Liver Mitochondrial Enzyme Research Articles

Effects of phytase enzyme supplementation on growth performance, intestinal morphologyand metabolism in Nile tilapia (Oreochromis niloticus).

Phytase is crucial in enhancing the bioavailability and release of phosphorus and other nutrients bound to phytic acid, making them more bioavailable for animal absorption. This study was carried out to inspect the effect of supplementing low phosphorus (P) diet with di-calcium phosphate (DCP) and liquid phytase enzyme (LP), which contains 1500 FTU/kg, on growth performance, intestinal morphometry, proximate body chemical composition, blood profile, immunity status, liver mitochondrial enzyme activities, the expression responseand economic returns of Nile tilapia (Oreochromis niloticus). Three triplicate groups of fish (initial weight 5.405 ± 0.045 g, N = 90) were fed on three different diets for 90 days. The first was a control diet with zero DCP; the second was a control diet supplemented with 0.71% DCP; the third was a control diet supplemented with 0.03% LP. The groups were designated as CG, DCPand LP, respectively. Results showed that LP induced considerable improvements (p < 0.05) in FBW, body weight gain, weight gain rate, specific growth rate, HIS, viscero-somatic index, spleen-somatic index, feed conversion ratio, blood parametersand the histomorphometry assessment of intestinal villi absorptive capacity, compared with the other groups. Also, whole-body protein and lipid contents pointedly (p < 0.05) increased by LP, compared with the DCP group. A positive response (p < 0.05) to the phytase enzyme was noted in complexes I, IIIand IV of the mitochondrial liver complex enzyme activity. Likewise, the relative gene expression levels of (GHr-1, IGF-1, FASand LPL) were notably (p < 0.05) upregulated by phytase enzyme, associated with DCP and control groups. Further, phytase recorded the highest total return and profit percentage. It can be concluded that Nile tilapia benefits from using phytase enzyme 1500 FTU/kg at 0.03% without adding DCPin terms of good performance and profits.

Impact of nucleotide enriched diets on the production of gilthead seabream, Sparus aurata fingerlings by modulation of liver mitochondrial enzyme activitity, antioxidant status, immune gene expression, and gut microbial ecology

The objective of this study was to evaluate the effect of fish meal replacement with a mixture of plant, and animal protein supplemented with a commercial nucleotide NucleoforceFish™, on growth, feed utilization, whole-body composition, biometric indices; liver mitochondrial enzyme activities, selected gene expression and influence on the gut microbiome of gilthead seabream, Sparus aurata. Six hundred and thirty gilthead seabream with an average initial body weight of 0.358 ± 0.002 g/fish, were used. Six experimental diets were formulated to be isonitrogenous, 45% crude protein (CP) and isocaloric 20 MJ/kg gross energy (GE). The control diet (D1) contained 25% of fish meal (FM25) and had no NucleoforceFish™ added (FM25/0), diets 2-3 contained FM at a level of 25% and supplemented with 250 (FM25/250) and 500 (FM25/500) mg/kg NucleoforceFish™, respectively. Diets 4-6 were a non-fishmeal formulation and had no NucleoforceFish™, (diet 4, FM0/0), 0%FM with 250mg/kg NucleoforceFish™ (diet 5, FM0/250), and 0%FM with 500mg/kg NucleoforceFish™ (diet 6, FM0/500). The results revealed that dietary nucleotides supplemented at 250mg/kg or 500mg/kg enhanced final body weight, weight gain, and specific growth rate of seabream either with dietary level of 25% FM diet (FM25) or non-fishmeal diet (FM0). All mitochondrial liver complex enzyme activities were recorded as having a significant positive response to the nucleotide dietary supplement. Gilthead seabream fingerlings fed FM0/250 diet for 150 days had significantly elevated liver mitochondrial enzyme (Complex III, Complex IV, Complex I and II) activities. In addition, gilthead seabream fed FM0/500 had significantly increased the gene expression of TCR-B and IL1-B, while fish fed FM25/250 increased significantly the gene expression of IGF1, while fish fed FM25/500 increased significantly the gene expression of Hepcidin (Hep) and the iron homeostasis hormone. There was a marked influence on specific bacteria comprising the gut microbiome namely Photobacterium; damselae; Vibrio anguillarum; Lactobacillus plantarum; Lactobacillus Enterococcus faecium subtilis. It can be concluded that FM25/250 and FM25/500 diets are expressing the best result, which explains that FM must be in the feed of gilthead seabream, and addition of NucleoforceFish™ at 250 mg/kg feed may further increase the performance.

