Decrease In ATP Content Research Articles

Choline dehydrogenase polymorphism rs12676 is a functional variation associated with changes in human sperm cell function

Approximately 15% of couples are affected by infertility and up to half of these cases arise from male factor infertility. Unidentified genetic aberrations such as chromosomal deletions, translocations and single nucleotide polymorphisms (SNPs) may be the underlying cause of many cases of idiopathic male infertility. Choline dehydrogenase (CHDH), a mitochondrial enzyme, catalyzes the conversion of choline to betaine, a methyl group donor and organic osmolyte. Deletion of the Chdh gene in mice results in decreased male fertility due to diminished sperm motility; sperm from Chdh−/− males have decreased ATP concentrations likely stemming from abnormal sperm mitochondrial morphology and function in these cells. SNPs have been identified in the human CHDH gene that may result in altered CHDH enzymatic activity. rs12676 (G233T), a non‐synonymous SNP located in the CHDH coding region, is associated with increased susceptibility to dietary choline deficiency and risk of breast cancer. We now report evidence that this SNP is also associated with altered sperm motility patterns and dysmorphic mitochondrial structure in sperm. Sperm produced by men who are GT or TT for rs12676 have 40% and 73% lower ATP concentrations, respectively, in their sperm. rs12676 is associated with decreased CHDH protein in human hepatocytes. We hypothesize that nutritional intervention involving dietary betaine supplementation may improve the function of sperm produced by men with the rs12676 SNP. This research was funded by NIH grants DK055865 and DK056350.Grant Funding Source: National Institutes of Health

Silencing of FAD synthase gene in Caenorhabditis elegans upsets protein homeostasis and impacts on complex behavioral patterns

BackgroundFAD synthase is a ubiquitous enzyme that catalyses the last step of FAD biosynthesis, allowing for the biogenesis of several flavoproteins. In humans different isoforms are generated by alternative splicing, isoform 1 being localized in mitochondria. Homology searching in Caenorabditis elegans leads to the identification of two human FAD synthase homologues, coded by the single copy gene R53.1. MethodsThe C. elegans R53.1 gene was silenced by feeding. The expression level of transcripts was established by semi-quantitative RT-PCR. Overall protein composition was evaluated by two-dimensional electrophoresis. Enzymatic activities were measured by spectrophotometry and oxygen consumption by polarography on isolated mitochondria. ResultsFrom R53.1 two transcripts are generated by trans-splicing. Reducing by 50% the transcription efficiency of R53.1 by RNAi results in a 50% reduction in total flavin with decrease in ATP content and increase in ROS level. Significant phenotypical changes are noticed in knock-down nematodes. Among them, a significant impairment in locomotion behaviour possibly due to altered cholinergic transmission. At biochemical level, impairment of flavoenzyme activities and of some KCN-insensitive oxygen-consuming enzymes is detected. At proteomic level, at least 15 abundant proteins are affected by R53.1 gene silencing, among which superoxide dismutases. Conclusion and General SignificanceFor the first time we addressed the existence of different isoforms of FAD-metabolizing enzymes in nematodes. A correlation between FAD synthase silencing and flavoenzyme derangement, energy shortage and redox balance impairment is apparent. In this aspect R53.1-interfered nematodes could provide an animal model system for studying human pathologies with alteration in flavin homeostasis/flavoenzyme biogenesis.

