Tricarboxylic Cycle Enzymes Research Articles

Radiation-induced bystander effect on the brain after fractionated spinal cord irradiation of aging rats

We investigated the influence of the so-called bystander effect on metabolic and histopathological changes in the rat brain after fractionated spinal cord irradiation. The study was initiated with adult Wistar male rats (n = 20) at the age of 9 months. The group designated to irradiation (n = 10) and the age-matched control animals (n = 10) were subjected to an initial measurement using in vivo proton magnetic resonance spectroscopy (1H MRS) and magnetic resonance imaging (MRI). After allowing the animals to survive until 12 months, they received fractionated spinal cord irradiation with a total dose of 24 Gy administered in 3 fractions (8 Gy per fraction) once a week on the same day for 3 consecutive weeks. 1H MRS and MRI of brain metabolites were performed in the hippocampus, corpus striatum, and olfactory bulb (OB) before irradiation (9-month-old rats) and subsequently 48 h (12-month-old) and 2 months (14-month-old) after the completion of irradiation. After the animals were sacrificed at the age of 14 months, brain tissue changes were investigated in two neurogenic regions: the hippocampal dentate gyrus (DG) and the rostral migratory stream (RMS). By comparing the group of 9-month-old rats and individuals measured 48 h (at the age of 12 months) after irradiation, we found a significant decrease in the ratio of total N-acetyl aspartate to total creatine (tNAA/tCr) and gamma-aminobutyric acid to tCr (GABA/tCr) in OB and hippocampus. A significant increase in myoinositol to tCr (mIns/tCr) in the OB persisted up to 14 months of age. Proton nuclear magnetic resonance (1H NMR)-based plasma metabolomics showed a significant increase in keto acids and decreased tyrosine and tricarboxylic cycle enzymes. Morphometric analysis of neurogenic regions of 14-month-old rats showed well-preserved stem cells, neuroblasts, and increased neurodegeneration. The radiation-induced bystander effect more significantly affected metabolite concentration than the distribution of selected cell types.

Protists: Eukaryotic single-celled organisms and the functioning of their organelles.

Organelles are membrane bound structures that compartmentalize biochemical and molecular functions. With improved molecular, biochemical and microscopy tools the diversity and function of protistan organelles has increased in recent years, providing a complex panoply of structure/function relationships. This is particularly noticeable with the description of hydrogenosomes, and the diverse array of structures that followed, having hybrid hydrogenosome/mitochondria attributes. These diverse organelles have lost the major, at one time, definitive components of the mitochondrion (tricarboxylic cycle enzymes and cytochromes), however they all contain the machinery for the assembly of Fe-S clusters, which is the single unifying feature they share. The plasticity of organelles, like the mitochondrion, is therefore evident from its ability to lose its identity as an aerobic energy generating powerhouse while retaining key ancestral functions common to both aerobes and anaerobes. It is interesting to note that the apicoplast, a non-photosynthetic plastid that is present in all apicomplexan protozoa, apart from Cryptosporidium and possibly the gregarines, is also the site of Fe-S cluster assembly proteins. It turns out that in Cryptosporidium proteins involved in Fe-S cluster biosynthesis are localized in the mitochondrial remnant organelle termed the mitosome. Hence, different organisms have solved the same problem of packaging a life-requiring set of reactions in different ways, using different ancestral organelles, discarding what is not needed and keeping what is essential. Don't judge an organelle by its cover, more by the things it does, and always be prepared for surprises.