Age- and Organ-Specific Differences in Mitochondrial Bioenergetics in Brown Norway Rats.

Mitochondria play a central role in energy homeostasis and act as regulatory checkpoints for downstream metabolic responses and cell senescence processes during an entire life span. Acute or chronic environmental toxicant exposures have shown deleterious organ-specific human health issues at various life stages. Since mitochondria are a prime target for ensuing cellular bioenergetics responses and senescence, it is essential to understand mitochondrial bioenergetic responses in different organs over multiple life stages. Therefore, in the present study, we evaluated mitochondrial bioenergetic parameters in the liver, lung, and heart in four diverse age groups (young: 1 month; adult: 4 months; middle-aged: 12 months; old-aged: 24 month) using male Brown Norway rats as a model of aging (n = 5 sample size/organ/age group) and compared them with our previously published results on brain. Real-time mitochondrial bioenergetic parameters (i.e., State III, State IV, and State V) were measured using the Seahorse Extracellular Flux Analyzer. Additionally, mitochondrial enzyme pyruvate dehydrogenase complex (PDHC), Complex I, Complex II, and Complex IV activities were measured using Synergy HT plate reader. Our results indicated that nearly in all parameters, significant age- and organ-specific interactions were observed. We observed age-specific declines in State III (i.e., ATP synthesis rate) responses in both the heart and lung, where opposite was observed in the liver as age advances. Across the age, the heart has highest enzyme activities than the liver and lung. Interestingly, heart and liver mitochondrial bioenergetic rates and enzyme activities remain higher than the lung, which specifies their higher metabolic capabilities than the lung. Amongst all, bioenergetic rates and enzyme activities in the lung remain lowest suggesting the lung may display higher vulnerability and lower resilience to environmental toxicants during aging than other organs tested here. Overall, these age- and organ-specific findings may facilitate a more contextualized understanding of mitochondrial bioenergetic outcomes when considering the interactions of age-related sensitivities with exposure to chemical stressors from the environment.

Prior reproduction alters how mitochondria respond to an oxidative event.

An animal's pace of life is mediated by the physiological demands and stressors it experiences (e.g. reproduction) and one likely mechanism that underlies these effects is oxidative stress. Reproduction has been shown to increase or reduce oxidative stress under different conditions and to modify mitochondrial performance. We hypothesized that the changes associated with reproduction can alter how animals respond to future oxidative stressors. We tested this theory by comparing the organ-specific mitochondrial response in wild-derived female house mice. Specifically, we examined the effect of an oxidant (X-irradiation) on virgin mice and on mice that had reproduced. We measured liver and skeletal muscle mitochondrial density, respiratory performance, enzyme activity and oxidant production, as well as markers of oxidative damage to tissues. In the liver, prior reproduction prevented a radiation-induced reduction in mitochondrial density and increased mitochondrial respiratory performance. In skeletal muscle, prior reproduction resulted in a radiation-induced decline in mitochondrial density which could reduce the bioenergetic capacity of skeletal muscle mitochondria. Yet, electron transport chain complex I activity in skeletal muscle, which dropped after reproduction, returned to control levels following oxidant exposure. The results of this investigation indicate that prior reproduction alters the response of mitochondria to an oxidative challenge in an organ-specific manner. Such changes could have differential effects on future reproductive performance and risk of death.

