Mitochondrial Electron Transfer System Research Articles

Imaging of Mitochondria Metabolic Change after Radiation Treatment using Dynamic Nuclear Polarization (DNP)-MRI

In vivo dynamic nuclear polarization-magnetic resonance imaging (DNP-MRI) enables the anatomical distribution of free radical species to be monitored. DNP enhances the MRI signal from nuclei such as 1H by irradiating tissue at the electron paramagnetic resonance (EPR) frequency of the free radical, before applying the MRI pulse sequence, thus increasing the image intensity where free radicals are present. Low-molecular-weight, stable nitroxyl compounds, including carbamoyl-PROXYL (CmP), are organic free radicals that possess SOD-like activity, and they have been used as probes in a variety of biophysical and biochemical experiments. The reduction rate of the radical probe depends on the tissue redox environment, which is influenced by factors such as overproduction of ROS and decreased antioxidant defences. In this study, we evaluated the feasibility of mitochondrial redox metabolic imaging, assessed by DNP-MRI with CmP as a molecular imaging probe, for detecting the metabolic change after radiation treatment to the tissue homogenate solution. Redox imaging of mitochondrial metabolism was performed with a low magnetic field using an in vivo DNP-MRI system (Keller, Japan Redox Inc.). The external magnetic field (B0) for EPR irradiation and MRI was fixed at 15 mT, and the radiofrequencies for EPR irradiation and MRI were 455 MHz and 683 kHz, respectively. DNP-MRI images of liver homogenate solution with or without radiation (10 or 20 gray) were obtained at 3, 5, 10, 15, 20, 25, 30, 35 and 40 min after starting measurement. The redox map was obtained by the slope of the enhanced DNP image intensity of each pixel from four pharmacokinetic images using a custom Excel macro program. Phantom redox experiments were conducted to examine the ability of CmP to participate in the mitochondrial redox reaction, and time-dependent changes in image contrast were assessed using liver tissue homogenate and KCN, an inhibitor of mitochondrial electron transfer chain. The DNP images clearly showed the significant enhancement in tissue homogenate solution by CmP. The enhancement of DNP-image was gradually reduced by liver homogenate solution due to reduction of CmP and the reduction rate of CmP was increased by the treatment of radiation. Interestingly, reduction of CmP in both homogenates with or without radiation were completely inhibited by KCN treatment. It is suggested that enhancement of CmP reduction is related in mitochondrial electron transfer system. We demonstrated that the mitochondrial redox metabolic change in the electron transfer chain after radiotherapy could be monitored by DNP-MRI with CmP as a redox probe.

Oxygen conserving mitochondrial adaptations in the skeletal muscles of breath hold divers.

BackgroundThe performance of elite breath hold divers (BHD) includes static breath hold for more than 11 minutes, swimming as far as 300 m, or going below 250 m in depth, all on a single breath of air. Diving mammals are adapted to sustain oxidative metabolism in hypoxic conditions through several metabolic adaptations, including improved capacity for oxygen transport and mitochondrial oxidative phosphorylation in skeletal muscle. It was hypothesized that similar adaptations characterized human BHD. Hence, the purpose of this study was to examine the capacity for oxidative metabolism in skeletal muscle of BHD compared to matched controls.MethodsBiopsies were obtained from the lateral vastus of the femoral muscle from 8 Danish BHD and 8 non-diving controls (Judo athletes) matched for morphometry and whole body VO2max. High resolution respirometry was used to determine mitochondrial respiratory capacity and leak respiration with simultaneous measurement of mitochondrial H2O2 emission. Maximal citrate synthase (CS) and 3-hydroxyacyl CoA dehydrogenase (HAD) activity were measured in muscle tissue homogenates. Western Blotting was used to determine protein contents of respiratory complex I-V subunits and myoglobin in muscle tissue lysates.ResultsMuscle biopsies of BHD revealed lower mitochondrial leak respiration and electron transfer system (ETS) capacity and higher H2O2 emission during leak respiration than controls, with no differences in enzyme activities (CS and HAD) or protein content of mitochondrial complex subunits myoglobin, myosin heavy chain isoforms, markers of glucose metabolism and antioxidant enzymes.ConclusionWe demonstrated for the first time in humans, that the skeletal muscles of BHD are characterized by lower mitochondrial oxygen consumption both during low leak and high (ETS) respiration than matched controls. This supports previous observations of diving mammals demonstrating a lower aerobic mitochondrial capacity of the skeletal muscles as an oxygen conserving adaptation during prolonged dives.