Tanshinone IIA Stimulates Erythrocyte Phosphatidylserine Exposure

Tanshinone IIA, an antimicrobial, antioxidant, antianaphylactic, antifibrotic, vasodilating, antiatherosclerotic, organo-protective and antineoplastic component from the rhizome of Salvia miltiorrhiza, is known to trigger apoptosis of tumor cells. Tanshinone IIA is effective in part through mitochondrial depolarization and altered gene expression. Erythrocytes lack mitochondria and nuclei but may undergo eryptosis, an apoptosis-like suicidal cell death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Eryptosis is triggered by increase of cytosolic Ca²⁺ activity, ATP depletion and ceramide formation. The present study explored, whether tanshinone IIA elicits eryptosis. Cytosolic Ca²⁺-concentration was determined from Fluo3-fluorescence, cell volume from forward scatter, phosphatidylserine exposure from binding of fluorescent annexin V, hemolysis from hemoglobin concentration in the supernatant, ATP concentration utilizing luciferin–luciferase and ceramide formation utilizing fluorescent anticeramide antibodies. Clearance of circulating erythrocytes was estimated by CFSE-labeling. A 48 h exposure to tanshinone IIA (≥10 µM) significantly increased cytosolic Ca²⁺-concentration, decreased ATP concentration (25 µM), increased lactate concentration (25 µM), increased ceramide formation (25 µM), decreased forward scatter, increased annexin-V-binding and increased (albeit to a much smaller extent) hemolysis. The effect of 25 µM tanshinone IIA on annexin-V binding was partially reversed in the nominal absence of Ca²⁺. Labelled tanshinone IIA-treated erythrocytes were more rapidly cleared from the circulating blood in comparison to untreated erythrocytes. The present observations reveal a completely novel effect of tanshinone IIA, i.e. triggering of Ca²⁺ entry, ATP depletion and ceramide formation in erythrocytes, events eventually leading to eryptosis with cell shrinkage and cell membrane scrambling.

Resveratrol plus ethanol counteract the ethanol-induced impairment of energy metabolism: 31P NMR study of ATP and sn-glycerol-3-phosphate on isolated and perfused rat liver

The effects of trans-resveratrol (RSV) combined with ethanol (EtOH) were evaluated by 31P NMR on total ATP and sn-glycerol-3-phosphate (sn-G3P) contents measured in real time in isolated and perfused whole liver of the rat. Mitochondrial ATP turnover was assessed by using specific inhibitors of glycolytic and mitochondrial ATP supply (iodacetate and KCN, respectively). In RSV alone, the slight decrease in ATP content (−14±5% of the initial content), sn-G3P content and ATP turnover were similar to those in the Krebs-Henseleit buffer control. Compared to control, EtOH alone (14 or 70mmol/L) induced a decrease in ATP content (−24.95±2.95% of initial content, p<0.05) and an increase in sn-G3P (+158±22%), whereas ATP turnover tended to be increased. RSV (20μmol/L) combined with EtOH, (i) maintained ATP content near 100%, (ii) induced a 1.6-fold increase in mitochondrial ATP turnover (p=0.049 and p=0.004 vs EtOH 14 and 70mmol/L alone, respectively) and (iii) led to an increase in sn-G3P (+49±9% and +81±6% for 14 and 70mmol/L EtOH, respectively). These improvements were obtained only when glycolysis was efficient at the time of addition of EtOH+RSV. Glycolysis inhibition by iodacetate (IAA) evidenced an almost 21% contribution of this pathway to ATP content. RSV alone or RSV+EtOH prevented the ATP decrease induced by IAA addition (p<0.05 vs control). This is the first demonstration of the combined effects of RSV and EtOH on liver energy metabolism. RSV increased (i) the flux of substrates through ATP producing pathways (glycolysis and phosphorylative oxidation) probably via the activation of AMPkinase, and (ii) maintained the glycolysis deviation to sn-G3P linked to NADH+H+ re-oxidation occurring during EtOH detoxication, thus reducing the energy cost due to the latter.

Apigenin-Induced Suicidal Erythrocyte Death

Apigenin, a flavone in fruits and vegetables, stimulates apoptosis and thus counteracts cancerogenesis. Erythrocytes may similarly undergo suicidal cell death or eryptosis, characterized by cell shrinkage and phosphatidylserine exposure at the cell surface. Triggers of eryptosis include increase of cytosolic Ca(2+) activity ([Ca(2+)](i)), ceramide formation and ATP depletion. The present study explored the effect of apigenin on eryptosis. [Ca(2+)](i) was estimated from Fluo3-fluorescence, cell volume from forward scatter, phosphatidylserine exposure from annexin V binding, hemolysis from hemoglobin release, ceramide utilizing antibodies, and cytosolic ATP with luciferin-luciferase. A 48 h exposure to apigenin significantly increased [Ca(2+)](i) (≥ 1 μM), increased ceramide formation (15 μM), decreased ATP concentration (15 μM), decreased forward scatter (≥ 1 μM), and increased annexin V binding (≥ 5 μM) but did not significantly modify hemolysis. The effect of 15 μM apigenin on annexin V binding was blunted by Ca(2+) removal. The present observations reveal novel effects of apigenin, i.e. stimulation of Ca(2+) entry, ceramide formation and ATP depletion in erythrocytes with subsequent triggering of suicidal erythrocyte death, paralleled by cell shrinkage and phosphatidylserine exposure.