Bioactivity, toxicity and detoxification assessment of Dioscorea bulbifera L.: a comprehensive review

Dioscorea bulbifera L. (Chinese: Huangdu, common name: air potato) is a traditional herbal medicine in China, and it is also one of the most widely consumed yam species, especially in West Africa. Studies have verified that D. bulbifera is effective in treating a wide range of diseases, such as pharyngitis, goitre, pyogenic skin infections, orchitis and cancer. However, more and more studies have also reported liver and kidney damage caused by D. bulbifera. To promote understanding of the bioactivity, toxicity and methods for detoxification of this medicinal and edible plant, the present article reviews the most valuable recent reports on its phytochemistry and pharmacological effects. The possible reasons for its toxicity include the toxic effects of diosbulbin B and D on hepatocytes, the inhibition of antioxidant enzymes in liver mitochondria, and inhibition of enzymes that ordinarily metabolize the herb’s components. Synergistic compatibility detoxification may help to reduce toxic effects and improve therapeutic effects. More clinical trials are also required to fully achieve its therapeutic potential.

Investigation of the effects of purification and characterization of turkey (Meleagris gallopavo) liver mitochondrial thioredoxin reductase enzyme and some metal ions on enzyme activity

The thioredoxin system, found in all living creatures, consists of thioredoxin protein (Trx), thioredoxin reductase enzyme (TrxR), and NADPH. In this study, turkey liver mitochondrial TrxR enzyme with 3.07 EU $\times $ mg$^{-1}$ specific activity was purified 990-fold in a yield of 2.05{\%} using 2',5'-ADP Sepharose 4B affinity chromatography. The purity of the enzyme was measured and the molecular weight of its subunits was determined to be 45.5 kDa by SDS-PAGE. The molecular mass of the enzyme's natural state was found to be 88 kDa using Sephadex-G 150 gel filtration chromatography. In addition, characteristic and kinetic properties of the enzyme were determined. Then the inhibitory effects of some heavy metal ions (Ag$^{+}$, Fe$^{3+}$, Cd$^{2+}$, Cu$^{2+}$, Zn$^{2+}$, Pb$^{2+}$, Ni$^{2+}$, and Co$^{2+})$ on the activity of TrxR enzyme were examined under in vitro conditions. IC$_{50}$ values were found with the heavy metal concentration with which 50{\%} of the activity of the TrxR enzyme was inhibited. Finally, K$_{i}$ values for these substances were calculated from the Lineweaver--Burk plots. It was determined that Ag$^{+}$, Fe$^{3+}$, Cd$^{2+}$, Cu$^{2+}$, and Zn$^{2+}$ ions inhibited TrxR enzyme, Pb$^{2+}$ ion increased enzyme activity, and Ni$^{2+}$ and Co$^{2+}$ ions had no effect on enzyme activity.

Molecular cloning of rat acss3 and characterization of mammalian propionyl-CoA synthetase in the liver mitochondrial matrix.

Among the three acyl-CoA synthetase short-chain family members (ACSS), ACSS3 is poorly characterized. To characterize ACSS3, we performed molecular cloning and protein expression of rat acss3 and determined its intracellular localization, tissue distribution, and substrate specificity. Transient expression of rat ACSS3 in HeLa cells resulted in a 10-fold increase of acetyl-CoA synthetase activity compared with that in control cells. The acss3 transcripts are expressed in a wide range of tissues, with the highest levels observed in liver tissue followed by kidney tissue. Subcellular fractionation using liver tissue showed that ACSS3 is localized into the mitochondrial matrix. Among the short-chain fatty acids examined, recombinant ACSS3, purified from Escherichia coli cells transformed with the plasmid containing rat acss3, preferentially utilized propionate with a KM value of 0.19 mM. Knockdown of acss3 in HepG2 cells resulted in a significant decrease of ACSS3 expression level and propionyl-CoA synthetase activity in cell lysates. Levels of ACSS3 in the liver and the activity of propionyl-CoA synthetase in the mitochondria were significantly increased by fasting. These results suggested that ACSS3 is a liver mitochondrial matrix enzyme with high affinity to propionic acid, and its expression level is upregulated under ketogenic conditions.

