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Mitochondrial Activity (mitochondrial + activity)
Selected AbstractsMitochondrial Activity, Distribution and Segregation in Bovine Oocytes and in Embryos Produced in VitroREPRODUCTION IN DOMESTIC ANIMALS, Issue 1 2006AM Tarazona Contents Bovine oocytes and embryos produced in vitro were studied to determine the mitochondrial pattern of distribution, segregation and activity using DIOC 6 and Jc-1 fluorescence. The highest fluorescence level observed in mature oocytes was taken as 100% activity and six activity levels were estimated as follows: (1) 0%, (2) 1,15%, (3) 16,30%, (4) 31,50%, (5) 51,75% and (6) 76,100%. Three patterns of mitochondrial distribution were found: (1) diffused throughout the cytoplasm in oocytes and embryos, (2) pericytoplasmic in oocytes and embryos, and (3) perinuclear only in embryos. The segregation of mitochondria in blastomeres showed two distinct patterns: (1) symmetrical with an even mitochondrial population, and (2) asymmetrical with different numbers of mitochondria in each blastomere. In immature oocytes, mitochondrial activity was very low and the distribution was diffuse or negligible, while in mature oocytes the activity was high and the distribution was diffuse or pericytoplasmic. Competent embryos up to the 16-cell stage showed intermediate levels of activity (16,50%) but activity decreased thereafter up to the blastocyst stage. Non-competent embryos showed low levels of activity (1,15%) at all stages. These results suggest that mitochondria might play an important role during early development and that a minimum threshold of activity regulates the potential competence for reaching the blastocyst stage. [source] Correlation of the Mitochondrial Activity of Two-Cell Embryos Produced In Vitro and the Two-Cell Block In Kunming and B6C3F1 MiceTHE ANATOMICAL RECORD : ADVANCES IN INTEGRATIVE ANATOMY AND EVOLUTIONARY BIOLOGY, Issue 5 2009Shie Wang Abstract The correlation between the early embryonic block to development and mitochondrial activity was investigated comparing two-cell embryos produced in vitro from Kunming (KM) and B6C3F1 mice. One-cell embryos were obtained from two species of hybrids (female KM mice mated with KM males and female B6C3F1 mice mated with KM males) and cultured for 84 hr in M16 media. The mitochondrial membrane potential, ATP content, and reactive oxygen species levels were measured in the resulting KM and B6C3F1 two-cell embryos. Mitochondrial membrane potential and ATP content were also determined in KM and B6C3F1 metaphase II eggs. The results showed that the two-cell block was observed in cultured KM embryos but not in B6C3F1 embryos. Mitochondrial membrane potential and ATP content of KM two-cell embryos were significantly lower than in B6C3F1 two-cell embryos (P < 0.01). Interestingly, the reactive oxygen species levels of KM two-cell embryos were significantly lower than their B6C3F1 counterparts (P < 0.01). There was no difference in mitochondrial membrane potential and ATP content between KM and B6C3F1 metaphase II eggs. It is concluded that KM mice have an early two-cell embryo block and that a possible "blocking" mechanism is the lower mitochondrial membrane potential and ATP content in these embryos. The results suggest a new approach for overcoming early embryonic development block, that of manipulating mitochondrial activity. Anat Rec, 292:661,669, 2009. © 2009 Wiley-Liss, Inc. [source] Liver damage underlying unexplained transaminase elevation in human immunodeficiency virus-1 mono-infected patients on antiretroviral therapy,HEPATOLOGY, Issue 2 2009Patrick Ingiliz Liver damage associated with chronic unexplained high serum transaminases in human immunodeficiency virus (HIV)-infected patients under combined antiretroviral therapy is unknown. Liver histology was prospectively investigated in patients presenting serum transaminase elevation for more than 6 months, after exclusion of alcohol abuse, hepatitis C virus (HCV) or hepatitis B virus (HBV) infection, autoimmune, and genetic liver diseases. In a subgroup of patients, liver mitochondrial activities were measured by spectrophotometry and mitochondrial DNA (mtDNA) by real-time polymerase chain reaction (PCR). Thirty patients were included with median values of alanine aminotransferase (ALT) levels: 80 U/L, age: 46 years, body mass index: 23 kg/m2, HIV RNA: 200 copies/mL, CD4 count: 365/mm3, duration of HIV infection: 13 years, and duration of treatment exposure: 118, 41, and 53 months for nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and protease inhibitors, respectively. Histological anomalies were found in 22 of 30 patients. Steatosis was present in 18 patients, severe in nine patients, and associated with inflammation in 16 patients with a diagnosis of non-alcoholic steatohepatitis (NASH). Fibrosis was found in 18 patients, severe in six patients and associated with steatosis in 13 patients. Significant liver respiratory complex I defect, contrasting with high complex IV activity and normal mitochondrial DNA content, was observed in the group of patients compared with controls. The presence of NASH was correlated with high fasting glycemia and insulin levels, not with liver mitochondrial function or mitochondrial DNA content. Conclusions: HIV-infected patients on combined antiretroviral therapy with chronic transaminase elevation of unknown origin have a high rate of liver lesions, mostly consistent with NASH related to insulin resistance. (HEPATOLOGY 2008.) [source] Estrogenic control of mitochondrial function and biogenesisJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2008Carolyn M. Klinge Abstract Estrogens have cell-specific effects on a variety of physiological endpoints including regulation of mitochondrial biogenesis and activity. Estrogens regulate gene transcription by the classical genomic mechanism of binding to estrogen receptors , and , (ER, and ER,) as well as the more recently described nongenomic pathways involving plasma membrane-associated ERs that activate intracellular protein kinase-mediated phosphorylation signaling cascades. Here I will review the rapid and longer-term effects of estrogen on mitochondrial function. The identification of ER, and ER, within mitochondria of various cells and tissues is discussed with a model of estrogen regulation of the transcription of nuclear respiratory factor-1 (NRF-1, NRF1). NRF-1 subsequently promotes transcription of mitochondrial transcription factor Tfam (mtDNA maintenance factor, also called mtTFA) and then Tfam targets mtDNA-encoded genes. The nuclear effects of estrogens on gene expression directly controlling mitochondrial biogenesis, oxygen consumption, mtDNA transcription, and apoptosis are reviewed. Overall, we are just beginning to evaluate the many direct and indirect effects of estrogens on mitochondrial activities. J. Cell. Biochem. 