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Spectroscopy in vacuum ultraviolet (VUV) and visible ranges plays an important role in the investigation and diagnosis of tokamak plasmas. However, under harsh environmental conditions of fusion grade devices, such as ITER, VUV–visible systems encounter many issues due to the degradation of optical components used in such systems. Here, near-infrared (NIR) spectroscopy has become an effective tool in understanding the edge plasma dynamics. Considering its importance, a NIR spectroscopic diagnostic has been developed and installed on the ADITYA-U tokamak. The system consists of a 0.5 m spectrometer having three gratings with different groove densities, and it is coupled with a linear InGaAs photodiode array. Radiation from the ADITYA-U edge plasma has been collected using a collimating lens and optical fiber combination and transported to the spectrometer. The spectrum in the NIR range from the ADITYA-U plasma has been recorded using this system, in which Paβ and Paγ along with many spectral lines from neutral and singly ionized impurities have been observed. The influxes of H and C have been estimated from measurements. The H influx value is found to be 2.8 × 1016 and 1.9 × 1016 particles cm−2 s−1 from neutral hydrogen lines Hα and Paβ, respectively, and the C influx value is found to be 3.5 × 1015 and 2.9 × 1015 particles cm−2 s−1 from the neutral carbon and singly ionized carbon, respectively. A good agreement is seen between these results and the results obtained by using a routine photomultiplier tube based diagnostic. [ABSTRACT FROM AUTHOR]

In this paper, the performance of p-type NiO pocket on Ga2O3/Graphene and Ga2O3/Black phosphorous hetero-MOSFET has been investigated to find out its applicability in the wireless applications. To show the utility of the proposed devices, its analog/RF characteristics have been studied and compared to those of the experimentally demonstrated conventional Ga2O3 MOSFET. The large signal RF performances analysis has also been carried out by considering CW Class-A power measurements at 0.8 GHz using passive source and load tuning. The important figure of merits (FOMs) used in the analysis are intrinsic capacitances CGS and CGD, cutoff frequency fT, output power gain GP, and power-added efficiency. The key idea behind this work is to propose a device which is efficient and shows low leakage current. All the analysis of proposed devices has been carried out using ATLAS TCAD simulator. [ABSTRACT FROM AUTHOR]

Background: The incorporation of leaf litters in agroforestry systems can meet a significant share of nutrients demand and improve crop yield. The Citrus aurontifolia, Aegle marmelos and Cordia myxa (among fruit trees) and Colophospermum mopane, Acacia senegal, Acacia tortilis, Dalbergia sissoo (among trees) are well-adapted species to the arid environment. Unfortunately, little work has been done in the past to study the influence of leaf litters of these species on yield and nutrition of crops grown in arid region. Therefore, the present experiment was planned to study the influence of leaf litters of Colophospermum mopane, Acacia senegal, Acacia tortilis, Dalbergia sissoo, Citrus aurontifolia, Aegle marmelos and Cordia myxa on yield and uptake of nutrients in moth bean and Indian mustard in arid region. Methods: Field experiment was conducted at ICAR-Central Arid Zone Research Institute, Regional Research Station, Bikaner in moth bean-mustard rotation during 2010-11 and 2011-12 by incorporating leaf litters of seven tree species i.e. Mopane (Colophospermum mopane), Gum acacia (Acacia Senegal), Umbrella tree (Acacia tortilis), Indian rosewood (Dalbergia sissoo), Sour lime (Citrus aurantifolia), Assyrian plum (Cordia myxa) and Bengal quince (Aegle marmelos) in randomized block design with three replications. Grain and straw yield was recorded and analysed for N, P and K content. Result: The maximum grain yield of moth bean and its residual effect on mustard was observed in the soils amended with leaf litters of Citrus aurontifolia followed by Aegle marmelos and Dalbergia sissoo. The total uptake of N, P and K was significantly higher in the treatments of Citrus aurontifolia and Aegle marmelos, which was due to the higher dry matter production of crops, faster rate of litter decomposition and higher release of nutrients. [ABSTRACT FROM AUTHOR]

The spectroscopic studies of medium and high Z impurities have been the subject of interest in fusion research due to their role in mitigating plasma disruption and reducing heat load on the plasma facing components. Line emissions from these impurities provide the rotation velocity and ion temperature measurements along with the understanding of the overall impurity behavior in plasma. In the Aditya-U tokamak, the spatially resolved Ar II line emissions have been observed using a high resolution multi-track spectroscopic diagnostic consisting of a 1 m Czerny–Turner spectrometer coupled with a charge coupled device (CCD) detector using seven lines of sight viewing plasma tangentially along the toroidal direction. The spatially resolved Ar II lines at 458.96 nm have been observed. The singly ionized Ar emission peaks at the radial location of ρ = 0.8 of the plasma having a minor radius of 25 cm. Moreover, a 0.5 m UV–visible spectrometer coupled with a CCD detector and having a line of sight passing through the plasma midplane from the radial port was used to record visible Ar survey spectra within the 670–810 nm wavelength range, and all these lines have been identified for further analysis. [ABSTRACT FROM AUTHOR]

A two year field experiment was conducted in hot arid region at Bikaner, Rajasthan to investigate the effects of application of sulphydryl compounds (thioglycollic acid, TGA; thiourea TU) on growth traits, photosynthetic efficiency, yield components and yield of moth bean under water deficit conditions. The experiment comprised seven treatments (water spray, TGA 200 mg L-1, TGA 300 mg L-1, TGA 400 mg L-1, TU 500 mg L-1, TU 750 mg L-1 and TU 1000 mg L-1) and laid out in randomized block design with three replications. Application of sulphydryl compounds had significant effects on growth, photosynthetic traits, activities of anti-oxidant enzymes, and yield of moth bean. The sulphydryl compounds sprayed plants had 24-70, 13-40 and 13-20% higher activities of antioxidant enzymes (glutathione reductase, GR; gluthathione-s-transferase, GST), photosynthetic parameters (net photosynthetic rate, PN ; stomatal conductance, gs ; transpiration rate, E) and seed yield respectively compared to unsprayed plants. The results suggest that application of sulphydryl compounds mitigate moisture deficit induced negative effects on growth and photosynthetic parameters which lead to better seed yield of moth bean. [ABSTRACT FROM AUTHOR]

