Search

Although purinergic signalling has been a well-established and accepted mechanism of chemical communication for many years, it remains important to measure the extracellular concentration of ATP and adenosine in real time. In this review I summarize the reasons why such measurements are still needed, how they provide additional mechanistic insight and give an overview of the techniques currently available to make spatially localised measurements of ATP and adenosine in real time. To illustrate the impact of direct real-time measurements, I explore CO 2 and nutrient sensing in the medulla oblongata and hypothalamus. In both of these examples, the sensing involves hemichannel mediated ATP release from glial cells. For CO 2 the hemichannels involved, connexin26, are directly CO 2 -sensitive. This mechanism contributes to the chemosensory control of breathing. In the hypothamalus, specialised glial cells, tanycytes, directly contact the cerebrospinal fluid in the 3rd ventricle and sense nutrients via sweet and umami taste receptors. Nutrient sensing by tanycytes is likely to contribute to the control of body weight as their selective stimulation alters food intake. To illustrate the importance of direct adenosine measurements, I consider the complex and multiple mechanisms of activity-dependent adenosine release in different brain regions. This activity dependent release of adenosine is likely to mediate important feedback regulation and may also be involved in controlling the sleep-wake state. I finish by briefly considering the potential of whole blood purine measurements in clinical practice., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The dynamic process of membrane fouling was characterized during relatively long-term (30 d) continuous nanofiltration (NF) of a real wastewater secondary effluent, with the roles of organic, inorganic and biological foulants quantified via statistical analyses. The analyses were based on time-series data of physical properties (morphology, roughness, hydrophilicity and charge), chemical compositions (X-ray and infrared responses) and biomass (adenosine triphosphate, ATP) on the membrane surface during fouling evolution. The individual and interactive contributions of organic factor (typical functional groups), inorganic factor (Ca as a representative) and biological factor (ATP amount) to fouling were quantified via multiple linear regression coupled with variance partitioning analysis. About 78% of the variance of filtration resistance can be explained by these factors, among which 16% was contributed by individual effect of organics (via e.g. physical adsorption), 21% by organic-inorganic binary effect (in the form of e.g. Ca-complex), 13% by organic-biological binary effect (organics as the nutrient/product of microorganisms), and 24% by organic-inorganic-biological ternary interaction. Organic matter was universally involved in these effects. The interrelations among fouling factors, foulant layer properties and filtration time were comprehensively explored via redundancy analysis, which clearly delineated the fouling evolution into three major stages: Stage I (0-1 d) for initial fouling mainly due to rapid organic adsorption; Stage II (1-10 d) mainly for the gradual growth of Ca-organic combined fouling; and Stage III (10-30 d) for the eventual maturation of biofouling. These may provide foundations for a targeted fouling control based on foulant type or fouling stage., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Liver fibrosis results from continuous inflammation and injury. Despite its high prevalence worldwide, no approved antifibrotic therapies exist. Omipalisib is a selective inhibitor of the PI3K/mTOR pathway that controls nutrient metabolism, growth and proliferation. It has shown antifibrotic properties in vitro. While clinical trials for idiopathic pulmonary fibrosis have been initiated, an in-depth preclinical evaluation is lacking. We evaluated omipalisib's effects on fibrogenesis using the ex vivo model of murine and human precision-cut tissue slices (PCTS)., Methods: Murine and human liver and jejunum PCTS were incubated with omipalisib up to 10 μM for 48 h. PI3K pathway activation was assessed through protein kinase B (Akt) phosphorylation and antifibrotic efficacy was determined via a spectrum of fibrosis markers at the transcriptional and translational level., Results: During incubation of PCTS the PI3K pathway was activated and incubation with omipalisib prevented Akt phosphorylation (IC 50  = 20 and 1.5 nM for mouse and human, respectively). Viability of mouse and human liver PCTS was compromised only at the high concentration of 10 and 1-5 μM, respectively. However, viability of jejunum PCTS decreased with 0.1 (mouse) and 0.01 μM (human). Spontaneously increased fibrosis related genes and proteins were significantly and similarly suppressed in mouse and in human liver PCTS., Conclusions: Omipalisib has antifibrotic properties in ex vivo mouse and human liver PCTS, but higher concentrations showed toxicity in jejunum PCTS. While the PI3K/mTOR pathway appears to be a promising target for the treatment of liver fibrosis, PCTS revealed likely side effects in the intestine at higher doses., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

That adenosine 5' triphosphate (ATP) functions as an extracellular signaling molecule has been established since the 1970s. Ubiquitous throughout the body as the principal molecular store of intracellular energy, ATP has a short extracellular half-life and is difficult to measure directly. Extracellular ATP concentrations are dependent both on the rate of cellular release and of enzymatic degradation. Some findings from in vitro studies suggest that extracellular ATP concentrations increase during high levels of neuronal activity and seizure-like events in hippocampal slices. Pharmacological studies suggest that antagonism of ATP-sensitive purinergic receptors can suppress the severity of seizures and block epileptogenesis. Directly measuring extracellular ATP concentrations in the brain, however, has a number of specific challenges, notably, the rapid hydrolysis of ATP and huge gradient between intracellular and extracellular compartments. Two studies using microdialysis found no change in extracellular ATP in the hippocampus of rats during experimentally-induced status epilepticus. One of which demonstrated that ATP increased measurably, only in the presence of ectoATPase inhibitors, with the other study demonstrating increases only during later spontaneous seizures. Current evidence is mixed and seems highly dependent on the model used and method of detection. More sensitive methods of detection with higher spatial resolution, which induce less tissue disruption will be necessary to provide evidence for or against the hypothesis of seizure-induced elevations in extracellular ATP. Here we describe the current hypothesis for ATP release during seizures and its role in epileptogenesis, describe the technical challenges involved and critically examine the current evidence., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The U L 47 gene product, VP8, is a major tegument protein of BoHV-1. While VP8 is not essential for virus replication in cell culture, a U L 47-deleted virus exhibits a smaller tegument structure and is avirulent in cattle. To obtain pure VP8 protein for structural analysis, we expressed a N-terminally truncated version of VP8 in Eschericia coli. However, the recombinant VP8 was consistently co-purified with a tightly associated bacterial protein; this protein was identified by mass spectrometry as GroEL, which has considerable homology with mammalian heat shock protein-60 (HSP60), thus suggesting a new role for VP8 in virus-host interaction. A physical interaction of HSP60 and VP8 in both VP8-transfected and BoHV-1-infected cells was demonstrated by immunoprecipitation. Analysis of different truncated VP8 constructs revealed that amino acids 259-482 and 632-741 are involved in binding to HSP60. Full-length VP8 and VP8 219-741 (containing both interacting domains, 259-482 and 632-741) co-localized with HSP60 and mitochondria. VP8 was localized in the mitochondria from 2 to 14 h post infection in BoHV-1-infected cells. The mitochondrial membrane potential was reduced in both VP8-transfected and BoHV-1-infected cells and was further diminished by overexpression of HSP60 in the presence of VP8. In addition, VP8 expression decreased the ATP concentration during transfection, as well as BoHV-1 infection. Thus, VP8 may play a role in the deregulation of mitochondrial function through interaction with HSP60. This is consistent with the fact that BoHV-1 infection is known to promote mitochondrial dysfunction., (Copyright © 2018 Elsevier B.V. All rights reserved.)