105: 1342,1351, 2008. © 2008 Wiley-Liss, Inc. [source] Tocopheryl quinones and mitochondriaMOLECULAR NUTRITION & FOOD RESEARCH (FORMERLY NAHRUNG/FOOD), Issue 5 2010Lars Gille Abstract In the past, the role of tocopherols and tocopheryl hydroquinones as antioxidants in mitochondria has been examined. However, structural properties of tocopherols and tocopheryl quinones (arrangement of polar/apolar moieties) have also been recognized as being crucial for the selective transport of RRR-,-congeners compared with other tocopherols in the cell, suggesting that these properties might be generally important for the binding of vitamin E-related compounds to proteins and enzymes in mitochondria. Therefore, direct modulation of mitochondrial activities, such as bioenergetics, production of reactive oxygen species and apoptosis, not exclusively related to the redox activity of these compounds is increasingly studied. This overview focuses on the influence of ,-/,-tocopheryl quinones and their parent ,-/,-tocopherols on mitochondrial functions, including formation of tocopheryl quinones, their analytical aspects, their potential as alternative substrates and their inhibitory activity for some mitochondrial functions. It is shown that the understanding of how tocopheryl quinones and tocopherols interfere with mitochondrial functions on the molecular level is still incomplete and that a better comprehension requires further research activities. [source] Purification and enzymatic activity of an NADH-fumarate reductase and other mitochondrial activities of Leishmania parasites,APMIS, Issue 12 2001M. CHEN A 65 kD membrane-associated NADH-fumarate reductase subunit, which has a molecular weight similar to that of one of the enzyme subunits from bacteria, was purified from Leishmania donovani promastigotes. NADH-fumarate reductase and other mitochondrial enzymatic activities of L. major and L. donovani promastigotes and amastigotes were investigated. The presence of NADH-fumarate reductase was demonstrated in digitonin-permeabilized L. major promastigotes and mitochondria of L. major and L. donovani promastigotes and amastigotes. The activity of solubilized NADH-fumarate reductase was measured in L. major and L. donovani promastigotes. Succinate exhibited a clear concentration-dependent inhibitory effect on fumarate reductase, whereas fumarate also exhibited a clear concentration-dependent inhibitory effect on succinate dehydrogenase. The data indicate that fumarate reductase is an obligatory component of the respiratory chain of the parasite. Since the enzyme is an important component in the intermediate metabolism in the Leishmania parasite and is absent in mammalian cells, it could be a potential target for antileishmanial drugs. [source] Walker tumor cells express larger amounts of the antiapoptotic protein Bcl-2 and presents higher resistance to toxic concentrations of Ca2+ than the tumor cells K 562DRUG DEVELOPMENT RESEARCH, Issue 4 2001Graziela Milani Abstract Ca2+ homeostasis was studied in two tumor cell lines (Walker 256 and K 562) previously shown to exhibit different mitochondrial Ca2+ accumulation capacity. When intact, both cells present cytosolic Ca2+ concentrations within the range expected for mammalian cells, as determined through fura-2 fluorescence ratios. In order to study intracellular Ca2+ distribution, digitonin was used to permeabilize the plasma membrane without affecting intracellular organelle structure, as assessed using electron microscopy. Digitonin-permeabilized Walker 256 cells incubated with Ca2+ presented uptake of the cation exclusively through mitochondrial activity. In addition, very large Ca2+ loads were necessary to promote a disruption of Walker 256 mitochondrial membrane potential. K 562 cells presented active Ca2+ uptake through both nonmitochondrial and mitochondrial compartments and suffered disruption of mitochondrial membrane potential at lower Ca2+ loads than Walker 256 mitochondria. The higher Ca2+ resistance in Walker 256 cells could be attributed to Bcl-2 overexpression, as evidenced by immunocytochemical staining. Thus, we correlate natural Bcl-2 overexpression, observed in Walker 256 cells, with higher resistance to mitochondrial Ca2+ overload, as was shown previously in mitochondria from cells transfected with the bcl-2 gene. Drug Dev. Res. 52:508,514, 2001. © 2001 Wiley-Liss, Inc. [source] Oxylipin studies expose aspirin as antifungalFEMS YEAST RESEARCH, Issue 8 2007Johan L. F. Kock Abstract The presence of aspirin-sensitive 3-hydroxy fatty acids (i.e. 3-OH oxylipins) in yeasts was first reported in the early 1990s. Since then, these oxidized fatty acids have been found to be widely distributed in yeasts. 3-OH oxylipins may: (1) have potent biological activity in mammalian cells; (2) act as antifungals; and (3) assist during forced spore release from enclosed sexual cells (asci). A link between 3-OH oxylipin production, mitochondria and aspirin sensitivity exists. Research suggests that: (1) 3-OH oxylipins in some yeasts are probably also produced by mitochondria through incomplete ,-oxidation; (2) aspirin inhibits mitochondrial ,-oxidation and 3-OH oxylipin production; (3) yeast sexual stages, which are probably more dependent on mitochondrial activity, are also characterized by higher 3-OH oxylipin levels as compared to asexual stages; (4) yeast sexual developmental stages as well as cell adherence/flocculation are more sensitive to aspirin than corresponding asexual growth stages; and (5) mitochondrion-dependent asexual yeast cells with a strict aerobic metabolism are more sensitive to aspirin than those that can also produce energy through an alternative anaerobic