A field experiment was conducted at research farm, Agricultural Research Sub-Station, Hanumangarh, Swami Keshwanand Rajasthan Agricultural University, Bikaner, Rajasthan during kharif, 2016 to study the effect of water and nitrogen levels on yield attributes, water productivity and economics of cluster bean [Cyamopsis tetragonoloba (L.) Taub.].Cluster bean variety RGC-1055 with seed rate of 16 kg/ha was planted using 3 levels of irrigation (100, 200 and 300 mm) and 4 level of nitrogen (0, 20, 40 and 60 kg/ha) and analyzed in split plot design with three replication. Results showed that irrigation at 200 mm significantly increased number of pods/plant (44.7), number of seeds/pod (7.8), test weight (28.7g), net returns (Rs 31179/ha) and B:C ratio (2.1) over 100 mm irrigation level. However, water productivity (0.25 kg m-3) was highest at 100 mm irrigation level. Nitrogen level at 40 kg/ha significantly increased number of pods/plant (48.3), number of seeds/pod (8.4), test weight (32.2 g), net returns (Rs. 32273/ha), B:C ratio (2.12) and water productivity (0.27 kg m-3) over no application of N. [ABSTRACT FROM AUTHOR]

An experiment entitled “Evaluation of CropSyst model for yield and water productivity of chickpea” was conducted on farmers field during rabi 2012-13 at village Mainawali in Hanumangarh district of Rajasthan. The soils of the area are alluvial and calcareous in nature formed under arid and semi arid climate. The soils of site are brown to grayish brown and dark gray in colour, besides being calcareous and slightly alkaline in reaction having 67.7, 11.1 and 21.0 % of sand, clay and silt, respectively in 0-15 cm soil depth with pH 8.09 and low soil organic matter content. The simulated and observed green area index differs with field measurements at all stages. The simulate yield of chickpea were closer to the observed yield. The total water applied in chickpea was 415 mm out of this 356.5 mm consumed in ET. Thus, ET constituted 86% of total water applied and deep drainage constituted 12% and rest 2% stored as residual soil moisture. [ABSTRACT FROM AUTHOR]

CropSyst is one of the most important process-oriented simulation models largely used for field crops all over the world to study the effect of climate, soil and management practices on crop productivity. In the present study, we have calibrated and validated the CropSyst model for groundnut crop grown at farmer's field in IGNP Stage-II of Bikaner. CropSyst model was calibrated using the experimental data of crop parameters, soil profile data and observed daily weather data of experimental site for 2012 and validated the experimental data of crop growth and yield parameters for 2013. The results of the study showed that the CropSyst model simulated the crop growth parameter data viz. green area index, seed yield, above ground biomass and N-uptake of groundnut reasonably well. The seed yield, above ground biomass and N- uptake was validated well by the model with relative error of 3.3, 2.2 and 8.4%, respectively. The total water applied in groundnut was 728.9 and 619.6 mm in 2012 and 2013, respectively out of this 664.9 and 530.5mm consumed in evapotranspiration. [ABSTRACT FROM AUTHOR]

The study on "Evaluation of Cropsyst model for yield and water productivity of clusterbean" was conducted on farmers field during kharif 2012 at village Mainawali in Hanumangarh district of Rajasthan. The soils of the area are alluvial and calcareous in nature formed under arid and semi arid climate. The soils of site are brown to greyish brown and dark grey in colour, besides being calcareous and slightly alkaline in reaction having 67.7, 11.1 and 21.0% of sand, clay and silt, respectively in 0-15 cm soil depth with pH 8.09 and low soil organic matter content. The simulate yield of clusterbean were closer to the observed clusterbean yield. Simulations of early clusterbean above ground biomass development matched the field data reasonably well. Final above ground biomass, however, was over estimated by the model. The total water applied in clusterbean was 405.8 mm out of this 326.7 mm consumed in ET. Thus, ET constituted 81% of total water applied and deep drainage constituted 13% and rest 6% stored as residual soil moisture. [ABSTRACT FROM AUTHOR]

Water stress is one of the main environmental stress conditions that adversely affect growth and yield of crops. Sulphydryl (-SH) compounds have been reported to induce tolerance to abiotic stress conditions. A two-year (2010 and 2011) experiment was conducted at Bikaner, Rajasthan, India to test whether the exogenous applications of thiourea (500, 750 and 1000 mg L-1) and thioglycolic acid (200, 300 and 400 mg L-1) as a foliar spray would alleviate deleterious effects of water stress on clusterbean (Cyamopsis tetragonoloba L.). The -SH-treated plants showed higher membrane stability index, photosynthetic pigment content, photosynthetic capacity and antioxidant enzyme activities and lower lipid peroxidation compared with untreated plants. Compared with untreated plants, the -SH-treated plants had 11-18, 18-30, 17-57, 25-47, 14-22% higher membrane stability index, total chlorophyll content, antioxidant enzyme activities, net photosynthetic rate and seed yield, respectively; whereas the malondialdehyde content was 10-19% lower. These data suggest that under water deficit stress, exogenous-SH compound application improves photosynthesis by increasing photosynthetic pigment, protects plants against oxidative damage by scavenging reactive oxygen species and minimizing lipid peroxidation by elevated antioxidant enzyme activities. These results indicated the role of -SH compounds in diminishing the negative effects of water deficit on clusterbean and suggest that -SH compounds could be used as a potential bioregulator to improve plant growth and yield under water deficit conditions. [ABSTRACT FROM AUTHOR]

A field experiment was conducted at Central Arid Zone Research Institute, Regional Research Station , Bikaner during Kharif seasons of 2012 and 2013 on Squash Melon (Tinda) crop under rainfed conditions to evaluate four treatments of Farm Yard Manure viz.,0,20,30 and 40 t ha-1 and three treatment of mulching viz., no mulch, straw mulch and plastic mulch. Experiment was conducted in split - plot design with three replications using the Tinda cv. Selection 1. Vine length, numbers of fruits per plant and yield of squash melon (Tinda) crop were significantly influenced by FYM, mulching and their interaction during both the years of experiment. Application of FYM recorded significant improvement in yield up to 30 t ha-1. Averaged across mulching, the yield with application of FYM @ 20, 30 and 40 t ha-1 had 43.3, 83.9 and 87.4 % higher yield compared to control. Application of mulch brought significant improvement in yield and its component. The straw mulch recorded highest vine length, numbers of fruits per plant and yield. Plastic and straw mulch had 24.80 and 42.25 % higher yield than no-mulch. The response of mulch varied with level of FYM application. The highest yield was recorded with application of 40 FYM t ha-1 combined with straw mulch, however the difference between FYM 30 t ha-1 and 40 t ha-1 was non-significant under both straw and plastic mulching. [ABSTRACT FROM AUTHOR]