Fumarate exerted an antihypertensive effect and reduced kidney injury molecule (KIM)-1 expression in deoxycorticosterone acetate-salt hypertension

ABSTRACT Background: The tricarboxylic (TCA) acid cycle provides the energy needed for regulatory functions in the cardio-renal system. Recently, a genetic defect in the TCA cycle enzyme, fumarase hydratase, altered L-arginine metabolism and exacerbated hypertension in salt-sensitive rats. This study evaluated the effect of fumarate and its possible link to L-arginine metabolism in deoxycorticosterone (DOCA)-salt hypertension, a non-genetic model of hypertension.Method: Hypertension was induced with DOCA (25 mg/kg s.c, twice weekly) + 1% NaCL in uninephrectomised rats placed on fumarate (1 g/L, ad libitum). Blood pressure was measured in conscious rats via carotid cannulation. Biochemical and western blot analyses were carried out on kidney fractions.Results: Fumarate reduced mean blood pressure (198 ± 5 vs 167 ± 7 mmHg, p < .01), increased nitric oxide levels in the renal cortex (36.1 ± 2 vs 61.3 ± 4 nM/µg) and medulla (27.4 ± 1 vs 54.1 ± 2 nM/µg) of DOCA-salt rats (p < .01). Consistent with this, arginase activity was reduced (threefold) in the renal medulla but not cortex of DOCA-salt rats. Fumarate increased superoxide dismutase activity in the medulla (p < .001) of DOCA-hypertensive rats. However, catalase activity was exacerbated by fumarate in both renal cortex (4.5 ± 1 vs 11.2 ± 1) and medulla (3.7 ± 1 vs 16.3 ± 1 units/mg) of DOCA-salt rats (p < .001). Proteinuria (64.6%), kidney injury molecule-1 expression and kidney weight were reduced in DOCA-hypertensive rats treated with fumarate (p< .05). However, there was a paradoxical increase in TGF-β expression in fumarate-treated DOCA-salt rats. Conclusion: These data show that fumarate attenuated hypertension, renal injury and improved the redox state of the kidney in DOCA/salt hypertension by mechanisms involving selective reduction of L-arginine metabolism.

Fumarate Reduced Mean Arterial Blood Pressure and Improved Renal Outcomes in DOCA/salt Hypertension

The mitochondria, through the tricarboxylic (TCA) acid cycle, converts organic molecules into energy that drives intricate renal blood‐pressure regulatory functions. The TCA cycle intermediaries regulate the metabolic state of the mitochondria and by extension, the redox status of the cell. Recently, fumarase, a major TCA cycle enzyme that converts fumarate to malate, has been linked to L‐arginine metabolism and implicated in the pathophysiology of Dahl‐salt sensitive hypertension. This study aimed to investigate the role of fumarase and L‐arginine in deoxycorticosterone (DOCA)/salt hypertension, a non‐genetic model of hypertension. Hypertension was induced in uninephrectomised rats with DOCA (25 mg/kg) + 1 % NaCL. Mean arterial blood pressure was measured in conscious rats via carotid cannulation. Biochemical and western blot analyses were carried out on homogenized kidney fractions. Fumarate reduced arterial blood pressure (198±5 vs 167±7 mmHg, P&lt;0.01). There were no changes in urinary output and sodium excretion in fumarate‐treated DOCA rats (P&gt;0.05). Superoxide dismutase and catalase activities were higher in the cortex, but not the medulla of DOCA‐hypertensive rats and were exacerbated by fumarate (P&lt;0.01). There was a selective increase in catalase activity in the medulla of fumarate‐treated DOCA‐hypertensive rats (P&lt;0.01). Fumarate also reduced urinary protein excretion in DOCA‐hypertensive rats (23.6 %, P&lt;0.05). Fumarate evoked a selective increase in nitric oxide in the medulla (34.1±2.9 vs 47.3±2.8 nM/μg, P&lt;0.05) of DOCA‐hypertensive rats. Consistent with this, Fumarate also tended to increase arginase activity in the cortex but not the medulla of DOCA‐hypertensive rats. However, there was an increase in expression TGF‐β, an index of fibrosis, in DOCA‐hypertensive rats, which was paradoxically increased in fumarate‐treated rats (P&gt;0.05). In conclusion, fumarate attenuated the hypertension and renal injury but not the fibrosis in DOCA/salt hypertension by mechanisms involving selective reduction of L‐arginine metabolism in the medulla and amelioration of renal oxidative stress.Support or Funding InformationNational Institute of Health grant HL03674 and G12 MD0076056Mean arterial blood changes after 21‐day administration of fumarate to DOCA/salt induced hypertensive rats. Control (n=8), DOCA (n=8), fumarate (n=8). a=P&lt;0.001 when compared to control; b=P&lt;0.01 when compared to DOCA group.Figure 1Nitric oxide produced in homogenized kidneys (cortex and medulla) of DOCA salt induced hypertensive rats after 21‐day fumarate (1 mg/kg) administrationa=P&lt;0.05 when compared to control.Figure 2

