potential, Em(8.0) = +280 mV), The organic component of the molybdenum cofactor was identified as molybdopterin contained in a 1:1 ratio to the Mo content of the enzyme. Electron paramagnetic resonance spectroscopy revealed the presence of a sulfite-inducible Mo(V) signal characteristic of sulfite:acceptor oxidoreductases. However, pH-dependent changes in the electron paramagnetic resonance signal were not detected. Kinetic studies showed that the enzyme exhibits a ping-pong mechanism involving two reactive sites. K-m values for sulfite and cytochrome c(550) were determined to be 27 and 4 mu M, respectively; the enzyme was found to be reversibly inhibited by sulfate and various buffer ions. The sorAB genes, which encode the enzyme, appear to form an operon, which is preceded by a putative extracytoplasmic function-type promoter and contains a hairpin loop termination structure downstream of sorB. While SorA exhibits significant similarities to known sequences of eukaryotic and bacterial sulfite:acceptor oxidoreductases, SorB does not appear to be closely related to any known c-type cytochromes.

Discovery and characterization of a family of insecticidal neurotoxins with a rare vicinal disulfide bridge

We have isolated a family of insect-selective neurotoxins from the venom of the Australian funnel-web spider that appear to be good candidates for biopesticide engineering. These peptides, which we have named the Janus-faced atracotoxins (J-ACTXs), each contain 36 or 37 residues, with four disulfide bridges, and they show no homology to any sequences in the protein/DNA databases. The three-dimensional structure of one of these toxins reveals an extremely rare vicinal disulfide bridge that we demonstrate to be critical for insecticidal activity. We propose that J-ACTX comprises an ancestral protein fold that we refer to as the disulfide-directed beta-hairpin.

Muscle fibre types and size distribution in sub-antarctic notothenioid fishes

The presumptive tonic muscles fibres of Cottoperca gobio, Champsocephalus esox, Harpagifer bispinis, Eleginops maclovinus, Patagonothen tessellata, P. cornucola and Paranotothenia magellanica stained weakly or were unstained for glycogen, lipid, succinic dehydrogenase (SDHase) and myosin ATPase (mATPase) activity. Slow, intermediate and fast twitch muscle fibres, distinguished on the basis of the pH stability of their mATPases, showed intense, moderate and low staining activity for SDHase, respectively. Slow fibres were the major component of the pectoral fin adductor profundis muscle. The proportion of different muscle fibre types varied from the proximal to distal end of the muscle, but showed relatively little variation between species. The myotomes contained a lateral superficial strip of red muscle composed of presumptive tonic, slow twitch and intermediate fibres, thickening to a major wedge at the horizontal septum. All species also had characteristic secondary dorsal and ventral wedges of red muscle. The relative abundance and localization of muscle fibre types in the red muscle varied between species and with body size in the protandric hermaphrodite E. maclovinus. The frequency distribution of diameters for fast twitch muscle fibres, the major component of deep white muscle, was determined in fish of a range of body sizes. The absence of fibres <20 mu m diameter was used as a criterion for the cessation of muscle fibre recruitment. Fibre recruitment had stopped in P, tessellata of 13.8 cm L-T and E, maclovinus of 32.8 cm L-T, equivalent to 49 and 36.5% of their recorded maximum sizes respectively. As a result in 20-cm P. tessellata, the maximum fibre diameter was 300 mu m and 36% of fibres were in excess of 200 mu m The unusually large maximum fibre diameter, the general arrangement of the red muscle layer and the extreme pH lability of the mATPase of fast twitch fibres are all common characters of the sub-Antarctic and Antarctic Notothenioids, including Cottoperca gobio, the suggested sister group to the Notothenidae. (C) 2000 The Fisheries Society of the British Isles.

Enzymology and molecular biology of prokaryotic sulfite oxidation

Despite its toxicity, sulfite plays a key role in oxidative sulfur metabolism and there are even some microorganisms which can use it as sole electron source. Sulfite is the main intermediate in the oxidation of sulfur compounds to sulfate, the major product of most dissimilatory sulfur-oxidizing prokaryotes. Two pathways of sulfite oxidation are known: (1) direct oxidation to sulfate catalyzed by a sulfite: acceptor oxidoreductase, which is thought to be a molybdenum-containing enzyme; (2) indirect oxidation under the involvement of the enzymes adenylylsulfate (APS) reductase and ATP sulfurylase and/or adenylylsulfate phosphate adenylyltransferase with APS as an intermediate. The latter pathway allows substrate phosphorylation and occurs in the bacterial cytoplasm. Direct oxidation appears to have a wider distribution; however, a redundancy of pathways has been described for diverse photo- or chemotrophic, sulfite-oxidizing prokaryotes. In many pro- and also eukaryotes sulfite is formed as a degradative product from molecules containing sulfur as a heteroatom. In these organisms detoxification of sulfite is generally achieved by direct oxidation to sulfate. (C) 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.