A field experiment was conducted during the rabi season of 2009-10 and 2010-11. The experiment consisted of two methods of irrigation viz. drip and furrow methods and four type of mulching material treatments viz. plastic mulch (black polyethylene-25micron), hessian cloth mulch (50% shade jute cloth), indigenous plant material (laptodoniaspps.@ lOt/ha) mulch and no mulch (control). Method of irrigation, mulching and their interaction had significant effects on growth, yield attributes and yield. Mean yield averaged across the mulching treatments was 18.3 % higher under drip irrigation than furrow method. Mulching improved the yield from 40 to 71.8 % over no mulch treatment. Among the tested mulching materials, the plastic mulch had the highest improvement in yield (71.8 %) followed by hessian cloth (54.1 %) and indigenous plant material mulch (40 %) over no mulch. Interaction effect between drip irrigation with plastic mulch resulted in significant response in growth, yield attributes and yield of pea as compared to all other treatment combinations. Number of branches per plant, canopy cover and survival percentage of ber was higher in ber + pea cropping system than sole ber. [ABSTRACT FROM AUTHOR]

Abstract: There have been several reports on the phosphorylation of various subunits of NADH-ubiquinone oxidoreductase (complex I) in mammalian mitochondria. The effects of phosphorylation on assembly or activity of these subunits have not been investigated directly. The cAMP-dependent phosphorylation of the MWFE and ESSS subunits in isolated bovine heart mitochondria has been recently reported. We have investigated the significance of potential phosphorylation of these two subunits in complex I assembly and function by mutational analysis of the phosphorylation sites. Chinese hamster mutant cell lines missing either the MWFE or the ESSS subunits were transfected and complemented with the corresponding wild type and mutant cDNAs made by site-directed mutagenesis. In MWFE the serine 55 was substituted by alanine, glutamate, glutamine, and aspartate (S55A, S55E, S55Q, and S55D, respectively). The glutamate substitutions might be expected to mimic the phosphorylated state of the protein. With the exception of the MWFE(S55A) mutant protein the assembly of complex I was completely blocked, and no activity could be detected. Various substitutions in the ESSS protein (S2A, S2E, S8A, S8E, T21A, T21E, S30A, S30E) appeared to cause lower levels of mature protein and a significantly reduced complex I activity measured polarographically. The ESSS (S2/8A) double mutant protein caused a complete failure to assemble. These mutational analyses suggest that if phosphorylation occurs in vivo, the effects on complex I activity are significant. [Copyright &y& Elsevier]

Formation of somatic embryoids was noted in cell suspensions of Plantago ovata Forsk. The callus was induced on a solid Murashige and Skoog medium supplemented with 2,4-dichlorophenoxyacetic acid (1 µM) and kinetin (2 µM) using leaf explants of 12 day old seedlings. After two subcultures on the same medium, embryogenic cell suspensions with various stages of embryoids were obtained. Addition of higher levels of kinetin caused these embryos to undergo normal germination. [ABSTRACT FROM PUBLISHER]

Background: Sepsis-induced cardiomyopathy (SIC) is a major contributing factor for morbidity and mortality in sepsis. Accumulative evidence has suggested that cardiac mitochondrial oxidative phosphorylation is attenuated in sepsis, but the underlying molecular mechanisms remain incompletely understood., Methods: Adult male mice of 9 to 12 weeks old were subjected to sham or cecal ligation and puncture procedure. Echocardiography in vivo and Langendorff-perfused hearts were used to assess cardiac function 24 h after the procedures. Unbiased proteomics analysis was performed to profile mitochondrial proteins in the hearts of both sham and SIC mice. Seahorse respirator technology was used to evaluate oxygen consumption in purified mitochondria., Results: Of the 665 mitochondrial proteins identified in the proteomics assay, 35 were altered in septic mice. The mitochondrial remodeling involved various energy metabolism pathways including subunits of the electron transport chain, fatty acid catabolism, and carbohydrate oxidative metabolism. We also identified a significant increase of pyruvate dehydrogenase (PDH) kinase 4 (PDK4) and inhibition of PDH activity in septic hearts. Furthermore, compared to sham mice, mitochondrial oxygen consumption of septic mice was significantly reduced when pyruvate was provided as a substrate. However, it was unchanged when PDH was bypassed by directly supplying the Complex I substrate NADH, or by using the Complex II substrate succinate, or using Complex IV substrate, or by providing the beta-oxidation substrate palmitoylcarnitine, neither of which require PDH for mitochondrial oxygen consumption., Conclusions: These data demonstrate a broad mitochondrial protein remodeling, PDH inactivation and impaired pyruvate-fueled oxidative phosphorylation during SIC, and provide a molecular framework for further exploration., Competing Interests: The authors report no conflicts of interests., (Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the Shock Society.)

Cancer cells survive and adapt to many types of stress including hypoxia, nutrient deprivation, metabolic, and oxidative stress. These stresses are sensed by diverse cellular signaling processes, leading to either degradation of mitochondria or alleviation of mitochondrial stress. This review discusses signaling during sensing and mitigation of stress involving mitochondrial communication with the endoplasmic reticulum, and how retrograde signaling upregulates the mitochondrial stress response to maintain mitochondrial integrity. The importance of the mitochondrial unfolded protein response, an emerging pathway that alleviates cellular stress, will be elaborated with respect to cancer. Detailed understanding of cellular pathways will establish mitochondrial stress response as a key mechanism for cancer cell survival leading to cancer progression and resistance, and provide a potential therapeutic target in cancer., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Cell-based respirometers, such as the Seahorse Extracellular Flux Analyzer, are valuable tools to assess the functionality of mitochondria within adherent neurons, as well as other cell types. The Mito Stress Test is the most frequently employed protocol of drug additions to evaluate mitochondrial bioenergetic function. Sequential exposure of cells to an ATP synthase inhibitor such as oligomycin and an uncoupler such as FCCP cause changes in oxygen consumption rate that allow estimation of the cellular efficiency and capacity for mitochondrial ATP synthesis. While a useful first step in assessing whether an experimental treatment or genetic manipulation affects mitochondrial energetics, the Mito Stress Test does not identify specific sites of altered respiratory chain function. This article discusses limitations of the Mito Stress Test, proposes a refined protocol for comparing cell populations that requires independent drug titrations at multiple cell densities, and describes a stepwise series of respirometry-based assays that "map" locations of electron transport deficiency. These include strategies to test for cytochrome c release, to probe the functionality of specific electron transport chain complexes within intact or permeabilized cells, and to measure NADH oxidation by the linked activity of Complexes I, III, and IV. To illustrate utility, we show that although UK5099 and ABT-737 each decrease the spare respiratory capacity of cortical neurons, the stepwise assays reveal different underlying mechanisms consistent with their established drug targets: deficient Complex I substrate supply induced by the mitochondrial pyruvate carrier inhibitor UK5099 and cytochrome c release induced by the anti-apoptotic BCL-2 family protein inhibitor ABT-737., Competing Interests: Declaration of Competing Interest N.Y. receives royalties from Agilent Technologies for U.S. Patent US9915647B2. Agilent Technologies had no role in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Mitochondria are therapeutic targets in many diseases including cancer, metabolic disorders, and neurodegenerative diseases. Therefore, strategies to deliver therapeutics of interest to mitochondria are important for therapeutic development. As delocalized lipophilic cations (DLCs) preferentially accumulate in mitochondria, DLC-conjugation has been utilized to facilitate therapeutic delivery systems with mitochondrial targeting capability. Here we report that upon DLC-conjugation, anionic polymers exhibit significantly improved mitochondrial targeting when compared to cationic polymers and charge-neutral polymers. Considering that the cell membrane generally bears a net negative charge, the observed phenomenon is unexpected. Notably, the DLC-conjugated anionic polymers circumvent endosomal entrapment. The rapid mitochondrial accumulation of DLC-conjugated anionic polymers is likely a membrane-potential-driven process, along with the involvement of the mitochondrial pyruvate carrier. Moreover, the structural variations on the side chain of DLC-conjugated anionic polymers do not compromise the overall mitochondrial targeting capability, widely extending the applicability of anionic macromolecules in therapeutic delivery systems.