Rotenone decreases ischemia-induced injury by inhibiting mitochondrial permeability transition in mature brains

The mitochondrial permeability transition pore (mPTP) is thought to be implicated in brain ischemia-induced cell death. Here we sought to determine whether complex I (CI) of the mitochondrial electron transfer system may be involved in regulation of mPTP opening during ischemia and whether a specific inhibitor of this complex – rotenone can protect against ischemia-induced cell death in an experimental model of total ischemia in adult rat brains. Anesthetized Wistar rats were administered a single injection of rotenone (0.01mg/kg) to the tail vein and brains were removed and subjected to 120min ischemia. We found that intravenous injection of rotenone 20min before ischemia increased resistance to Ca2+-induced mPTP opening and decreased production of reactive oxygen species (ROS) in mitochondria isolated from ischemia-damaged cortex and cerebellum. Rotenone administration before ischemia decreased infarct size in both brain regions (cortex and cerebellum). Rotenone added directly to normal, non-ischemic cortical or cerebellar mitochondria increased their resistance to Ca2+-induced mPTP opening at concentration which fully inhibited NAD-dependent mitochondrial respiration. Our data demonstrate that rotenone used intravenously may be protective against acute brain ischemia-induced injuries by inhibition of mPTP opening and ROS production. These findings suggest that CI of mitochondrial electron transfer system plays a role in mPTP regulation during cerebral ischemia in mature brains and that agents acting on CI activity may be clinically useful for stroke therapy.

Bioaccumulation of gadolinium in freshwater bivalves.

In this study, the presence of anthropogenic gadolinium (Gd) was evaluated in rivers, close to wastewater treatment plant outputs. Then, one site was selected for in situ experiments to assess the bioaccumulation of Gd in the digestive gland and in the gills of two bivalves (Dreissena rostriformis bugensis and Corbicula fluminea). For both organisms, the results suggested that the bioaccumulation of Gd can be observed when organisms are exposed in a geogenic and anthropogenic Gd mixture. In order to observe if Gd can bioaccumulate in tissues of bivalves when the ion is only present as the main anthropogenic speciation of Gd, i.e., Gd-contrast agents (Gd-CAs), the gadoteric acid was used for a laboratory experiment. In this case, the presence of Gd was clearly detected in a significant amount in the digestive glands of D. rostriformis bugensis and C. fluminea while low concentrations are measured in the gills. For the first time, these results clearly showed that Gd can bioaccumulate in bivalve tissues even when it is only present as Gd-CAs. Biochemical activities were measured in the digestive gland and in the gills of the bivalves to assess the effects of Gd-CA bioaccumulation. No significant variations were observed in the gills. Concerning the digestive gland, after 7days of exposure at 10μgL-1 of Gd as Gd-CA speciation, GST activity in D. rostriformis bugensis and lipid hydroperoxide and mitochondrial electron transfer system in C. fluminea had increased. The results suggest an acclimation of the organisms to the presence of Gd-CAs in the medium within less than 21days.

Oxidative phosphorylation and mitochondrial function differ between human prostate tissue and cultured cells.

