Mitochondrial Electron Transport Activity Research Articles

A CRISPR screen identifies redox vulnerabilities for KEAP1/NRF2 mutant non-small cell lung cancer

The redox regulator NRF2 is hyperactivated in a large percentage of non-small cell lung cancer (NSCLC) cases, which is associated with chemotherapy and radiation resistance. To identify redox vulnerabilities for KEAP1/NRF2 mutant NSCLC, we conducted a CRISPR-Cas9-based negative selection screen for antioxidant enzyme genes whose loss sensitized cells to sub-lethal concentrations of the superoxide (O2•–) -generating drug β-Lapachone. While our screen identified expected hits in the pentose phosphate pathway, the thioredoxin-dependent antioxidant system, and glutathione reductase, we also identified the mitochondrial superoxide dismutase 2 (SOD2) as one of the top hits. Surprisingly, β-Lapachone did not generate mitochondrial O2•– but rather SOD2 loss enhanced the efficacy of β-Lapachone due to loss of iron-sulfur protein function, loss of mitochondrial ATP maintenance and deficient NADPH production. Importantly, inhibition of mitochondrial electron transport activity sensitized cells to β-Lapachone, demonstrating that these effects may be translated to increase ROS sensitivity therapeutically.

Bioenergetic and Metabolic Adaptation in Tumor Progression and Metastasis.

The ability of cancer cells to adjust their metabolism in response to environmental changes is a well-recognized hallmark of cancer. Diverse cancer and non-cancer cells within tumors compete for metabolic resources. Metabolic demands change frequently during tumor initiation, progression and metastasis, challenging our quest to better understand tumor biology and develop novel therapeutics. Vascularization, physical constraints, immune responses and genetic instability promote tumor evolution resulting in immune evasion, opportunities to breach basement membrane barriers and spread through the circulation and lymphatics. In addition, the unfolded protein response linked to the ubiquitin proteasome system is a key player in addressing stoichiometric imbalances between nuclear and mitochondrially-encoded protein subunits of respiratory complexes, and nuclear-encoded mitochondrial ribosomal protein subunits. While progressive genetic changes, some of which affect metabolic adaptability, contribute to tumorigenesis and metastasis through clonal expansion, epigenetic changes are also important and more dynamic in nature. Understanding the role of stromal and immune cells in the tumor microenvironment in remodeling cancer cell energy metabolism has become an increasingly important area of research. In this perspective, we discuss the adaptations made by cancer cells to balance mitochondrial and glycolytic energy metabolism. We discuss how hypoxia and nutrient limitations affect reductive and oxidative stress through changes in mitochondrial electron transport activity. We propose that integrated responses to cellular stress in cancer cells are central to metabolic flexibility in general and bioenergetic adaptability in particular and are paramount in tumor progression and metastasis.

Optimization of measurement of mitochondrial electron transport activity in postmortem human brain samples and measurement of susceptibility to rotenone and 4-hydroxynonenal inhibition

Mitochondrial function is required to meet the energetic and metabolic requirements of the brain. Abnormalities in mitochondrial function, due to genetic or developmental factors, mitochondrial toxins, aging or insufficient mitochondrial quality control contribute to neurological and psychiatric diseases. Studying bioenergetics from postmortem human tissues has been challenging due to the diverse range of human genetics, health conditions, sex, age, and postmortem interval. Furthermore, fresh tissues that were in the past required for assessment of mitochondrial respiratory function were rarely available. Recent studies established protocols to use in bioenergetic analyses from frozen tissues using animal models and cell cultures. In this study we optimized these methods to determine the activities of mitochondrial electron transport in postmortem human brain. Further we demonstrate how these samples can be used to assess the susceptibility to the mitochondrial toxin rotenone and exposure to the reactive lipid species 4-hydroxynonenal. The establishment of such an approach will significantly impact translational studies of human diseases by allowing measurement of mitochondrial function in human tissue repositories.