Recent advances in our understanding of tumor cell mitochondrial metabolism suggest it may be an attractive therapeutic target. Mitochondria are central hubs of metabolism that provide energy during the differentiation and maintenance of immune cell phenotypes. Mitochondrial membranes harbor several enzyme complexes that are involved in the process of oxidative phosphorylation, which takes place during energy production. Data suggest that, among these enzyme complexes, deficiencies in electron transport complex I may differentially affect immune responses and may contribute to the pathophysiology of several immunological conditions. Once activated by T cell receptor signaling, along with co-stimulation through CD28, CD4 T cells utilize mitochondrial energy to differentiate into distinct T helper (Th) subsets. T cell signaling activates Notch1, which is cleaved from the plasma membrane to generate its intracellular form (N1ICD). In the presence of specific cytokines, Notch1 regulates gene transcription related to cell fate to modulate CD4 Th type 1, Th2, Th17, and induced regulatory T cell (iTreg) differentiation. The process of differentiating into any of these subsets requires metabolic energy, provided by the mitochondria. We hypothesized that the requirement for mitochondrial metabolism varies between different Th subsets and may intersect with Notch1 signaling. We used the organic pesticide rotenone, a well-described complex I inhibitor, to assess how compromised mitochondrial integrity impacts CD4 T cell differentiation into Th1, Th2, Th17, and iTreg cells. We also investigated how Notch1 localization and downstream transcriptional capabilities regulation may be altered in each subset following rotenone treatment. Our data suggest that mitochondrial integrity impacts each of these Th subsets differently, through its influence on Notch1 subcellular localization. Our work further supports the notion that altered immune responses can result from complex I inhibition. Therefore, understanding how mitochondrial inhibitors affect immune responses may help to inform therapeutic approaches to cancer treatment.

Outcomes of children with high grade neuroblastoma remain poor despite multi-agent chemotherapy regimens. Rhodiola crenulata extracts display anti-neoplastic properties against several cancers including breast cancer, melanoma, and glioblastoma. In this study, we evaluated the anti-neoplastic potential of Rhodiola crenulata extracts on human neuroblastoma cells. Through this work, cell viability and proliferation were evaluated following treatments with ethanol (vehicle control) or Rhodiola crenulata extract in neuroblastoma, NB-1691 or SK-N-AS cells, in vitro. HIF-1 transcriptional activity was evaluated using a dual luciferase assay. Quantitative real-time polymerase chain reaction was utilized to assess the expression of HIF-1 targets. Selected metabolic intermediates were evaluated for their ability to rescue cells from Rhodiola crenulata extract-induced death. Lactate dehydrogenase, pyruvate kinase, and pyruvate dehydrogenase activities and NAD + /NADH levels were assayed in vehicle and Rhodiola crenulata extract-treated cells. The effects of Rhodiola crenulata extracts on metabolism were assessed by respirometry and metabolic phenotyping/fingerprinting. Our results revealed striking cytotoxic effects upon Rhodiola crenulata extract treatment, especially prominent in NB-1691 cells. As a greater response was observed in NB-1691 cells therefore it was used for remaining experiments. Upon Rhodiola crenulata extract treatment, HIF-1 transcriptional activity was increased. This increase in activity correlated with changes in HIF-1 targets involved in cellular metabolism. Serendipitously, we observed that addition of pyruvate protected against the cytotoxic effects of Rhodiola crenulata extracts. Therefore, we focused on the metabolic effects of Rhodiola crenulata extracts on NB-1691 cells. We observed that while the activities of pyruvate kinase and pyruvate dehydrogenase activities were increased, the activity of lactate dehydrogenase activity was decreased upon Rhodiola crenulata extract treatment. We also noted a decline in the total NAD pool following Rhodiola crenulata extract treatment. This correlated with decreased cellular respiration and suppressed utilization of carbon substrates. Through this work, we observed significant cytotoxic effects of Rhodiola crenulata extract treatment upon treatment on NB-1691 cells, a human neuroblastoma cell line with MYCN amplification. Our studies suggest that these cytotoxic effects could be secondary to metabolic effect induced by treatment with Rhodiola crenulata extract.

Metabolic reprograming is a hallmark of cancer cells. However, the roles of pre-existing differences in normal cells metabolism toward cancer risk is not known. In order to assess pre-existing variations in normal cell metabolism, we have quantified the inter-individual variation in oxidative metabolism of normal primary human mammary epithelial cells (HMECs). We then assessed their response to selected cytokines such as insulin growth factor 1 (IGF1) and tumor necrosis factor alpha (TNFα), which are associated with breast cancer risk. Specifically, we compared the oxidative metabolism of HMECs obtained from women with breast cancer and without cancer. Our data show considerable inter-individual variation in respiratory activities of HMECs from different women. A bioenergetic parameter called pyruvate-stimulated respiration (PySR) was identified as a key distinguishing feature of HMECs from women with breast cancer and without cancer. Samples showing PySR over 20% of basal respiration rate were considered PySR +ve and the rest as PySR -ve . By this criterion, HMECs from tumor-affected breasts (AB) and non-tumor affected breasts (NAB) of cancer patients were mostly PySR -ve (88% and 89%, respectively), while HMECs from non-cancer patients were mostly PySR +ve (57%). This suggests that PySR -ve/+ve phenotypes are individual-specific and are not caused by field effects due to the presence of tumor. The effects of IGF1 and TNFα treatments on HMECs revealed that both suppressed respiration and extracellular acidification. In addition, IGF1 altered PySR -ve/+ve phenotypes. These results reveal individual-specific differences in pyruvate metabolism of normal breast epithelial cells and its association with breast cancer risk., (© 2017 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.)