We fabricated a quencher-free and enzyme-free fluorescence detection system by employing the DNA logic circuits as signal amplifier and 2-aminopurine as signal indicator, and applied it to detect DNA and adenosine triphosphate (ATP). The assay system consisted of three hairpin probes with a sequestered 2-aminopurine molecule in each stem domain which was defined as inputs A, B and C of the logic operation. These three hairpin inputs kept stability and coexisted in reaction solution without target. However, adding target to the system would break the stability and initiate a dynamic assembly of the three inputs through toehold mediated displacement, resulting in the formation of three way junction and the liberation of 2-aminopurine from duplex structure. The structural circumstance changes from duplex to single stand switched the signal from "off" to "on" due to the disarming of base stack interaction, thus attaining amplified fluorescence detection without any extra quencher and avoiding the limitation of distance-independent signal conversion in conventional methods. A limit of detection of 0.46 pM was achieved for target DNA with high discrimination capability. Moreover, the sensing system was expandable for ATP detection. Importantly, the method was simple and easy-to-operate. These features make the DNA logic circuits adaptable as an enzyme-free and quencher-free amplifier, and thus the proposed method offers a powerful platform for DNA and ATP determination, and even other biotargets in clinical diagnosis., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Objectives: Acquired aplastic anemia (AA) is a hematopoietic stem cell disease that leads to hematopoietic disorder and peripheral blood pancytopenia. We investigated whether nutritional support is helpful to AA recovery., Methods: We established a rat model with AA. A nutrient mixture was administered to rats with AA through different dose gavage once per day for 55 d. Animals in this study were assigned to one of five groups: normal control (NC; group includes normal rats); AA (rats with AA); high dose (AA + nutritional mixture, 2266.95 mg/kg/d); medium dose (1511.3 mg/kg/d); and low dose (1057.91 mg/kg/d). The effects of nutrition administration on general status and mitochondrial function of rats with AA were evaluated., Results: The nutrient mixture with which the rats were supplemented significantly improved weight, peripheral blood parameters, and histologic parameters of rats with AA in a dose-dependent manner. Furthermore, we observed that the number of mitochondria in the liver, spleen, kidney, and brain was increased after supplementation by transmission electron microscopy analysis. Nutrient administration also improved mitochondrial DNA content, adenosine triphosphate content, and membrane potential but inhibited oxidative stress, thus, repairing the mitochondrial dysfunction of the rats with AA., Conclusions: Taken together, nutrition supplements may contribute to the improvement of mitochondrial function and play an important role in the recuperation of rats with AA., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Effects of pre-gestational physical activity on the later development of embryos generated in vitro were evaluated. Kunming mice were divided into two groups, namely exercised and unexercised, with the former undergoing physical training on a motor-driven leveled treadmill over a period of 4weeks (5days/week and 60min/day). After that, following superovulation, collection of oocytes from both groups was performed for in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT). Notably and specifically, natural mating between the unexercised mice was also done for in vivo fertilization (IVIF). Observation on the preimplantation embryo development showed that SCNT embryos from exercised group (NEM) had significant higher rates of cleavage and formation of blastocyst containing more blastomeres compared to SCNT embryos from unexercised group (NCM); while IVF embryos in exercised group (FEM) showed a significant higher rate of cleavage and blastocyst formation. Further analysis of embryos from the trained group on the molecular level demonstrated a prominent increase in terms of ATP levels, mitochondria membrane potential (Δψm) and mtDNA copy number and an obvious decrease in H 2 O 2 concentration than those from the respective control group. Meanwhile, epigenetically, methylation levels of CpG sites on imprinting control regions of imprinted genes (Igf2, Igf2r, Meg 3 and H19) in the NEM embryos were normalized in exercise groups compared to the NCM embryos, which were comparable to the in vivo-derived embryos. Moreover, global DNA and histone methylation (H3K4m2, H3K9m3 and H3K36m) further confirmed that NEM-derived embryos and in vivo-derived ones stay in the same cluster, indicating that physical activity in pre-pregnancy facilitates the maintenance of epigenetic modifications. Generally, these results strongly suggest that physical exercise before pregnancy is in favor of the later embryo development., (Copyright © 2016 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

Interleukin-15 receptor alpha knockout (IL15RαKO) mice exhibit a greater skeletal muscle mitochondrial density with an altered mitochondrial morphology. However, the mechanism and functional impact of these changes have not been determined. In this study, we characterized the functional, proteomic, and genomic alterations in mitochondrial subpopulations isolated from the skeletal muscles of IL15RαKO mice and B6129 background control mice. State 3 respiration was greater in interfibrillar mitochondria and whole muscle ATP levels were greater in IL15RαKO mice supporting the increases in respiration rate. However, the state 3/state 4 ratio was lower, suggesting some degree of respiratory uncoupling. Proteomic analyses identified several markers independently in mitochondrial subpopulations that are associated with these functional alterations. Next Generation Sequencing of mtDNA revealed a high degree of similarity between the mitochondrial genomes of IL15RαKO mice and controls in terms of copy number, consensus coding and the presence of minor alleles, suggesting that the functional and proteomic alterations we observed occurred independent of alterations to the mitochondrial genome. These data provide additional evidence to implicate IL-15Rα as a regulator of skeletal muscle phenotypes through effects on the mitochondrion, and suggest these effects are driven by alterations to the mitochondrial proteome., (Copyright © 2015 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

Mitochondrial dysfunction has recently been implicated as an underlying factor to several common neurodegenerative diseases, including Parkinson's disease, Alzheimer's and amyotrophic lateral sclerosis (ALS). Valosin containing protein (VCP)-associated multisystem proteinopathy is a new hereditary disorder associated with inclusion body myopathy, Paget disease of bone (PDB), frontotemporal dementia (FTD) and ALS. VCP has been implicated in several transduction pathways including autophagy, apoptosis and the PINK1/Parkin cascade of mitophagy. In this report, we characterized VCP patient and mouse fibroblasts/myoblasts to examine their mitochondrial dynamics and bioenergetics. Using the Seahorse XF-24 technology, we discovered decreased spare respiratory capacity (measurement of extra ATP that can be produced by oxidative phosphorylation in stressful conditions) and increased ECAR levels (measurement of glycolysis), and proton leak in VCP human fibroblasts compared with age- and sex-matched unaffected first degree relatives. We found decreased levels of ATP and membrane potential, but higher mitochondrial enzyme complexes II+III and complex IV activities in the patient VCP myoblasts when compared to the values of the control cell lines. These results suggest that mutations in VCP affect the mitochondria's ability to produce ATP, thereby resulting in a compensatory increase in the cells' mitochondrial complex activity levels. Thus, this novel in vitro model may be useful in understanding the pathophysiology and discovering new drug targets of mitochondrial dynamics and physiology to modify the clinical phenotype in VCP and related multisystem proteinopathies (MSP)., (Copyright © 2015 © Elsevier B.V. and Mitochondria Research Society. Published by Elsevier B.V. All rights reserved.)

Ziram is a dimethyldithiocarbamate fungicide which can cause intraneuronal calcium (Ca(2+)) dysregulation and subsequently neuronal death. The signaling mechanisms underlying ziram-induced Ca(2+) dyshomeostasis and neurotoxicity are not fully understood. NCX3 is the third isoform of the sodium-calcium exchanger (NCX) family and plays an important role in regulating Ca(2+) homeostasis in excitable cells. We previously generated a mouse model deficient for the sodium-calcium exchanger 3 and showed that NCX3 is protective against ischemic damage. In the present study, we aim to examine whether NCX3 exerts a similar role against toxicological injury caused by the pesticide ziram. Our data show baby hamster kidney (BHK) cells stably transfected with NCX3 (BHK-NCX3) are more susceptible to ziram toxicity than cells transfected with the empty vector (BHK-WT). Increased toxicity in BHK-NCX3 was associated with a rapid rise in cytosolic Ca(2+) concentration [Ca(2+)]i induced by ziram. Profound mitochondrial dysfunction and ATP depletion were also observed in BHK-NCX3 cells following treatment with ziram. Lastly, primary dopaminergic neurons lacking NCX3 (NCX3(-/-)) were less sensitive to ziram neurotoxicity than wildtype control dopaminergic neurons. These results demonstrate that NCX3 genetic deletion protects against ziram-induced neurotoxicity and suggest NCX3 and its downstream molecular pathways as key factors involved in ziram toxicity. Our study identifies new molecular events through which pesticides (e.g. ziram) can lead to pathological features of degenerative diseases such as Parkinson's disease and indicates new targets to slow down neuronal degeneration., (Copyright © 2014 Elsevier Inc. All rights reserved.)

As the latest addition to the sub-strain specific WHO Reference Reagents of BCG vaccine, an international collaborative study was completed to evaluate the suitability of a candidate BCG Moreau-RJ sub-strain as a WHO Reference Reagent of BCG vaccine. This follows the recent replacement of the WHO 1st International Reference Preparation for BCG vaccine, by three sub-strain specific WHO Reference Reagents of BCG vaccine (Danish 1331, Tokyo 172-1 and Russian BCG-I) in order to complete the coverage of most predominant sub-strains used for BCG vaccine production and distribution for use worldwide. The study used cultural viable count and modified ATP assays to quantify the preparation and multiplex PCR to confirm the identity of the sub-strain. The establishment of this WHO Reference Reagent of BCG vaccine of Moreau-RJ sub-strain was approved by the WHO Expert Committee on Biological Standardization meeting in October 2012. This preparation is available for distribution by NIBSC-MHRA, UK. The data from real-time stability monitoring demonstrated that these Reference Reagents of BCG vaccine are very stable in storage condition at -20°C. They serve as the valuable source of BCG Reference Reagents for use as comparators (1) for viability assays (such as cultural viable count and modified ATP assays); (2) for in vivo assays (such as the absence of virulent mycobacteria, dermal reactivity and protection assays) in the evaluation of candidate TB vaccines in non-clinical models; (3) for identity assays using molecular biology techniques., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Mitochondrial dysfunction is associated with various forms of lung injury and disease that also involve alterations in pulmonary endothelial permeability, but the relationship, if any, between the two is not well understood. This question was addressed by perfusing isolated intact rat lung with a buffered physiological saline solution in the absence or presence of the mitochondrial complex I inhibitor rotenone (20 μM). Compared to control, rotenone depressed whole lung tissue ATP from 5.66 ± 0.46 (SEM) to 2.34 ± 0.15 µmol · g(-1) dry lung, with concomitant increases in the ADP:ATP and AMP:ATP ratios. Rotenone also increased lung perfusate lactate (from 12.36 ± 1.64 to 38.62 ± 3.14 µmol · 15 min(-1) perfusion · g(-1) dry lung) and the lactate:pyruvate ratio, but had no detectable impact on lung tissue GSH:GSSG redox status. The amphipathic quinone coenzyme Q1 (CoQ1; 50 μM) mitigated the impact of rotenone on the adenine nucleotide balance, wherein mitigation was blocked by NAD(P)H-quinone oxidoreductase 1 or mitochondrial complex III inhibitors. In separate studies, rotenone increased the pulmonary vascular endothelial filtration coefficient (Kf) from 0.043 ± 0.010 to 0.156 ± 0.037 ml · min(-1) · cm H2O(-1) · g(-1) dry lung, and CoQ1 protected against the effect of rotenone on Kf. A second complex I inhibitor, piericidin A, qualitatively reproduced the impact of rotenone on Kf and the lactate:pyruvate ratio. Taken together, the observations imply that pulmonary endothelial barrier integrity depends on mitochondrial bioenergetics as reflected in lung tissue ATP levels and that compensatory activation of whole lung glycolysis cannot protect against pulmonary endothelial hyperpermeability in response to mitochondrial blockade. The study further suggests that low-molecular-weight amphipathic quinones may have therapeutic utility in protecting lung barrier function in mitochondrial insufficiency., (Published by Elsevier Inc.)