Alpha-mangostin from mangosteen (Garcinia mangostana Linn.) pericarp extract reduces high fat-diet induced hepatic steatosis in rats by regulating mitochondria function and apoptosis.

BackgroundNon-alcoholic fatty liver disease (NAFLD) is caused by multiple factors including hepatic oxidative stress, lipotoxicity, and mitochondrial dysfunction. Obesity is among the risk factors for NAFLD alongside type 2 diabetes mellitus and hyperlipidemia. α- mangostin (α-MG) extracts from the pericarps of mangosteen (Garcinia mangostana Linn.) may regulate high fat diet-induced hepatic steatosis; however the underlying mechanisms remain unknown. The aim of this study was to investigate the regulatory effect of α-MG on high fat diet-induced hepatic steatosis and the underlying mechanisms related to mitochondrial functionality and apoptosis in vivo and in vitro.MethodsSprague Dawley (SD) rats were fed on either AIM 93-M control diet, a high-fat diet (HFD), or high-fat diet supplemented with 25 mg/day mangosteen pericarp extract (MGE) for 11 weeks. Thereafter, the following were determined: body weight change, plasma free fatty acids, liver triglyceride content, antioxidant enzymes (superoxide dismutase, SOD; glutathione, GSH; glutathione peroxidase, GPx; glutathione reductase GRd; catalase, CAT) and mitochondrial complex enzyme activities. In the in vitro study, primary liver cells were treated with 1 mM free fatty acid (FFA) (palmitate: oleate acid = 2:0.25) to induce steatosis. Thereafter, the effects of α-MG (10 μM, 20 μM, 30 μM) on total and mitochondria ROS (tROS, mitoROS), mitochondria bioenergetic functions, and mitochondrial pathway of apoptosis were examined in the FFA-treated primary liver cells.ResultsThe MGE group showed significantly decreased plasma free fatty acids and hepatic triglycerides (TG) and thiorbarbituric acid reactive substances (TBARS) levels; increased activities of antioxidant enzymes (SOD, GSH, GPx, GRd, CAT); and enhanced NADH-cytochrome c reductase (NCCR) and succinate-cytochrome c reductase (SCCR) activities in the liver tissue compared with HFD group. In the in vitro study, α-MG significantly increased mitochondrial membrane potential, enhanced cellular oxygen consumption rate (OCR), decreased tROS (total ROS) and mitoROS (mitochondrial ROS) levels ; reduced Ca2+ and cytochrome c (cyt c) release from mitochondria, and reduced caspases 9 and 3 activities compared with control group.ConclusionThese findings demonstrate α-MG attenuated hepatic steatosis in high fat-diet fed rats potentially through enhanced cellular antioxidant capacity and improved mitochondrial functions as well as suppressed apoptosis of hepatocytes. The findings of study represent a novel nutritional approach on the use of α-MG in the prevention and management of NAFLD.