The respiratory chain Complex I deficiencies are the most common cause of mitochondrial diseases. Complex I biogenesis is controlled by 58 genes and at least 47 of these cause mitochondrial disease in humans. Two of these are X-chromosome linked nuclear (nDNA) genes (NDUFA1 and NDUFB11), and 7 are mitochondrial (mtDNA, MT-ND1-6, -4L) genes, which may be responsible for sex-dependent variation in the presentation of mitochondrial diseases. In this study, we describe an X-chromosome linked mouse model (Ndufa1 S55A ) for systemic partial Complex I deficiency. By homologous recombination, a point mutation T > G within 55th codon of the Ndufa1 gene was introduced. The resulting allele Ndufa1 S55A introduced systemic serine-55-alanine (S55A) mutation within the MWFE protein, which is essential for Complex I assembly and stability. The S55A mutation caused systemic partial Complex I deficiency of ∼50% in both sexes. The mutant males (Ndufa1 S55A/Y ) displayed reduced respiratory exchange ratio (RER) and produced less body heat. They were also hypoactive and ate less. They showed age-dependent Purkinje neurons degeneration. Metabolic profiling of brain, liver and serum from males showed reduced heme levels in mutants, which correlated with altered expressions of Fech and Hmox1 mRNAs in tissues. This is the first genuine X-chromosome linked mouse model for systemic partial Complex I deficiency, which shows age-dependent neurodegeneration. The effect of Complex I deficiency on survival patterns of males vs. females was different. We believe this model will be very useful for studying sex-dependent predisposition to both spontaneous and stress-induced neurodegeneration, cancer, diabetes and other diseases., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Mitochondrial fission mediated by the GTPase dynamin-related protein 1 (Drp1) is an attractive drug target in numerous maladies that range from heart disease to neurodegenerative disorders. The compound mdivi-1 is widely reported to inhibit Drp1-dependent fission, elongate mitochondria, and mitigate brain injury. Here, we show that mdivi-1 reversibly inhibits mitochondrial complex I-dependent O 2 consumption and reverse electron transfer-mediated reactive oxygen species (ROS) production at concentrations (e.g., 50 μM) used to target mitochondrial fission. Respiratory inhibition is rescued by bypassing complex I using yeast NADH dehydrogenase Ndi1. Unexpectedly, respiratory impairment by mdivi-1 occurs without mitochondrial elongation, is not mimicked by Drp1 deletion, and is observed in Drp1-deficient fibroblasts. In addition, mdivi-1 poorly inhibits recombinant Drp1 GTPase activity (K i > 1.2 mM). Overall, these results suggest that mdivi-1 is not a specific Drp1 inhibitor. The ability of mdivi-1 to reversibly inhibit complex I and modify mitochondrial ROS production may contribute to effects observed in disease models., (Copyright © 2017 Elsevier Inc. All rights reserved.)

We have previously reported that neem limonoids (neem) induce multiple cancer cell death pathways. Here we dissect the underlying mechanisms of neem-induced apoptotic cell death in cancer. We observed that neem-induced caspase activation does not require Bax/Bak channel-mediated mitochondrial outer membrane permeabilization, permeability transition pore, and mitochondrial fragmentation. Neem enhanced mitochondrial DNA and mitochondrial biomass. While oxidative phosphorylation (OXPHOS) Complex-I activity was decreased, the activities of other OXPHOS complexes including Complex-II and -IV were unaltered. Increased reactive oxygen species (ROS) levels were associated with an increase in mitochondrial biomass and apoptosis upon neem exposure. Complex-I deficiency due to the loss of Ndufa1-encoded MWFE protein inhibited neem-induced caspase activation and apoptosis, but cell death induction was enhanced. Complex II-deficiency due to the loss of succinate dehydrogenase complex subunit C (SDHC) robustly decreased caspase activation, apoptosis, and cell death. Additionally, the ablation of Complexes-I, -III, -IV, and -V together did not inhibit caspase activation. Together, we demonstrate that neem limonoids target OXPHOS system to induce cancer cell death, which does not require upregulation or activation of proapoptotic Bcl-2 family proteins., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Introduction: Human mesenchymal stem cells (MSCs) are multipotent cells that can differentiate into fat, muscle, bone and cartilage cells. Exposure of subcutaneous abdominal adipose tissue derived AD-MSCs to high glucose (HG) leads to superoxide accumulation and up-regulation of inflammatory molecules. Our aim was to inquire how HG exposure affects MSCs differentiation and whether the mechanism is reversible., Methods: We exposed human adipose tissue derived MSCs to HG (25 mM) and compared it to normal glucose (NG, 5.5 mM) exposed cells at 7, 10 and 14 days. We examined mitochondrial superoxide accumulation (Mitosox-Red), cellular oxygen consumption rate (OCR, Seahorse) and gene expression., Results: HG increased reactive superoxide (ROS) accumulation noted by day 7 both in cytosol and mitochondria. The OCR between the NG and HG exposed groups however did not change until 10 days at which point OCR of HG exposed cells were reduced significantly. We noted that HG exposure upregulated mRNA expression of adipogenic (PPARG, FABP-4, CREBP alpha and beta), inflammatory (IL-6 and TNF alpha) and antioxidant (SOD2 and Catalase) genes. Next, we used AdSOD2 to upregulate SOD2 prior to HG exposure and thereby noted reduction in superoxide generation. SOD2 upregulation helped reduce mRNA over-expression of PPARG, FABP-4, IL-6 and TNFα. In a series of separate experiments, we delivered the eGFP and SOD2 upregulated MSCs (5 days post ex-vivo transduction) and saline intra-peritoneally (IP) to obese diabetic (db/db) mice. We confirmed homing-in of eGFP labeled MSCs, delivered IP, to different inflamed fat pockets, particularly omental fat. Mice receiving SOD2-MSCs showed progressive reduction in body weight and improved glucose tolerance (GTT) at 4 weeks, post MSCs transplantation compared to the GFP-MSC group (control)., Conclusions: High glucose evokes superoxide generation, OCR reduction and adipogenic differentiation. Mitochondrial superoxide dismutase upregulation quenches excess superoxide and reduces adipocyte inflammation. Delivery of superoxide dismutase (SOD2) using MSCs as a gene delivery vehicle reduces inflammation and improves glucose tolerance in vivo. Suppression of superoxide production and adipocyte inflammation using mitochondrial superoxide dismutase may be a novel and safe therapeutic tool to combat hyperglycemia mediated effects.

