ATP Pool Research Articles

Reconstructed Metabolic Network Models Predict Flux-Level Metabolic Reprogramming in Glioblastoma.

Developments in genome scale metabolic modeling techniques and omics technologies have enabled the reconstruction of context-specific metabolic models. In this study, glioblastoma multiforme (GBM), one of the most common and aggressive malignant brain tumors, is investigated by mapping GBM gene expression data on the growth-implemented brain specific genome-scale metabolic network, and GBM-specific models are generated. The models are used to calculate metabolic flux distributions in the tumor cells. Metabolic phenotypes predicted by the GBM-specific metabolic models reconstructed in this work reflect the general metabolic reprogramming of GBM, reported both in in-vitro and in-vivo experiments. The computed flux profiles quantitatively predict that major sources of the acetyl-CoA and oxaloacetic acid pool used in TCA cycle are pyruvate dehydrogenase from glycolysis and anaplerotic flux from glutaminolysis, respectively. Also, our results, in accordance with recent studies, predict a contribution of oxidative phosphorylation to ATP pool via a slightly active TCA cycle in addition to the major contributor aerobic glycolysis. We verified our results by using different computational methods that incorporate transcriptome data with genome-scale models and by using different transcriptome datasets. Correct predictions of flux distributions in glycolysis, glutaminolysis, TCA cycle and lipid precursor metabolism validate the reconstructed models for further use in future to simulate more specific metabolic patterns for GBM.

Anoxic metabolism and biochemical production in Pseudomonas putida F1 driven by a bioelectrochemical system.

BackgroundPseudomonas putida is a promising host for the bioproduction of chemicals, but its industrial applications are significantly limited by its obligate aerobic character. The aim of this paper is to empower the anoxic metabolism of wild-type Pseudomonas putida to enable bioproduction anaerobically, with the redox power from a bioelectrochemical system (BES).ResultsThe obligate aerobe Pseudomonas putida F1 was able to survive and produce almost exclusively 2–Keto-gluconate from glucose under anoxic conditions due to redox balancing with electron mediators in a BES. 2-Keto-gluconate, a precursor for industrial anti-oxidant production, was produced at an overall carbon yield of over 90 % based on glucose. Seven different mediator compounds were tested, and only those with redox potential above 0.207 V (vs standard hydrogen electrode) showed interaction with the cells. The productivity increased with the increasing redox potential of the mediator, indicating this was a key factor affecting the anoxic production process. P. putida cells survived under anaerobic conditions, and limited biofilm formation could be observed on the anode’s surface. Analysis of the intracellular pools of ATP, ADP and AMP showed that cells had an increased adenylate energy charge suggesting that cells were able to generate energy using the anode as terminal electron acceptor. The analysis of NAD(H) and NADP(H) showed that in the presence of specific extracellular electron acceptors, the NADP(H) pool was more oxidised, while the NAD(H) pool was unchanged. This implies a growth limitation under anaerobic conditions due to a shortage of NADPH and provides a way to limit biomass formation, while allowing cell maintenance and catalysis at high purity and yield.ConclusionsFor the first time, this study proved the principle that a BES-driven bioconversion of glucose can be achieved for a wild-type obligate aerobe. This non-growth bioconversion was in high yields, high purity and also could deliver the necessary metabolic energy for cell maintenance. By combining this approach with metabolic engineering strategies, this could prove to be a powerful new way to produce bio-chemicals and fuels from renewables in both high yield and high purity.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0452-y) contains supplementary material, which is available to authorized users.

Deficiency in Apoptosis-Inducing Factor Recapitulates Chronic Kidney Disease via Aberrant Mitochondrial Homeostasis.

