Loss Of ATP Research Articles

Ammonium trichloro [1,2-ethanediolato-O,O']-tellurate cures experimental visceral leishmaniasis by redox modulation of Leishmania donovani trypanothione reductase and inhibiting host integrin linked PI3K/Akt pathway.

In an endeavor to search for affordable and safer therapeutics against debilitating visceral leishmaniasis, we examined antileishmanial potential of ammonium trichloro [1,2-ethanediolato-O,O']-tellurate (AS101); a tellurium based non toxic immunomodulator. AS101 showed significant in vitro efficacy against both Leishmania donovani promastigotes and amastigotes at sub-micromolar concentrations. AS101 could also completely eliminate organ parasite load from L. donovani infected Balb/c mice along with significant efficacy against infected hamsters (˃93% inhibition). Analyzing mechanistic details revealed that the double edged AS101 could directly induce apoptosis in promastigotes along with indirectly activating host by reversing T-cell anergy to protective Th1 mode, increased ROS generation and anti-leishmanial IgG production. AS101 could inhibit IL-10/STAT3 pathway in L. donovani infected macrophages via blocking α4β7 integrin dependent PI3K/Akt signaling and activate host MAPKs and NF-κB for Th1 response. In silico docking and biochemical assays revealed AS101's affinity to form thiol bond with cysteine residues of trypanothione reductase in Leishmania promastigotes leading to its inactivation and inducing ROS-mediated apoptosis of the parasite via increased Ca2+ level, loss of ATP and mitochondrial membrane potential along with metacaspase activation. Our findings provide the first evidence for the mechanism of action of AS101 with excellent safety profile and suggest its promising therapeutic potential against experimental visceral leishmaniasis.

Antioxidant defense in quiescent cells determines selectivity of electron transport chain inhibition-induced cell death

Mitochondrial electron transport chain (ETC) targeting shows a great promise in cancer therapy. It is particularly effective in tumors with high ETC activity where ETC-derived reactive oxygen species (ROS) are efficiently induced. Why modern ETC-targeted compounds are tolerated on the organismal level remains unclear. As most somatic cells are in non-proliferative state, the features associated with the ETC in quiescence could account for some of the specificity observed. Here we report that quiescent cells, despite increased utilization of the ETC and enhanced supercomplex assembly, are less susceptible to cell death induced by ETC disruption when glucose is not limiting. Mechanistically, this is mediated by the increased detoxification of ETC-derived ROS by mitochondrial antioxidant defense, principally by the superoxide dismutase 2 – thioredoxin axis. In contrast, under conditions of glucose limitation, cell death is induced preferentially in quiescent cells and is correlated with intracellular ATP depletion but not with ROS. This is related to the inability of quiescent cells to compensate for the lost mitochondrial ATP production by the upregulation of glucose uptake. Hence, elevated ROS, not the loss of mitochondrially-generated ATP, are responsible for cell death induction by ETC disruption in ample nutrients condition, e.g. in well perfused healthy tissues, where antioxidant defense imparts specificity. However, in conditions of limited glucose, e.g. in poorly perfused tumors, ETC disruption causes rapid depletion of cellular ATP, optimizing impact towards tumor-associated dormant cells. In summary, we propose that antioxidant defense in quiescent cells is aided by local glucose limitations to ensure selectivity of ETC inhibition-induced cell death.

The Role of Light-Dark Regulation of the Chloroplast ATP Synthase.