Environmental cocaine places mussels at risk by increasing neurotransmitters

When going to the beach for a swim, probably the last thing on your mind is whether you are swimming in a thin soup of drug traces. Whether prescribed over the counter or illegal, many drugs taken by people wind up in our waterways. These drugs cannot be removed by wastewater treatment plants and thus get released back into the environment. A famous coastal region in Brazil, the Bay of All Saints (Baía de Todos os Santos in Portuguese), has been the subject of multiple environmental contaminant studies and researchers have found measurable quantities of cocaine in the seawater, sediment and mussels (Perna perna) of the bay. Although the environmental concentrations are too low to affect humans, whether cocaine at these levels has an effect on the nervous system of aquatic species that reside in the bay was unknown. So, Mayana Fontes and a team of researchers from Universidade Estadual Paulista Júlio de Mesquita Filho, Brazil, and other universities in Brazil investigated how levels of cocaine in the seawater affect the nervous system of P. perna mussels.Fontes and colleagues placed adult mussels in tanks containing seawater with two different concentrations of cocaine that were realistic for the Bay of All Saints (0.2 and 2 μg l−1) for 2, 4 or 7 days. Then, they collected the adductor muscles, which open and close the shell, and gonads of these mussels. In the adductor muscle, they measured the activity of acetylcholine esterase, an enzyme essential for a functioning nervous system. In the gonads, they measured the levels of two major neurotransmitters, serotonin and dopamine, the activity of the enzyme that breaks down these neurotransmitters, monoamine oxidase, and the activity of cyclooxygenase, an enzyme that is a sign of inflammation and is important for egg release in mussels. In addition, the authors also measured how much energy the mussels spent during the exposure to cocaine by measuring mitochondrial electron transport activity, and how much energy they set aside by measuring the level of total lipids in the gonads.The team found that mussels exposed to cocaine had a decreased acetylcholine esterase activity in the adductor muscle, which shows that cocaine disrupted the normal activity of the nervous system. They also found that mussels exposed to cocaine had raised levels of dopamine and serotonin. These neurotransmitters are essential for reproduction, and changes could lead to an unsustainable population in the wild. The mussels also increased cyclooxygenase activity in the gonads, which shows an inflammatory response to low doses of cocaine. In addition, the scientists found changes in the mitochondrial electron transport chain – which converts the energy from oxygen into ATP in the mitochondria – directly increasing the amount of energy the mussels spent after exposure to the drug, which decreases the amount of energy they can access for reproduction as energy is limited. Finally, they also found that there was an increase in levels of total lipid, which act as a convenient and easy to access energy source for the animals.Fontes and colleagues have shown that the current concentration of cocaine in the seawater around the Bay of All Saints can negatively impact the area's mussels in complex ways and this may put them at a greater risk from other chemicals that we dump into the environment.

Prostate cancer cells survive anti-androgen and mitochondrial metabolic inhibitors by modulating glycolysis and mitochondrial metabolic activities.

Most cancer cells are more glycolytic even under aerobic conditions compared with their normal counterparts. Recent evidence of tumor cell metabolism, however, shows that some tumors also increase mitochondrial oxidative phosphorylation (ox-phos) at some disease states during progression and/or development of drug resistance. Our data show that anti-androgen enzalutamide (ENZA) resistant prostate cancer (PCa) cells use more mitochondrial metabolism leading to higher ox-phos as compared to the ENZA-sensitive cells and can become vulnerable to mitochondrial metabolism targeted therapies. Seahorse assay, mass spectrometry and high resolution fluorescence confocal microscopy coupled with image analysis has been used to compare mitochondrial metabolism in ENZA-treated and -untreated anti-androgen-sensitive LNCaP and -resistant C4-2, CWR22ν1, and PCa2b cells. Ex vivo fluorescence microscopy and image analysis has been standardized to monitor mitochondrial electron transport (ETS) activity that likely increases ox-phos in circulating tumor cells (CTCs) isolated fom patients undergoing AR-targeted therapies. Our data show that PCa cells that are resistant to anti-androgen ENZA switch from glycolysis to ox-phos leading to an increased ETS activity. ENZA pretreated cells are more vulnerable to ETS component complex I inhibitor IACS-010759 (IACS) and mitochondrial glutaminase inhibitor CB-839 that reduces glutamate supply to tricarboxylic acid cycle. CTCs isolated from 6 of 20 patient blood samples showed relatively higher ETS activity than the rest of the patients. All six patients have developed ENZA resistance within less than 6 months of the sample collection. The enhanced growth inhibitory effects of mitochondrial metabolic inhibitors IACS and CB-839 in ENZA pretreated PCa cells provides a rationale for designing a drug combination trial. Patients can be selected for such trials by monitoring the mitochondrial ETS activities in their CTCs to maximize success.