Folate deficiency affects histone methylation

Formaldehyde is extremely toxic reacting with proteins to crosslinks peptide chains. Formaldehyde is a metabolic product in many enzymatic reactions and the question of how these enzymes are protected from the formaldehyde that is generated has largely remained unanswered. Early experiments from our laboratory showed that two liver mitochondrial enzymes, dimethylglycine dehydrogenase (DMGDH) and sarcosine dehydrogenase (SDH) catalyze oxidative demethylation reactions (sarcosine is a common name for monomethylglycine). The enzymatic products of these enzymes were the demethylated substrates and formaldehyde, produced from the removed methyl group. Both DMGDH and SDH contain FAD and both have tightly bound tetrahydrofolate (THF), a folate coenzyme. THF binds reversibly with formaldehyde to form 5,10-methylene-THF. At that time we showed that purified DMGDH, with tightly bound THF, reacted with formaldehyde generated during the reaction to form 5,10-methylene-THF. This effectively scavenged the formaldehyde to protect the enzyme.Recently, post-translational modifications on histone tails have been shown to be responsible for epigenetic regulation of gene expression. One of these modifications is methylation of lysine residues. The first enzyme discovered to accomplish demethylation of these modified histones was histone lysine demethylase (LSD1). LSD1 specifically removes methyl groups from di- and mono-methylated lysines at position 4 of histone 3. This enzyme contained tightly bound FAD and the products of the reaction were the demethylated lysine residue and formaldehyde. The mechanism of LSD1 demethylation is analogous to the mechanism previously postulated for DMGDH, i.e. oxidation of the N-methyl bond to the methylene imine followed by hydrolysis to generate formaldehyde. This suggested that THF might also be involved in the LSD1 reaction to scavenge the formaldehyde produced.Our hypotheses are that THF is bound to native LSD1 by analogy to DMGDH and SDH and that the bound THF serves to protect the FAD class of histone demethylases from the destructive effects of formaldehyde generation by formation of 5,10-methylene-THF. We present pilot data showing that decreased folate in livers as a result of dietary folate deficiency is associated with increased levels of methylated lysine 4 of histone 3. This can be a result of decreased LSD1 activity resulting from the decreased folate available to scavenge the formaldehyde produced at the active site caused by the folate deficiency. Because LSD1 can regulate gene expression this suggests that folate may play a more important role than simply serving as a carrier of one-carbon units and be a factor in other diseases associated with low folate.

Is Liver Enzyme Release Really Associated with Cell Necrosis Induced by Oxidant Stress?

Hepatic diseases are a major concern worldwide. Increased specific plasma enzyme activities are considered diagnostic features for liver diseases, since enzymes are released into the blood compartment following the deterioration of the organ. Release of liver mitochondrial enzymes is considered strong evidence for hepatic necrosis, which is associated with an increased production of ROS, often leading to greater hepatic lipid peroxidation. Lipotoxic mediators and intracellular signals activated Kupffer cells, which provides evidence strongly suggesting the participation of oxidant stress in acute liver damage, inducing the progression of liver injury to chronic liver damage. Elevated transaminase activities are considered as an index marker of hepatotoxicity, linked to oxidant stress. However, a drastic increase of serum activities of liver enzyme markers ought not necessarily to reflect liver cell death. In fact, increased serum levels of cytoplasmic enzymes have readily been observed after partial hepatectomy (PH) in the regenerating liver of rats. In this regard, we are now showing that in vitro modifications of the oxidant status affect differentially the release of liver enzymes, indicating that this release is a strictly controlled event and not directly related to the onset of oxidant stress of the liver.

Unacylated ghrelin does not alter mitochondrial function, redox state and triglyceride content in rat liver in vivo

Summary Changes in liver mitochondrial function with more oxidized redox state and enhanced inflammation may contribute to the onset of obesity- and insulin resistance-associated hepatic complications, including non-alcoholic fatty liver disease and steato-hepatitis. Unacylated ghrelin (UnAG) is a gastric hormone reported to be associated with lower oxidative stress in different cell types, but its potential effects on liver mitochondrial function, redox state and inflammation in vivo remains undetermined. We investigated the impact of chronic UnAG overexpression (Tg Myh6/Ghrl) leading to systemic upregulation of circulating hormone on mitochondrial ATP production, redox state (oxidized-to-total glutathione) and inflammation markers in lean mice. Compared to wild-type animals (wt), Tg Myh6/Ghrl had superimposable liver weight, triglyceride content and plasma lipid profile. Liver mitochondrial enzyme activities and ATP production as well as oxidized-to-total glutathione were also similar in the two groups. In addition, no differences were observed in tissue inflammation marker TNF-alpha between wild-type and Tg Myh6/Ghrl animals. Thus, chronic systemic UnAG upregulation does not alter liver triglyceride content, mitochondrial function, redox state and inflammation markers in lean mice. These findings do not support a major role of UnAG as a physiological modulator of in vivo liver oxidative-lipid metabolism and inflammation.