Hexavalent chromium-induced erythrocyte membrane phospholipid asymmetry

Hexavalent (VI) chromium is a global contaminant with cytotoxic activity. Chromium (VI) induces oxidative stress, inflammation, cell proliferation, malignant transformation and may trigger carcinogenesis and at the same time apoptosis. The toxic effects of chromium (VI) at least partially result from mitochondrial injury and DNA damage. Erythrocytes lack mitochondria and nuclei but may experience an apoptosis-like suicidal cell death, i.e. eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Eryptosis may result from increase of cytosolic Ca(2+) activity, ATP depletion and/or ceramide formation. The present study explored, whether chromium (VI) triggers eryptosis. Fluo-3-fluorescence was employed to determine cytosolic Ca(2+)-concentration, forward scatter to estimate cell volume, binding of fluorescent annexin V to detect phosphatidylserine exposure, hemoglobin concentration in the supernatant to quantify hemolysis, luciferin-luciferase to determine cytosolic ATP concentration and fluorescent anti-ceramide antibodies to uncover ceramide formation. A 48 h exposure to chromium (VI) (≥10 μM) significantly increased cytosolic Ca(2+)-concentration, decreased ATP concentration (20 μM), decreased forward scatter, increased annexin V-binding and increased (albeit to a much smaller extent) hemolysis. Chromium (VI) did not significantly modify ceramide formation. The effect of 20 μM chromium (VI) on annexin V binding was partially reversed in the nominal absence of Ca(2+). The present observations disclose a novel effect of chromium (VI), i.e. Ca(2+) entry and cytosolic ATP depletion in erythrocytes, effects resulting in eryptosis with cell shrinkage and cell membrane scrambling.

Magnesium deficiency suppresses cell cycle progression mediated by increase in transcriptional activity of p21Cip1 and p27Kip1 in renal epithelial NRK-52E cells

Lack of magnesium suppresses cell growth, but the molecular mechanism is not examined in detail. We examined the effect of extracellular magnesium deficiency on cell cycle progression and the expression of cell cycle regulators in renal epithelial NRK-52E cells. In synchronized cells caused by serum-starved method, over 80% cells were distributed in G1 phase. Cell proliferation and percentage of the cells in S phase in the presence of MgCl(2) were higher than those in the absence of MgCl(2) , suggesting that magnesium is involved in the cell cycle progression from G1 to S phase. After serum addition, the expression levels of p21(Cip1) and p27(Kip1) in the absence of MgCl(2) were higher than those in the presence of MgCl(2) . The exogenous expression of p21(Cip1) or p27(Kip1) increased the percentage in G1 phase, whereas it decreased that in S phase. The mRNA levels and promoter activities of p21(Cip1) and p27(Kip1) in the absence of MgCl(2) were higher than those in the presence of MgCl(2) . The phosphorylated p53 (p-p53) level was decreased by MgCl(2) addition. Pifithrin-α, a p53 inhibitor, decreased the p-p53, p21(Cip1) and p27(Kip1) levels, and the percentage in G1 phase in the absence of MgCl(2) . Rotenone, a mitochondrial respiratory inhibitor, decreased ATP content and increased the p-p53 level in the presence of MgCl(2) . Together, lack of magnesium may increase p21(Cip1) and p27(Kip1) levels mediated by the decrease in ATP content and the activation of p53, resulting in the suppression of cell cycle progression from G1 to S phase in NRK-52E cells.