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein with dual roles in redox signaling and programmed cell death. Deficiency in AIF is known to result in defective oxidative phosphorylation (OXPHOS), via loss of complex I activity and assembly in other tissues. Because the kidney relies on OXPHOS for metabolic homeostasis, we hypothesized that a decrease in AIF would result in chronic kidney disease (CKD). Here, we report that partial knockdown of Aif in mice recapitulates many features of CKD, in association with a compensatory increase in the mitochondrial ATP pool via a shift toward mitochondrial fusion, excess mitochondrial reactive oxygen species production, and Nox4 upregulation. However, despite a 50% lower AIF protein content in the kidney cortex, there was no loss of complex I activity or assembly. When diabetes was superimposed onto Aif knockdown, there were extensive changes in mitochondrial function and networking, which augmented the renal lesion. Studies in patients with diabetic nephropathy showed a decrease in AIF within the renal tubular compartment and lower AIFM1 renal cortical gene expression, which correlated with declining glomerular filtration rate. Lentiviral overexpression of Aif1m rescued glucose-induced disruption of mitochondrial respiration in human primary proximal tubule cells. These studies demonstrate that AIF deficiency is a risk factor for the development of diabetic kidney disease.

Intracellular ATP Decrease Mediates NLRP3 Inflammasome Activation upon Nigericin and Crystal Stimulation.

Activation of the nucleotide-binding oligomerization domain-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome initiates an inflammatory response, which is associated with host defense against pathogens and the progression of chronic inflammatory diseases such as gout and atherosclerosis. The NLRP3 inflammasome mediates caspase-1 activation and subsequent IL-1β processing in response to various stimuli, including extracellular ATP, although the roles of intracellular ATP (iATP) in NLRP3 activation remain unclear. In this study, we found that in activated macrophages artificial reduction of iATP by 2-deoxyglucose, a glycolysis inhibitor, caused mitochondrial membrane depolarization, leading to IL-1β secretion via NLRP3 and caspase-1 activation. Additionally, the NLRP3 activators nigericin and monosodium urate crystals lowered iATP through K(+)- and Ca(2+)-mediated mitochondrial dysfunction, suggesting a feedback loop between iATP loss and lowering of mitochondrial membrane potential. These results demonstrate the fundamental roles of iATP in the maintenance of mitochondrial function and regulation of IL-1β secretion, and they suggest that maintenance of the intracellular ATP pools could be a strategy for countering NLRP3-mediated inflammation.

Comparative Genomics of Candidate Phylum TM6 Suggests That Parasitism Is Widespread and Ancestral in This Lineage.

Candidate phylum TM6 is a major bacterial lineage recognized through culture-independent rRNA surveys to be low abundance members in a wide range of habitats; however, they are poorly characterized due to a lack of pure culture representatives. Two recent genomic studies of TM6 bacteria revealed small genomes and limited gene repertoire, consistent with known or inferred dependence on eukaryotic hosts for their metabolic needs. Here, we obtained additional near-complete genomes of TM6 populations from agricultural soil and upflow anaerobic sludge blanket reactor metagenomes which, together with the two publicly available TM6 genomes, represent seven distinct family level lineages in the TM6 phylum. Genome-based phylogenetic analysis confirms that TM6 is an independent phylum level lineage in the bacterial domain, possibly affiliated with the Patescibacteria superphylum. All seven genomes are small (1.0–1.5 Mb) and lack complete biosynthetic pathways for various essential cellular building blocks including amino acids, lipids, and nucleotides. These and other features identified in the TM6 genomes such as a degenerated cell envelope, ATP/ADP translocases for parasitizing host ATP pools, and protein motifs to facilitate eukaryotic host interactions indicate that parasitism is widespread in this phylum. Phylogenetic analysis of ATP/ADP translocase genes suggests that the ancestral TM6 lineage was also parasitic. We propose the name Dependentiae (phyl. nov.) to reflect dependence of TM6 bacteria on host organisms.

Manipulation of the ATP pool as a tool for metabolic engineering.

Cofactor engineering has been long identified as a valuable tool for metabolic engineering. Besides interventions targeting the pools of redox cofactors, many studies addressed the adenosine pools of microorganisms. In this mini-review, we discuss interventions that manipulate the availability of ATP with a special focus on ATP wasting strategies. We discuss the importance to fine-tune the ATP yield along a production pathway to balance process performance parameters like product yield and volumetric productivity.