The influence of surface coatings on the toxicity of silver nanoparticle in rainbow trout

Silver nanoparticles (nAg) are often produced with different coatings that could influence bioavailability and toxicity in aquatic organisms. The purpose of this study was to examine the influence of 4 surface coatings of nAg of the same core size towards bioavailability and toxicity in juvenile rainbow trout (Oncorhynchus mykiss). Juveniles were exposed to 50 μg/L of 50 nm diameter nAg for 96 h at 15 °C with the following coatings: branched polyethylenimine (bPEI), citrate, polyvinylpyrrolidone (PVP) and silicate (Si). The data revealed that the coatings influenced hepatic Ag loadings in the following trend PVP > citrate > bPEI and Si with estimated bioavailability factors of 28, 18, 6 and 2 L/kg respectively. Hepatic Ag levels were significantly associated with DNA damage and inflammation as determined by arachidonate cyclooxygenase activity. The bPEI and citrate-coated nAg consistently produced the observed effects above in addition to increased mitochondrial electron transport activity and glutathione S-transferase activity. The absence of metallothionein and lipid peroxidation suggests that mechanisms other than the liberation of Ag+ were at play. In conclusion, surface coatings were shown to significantly influence bioavailability and toxic properties of nAg to rainbow trout juveniles.

Short Variant of Mitochondrial Dynamin OPA1 Renders Improved Cell Survival under Stress Conditions

The protein optic atrophy 1 (OPA1) is a dynamin‐related membrane remodeling protein associated with the inner mitochondrial membrane. OPA1 is a critical factor for mitochondrial structure and function by mediating mitochondrial inner membrane fusion and maintaining cristae structure. Under cellular stress associated with mitochondrial dysfunction, the inner membrane‐anchored long OPA1 (L‐OPA1) undergoes proteolytic cleavage to form short soluble OPA1 (S‐OPA1) lacking the transmembrane domain. The prevalent thought has been that S‐OPA1 is a functionally insignificant proteolytic product of L‐OPA1. Hence, the loss of L‐OPA1 and accumulation of S‐OPA1 by OPA1 cleavage under stress conditions is thought to be detrimental to cell survival. However, we recently demonstrated that S‐OPA1 is fully competent for maintaining energetics and cristae structure. Therefore, in this study, we used cells exclusively expressing L‐, S‐OPA1, or both, and tested the capacity of L‐ and S‐OPA1 in supporting cell survival under stress conditions. Under hydrogen peroxide‐mediated oxidant stress, we found that S‐OPA1‐expressing cells have better survival than L‐OPA1 cells. Interestingly, this difference in cell survival under oxidant stress was only observed in glucose‐free OXPHOS media but not in the presence of glucose, suggesting that S‐OPA1 may confer higher mitochondrial energetic activity compared with L‐OPA1. We also observed better survival of S‐OPA1‐expressing cells under nutrient starvation. Under apoptotic stress by treating cells with the transcription inhibitor actinomycin D, S‐OPA1 cells showed greatly decreased cytochrome c release compared to L‐OPA1 cells. These observations suggest that S‐OPA1 is more protective during apoptosis than L‐OPA1. Mechanistically, we found that S‐OPA1 cells have higher mitochondrial electron transport activity in nutrient depletion. Furthermore, by testing mitochondrial calcium retention capacity, we found that S‐OPA1 cells show increased resistance to mitochondrial permeability transition induced by mitochondrial calcium overload, compared with L‐OPA1 only cells. These data indicate that S‐OPA1 confers more efficient energy metabolism and more resistance to mitochondrial stress, allowing prolonged cell survival under stress.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Commitment to Aerobic Glycolysis Sustains Immunosuppression of Human Mesenchymal Stem Cells.