Altered mitochondrial metabolism plays a pivotal role in the development and progression of various diseases, including cancer. Cell lines are frequently used as models to study mitochondrial (dys)function, but little is known about their mitochondrial respiration and metabolic properties in comparison to the primary tissue of origin. We have developed a method for assessment of oxidative phosphorylation in prostate tissue samples of only 2 mg wet weight using high-resolution respirometry. Reliable protocols were established to investigate the respiratory activity of different segments of the mitochondrial electron transfer system (ETS) in mechanically permeabilized tissue biopsies. Additionally, the widely used immortalized prostate epithelial and fibroblast cell lines, RWPE1 and NAF, representing the major cell types in prostate tissue, were analyzed and compared to the tissue of origin. Our results show that mechanical treatment without chemical permeabilization agents or sample processing constitutes a reliable preparation method for OXPHOS analysis in small amounts of prostatic tissue typically obtained by prostate biopsy. The cell lines represented the bioenergetic properties of fresh tissue to a limited extent only. Particularly, tissue showed a higher oxidative capacity with succinate and glutamate, whereas pyruvate was a substrate supporting significantly higher respiratory activities in cell lines. Several fold higher zinc levels measured in tissue compared to cells confirmed the role of aconitase for prostate-specific metabolism in agreement with observed respiratory properties. In conclusion, combining the flexibility of cell culture models and tissue samples for respirometric analysis are powerful tools for investigation of mitochondrial function and tissue-specific metabolism.

On the regulative role of the glutamate receptor in mitochondria.

The purpose of this work was to study the regulative role of the glutamate receptor found earlier in the brain mitochondria. In the present work a glutamate-dependent signaling system with similar features was detected in mitochondria of the heart. The glutamate-dependent signaling system in the heart mitochondria was shown to be suppressed by γ-aminobutyric acid (GABA). The GABA receptor presence in the heart mitochondria was shown by golding with the use of antibodies to α- and β-subunits of the receptor. The activity of glutamate receptor was assessed according to the rate of synthesis of hydrogen peroxide. The glutamate receptor in mitochondria could be activated only under conditions of hypoxic stress, which in model experiments was imitated by blocking Complex I by rotenone or fatty acids. The glutamate signal in mitochondria was shown to be calcium- and potential-dependent and the activation of the glutamate cascade was shown to be accompanied by production of hydrogen peroxide. It was discovered that H2O2 synthesis involves two complexes of the mitochondrial electron transfer system - succinate dehydrogenase (SDH) and fatty acid dehydrogenase (ETF:QO). Thus, functions of the glutamate signaling system are associated with the system of respiration-glycolysis switching (the Pasteur-Crabtree) under conditions of hypoxia.

Redox Molecular Imaging Using ReMI

Tissue redox status is one of the most important parameters to maintain homeostasis in the living body. Numerous redox reactions are involved in metabolic processes, such as energy production in the mitochondrial electron transfer system. A variety of intracellular molecules such as reactive oxygen species, glutathione, thioredoxins, NADPH, flavins, and ascorbic acid may contribute to the overall redox status in tissues. Breakdown of redox balance may lead to oxidative stress and can induce many pathological conditions such as cancer, neurological disorders, and aging. Therefore imaging of tissue redox status and monitoring antioxidant levels in living organisms can be useful in the diagnosis of disease states and assessment of treatment response. In vivo redox molecular imaging technology such as electron spin resonance imaging (ESRI), magnetic resonance imaging (MRI), and dynamic nuclear polarization (DNP)-MRI (redox molecular imaging; ReMI) is emerging as a viable redox status imaging modality. This review focuses on the application of magnetic resonance technologies using MRI or DNP-MRI and redox-sensitive contrast agents.

Higd1a is a positive regulator of cytochrome c oxidase.

Cytochrome c oxidase (CcO) is the only enzyme that uses oxygen to produce a proton gradient for ATP production during mitochondrial oxidative phosphorylation. Although CcO activity increases in response to hypoxia, the underlying regulatory mechanism remains elusive. By screening for hypoxia-inducible genes in cardiomyocytes, we identified hypoxia inducible domain family, member 1A (Higd1a) as a positive regulator of CcO. Recombinant Higd1a directly integrated into highly purified CcO and increased its activity. Resonance Raman analysis revealed that Higd1a caused structural changes around heme a, the active center that drives the proton pump. Using a mitochondria-targeted ATP biosensor, we showed that knockdown of endogenous Higd1a reduced oxygen consumption and subsequent mitochondrial ATP synthesis, leading to increased cell death in response to hypoxia; all of these phenotypes were rescued by exogenous Higd1a. These results suggest that Higd1a is a previously unidentified regulatory component of CcO, and represents a therapeutic target for diseases associated with reduced CcO activity.