3-Bromopyruvate induces rapid human prostate cancer cell death by affecting cell energy metabolism, GSH pool and the glyoxalase system.

3-bromopyruvate (3-BP) is an anti-tumour drug effective on hepatocellular carcinoma and other tumour cell types, which affects both glycolytic and mitochondrial targets, depleting cellular ATP pool. Here we tested 3-BP on human prostate cancer cells showing, differently from other tumour types, efficient ATP production and functional mitochondrial metabolism. We found that 3-BP rapidly induced cultured androgen-insensitive (PC-3) and androgen-responsive (LNCaP) prostate cancer cell death at low concentrations (IC(50) values of 50 and 70 μM, respectively) with a multimodal mechanism of action. In particular, 3-BP-treated PC-3 cells showed a selective, strong reduction of glyceraldeide 3-phosphate dehydrogenase activity, due to the direct interaction of the drug with the enzyme. Moreover, 3-BP strongly impaired both glutamate/malate- and succinate-dependent mitochondrial respiration, membrane potential generation and ATP synthesis, concomitant with the inhibition of respiratory chain complex I, II and ATP synthase activities. The drastic reduction of cellular ATP levels and depletion of GSH pool, associated with significant increase in cell oxidative stress, were found after 3-BP treatment of PC-3 cells. Interestingly, the activity of both glyoxalase I and II, devoted to the elimination of the cytotoxic methylglyoxal, was strongly inhibited by 3-BP. Both N-acetylcysteine and aminoguanidine, GSH precursor and methylglyoxal scavenger, respectively, prevented 3-BP-induced PC-3 cell death, showing that impaired cell antioxidant and detoxifying capacities are crucial events leading to cell death. The provided information on the multi-target cytotoxic action of 3-BP, finally leading to PC-3 cell necrosis, might be useful for future development of 3-BP as a therapeutic option for prostate cancer treatment.

Targeting metabolic flexibility by simultaneously inhibiting respiratory complex I and lactate generation retards melanoma progression.

Melanoma is a largely incurable skin malignancy owing to the underlying molecular and metabolic heterogeneity confounded by the development of resistance. Cancer cells have metabolic flexibility in choosing either oxidative phosphorylation (OXPHOS) or glycolysis for ATP generation depending upon the nutrient availability in tumor microenvironment. In this study, we investigated the involvement of respiratory complex I and lactate dehydrogenase (LDH) in melanoma progression. We show that inhibition of complex I by metformin promotes melanoma growth in mice via elevating lactate and VEGF levels. In contrast, it leads to the growth arrest in vitro because of enhanced extracellular acidification as a result of increased glycolysis. Inhibition of LDH or lactate generation causes decrease in glycolysis with concomitant growth arrest both in vitro and in vivo. Blocking lactate generation in metformin-treated melanoma cells results in diminished cell proliferation and tumor progression in mice. Interestingly, inhibition of either LDH or complex I alone does not induce apoptosis, whereas inhibiting both together causes depletion in cellular ATP pool resulting in metabolic catastrophe induced apoptosis. Overall, our study suggests that LDH and complex I play distinct roles in regulating glycolysis and cell proliferation. Inhibition of these two augments synthetic lethality in melanoma.

Efficient entry of cell-penetrating peptide nona-arginine into adherent cells involves a transient increase in intracellular calcium.