Human mesenchymal stem cells (hMSCs) promote endogenous tissue repair in part by coordinating multiple components of the host immune system in response to environmental stimuli. Recent studies have shown that hMSCs are metabolically heterogeneous and actively reconfigure metabolism to support the biochemical demands of tissue repair. However, how hMSCs regulate their energy metabolism to support their immunomodulatory properties is largely unknown. This study investigates hMSC metabolic reconfiguration during immune activation and provides evidence that the hMSC metabolic state significantly influences their immunomodulatory properties. Specifically, hMSC immune polarization by interferon‐gamma (IFN‐γ) treatment leads to remodeling of hMSC metabolic pathways toward glycolysis, which is required to sustain the secretion of immunosuppressive factors. IFN‐γ exposure also inhibited mitochondrial electron transport activity, and the accumulation of mitochondrial reactive oxygen species plays an important signaling role in this metabolic reconfiguration. The results also show that activation of the Akt/mTOR signaling pathway is required for metabolic reconfiguration during immune polarization and that interruption of these metabolic changes alters the immune response in IFN‐γ licensed hMSCs. The results demonstrate the potential of altering hMSC metabolism to enhance their immunomodulatory properties and therapeutic efficacy in various diseases. Stem Cells Translational Medicine 2019;8:93–106

P-211 - Oleic acid induces the novel Apolipoprotein O and reduces mitochondrial membrane potential in chicken and human hepatoma cells

Non-alcoholic fatty liver disease (NAFLD) reflects a spectrum of chronic liver diseases characterized by hepatic fat accumulation. Apolipoprotein O (ApoO) is a new member of the apolipoprotein family that may play a role in lipid metabolism and mitochondrial electron transport activity. We hypothesized that hepatic expression of ApoO is tightly linked not only to diet-induced hepatosteatosis, but also to oxidative stress and hormones. Therefore, we compared the effects of lipid loading on ApoO regulation in chicken (LMH) with those in human (HepG2) hepatoma cells. Incubation with oleic acid (OA) induced triglyceride accumulation but did not affect cell viability. RT-qPCR and Western blot analyses showed significant increase in ApoO transcript and protein levels in both cell lines. Oxidative stress applied by H2O2 revealed induction of ApoO in the same or even higher extent as monitored by OA. ApoO increased upon treatment with estrogen supporting the assumption that estrogen affects lipoprotein metabolism. Furthermore, both cell lines showed a significant decrease of the mitochondrial membrane potential upon incubation with OA. We assume that our findings support a role of ApoO as an effector of compromised mitochondrial function that likely accompanies the onset of NAFLD.

Calcium uptake and cytochrome c release from normal and ischemic brain mitochondria

At abnormally elevated levels of intracellular Ca2+, mitochondrial Ca2+ uptake may compromise mitochondrial electron transport activities and trigger membrane permeability changes that allow for release of cytochrome c and other mitochondrial apoptotic proteins into the cytosol. In this study, a clinically relevant canine cardiac arrest model was used to assess the effects of global cerebral ischemia and reperfusion on mitochondrial Ca2+ uptake capacity, Ca2+ uptake-mediated inhibition of respiration, and Ca2+-induced cytochrome c release, as measured in vitro in a K+-based medium in the presence of Mg2+, ATP, and NADH-linked oxidizable substrates. Maximum Ca2+ uptake by frontal cortex mitochondria was significantly lower following 10 min cardiac arrest compared to non-ischemic controls. Mitochondria from ischemic brains were also more sensitive to the respiratory inhibition associated with accumulation of large levels of Ca2+. Cytochrome c was released from brain mitochondria in vitro in a Ca2+-dose-dependent manner and was more pronounced following both 10 min of ischemia alone and following 24 h reperfusion, in comparison to mitochondria from non-ischemic Shams. These effects of ischemia and reperfusion on brain mitochondria could compromise intracellular Ca2+ homeostasis, decrease aerobic and increase anaerobic cerebral energy metabolism, and potentiate the cytochrome c-dependent induction of apoptosis, when re-oxygenated mitochondria are exposed to abnormally high levels of intracellular Ca2+.