Dynamic Function of the Alkyl Spacer of Acetogenins as Potent Inhibitors of Mitochondrial Complex I. A Molecular Dynamics Simulation Approach

Acetogenins are among the most potent of the known inhibitors of complex I (NADH-ubiquinone oxidoreductase) in mitochondrial electron transfer system. Elucidation of the dynamic function of the alkyl spacer linking the two toxophores (i.e., the hydroxylated tetrahydrofuran and the γ-lactone rings) is critical for fully understanding their inhibition mechanism. To this end, using molecular dynamics simulations a structure-activity relationship study of a series of acetogenins was performed for the first time using this approach. Our results clearly indicated that both, the length and the molecular flexibility of the spacer, were crucial for taking an active conformation. A partially folded conformation with an optimal length (bis-tetrahydrofuran rings and 13 carbon atoms) of about 16 Å with a high molecular flexibility might depict an active form of the spacer. In addition, we demonstrated that the bis-tetrahydrofuran derivatives are able to overcome the shortage of the length of the spacer more efficiently than the mono-tetrahydrofuran derivatives with the help of the additional tetrahydrofuran, which acts as a pseudospacer. Our results obtained from molecular dynamics calculations supported the use of a combined decane/water system as a good solvent model to simulate the biological environment of acetogenins acting as inhibitor of complex I.

Abstract 766: Phenformin is a MYCN/MYC destabilizer that can specifically target neural crest derived tumors.

Abstract We have been interested in identifying MYCN/MYC destabilizers that can specifically target neural crest derived tumors (neuroblastoma, melanoma, and malignant pheochromocytoma). MYCN amplification and its over-expression are associated with the worst neuroblastoma disease outcome. Non-MYCN amplified neuroblastomas express high-levels of MYC, and prognosis of these neuroblastomas is very poor. Melanoma and malignant pheochromocytoma are also known for high MYC expression. Interestingly, these neural crest derived tumors characteristically express the norepinephrine transporter (NET). This feature can be used to specifically target the tumor cells with agonists of NET, such as Meta-Iodo-BenzylGuanidine (MIBG). In the previous AACR meeting, we presented our finding that non-radio-labeled MIBG can suppress growth of neuroblastoma cells in vitro, and this effect is in part related to its ability to destabilize MYC and MYCN. We have also shown that FCCP, a well-known mitochondrial respiration inhibitor, destabilized MYCN and MYC in neuroblastoma cells and caused growth suppression. Recently, it has been reported that Phenformin, a Type II diabetic drug, binds NET, suggesting that NET positive cells preferentially uptake Phenformin. It is also known that Phenformin inhibits the mitochondrial electron transfer system. In this study, we have investigated a possible effect of Phenformin on growth and MYC/MYCN stability in neuroblastoma cells in vitro. SKNBE(2)C and CHP134 neuroblastoma cell lines were treated with various doses (0-2.5mM) of Phenformin for two days. MTS assay was used to assess the effect of Phenformin on growth of the cells. There was a dose-dependent growth suppression of the NB cell lines tested. Phenformin was more effective against growth of SKNBE(2)C than CHP134 cells. The effect of Phenformin on MYC/MYCN expression in four neuroblastoma cell lines [SKNBE(2)C, CHP134, SKNAS, and SY5Y] was also examined by Western blot assay. SKNBE(2)C and CHP134 express MYCN, whereas SKNAS and SY5Y express MYC at high levels. Treatment of these cells with Phenformin at the dose of 1mM for 24 hours resulted in destabilization of MYCN/MYC, suggesting that the growth suppressive effect of Phenformin on neuroblastoma cells was in part due to reduction in MYC/MYCN expression. We are currently examining whether the above observations made in neuroblastoma are also applicable to other neural crest derived tumors. In addition, we are investigating how Phenformin-related mitochondrial dysfunction causes destabilization of MYC/MYCN in neuroblastoma cells. Citation Format: Naohiko Ikegaki, Srah Lomahan, Xao X. Tang. Phenformin is a MYCN/MYC destabilizer that can specifically target neural crest derived tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 766. doi:10.1158/1538-7445.AM2013-766