The chloroplast ATP synthase catalyzes the light-driven synthesis of ATP and is activated in the light and inactivated in the dark by redox-modulation through the thioredoxin system. It has been proposed that this down-regulation is important for preventing wasteful hydrolysis of ATP in the dark. To test this proposal, we compared the effects of extended dark exposure in Arabidopsis lines expressing the wild-type and mutant forms of ATP synthase that are redox regulated or constitutively active. In contrast to the predictions of the model, we observed that plants with wild-type redox regulation lost photosynthetic capacity rapidly in darkness, whereas those expressing redox-insensitive form were far more stable. To explain these results, we propose that in wild-type plants, down-regulation of ATP synthase inhibits ATP hydrolysis, leading to dissipation of thylakoid proton motive force (pmf) and subsequent inhibition of protein transport across the thylakoid through the twin arginine transporter (Tat)-dependent and Sec-dependent import pathways, resulting in the selective loss of specific protein complexes. By contrast, in mutants with a redox-insensitive ATP synthase, pmf is maintained by ATP hydrolysis, thus allowing protein transport to maintain photosynthetic activities for extended periods in the dark. Hence, a basal level of Tat-dependent, as well as, Sec-dependent import activity, in the dark helps replenishes certain components of the photosynthetic complexes and thereby aids in maintaining overall complex activity. However, the influence of a dark pmf on thylakoid protein import, by itself, could not explain all the effects we observed in this study. For example, we also observed in wild type plants a large transient buildup of thylakoid pmf and nonphotochemical exciton quenching upon sudden illumination of dark adapted plants. Therefore, we conclude that down-regulation of the ATP synthase is probably not related to preventing loss of ATP per se. Instead, ATP synthase redox regulation may be impacting a number of cellular processes such as (1) the accumulation of chloroplast proteins and/or ions or (2) the responses of photosynthesis to rapid changes in light intensity. A model highlighting the complex interplay between ATP synthase regulation and pmf in maintaining various chloroplast functions in the dark is presented.Significance Statement: We uncover an unexpected role for thioredoxin modulation of the chloroplast ATP synthase in regulating the dark-stability of the photosynthetic apparatus, most likely by controlling thylakoid membrane transport of proteins and ions.

Abstract 14: Identification of a Major Role for Cytochrome B5 Reductase 3 in Cardiomyocyte Metabolism and Function

Cytochrome B 5 Reductase 3 (Cyb5R3) also known as methemoglobin reductase regulates redox signaling in erythrocytes and endothelial cells by maintaining heme iron in the reduced (Fe 2+ ) state. Knowing the importance of highly regulated redox signaling in other hematopoietic and somatic lineages, we conducted a pharmacological inhibition study using a CyB5R3 inhibitor in mice. Inhibition resulted in dilated cardiomyopathy (DCM) after 2 weeks. To determine the potential for human relevance, a high frequency point mutation (T117S) in African American populations was studied and served as a model to understand the impact of mutated CyB5R3 in human heart failure. We found T117S individuals associated with accelerated time to first acute cardiac events and time to death. With this evidence and the unknown function of CyB5R3 in cardiomyocytes, we created the first CyB5R3, cardiomyocyte specific inducible knockout (i-cKO) (Myh6-Cre ERT2 - flox/flox) and we observed >50% lethality 15 days post-last tamoxifen injection. These mice recapitulated a DCM phenotype similar to the pharmacological study. Hemodynamic measurements showed increased left and right ventricular stroke volumes and decreased ejection fractions. Histology displayed myocardial inflammation and early stage fibrosis while electron microscopy revealed myofibril dystrophy. With these results, we hypothesized CyB5R3 i-cKO mice develop impaired metabolism and bioenergetics due to loss of CyB5R3 mediated heme reduction. I-cKO animals had smaller mitochondrial size, a 30% loss of total ATP and a rise in lactate production, indicating glycolytic shift from oxidative phosphorylation. RNAseq analysis showed decreased transcription in mitochondrial complexes I, II and IV and decreased complex IV activity. Since oxygen is the final electron acceptor for complex IV, we hypothesized the loss of CyB5R3 impaired oxygen delivery to the mitochondria. Therefore, a “psuedohypoxic state” was created, which was supported via Hypoxyprobe staining, by labeling cardiomyocytes specifically with low pO 2 . Collectively, the results provide an important breakthrough in cardiomyocyte biology by identifying Cyb5R3 as the first heme iron reductase critical for regulating cardiac metabolism.

Abstract LB-308: KPT-9274 inhibits cellular NAD and synergizes with doxorubicin to treat dogs with lymphoma