L-Carnitine rescues ketamine-induced attenuated heart rate and MAPK (ERK) activity in zebrafish embryos

Ketamine, an antagonist of the N-methyl-d-aspartate (NMDA)-type glutamate receptors, is a pediatric anesthetic. Ketamine has been shown to be neurotoxic and cardiotoxic in mammals. Here, we show that after 2h of exposure, 5mM ketamine significantly reduced heart rate in 26h old zebrafish embryos. In 52h old embryos, 1mM ketamine was effective after 2h and 0.5mM ketamine at 20h of exposure. Ketamine also induced significant reductions in activated MAPK (ERK) levels. Treatment of the embryos with the ERK inhibitor, PD 98059, also significantly reduced heart rate whereas the p38/SAPK inhibitor, SB203580, was ineffective. Ketamine is known to inhibit lipolysis and a decrease of ATP content in the heart. Co-treatment with l-carnitine that enhances fatty acid metabolism effectively rescued ketamine-induced attenuated heart rate and ERK activity. These findings demonstrate that l-carnitine counteracts ketamine's negative effects on heart rate and ERK activity in zebrafish embryos.

Stress-Resistant Neural Stem Cells Positively Influence Regional Energy Metabolism After Spinal Cord Injury in Mice

The importance of stem cells to ameliorate the devastating consequences of traumatic injuries in the adult mammalian central nervous system calls for improvements in the capacity of these cells to cope, in particular, with the host response to the injury. We have previously shown, however, that in the acutely traumatized spinal cord local energy metabolism led to decreased ATP levels after neural stem cell (NSC) transplantation. As this might counteract NSC-mediated regenerative processes, we investigated if NSC selected for increased oxidative stress resistance are better suited to preserve local energy content. For this purpose, we exposed wild-type (WT) NSC to hydrogen peroxide prior to transplantation. We demonstrate here that transplantation of WT-NSC into a complete spinal cord compression injury model even lowers the ATP content beyond the level detected in spinal cord injury-control animals. Compared to WT-NSC, stress-resistant (SR) NSC did not lead to a further decrease in ATP content. These differences between WT- and SR-NSC were observed 4h after the lesion with subsequent transplantation. At 24h after lesioning, these differences were no more as obvious. Thus, in contrast to native NSC, transplantation of NSC selected for oxidative stress resistance can positively influence local energy metabolism in the first hours after spinal cord compression. The functional relevance of this observation has to be tested in further experiments.

Altering trehalose-6-phosphate content in transgenic potato tubers affects tuber growth and alters responsiveness to hormones during sprouting.

Trehalose-6-phosphate (T6P) is a signaling metabolite that regulates carbon metabolism, developmental processes, and growth in plants. In Arabidopsis (Arabidopsis thaliana), T6P signaling is, at least in part, mediated through inhibition of the SNF1-related protein kinase SnRK1. To investigate the role of T6P signaling in a heterotrophic, starch-accumulating storage organ, transgenic potato (Solanum tuberosum) plants with altered T6P levels specifically in their tubers were generated. Transgenic lines with elevated T6P levels (B33-TPS, expressing Escherichia coli osmoregulatory trehalose synthesis A [OtsA], which encodes a T6P synthase) displayed reduced starch content, decreased ATP contents, and increased respiration rate diagnostic for high metabolic activity. On the other hand, lines with significantly reduced T6P (B33-TPP, expressing E. coli OtsB, which encodes a T6P phosphatase) showed accumulation of soluble carbohydrates, hexose phosphates, and ATP, no change in starch when calculated on a fresh weight basis, and a strongly reduced tuber yield. [¹⁴C]glucose feeding to transgenic tubers indicated that carbon partitioning between starch and soluble carbohydrates was not altered. Transcriptional profiling of B33-TPP tubers revealed that target genes of SnRK1 were strongly up-regulated and that T6P inhibited potato tuber SnRK1 activity in vitro. Among the SnRK1 target genes in B33-TPP tubers, those involved in the promotion of cell proliferation and growth were down-regulated, while an inhibitor of cell cycle progression was up-regulated. T6P-accumulating tubers were strongly delayed in sprouting, while those with reduced T6P sprouted earlier than the wild type. Early sprouting of B33-TPP tubers correlated with a reduced abscisic acid content. Collectively, our data indicate that T6P plays an important role for potato tuber growth.