Trigonelline Ameliorates Oxidative Stress in Type 2 Diabetic Goto-Kakizaki Rats

Previously, we showed that trigonelline (TRG) exerts antidiabetic effects in type 2 diabetic Goto-Kakizaki (GK) rats and also lowers blood and liver thiobarbituric acid reactive substances and urinary 8-hydroxydeoxyguanosine when compared with those levels in GK control rats without TRG. These results suggested that TRG also mitigates oxidative stress, which accelerates diabetes. In this study, the mechanisms of TRG prevention of oxidative stress were determined by measuring erythrocyte and liver antioxidant enzyme activities, and expressions of genes associated with reactive oxygen species production, and carbohydrate and lipid metabolisms by DNA microarray. Erythrocyte and liver glutathione peroxidase, and liver catalase activities in the GK rats fed with TRG were significantly lower than those of the GK control rats. TRG downregulated the gene expressions involved with NADPH oxidase and mitochondrial electron transfer system when compared with those of the GK control group. These results suggested that mitigation of diabetes by TRG is mediated by its ameliorating effects on oxidative stress.

Computational analysis of a novel mutation in ETFDH gene highlights its long-range effects on the FAD-binding motif

BackgroundMultiple acyl-coenzyme A dehydrogenase deficiency (MADD) is an autosomal recessive disease caused by the defects in the mitochondrial electron transfer system and the metabolism of fatty acids. Recently, mutations in electron transfer flavoprotein dehydrogenase (ETFDH) gene, encoding electron transfer flavoprotein:ubiquinone oxidoreductase (ETF:QO) have been reported to be the major causes of riboflavin-responsive MADD. To date, no studies have been performed to explore the functional impact of these mutations or their mechanism of disrupting enzyme activity.ResultsHigh resolution melting (HRM) analysis and sequencing of the entire ETFDH gene revealed a novel mutation (p.Phe128Ser) and the hotspot mutation (p.Ala84Thr) from a patient with MADD. According to the predicted 3D structure of ETF:QO, the two mutations are located within the flavin adenine dinucleotide (FAD) binding domain; however, the two residues do not have direct interactions with the FAD ligand. Using molecular dynamics (MD) simulations and normal mode analysis (NMA), we found that the p.Ala84Thr and p.Phe128Ser mutations are most likely to alter the protein structure near the FAD binding site as well as disrupt the stability of the FAD binding required for the activation of ETF:QO. Intriguingly, NMA revealed that several reported disease-causing mutations in the ETF:QO protein show highly correlated motions with the FAD-binding site.ConclusionsBased on the present findings, we conclude that the changes made to the amino acids in ETF:QO are likely to influence the FAD-binding stability.

バンレイシ科アセトゲニン類の合成とミトコンドリアｃｏｍｐｌｅｘ Ｉに対する阻害活性

Annonaceous acetogenins are a large family of natural polyketides. So far, more than 430 related compounds have been isolated. These compounds exhibit a number of interesting biological activities including cytotoxic, antitumor, pesticidal, anti-infective properties. From a view point of mechanism of action, they are among the most potent of the known inhibitors of complex I (NADH-ubiquinone oxidoreductase) in mitochondrial electron transfer system. We report here the asymmetric synthesis of mono-tetrahydrofuran (mono-THF) acetogenins, non-tetrahydrofuran acetogenins, and tetrahydropyran acetogenins. We also wish to report structure-activity relationship (SAR) study of the inhibitory action with bovine heart mitochondrial complex I.