glycolytic fermentative pathway in which mitochondria are not involved. This review interprets a wide network of studies that reveal aspirin to be a novel antifungal. [source] Experimental design comparison of studies evaluating doxorubicin nanoparticles in breast cancer therapyHUMAN FACTORS AND ERGONOMICS IN MANUFACTURING & SERVICE INDUSTRIES, Issue 3 2008Farman A. Moayed Background The unique properties of nanoparticles (NP) qualify these colloidal systems for a wide range of medical applications, including diagnosis and treatment. Particularly in cancer therapy, NP have significantly enhanced the potential of conventional imaging, radiotherapy, and chemotherapy and, consequently, offered new avenues for early interventions. So far, breast cancer has been one of the most studied cancer types with NP research, which can benefit the occupational breast cancer for the increasing number of women in the labor force in industry. Objectives The objective of this study is to compare the experimental designs of preclinical studies that assessed the effect of doxorubicin NP (DOX-NP) on the estrogen-dependent MCF-7 breast cancer cell line using a recently established quantitative Experimental Appraisal Instrument (ExpAI). Methods A systematic review of research articles published between August 2004 and August 2005 on NP and breast cancer treatment with doxorubicin was performed using various online databases and indexes available through the University of Cincinnati. Restrictive inclusion and exclusion criteria were defined leading to selection of four relevant articles that used comparable experimental designs. Critical appraisal of those studies was performed by five independent assessors using the ExpAI version 2.0 and the results were summarized in a table of evidence. Results The study design in the selected articles was either between groups or mixed, with sample sizes varying from n = 3,6, and the evaluation of the effect of DOX-NP either in vitro or in vivo. The cytotoxic drug doxorubicin was the input variable in all studies, whereas different end points such as pharmacokinetic parameters, cytotoxicity surrogates (e.g., growth inhibition, mitochondrial activity), and quantitative analysis of messenger RNA were used as output variables. Conclusions Although the articles assessed in this article were preclinical experimental studies, the results showed that doxorubicin NP drugs can be used effectively to enhance the delivery process in MCF-7 breast cancer cells by increasing the circulation time and targeting the tumor tissues. Considering the rising number of women in the labor force and the risk of occupational breast cancer, it can be concluded that DOX-NP may potentially be used as an effective anticancer drug on humans, but further research and studies are required to understand how DOX-NP drugs might react in the human body before using it on breast cancer patients. © 2008 Wiley Periodicals, Inc. [source] Enzymatic oxidation products of spermine induce greater cytotoxic effects on human multidrug-resistant colon carcinoma cells (LoVo) than on their wild-type counterpartsINTERNATIONAL JOURNAL OF CANCER, Issue 1 2002Annarica Calcabrini Abstract The occurrence of resistance to cytotoxic agents in tumor cells, associated with several phenotypic alterations, is one of the major obstacles to successful anticancer chemotherapy. A new strategy to overcome MDR of human cancer cells was studied, using BSAO, which generates cytotoxic products from spermine, H2O2 and aldehyde(s). The involvement of these products in causing cytotoxicity was investigated in both drug-sensitive (LoVo WT) and drug-resistant (LoVo DX) colon adenocarcinoma cells. Evaluation of clonogenic cell survival showed that LoVo DX cells are more sensitive than LoVo WT cells. Fluorometric assay and treatments performed in the presence of catalase demonstrated that the cytotoxicity was due mainly to the presence of H2O2. Cytotoxicity was eliminated in the presence of both catalase and ALDH. Transmission electron microscopic observations showed more pronounced mitochondrial modifications in drug-resistant than in drug-sensitive cells. Mitochondrial functionality studies performed by flow cytometry after JC-1 labeling revealed basal hyperpolarization of the mitochondrial membrane in LoVo DX cells. After treatment with BSAO and spermine, earlier and higher mitochondrial membrane depolarization was found in LoVo DX cells than in drug-sensitive cells. In addition, higher basal ROS production in LoVo DX cells than in drug-sensitive cells was detected by flow-cytometric analysis, suggesting increased mitochondrial activity in drug-resistant cells. Our results support the hypothesis that mitochondrial functionality affects the sensitivity of cells to the cytotoxic enzymatic oxidation products of spermine, which might be promising anticancer agents, mainly against drug-resistant tumor cells. © 2002 Wiley-Liss, Inc. [source] DROUGHT STRESS: Role of Carbohydrate Metabolism in Drought-Induced Male Sterility in Rice Anthers,JOURNAL OF AGRONOMY AND CROP SCIENCE, Issue 5 2010G. N. Nguyen Abstract Rice plants exposed to three consecutive days of water stress (,0.5 MPa) show a reduction in male fertility and grain set, which is attributed to increased levels of reactive oxygen species (ROS) and activation of a programmed cell death. This current research was conducted to further investigate the association of sugar metabolism with microspore abortion in rice anthers. Biochemical assays showed that sucrose, glucose and fructose contents were found to be significantly increased in anthers from water stressed plants compared with the control. qRT-PCR analyses and in situ hybridization of metabolic genes (sugar transporters, invertase and phosphotransferase/kinases) demonstrated that the supply of sugars for developing microspores and the initial steps of sugar utilization e.g. glycolysis, were not repressed. However, it appears that the accumulation of sugars in stressed anthers might involve a reduction of mitochondrial activity during the tricarboxylic acid cycle, which could result in excessive production of ROS and a depletion of the ATP pool. These results also suggest that higher levels of sugars at all stages of anther development seemed to be associated