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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]

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]

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.)

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.

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.)

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.

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.

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.

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.

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 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.

We developed a conditional complex I assembly system in a Chinese hamster fibroblast mutant line, CCL16-B2, that does not express the NDUFA1 gene (encoding the MWFE protein). In this mutant, a hemagglutinin (HA) epitope-tagged MWFE protein was expressed from a doxycycline-inducible promoter. The expression of the protein was absolutely dependent on the presence of doxycycline, and the gene could be turned off completely by removal of doxycycline. These experiments demonstrated a key role of MWFE in the pathway of complex I assembly. Upon induction the MWFE.HA protein reached steady-state levels within 24 h, but the appearance of fully active complex I was delayed by another approximately 24 h. The MWFE appeared in a precomplex that probably includes one or more subunits encoded by mtDNA. The fate of MWFE and the stability of complex I were themselves very tightly linked to the activity of mitochondrial protein synthesis and to the assembly of subunits encoded by mtDNA (ND1-6 and ND4L). This novel conditional system can shed light not only on the mechanism of complex I assembly but emphasizes the role of subunits previously thought of as "accessory." It promises to have broader applications in the study of cellular energy metabolism and production of reactive oxygen species and related processes.

The MWFE protein (70 amino acids) is highly conserved in evolution, but the human protein (80% identical to hamster) does not complement a null mutation in Chinese hamster cells. We have identified a small protein segment where significant differences exist between rodents and primates, illustrating very specifically the need for compatibility of the nuclear and mitochondrial genomes in the assembly of complex I. The segment between amino acids 39 and 46 appears to be critical for species-specific compatibility. Amino acid substitutions in this region were tested that caused a reduction of activity of the hamster protein or converted the inactive human protein into a partially active one. Such mutations could be useful in making mice with partial complex I activity as models for mitochondrial diseases. Their potential as dominant negative mutants was explored. More deleterious mutations in the NDUFA1 gene were also characterized. A conservative substitution, R50K, or a short C-terminal deletion makes the protein completely inactive. In the absence of MWFE, no high molecular weight complex was detectable by Blue Native-gel electrophoresis. The MWFE protein itself is unstable in the absence of assembled mitochondrially encoded integral membrane proteins of complex I.

FbxA is a novel member of a family of proteins that contain an F-box and WD40 repeats and that target specific proteins for degradation via proteasomes. In fruiting bodies formed from cells where the fbxA gene is disrupted (fbxA(-) cells), the spore mass fails to fully ascend the stalk. In addition, fbxA(-) slugs continue to migrate under environmental conditions where the parental strain immediately forms fruiting bodies. Consistent with this latter behaviour, the development of fbxA(-) cells is hypersensitive to ammonia, the signaling molecule that regulates the transition from the slug stage to terminal differentiation. The slug comprises an anterior prestalk region and a posterior prespore region and the fbxA mRNA is highly enriched in the prestalk cells. The prestalk zone of the slug is further subdivided into an anterior pstA region and a posterior pstO region. In fbxA(-) slugs the pstO region is reduced in size and the prespore region is proportionately expanded. Our results indicate that FbxA is part of a regulatory pathway that controls cell fate decisions and spatial patterning via regulated protein degradation., (Copyright 2000 Academic Press.)