High resolution melting analysis facilitates mutation screening of ETFDH gene: Applications in riboflavin-responsive multiple acyl-CoA dehydrogenase deficiency

Background Multiple acyl-CoA dehydrogenase deficiency (MADD) or gluaric aciduria type II is an autosomal recessive disease caused by defects in mitochondrial electron transfer system and metabolism of fatty acid. Recently, ETFDH mutations were reported to be major causes of riboflavin-responsive MADD. The present study is aimed at screening ETFDH mutations. Methods High resolution melting (HRM) analysis was performed to screen ETFDH mutations. Genomic DNA was extracted from peripheral blood samples of the 9 patients with MADD and normal controls. Total 13 exons of ETFDH were screened by HRM analysis. The results were subsequently confirmed by direct DNA sequencing. Results This diagnostic strategy proved to be feasible in detecting 3 known (c.250G > A, c380T > A, c.524G > T) and 1 novel (c.1831G > A) ETFDH mutations. Each mutation could be readily and accurately identified in the difference plot curves. We estimated the carrier frequency of the hotspot mutation, c.250G > A, in the Taiwanese population to be 1:125 (0.8%). Conclusions HRM analysis can be successfully applied to screen ETFDH mutations. Since riboflavin-responsive MADD is often treatable, especially with mutations in ETFDH, identifying ETFDH mutations is crucial for these patients.

Bovine Heart NADH−Ubiquinone Oxidoreductase Contains One Molecule of Ubiquinone with Ten Isoprene Units as One of the Cofactors

NADH-ubiquinone oxidoreductase (Complex I) is located at the entrance of the mitochondrial electron transfer chain and transfers electrons from NADH to ubiquinone with 10 isoprene units (Q(10)) coupled with proton pumping. The composition of Complex I, the largest and most complex proton pump in the mitochondrial electron transfer system, especially the contents of Q(10) and phospholipids, has not been well established. An improved purification method including solubilization of mitochondrial membrane with deoxycholate followed by sucrose gradient centrifugation and anion-exchange column chromatography provided reproducibly a heme-free preparation containing 1 Q(10), 70 phosphorus atoms of phospholipids, 1 zinc ion, 1 FMN, 30 inorganic sulfur ions, and 30 iron atoms as the intrinsic constituents. The rotenone-sensitive enzymatic activity of the Complex I preparation was comparable to that of Complex I in the mitochondrial membrane. It has been proposed that Complex I has two Q(10) binding sites, one involved in the proton pump and the other functioning as a converter between one and two electron transfer pathways [Ohnishi, T., Johnson, J. J. E., Yano, T., LoBrutto, R., and Widger, R. W. (2005) FEBS Lett. 579, 500-506]. The existence of one molecule of Q(10) in the fully oxidized Complex I suggests that the affinity of Q(10) to one of the two Q(10) sites is greatly dependent on the oxidation state and/or the membrane potential and that the Q(10) in the present preparation functions as the converter of the electron transfer pathways which should be present in any oxidation state.

Current Topics of Organic and Biological Chemistry of Annonaceous Acetogenins and their Synthetic Mimics

Annonaceous acetogenins are a large family of natural polyketides. So far, more than 430 compounds have been isolated. Biologically, they are among the most potent of the known inhibitors of complex I (NADH-ubiquinone oxidoreductase) in mitochondrial electron transfer system. Herein, we would like to conduct an overview on the progress of the total synthesis, structural revisions, structure activity relationship for the inhibition of complex I, and action mechanism.

An Assembled Complex IV Maintains the Stability and Activity of Complex I in Mammalian Mitochondria

In the mammalian mitochondrial electron transfer system, the majority of electrons enter at complex I, go through complexes III and IV, and are finally delivered to oxygen. Previously we generated several mouse cell lines with suppressed expression of the nuclearly encoded subunit 4 of complex IV. This led to a loss of assembly of complex IV and its defective function. Interestingly, we found that the level of assembled complex I and its activity were also significantly reduced, whereas levels and activity of complex III were normal or up-regulated. The structural and functional dependence of complex I on complex IV was verified using a human cell line carrying a nonsense mutation in the mitochondrially encoded complex IV subunit 1 gene. Our work documents that, although there is no direct electron transfer between them, an assembled complex IV helps to maintain complex I in mammalian cells.