Cadmium Alters Mitochondrial Membrane Potential, Inhibits Electron Transport Chain Activity and Induces Callose Deposition in Rice Seedlings

In the present study, seedlings of indica rice (Oryza sativa L.) cultivars were subjected to 100 and 200 µM Cd(NO3)2 treatment in hydroponics for 10 days which resulted in a marked decline in the growth of the seedlings compared to untreated controls. The integrity of the root cell plasma membrane was significantly decreased and high callose deposition in roots was observed. We observed that mitochondrial functioning was adversely affected in Cd-treated seedling marked by alteration of mitochondrial membrane potential and inhibition of electron transport chain activity. In particular, the activity of the electron transport chain Complex II was more inhibited with Cd treatment compared to the activities of Complex I and Complex IV. Transmission electron microscopic (TEM) analysis of rice leaves from control and Cd-treated seedlings showed significant alteration in membrane composition. Cd treatment at 100 and 200 µM resulted in partial to full disintegration of the mitochondrial membrane, respectively. The increased callose deposition might be a defense response against Cd toxicity. The findings indicate that Cd exposure to rice seedlings causes damage to mitochondria and inhibition of mitochondrial electron transport activity which would ultimately contribute to inhibited growth of rice plants in Cd-polluted soils.

Urban Pollution Increases Sensitivity to Temperature Changes in Elliptio complanata Mussels Exposed to Municipal Wastewaters

The cumulative effects of pollution in the context of global warming are not well understood at the present time. The purpose of this study was to determine whether organisms exposed to urban pollution are more susceptible to temperature changes. Feral Elliptio complanata freshwater mussels were caged and immersed for 1, 12 and 24 days in the final aeration pond treating domestic wastewaters from a township with a population of about 25,000. Mussels were also placed in the river upstream from the township to serve as reference. Following each exposure times, mussels were collected for temperature-dependent mitochondrial electron transport (METT) activity assessments. Other biomarkers were used, including xenobiotic metabolism (cytochrome P4503A4, glutathione S-tranferase and metallothioneins), energy budget (MET at 20oC and total lipids) and gonad status endpoints (gonadosomatic index [GSI], lipid peroxidation [LPO] and vitellogenin-like proteins). The data revealed that mussels exposed to the aeration pond had elevated METT and MET activities, suggesting that they were more sensitive to temperature changes and spent more energy than the controls. METT activity was negatively correlated with lipid stores in gonad, pointing to a negative energy budget. METT activity was also influenced by all xenobiotic metabolism and gonad status markers, with the exception of GSI. This suggests that temperature sensitivity is increased in mussels exposed to urban pollution and involves the expression of xenobiotic biotransformation, oxidative stress and endocrine disruption.

Mitochondrial electron transport activity and metabolism of experimentally bleached hermatypic corals

Bleached corals (Porites cylindrica and Galaxea fascicularis) were obtained through extended incubation (over 45days) under light depletion and privation: low light and dark conditions, and heat stress (32°C). The colonies in the different treatments became bleached and had reduced metabolic rates, photosynthesis, calcification and respiration; reduced biomass, zooxanthellae density, and chlorophyll a concentrations; and reduced mitochondrial electron transport system activity, which represent potential respiration rates. The most important reduction in mitochondrial electron transport activity was shown when the activities were normalized by the unit of surface and not by the unit of host protein. This result indicates that the reduction in activity could be mainly explained by the reduction of biomass and tissue thickness. However, increased Manganese Superoxide dismutase (Mn SOD) activity, a mitochondrial SOD, suggests that ROS production occurs in the mitochondria under heat stress with the consequence of potentially damaging the electron transport system. The reduced calcification rates observed are hypothesized to be the results of a decrease in the energy available for calcification due to the reduced photosynthetic rates, limiting the availability of substrates for respiration and therefore the energy production, and the decreased in the number of active mitochondrial electron transport system. Electron transport system activity associated with respiration is the basis of all metabolic processes and is not biased by incubation like traditional measurements of respiration in an aquarium. Therefore, ETSA could be used as an overall indicator of coral health, especially for host animal health.