Effects of Acute Malathion Exposure on Liver Mitochondrial Respiratory Chain Enzymes in Rats

Malathion is an organophosphorus insecticide widely used to control pests. Previous toxicological studies attributed to the ability of Malathion to effect on cellular metabolism. The main objective of present study was to find out what actually happens on the activities of key enzymes of the mitochondrial respiratory chain after acute exposure to Malathion. To design an acute toxicity study, all male Wistar rats were given Malathion at a single oral dose of 400 mg/kg. Activities of plasma acetylcholinesterase (AchE) and liver mitochondrial respiratory chain enzymes were measured after 24 h. Inhibition of AchE was accompanied by depressed nicotinamide adenine dinucleotide-ubiquinone oxidoreductase, and cytochrome C oxidase enzymes activity in the mitochondrial respiratory chain. On the other hand, Malathion did not significantly change succinate-ubiquinone oxidoreductase, and adenosine triphosphate synthase enzymes activity. It can be concluded that Malathion by extra energy demand in cholinergic storm phase reduced glycogen content of liver to supply glucose. On the other hand, inhibition of the mitochondrial respiratory chain key enzymes activity shift cellular glucose metabolism from mitochondrial respiration to anaerobic glycolysis pathway to provide demanded energy.

Effect of Solanum surattense on mitochondrial enzymes in diabetic rats and in vitro glucose uptake activity in L6 myotubes.

Background:S. surattense is widely used in Siddha medicine for various ailments.Objective:The aim was to evaluate the impact of alcoholic leaf-extract of S. surattense on mitochondrial enzymes in streptozotocin (STZ) induced diabetic rats and to study the in vitro muscle glucose uptake activity on L6 myotubes.Materials and Methods:The male albino Wistar rats were randomly divided into five groups of six animals each. Diabetes was induced by intraperitoneal injection of STZ (40 mg/kg body weight). After being confirmed the diabetic rats were treated with alcoholic leaf-extract of S. surattense (100 mg/kg body weight) for 45 days. The biochemical estimations (liver mitochondrial enzymes, antioxidants, thiobarbituric acid reactive substances [TBARS]) and histopathological studies were performed. Further, the in vitro muscle glucose uptake activity in L6 myotubes and messenger RNA (mRNA) expression of glucose transporter-4 (GLUT-4) was performed.Results:In diabetic rats, the activities of liver mitochondrial enzymes were found to be significantly lowered. The mitochondrial TBARS level increased, whereas the activities/level of enzymatic and non-enzymatic antioxidants decreased in diabetic rats. Administration of S. surattense to diabetic rats significantly reversed the above parameters toward normalcy. Furthermore in diabetic rats, the histopathological studies showed growth of adipose tissue and shrinkage of islets in the pancreas, liver showed fatty change with mild inflammation of portal triad, and kidney showed messangial capillary proliferation of glomeruli and fatty infiltration of tubules. Treatment with S. surattense brought back these changes to near normalcy. The extract was analyzed for in vitro muscle glucose uptake activity in L6 myotubes and mRNA expression of GLUT-4 by semi-quantitative reverse transcriptase-polymerase chain reaction. One nano gram per millilitre of S. surattense leaf-extract gave 115% glucose uptake on L6 myotubes. It also showed elevated levels of GLUT-4 mRNA transcripts, when compared with control cells.Conclusion:These studies strongly support the anti-diabetic nature of S. surattense.

The influence of dietary lipid composition on liver mitochondria from mice following 1 month of calorie restriction

To investigate the role mitochondrial membrane lipids play in the actions of CR (calorie restriction), C57BL/6 mice were assigned to four groups (control and three 40% CR groups) and the CR groups were fed diets containing soya bean oil (also in the control diet), fish oil or lard. The fatty acid composition of the major mitochondrial phospholipid classes, proton leak and H2O2 production were measured in liver mitochondria following 1 month of CR. The results indicate that mitochondrial phospholipid fatty acids reflect the PUFA (polyunsaturated fatty acid) profile of the dietary lipid sources. CR significantly decreased the capacity of ROS (reactive oxygen species) production by Complex III but did not markedly alter proton leak and ETC (electron transport chain) enzyme activities. Within the CR regimens, the CR-fish group had decreased ROS production by both Complexes I and III, and increased proton leak when compared with the other CR groups. The CR-lard group showed the lowest proton leak compared with the other CR groups. The ETC enzyme activity measurements in the CR regimens showed that Complex I activity was decreased in both the CR-fish and CR-lard groups. Moreover, the CR-fish group also had lower Complex II activity compared with the other CR groups. These results indicate that dietary lipid composition does influence liver mitochondrial phospholipid composition, ROS production, proton leak and ETC enzyme activities in CR animals.