with some measure of protection to the anthers against oxidative stress. Induced expression of sugar transporter genes might have maintained the high levels of sugar in the tapetum and the locules, which alleviated oxidant damage caused by excessive ROS generation. Thus, the increased level of sugars might potentially be a natural response in providing protection against oxidant damage by strengthening the antioxidant system in anthers. [source] Neuronal expression of a single-subunit yeast NADH,ubiquinone oxidoreductase (Ndi1) extends Drosophila lifespanAGING CELL, Issue 2 2010Sepehr Bahadorani Summary The ,rate of living' theory predicts that longevity should be inversely correlated with the rate of mitochondrial respiration. However, recent studies in a number of model organisms, including mice, have reported that interventions that retard the aging process are, in fact, associated with an increase in mitochondrial activity. To better understand the relationship between energy metabolism and longevity, we supplemented the endogenous respiratory chain machinery of the fruit fly Drosophila melanogaster with the alternative single-subunit NADH,ubiquinone oxidoreductase (Ndi1) of the baker's yeast Saccharomyces cerevisiae. Here, we report that expression of Ndi1 in fly mitochondria leads to an increase in NADH,ubiquinone oxidoreductase activity, oxygen consumption, and ATP levels. In addition, exogenous Ndi1 expression results in increased CO2 production in living flies. Using an inducible gene-expression system, we expressed Ndi1 in different cells and tissues and examined the impact on longevity. In doing so, we discovered that targeted expression of Ndi1 in fly neurons significantly increases lifespan without compromising fertility or physical activity. These findings are consistent with the idea that enhanced respiratory chain activity in neuronal tissue can prolong fly lifespan. [source] The efficiency of mitochondrial electron transport chain is increased in the long-lived mrg19 Saccharomyces cerevisiaeAGING CELL, Issue 6 2009Nitish Mittal Summary Integrity of mitochondrial functionality is a key determinant of longevity in several organisms. In particular, reduced mitochondrial ROS (mtROS) production leading to decreased mtDNA damage is believed to be a crucial aspect of longevity. The generation of low mtROS was thought to be due to low mitochondrial oxygen consumption. However, recent studies have shown that higher mitochondrial oxygen consumption could still result in low mtROS and contribute to longevity. This increased mitochondrial efficiency (i.e. low mtROS generated despite high oxygen consumption) was explained as a result of mitochondrial biogenesis, which provides more entry points for the electrons to the electron transport chain (ETC), thereby resulting in low mtROS production. In this study, we provide evidence for the existence of an alternative pathway to explain the observed higher mitochondrial efficiency in the long-lived mrg19 mutant of Saccharomyces cerevisiae. Although we observe similar amounts of mitochondria in mrg19 and wild-type (wt) yeast, we find that mrg19 mitochondria have higher expression of ETC components per mitochondria in comparison with the wt. These findings demonstrate that more efficient mitochondria because of increased ETC per mitochondria can also produce less mtROS. Taken together, our findings provide evidence for an alternative explanation for the involvement of higher mitochondrial activity in prolonging lifespan. We anticipate that similar mechanisms might also exist in eukaryotes including human. [source] Muscle mitochondrial activity increases rapidly after an endotoxin challenge in human volunteersACTA ANAESTHESIOLOGICA SCANDINAVICA, Issue 3 2009K. FREDRIKSSON Background: Mitochondrial derangements in muscle of patients suffering from sepsis have been established in several studies and have been related to muscle dysfunction and organ failure. It is not possible to study the early phase of sepsis in patients; therefore, we used a human endotoxaemia model to study the effect of early sepsis on muscle mitochondria. Methods: Seven healthy male volunteers received a standardised endotoxin challenge. Muscle biopsies were obtained immediately before the challenge, and at 2 and 4 h following the endotoxin challenge. The muscle biopsies were analysed for maximal activities of citrate synthase and complexes I and IV of the respiratory chain. In addition, total and mitochondrial superoxide dismutase (SOD) activities were analysed. The concentrations of ATP, creatine phosphate and lactate were analysed to assess the cellular energy status. Total and phosphorylated AMP-activated protein kinase (AMPK-P), a key regulator in intracellular energy metabolism, was measured. Results: Activities of citrate synthase and complex I were significantly increased 2 h after the endotoxin challenge. SOD activities were unaffected by the endotoxin challenge. No changes in ATP, creatine phosphate or lactate were observed. Neither total nor AMPK-P changed. Conclusions: An endotoxin challenge given to healthy volunteers rapidly increases mitochondrial enzyme activity in skeletal muscle. The results of this human model indicate that possibly early during sepsis, mitochondrial activity might be increased in contrast to what has been shown in the later phases of sepsis. It is possible that this early activation leads to exhaustion of the mitochondria and a decreased function later during sepsis. [source] Insensitivity to glutamate neurotoxicity mediated by NMDA receptors in association with delayed mitochondrial membrane potential disruption in cultured rat cortical neuronsJOURNAL OF NEUROCHEMISTRY, Issue 5 2008Yuki Kambe Abstract We have attempted to elucidate mechanisms underlying differential vulnerability to glutamate (Glu) using cultured neurons prepared from discrete structures of embryonic rat brains. Brief exposure to Glu led to a significant decrease in the mitochondrial activity in hippocampal neurons cultured for 9 or 12 days at 10 ,M to 1 mM with an apoptosis-like profile, without markedly affecting that in cortical neurons. Brief exposure to Glu also increased lactate dehydrogenase release along with a marked decrease in the number of cells immunoreactive for a