Inhibition of Alanine Aminotransferase in Silico and in Vivo Promotes Mitochondrial Metabolism to Impair Malignant Growth

Cancer cells commonly exhibit increased nonoxidative d-glucose metabolism whereas induction of mitochondrial metabolism may impair malignant growth. We have first used an in silico method called elementary mode analysis to identify inhibition of ALAT (l-alanine aminotransferase) as a putative target to promote mitochondrial metabolism. We then experimentally show that two competitive inhibitors of ALAT, l-cycloserine and β-chloro-l-alanine, inhibit l-alanine production and impair d-glucose uptake of LLC1 Lewis lung carcinoma cells. The latter inhibition is linked to an initial energy deficit, as quantified by decreased ATP content, which is then followed by an activation of AMP-activated protein kinase and subsequently increased respiration rates and mitochondrial production of reactive oxygen species, culminating in ATP replenishment in ALAT-inhibited LLC1 cells. Moreover, we observe altered phosphorylation of p38 MAPK (mitogen-activated protein kinase 14), ERK (extracellular signal-regulated kinase 1/2), and Rb1 (retinoblastoma 1) proteins, as well as decreased expression of Cdc25a (cell decision cycle 25 homolog A) and Cdk4 (cyclin-dependent kinase 4). Importantly, these sequelae of ALAT inhibition culminate in similarly reduced anchorage-dependent and anchorage-independent growth rates of LLC1 cells, together suggesting that inhibition of ALAT efficiently impairs cancer growth by counteracting the Warburg effect due to compensatory activation of mitochondrial metabolism.

Regional Mitochondrial Depolarization Causes Spontaneous Ventricular Arrhythmia in Cardiac Tissue

Sudden cardiac death (SCD) resulting from ventricular arrhythmia remains a leading cause of death in the USA. The underlying molecular and structural mechanisms of arrhythmic SCD, however, are still unclear. We have recently reported that oxidative stress can trigger the abrupt collapse of mitochondrial inner membrane potential (ΔΨm), reverse of ATP synthase and decrease of ATP concentration. This could activate the sarcolemmal ATP sensitive potassium channel (KATP), affecting cellular electrical excitability and electrical wave propagation in the cardiac tissue. To test this hypothesis, we investigated the effect of regional mitochondrial depolarization on electrical wave propagation using combined optical mapping and computer simulations. The regional mitochondrial depolarization was induced in the center of cardiac cell monolayer by oxidative stress or mitochondrial uncoupler. The results show a coupling between ΔΨm depolarization, activation of KATP current and shortening of action potentials. When this effect is amplified, regional mitochondrial depolarization could form a metabolic current sink, preventing electrical wave propagation on the cardiac tissue. The overall influence involves a combination of a decrease in the action potential amplitude and shortening of the action potential duration. This mechanism introduces both temporal and spatial dispersion of electrical excitability and heterogeneous conduction, leading to wave breaks and spontaneous arrhythmia when the mitochondrial energetics within the metabolic current sink zone is recovering. In summary, this study illustrates the mechanism by which “metabolic” sinks can contribute the formation of fatal reentrant arrhythmias and reveal a novel way that reentry may be triggered in tissues recovering from metabolic inhibition (e.g., ischemia-reperfusion). The findings underscore the importance of considering mitochondrial targets for developing new therapies for SCD in the context of cardiovascular disease.