Abstract Nicotinamide adenine dinucleotide (NAD), an essential metabolite and cofactor of several biological processes (e.g. genomic stability), undergoes significant alterations during malignant transformation. In cancer cells, the high metabolic demands of proliferating cells and increased activity of NAD consuming enzymes (i.e. SIRT1 and PARP1) lead to rapid NAD turnover. NAD can be generated de novo from tryptophan or regenerated by nicotinamide phosphoribosyl transferase (NAMPT) or nicotinate phosphoribosyl transferase 1 (NAPRT1) in NAD salvage pathways. However, cancer cells rely mainly on the NAMPT-dependent pathway, making NAD depletion a promising anti-cancer therapy. KPT-9274 is a novel first-in-class orally bioavailable dual inhibitor of p21-activated kinase 4 (PAK4) and NAMPT that demonstrates potent anti-tumor activity in a variety of cancer cell lines. KPT-9274 treatment rapidly depletes cellular NAD levels, leading to ATP loss and cell death. Similarly, hyper activation of PARP1 through DNA damaging agents (e.g. doxorubicin; DOX) can lead to apoptosis. Here we present results from a phase 1 study of KPT-9274 as a single agent and in combination with DOX for the treatment of dogs with solid tumors or lymphomas. Methods: Dogs with cancer (lymphoma, n=4; sarcoma n=6; mast cell tumor n=2) were enrolled into a prospective phase 1 dose escalation study of KPT-9274 given qod x 3 per week. Plasma samples were used for pharmacokinetics (PK) and tumor biopsies were used to assess pharmacodynamics (PDn; i.e. PAK4, NAMPT and downstream effectors). In an expansion cohort, dogs with lymphoma (n=6) received 4 doses of KPT-9274 at 2 mg/kg prior to administration of a single dose of DOX. Results: Doses up to 4 mg/kg KPT-9274 were well tolerated with no grade ≥3 toxicities. At 4.5 mg/kg one dog exhibited severe vomiting, diarrhea, collapse, anemia and thrombocytopenia, establishing 4 mg/kg as MTD. The PK of KPT-9274 was dose proportional and in agreement with healthy dogs. PDn markers (NAD levels) in tumors showed target engagement through NAD depletion as well as changes in PAK4 pathway biomarkers using IHC. Four dogs exhibited stable disease during treatment (3 soft tissue sarcomas, 1 mast cell tumor) at doses ranging from 3 - 4.5 mg/kg. Of the 6 dogs with naïve lymphoma that received KPT-9274 and a single dose of DOX, 5 achieved a complete response, one of which lasted for over 3 months. No unexpected toxicities were noted with the combination when compared to those expected from DOX alone. Conclusions: KPT-9274 exhibits single agent activity in canine spontaneous cancers. Moreover, the combination of KPT-9274 and DOX has substantial biologic activity against canine Non-Hodgkin lymphoma, likely through the activation of NAD consuming enzymes such as PARP1 by DOX. Importantly, the drug combination was safe with no enhanced toxicity over DOX alone. This data could be applied directly to the current trial of KPT-9274 in a Phase 1 clinical trial in patients with advanced solid malignancies or NHL (NCT02702492). Citation Format: Cheryl London, Megan Brown, Emma Warry, Elizabeth Schuh, William T. Senapedis, Christian Argueta, Trinayan Kashyap, Hua Chang, Joel Ellis, Sharon Shacham, Erkan Baloglu. KPT-9274 inhibits cellular NAD and synergizes with doxorubicin to treat dogs with lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-308. doi:10.1158/1538-7445.AM2017-LB-308

Arctic ground squirrel hippocampus tolerates oxygen glucose deprivation independent of hibernation season even when not hibernating and after ATP depletion, acidosis, and glutamate efflux.

Cerebral ischemia/reperfusion (I/R) triggers a cascade of uncontrolled cellular processes that perturb cell homeostasis. The arctic ground squirrel (AGS), a seasonal hibernator resists brain damage following cerebral I/R caused by cardiac arrest and resuscitation. However, it remains unclear if tolerance to I/R injury in AGS depends on the hibernation season. Moreover, it is also not clear if events such as depletion of ATP, acidosis, and glutamate efflux that are associated with anoxic depolarization are attenuated in AGS. Here, we employ a novel microperfusion technique to test the hypothesis that tolerance to I/R injury modeled in an acute hippocampal slice preparation in AGS is independent of the hibernation season and persists even after glutamate efflux. Acute hippocampal slices were harvested from summer euthermic AGS, hibernating AGS, and interbout euthermic AGS. Slices were subjected to oxygen glucose deprivation (OGD), an invitro model of I/R injury to determine cell death marked by lactate dehydrogenase (LDH) release. ATP was assayed using ENLITEN ATP assay. Glutamate and aspartate efflux was measured using capillary electrophoresis. For acidosis, slices were subjected to pH 6.4 or ischemic shift solution (ISS). Acute hippocampal slices from rats were used as a positive control, susceptible to I/R injury. Our results indicate that when tissue temperature is maintained at 36°C, hibernation season has no influence on OGD-induced cell death in AGS hippocampal slices. Our data also show that tolerance to OGD in AGS hippocampal slices occurs despite loss of ATP and glutamate release, and persists during conditions that mimic acidosis and ionic shifts, characteristic of cerebral I/R. Read the Editorial Comment for this article on page 10.