neuronal marker protein in hippocampal, but not cortical, neurons. Similar insensitivity was seen to the cytotoxicity by NMDA, but not to that by tunicamycin, 2,4-dinitrophenol, hydrogen peroxide or A23187, in cortical neurons. However, NMDA was more efficient in increasing intracellular free Ca2+ levels in cortical neurons than in hippocampal neurons. Antagonists for neuroprotective metabotropic Glu receptors failed to significantly affect the insensitivity to Glu, while NMDA was more effective in disrupting mitochondrial membrane potentials in hippocampal than cortical neurons. These results suggest that cortical neurons would be insensitive to the apoptotic neurotoxicity mediated by NMDA receptors through a mechanism related to mitochondrial membrane potentials, rather than intracellular free Ca2+ levels, in the rat brain. [source] Regional and cellular distribution of mitochondrial ferritin in the mouse brainJOURNAL OF NEUROSCIENCE RESEARCH, Issue 14 2010Amanda M. Snyder Abstract Iron and mitochondrial dysfunction are important in many neurodegenerative diseases. Several iron transport proteins have been identified that are associated with mitochondria, most recently mitochondrial ferritin. Here we describe the cellular distribution of mitochondrial ferritin in multiple regions of the brain in C57/BL6 mice. Mitochondrial ferritin was found in all regions of the brain, although staining intensity varied between regions. Mitochondrial ferritin was detected throughout the layers of cerebral cortex and in the cerebellum, hippocampus, striatum, choroid plexus, and ependymal cells. The cell type in the brain that stains most prominently for mitochondrial ferritin is neuronal, but oligodendrocytes also stain strongly in both gray matter and in white matter tracts. Mice deficient in H-ferritin do not differ in the mitochondrial ferritin staining pattern or intensity compared with C57/BL6 mice, suggesting that there is no compensatory expression of these proteins. In addition, by using inbred mouse strains with differing levels of iron content, we have shown that regional brain iron content does not affect expression of mitochondria ferritin. The expression of mitochondria ferritin appears to be more influenced by mitochondrial density. Indeed, at an intracellular level, mitochondrial ferritin immunoreaction product is strongest where mitochondrial density is high, as seen in the ependymal cells. Given the importance and relationship between iron and mitochondrial activity, understanding the role of mitochondrial ferritin can be expected to contribute to our knowledge of mitochondrial dysfunction and neurodegenerative disease. © 2010 Wiley-Liss, Inc. [source] Mercury compounds disrupt neuronal glutamate transport in cultured mouse cerebellar granule cellsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 4 2005Elena Fonfrķa Abstract Cerebellar granule cells are targeted selectively by mercury compounds in vivo. Despite the affinity of mercury for thiol groups present in all cells, the molecular determinant(s) of selective cerebellar degeneration remain to be elucidated fully. We studied the effect of mercury compounds on neuronal glutamate transport in primary cultures of mouse cerebellar granule cells. Immunoblots probed with an antibody against the excitatory amino acid transporter (EAAT) neuronal glutamate transporter, EAAT3, revealed the presence of a specific band in control and mercury-treated cultures. Micromolar concentrations of both methylmercury and mercuric chloride increased the release of endogenous glutamate, inhibited glutamate uptake, reduced mitochondrial activity, and decreased ATP levels. All these effects were completely prevented by the nonpermeant reducing agent Tris-(2-carboxyethyl)phosphine (TCEP). Reduction of mitochondrial activity by mercuric chloride, but not by methylmercury, was inhibited significantly by 4,4,-diisothiocyanato-stilbene-2,2,-disulfonic acid (DIDS) and by reduced extracellular Cl, ion concentration. In addition, DIDS and low extracellular Cl, completely inhibited the release of glutamate induced by mercuric chloride, and produced a partial although significant reduction of that induced by methylmercury. We suggest that a direct inhibition of glutamate uptake triggers an imbalance in cell homeostasis, leading to neuronal failure and Cl, -regulated cellular glutamate efflux. Our results demonstrate that neuronal glutamate transport is a novel target to be taken into account when assessing mercury-induced neurotoxicity. © 2005 Wiley-Liss, Inc. [source] The intensity of the 1602 cm,1 band in human cells is related to mitochondrial activityJOURNAL OF RAMAN SPECTROSCOPY, Issue 5 2009Vishnu Vardhan Pully Abstract We report a Raman band at 1602 cm,1 in the spectra of human cells, which previously had only been observed in mitochondria of yeast cells. This band, which has not yet been assigned to a particular molecular species, was found to occur in HeLa cells, peripheral blood lymphocytes, human mesenchymal stem cells and bovine chondrocytes. The band is proposed as an indicator of the activity of mitochondria in cells. Cells were cultured with and without serum or temporarily deprived of serum. The band can be observed for all these variations in cell culture methodology. The band intensity decreases under the influence of an increase of the calcium ion concentration in the surrounding medium. Copyright © 2009 John Wiley & Sons, Ltd. [source] Cultured epithelial cells response to phototherapy with low intensity laser,LASERS IN SURGERY AND MEDICINE, Issue 4 2007Fernanda P. Eduardo PhD Abstract Background and Objectives Little is known about the intracellular response of epithelial cells to phototherapy. The aim of this in vitro study was to analyze the effect of phototherapy with low-energy lasers with different wavelengths and powers on cultured epithelial cell growth under different nutritional conditions. Study Design/Materials and Methods Epithelial cell cultures (Vero cell line) grown in nutritional deficit in culture medium supplemented with 2% fetal bovine serum (FBS) were irradiated with low-energy laser from one to three times with a GaAlAs laser (660 nm) and InGaAlP (780 nm), 40 and 70 mW, respectively, with 3 or 5 J/cm2. Cell growth was indirectly assessed