The effects of long‐term storage of human red blood cells on the glutathione synthesis rate and steady‐state concentration

Banked red blood cells (RBCs) undergo changes that reduce their viability after transfusion. Dysfunction of the glutathione (GSH) antioxidant system may be implicated. We measured the rate of GSH synthesis in stored RBCs and applied a model of GSH metabolism to identify storage-dependent changes that may affect GSH production. RBC units (n = 6) in saline-adenine-glucose-mannitol (SAGM) solution were each divided into four transfusion bags and separate treatments were applied: 1) SAGM (control), 2) GSH precursor amino acids, 3) aminoguanidine, and 4) glyoxal. RBCs were sampled during 6 weeks of storage. Rejuvenated RBCs were also analyzed. After 6 weeks, the ATP concentration declined to 50 ± 5.5% (p < 0.05) of that in the fresh RBCs. For control RBCs, the GSH concentration decreased by 27 ± 6.5% (p < 0.05) and the rate of GSH synthesis by 45 ± 8% (p < 0.05). The rate of GSH synthesis in rejuvenated and amino acid-treated RBCs was unchanged after 6 weeks. Modeling identified that the decline in GSH synthesis was due to decreased intracellular substrate concentrations and reduced amino acid transport, secondary to decreased ATP concentration. This study has uniquely shown that the glutathione synthesis rate decreased significantly after 6 weeks in stored RBCs. Our results have identified potential opportunities for improvement of banked blood storage.

Time-resolved Measurements of Intracellular ATP in the Yeast Saccharomyces cerevisiae using a New Type of Nanobiosensor

Adenosine 5'-triphosphate is a universal molecule in all living cells, where it functions in bioenergetics and cell signaling. To understand how the concentration of ATP is regulated by cell metabolism and in turn how it regulates the activities of enzymes in the cell it would be beneficial if we could measure ATP concentration in the intact cell in real time. Using a novel aptamer-based ATP nanosensor, which can readily monitor intracellular ATP in eukaryotic cells with a time resolution of seconds, we have performed the first on-line measurements of the intracellular concentration of ATP in the yeast Saccharomyces cerevisiae. These ATP measurements show that the ATP concentration in the yeast cell is not stationary. In addition to an oscillating ATP concentration, we also observe that the concentration is high in the starved cells and starts to decrease when glycolysis is induced. The decrease in ATP concentration is shown to be caused by the activity of membrane-bound ATPases such as the mitochondrial F(0)F(1) ATPase-hydrolyzing ATP and the plasma membrane ATPase (PMA1). The activity of these two ATPases are under strict control by the glucose concentration in the cell. Finally, the measurements of intracellular ATP suggest that 2-deoxyglucose (2-DG) may have more complex function than just a catabolic block. Surprisingly, addition of 2-DG induces only a moderate decline in ATP. Furthermore, our results suggest that 2-DG may inhibit the activation of PMA1 after addition of glucose.

Effect of root-applied spermidine on growth and respiratory metabolism in roots of cucumber (Cucumis sativus) seedlings under hypoxia

The effects of exogenous spermidine (Spd) application to hypoxic nutrient solution on the contents of endogenous polyamines (PAs) and respiratory metabolism in the roots of cucumber (Cucumis sativus L.) seedlings were investigated. Cucumber seedlings were grown hydroponically in control and hypoxic nutrient solutions with and without addition of Spd at a concentration of 0.05 mM. The activities of key enzymes involved in the tricarboxylic acid cycle (TCAC), such as succinate dehydrogenase (SDH) and isocitrate dehydrogenase (IDH), were significantly inhibited under root-zone hypoxia with dissolved oxygen (DO) at 1 mg/l. In contrast, the activities of enzymes involved in the process of fermentation, such as pyruvate decarboxylase (PDC), alcohol dehydrogenase (ADH), lactate dehydrogenase (LDH), and alanine aminotransferase (AlaAT), were significantly increased. Thus, aerobic respiration was inhibited and fermentation was enhanced in the roots of cucumber seedlings as a result of decreasing ATP content to inhibit the dry weight of seedlings under hypoxic stress. Moreover, the contents of free, soluble conjugated, and insoluble bound putrescine (Put), Spd, and spermine (Spm) in the roots of cucumber seedlings were significantly increased under hypoxia stress. Interestingly, application of Spd to hypoxic roots markedly suppressed the accumulation of free Put and, in contrast, promoted an increase in free Spd and Spm, as well as soluble conjugated and insoluble bound Put, Spd, and Spm contents. From these data, we deduced that exogenous Spd promotes the conversion of free Put into free Spd and Spm, and soluble conjugated and insoluble bound PAs under hypoxia stress. Furthermore, the activities of LDH, PDC, and ADH were suppressed and, in contrast, the activities of SDH and IDH were enhanced by application of exogenous Spd to hypoxic roots. As a result, aerobic respiration was enhanced but fermentation metabolism was inhibited in the roots of cucumber seedlings, leading to an increase in ATP content to alleviate the inhibited dry weight of seedlings due to hypoxia stress. These results suggest that application of Spd to hypoxic nutrient solution promoted conversion of free Put into free Spd and Spm as well as soluble conjugated and insoluble bound PAs, further enhanced IDH and SDH activities, and inhibited ethanol fermentation and lactate fermentation, resulting in increased ATP content and eventually enhanced tolerance of cucumber plants to root-zone hypoxia.