Glutamate oxaloacetate transaminase enables anaplerotic refilling of TCA cycle intermediates in stroke-affected brain.

Ischemic stroke results in excessive release of glutamate, which contributes to neuronal cell death. Here, we test the hypothesis that otherwise neurotoxic glutamate can be productively metabolized by glutamate oxaloacetate transaminase (GOT) to maintain cellular energetics and protect the brain from ischemic stroke injury. The GOT-dependent metabolism of glutamate was studied in primary neural cells and in stroke-affected C57-BL6 mice using magnetic resonance spectroscopy and GC-MS. Extracellular Glu sustained cell viability under hypoglycemic conditions and increased GOT-mediated metabolism in vitro Correction of stroke-induced hypoxia using supplemental oxygen in vivo lowered Glu levels as measured by 1H magnetic resonance spectroscopy. GOT knockdown abrogated this effect and caused ATP loss in the stroke-affected brain. GOT overexpression increased anaplerotic refilling of tricarboxylic acid cycle intermediates in mouse brain during ischemic stroke. Furthermore, GOT overexpression not only reduced ischemic stroke lesion volume but also attenuated neurodegeneration and improved poststroke sensorimotor function. Taken together, our results support a new paradigm that GOT enables metabolism of otherwise neurotoxic extracellular Glu through a truncated tricarboxylic acid cycle under hypoglycemic conditions.-Rink, C., Gnyawali, S., Stewart, R., Teplitsky, S., Harris, H., Roy, S., Sen, C. K., Khanna, S. Glutamate oxaloacetate transaminase enables anaplerotic refilling of TCA cycle intermediates in stroke-affected brain.

Neonatal seizures and disruption to neurotransmitter systems

Neonatal seizures and disruption to neurotransmitter systems

Alteration in Escherichia coli and Streptococcus faecalis cells induced by ozone

To elucidate ozone action mechanism, cell suspensions of E. coli 8099 and S. faecalis ATCC29212 were initially exposed to ozone for 5 min, and then treated with sodium dodecyl sulfate, sodium hydroxide, and proteinase K. those of S. faecalis were 7.95±2.81%–55.85±1.43%. After ozone pre-treatment, OD600 value of E. coli cells, sequently treated with SDS, NaOH, and PK, decreased by 8.83±1.86%, 34.72±0.42%, and 10.42±2.08%, respectively. And that of S. feacalis decreased by 2.85±3.63%, 71.30±1.08%, and 7.00±2.47%, respectively. Therefore, ozone pre-treatment made E. coli cells be sensitive to SDS and NaOH, not susceptible to PK. It was confirmed by transmission electron microscopy of E. coli cells. To S. faecalis, ozone pre-treatment activated the effects of SDS, NaOH, and PK on cells. ATP loss from S. faecalis cells indicated that ozone damaged cell membrane and increased membrane permeability. Because SDS-, NaOH-, and PK-induced cell lyses were respectively due to protein denaturation, cell membrane damage, and the reactions between PK and proteins or conjugated proteins, it was concluded that protein changes and cell membrane damage generated by ozone are responsible for E. coli and S. faecalis inactivation. Results showed that, after ozone treatment, OD600 reduction rates of E. coli cells were 6.11±1.29%–7.06±1.23%.