by measuring the cell mitochondrial activity. Results Nonirradiated cell cultures grown in nutritional regular medium supplemented with 10% FBS produced higher cell growth than all cultures grown in nutritional deficit irradiated or not. The overall cell growth of cultures grown under nutritionally deficit conditions was significantly improved especially when irradiated with 780 nm for three times. Conclusions Phototherapy with the laser parameters tested increases epithelial cell growth rate for cells stressed by growth under nutritionally deficient states. This cell growth improvement is directly proportional to the number of irradiations; however, was not enough to reach the full cell growth potential rate of Vero epithelial cell line observed when growing under nutritional regular condition. Lasers Surg. Med. 39: 365,372, 2007. © 2007 Wiley-Liss, Inc. [source] Induction of apoptosis in Caco-2 and HT-29 human intestinal epithelial cells by enterohemolysin produced by classic enteropathogenic Escherichia coliLETTERS IN APPLIED MICROBIOLOGY, Issue 4 2007P.M.S. Figueiredo Abstract Aims:, Detect the cytotoxic effects of the Enterohemolysin from enteropathogenic Escherichia coli C3888 (O 26: H,) on Caco 2 and HT-29-human epithelial intestinal cells. Methods and Results:, The Caco 2 and HT-29 cells, which were treated with Enterohemolysin (EHly) within 10,15 min, became round, lost attachment to substrate, showed extensive surface blebbing, nucleus shrank, and the chromatin became more compact. After 10 min of exposure to the EHly, the cells showed lactate dehydrogenase (LDH) leakage and reduction of mitochondrial activity. The cells showed disorganization of the actin fibers at 15 min. The death of these human epithelial intestinal cells by apoptosis was confirmed by annexin V. Conclusions:, Enterohemolysin induced apoptosis on human epithelial intestinal cells. Significance and Impact of the Study:, The finding of EHly cytotoxic activity suggests the involvement of this hemolysin in the (Enteropathogenic Escherichia coli) EPEC infection mechanism and may facilitate the understanding of the diarrhea caused by EPEC. [source] Molecular control of mitochondrial function in preimplantation mouse embryosMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 4 2005Jacob Thundathil Abstract Mitochondria play a key role in a number of physiological events during all stages of life, including the very first stages following fertilization. It is, therefore, important to understand the mechanisms controlling mitochondrial activity during early embryogenesis to determine their role in development outcome. The objective of this study was to investigate the molecular control of mitochondrial transcription and mitochondrial DNA (mtDNA) replication in mouse preimplantation embryos. We estimated the mtDNA copy number and characterized the expression patterns of two mitochondrial genes and several nuclear genes that encode mitochondrial transcription and replication factors throughout preimplantation development. Mitochondrial gene transcripts were present in larger quantities in morula and blastocyst stage embryos relative to other stages. A significant increase in the amount of mRNA for nuclear genes encoding mtDNA transcription factors was observed in eight-cell stage embryos. Although a similar increase in the mRNA levels of nuclear genes encoding mtDNA replication factors was observed in morula and blastocyst stage embryos, the number of mtDNA molecules remained stable during preimplantation stages, suggesting that nuclear-encoded mitochondrial transcription factors are involved in the regulation of mtDNA transcription during early development. Although transcripts of replication factors are abundant at the morula and blastocyst stage, mtDNA replication did not occur until the blastocyst stage, suggesting that the inhibition of mtDNA replication is controlled at the post-transcriptional level during early embryogenesis. Mol. Reprod. Dev. © 2005 Wiley-Liss, Inc. [source] Resveratrol induces apoptosis and inhibits adipogenesis in 3T3-L1 adipocytes ,PHYTOTHERAPY RESEARCH, Issue 10 2008Srujana Rayalam Abstract Resveratrol, a phytoallexin, has recently been reported to slow aging by acting as a sirtuin activator. Resveratrol also has a wide range of pharmacological effects on adipocytes. In this study, we investigated the effects of resveratrol on adipogenesis and apoptosis using 3T3-L1 cells. In mature adipocytes, 100 and 200 µM resveratrol decreased cell viability dose-dependently by 23 ± 2.7%, and 75.3 ± 2.8% (p < 0.0001), respectively, after 48 h treatment, and 100 µM resveratrol increased apoptosis by 76 ± 8.7% (p < 0.0001). Resveratrol at 25 and 50 µM decreased lipid accumulation in maturing preadipocytes significantly by 43 ± 1.27% and 94.3 ± 0.3% (p < 0.0001) and decreased cell viability by 25 ± 1.3% and 70.4 ± 1.6% (p < 0.0001), respectively. In order to understand the anti-adipogenic effects of resveratrol, maturing 3T3-L1 preadipocytes were treated with 25 µM resveratrol and the change in the expression of several adipogenic transcription factors and enzymes was investigated using real-time RT-PCR. Resveratrol down-regulated the expression of PPAR,, C/EBP,, SREBP-1c, FAS, HSL, LPL and up-regulated the expression of genes regulating mitochondrial activity (SIRT3, UCP1 and Mfn2). These results indicate that resveratrol may alter fat mass by directly affecting cell viability and adipogenesis in maturing preadipocytes and inducing apoptosis in adipocytes and thus may have applications for the treatment of obesity. Copyright © 2008 John Wiley & Sons, Ltd. [source] Mitochondrial Activity, Distribution and Segregation in Bovine Oocytes and in Embryos Produced in VitroREPRODUCTION IN DOMESTIC ANIMALS, Issue 1 2006AM Tarazona Contents Bovine oocytes and embryos produced in vitro were studied to determine the mitochondrial pattern of distribution, segregation and activity using DIOC 6 and Jc-1 fluorescence. The highest fluorescence level observed in mature oocytes was taken as 100% activity and six activity levels were estimated as follows: (1) 0%, (2) 1,15%, (3) 16,30%, (4) 31,50%, (5) 51,75% and (6) 76,100%. Three patterns of mitochondrial distribution were found: (1) diffused throughout the cytoplasm in oocytes and