The lifespan of each individual, even in an isogenic cohort and a uniform environment, is quite different. The genetic factors influencing the lifespan in humans as well as animal models are few. The balance is attributed to "chance" variations. In this study, we focus on a third factor, noise or chance variations, as well as on genetic and environmental factors and examine how biodemographic aging is related to stochastic fluctuations, or noise. To elucidate the third factor in relation to aging and lifespan, we employed the nematode Caenorhabditis elegans, which can provide an ideal system for analyzing the mathematical and biophysical models. An amplification of ATP noise was clearly evident from around the onset of biodemographic aging (t(0)) as if the t(0) was synchronized with or derived from the amplification of noise. Furthermore, the expression noise of the unc-54 gene, which encodes the myosin heavy chain, increased from around the t(0). In contrast, the noise of genes related to the mitochondrial respiratory chain was almost constant with aging. There is a high energy barrier between life and death. Here we propose that the transition from living to dying may be facilitated by noise amplification. The finite value (or non-zero) of t(0) is essential to the lifespan equation derived from the diffusion model., (Copyright © 2013 The Author. Published by Elsevier Inc. All rights reserved.)

Adenosine nucleotides are involved as substrates or co-factors in several biochemical reactions, catalyzed by enzymes, which modulate energy production, signal transduction and cell proliferation. We here report the development and optimization of an ion exchange liquid chromatography (LC) method for the determination of ATP, ADP and AMP. This method is specifically aimed at the determination of the ATP-ase activity of human heat shock protein 90 (Hsp90), a molecular chaperone that has emerged as target enzyme in cancer therapy. Separation of the three nucleotides was achieved in a 15-min run by using a disk shaped monolithic ethylene diamine stationary phase of small dimensions (2mm×6mm i.d.), under a three-solvent gradient elution mode and UV detection at 256nm. The described direct LC method resulted highly specific as a consequence of the baseline separation of the three adenosine nucleotides and could be applied to the determination of the enzymatic activity of ADP/ATP generating or consuming enzymes (such as kinases). Furthermore, comparison of the LOD and LOQ values of the LC method with those obtained with the malachite green assay, which is one of the most used indirect screening methodologies for ATP-ase activity, showed that the LC method has a similar range of application without presenting the drawbacks related to contamination by inorganic phosphate ions and glycerol, which are present in Hsp90 commercial samples., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Mitochondrial chronic stress that originates from defective mitochondria is implicated in a growing list of human diseases. To enhance understanding of pathophysiology of chronic mitochondrial dysfunction we investigated human osteosarcoma cells with 2 types of chronic stress: corresponding to the mutation in ATP synthase subunit 6 encoded by mtDNA (NARP syndrome-mild stress) and to a total lack of mtDNA (Rho0 cells-heavy stress). We previously found that selenium influenced mitochondrial stress response and lowered ROS production. Therefore, in this study effect of selenite on other mitochondrial parameters was investigated. We showed that presence of selenium improved survival of starved cells, modified organization of mitochondrial network in NARP cybrids and decreased cytosolic calcium level in NARP and Rho0 cells. Selenium did not affect mitochondrial membrane potential, ATP level, activity of ATP synthase and activity of complex II of the respiratory chain., (Copyright © 2011 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

Neuronal signaling consumes much of the brain energy, mainly through the restoration of the membrane potential (MP) by ATP-consuming ionic pumps. We have reported that, compared with waking, ATP levels increase during the initial hours of natural slow-wave sleep, a time with prominent electroencephalogram (EEG) delta oscillations (0.5-4.5 Hz). We have hypothesized that there is a delta oscillation-ATP increase coupling, since, during delta waves, neurons exhibit a prolonged hyperpolarizing phase followed by a very brief phase of action potentials. However, direct proof of this hypothesis is lacking, and rapid changes in EEG/neuronal activity preclude measurement in the naturally sleeping brain. Thus, to induce a uniform state with pure delta oscillations and one previously shown to be accompanied by a similar pattern of neuronal activity during delta waves as natural sleep, we used ketamine-xylazine treatment in rats. We here report that, with this treatment, the high-energy molecules ATP and ADP increased in frontal and cingulate cortices, basal forebrain, and hippocampus compared with spontaneous waking. Moreover, the degree of ATP increase positively and significantly correlated with the degree of EEG delta activity. Supporting the hypothesis of decreased ATP consumption during delta activity, the ATP-consuming Na+-K+-ATPase mRNA levels were significantly decreased, whereas the mRNAs for the ATP-producing cytochrome c oxidase (COX) subunits COX III and COX IVa were unchanged. Taken together, these data support the hypothesis of a cortical delta oscillation-dependent reduction in ATP consumption, thus providing the brain with increased ATP availability, and likely occurring because of reduced Na+-K+-ATPase-related energy consumption., (Published by Elsevier Ltd.)

The era of highly active antiretroviral therapy (HAART) has controlled AIDS and its related disorders considerably; however, the prevalence of HIV-1-associated neurocognitive disorders has been on the rise in the post-HAART era. In view of these developments, we investigated whether a HAART drug combination of 3'-azido-2',3'-deoxythymidine (AZT) and indinavir (IDV) can alter the functionality of the blood-brain barrier (BBB) endothelial cells, thereby exacerbating this condition. The viability of hCMEC/D3 cells (in vitro model of BBB) that were exposed to these drugs was significantly reduced after 72h treatment, in a dose-dependent manner. Reactive oxygen species were highly elevated after the exposure, indicating that mechanisms that induce oxidative stress were involved. Measures of oxidative stress parameters, such as glutathione and malondialdehyde, were altered in the treated groups. Loss of mitochondrial membrane potential, as assessed by fluorescence microscopy and decreased levels of ATP, indicated that cytotoxicity was mediated through mitochondrial dysfunction. Furthermore, AZT+IDV treatment caused apoptosis in endothelial cells, as assessed by the expression of cytochrome c and procaspase-3 proteins. Pretreatment with the thiol antioxidant N-acetylcysteine amide reversed some of the pro-oxidant effects of AZT+IDV. Results from our in vitro studies indicate that the AZT+IDV combination may affect the BBB in HIV-infected individuals treated with HAART drugs., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Impaired energy metabolism is considered a possible cause of fatigue. The thiamine derivative, thiamine tetrahydrofurfuryl disulfide (TTFD), is prescribed and is also an over-the-counter drug for the attenuation of fatigue. It is readily absorbed from the intestinal tract and converted into thiamine pyrophosphate (TPP), which plays an important role as a cofactor for enzymes of metabolic pathways involved in the production of adenosine triphosphate (ATP). We postulated that TTFD has an anti-fatigue effect by improving energy metabolism during physical-fatigue loading. Here, we initially used the forced swimming test to determine whether daily TTFD or thiamine for 5 days has anti-fatigue effects on weight-loaded rats. The swimming duration of TTFD-, but not of thiamine-treated rats, was significantly longer than that of control rats (P < .05). Based on these findings, we examined changes in the levels of thiamine and its phosphate esters in various organs and the effect of TTFD on ATP levels in skeletal muscle after forced swimming, to determine the cellular mechanisms of the anti-fatigue effect of TTFD. Daily TTFD resulted in a characteristic distribution of thiamine and its phosphate esters in rat skeletal muscle, liver, kidney, heart, brain, and plasma. Furthermore, daily TTFD attenuated the decrease in ATP content in the skeletal muscle caused by forced swimming with a weight load for a defined period (150 s). These results indicate that TTFD exerts anti-fatigue effects by improving energy metabolism during physical fatigue.