Cancer cells tend to develop resistance to various types of anticancer agents, whether they adopt similar or distinct mechanisms to evade cell death in response to a broad spectrum of cancer therapeutics is not fully defined. Current study concludes that DNA-damaging agents (etoposide and doxorubicin), ER stressor (thapsigargin), and histone deacetylase inhibitor (apicidin) target oxidative phosphorylation (OXPHOS) for apoptosis induction, whereas other anticancer agents including staurosporine, taxol, and sorafenib induce apoptosis in an OXPHOS-independent manner. DNA-damaging agents promoted mitochondrial biogenesis accompanied by increased accumulation of cellular and mitochondrial ROS, mitochondrial protein-folding machinery, and mitochondrial unfolded protein response. Induction of mitochondrial biogenesis occurred in a caspase activation-independent mechanism but was reduced by autophagy inhibition and p53-deficiency. Abrogation of complex-I blocked DNA-damage-induced caspase activation and apoptosis, whereas inhibition of complex-II or a combined deficiency of OXPHOS complexes I, III, IV, and V due to impaired mitochondrial protein synthesis did not modulate caspase activity. Mechanistic analysis revealed that inhibition of caspase activation in response to anticancer agents associates with decreased release of mitochondrial cytochrome c in complex-I-deficient cells compared with wild type (WT) cells. Gross OXPHOS deficiencies promoted increased release of apoptosis-inducing factor from mitochondria compared with WT or complex-I-deficient cells, suggesting that cells harboring defective OXPHOS trigger caspase-dependent as well as caspase-independent apoptosis in response to anticancer agents. Interestingly, DNA-damaging agent doxorubicin showed strong binding to mitochondria, which was disrupted by complex-I-deficiency but not by complex-II-deficiency. Thapsigargin-induced caspase activation was reduced upon abrogation of complex-I or gross OXPHOS deficiency whereas a reverse trend was observed with apicidin. Together, these finding provide a new strategy for differential mitochondrial targeting in cancer therapy.

Several flavin-dependent enzymes of the mitochondrial matrix utilize NAD(+) or NADH at about the same operating redox potential as the NADH/NAD(+) pool and comprise the NADH/NAD(+) isopotential enzyme group. Complex I (specifically the flavin, site IF) is often regarded as the major source of matrix superoxide/H2O2 production at this redox potential. However, the 2-oxoglutarate dehydrogenase (OGDH), branched-chain 2-oxoacid dehydrogenase (BCKDH), and pyruvate dehydrogenase (PDH) complexes are also capable of considerable superoxide/H2O2 production. To differentiate the superoxide/H2O2-producing capacities of these different mitochondrial sites in situ, we compared the observed rates of H2O2 production over a range of different NAD(P)H reduction levels in isolated skeletal muscle mitochondria under conditions that favored superoxide/H2O2 production from complex I, the OGDH complex, the BCKDH complex, or the PDH complex. The rates from all four complexes increased at higher NAD(P)H/NAD(P)(+) ratios, although the 2-oxoacid dehydrogenase complexes produced superoxide/H2O2 at high rates only when oxidizing their specific 2-oxoacid substrates and not in the reverse reaction from NADH. At optimal conditions for each system, superoxide/H2O2 was produced by the OGDH complex at about twice the rate from the PDH complex, four times the rate from the BCKDH complex, and eight times the rate from site IF of complex I. Depending on the substrates present, the dominant sites of superoxide/H2O2 production at the level of NADH may be the OGDH and PDH complexes, but these activities may often be misattributed to complex I.

The mitochondrial metabolism of β cells is thought to be highly specialized. Its direct comparison with other cells using isolated mitochondria is limited by the availability of islets/β cells in sufficient quantity. In this study, we have compared mitochondrial metabolism of INS1E/β cells with other cells in intact and permeabilized states. To selectively permeabilize the plasma membrane, we have evaluated the use of perfringolysin-O (PFO) in conjunction with microplate-based respirometry. PFO is a protein that binds membranes based on a threshold level of active cholesterol. Therefore, unless active cholesterol reaches a threshold level in mitochondria, they are expected to remain untouched by PFO. Cytochrome c sensitivity tests showed that in PFO-permeabilized cells, the mitochondrial integrity was completely preserved. Our data show that a time-dependent decline of the oligomycin-insensitive respiration observed in INS1E cells was due to a limitation in substrate supply to the respiratory chain. We predict that it is linked with the β cell-specific metabolism involving metabolites shuttling between the cytoplasm and mitochondria. In permeabilized β cells, the Complex l-dependent respiration was either transient or absent because of the inefficient TCA cycle. The TCA cycle insufficiency was confirmed by analysis of the CO 2 evolution. This may be linked with lower levels of NAD + , which is required as a co-factor for CO 2 producing reactions of the TCA cycle. β cells showed comparable OxPhos and respiratory capacities that were not affected by the inorganic phosphate (Pi) levels in the respiration medium. They showed lower ADP-stimulation of the respiration on different substrates. We believe that this study will significantly enhance our understanding of the β cell mitochondrial metabolism.

Facilitated pyruvate transport across the mitochondrial inner membrane is a critical step in carbohydrate, amino acid, and lipid metabolism. We report that clinically relevant concentrations of thiazolidinediones (TZDs), a widely used class of insulin sensitizers, acutely and specifically inhibit mitochondrial pyruvate carrier (MPC) activity in a variety of cell types. Respiratory inhibition was overcome with methyl pyruvate, localizing the effect to facilitated pyruvate transport, and knockdown of either paralog, MPC1 or MPC2, decreased the EC50 for respiratory inhibition by TZDs. Acute MPC inhibition significantly enhanced glucose uptake in human skeletal muscle myocytes after 2 h. These data (i) report that clinically used TZDs inhibit the MPC, (ii) validate that MPC1 and MPC2 are obligatory components of facilitated pyruvate transport in mammalian cells, (iii) indicate that the acute effect of TZDs may be related to insulin sensitization, and (iv) establish mitochondrial pyruvate uptake as a potential therapeutic target for diseases rooted in metabolic dysfunction.