Understanding the mechanism of entry of cationic peptides such as nona-arginine (R9) into cells remains an important challenge to their use as efficient drug-delivery vehicles. At nanomolar to low micromolar R9 concentrations and at physiological temperature, peptide entry involves endocytosis. In contrast, at a concentration ≥10μM, R9 induces a very effective non-endocytic entry pathway specific for cationic peptides. We found that a similar entry pathway is induced at 1-2μM concentrations of R9 if peptide application is accompanied by a rapid temperature drop to 15°C. Both at physiological and at sub-physiological temperatures, this entry mechanism was inhibited by depletion of the intracellular ATP pool. Intriguingly, we found that R9 at 10-20μM and 37°C induces repetitive spikes in intracellular Ca(2+) concentration. This Ca(2+) signalling correlated with the efficiency of the peptide entry. Pre-loading cells with the Ca(2+) chelator BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) inhibited both Ca(2+) spikes and peptide entry, suggesting that an increase in intracellular Ca(2+) precedes and is required for peptide entry. One of the hallmarks of Ca(2+) signalling is a transient cell-surface exposure of phosphatidylserine (PS), a lipid normally residing only in the inner leaflet of the plasma membrane. Blocking the accessible PS with the PS-binding domain of lactadherin strongly inhibited non-endocytic R9 entry, suggesting the importance of PS externalization in this process. To conclude, we uncovered a novel mechanistic link between calcium signalling and entry of cationic peptides. This finding will enhance our understanding of the properties of plasma membrane and guide development of future drug-delivery vehicles.

Salvage of nicotinamide adenine dinucleotide plays a critical role in the bioenergetic recovery of post-hypoxic cardiomyocytes.

Ischaemic heart disease can lead to serious, life-threatening complications. Traditional therapies for ischaemia aim to increase oxygen delivery and reduce the myocardial ATP consumption by increasing the coronary perfusion and by suppressing cardiac contractility, heart rate or blood pressure. An adjunctive treatment option for ischaemia is to improve or optimize myocardial metabolism. Metabolic suppression in the ischaemic heart is characterized by reduced levels of high-energy molecules: ATP and NAD(+) . Because NAD(+) is required for most metabolic processes that generate ATP, we hypothesized that restoration of NAD(+) would be a prerequisite for ATP regeneration and examined the role of the major NAD(+) anabolic and catabolic pathways in the bioenergetic restoration process following oxygen-glucose deprivation injury in a cardiomyocyte cell line (H9c2 cells). Salvage of NAD(+) via nicotinamide phosphoribosyl transferase was essential for bioenergetic recovery in cardiomyocytes. Blockade of nicotinamide phosphoribosyl transferase prevented the restoration of the cellular ATP pool following oxygen-glucose deprivation injury by inhibiting both the aerobic and anaerobic metabolism in the cardiomyocytes. NAD(+) consumption by PARP-1 also undermined the recovery processes, and PARP inhibition significantly improved the metabolism and increased cellular ATP levels in cardiomyocytes. We conclude that the NAD(+) salvage pathway is essential for bioenergetic recovery in post-hypoxic cardiomyocytes and PARP inhibition may represent a potential future therapeutic intervention in ischaemic heart disease.

Piezo1 regulates mechanotransductive release of ATP from human RBCs

Piezo proteins (Piezo1 and Piezo2) are recently identified mechanically activated cation channels in eukaryotic cells and associated with physiological responses to touch, pressure, and stretch. In particular, human RBCs express Piezo1 on their membranes, and mutations of Piezo1 have been linked to hereditary xerocytosis. To date, however, physiological functions of Piezo1 on normal RBCs remain poorly understood. Here, we show that Piezo1 regulates mechanotransductive release of ATP from human RBCs by controlling the shear-induced calcium (Ca(2+)) influx. We find that, in human RBCs treated with Piezo1 inhibitors or having mutant Piezo1 channels, the amounts of shear-induced ATP release and Ca(2+) influx decrease significantly. Remarkably, a critical extracellular Ca(2+) concentration is required to trigger significant ATP release, but membrane-associated ATP pools in RBCs also contribute to the release of ATP. Our results show how Piezo1 channels are likely to function in normal RBCs and suggest a previously unidentified mechanotransductive pathway in ATP release. Thus, we anticipate that the study will impact broadly on the research of red cells, cellular mechanosensing, and clinical studies related to red cell disorders and vascular disease.

Blowing Off Steam: Virus Inhibition of cGAS DNA Sensing.