Comparison of Uptake between PureSorb-Q40 and Regular Hydrophobic Coenzyme Q10 in Rats and Humans after Single Oral Intake

Coenzyme Q10 (CoQ10) is a lipid-soluble antioxidant and essential component of the mitochondrial electron transfer system in the body, and is in wide use as a functional food material and cosmetic raw material. However, as CoQ10 is extremely lipid-soluble, absorption by the body is not easy. In general, people use soft-gel capsules in which CoQ10 is suspended in oil, and take these capsules with food. PureSorb-Q40 (P40) was developed to improve CoQ10 processability and absorption when taken without food, and the present study compared the effects of food on absorption between P40 and conventional lipid-soluble CoQ10 in rats and humans. The results of a rat study showed higher uptake when P40 was administered in the fasting state or with food compared to lipid-soluble CoQ10. The results of a human study showed that uptake was favorable when P40 was administered in the fasting state, and even when administered postprandially, a significant difference was noted in uptake rate up to 6 h after intake and uptake volume up to 8 h after intake when compared to lipid-soluble CoQ10. These results show that any CoQ10 product using P40 can be quickly and reliably absorbed by the body regardless of dosage form or intake time.

Metabolic regulation at the tricarboxylic acid and glyoxylate cycles of the lignin‐degrading basidiomycete Phanerochaete chrysosporium against exogenous addition of vanillin

A proteomic differential display technique was utilized to study cellular responses of Phanerochaete chrysosporium exposed to vanillin, one of the key intermediates found during lignin biodegradation. Intracellular proteins were resolved by 2-DE and target protein spots were identified using MALDI-MS after in-gel tryptic digestions. Upon addition of vanillin to P. chrysosporium, up-regulation of homogentisate 1,2-dioxygenase, 1,4-benzoquinone reductases, aldehyde dehydrogenase, and aryl-alcohol dehydrogenase, which seem to play roles in vanillin metabolism, was observed. Furthermore, enzymes involved in glycolysis, the tricarboxylic acid cycle, the pentose-phosphate cycle, and heme biosynthesis were also activated. Up-regulation of extracellular peroxidase was also observed. One of the most unique phenomena against exogenous vanillin was a switch from the glyoxylate cycle to the tricarboxylic acid cycle, where a drastic increase in isocitrate dehydrogenase activity was observed. The exogenous addition of other aromatic compounds also caused an increase in its activity, which in turn triggered NAD(P)H production via the action of dehydrogenases in the tricarboxylic acid cycle, heme biosynthesis via the action of aminolevulinic acid synthase on succinyl-CoA, and energy production via activation of the mitochondrial electron transfer system. These metabolic shifts seem to be required for activating a metabolic system for aromatic compounds.

Artificial Control of Enzyme Reactions by the Photoexcitation of Hematoporphyrin Adsorbed on Living Yeast Cells

Abstract The objective of our research was to establish control of biological functions by the illumination of a photosensitizer of hematoporphyrin that was externally provided in a biological system. We found using an ESR method that the reduction of 4-hydroxy-2,2,6,6-tetramethylpiperidinyl-1-oxyl (TEMPOL), as a reduction probe, caused by enzyme reactions can be controlled through the photoexcitation of hematoporphyrin adsorbed on the living yeast cells (HP/Yeast system). On the basis of the effects of some electron-transfer blockers through its reduction rate, it has been postulated that the reduction of TEMPOL is mainly conducted through ferredoxin (ferredoxin-NAD(P)+ reductase), which could be controlled through the illumination of HP connected with a mitochondrial electron-transfer system. A possible mechanism for controlling the reduction of TEMPOL is discussed in connection with the energy levels of HP and the redox potentials of the components in yeast cell participating in this phenomenon.