Mitochondrial oxidative metabolism plays a key role in meeting energetic demands of cells by oxidative phosphorylation (OxPhos). Here, we have briefly discussed (a) the dynamic relationship that exists among glycolysis, the tricarboxylic acid (TCA) cycle, and OxPhos; (b) the evidence of impaired OxPhos (i.e. mitochondrial dysfunction) in breast cancer; (c) the mechanisms by which mitochondrial dysfunction can predispose to cancer; and (d) the effects of host and environmental factors that can negatively affect mitochondrial function. We propose that impaired OxPhos could increase susceptibility to breast cancer via suppression of the p53 pathway, which plays a critical role in preventing tumorigenesis. OxPhos is sensitive to a large number of factors intrinsic to the host (e.g. inflammation) as well as environmental exposures (e.g. pesticides, herbicides and other compounds). Polymorphisms in over 143 genes can also influence the OxPhos system. Therefore, declining mitochondrial oxidative metabolism with age due to host and environmental exposures could be a common mechanism predisposing to cancer.

Recently, mitochondria have been suggested to act in tumor suppression. However, the underlying mechanisms by which mitochondria suppress tumorigenesis are far from being clear. In this study, we have investigated the link between mitochondrial dysfunction and the tumor suppressor protein p53 using a set of respiration-deficient (Res(-)) mammalian cell mutants with impaired assembly of the oxidative phosphorylation machinery. Our data suggest that normal mitochondrial function is required for γ-irradiation (γIR)-induced cell death, which is mainly a p53-dependent process. The Res(-) cells are protected against γIR-induced cell death due to impaired p53 expression/function. We find that the loss of complex I biogenesis in the absence of the MWFE subunit reduces the steady-state level of the p53 protein, although there is no effect on the p53 protein level in the absence of the ESSS subunit that is also essential for complex I assembly. The p53 protein level was also reduced to undetectable levels in Res(-) cells with severely impaired mitochondrial protein synthesis. This suggests that p53 protein expression is differentially regulated depending upon the type of electron transport chain/respiratory chain deficiency. Moreover, irrespective of the differences in the p53 protein expression profile, γIR-induced p53 activity is compromised in all Res(-) cells. Using two different conditional systems for complex I assembly, we also show that the effect of mitochondrial dysfunction on p53 expression/function is a reversible phenomenon. We believe that these findings will have major implications in the understanding of cancer development and therapy.

Respirometry using modified cell culture microplates offers an increase in throughput and a decrease in biological material required for each assay. Plate based respirometers are susceptible to a range of diffusion phenomena; as O(2) is consumed by the specimen, atmospheric O(2) leaks into the measurement volume. Oxygen also dissolves in and diffuses passively through the polystyrene commonly used as a microplate material. Consequently the walls of such respirometer chambers are not just permeable to O(2) but also store substantial amounts of gas. O(2) flux between the walls and the measurement volume biases the measured oxygen consumption rate depending on the actual [O(2)] gradient. We describe a compartment model-based correction algorithm to deconvolute the biological oxygen consumption rate from the measured [O(2)]. We optimize the algorithm to work with the Seahorse XF24 extracellular flux analyzer. The correction algorithm is biologically validated using mouse cortical synaptosomes and liver mitochondria attached to XF24 V7 cell culture microplates, and by comparison to classical Clark electrode oxygraph measurements. The algorithm increases the useful range of oxygen consumption rates, the temporal resolution, and durations of measurements. The algorithm is presented in a general format and is therefore applicable to other respirometer systems.

Apoptosis-inducing factor (AIF)-deficient harlequin (Hq) mice undergo neurodegeneration associated with a 40-50% reduction in complex I level and activity. We tested the hypothesis that AIF and complex I regulate reactive oxygen species (ROS) production by brain mitochondria. Isolated Hq brain mitochondria oxidizing complex I substrates displayed no difference compared to wild type (WT) in basal ROS production, H2O2 removal, or ROS production stimulated by complex I inhibitors rotenone or 1-methyl-4-phenylpyridinium. In contrast, ROS production caused by reverse electron transfer to complex I was attenuated by approximately 50% in Hq mitochondria oxidizing the complex II substrate succinate. Basal and rotenone-stimulated rates of H2O2 release from in situ mitochondria did not differ between Hq and WT synaptosomes metabolizing glucose, nor did the level of in vivo oxidative protein carbonyl modifications detected in synaptosomes, brain mitochondria, or homogenates. Our results suggest that AIF does not directly modulate ROS release from brain mitochondria. In addition, they demonstrate that in contrast to ROS produced by mitochondria oxidizing succinate, ROS release from in situ synaptosomal mitochondria or from isolated brain mitochondria oxidizing complex I substrates is not proportional to the amount of complex I. These findings raise the important possibility that complex I contributes less to physiological ROS production by brain mitochondria than previously suggested.

Studies of both survival after sepsis and sperm motility in human populations have shown significant associations with common European mitochondrial DNA haplogroups, and have led to proposals that mitochondria bearing haplogroup H have different bioenergetic capacities than those bearing haplogroup T. However, the validity of such associations assumes that there are no non-random influences of nuclear genes or other factors. Here, we removed the effect of any differences in nuclear genes by constructing transmitochondrial cybrids harbouring mitochondria with either haplogroup H or haplogroup T in cultured A549 human lung carcinoma cells with identical nuclear backgrounds. We compared the bioenergetic capacities and coupling efficiencies of mitochondria isolated from these cells, and of mitochondria retained within the cells, as a critical experimental test of the hypothesis that these haplogroups affect mitochondrial bioenergetics. We found that there were no functionally-important bioenergetic differences between mitochondria bearing these haplogroups, using either isolated mitochondria or mitochondria within cells.

The pathologic activation of NMDA receptors by glutamate is a major contributor to neuronal cell death after stroke. Receptor activation causes a massive influx of calcium into the neuron that is accumulated by the mitochondria. The favored hypothesis is that the calcium loaded mitochondria generate reactive oxygen species that damage and ultimately killed the neuron. In this review this hypothesis is critically re-examined with an emphasis on the role played by deficits in ATP generation. Novel techniques are developed to monitor the bioenergetic status of in situ mitochondria in cultured neurons. Applying these techniques to a model of glutamate excitotoxicity suggests that enhanced reactive oxygen species are a consequence rather than a cause of failed cytoplasmic calcium homeostasis (delayed calcium deregulation, [DCD]), but that prior oxidative damage facilitates DCD by damaging mitochondrial ATP generation. This impacts on current hypotheses relating to the neuroprotective effects of mild mitochondrial uncoupling.