Itraconazole, an Oral Antifungal Drug, Inhibits Tumor Growth and Enhances Therapeutic Agent Activity in Double Hit Lymphoma

Background: Among B-cell lymphoma, double hit lymphoma (DHL) is defined by a MYC/8q24 combined with another recurrent BCL/18q21 breakpoint. DHL most affects elderly patients (median age ranged from 51 to 65 years) and have a poor prognosis with a median overall survival of 0.2-1.5 year. R+CHOP or high-intensity treatment including high-dose chemotherapy followed stem cell transplant yield to suboptimal outcomes. Therefore, there is a need to discover alternative effective agents. Itraconazole, an oral antifungal drug, was reported had potent anticancer activity in basal cell carcinoma, non-small cell lung cancer and prostate cancer by interfering with the apoptotic threshold of cancer cells. Its activity in DHL has yet to be defined.Methods: Double hit lymphoma cell lines: DOHH2, VAL and ROS50 were exposed to escalating doses of itraconazole (0-20μM) for 24, 48 and 72h. Changes in cell viability and cell cycle distribution were evaluated using the Presto Blue assay and flow cytometry respectively. IC50 was calculated by Graphpad Prism 6 software. Loss of ATP, Caspase 3/7 activation and loss of mitochondrial membrane potential (Δψm) following itraconazole exposure were assessed by Cell Titer Glo, Caspase3 /7 detection kit and DiOC6 staining, respectively. DHL cells were exposed to itraconazole or vehicle combined with various inhibitors, such as BCL-2 inhibitor ABT199, c-MYC inhibitor JQ1, bruton’s kinase inhibitor ibrutinib and different generations of proteasome inhibitors (carfilzomib or MLN4924) for 48h and cell viability was determined by Presto Blue assay. Coefficient of synergy index was calculated using the CalcuSyn software.Result: Itraconazole consistently showed potent, specific, dose-and time- dependent inhibition in DHL. In vitro exposure cells to itraconazole resulted in a loss of ATP, loss of mitochondrial membrane potential and activation of caspase 3/7 activities. Itraconazole caused G1 cell cycle arrest and decrease S phase in DHL. Moreover, itraconazole had a strong synergistic anti-tumor effect combined with various inhibitors, including BCL-2 inhibitor ABT199, c-MYC inhibitor JQ1, bruton's kinase inhibitor ibrutinib and different generations of proteasome inhibitors (bortezomib, carfilzomib, Ixazomib or MLN4924).Conclusion: Taking together, our data suggest that itraconazole had a potent anti-tumor activity against DHL. Disruption of mitochondrial membrane potential and activation of caspases may contribute its antitumor activity against DHL. It is worth noticing that combined itraconazole with DHL specific target inhibitors ABT199 or JQ1 had a strong synergistic efficacy. Our findings offered the first assessment of the efficacy of itraconazole as a novel anticancer agent in double hit lymphoma. The molecular mechanism of action of itraconazole need to be further investigated in the near further. DisclosuresNo relevant conflicts of interest to declare.

15 - Cytotoxic Mechanism of Supramolecular Helicates

Proteins, DNA and RNA adopt stable tertiary conformations which are characterised by common structural motifs including loops, sheets, hairpins, fingers and helices. A promising approach to develop new drugs is to mimic these naturally occurring three dimensional architectures using large assemblies of organic molecules, termed supramolecules. We have developed a family of supramolecules which consist of four organic strands wound around central metal ions to form structures termed helicates, molecules which mimic naturally occurring helices. Of these helicates, [Pd 2 (hextrz) 4 ] 4+ exhibited rapid cytotoxicity against a panel of cancer cell lines at concentrations 2 (hextrz) 4 ] 4+ operates via a non-classical cell death pathway which features nuclear condensation and mitochondrial damage, accompanied by disruptions to cellular bioenergetics and loss of ATP. Analysis with the membrane probe DiI revealed that the helicate also initiates re-organization of membrane structure, suggesting that [Pd 2 (hextrz) 4 ] 4+ initially interacts with lipids to disrupt cellular compartmentalization, followed by changes to metal ion homeostasis and organelle function. These data provide the first indication that quadruply stranded helicates interact with cells via a different mechanism to other supramolecular complexes, raising the possibility that they may become therapeutic alternatives to current cytotoxic agents.

Cardiotoxicity Associated with Nicotinamide Phosphoribosyltransferase Inhibitors in Rodents and in Rat and Human-Derived Cells Lines.