The toxic mechanism of high lethality of herbicide butachlor in marine flatfish flounder, Paralichthys olivaceus

The toxic mechanism of herbicide butachlor to induce extremely high lethality in marine flatfish flounder, Paralichthys Olivaceus, was analyzed by histopathological examination, antioxidant enzymes activities and ATP content assay. Histopathological examination of gill, liver and kidney of exposed fishes showed that gill was a target organ of butachlor. The butachlor seriously impaired the respiration of gills by a series of lesions such as edema, lifting and detachment of lamellar epithelium, breakdown of pillar cells, and blood congestion. The dysfunction of gill respiration caused suffocation to the exposed flounder with extremely high acute lethality. Antioxidant enzyme activity assay of the in vitro cultured flounder gill (FG) cells exposed to butachlor indicated that butachlor markedly inhibited the antioxidant enzyme activities of Superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX). Furthermore, along with the decline of antioxidant enzyme activities, ATP content in the exposed FG cells decreased, too. This infers that the oxidative stress induced by butachlor can inhibit the production of cellular ATP. Similar decrease of ATP content was also observed in the exposed flounder gill tissues. Taken together, as in FG cells, butachlor possibly induced a short supply of ATP in pillar cells by inhibiting the antioxidant enzyme activities and then affecting the contractibility of the pillar cells, which in turn resulted in the blood congestion and suffocation of exposed flounder.

Biochemical changes in oyster tissues and hemolymph during long-term air exposure

The Pacific oyster Crassostrea gigas is a sessile bivalve that inhabits the intertidal zone and therefore frequently exposed to air during the tidal cycle. It is highly adaptive to hypoxic conditions. We have studied the physiological state of oysters during long-term exposure to air. The oysters became hypoxic when exposed to air or hypoxic seawater. The 50% lethal time of oysters exposed to air at 4, 15 and 20°C was 47.8, 15.9 and 12.2 days, respectively. The hemolymph pH decreased by day 3; however, it showed a slight increase by day 5 at both 4 and 20°C. The adenylate energy charge (AEC) values decreased rapidly on the first day of air exposure in the adductor muscle, mantle, gill and body trunk, and these decreases were accompanied by decreases in ATP concentrations and increases in AMP concentrations. The AEC values in all of the tissues had fallen to below 30% by day 50 of air exposure at 4°C. These data suggest that the energy state of oysters deteriorates rapidly with air exposure. Consequently, AEC values may be useful indices of the physiological state of the oyster during long-term exposure to air.

Indirubin-3′-oxime prevents hepatic I/R damage by inhibiting GSK-3β and mitochondrial permeability transition