Partial inhibition of mitochondrial respiratory complex I by rotenone reproduces aspects of Parkinson's disease in rodents. The hypothesis that rotenone enhancement of neuronal cell death is attributable to oxidative stress was tested in an acute glutamate excitotoxicity model using primary cultures of rat cerebellar granule neurons. As little as 5 nM rotenone increased mitochondrial superoxide (O2*-) levels and potentiated glutamate-induced cytoplasmic Ca2+ deregulation, the first irreversible stage of necrotic cell death. However, the potent cell-permeant O2*- trap manganese tetrakis (N-ethylpyridinium-2yl) porphyrin failed to prevent the effects of the inhibitor. The bioenergetic consequences of rotenone addition were quantified by monitoring cell respiration. Glutamate activation of NMDA receptors used the full respiratory capacity of the in situ mitochondria, and >80% of the glutamate-stimulated respiration was attributable to increased cellular ATP demand. Rotenone at 20 nM inhibited basal and carbonyl cyanide p-trifluoromethoxyphenylhydrazone-stimulated cell respiration and caused respiratory failure in the presence of glutamate. ATP synthase inhibition by oligomycin was also toxic in the presence of glutamate. We conclude that the cell vulnerability in the rotenone model of partial complex I deficiency under these specific conditions is primarily determined by spare respiratory capacity rather than oxidative stress.

Basal cell carcinoma (BCC) is the most common cutaneous malignancy that, like other tumours, possesses a heterogeneous genetic composition. In order to select genes with consistent changes in expression among these tumours, we analysed BCC microarray expression data by using a novel approach, termed correlative analysis of microarrays (CAM). CAM is a nested, non-parametric method designed to qualitatively select candidates based on their individual, similar effects upon an array-wide closeness measure. We applied the CAM method to expression data generated by two-channel cDNA microarray experiments, where 21 BCC and patient-matched normal skin specimens were examined. Fifteen candidate genes were selected, with six overexpressed and nine underexpressed in BCC vs. normal skin. Five of the nine consistently downregulated genes in the tumour samples are involved in mitochondrial function and the oxidative phosphorylation (OXPHOS) pathway. One of these genes was the 7.5-kDa subunit, NADH dehydrogenase (ubiquinone) alpha subcomplex-1 (NDUFA1), an accessory component of OXPHOS complex-I that is essential for respiratory activity. These findings support the hypothesis that irregularities in mitochondrial function are involved in neoplasia. Suppression of NDUFA1 expression could represent a key pathogenic mechanism in the development of BCC.

The work from our laboratory on complex I-deficient Chinese hamster cell mutants is reviewed. Several complementation groups with a complete defect have been identified. Three of these are due to X-linked mutations, and the mutated genes for two have been identified. We describe null mutants in the genes for the subunits MWFE (gene: NDUFA1) and ESSS. They represent small integral membrane proteins localized in the Ialpha (Igamma) and Ibeta subcomplexes, respectively [J. Hirst, J. Carroll, I.M. Fearnley, R.J. Shannon, J.E. Walker. The nuclear encoded subunits of complex I from bovine heart mitochondria. Biochim. Biophys. Acta 1604 (7-10-2003) 135-150.]. Both are absolutely essential for assembly and activity of complex I. Epitope-tagged versions of these proteins can be expressed from a poly-cistronic vector to complement the mutants, or to be co-expressed with the endogenous proteins in other hamster cell lines (mutant or wild type), or human cells. Structure-function analyses can be performed with proteins altered by site-directed mutagenesis. A cell line has been constructed in which the MWFE subunit is conditionally expressed, opening a window on the kinetics of assembly of complex I. Its targeting, import into mitochondria, and orientation in the inner membrane have also been investigated. The two proteins have recently been shown to be the targets for a cAMP-dependent kinase [R. Chen, I.M. Fearnley, S.Y. Peak_Chew, J.E. Walker. The phosphorylation of subunits of complex I from bovine heart mitochondria. J. Biol. Chem. xx (2004) xx-xx.]. The epitope-tagged proteins can be cross-linked with other complex I subunits.

The ESSS protein is a recently identified subunit of mammalian mitochondrial complex I. It is a relatively small integral membrane protein (122 amino acids) found in the beta-subcomplex. Genomic sequence database searches reveal its localization to the X-chromosome in humans and mouse. The ESSS cDNA from Chinese hamster cells was cloned and shown to complement one complementation group of our previously described mutants with a proposed X-linkage. Sequence analyses of the ESSS cDNA in these mutants revealed chain termination mutations. In two of these mutants the protein is truncated at the C-terminus of the targeting sequence; the mutants are null mutants for the ESSS subunit. There is no detectable complex I assembly and activity in the absence of the ESSS subunit as revealed by blue native polyacrylamide gel electrophoresis (BN/PAGE) analysis and polarography. Complex I activity can be restored with ESSS subunits tagged with either hemagglutinin (HA) or hexahistidine (His6) epitopes at the C-terminus. Although, the accumulation of ESSS-HA is not dependent upon the presence of mtDNA-encoded subunits (ND1-6,4 L), it is incorporated into complex I only in presence of compatible complex I subunits from the same species.

Mitochondrial outer membrane permeabilization and cytochrome c release promote caspase activation and execution of apoptosis through cleavage of specific caspase substrates in the cell. Among the first targets of activated caspases are the permeabilized mitochondria themselves, leading to disruption of electron transport, loss of mitochondrial transmembrane potential (DeltaPsim), decline in ATP levels, production of reactive oxygen species (ROS), and loss of mitochondrial structural integrity. Here, we identify NDUFS1, the 75 kDa subunit of respiratory complex I, as a critical caspase substrate in the mitochondria. Cells expressing a noncleavable mutant of p75 sustain DeltaPsim and ATP levels during apoptosis, and ROS production in response to apoptotic stimuli is dampened. While cytochrome c release and DNA fragmentation are unaffected by the noncleavable p75 mutant, mitochondrial morphology of dying cells is maintained, and loss of plasma membrane integrity is delayed. Therefore, caspase cleavage of NDUFS1 is required for several mitochondrial changes associated with apoptosis.

The MWFE subunit of the mitochondrial NADH-ubiquinone oxidoreductase (complex I) is a small, essential membrane protein of 70 amino acids that is made in the cytosol, imported into mitochondria, and assembled without further proteolytic processing. The experiments identify the first approximately 30 amino acids as a minimal mitochondrial targeting sequence, and establish its orientation in the inner membrane and in complex I. This sequence has a highly conserved glutamate at position 4, which is not typical of a mitochondrial targeting signal. However, it is not essential for MWFE function. Within this sequence there is also a 'stop-transfer' signal. The membrane anchor cannot be replaced by that from another subunit within complex I.