Detection of viral DNA is essential for eliciting mammalian innate immunity. However, viruses have acquired effective mechanisms for blocking host defense. Indeed, in this issue of Cell Host & Microbe, Wu et al. (2015) discover a herpesviral strategy for inhibiting the prominent host sensor of viral DNA, cGAS.

Abstract 5462: Extracellular ATP promotes cancer cell drug resistance to tyrosine kinase inhibitors by competing for the ATP-binding site of tyrosine kinase

Abstract ATP plays important roles in cellular processes in cancer cells, which are a heterogeneous population in tumors regarding their genetic background and oxygen and energy supplies. The fact that cancer cells undergo rapid proliferation suggests that they must possess mechanisms, which may not be present in normal cells, to meet their high energy needs. Studies by others revealed that extracellular ATP levels of various cancer types are 10^3 to 10^4 times higher than those in their corresponding normal tissues (1). We recently reported that extracellular ATP can be taken up directly by cancer cells by macropinocytosis (2). Based on these results, we hypothesized that the internalized extracellular ATP not only promotes cancer cell growth and survival, but also drug resistance. In this study, human lung cancer A549 and breast cancer MCF7 cells were used as cell models to determine the effects of extracellular ATP on drug resistance to tyrosine kinase inhibitors (TKIs) in cancer cells. Cancer cell viability and intracellular ATP measurement results showed that extracellular ATP substantially increased intracellular ATP levels and promoted cancer cell drug resistance to TKIs. Viability of TKI treated cancer cells was significantly increased in the presence of extracellular ATP, compared to cancer cells with TKI treatment alone. Administration of macropinocytosis inhibitors significantly reduced the drug resistance induced by extracellular ATP, suggesting that extracellular ATP promoted drug resistance by contributing to the intracellular ATP pool. Protein and protein phosphorylation analyses results suggested that the increased intracellular ATP reversed the inhibition of TKIs by competing with the inhibitor for the ATP-binding site of platelet-derived growth factor receptor (PDGFR) and enhancing Akt and MAPK signaling pathways. These findings identify, for the first time, new biological functions of extracellular ATP in tumors and may lead to novel anti-cancer strategies combating drug resistance in cancers. 1. Pellegatti P, et al. PLoS ONE 2008;3:e2599. 2. Qian Y, et al. Cancer Lett 2014;351:242-51. Citation Format: Xuan Wang, Yanrong Qian, Yunsheng Li, Xiaozhuo Chen. Extracellular ATP promotes cancer cell drug resistance to tyrosine kinase inhibitors by competing for the ATP-binding site of tyrosine kinase. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5462. doi:10.1158/1538-7445.AM2015-5462

Compartment‐specific metabolomics for CHO reveals that ATP pools in mitochondria are much lower than in cytosol

Mammalian cells show a compartmented metabolism. Getting access to subcellular metabolite pools is of high interest to understand the cells' metabolomic state. Therefore a protocol is developed and applied for monitoring compartment-specific metabolite and nucleotide pool sizes in Chinese hamster ovary (CHO) cells. The approach consists of a subtracting filtering method separating cytosolic components from physically intact mitochondrial compartments. The internal standards glucose-6-phosphate and cis-aconitate were chosen to quantify cytosolic secession and mitochondrial membrane integrity. Extracts of related fractions were studied by liquid chromatography-isotope dilution mass spectrometry for the absolute quantification of a subset of glycolytic and tricarboxylic acid cycle intermediates together with the adenylate nucleotides ATP, ADP and AMP. The application of the protocol revealed highly dynamic changes in the related pool sizes as a function of distinct cultivation periods of IgG1 producing CHO cells. Mitochondrial and cytosolic pool dynamics were in agreement with anticipated metabolite pools of independent studies. The analysis of adenosine phosphate levels unraveled significantly higher ATP levels in the cytosol leading to the hypothesis that mitochondria predominantly serve for fueling ATP into the cytosol where it is tightly controlled at physiological adenylate energy charges about 0.9.