Antihyperglycemic Potential of Back Tea Extract Attenuates Tricarboxylic AcidCycle Enzymes by Modulating Carbohydrate Metabolic Enzymes in Streptozotocin-Induced Diabetic Rats.

The present study was aimed to investigate the effect of black tea extract on blood glucose, plasma insulin, Hemoglobin, carbohydrate metabolic enzymes and tricarboxylic enzymes in streptozotocin (STZ) induced diabetic rats. Diabetes was induced in male albino Wistar rats by intraperitoneal administration of STZ (40mg/kgbwt). Black tea extract was administered to diabetic rats at a dose of 25, 50 and 100mg/kg body weight for 30days. The effects of black tea extract on glucose, insulin and HbA1c levels were analyzed to confirm the effective dose. Administration of black tea extract to diabetic rats was significantly decreased the level of glucose, glycated hemoglobin and increased the levels of insulin in a dose dependent manner. The black tea extracts at a dose of 100mg/kgbwt showed a highly significant effect compared to other two doses (25 and 50mg/kgbwt). The effect produced by black tea extract (100mg/kgbwt) was comparable to that of glibenclamide (5mg/kgbwt) a reference anti diabetic drug. Therefore, 100mg/kgbwt was fixed as an effective dose and used for further analyses. Black tea extract was administered to diabetic rats at a dose of 100mg/kgbwt for 30days reinstated the altered levels of the plasma glucose, insulin, hemoglobin, glycosylated hemoglobin, carbohydrate metabolizing enzymes and tricarboxylic cycle enzymes in diabetic rats. Black tea extract administered to diabetic rats at a dose of 100mg/kg bwt for 30days reinstated the altered levels of the plasma glucose, insulin, hemoglobin, glycosylated hemoglobin, carbohydrate metabolizing enzymes and tricarboxylic cycle enzymes in diabetic rats. The effect produced by black tea extract of all the biochemical parameters were comparable with glibenclamide-used as a reference drug.

In vitro proteomic analysis of methapyrilene toxicity in rat hepatocytes reveals effects on intermediary metabolism.

The antihistaminic drug methapyrilene was withdrawn from the market in 1979 because of hepatocarcinogenicity in rats. Since then, the drug has been used as a model hepatotoxin especially for transcriptomic analyses using material from in vivo studies. Much less transcriptomics data are available from in vitro studies, and no studies have investigated proteomic effects of methapyrilene in vitro. Thus, the present study was aimed to characterize the proteomic response of primary rat hepatocytes to methapyrilene, to broaden our knowledge on the molecular mechanisms of methapyrilene toxicity, and to compare the results of collagen sandwich-cultured hepatocytes to in vivo data. In vitro methapyrilene concentrations (0.39µM, 6.25µM, and 100µM) were chosen to cover an in vivo-relevant range. Based on published pharmacokinetic data they correspond to concentrations in portal vein blood for previously in vivo-tested doses of methapyrilene, up to a concentration showing slight cytotoxicity. Analysis of proteomic alterations by two-dimensional gel electrophoresis and mass-spectrometric protein identification demonstrated consistent and concentration-dependent effects of methapyrilene, in particular on mitochondrial proteins. Data suggest substantial deregulation of amino acid and ammonia metabolism and effects on mitochondrial energy supply pathways. The effects identified in vitro concur well with into previous in vivo observations. Several effects, for example, the influence of methapyrilene on S-adenosylmethionine metabolism, have not been described previously. The data suggest that already non-toxic concentrations of methapyrilene alter components of the intermediary metabolism, such as branched-chain amino acid metabolism, as well as urea and tricarboxylic cycle enzymes. In summary, data substantially add to our knowledge on molecular mechanisms of methapyrilene hepatotoxicity at the protein level.