Liver Mitochondrial Enzyme System In The Dynamics Of Experimental Leukemia

In rats with leukemia activity of liver mitochondrial enzymes are decreased at 6 months. Later, we noted compensatory activation of complexes II and III of the respiratory chain and transition to decompensation stage by final deadline. The activity of complex IV of the respiratory chain remains high. These changes result from increased lipid peroxidation and decreased activity of antioxidant protection enzymes.

Biotechnological Approach to Evaluate the Immunomodulatory Activity of Ethanolic Extract of Tinospora cordifolia Stem (Mango Plant Climber).

The present study was designed to investigate the immunomodulatory activity of the ethanolic extract of Tinospora cordifolia (Family: Menispermaceae) stem (climbing shrub, mango plant) at cellular level. For antioxidant study, the liver mitochondria were separated and the concentration of enzymes like lipid peroxidation (LPO), reduced glutathione (GSH), catalase (CAT) and superoxide Dismutase (SOD) were estimated; melatonin secretion characterization was carried out through SDS-PAGE. The spleen lymphocyte proliferation assay was performed through measuring its optical density at 570 nm using Elisa Reader. The cytokines viz. IL-2, IL-10 and TNF-α expression in spleen cells were determined through Real Time PCR. Tinospora cordifolia (Tc) ethanolic extract (100 mg/Kg/p.o.) increased the level of liver mitochondrial enzymes like GSH, CAT and SOD but decreased the level of LPO in liver as compared to the vehicle, SRBC and cyclophosphamide-treated groups. The secretion of melatonin via pineal gland was enhanced with Tc treatment. The extract also increased the spleen lymphocyte proliferation. In RT-PCR analysis, the expression of cytokines viz. IL-2, IL-10 and TNF-α was more in Tc-treated animals than vehicle and cyclophosphamide treatment. Hence, the study confirms the immunomodulatory activity of Tc stem through altering the concentration of antioxidant enzymes, increasing T and B cells and antibody which play an important role in immunity, enhancing the concentration of melatonin in pineal gland and increasing the level of cytokines like IL-2, IL-10 and TNF-α which plays an important role in immunity.

Immunomodulatory Activity of Alcohol Extract of &lt;i&gt;Terminalia chebula&lt;/i&gt; Retz Combretaceae

Purpose: To investigate the immunomodulatory activity of the alcohol extract of Terminalia chebula Retz (Combretaceae) dried ripe fruits at the cellular level. Methods: For antioxidant study, the liver mitochondria were separated and used for the estimation of enzymes catalase (CAT) and superoxide dismutase (SOD) - as well as lipid peroxidation (LPO) and reduced glutathione (GSH); Melatonin secretion was characterized using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) while spleen lymphocyte proliferation assay was performed by measuring optical density at 570 nm using ELISA reader. The cytokines, namely, IL-2, IL-10 and TNF-α expression in spleen cells, were determined by real time polymerase chain reaction (RTPCR) Results: Terminalia chebula extract (100 mg/kg/p.o.) increased the level of liver mitochondrial enzymes CAT and SO) as well as GSH but decreased the level of LPO in the liver when compared to the vehicle, sheep red blood cells (SRBC) and cyclophosphamide-treated groups. Secretion of melatonin by pineal gland was enhanced by T. chebula treatment. The extract also increased spleen lymphocyte proliferation. Based on RT-PCR analysis, the expression of cytokines, viz, IL-2, IL-10 and TNF-α, was more in T. chebula -treated than in vehicle- and cyclophosphamide- treated groups. Conclusion: This study confirms the immunomodulatory activity of ripe T. chebula fruits as evidenced by increase in the concentration of antioxidant enzymes, GSH, T and B cells, the proliferation of which play important roles in immunity. This phenomenon also enhances the concentration of melatonin in pineal gland as well as the levels of cytokines, such as IL-2,IL-10 and TNF-α, which play important roles in immunity. Keywords: Terminalia chebula , Antioxidants, SDS-PAGE, Lymphocytes proliferation, Real-time PCR, Immunity