Sirtuins Function as the Modulators in Aging-related Diseases in Common or Respectively.

Sirtuins Function as the Modulators in Aging-related Diseases in Common or Respectively.

UV-B stress induced metabolic rearrangements explored with comparative proteomics in three Anabaena species

Comparative proteomics together with physiological variables revealed different responses among three species of diazotrophic cyanobacterium Anabaena exposed to UV-B stress at the same time points. Perceptible decline in PSII activity, ATP pool, nitrogenase activity and respiration rate was observed for all the three species; this being maximum in Anabaena doliolum, followed by Anabaena sp. PCC 7120 and minimum in Anabaena L31. Statistical analysis of the protein abundance divided majority of them as early accumulated in A. L31, late accumulated in A. sp. PCC 7120 and downregulated in A. doliolum. Tolerance of A. L31 may be ascribed to post-translational modification reflected through the highest number of protein isoforms in its proteome followed by A. PCC 7120 and A. doliolum. Furthermore, increase in abundance of cyanophycinase, glutamine synthetase and succinate semialdehyde dehydrogenase in A. L31 suggests operation of an alternate pathway for assimilation of nitrogen and carbon under UV-B stress. An early accumulation of four proteins viz., glutamate ammonia ligase (Alr2328), transketolase (Alr3344), inorganic pyrophosphatase (All3570), and trigger protein (Alr3681) involved respectively in amino acid metabolism, energy metabolism, biosynthesis of cofactor and trigger protein and chaperone like activity across three species, suggests them to be marker of UV-B stress in Anabaena spp.This article is part of a Special Issue entitled: Proteomics in India.

Genetics and Physiology of Acetate Metabolism by the Pta-Ack Pathway of Streptococcus mutans.

In the dental caries pathogen Streptococcus mutans, phosphotransacetylase (Pta) catalyzes the conversion of acetyl coenzyme A (acetyl-CoA) to acetyl phosphate (AcP), which can be converted to acetate by acetate kinase (Ack), with the concomitant generation of ATP. A ΔackA mutant displayed enhanced accumulation of AcP under aerobic conditions, whereas little or no AcP was observed in the Δpta or Δpta ΔackA mutant. The Δpta and Δpta ΔackA mutants also had diminished ATP pools compared to the size of the ATP pool for the parental or ΔackA strain. Surprisingly, when exposed to oxidative stress, the Δpta ΔackA strain appeared to regain the capacity to produce AcP, with a concurrent increase in the size of the ATP pool compared to that for the parental strain. The ΔackA and Δpta ΔackA mutants exhibited enhanced (p)ppGpp accumulation, whereas the strain lacking Pta produced less (p)ppGpp than the wild-type strain. The ΔackA and Δpta ΔackA mutants displayed global changes in gene expression, as assessed by microarrays. All strains lacking Pta, which had defects in AcP production under aerobic conditions, were impaired in their abilities to form biofilms when glucose was the growth carbohydrate. Collectively, these data demonstrate the complex regulation of the Pta-Ack pathway and critical roles for these enzymes in processes that appear to be essential for the persistence and pathogenesis of S. mutans.

In vitro evaluation of Schisandra extracts (SE) herb-drug interaction potential utilizing B-Clear® sandwich-culture human hepatocytes (SCHH)