Activation of mitochondrial STAT-3 and reduced mitochondria damage during hypothermia treatment for post-cardiac arrest myocardial dysfunction.

While therapeutic hypothermia improves the outcomes of individuals in cardiac arrest, the hemodynamic responses and mechanisms which underlie hypothermia-induced cardioprotection are not fully understood. Therefore, we investigated the mechanism by which induced hypothermia preserves cardiac function and protects against mitochondrial damage following cardiac arrest. Cardiac arrest was induced in adult male Wistar rats by asphyxiation for 8.5min. Following resuscitation, the animals were randomly assigned to a hypothermia (32°C) or normothermia (37°C) group. Monitoring results showed that cardiac output at the fourth hour after resuscitation was significantly better in rats treated with hypothermia when compared to rats treated with normothermia (P<0.01). Examinations by transmission electron microscopy showed that mitochondria in the left ventricle of rats in the hypothermia group were significantly less swollen compared to such mitochondria in the normothermia group (P<0.001). Additionally, opening of mitochondrial permeability transition pores occurred less frequently in the hypothermic group. While complex I/III activity in the electron transport reaction was damaged after cardiac arrest and resuscitation, the degree of injury was ameliorated by hypothermia treatment (P<0.05). The amount of STAT-3 phosphorylated at tyrosine 705 and its expression in mitochondria were significantly higher under hypothermia treatment compared to normothermia treatment. In vitro studies showed that inhibition STAT-3 activation abolished the ability of hypothermia to protect H9C2 cardiomyocytes against injury produced by simulated ischemia and reperfusion. Therapeutic hypothermia treatment can ameliorate cardiac dysfunction and help preserve both mitochondrial integrity and electron transport activity.

Adverse effects of wastewater discharges in reproduction, energy budget, neuroendocrine and inflammation processes observed in marine clams Ruditapes philippinarum

The present study investigated possible adverse outcomes in the marine clams Ruditapes philippinarum exposed to sediment affected by wastewater discharges at the Bay of Cádiz (SW, Spain). Six locations representing five cities were chosen for the sediment sampling during winter and summer seasons: P1 – Chiclana de la Frontera, P2 – Puerto Real, P3 – Cádiz, P4 and P5 – El Puerto de Santa María, P6 – Rota (reference site). Biochemical biomarkers were explored in clams after 14-days of exposure under controlled conditions, that included changes in cellular energy status (total lipids content – TLP and mitochondrial electron transport activity – MET), gametogenic activity (dopamine and ALP levels), metabolism of monoamines (monoamine oxidase activity – MAO), inflammation and spawning properties (cyclooxygenase activity – COX). Wastewater discharges induced energy budget alterations, as suggested by MET decrease (P4 and P5) and accumulation of TLP (P1, P2 and P3) in gonads. ALP levels (P1, P2 and P3), dopamine (P2) and COX activity (P1, P2, P3, P4 and P5) decreased in clams after the exposure to summer sediments. MAO increased in clams exposed to winter (P1 and P2) and summer (P3 and P4) sediments. Wastewater discharges composition changed between different seasons, mainly leading to oxidative stress, inflammation (COX activity and ALP levels) and spawning delay in summer. This study highlights the importance of considering reproduction of marine biota when assessing adverse effects of wastewater discharges. Continuous release of wastewater adequately threated or not, in aquatic ecosystems may culminate in adverse effects to the local benthic biota.