Effect of beer consumption on levels of complex I and complex IV liver and heart mitochondrial enzymes and coenzymes Q9 and Q10 in adriamycin-treated rats

There is increasing evidence indicating that the dietary intake of food with high antioxidant capacity may protect mitochondria from damage and exert positive effects on different pathogenic processes. The present study was designed to evaluate the possible protective effect of alcohol-free beer intake on chain components dysfunction of liver and heart mitochondria, and to compare with the effect of alcohol beer intake. The study was carried out in rat heart and liver mitochondria by inducing with Adriamycin the dysfunction of the respiratory chain. Heart and liver mitochondria were isolated from rats and subjected to oxidative stress with two doses of Adriamycin (5 mg/Kg) 7 days from the beginning of consumption of both alcohol-free and alcohol beer during 31 days. Complexes I and IV and the levels of coenzymes Q(9) and Q(10) were evaluated and compared with a control group. Liver and heart mitochondria isolated from rats treated with Adryamicin showed a decrease in levels of complex I and complex IV enzymatic activity and in levels of coenzymes Q(9) and Q(10). Beer intake for itself does not affect any of the studied parameters. Therefore, the consumption of both alcohol and alcohol-free beer by rats treated with Adriamycin prevents the inhibition of enzymatic activities of complexes I and IV and the oxidation of coenzymes Q(9) and Q(10) in rat heart and liver mitochondria. These results indicate that alcohol-free beer prevents adriamycin-induced damage to mitochondrial chain components and, therefore, helps to prevent mitochondrial dysfunction.

De Novo Mutations in POLG Presenting with Acute Liver Failure or Encephalopathy

De Novo Mutations in <i>POLG</i> Presenting with Acute Liver Failure or Encephalopathy

Human liver mitochondrial cytochrome P450 2D6 – individual variations and implications in drug metabolism

Constitutively expressed human cytochrome P450 2D6 (CYP2D6; EC 1.14.14.1) is responsible for the metabolism of approximately 25% of drugs in common clinical use. It is widely accepted that CYP2D6 is localized in the endoplasmic reticulum of cells; however, we have identified this enzyme in the mitochondria of human liver samples and found that extensive inter-individual variability exists with respect to the level of the mitochondrial enzyme. Metabolic assays using 7-methoxy-4-aminomethylcoumarin as a substrate show that the human liver mitochondrial enzyme is capable of oxidizing this substrate and that the catalytic activity is supported by mitochondrial electron transfer proteins. In the present study, we show that CYP2D6 contains an N-terminal chimeric signal that mediates its bimodal targeting to the endoplasmic reticulum and mitochondria. In vitro mitochondrial import studies using both N-terminal deletions and point mutations suggest that the mitochondrial targeting signal is localized between residues 23-33 and that the positively-charged residues at positions 24, 25, 26, 28 and 32 are required for mitochondrial targeting. The importance of the positively-charged residues was confirmed by transient transfection of a CYP2D6 mitochondrial targeting signal mutant in COS-7 cells. Both the mitochondria and the microsomes from a CYP2D6 stable expression cell line contain the enzyme and both fractions exhibit bufuralol 1'-hydroxylation activity, which is completely inhibited by CYP2D6 inhibitory antibody. Overall, these results suggest that the targeting of CYP2D6 to mitochondria could be an important physiological process that has significance in xenobiotic metabolism.