Redox Regulation of Arabidopsis Mitochondrial Citrate Synthase

Citrate synthase has a key role in the tricarboxylic (TCA) cycle of mitochondria of all organisms, as it catalyzes the first committed step which is the fusion of a carbon-carbon bond between oxaloacetate and acetyl CoA. The regulation of TCA cycle function is especially important in plants, since mitochondrial activities have to be coordinated with photosynthesis. The posttranslational regulation of TCA cycle activity in plants is thus far almost entirely unexplored. Although several TCA cycle enzymes have been identified as thioredoxin targets in vitro, the existence of any thioredoxin-dependent regulation as known for the Calvin cycle, yet remains to be demonstrated. Here we have investigated the redox regulation of the Arabidopsis citrate synthase enzyme by site-directed mutagenesis of its six cysteine residues. Our results indicate that oxidation inhibits the enzyme activity by the formation of mixed disulfides, as the partially oxidized citrate synthase enzyme forms large redox-dependent aggregates. Furthermore, we were able to demonstrate that thioredoxin can cleave diverse intra- as well as intermolecular disulfide bridges, which strongly enhances the activity of the enzyme. Activity measurements with the cysteine variants of the enzyme revealed important cysteine residues affecting total enzyme activity as well as the redox sensitivity of the enzyme.

Mitochondrial DNA polymorphisms specifically modify cerebral β-amyloid proteostasis

Several lines of evidence link mutations and deletions in mitochondrial DNA (mtDNA) and its maternal inheritance to neurodegenerative diseases in the elderly. Age-related mutations of mtDNA modulate the tricarboxylic cycle enzyme activity, mitochondrial oxidative phosphorylation capacity and oxidative stress response. To investigate the functional relevance of specific mtDNA polymorphisms of inbred mouse strains in the proteostasis regulation of the brain, we established novel mitochondrial congenic mouse lines of Alzheimer's disease (AD). We crossed females from inbred strains (FVB/N, AKR/J, NOD/LtJ) with C57BL/6 males for at least ten generations to gain specific mitochondrial conplastic strains with pure C57BL/6 nuclear backgrounds. We show that specific mtDNA polymorphisms originating from the inbred strains differentially influence mitochondrial energy metabolism, ATP production and ATP-driven microglial activity, resulting in alterations of cerebral β-amyloid (Aβ) accumulation. Our findings demonstrate that mtDNA-related increases in ATP levels and subsequently in microglial activity are directly linked to decreased Aβ accumulation in vivo, implicating reduced mitochondrial function in microglia as a causative factor in the development of age-related cerebral proteopathies such as AD.

Differential metabolic consequences of fumarate hydratase and respiratory chain defects

Defects of the oxidative ATP production pathway lead to an amazing variety of disease phenotypes, ranging from childhood encephalomyopathies to hereditary tumor formation. A key enzyme of tricarboxylic cycle, fumarate hydratase (FH), is involved in encephalopathies, but also in leiomyoma formation, and occasionally also in various types of cancer. MELAS (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes) and NARP (neuropathy ataxia retinitis pigmentosa) are progressive neurological disorders, caused by mitochondrial DNA mutations and respiratory chain (RC) deficiency. These diseases lead to disability and premature death, but not to tumorigenesis. We studied the cellular consequences of FH and RC deficiencies, aiming to identify general responses to energy metabolism defect and those specific for FH-deficiency, suggestively connected to tumorigenesis. Unlike in RC deficiency, the FH-deficient diploid human fibroblasts showed no signs of oxidative stress, but had a reduced redox state with high glutathione levels. The cytoplasmic FH isoform, previously described, but with an unknown function, was completely lacking in all FH-deficient lines. Fumarate was increased in two of our FH-lines, but accumulation of HIF-1α was not detected. Glycolysis was induced in both MELAS and in FH-deficiency. Accumulation of fumarate in primary fibroblasts did not activate a hypoxia response, suggesting that hypoxia activation due to fumarate accumulation may be a tissue-specific response. The lack of cytoplasmic form of FH and the reduced redox environment were typical for all FH-mutant lines, and their role in FH-related tumorigenesis requires further attention.