Toxicological evaluation of sediment samples spiked with human pharmaceutical products: Energy status and neuroendocrine effects in marine polychaetes Hediste diversicolor

There is a lack of studies about the ecotoxicology of pharmaceutical products on marine environment. To predict possible adverse effects of pharmaceutical products on benthic biota, polychaetes Hediste diversicolor were exposed for 14-days to pharmaceutical-spiked sediments under laboratory conditions. Carbamazepine (CBZ), ibuprofen (IBP) and propranolol (PRO) at concentrations of 500ngg−1, 50ngg−1, 5ngg−1, 0.5ngg−1 and 0.05ngg−1, fluoxetine (FX) and 17α-ethynylestradiol (EE2) at concentrations of 100ngg−1, 10ngg−1, 1ngg−1, 0.1ngg−1 and 0.01ngg−1, including environmental concentrations (underlined), were spiked in marine sediment samples. After the exposure, cellular energy status (total lipids content – TLP; and mitochondrial electron transport activity – MET), metabolism of monoamines (monoamine oxidase activity – MAO) and inflammation properties (cyclooxygenase activity – COX) were observed in polychaetes. CBZ increased TLP content and MET activity, and decreased MAO activity in polychaetes. IBP did not interfere on the TLP level, but on the MET and MAO activities (environmental concentrations). FX did not cause changes in the energy status. Therefore, environmental concentration diminished MAO activity. EE2 did not affect the energy status, however, MAO activity was significantly lower in polychaetes exposed to environmental concentration. PRO increased TLP level in polychaetes, but not MET activity. MAO activity was significantly lower for polychaetes exposed to environmental concentration. Except FX, all pharmaceuticals showed anti-inflammatory properties confirmed by the decrease of COX activity. Pharmaceutical products affected H. diversicolor physiology and health. As a benthic top predator, adverse effects on sea-worms can potentially culminate in ecosystem perturbations.

Functional Properties of Mitochondria in the Type-1 Cell and Their Role in Oxygen Sensing.

The identity of the oxygen sensor in arterial chemoreceptors has been the subject of much speculation. One of the oldest hypotheses is that oxygen is sensed through oxidative phosphorylation. There is a wealth of data demonstrating that arterial chemoreceptors are excited by inhibitors of oxidative phosphorylation. These compounds mimic the effects of hypoxia inhibiting TASK1/3 potassium channels causing membrane depolarisation calcium influx and neurosecretion. The TASK channels of Type-I cells are also sensitive to cytosolic MgATP. The existence of a metabolic signalling pathway in Type-1 cells is thus established; the contentious issue is whether this pathway is also used for acute oxygen sensing. The main criticism is that because cytochrome oxidase has a high affinity for oxygen (P50 ≈ 0.2 mmHg) mitochondrial metabolism should be insensitive to physiological hypoxia. This argument is however predicated on the assumption that chemoreceptor mitochondria are analogous to those of other tissues. We have however obtained new evidence to support the hypothesis that type-1 cell mitochondria are not like those of other cells in that they have an unusually low affinity for oxygen (Mills E, Jobsis FF, J Neurophysiol 35(4):405-428, 1972; Duchen MR, Biscoe TJ, J Physiol 450:13-31, 1992a). Our data confirm that mitochondrial membrane potential, NADH, electron transport and cytochrome oxidase activity in the Type-1 cell are all highly sensitive to hypoxia. These observations not only provide exceptionally strong support for the metabolic hypothesis but also reveal an unknown side of mitochondrial behaviour.

Comparative transcriptome analysis of short fiber mutants Ligon-lintless 1 and 2 reveals common mechanisms pertinent to fiber elongation in cotton (Gossypium hirsutum L.).