Herb-drug interaction (HDI) potential should be evaluated given the dramatic increase in the sale and use of alternative medicines. A fully integrated hepatic cell system that includes drug metabolism, drug transport, and key regulatory pathways was utilized to assess SE HDI potential. We investigated SE for hepatocyte cytotoxicity using ATP and LDH assays following 24 or 72 hrs exposure. After 24 hrs exposure with S. chinensis extract (SCE) or S. sphenanthera extract (SSE), no marked changes were observed in either assay at the concentrations examined. However, ATP pools were modestly reduced in hepatocytes following 72 hrs of exposure at the highest concentrations. Next, we examined the SE potential to inhibit CYP3A4/5 enzyme activity and P-gP function in our system. Significant inhibition of OH-midazolam formation was observed in SCHH by SCE (IC50 = 4.4 µg/mL) and SPE (IC50 = 0.8 µg/mL). Pre-incubation of SCE in SCHH prior to midazolam exposure yielded a 3-fold decrease in the CYP3A4/5 IC50 providing evidence of time-dependent inhibition; an effect not observed with SPE. Significant inhibition of digoxin efflux was observed with increasing concentrations of both SE, reducing the digoxin hepatobiliary clearance significantly. Finally, we examined the potential of SE to modulate the expression of P450 or drug transporter proteins in SCHH following 72 hr of exposure to SCE or SPE. St. John's Wort (20 µg/mL), a positive control inducer, induced CYP2B6 and CYP3A4 mRNA and enzyme activity demonstrating that key regulatory pathways in SCHH were functional. Changes were observed in gene expression and functional assays in response to SE. Our results of SPE inhibition of CYP3A4/5 and P-gP are consistent with clinical data that demonstrated significant SPE drug interactions resulting in > 60% increase in AUC of tacrolimus. In summary, in vitro results demonstrated that SCE and SPE both had similar inhibition profiles of CYP3A4/5 and P-gP but SCE has a higher potential to cause clinically relevant HDIs as compared to the SPE varietal because of time-dependent inhibition.

Expressing AtNHX1 in barley (Hordium vulgare L.) does not improve plant performance under saline conditions

Salinity is one of the main causes of yield loss in agricultural crop production worldwide. Numerous attempts have been made to produce salinity tolerant crops by manipulating the expression of genes involved in ion transport; yet, the reported results are often rather controversial. In this work, Arabidopsis AtNHX1 gene encoding vacuolar Na+/H+ exchanger was introduced into barley (Horduem vulgare L. cv. Golden Promise) plants grown under saline conditions. Transgenic plants were compared to null segregants for biomass, water content, gas exchange, and Na+ and K+ content of the leaf. It was shown that most of the lines expressing AtNHX1 have no significant difference to null segregants, suggesting the lack of beneficial effect of expressing the AtNHX1 gene in barley, at least under our experimental conditions. The lack of phenotype might be explained by: (1) low level of activity of vacuolar H+-inorganic pyrophosphatase and vacuolar H+-ATPase resulting in an insufficient proton gradient required for the vacuolar Na+/H+ exchanger; (2) the inability of transgenic plants to prevent a passive leak of sodium via Na+ permeable slow activating and fast activating channels; (3) insufficient ATP pool to support H+ pumping activity; and (4) the fact that the AtNHX1 protein might not been properly folded, inactive or incorrectly targeted. Overall, this study suggests that, while targeting vacuolar Na+ sequestration, at least several genes need to be pyramided to ensure the efficiency of the above process.

Creatine kinase B is necessary to limit myoblast fusion during myogenesis.

Myoblast fusion is critical for proper muscle growth and regeneration. During myoblast fusion, the localization of some molecules is spatially restricted; however, the exact reason for such localization is unknown. Creatine kinase B (CKB), which replenishes local ATP pools, localizes near the ends of cultured primary mouse myotubes. To gain insights into the function of CKB, we performed a yeast two-hybrid screen to identify CKB-interacting proteins. We identified molecules with a broad diversity of roles, including actin polymerization, intracellular protein trafficking, and alternative splicing, as well as sarcomeric components. In-depth studies of α-skeletal actin and α-cardiac actin, two predominant muscle actin isoforms, demonstrated their biochemical interaction and partial colocalization with CKB near the ends of myotubes in vitro. In contrast to other cell types, specific knockdown of CKB did not grossly affect actin polymerization in myotubes, suggesting other muscle-specific roles for CKB. Interestingly, knockdown of CKB resulted in significantly increased myoblast fusion and myotube size in vitro, whereas knockdown of creatine kinase M had no effect on these myogenic parameters. Our results suggest that localized CKB plays a key role in myotube formation by limiting myoblast fusion during myogenesis.