As part of the World Health Organisation (WHO) initiative to update the current requirements for BCG vaccine a collaborative study was carried out to establish the robustness, reproducibility and the suitability of the modified ATP assay. This assay was developed by Statens Serum Institut, Denmark, as a potential replacement of the method for detection of viable counts of BCG vaccine which is routinely used as a quality control test for lot release. Two BCG preparations, of same strain but different production methods, were tested. For each preparation, two different storage conditions of -20 or 37 degrees C were used in order to establish the suitability of this assay for testing heat-treated BCG vaccine as in the temperature stability test. The lyophilised BCG samples were tested using the ATP reagents from the same source and same principle of testing but some procedural modifications were allowed to accommodate different equipment and resource availability in different laboratories. Data from four laboratories showed that the heat-treated BCG samples contained significantly lower ATP content per sample than the untreated control stored at -20 degrees C. Three laboratories gave consistent mean ATP contents, especially for control samples, even with variations in testing protocol. The present study showed that this modified ATP assay is very robust and can be reproducible. Once the correlation of cultural viable count and ATP content of a BCG vaccine product has been established, this rapid alternative assay may be used to monitor BCG viable count. Due to the fact that this study was small, further investigation is planned. A collaborative study will be carried out using this modified ATP assay in parallel with the cultural viable count method in the establishment of the replacement of the WHO International Reference Preparation of BCG vaccine.

In the present study, we reported the metabolic effects of salidroside, one of the active components of Rhodiola Rosea, on skeletal muscle cells. Salidroside dose-dependently stimulated glucose uptake in differentiated L6 rat myoblast cells. Inhibitor of AMP-activated protein kinase (AMPK) by pretreating the cells with compound C potently reduced salidroside-stimulated glucose uptake, while inhibition of phosphatidylinositol 3-kinase (PI3K) by wortmannin exhibited no significant inhibitory effect on salidroside-mediated glucose transport activation. Western blotting analyses revealed that salidroside increased the phosphorylation level of AMPK and acetyl-CoA carboxylase (ACC). In addition, salidroside enhanced insulin-mediated Akt activation and glucose uptake, and such enhancement can be specifically inhibited by compound C. In summary, AMPK activation was involved in the effects of salidroside on glucose transport activation and insulin sensitivity. Salidroside can be further developed as potential compound for the anti-diabetic therapy.

Mitochondria likely play a role in Parkinson's disease (PD) neurodegeneration. We modelled PD by creating cytoplasmic hybrid (cybrid) cell lines in which endogenous mitochondrial DNA (mtDNA) from PD or control subject platelets was expressed within human teratocarcinoma (NT2) cells previously depleted of endogenous mtDNA. Complex I activity was reduced in both PD cybrid lines and in the platelet mitochondria used to generate them. Under basal conditions PD cybrids had less ATP, more LDH release, depolarized mitochondria, less mitochondrial cytochrome c, and higher caspase 3 activity. Equivalent MPP+ exposures are more likely to trigger programmed cell death in PD cybrid cells than in control cybrid cells. Our data support a relatively upstream role for mitochondrial dysfunction in idiopathic PD.

Mitochondrial dysfunction may play an important role in the pathogenesis of ageing and age-neurodegenerative diseases such as Alzheimer's disease (AD). Platelet mitochondrial membrane potential (reflected by measurement of JC-1 fluorescence ratio) and adenosine 5'-triphosphate (ATP) contents of 24 moderate probable AD patients, 20 age-matched control subjects and 20 young control subjects were measured. Also, a beta-amyloid peptide (Abeta)-induced damage model of platelets was established. After the addition of Abeta, platelet JC-1 fluorescence ratio and ATP content of platelets were measured in 16 AD patients, 20 aged and 20 young control subjects. Young control subjects had higher JC-1 fluorescence ratio than both AD patients and aged control subjects. No significant differences in platelet ATP contents were found among AD patients, aged and young control subjects. After the addition of Abeta, platelet JC-1 fluorescence ratio and ATP content of aged and young control subjects lowered markedly, but no obvious decrease of platelet JC-1 fluorescence ratio of AD patients was found compared with those of aged and young control subjects. Decrease of platelet JC-1 fluorescence ratio of aged control subjects was lower than that of young control subjects following the addition of Abeta. These results indicated that mitochondrial dysfunction may occur during ageing and platelet mitochondria of AD patients and aged subjects showed a tolerance to Abeta-induced damage. Therefore, blood platelets might serve as a biomarker for detection of mitochondrial function and age-related disease.

Oxidant-mediated apoptosis has been implicated in renal injury due to ischemia reperfusion (IR); however, the apoptotic signaling pathways following IR have been incompletely defined. The purpose of this study was to examine the role of oxidants on cell death in a model of in vitro simulated IR injury in renal proximal tubular epithelial cells by analyzing the effects of a cell-permeable superoxide dismutase mimetic, manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin pentachloride (MnTmPyP). Renal proximal tubular epithelial cells were ATP depleted for 2, 4, or 6 h, followed by 2 h of recovery. We found that MnTmPyP was effective in attenuating cytotoxicity (P<0.001) and decreasing steady-state oxidant levels (P<0.001) and apoptotic cell death (P<0.001) following ATP depletion-recovery. MnTmPyP treatment prevented the early cytosolic release of cytochrome c and increased Bcl-2 protein levels following short durations of ATP depletion-recovery. After longer periods of ATP depletion-recovery, we observed a significant increase in TNF-alpha protein levels (P<0.001) and caspase-8 activation (P<0.001), both of which were decreased (P<0.001) by treatment with MnTmPyP. Our results suggest that oxidant mediated apoptosis via the mitochondrial pathway during the early phase of ATP depletion and by activation of the receptor-mediated apoptotic pathway following longer durations of injury.

Hypoxanthine, a nucleobase, serves as the major source of the essential purine group for the intraerythrocytic malaria parasite. In this study we have measured the uptake of hypoxanthine, and that of the related purine nucleobase adenine, by mature blood-stage Plasmodium falciparum parasites isolated from their host cells by saponin-permeabilisation of the erythrocyte and parasitophorous vacuole membranes. The uptake of both [3H]hypoxanthine and [3H]adenine was comprised of at least two components; in each case there was a rapid equilibration of the radiolabel between the intra- and extracellular solutions via a low-affinity transport mechanism, and an accumulation of radiolabel (such that the estimated intracellular concentration exceeded the extracellular concentration) via a higher-affinity process. The uptake of [3H]adenine was studied in more detail. The rapid, low-affinity equilibration of [3H]adenine between the intra-and extracellular solution was independent of the energy status of the parasite whereas the higher-affinity accumulation of the radiolabel was ATP-dependent. A kinetic analysis of adenine uptake revealed that the low-affinity (equilibrative) process had a Km of approximately 1.2mM, similar to the value of 0.82 mM estimated here (using the Xenopus laevis oocyte expression system) for the Km for the transport of adenine by PfENT1, a parasite-encoded member of the 'equilibrative nucleoside/nucleobase transporter' family. The results indicate that nucleobases enter the intraerythrocytic parasite via a rapid, equilibrative process that has kinetic characteristics similar to those of PfENT1.

Mechanistic aberrations leading to Gemcitabine (2',2'-dFdCyd,2,2-difluorodeoxycytidine, Gem) resistance may include alteration in its transport, metabolism and incorporation into DNA. To explore the mechanism of Gem resistance, the restriction fragment differential display PCR (RFDD-PCR) was employed to compare the mRNA expression patterns of KBGem (Gem resistant), KBHURs (hydroxyurea resistant) and KBwt (parental KB cell). Nine gene fragments were overexpressed specifically in the KBGem clone. Sequencing and BLAST results showed that three fragments represent cytochrome C oxidase (CCOX, respiration complex IV) subunit III (CCOX3). The cDNA microarray confirmed that the mRNAs of CCOX and ATP synthase subunits were upregulated in KBGem as compared to KBwt and KBHURs. The increase in CCOX1 protein and activity led to the increase of free ATP concentration, which is consistent with the gene expression profile of KBGem. Furthermore, the sensitivity to Gem could be reversed by sodium azide, a CCOX inhibitor. Following the treatment of sodium azide, the cellular accumulation of [3H]-Gem increased in a dose (of azide)-dependent manner, which is associated with increase of [3H]-Gem incorporation into DNA in KBGem. In summary, an increase of CCOX activity and free ATP level may reduce the transport, metabolism and DNA incorporation of Gem, resulting in Gem resistance.

Cell mediated immunity (CMI) was assessed by the ImmuKnow assay in 12 patients after kidney transplantation, who presented with viral infection. Treatment included lowering of immunosuppression in all cases and antiviral treatment if indicated. The assay was repeated during the follow up. The ImmuKnow assay at time of presentation of viral infections was 56.8+/-58.2 (range 3-178; median 22) ATP ng/ml. With the clearance of viral infection and lowering of immunosuppression, the assay showed an increase in the level of CMI at 194.5+/-118.9 (range 53-409; median 150) ATP ng/ml. There was viral clearance or stabilization in all cases and there was no incidence of allograft rejection. The ImmuKnow assay of CMI can be used to titrate initial immunosuppression reduction and its subsequent increase, in patients with viral infection after transplantation.