Understanding the molecular processes affecting cotton (Gossypium hirsutum) fiber development is important for developing tools aimed at improving fiber quality. Short fiber cotton mutants Ligon-lintless 1 (Li1) and Ligon-lintless 2 (Li2) are naturally occurring, monogenic mutations residing on different chromosomes. Both mutations cause early cessation in fiber elongation. These two mutants serve as excellent model systems to elucidate molecular mechanisms relevant to fiber length development. Previous studies of these mutants using transcriptome analysis by our laboratory and others had been limited by the fact that very large numbers of genes showed altered expression patterns in the mutants, making a targeted analysis difficult or impossible. In this research, a comparative microarray analysis was conducted using these two short fiber mutants and their near isogenic wild type (WT) grown under both field and greenhouse environments in order to identify key genes or metabolic pathways common to fiber elongation. Analyses of three transcriptome profiles obtained from different growth conditions and mutant types showed that most differentially expressed genes (DEGs) were affected by growth conditions. Under field conditions, short fiber mutants commanded higher expression of genes related to energy production, manifested by the increasing of mitochondrial electron transport activity or responding to reactive oxygen species when compared to the WT. Eighty-eight DEGs were identified to have altered expression patterns common to both short fiber mutants regardless of growth conditions. Enrichment, pathway and expression analyses suggested that these 88 genes were likely involved in fiber elongation without being affected by growth conditions.

Substrate ‐dependent Action of Isoflurane on Electron Transport Chain Complexes

The volatile anesthetic isoflurane (ISO) alters mitochondrial electron transport activity, which, in part, may underlie ISO‐induced cell protection. To unravel the mechanism of ISO effects on mitochondrial bioenergetics (ΔΨm, NADH and respiration), rat isolated cardiac mitochondria were energized with complex I (K+ ‐ pyruvate/malate; KPM) or complex II (K+ ‐succinate; KSuc) substrates ± sub‐maximal concentrations of ISO and the complex I and complex III blockers rotenone (ROT) and antimycin A (AA). Results showed that: (a) ISO decreased state 3 NADH oxidation and respiration, and increased the duration of state 3 ΔΨm with KPM; (b) ISO decreased state 3 respiration and the fall in ΔΨm, but did not decrease NADH oxidation with KSuc; (c) ISO increased state 3 ΔΨm duration but enhanced respiration with KSuc and ROT. These data indicate that: (1) ISO decreases the magnitude, while enhancing the duration, of state 3 NADH oxidation, ΔΨm and respiration, in part by inhibiting complex I activity with KPM; (2) ISO decreases KSuc supported production of NADH by attenuating complex I activity; (3) ISO increases state 3 ΔΨm duration but enhances respiration by attenuating complex III activity), with KSuc and ROT. Thus, isoflurane direct substrate‐dependent attenuation of complex I and complex III activities suggests a mitochondrial bioenergetic mechanism of cardioprotection during anesthetic exposure. (NIH Grants)

Energy status and immune system alterations in Elliptio complanata after ingestion of cyanobacteria Anabaena flos-aquae

Cyanobacteria have often been described as nutritionally poor for herbivorous organisms. To gain additional information on the potential impacts of invertebrates feeding on cyanobacteria, we fed Elliptio complanata mussels with two types of algae: Anabaena flos-aquae (cyanobacteria) and Pseudokirchneriella subcapitata (green algae). Physiological parameters were examined at the energy status, immune system and oxidative stress levels. Energy status was examined by following the rate of electron transport activity in mitochondria (a measure of cellular energy expense) and lipid/sugar stores in the visceral mass. The cyanobacteria were not actively producing toxins. Based on the digestive gland index, the mussels fed equally on either regime. However, the energy status in mussels fed A. flos-aquae revealed that the total sugar was lower in the digestive gland, whereas mitochondrial electron transport activity (MET), once corrected against the digestive gland somatic index, showed increased energy expenses. Acetylcholinesterase activity and lipid peroxidation (LPO) were also higher in mussels fed with A. flos-aquae compared with mussels fed with P. subcapitata. LPO was correlated by mitochondrial activity in both the digestive gland and gills, suggesting that oxidative stress resulted from metabolic respiration. Immunocompetence (phagocytic activity, natural killer cell-like activity, haemocyte count and viability) and humoral level of lysozyme were not affected in mussels by the algae or cyanobacteria regime. Moreover, the xenobiotic conjugating enzyme, glutathione S-transferase, hemoprotein oxidase and vitellogenin-like proteins were not affected in mussel organs via ingestion of A. flos-aquae. Our study suggests that ingestion of cyanobacteria leads to increased energy expenses, oxidative stress and increased acetylcholine turnover in mussels.