Nicotinamide phosphoribosyltransferase (NAMPT) is a pleiotropic protein that functions as an enzyme, cytokine, growth factor and hormone. As a target for oncology, NAMPT is particularly attractive, because it catalyzes the rate-limiting step in the salvage pathway to generate nicotinamide adenine dinucleotide (NAD), a universal energy- and signal-carrying molecule involved in cellular energy metabolism and many homeostatic functions. Inhibition of NAMPT generally results in NAD depletion, followed by ATP reduction and loss of cell viability. Herein, we describe NAMPT inhibitor (NAMPTi)-induced cardiac toxicity in rodents following short-term administration (2-7days) of NAMPTi's. The cardiac toxicity was interpreted as a functional effect leading to congestive heart failure, characterized by sudden death, thoracic and abdominal effusion, and myocardial degeneration. Based on exposures in the initial in vivo safety rodent studies and cardiotoxicity observed, we conducted studies in rat and human in vitro cardiomyocyte cell systems. Based on those results, combined with human cell line potency data, we demonstrated the toxicity is both on-target and likely human relevant. This toxicity was mitigated in vitro by co-administration of nicotinic acid (NA), which can enable NAD production through the NAMPT-independent pathway; however, this resulted in only partial mitigation in in vivo studies. This work also highlights the usefulness and predictivity of in vitro cardiomyocyte assays using human cells to rank-order compounds against potency in cell-based pharmacology assays. Lastly, this work strengthens the correlation between cardiomyocyte cell viability and functionality, suggesting that these assays together may enable early assessment of cardiotoxicity in vitro prior to conduct of in vivo studies and potentially reduce subsequent attrition due to cardiotoxicity.

Synergistic Anticancer Action of Lysosomal Membrane Permeabilization and Glycolysis Inhibition

We investigated the in vitro and in vivo anticancer effect of combining lysosomal membrane permeabilization (LMP)-inducing agent N-dodecylimidazole (NDI) with glycolytic inhibitor 2-deoxy-d-glucose (2DG). NDI-triggered LMP and 2DG-mediated glycolysis block synergized in inducing rapid ATP depletion, mitochondrial damage, and reactive oxygen species production, eventually leading to necrotic death of U251 glioma cells but not primary astrocytes. NDI/2DG-induced death of glioma cells was partly prevented by lysosomal cathepsin inhibitor E64 and antioxidant α-tocopherol, suggesting the involvement of LMP and oxidative stress in the observed cytotoxicity. LMP-inducing agent chloroquine also displayed a synergistic anticancer effect with 2DG, whereas glucose deprivation or glycolytic inhibitors iodoacetate and sodium fluoride synergistically cooperated with NDI, thus further indicating that the anticancer effect of NDI/2DG combination was indeed due to LMP and glycolysis block. The two agents synergistically induced ATP depletion, mitochondrial depolarization, oxidative stress, and necrotic death also in B16 mouse melanoma cells. Moreover, the combined oral administration of NDI and 2DG reduced in vivo melanoma growth in C57BL/6 mice by inducing necrotic death of tumor cells, without causing liver, spleen, or kidney toxicity. Based on these results, we propose that NDI-triggered LMP causes initial mitochondrial damage that is further increased by 2DG due to the lack of glycolytic ATP required to maintain mitochondrial health. This leads to a positive feedback cycle of mitochondrial dysfunction, ATP loss, and reactive oxygen species production, culminating in necrotic cell death. Therefore, the combination of LMP-inducing agents and glycolysis inhibitors seems worthy of further exploration as an anticancer strategy.

Promoting anti‐listeria activity of lemongrass oil on pork loin by cold nitrogen plasma assist

AbstractThis work aims to evaluate the synergetic antimicrobial effects of cold nitrogen plasma (CNP) and lemongrass oil against L. monocytogenes on pork loin. Their possible effects on the physicochemical properties of pork were investigated as well. Lemongrass oil displayed anticipant antibacterial activities against L. monocytogenes at high dose, which could damage the bacterial membrane, leading to the losses of intracellular constituents, including DNA, protein and ATP. Compared with control groups, the number of L. monocytogenes on pork samples was reduced by 2.80 log10 steps after synergy treatment of CNP (500 W, 120 s) and lemongrass oil (5 mg/mL, 30 min). With the assistance of CNP, the satisfactory antibacterial effect for L. monocytogenes on pork was achieved after treatment for low concentration of lemongrass oil. In addition, the potential impacts on sensory attributes of pork were also minimized via combined treatment.Practical applicationsUnder low concentration, lemongrass oil achieved the desired antibacterial effect for L. monocytogenes on pork loin with the assistance of CNP. The potential impacts on sensory attributes of pork loin were minimized via synergetic treatment as well. The synergetic treatment provides a new way for application of plant essential oil in food preservation.