ADP is the endogenous agonist for both P2Y(1) and P2Y(12) receptors, which are important therapeutic targets. It was previously demonstrated that ADP and a synthetic agonist, 2-methylthioadenosine 5'-diphosphate (2MeSADP), can induce apoptosis by activating the human P2Y(1) receptor heterologously expressed in astrocytoma cells. However, it was not known whether the P2Y(12) receptor behaved similarly. We demonstrated here that, unlike with the G(q)-coupled P2Y(1) receptor, activation of the G(i)-coupled P2Y(12) receptor does not induce apoptosis. Furthermore, activation of the P2Y(12) receptor by either ADP or 2MeSADP significantly attenuates the tumor necrosis factor alpha (TNFalpha)-induced apoptosis in 1321N1 human astrocytoma cells. This protective effect was blocked by the P2Y(12) receptor antagonist 2-methylthioAMP and by inhibitors of phospholipase C (U73122) and protein kinase C (chelerythrin), but not by the P2Y(1) receptor antagonist MRS2179. Toward a greater mechanistic understanding, we showed that hP2Y(12) receptor activation by 10nM 2MeSADP, activates Erk1/2, Akt, and JNK by phosphorylation. However, at a lower protective concentration of 100pM 2MeSADP, activation of the hP2Y(12) receptor involves only phosphorylated Erk1/2, but not Akt or JNK. This activation is hypothesized as the major mechanism for the protective effect induced by P2Y(12) receptor activation. Apyrase did not affect the ability of TNFalpha to induce apoptosis in hP2Y(12)-1321N1 cells, suggesting that the endogenous nucleotides are not involved. These results may have important implications for understanding the signaling cascades that follow activation of P2Y(1) and P2Y(12) receptors and their opposing effects on cell death pathways.

We have previously found that uridine 5'-triphosphate (UTP) significantly reduced cardiomyocyte death induced by hypoxia via activating P2Y(2) receptors. To explore the effect of UTP following myocardial infarction (MI) in vivo we studied four groups: sham with or without LAD ligation, injected with UTP (0.44microg/kg i.v.) 30min before MI, and UTP injection (4.4microg/kg i.v.) 24h prior to MI. Left ventricular end diastolic area (LVEDA), end systolic area (LVESA) fractional shortening (FS), and changes in posterior wall (PW) thickness were performed by echocardiography before and 24h after MI. In addition, we measured different biochemical markers of damage and infarct size using Evans blue and TTC staining. The increase in LVEDA and LVESA of the treated animals was significantly smaller when compared to the MI rats (p<0.01). Concomitantly, FS was higher in groups pretreated with UTP 30min or 24h (56+/-14.3 and 36.7+/-8.2%, p<0.01, respectively). Ratio of infarct size to area at risk was smaller in the UTP pretreated hearts than MI rats (22.9+/-6.6, 23.1+/-9.1%, versus 45.4+/-7.6%, respectively, p<0.001). Troponin T and ATP measurements, demonstrated reduced myocardial damage. Using Rhod-2-AM loaded cardiomyocytes, we found that UTP reduced mitochondrial calcium levels following hypoxia. In conclusion, early or late UTP preconditioning is effective, demonstrating reduced infarct size and superior myocardial function. The resulting cardioprotection following UTP treatment post ischemia demonstrates a reduction in mitochondrial calcium overload, which can explain the beneficial effect of UTP.

Objective: Glutamine synthetase (GS), a key enzyme in the production of glutamine, is preserved in skeletal muscle during early aging (<24 mo). Because the effects of advanced age on GS are unknown, we investigated the effect of advanced age (>24 mo) on GS activity in skeletal muscle. We hypothesized that advanced age would enhance muscle GS activity., Methods: Muscle GS activities were assessed in adult (8 mo), mature adult (15 mo), aged (20-22 mo), advanced age (25-27 mo), or very advanced age (29-32 mo) female Wistar rats. Male Wistar (6-27 mo) were used to investigate the effect of gender on this activity., Results: Glutamine synthetase activity remained low and unaltered in rats from 8 to 22 mo of age, as previously demonstrated. In contrast, GS activity was high ( approximately 75% of individual values were higher than the low value mean) in 25-mo to 27-mo-old rats. In very-old-aged rats (29-32 mo), approximately 55% of GS activity data points exhibited low values. Changes in GS protein content paralleled those in GS activities. In male rats, GS activity was also high ( approximately 80% of individual values were higher than the mean value of 6-mo to 19-mo-old rats) at the upper limit of life expectancy (27 mo)., Conclusion: There is enhanced GS activity in old female and male rats suggesting a greater need for glutamine. In some very old rats, low GS activity may be associated with longevity or reflect a limitation in glutamine production due to extremely advanced age per se.

Cardiomyocytes express one or more subtypes of P2 receptors for extracellular nucleotides. P2 purinoceptors, which are activated by nucleotides, are classified as P2X or P2Y: P2X receptors are ligand-gated intrinsic ion channels, and P2Y receptors are G protein-coupled receptors. Extracellular pyrimidine and purine nucleotides are released from the heart during hypoxia. Although the cardioprotective effects of purines acting via purinoceptors were studied intensively, the physiological role of uracil nucleotide-responsive P2Y2, P2Y4, P2Y6, and P2Y14 receptors is still unclear, especially in the cardiovascular system. This study revealed that uridine-5'-triphosphate (UTP) protected cultured rat cardiomyocytes during hypoxia and explored the UTP signaling pathway leading to this cardioprotection. We found that UTP, but not UDP or uridine, significantly reduced cardiomyocyte death induced by hypoxia. Incubation with UTP for 1 h, before exposure to hypoxic conditions, protected the cells 24 h later. The cardioprotective effect of UTP was reduced in the presence of the P2 antagonist suramin. In addition, UTP caused a transient increase of [Ca2+]i in cardiomyocytes. Pyridoxal-5'-phosphate-6-azophenyl-2,4-disulfonate (PPADS) or Reactive blue 2 (RB-2), other antagonists of P2 receptors, abolished the [Ca2+]i elevation caused by UTP. We used various inhibitors of the Ca2+ signaling pathway to show that UTP elevated levels of [Ca2+]i, originating from intracellular sources, via activation of phospholipase C and the IP3 receptor. Interestingly, these inhibitors of the Ca2+ signaling pathway did not prevent the immediate protective effect caused by UTP. Although mitochondrial KATP channels are involved in other preconditioning mediator pathways, the involvement of these channels in the cardioprotective effect induced by UTP was ruled out, because 5-hydroxydecanoic acid (5-HD), a specific inhibitor of these channels, did not prevent the protection.

Male bluegill displays one of two life history tactics. Some males (termed "parentals") delay reproduction until ca. 7 years of age, at which time they build nests and actively courts females. Others mature precociously (sneakers) and obtain fertilizations by cuckolding parental males. In the current study, we studied the relations among sperm motility, ATP levels, and metabolic enzyme activity in parental and sneaker bluegill. In both reproductive tactics, sperm swimming speed and ATP levels declined in parallel over the first 60 s of motility. Although sneaker sperm initially had higher ATP levels than parental sperm, by approximately 30 s postactivation, no differences existed between tactics. No differences were noted between tactics in swimming speed, percent motility, or the activities of key metabolic enzymes, although sperm from parentals had a higher ratio of creatine phosphokinase (CPK) to citrate synthase (CS). In both tactics, with increasing CPK and CS activity, sperm ATP levels increased at 20 s postactivation, suggesting that capacities for phosphocreatine hydrolysis and aerobic metabolism may influence interindividual variation in rates of ATP depletion. Nonetheless, there was no relation between sperm ATP levels and either swimming speed or percent of sperm that were motile. This suggests that interindividual variation in ATP levels may not be the primary determinant of variation in sperm swimming performance in bluegill.

Red blood cells of marine fish have been used in suitable biological assays to study the (eco)toxicity of wastewater treatment plant effluents. The aim of the present work was to draw upon their more relevant effects on cell hemolysis, ATP content, osmotic resistance and cell volume regulation. Following physico-chemical treatment, treatment plant effluents showed a residual toxicity resulting from multiple impairment of cell metabolism and structures. The earliest and most sensitive effects were related to the regulation of intracellular osmotic pressure leading to decreased cell water volume. Such effects were also observed following short-term incubation in 10-fold diluted effluent. Other damages were found following incubation in non-diluted effluent. Membrane structure was affected leading to increased osmotic resistance. Later, a decrease of the intracellular ATP level was found, followed by hemolysis. The presence of glucose in the incubation medium lessened the fall in ATP content and hemolysis in the treated cells but also in control cells.