embryos, (2) pericytoplasmic in oocytes and embryos, and (3) perinuclear only in embryos. The segregation of mitochondria in blastomeres showed two distinct patterns: (1) symmetrical with an even mitochondrial population, and (2) asymmetrical with different numbers of mitochondria in each blastomere. In immature oocytes, mitochondrial activity was very low and the distribution was diffuse or negligible, while in mature oocytes the activity was high and the distribution was diffuse or pericytoplasmic. Competent embryos up to the 16-cell stage showed intermediate levels of activity (16,50%) but activity decreased thereafter up to the blastocyst stage. Non-competent embryos showed low levels of activity (1,15%) at all stages. These results suggest that mitochondria might play an important role during early development and that a minimum threshold of activity regulates the potential competence for reaching the blastocyst stage. [source] Assessment of Different Sperm Quality Parameters to Predict in vitro Fertility of BullsREPRODUCTION IN DOMESTIC ANIMALS, Issue 3 2002S Tanghe Contents Frozen-thawed semen from six bulls with high (> 60%) and low (20,35%) in vitro fertility was used for studying the predictive value of simple sperm quality tests with respect to in vitro fertilization (IVF) outcome as assessed by pronucleus (PN) formation ability. Sperm quality parameters, such as sperm concentration, motility, progressive motility, live-dead sperm ratio, morphology, membrane integrity, mitochondrial activity and acrosomal status were analysed using both conventional and automatic techniques at three time points during the IVF process, namely after sperm thawing, Percoll differential gradient centrifugation and IVF. Associations between the sperm quality parameters before and after IVF, and PN formation ability were assessed by using linear regression analyses. The percentages of motility, progressive motility and normal morphology determined after sperm thawing, and the percentage of live spermatozoa assessed after Percoll preparation by using nigrosin-eosin (N-E) staining showed a good correlation with PN formation ability, but the regression parameters were borderline not significant. These parameters formed the most reliable basis for predicting IVF outcome. After IVF, the percentage of live spermatozoa determined by using N-E staining was the only sperm quality parameter showing a significant association with the PN formation ability of a given bull. This sperm quality test can be used as a non-invasive method to estimate the PN formation ability of oocytes which are further cultured to assess embryonic development. [source] Correlation of the Mitochondrial Activity of Two-Cell Embryos Produced In Vitro and the Two-Cell Block In Kunming and B6C3F1 MiceTHE ANATOMICAL RECORD : ADVANCES IN INTEGRATIVE ANATOMY AND EVOLUTIONARY BIOLOGY, Issue 5 2009Shie Wang Abstract The correlation between the early embryonic block to development and mitochondrial activity was investigated comparing two-cell embryos produced in vitro from Kunming (KM) and B6C3F1 mice. One-cell embryos were obtained from two species of hybrids (female KM mice mated with KM males and female B6C3F1 mice mated with KM males) and cultured for 84 hr in M16 media. The mitochondrial membrane potential, ATP content, and reactive oxygen species levels were measured in the resulting KM and B6C3F1 two-cell embryos. Mitochondrial membrane potential and ATP content were also determined in KM and B6C3F1 metaphase II eggs. The results showed that the two-cell block was observed in cultured KM embryos but not in B6C3F1 embryos. Mitochondrial membrane potential and ATP content of KM two-cell embryos were significantly lower than in B6C3F1 two-cell embryos (P < 0.01). Interestingly, the reactive oxygen species levels of KM two-cell embryos were significantly lower than their B6C3F1 counterparts (P < 0.01). There was no difference in mitochondrial membrane potential and ATP content between KM and B6C3F1 metaphase II eggs. It is concluded that KM mice have an early two-cell embryo block and that a possible "blocking" mechanism is the lower mitochondrial membrane potential and ATP content in these embryos. The results suggest a new approach for overcoming early embryonic development block, that of manipulating mitochondrial activity. Anat Rec, 292:661,669, 2009. © 2009 Wiley-Liss, Inc. [source] Enhancement of recombinant protein production in Chinese hamster ovary cells through anti-apoptosis engineering using 30Kc6 geneBIOTECHNOLOGY & BIOENGINEERING, Issue 3 2006Shin Sik Choi Abstract It was previously reported that silkworm hemolymph (SH) inhibits apoptosis and increases the production of recombinant human erythropoietin (EPO) in Chinese hamster ovary (CHO) cells. The apoptosis-inhibiting component in SH is a member of 30K protein family. In this study, the CHO cell line producing EPO was manipulated genetically to express the 30Kc6 gene encoding a 30K protein in the hemolymph of the silkworm, Bombyx mori. The transient expression of 30Kc6 significantly suppressed the cell death induced by serum deprivation. A stable cell line expressing 30Kc6 with an anti-apoptotic property was established. The stable expression of 30Kc6 inhibited serum-deprivation-induced apoptosis and increased the cell density and EPO titer by 5- and 10-fold, respectively. The positive effects of the 30Kc6 expression on cell viability and productivity were due to the stable maintenance of the mitochondrial activity. The 30Kc6 expression efficiently suppressed the depolarization of the mitochondrial membrane and subsequently balanced the generation/consumption of ATP. The use of the 30Kc6 gene is expected to provide a new method of host cell engineering for improving the productivity of the recombinant protein. © 2006 Wiley Periodicals, Inc. [source] SUMOylation and cell signallingBIOTECHNOLOGY JOURNAL, Issue 12 2009Artemisia M. Andreou Abstract SUMOylation is a highly transient post-translational protein modification. Attachment of SUMO to target proteins occurs via a number of specific activating and ligating enzymes that form the SUMO-substrate complex, and other SUMO-specific proteases that cleave the covalent bond, thus leaving both SUMO and target protein free for the next round of