MiR-570 interacts with mitochondrial ATPase subunit g (ATP5L) encoding mRNA in stored platelets

Loss of platelet quality during ex vivo storage is a major concern in the transfusion medicine field and it has been known that platelet mitochondrial dysfunction is associated with storage time. In the last decade, small noncoding RNAs also known as microRNAs (miRNAs) have been reported to regulate key cellular processes through their target sequence interactions with selected mRNAs. In this study, we focused on understanding the mechanisms of platelet mitochondrial dysfunction during storage through miRNA regulation of mRNAs. RNA was isolated from day 0, day 5, and day 9 of stored human leukocyte-depleted platelets and subjected to differential miRNA and mRNA profiling. The miRNA profiling identified several miRNAs at low levels including a set of 12 different miR-548 family members (miR-548a-3p, miR-548aa, miR-548x, miR-548ac, miR-548c-3p, miR-603, miR-548aj, miR-548ae, miR-548z, miR-548u, miR-548al, and miR-570-3p). The mRNA profiling identified, among many, the mitochondrial ATP synthase subunit g (ATP5L) mRNA at high levels during storage. Target Scan algorithm for potential targets of miR-570-3p also identified ATP5L as one of its targets. We further identified two target sites for miR-570-3p in the 3′ untranslated region (3′UTR) of ATP5L mRNA. While ATP5L is a subunit of F0ATPase complex, its function is not established yet. Overexpression of miR-570-3p in platelets resulted in reduced levels of ATP5L mRNA and concomitant ATP loss. These experimental results provide first-time insights into the miRNA–mRNA interactions underlying mitochondrial dysfunction in ex vivo stored platelets and warrants further investigation.

Destabilization of mitochondrial functions as a target against breast cancer progression: Role of TPP+-linked-polyhydroxybenzoates

Mitochondrion is an accepted molecular target in cancer treatment since it exhibits a higher transmembrane potential in cancer cells, making it susceptible to be targeted by lipophilic-delocalized cations of triphenylphosphonium (TPP+). Thus, we evaluated five TPP+-linked decyl polyhydroxybenzoates as potential cytotoxic agents in several human breast cancer cell lines that differ in estrogen receptor and HER2/neu expression, and in metabolic profile. Results showed that all cell lines tested were sensitive to the cytotoxic action of these compounds. The mechanism underlying the cytotoxicity would be triggered by their weak uncoupling effect on the oxidative phosphorylation system, while having a wider and safer therapeutic range than other uncouplers and a significant lowering in transmembrane potential. Noteworthy, while the TPP+-derivatives alone led to almost negligible losses of ATP, when these were added in the presence of an AMP-activated protein kinase inhibitor, the levels of ATP fell greatly. Overall, data presented suggest that decyl polyhydroxybenzoates-TPP+ and its derivatives warrant future investigation as potential anti-tumor agents.

Attenuation of transcriptional and signaling responses limits viability of ρ0Saccharomyces cerevisiae during periods of glucose deprivation

BackgroundThe maintenance of viability during periods when a glycolytic carbon source is limited (or absent) is a major obstacle for cells whose mitochondrial DNA (mtDNA) has been damaged or lost. MethodsWe utilized genome wide transcriptional profiling and in gel mobility analyses to examine the transcriptional response and characterize defects in the phosphorylation dependent signaling events that occur during acute glucose starvation in ρ0 cells that lack mtDNA. Genetic and pharmacological interventions were employed to clarify the contribution of nutrient responsive kinases to regulation of the transcription factors that displayed abnormal phosphoregulation in ρ0 cells. ResultsThe transcriptional response to glucose deprivation is dampened but not blocked in ρ0 cells. Genes regulated by the transcription factors Mig1, Msn2, Gat1, and Ume6 were noticeably affected and phosphorylation of these factors in response to nutrient depletion is abnormal in ρ0 cells. Regulation of the nutrient responsive kinases PKA and Snf1 remains normal in ρ0 cells. The phosphorylation defect results from ATP depletion and loss of the activity of kinases including GSK3β, Rim15, and Yak1. Interventions which rescue phosphoregulation of transcription factors bolster maintenance of viability in ρ0 cells during subsequent glucose deprivation. ConclusionsA subset of nutrient responsive kinases is especially sensitive to ATP levels and their misregulation may underlie regulatory defects presented by ρ0 cells.General significance: Abnormal regulation of mitochondrial function is implicated in numerous human disorders. This work illustrates that some signaling pathways are more sensitive than others to metabolic defects caused by mitochondrial dysfunction.