This study reports the follow-up results of 36 highly bacillated untreated BL/LL cases who were serially allocated to three treatment groups. Group I patients received a modified WHO regimen (Rifampicin 600 mg once a month supervised, 50 mg of Clofazimine and 100 mg of Dapsone daily unsupervised) and BCG 0.1 mg per dose 6 monthly; group II patients received the same multi-drug treatment (MDT) and Mw (2 x 10(8) killed bacilli per dose) 6 monthly: group III patients received the same MDT with 0.1 ml of distilled water 6 monthly and acted as a control. Treatment was continued till smear negativity. All these three groups were comparable by their initial clinical score, bacteriological index (BI), viable bacilli as assessed by the mouse foot pad (MFP), bacillary adenosine triphosphate (ATP) content and also histologically at the time of starting treatment. All these parameters were evaluated every 6 months. The vaccines were well tolerated. All the patients in group I became smear negative by 3.5 years, in group II in 3 years whereas those in group III took 5 years. The incidence of reactions was the same in all the groups during the first 2 years, however, patients of group III (MDT + placebo) continued to have reactions up to 3 years. No viable bacilli could be detected in the local and distal sites as estimated by MFP and bacillary ATP after 12 months in both the immunotherapy groups. These could be detected in patients on MDT alone up to 24 months of therapy. Histologically patients in both the immunotherapy groups (groups I and II) showed accelerated granuloma clearance, histological upgrading and non-specific healing without granuloma formation both at the local and distal sites and this was achieved much earlier compared to the MDT + placebo group. Thus, by the addition of immunotherapy the effective treatment period of achieving bacteriological negativity could be reduced by about 40%, time period of reactions reduced by 33% and there were no reactions and/or relapses in the 10-12 years post-treatment follow-up.

The dynamics of energy production and utilization in fish eggs before and shortly after fertilization may be critical for embryo survival. Therefore, the current study examined the turnover of adenosine triphosphate (ATP) as well as examined the possible role and localization of ATP in unfertilized steelhead (Oncorhynchus mykiss) eggs and early embryos. The mean ATP level in unfertilized steelhead eggs was 1.92+/-0.10 (mean+/-S.E.M., n=17) nmol ATP per egg. Exposure of the unfertilized egg to 10 degrees C water (water activation) and fertilization resulted in comparable and substantial decreases (approx. 20-50%) in egg ATP levels within 3 min. This suggests that the energy expended at fertilization is used in response to water activation rather than fertilization per se. Unfertilized eggs maintained in ovarian fluid for 9 days at 10 degrees C under air showed a progressive decline of fertility that reached zero after 6 days. In contrast, no significant changes were seen in ATP levels throughout this 9 days period. Thus, fertility does not positively correlate with egg ATP levels in stored eggs. In the unfertilized egg, the ATP stored in the yolk accounted for approximately 1.5% of the total egg ATP. After fertilization, the concentration of ATP in the yolk increased approximately seven-fold, with the yolk and blastoderm each now accounting for approximately 20% of the total remaining ATP. Finally, to estimate the changes in oxidative metabolism following fertilization, the cyanide (KCN)-sensitive decline in total ATP was determined for unfertilized eggs and 1 day embryos. In the presence of KCN, ATP levels declined to approximately 50% within 24 h in both unfertilized eggs as well as embryos; the rates of ATP decline were not different. Therefore, there was not a discernible increase in ATP generation by oxidative phosphorylation at the time of fertilization.

The present study tests the hypothesis that cerebral tissue hypoxia results in increased Ca(2+)/calmodulin (CaM) kinase kinase activity and that the administration of nitric oxide synthase inhibitors (N-nitro-l-arginine [NNLA], or 7-nitroindazole sodium [7-NINA]) prior to the onset of hypoxia will prevent the hypoxia-induced increase in the enzyme activity. To test this hypothesis, CaM kinase kinase and CaM kinase IV activities were determined in normoxic, hypoxic, NNLA-treated hypoxic, and 7-NINA-treated hypoxic piglets. Hypoxia was induced (FiO(2)=0.05-0.08x1 h) and confirmed biochemically by tissue levels of ATP and phosphocreatine. CaM kinase kinase activity was determined in a medium containing protein kinase and phosphatase inhibitors, calmodulin, and a specifically designed CaM kinase kinase target peptide. CaM kinase IV activity was determined by (33)P-incorporation into syntide-2 in a buffer containing protein kinase and phosphatase inhibitors. Compared with normoxic animals, ATP and phosphocreatine levels were significantly lower in all hypoxic piglets whether or not pretreated with nitric oxide synthase inhibitors. There was a significant difference among CaM kinase kinase activity (pmol/mg protein/min) in normoxic (76.84+/-14.1), hypoxic (138.86+/-18.2, P<0.05 vs normoxia), NNLA-pretreated hypoxic (91.34+/-19.3; P=NS vs normoxia, P<0.05 vs hypoxia) and 7-NINA-pretreated hypoxic animals (100.12+/-23.3; P=NS vs normoxia, P<0.05 vs hypoxia). There was a significant difference among CaM kinase IV activity (pmol/mg protein/min) in normoxia (1270.80+/-126.1), hypoxia (2680.80+/-136.7; P<0.05 vs normoxia), NNLA-pretreated hypoxia (1666.00+/-154.8; P<0.05 vs normoxia, P<0.05 vs hypoxia), and 7-NINA-pretreated hypoxic (1712.9+/-231.5; P=NS vs normoxia, P<0.05 vs hypoxia). We conclude that the hypoxia-induced increase in CaM kinase kinase and CaM kinase IV activity is mediated by neuronal NOS-derived NO.

Acetyl-L-carnitine (ALCAR) plays an integral role in the transport of long chain fatty acids across the inner mitochondrial membrane for oxidative phosphorylation. In non-human primates, administration of ALCAR was reported to prevent 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurological injury to the substantia nigra. The present study investigates the effects of ALCAR against the toxicity of 1-methyl-4-phenylpyridinium (MPP(+)), the neurotoxic metabolite of MPTP, in murine brain neuroblastoma cells. MPP(+), a potent mitochondrial toxin, induced a dose-dependent reduction in mitochondrial oxygen consumption and cell viability, corresponding to an accelerated rate of cellular glucose utilization. Treatment with ALCAR, but not L-carnitine, prevented MPP(+) toxicity and partially restored intracellular ATP concentrations, but did not reverse the MPP(+)-induced loss of mitochondrial oxygen consumption. These data indicate that protective effects are independent of oxidative phosphorylation. ALCAR had a substantial glucose sparing effect in both controls and MPP(+)-treated groups, demonstrating a potential role in enhancing glucose utilization through glycolysis. Antagonizing the entry of fatty acids into the mitochondria, with either insulin or malonyl CoA, did not interfere with ALCAR protection against MPP(+). On the contrary, insulin potentiated the protective effects of ALCAR. In conclusion, these data indicate that ALCAR protects against MPP(+) toxicity, independent of mitochondrial oxidative capacity or beta-oxidation of fatty acids. In contrast, the protective effects of ALCAR appear to involve potentiation of energy derived from glucose through anaerobic glycolysis.

Glucose is the main substrate that fulfills energy brain demands. However, in some circumstances, such as diabetes, starvation, during the suckling period and the ketogenic diet, brain uses the ketone bodies, acetoacetate and beta-hydroxybutyrate, as energy sources. Ketone body utilization in brain depends directly on its blood concentration, which is normally very low, but increases substantially during the conditions mentioned above. Glutamate neurotoxicity has been implicated in neurodegeneration associated with brain ischemia, hypoglycemia and cerebral trauma, conditions related to energy failure, and to elevation of glutamate extracellular levels in brain. In recent years substantial evidence favoring a close relation between glutamate neurotoxic potentiality and cellular energy levels, has been compiled. We have previously demonstrated that accumulation of extracellular glutamate after inhibition of its transporters, induces neuronal death in vivo during energy impairment induced by glycolysis inhibition. In the present study we have assessed the protective potentiality of the ketone body, acetoacetate, against glutamate-mediated neuronal damage in the hippocampus of rats chronically treated with the glycolysis inhibitor, iodoacetate, and in hippocampal cultured neurons exposed to a toxic concentration of iodoacetate. Results show that acetoacetate efficiently protects against glutamate neurotoxicity both in vivo and in vitro probably by a mechanism involving its role as an energy substrate.

Modification of biomaterial surfaces is one approach aimed at improving cellular interactions and the subsequent integration of medical devices into the body. However, by optimising surfaces to enhance mammalian cell adhesion there is the potential risk that adhesion of bacteria will also be increased. Bacterial colonisation of biomaterials can be problematic as infection often results in morbidity and the consequent removal of the failing device from the body. Currently, quantifying cellular adhesion from a mixed population of bacterial and mammalian cells can only be determined by slow and laborious methods such as microscopy. ATP is a key molecule in the metabolism of both mammalian and bacterial cells and can be used to quantify cell numbers. In this study, we have modified a bioluminescence-based ATP assay to enable the differential measurement of both mammalian and bacterial cell ATP levels within the same culture. Mixed populations of Staphylococcus epidermidis and 3T3 fibroblasts were assessed both in suspension and adhered to a surface. ATP levels from cultures in suspension were selectively extracted and measured from both cell types, revealing a linear trend that would enable the differentiation of cell numbers from a mixed population. The application of the assay to adhered mixed cultures also allowed differences in ATP levels from both cell types to be distinguished. The data presented reveals that this assay would be useful for the rapid screening of cellular adhesion to modified surfaces although, its use in detecting subtle differences in ATP levels may be limited due to natural interactions between the two cell types., (Copyright 2002 Elsevier Science Ltd.)