Indirubin-3′-oxime is an indirubin analogue that shows favorable inhibitory activity targeting glycogen synthase kinase 3β (GSK-3β). In this study, we evaluated if acute treatment with indirubin-3′-oxime (Ind) prevents hepatic ischemia/reperfusion (I/R) damage. Wistar rats were subjected to 150 min of 70% warm ischemia and 16 h of reperfusion. In the treated group 1 μM indirubin-3′-oxime was administered in the hepatic artery 30 min before ischemia. Acute treatment with Ind decreased serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) levels, comparatively to I/R livers. Bax translocation to the mitochondria and cytochrome c release were higher in I/R livers. Ind treatment significantly attenuated Bax translocation and preserved mitochondrial cytochrome c content. Ind also protected mitochondria from calcium-induced mitochondrial permeability transition (MPT), as well as the decrease in state 3 mitochondrial respiration, the delay in the repolarization after a phosphorylative cycle and the decrease in ATP content caused by I/R. By addressing GSK-3β activity and phosphorylated GSK-3β at Ser 9 content in liver homogenates and isolated mitochondria, data suggests that inhibition of GSK-3β by indirubin-3′-oxime prevents the increase in mitochondrial phosphorylated GSK-3β at Ser 9 induced by I/R, thus correlating with MPT inhibition and preservation of cytochrome c content. Pre-treatment with indirubin-3′-oxime in conditions of hepatic I/R, protects the liver by maintaining mitochondrial function and hepatic energetic balance.

Apoptosis-promoting effects of Sutherlandia frutescens extracts on normal human lymphocytes in vitro

Sutherlandia frutescens (SF), an indigenous medicinal plant to South Africa, is traditionally used to treat a diverse range of illnesses. More specifically, the immune-enhancing potential of SF has been recognised to the extent that SF extracts have been recommended as an adjuvant in HIV/AIDS treatment by the South African Ministry of Health, despite a lack of knowledge of its mechanism of action or potential immune toxicity. As yet, unsubstantiated data support the notion of immunostimulatory effects of SF extracts in HIV-infected patients. This was suggested by post-treatment recovery of CD4+ cells brought about by the reduction of the impact of virus-induced apoptosis. This study investigated the apoptotic effects of SF extracts on normal human lymphocytes in vitro. Initially, an acute cytotoxic profile of SF extract was formulated, from which an IC50 of 7.5 mg/mL was calculated and administered for 3 h, 6 h and 12 h to cell populations. At 12 h, SF caused a significant increase in apoptosis in the total lymphocyte population and CD4+ cells as evidenced by increased phosphatidylserine (PS) translocation, caspase-3/7 activity, and decreased ATP content. After 12 h, the SF extract initiated lymphocyte activation in both total lymphocyte and CD4+ subpopulations, indicated by a doubling of the number of cells expressing the CD69 activation marker. The apoptosis observed may thus be the result of activation-induced lymphocyte cell death (AICD). Our results are in conflict with preliminary clinical evidence which has suggested SF extracts are possibly beneficial in the treatment of HIV infection. More extensive evaluations of the effects of SF extracts on the immune system in such subjects are urgently needed.

Changes in motility, ATP content, morphology and fertilisation capacity during the movement phase of tetraploid Pacific oyster (Crassostrea gigas) sperm

Changes in sperm features during the movement phase are especially interesting to study in external fertilization species whose sperm duration movement is long because this implies a significant adaptation of moving cells to the external medium. This study describes the changes in tetraploid Pacific oyster sperm characteristics in relation to time post activation. Sperm individually collected on three tetraploid males were activated in seawater. Their features were analysed over a 24h period and compared to a sperm pool collected on three diploid males as a reference. The percentage of motile spermatozoa, the intracellular ATP content, and the fine structure of spermatozoa were studied in relation to time post activation. Furthermore, the fertilisation capacity of sperm individually collected on five diploid males was assessed after 1 and 24h post activation. A forward progressive movement was maintained for at least a 20h duration. Compared to diploid males, the percentage of motile spermatozoa was lower in tetraploid males. The intracellular ATP concentration was higher in spermatozoa from tetraploid males than in spermatozoa from diploid males. A decrease in ATP content was observed in the first 6h post activation and severe alterations were observed in sperm morphology after 24h. Then, a lower fertilisation capacity of sperm from diploid males was observed at the end of the movement phase. The cessation of Pacific oyster sperm motility was unlikely caused by ATP consumption as ATP concentration was still high at the end of sperm movement but rather caused by drastic changes in sperm morphology. Compared to sperm collected on diploid males, the lower quality of sperm from tetraploid males was emphasized by a shorter movement duration and deeper morphological alterations at the end of the movement phase.