Effect of Overproduction of Mitochondrial Uncoupling Protein 2 on Cos7 Cells: Induction of Senescent-like Morphology and Oncotic Cell Death

The maintenance of mitochondrial membrane potential is essential for cell growth and survival. Mitochondrial uncoupling protein 2 plays the most important roles in uncoupling oxidative phosphorylation and decreasing mitochondrial O2- production by regulating the mitochondrial membrane potential. We propose that mouse UCP2 has two glycine-rich motifs, motif 1: EGIRGLWKG (170-178) and a known Walker A-like motif 2: EGPRAFYKG (264-272). These motifs seem to be important for the function of UCP2. We investigated the biological effects of overproduced-UCP2 and its physiological consequence in Cos7 cells. We introduced several amino acid changes in the motif 1. The expression vectors of the green fluorescent protein (GFP)-fused UCP2 and mutant UCP2 were constructed and expressed in Cos7 cells. The UCP2-GFP-expressed cells significantly down-regulated the mitochondrial membrane potentials and induced the enlarged cell shapes. Next we generated the stably UCP2-GFP-expressed Cos7 cells by selection with the antibiotic Genecitin (G418). Within the first few weeks following G418-selection, the stably UCP2-GFP-expressed cells could not divide well and gradually manifested the irregular and enlarged senescent-like cell morphology. The UCP2/K177E- or UCP2/G174L-expressed cells did not induce the enlarged cell shapes. Hence, UCP2/K177E and UCP2/G174L produced the functional incompetence of the glycine-rich motif 1. The senescent-like cells significantly decreased the mitochondrial membrane potentials and finally died nearly one month. Overproduction of UCP2 irreversibly reduces the mitochondrial membrane potentials and induces the senescent-like morphology and finally oncotic cell death in Cos7 cells. These changes seem to occur from the irreversible metabolic changes following total loss of cellular ATP.

Engineering E. coli for large-scale production – Strategies considering ATP expenses and transcriptional responses

Microbial producers such as Escherichia coli are evolutionarily trained to adapt to changing substrate availabilities. Being exposed to large-scale production conditions, their complex, multilayered regulatory programs are frequently activated because they face changing substrate supply due to limited mixing. Here, we show that E. coli can adopt both short- and long-term strategies to withstand these stress conditions. Experiments in which glucose availability was changed over a short time scale were performed in a two-compartment bioreactor system. Quick metabolic responses were observed during the first 30s of glucose shortage, and after 70s, fundamental transcriptional programs were initiated. Since cells are fluctuating under simulated large-scale conditions, this scenario represents a continuous on/off switching of about 600 genes. Furthermore, the resulting ATP maintenance demands were increased by about 40–50%, allowing us to conclude that hyper-producing strains could become ATP-limited under large-scale production conditions. Based on the observed transcriptional patterns, we identified a number of candidate gene deletions that may reduce unwanted ATP losses. In summary, we present a theoretical framework that provides biological targets that could be used to engineer novel E. coli strains such that large-scale performance equals laboratory-scale expectations.

Oxidative stress, protein modification and Alzheimer disease.

Alzheimer disease (AD) is a progressive neurodegenerative disease that affects the elderly population with complex etiology. Many hypotheses have been proposed to explain different causes of AD, but the exact mechanisms remain unclear. In this review, we focus attention on the oxidative-stress hypothesis of neurodegeneration and we discuss redox proteomics approaches to analyze post-mortem human brain from AD brain. Collectively, these studies have provided valuable insights into the molecular mechanisms involved both in the pathogenesis and progression of AD, demonstrating the impairment of numerous cellular processes such as energy production, cellular structure, signal transduction, synaptic function, mitochondrial function, cell cycle progression, and degradative systems. Each of these cellular functions normally contributes to maintain healthy neuronal homeostasis, so the deregulation of one or more of these functions could contribute to the pathology and clinical presentation of AD. In particular, we discuss the evidence demonstrating the oxidation/dysfunction of a number of enzymes specifically involved in energy metabolism that support the view that reduced glucose metabolism and loss of ATP are crucial events triggering neurodegeneration and progression of AD.