The death of dopaminergic neurons induced by systemic administration of mitochondrial respiratory chain complex I inhibitors such as 1-methyl-4-phenylpyridinium (MPP(+); given as the prodrug 1-methyl-1,2,3,6-tetrahydropyridine) or the pesticide rotenone have raised the question as to whether this family of compounds are the cause of some forms of Parkinsonism. We have examined the neurotoxic potential of another complex I inhibitor, annonacin, the major acetogenin of Annona muricata (soursop), a tropical plant suspected to be the cause of an atypical form of Parkinson disease in the French West Indies (Guadeloupe). When added to mesencephalic cultures for 24 h, annonacin was much more potent than MPP(+) (effective concentration [EC(50)]=0.018 versus 1.9 microM) and as effective as rotenone (EC(50)=0.034 microM) in killing dopaminergic neurons. The uptake of [(3)H]-dopamine used as an index of dopaminergic cell function was similarly reduced. Toxic effects were seen at lower concentrations when the incubation time was extended by several days whereas withdrawal of the toxin after a short-term exposure (<6 h) arrested cell demise. Unlike MPP(+) but similar to rotenone, the acetogenin also reduced the survival of non-dopaminergic neurons. Neuronal cell death was not excitotoxic and occurred independently of free radical production. Raising the concentrations of either glucose or mannose in the presence of annonacin restored to a large extent intracellular ATP synthesis and prevented neuronal cell demise. Deoxyglucose reversed the effects of both glucose and mannose. Other hexoses such as galactose and fructose were not protective. Attempts to restore oxidative phosphorylation with lactate or pyruvate failed to provide protection to dopaminergic neurons whereas idoacetate, an inhibitor of glycolysis, inhibited the survival promoting effects of glucose and mannose indicating that these two hexoses acted independently of mitochondria by stimulating glycolysis. In conclusion, our study demonstrates that annonacin promotes dopaminergic neuronal death by impairment of energy production. It also underlines the need to address its possible role in the etiology of some atypical forms of Parkinsonism in Guadeloupe.

The effects of tributyltin (TBT) on the energy metabolism and motility of fish spermatozoa were investigated in vitro in African catfish and common carp. A significant (P<0.05) decrease of the duration and the intensity of motility was observed in catfish spermatozoa exposed to 0.27 microg/l TBT for 24 h. Exposure of catfish spermatozoa to 2.7-27 microg/l TBT caused an instant decrease in ATP content. In the presence of 27 microg/l TBT approximately 55% of the initial ATP concentration in catfish semen was lost after 60 min incubation while AMP concentrations increased and the total adenine nucleotide (TAN) pool remained unchanged. The reduction in sperm ATP levels could not be attributed to cell death since viability decreased only slightly over the period of exposure. In carp by contrast, none of the adenylates concentrations studied (ATP, ADP and AMP) were affected by TBT exposure at any experimental condition. However, carp sperm motility was significantly reduced by exposure to 2.7 microg/l TBT. Among the enzymes investigated only lactate dehydrogenase (LDH) in catfish sperm was significantly (P<0.01) affected by 27 microg/l TBT treatment with a reduction in activity of approximately 75%. Compared with carp sperm before TBT exposure, that of catfish had lower adenylate contents and overall lower enzymatic activities; this explains its slower sperm velocity and shorter duration of movement as measured by computer assisted sperm analysis (CASA). The present in vitro study shows that catfish spermatozoa are more sensitive to TBT exposure (and probably to other toxicants) than those of carp.

Objective: Glutamine enhances recovery from acute normothermic ischemia in isolated rat heart by a dose-dependent effect (Khogali et al. J Mol Cell Cardiol 1998;30:819). We compared the cardioprotective effects of equimolar concentrations of glutamine, glutamate, and aspartate in isolated rat heart. We also explored the potential cardioprotective effects of glutamine in patients with chronic stable angina., Methods: The isolated perfused working rat heart was subjected to ischemia, followed by reperfusion with or without an amino acid (2.5 mM). Patients with chronic stable angina received a single oral dose of glutamine (80 mg/kg) or placebo in a double-blind, random fashion 40 min before a standard Bruce exercise test., Results: Postischemic reperfusion of isolated rat heart with glutamine (but not with glutamate or aspartate) resulted in full recovery of cardiac output. Only glutamine prevented the decrease in the myocardial ratio between adenosine triphosphate to adenosine diphosphate and significantly enhanced the myocardial ratio of reduced to oxidized glutathione. A single oral dose of glutamine given to patients with chronic stable angina significantly increased plasma glutamine concentration from 419 to 649 microM and delayed time to onset of more than 1.0 mm of ST segment depression on the ECG by 38 s., Conclusion: Glutamine may be cardioprotective in patients with coronary heart disease.

Previous research has shown that the sulfonylurea derivative glibenclamide may improve post-ischemic cardiac functional recovery. Although K(ATP) channel blockade is a possible explanation for this observation, alternative mechanisms exist. Therefore, we simultaneously recorded cardiac function and the intracellular concentration of ATP, phosphocreatine, Pi and pH before and after ischemia in the presence of glibenclamide or vehicle. (31)Phosphorus magnetic resonance (MS) spectroscopy on erythrocyte-perfused, isolated working rat hearts was performed. Glibenclamide 4 micromol l(-1) or vehicle alone was tested (both n=5). The following protocol was used: 8 min performance assessment, 10 min drug treatment, 12 min global ischemia, 20 min reperfusion with drug treatment and 8 min functional recovery assessment. Compared with vehicle, glibenclamide significantly decreased coronary blood flow (59.5+/-7.0% vs. 94.3+/-1.3%, P=0.008), ischemia-induced cardiac functional loss (7.4+/-1.3% vs. 18.8+/-3.3%; P=0.019) as well as the ischemia-induced intracellular acidosis (6.75+/-0.01 vs. 6.43+/-0.03 for vehicle, P=0.03). In conclusion, glibenclamide is able to reduce the myocardial functional loss after ischemia while preserving pH but not ATP levels during ischemia. This suggests that the beneficial response to glibenclamide is probably not the result of myocardial K(ATP) channel blockade, but may be explained by inhibition of glycolysis.

3-Nitropropionic acid (3-NP), an irreversible inhibitor of succinate dehydrogenase, induced ATP depletion and both necrosis and apoptosis in human NT2-N neurons. Necrosis occurred predominantly within the first two days, and increased in a dose-dependent fashion with the concentration of 3-NP, whereas apoptosis was observed after 24 h or later at a similar rate in 0.1 mM and 5 mM 3-NP. We focused our efforts on intracellular calcium homeostasis during the first 48 h in 1 mM 3-NP, a period during which 10% of the neurons died by necrosis and 3% by apoptosis. All NT2-N neurons showed a stereotyped [Ca(2+)](i) rise, from 48+/-2 to 140+/-12 nM (mean +/-S.E.M.), during the first 2 h in 3-NP. Despite severe ATP depletion, however, [Ca(2+)](i) remained above 100 nM in only 17% and 25% of the NT2-N neurons after 24 and 48 h in 3-NP, respectively, indicating that most neurons were able to recover from acute [Ca(2+)](i) rise, and suggesting that chronic [Ca(2+)](i) dysregulation is a better indicator of subsequent necrosis. Blockade of N-methyl-D-aspartate-glutamate receptor by MK-801 substantially ameliorated 3-NP-induced ATP depletion, subsequent chronic [Ca(2+)](i) elevation, and survival. Moreover, xestospongin C, an inhibitor of endoplasmic reticulum Ca(2+) release, enhanced the capacity of NT2-N neurons to maintain [Ca(2+)](i) homeostasis and resist necrosis while subjected to sustained energy deprivation. As far as we know, this report is the first to employ human neurons to study the pathophysiology of 3-NP neurotoxicity.