modification. SUMO modification has major effects on numerous aspects of substrate function, including subcellular localisation, regulation of their target genes, and interactions with other molecules. The modified SUMO-protein complex is a very transient state, and it thus facilitates rapid response and actions by the cell, when needed. Like phosphorylation, acetylation and ubiquitination, SUMOylation has been associated with a number of cellular processes. In addition to its nuclear role, important sides of mitochondrial activity, stress response signalling and the decision of cells to undergo senescence or apoptosis, have now been shown to involve the SUMO pathway. With ever increasing numbers of reports linking SUMO to human disease, like neurodegeneration and cancer metastasis, it is highly likely that novel and equally important functions of components of the SUMOylation process in cell signalling pathways will be elucidated in the near future. [source] Insulin-like growth factor-induced signals activate mitochondrial respirationBIOTECHNOLOGY JOURNAL, Issue 6 2008Hermann Unterluggauer Abstract From experiments with lower eukaryotes it is known that the metabolic rate and also the rate of aging are tightly controlled by the insulin-like growth factor (IGF)/insulin signal transduction pathway. The mitochondrial theory of aging implies that an increased metabolic rate leads to increased mitochondrial activity; increased production of reactive oxygen species due to these alterations would speed up the aging process. To address the question if mitochondrial activity is influenced by insulin/IGF signaling, we have established an experimental system to determine the influence of IGF-I-dependent signaling on mitochondrial function. We used DU145 prostate cancer cells, known for the intact IGF signal transduction pathway, to address the influence of IGF receptor activation on mitochondrial function by high-resolution respirometry. These experiments revealed that indeed mitochondrial function is regulated by IGF signaling, and up-regulation of respiration seems to require phosphoinositide 3-kinase/AKT signaling, but is independent of IGF effects on cell cycle progression. Collectively these data establish a regulatory cross-talk between insulin/IGF signal transduction and mitochondrial function, two major pathways implicated in controlling the rate of aging. [source] Regulation of retinal ganglion cell gene expression by bHLH transcription factors in the developing and ischemic retinasACTA OPHTHALMOLOGICA, Issue 2009JM MATTER Purpose The loss of retinal ganglion cells (RGC) in the glaucomatous retina exhibits similarities to the pattern of neuronal degeneration detected after experimental ischemia. However, a short episode of retinal ischemia does not provoke damage but rather triggers an endogenous form of neuroprotection. HIFs are bHLH proteins that regulate hypoxic response in ischemic retinas and they are involved in neuroprotection. Hypoxic environments also occur in the developing embryo and create specific niches controlling cell differentiation. Genetic analyses of HIF functions have revealed the importance of oxygen as a key regulator of ontogeny. We have compared the transcriptomes of RGCs in ischemic versus developing retinas. Methods Genome-wide screens were conducted to identify genes which are expressed in newborn RGCs and growing optic nerve axons and which are up- or down-regulated after venal occlusion by photodynamic thrombosis in the rat retinas. Results Atoh7 is a bHLH protein which is central to the transcriptional network regulating the production of RGCs. Among the targets of Atoh7 there are genes involved in the general metabolism and energy supply , e.g., alpha-enolase (ENO1), glucose-6 -phosphate isomerase (GPI). These glycolytic enzymes are also targets of HIFs and they are upregulated during hypoxia. To investigate the linkage of glycolysis and mitochondrial activity in RGCs, we monitored by confocal time-lapse imaging the dynamic distribution of mitochondria in the cell bodies and axons of RGCs that express HIF/Atoh7 targets in developing and ischemic retinas. Conclusion Some gene expression programs involved in differentiating RGCs might be reinitiated in neuroprotection. [source] Gene Therapy in HIV-Infected Cells to Decrease Viral Impact by Using an Alternative Delivery MethodCHEMMEDCHEM, Issue 6 2010Teresa Gonzalo Dr. Abstract The ability of dendrimer 2G-[Si{O(CH2)2N(Me)2+(CH2)2NMe3+(I,)2}]8 (NN16) to transfect a wide range of cell types, as well as the possible biomedical application in direct or indirect inhibition of HIV replication, was investigated. Cells implicated in HIV infection such as primary peripheral blood mononuclear cells (PBMC) and immortalized suspension cells (lymphocytes), primary macrophages and dendritic cells, and immortalized adherent cells (astrocytes and trophoblasts) were analyzed. Dendrimer toxicity was evaluated by mitochondrial activity, cell membrane rupture, release of lactate dehydrogenase, erythrocyte hemolysis, and the effect on global gene expression profiles using whole-genome human microarrays. Cellular uptake of genetic material was determined using flow cytometry and confocal microscopy. Transfection efficiency and gene knockdown was investigated using dendrimer-delivered antisense oligonucleotides and small interfering RNA (siRNA). Very little cytotoxicity was detected in a variety of cells relevant to HIV infection and erythrocytes after NN16 dendrimer treatment. Imaging of cellular uptake showed high transfection efficiency of genetic material in all cells tested. Interestingly, NN16 further enhanced the reduction of HIV protein 24 antigen release by antisense oligonucleotides due to improved transfection efficiency. Finally, the dendrimer complexed with siRNA exhibited therapeutic potential by specifically inhibiting cyclooxygenase-2 gene expression in HIV-infected nervous system cells. NN16 dendrimers demonstrated the ability to transfect genetic material into a vast array of cells relevant to HIV pathology, combining high efficacy with low toxicity. These results suggest that NN16 dendrimers have the potential to be used as a versatile non-viral vector for gene therapy against HIV infection. [source] | |||||||||||||||||||||||||||||||