Macromolecular synthesis during microcycle sporogenesis of Bacillus cereus T.

Microcycle sporogenesis induced in Bacillus cereus T by phosphate limitation occurs over a narrow range of phosphate to spore inoculum ratios. Sufficient phosphate is required to satisfy the demands for a twofold increase in deoxyribonucleic acid; net ribonucleic acid synthesis is not required. The total ribonucleic acid content of the culture was variable, and deoxyribonucleic acid synthesis was restricted to a twofold increase. Developmental changes during outgrowth occurred synchronously, whereas enzyme synthesis was periodic. The timing of the synthesis of tricarboxylic cycle enzymes, extracellular protease, arginase, histidase, and alkaline phosphatase was measured. Histidase could be induced after 2.5 hr throughout microcycle sporogenesis. Several other features of macromolecular synthesis during microcycle sporogenesis are described. Differences between this pattern and those observed during outgrowth leading to cell division are discussed. A technique for accurately estimating the levels and time of synthesis of incompletely extractable, labile enzymes is also presented.

Tricarboxylic acid cycle enzymes and morphogenesis in Blastocladiella emersonii.

During exponential growth, ordinary colorless (OC) plants of Blastocladiella emersonii consumed little glucose and produced no lactic acid. Similarly, resistant sporangial (RS) plants did not utilize glucose or produce lactic acid during the first 24 hr of exponential growth. During the next 24 hr of RS development, glucose was consumed with the concomitant production of lactic acid which was then reutilized. Lactic acid gradually accumulated again at maturity. Enzyme studies on cell-free extracts indicated the presence of all tricarboxylic cycle enzymes except alpha-ketoglutarate dehydrogenase at all stages of development of both RS and OC plants. Included among the enzymes detected were an adenosine monophosphate-stimulated, nicotinamide adenine dinucleotide-isocitric dehydrogenase, and citrate-condensing enzyme. When measured on a per plant basis, tricarboxylic cycle enzyme levels increased during the exponential growth of both kinds of plants. Only after the bicarbonate ceased to have effect on RS plant morphogenesis was there a decrease in the levels of the tricarboxylic cycle enzymes when measured on a per plant basis. Specific activity measurements indicated some differences in the differential rates of synthesis among the enzymes studied previous to 36 hr. Preliminary studies utilizing short periods of (14)C-bicarbonate fixation in young RS plants indicated that during the first 4 min most of the label was located in aspartic acid. These results are discussed in terms of previous results and particularly Cantino's hypothesis concerning the relationship between bicarbonate induction and tricarboxylic-cycle enzymes in the morphogenesis of B. emersonii.

The incomplete tricarboxylic acid cycle in the blue-green alga Anabaena variabilis.

Summary: The presence of an incomplete tricarboxylic acid cycle in Anabaena varia-bilis and Anacystis nidulam is described. These blue-green algae lack both α-oxoglutarate dehydrogenase and succinyl-CoA synthetase. Succinyl-CoA was formed in extracts of A. variabilis by 3-ketoacyl CoA-transferase using acetoacetyl-CoA as CoA donor. The activities of the other tricarboxylic cycle enzymes were measured in extracts prepared from autotrophic organisms and from those grown in the presence of acetate. No alterations in activity indicative of enzyme repression or de-repression were observed. These results are discussed in relation to possible reasons for autotrophic behaviour.