The evolution of brain injury was examined in mice subjected to focal cerebral ischemia as induced by 30 min of intraluminar thread occlusion of the middle cerebral artery, followed by 3 h to 3 days of reperfusion. Metabolic dysfunctions were studied by 3H-leucine autoradiography for the measurement of cerebral protein synthesis and by regional ATP bioluminescent imaging. Metabolic changes were compared with responses of the genes c-fos, c-jun, heat-shock protein gene (hsp)72, p53-activated gene (pag)608 and caspase-3, which were investigated by in situ hybridization histochemistry and immunocytochemistry, and correlated with the degree of DNA fragmentation, as assessed by the terminal TdT-mediated dUTP-biotin nick end labeling method. Intraluminar thread occlusion led to a reproducible reduction of cerebral laser Doppler flow to 20-30% of control. Thread withdrawal was followed by a short-lasting post-ischemic hyperperfusion to approximately 120%. In non-ischemic control animals, fractional protein synthesis values of 0.81+/-0.26 and 0.94+/-0.23 were obtained. Thread occlusion resulted in a suppression of protein synthesis throughout the territory of the middle cerebral artery after 3 h of reperfusion (0.04+/-0.08 in caudate-putamen and 0.14+/-0.19 in somatosensory cortex, P<0.05). Protein synthesis partly recovered in the cortex after 24 h and 3 days (0.71+/-0.40 and 0.63+/-0.26, respectively), but remained suppressed in the caudate-putamen (0.14+/-0.22 and 0.28+/-0.28). Regional ATP levels did not show any major disturbances at the reperfusion times examined. Thread occlusion resulted in a transient increase of c-fos mRNA levels in ischemic and non-ischemic parts of the cortex and caudate-putamen at 3 h after ischemia, which suggests that spreading depressions were elicited in the tissue. At the same time, c-jun and hsp72 mRNAs were elevated only in ischemic brain areas showing inhibition of protein synthesis. C-fos and c-jun responses completely disappeared within 24 h of reperfusion. Hsp72 mRNA levels remained elevated in the cortex after 24 h, but decreased to basal values in the caudate-putamen. Twenty-four hours after reperfusion, pag608 and caspase-3 mRNA levels increased in the caudate-putamen, where protein synthesis rates were still reduced, and remained elevated even after 3 days. However, pag608 and caspase-3 mRNA levels did not increase in the cortex, where protein synthesis recovered. After 24 h and 3 days, functionally active p20 fragment of caspase-3 was detected in the caudate-putamen, closely associated with the appearance of DNA fragmented cells. Neither activated caspase-3 nor DNA fragmentation were noticed in the cortex.In summary, the suppression of protein synthesis is reversible in the ischemia-resistant cortex following 30 min of thread occlusion in mice, but persists in the vulnerable caudate-putamen. In the caudate-putamen, apoptotic programs are induced, closely in parallel with the manifestation of delayed cell death. Thus, the recovery of protein synthesis may be a major factor influencing tissue survival after transient focal ischemia.

The neurotoxic effects of low-level lead (Pb) during senescence are increasing interests of importance. We investigated the effects of low-level Pb on the brain in a normal condition and a pathophysiological condition of energy shortage that is commonly found in age-related neurological diseases. Middle-aged rats (15 months old) were exposed to 200 mg/l Pb acetate in drinking water for 2 months and thereafter received bilateral intracerebroventricular injections of streptozotocin (STZ). After 1 month's additional exposure to the same level of Pb solution as before the rats were sacrificed. Blood and brain Pb levels were measured by graphite furnace atomic absorption spectrophotometry. Energy-rich phosphate levels in the brain were determined by high-performance liquid chromatography equipped with a UV detector. Astroglial activation and glucose-regulated protein (GRP)94 expression were examined immunohistochemically. Exposure to Pb increased the blood Pb level to 10.8 microg/dl and the brain Pb level to 0.052 microg/g. But a significant additional increase in the brain Pb level, to 0.101 microg/g, became obvious in rats treated with Pb + STZ. Both Pb and STZ induced perturbation in brain energy metabolism, but no further alteration in energy metabolite levels was found in rats treated with Pb + STZ. Astroglial activation and GRP94-positive astrocytes and neurons were found only in the brains of Pb + STZ-treated rats. These results suggest that exposure to low-level Pb can perturb brain energy metabolism and the brain becomes more vulnerable to Pb when it is under energy stress.

We examined the effects of enalapril and 4'-[(1, 4'-dimethyl-2'-propyl-[2,6'-bi-1H-enzimidazole]-1'-yl)methyl]-[1, 1'-biphenyl]-2-carboxylic acid (BIBR-277), an angiotensin II receptor antagonist, on contractile dysfunction in the stunned myocardium. Dogs were subjected to 20-min ligation of the coronary artery, followed by 60-min reperfusion. Saline, enalapril (1 mg/kg or 3 mg/kg), or BIBR-277 (3 mg/kg) was injected i.v. 10 min before ligation. D-Arginyl-L-arginlyl-L-prolyl-trans-4-hydroxy-L-prolylglycyl -3-(2-thi enyl)-L-alanyl-L-seryl-D-1,2,3, 4-tetrahydro-3-isoquinolinecarbonyl-L-(2alpha, 3beta, 7abeta)-octahydro-1H-indole-2-carbonyl-L-arginine (Hoe-140), a bradykinin B(2) receptor antagonist, at 300 microg/kg was injected i. v. 10 min before drug injection. Contractile function was assessed on the basis of percentage segment shortening (%SS). ATP levels were measured in 60-min reperfused hearts. %SS significantly decreased during ischemia, and recovered during reperfusion, although the %SS was significantly less than the pre-ischemic level. Both enalapril at either dose and BIBR-277 significantly enhanced %SS recovery during reperfusion, an effect which was associated with a tendency toward energy preservation. Hoe-140 completely abolished the effect of enalapril at either dose, while it did not modify that of BIBR-277. Inhibition of angiotensin II formation and bradykinin breakdown may be separately related to the improvement of myocardial stunning.

Adenosine triphosphate (ATP) and catecholamine (CA) released from cultured porcine adrenal chromaffin cells were continuously measured with an ATP photometer (luciferin-luciferase method) and electrochemical detector, respectively. Application of acetylcholine (ACh, 0.1 mM) or high K+ (60 mM) caused increases of ATP and CA in perfused effluent with the same time course. The peak molar ratio of CA to ATP in the effluent was about 10 for ACh and high K+ stimulation. The high-performance liquid chromatographic (HPLC) analysis of adenine nucleotides in the collected effluent revealed that the relative amounts of ATP, ADP and AMP were almost the same throughout the period of stimulation, suggesting that ATP breakdown in the effluent was constant. Changes in the peak molar ratio of CA to ATP appearing in the effluent did not occur with repetitive high K+ or sustained Ba2+ stimulation (5 mM). The similarity between the time courses of ATP and CA appearing in the effluent suggests that releasable chromaffin granules have a constant molar ratio of CA to ATP. The on-line system developed is a simple and rapid method for examining ATP and CA secretion, simultaneously.

Photoactivated hypericin produces singlet oxygen and superoxide anion radical; however, the intracellular events contributing to toxicity are unknown. Clonogenic assays of oxygen-dependent hypericin phototoxicity to EMT6 cells have previously shown that 0.5 microM hypericin + 1.5 J cm(-2) fluorescent light is non-toxic and that 1.0 microM hypericin + 1.5 J cm(-2) fluorescent light produces LD40 toxicity. Intracellular events leading to toxicity were revealed at these doses. Lactate dehydrogenase leakage was elevated for both 0.5 microM and 1.0 microM hypericin + light immediately following irradiation. While values eventually returned to control levels for 0.5 microM hypericin + light, leakage increased over time for 1.0 microM hypericin indicating reversible and irreversible toxicity, respectively. Increases in lipid and protein oxidation were measured immediately following irradiation; however, these parameters return to control levels within 0.5 h for both doses. Both total cellular ATP levels and cellular respiration were depressed by approximately 50% of control values for 1.0 microM hypericin + light. These values were unchanged for 0.5 microM hypericin + light. Along with previously reported data demonstrating that light-activated hypericin can inhibit mitochondial succinoxidase in beef heart mitochondria in vitro, these data support oxidative stress-initiated mitochondrial damage as a key target in hypericin phototoxicity.

Anoxia/reoxygenation injury of isolated rat liver mitochondria was investigated. During anoxia of up to 60 min, the membrane potential was largely preserved and mitochondrial swelling was not observed. Reoxygenation of anoxic mitochondria rapidly caused swelling, cyclosporin A-sensitive Ca2+ efflux, [14C]sucrose trapping, and loss of the membrane potential along with increased generation of reactive oxygen intermediates (ROI). Although pretreatment with catalase and superoxide dismutase completely abolished reoxygenation-induced generation of ROI, mitochondrial damage was not prevented, as indicated by swelling, loss of the membrane potential, a decrease of the ATP content, and cyclosporin A-sensitive Ca2+ efflux. However, addition of the immunosuppressant cyclosporin A or addition of ADP completely prevented the mitochondrial damage induced by reoxygenation. The same protective effect was noted when Ca2+ cycling was prevented, either by chelating Ca2+ with EGTA or by inhibiting Ca2+ reuptake with ruthenium red. These findings indicate that mitochondrial anoxia/reoxygenation injury is caused by the cyclosporin A-sensitive and Ca2+-dependent membrane permeability transition. In contrast, reoxygenation injury does not appear to be triggered by the enhanced production of ROI.