Cytochrome Pathway Research Articles

Native organization of alternative NAD(P)H-dehydrogenases NDA and NDB in mitochondria of etiolated pea sprouts

Numerous biochemical and structural studies into the native organization of oxidative phosphorylation in the mitochondria of various eukaryotic organisms have convincingly shown that respiratory complexes can associate with one another to form higher-order structures referred to as supercomplexes. Plant mitochondria are distinguished by a more complicated organization of the respiratory chain due to the presence of a number of alternative oxidoreductases. It is considered that these enzymes do not physically interact with those of the cytochrome pathway. However, the available literature data obtained on yeast mitochondria suggests the possibility of such an association. In this regard, we aimed to study the native organization of alternative NAD(P)H-dehydrogenases NDA and NDB in plant mitochondria. The work was performed on six-day etiolated pea seedlings. The 2D BN/SDS-PAGE in combination with immunochemistry found that, in pea organelles, the main part of the populations of NDA and NDB alternative NAD(P)H dehydrogenases were included in superstructures with masses of 700, 780, and 900 kDa. Additionally, NDA was detected in the region of 1480 and 1600 kDa, and NDB was registered at values of 1330, 340, and 100–120 kDa. An analysis of subunit profiles of the observed associations and a colorimetric detection of ATPase activity in 1D BN-gel suggested that the major part of the NDA and NDB populations identified by the available antibodies was associated with ATP synthase and represented a heterogeneous population of ATP-synthasomes, assumably, with a NDA2/NDB2Va/b1-2 composition. The rest of the enzymes were likely to be part of the NDA2/NDB2III2IV and NDA2IV1Va2 supercomplexes. The physiological significance of the association of alternative NAD(P)H dehydrogenases with ATP synthase requires further study.

Is ATP a signaling regulator for postharvest chilling tolerance in fruits?

Low-temperature storage is used to extend the shelf life of fruits, but prolonged storage at temperatures below tolerable levels may cause postharvest chilling injury (PCI) in sensitive commodities. This review aims to highlight adenosine triphosphate (ATP) activation and the interplay of extracellular ATP (eATP) and intracellular ATP (iATP) in fruits and to find out its significance in mitigating PCI. Various pathways, such as the Embden-Meyerhof-Parnas pathway, the tricarboxylic acid cycle, the pentose phosphate pathway, the γ-aminobutyric acid shunt pathway, and the cytochrome pathway, are studied critically to elucidate their role in continuous ATP supply and maintaining the membrane fluidity and integrity. This review summarizes the treatments helpful in modulating energy metabolism in fruit. Additionally, this work provides insights into the energy status in attenuating chilling tolerance. Moreover, it states the potential of nicotinamide adenine dinucleotide in mitigating PCI. Furthermore, it discusses the role of eATP and its receptor DORN1 in mitigating chilling stress.

6-BA Delays the Senescence of Postharvest Cabbage Leaves by Inhibiting Respiratory Metabolism.

6-BA, a small molecule compound of cytokinins, has been proven to delay leaf senescence in different species, including Chinese flowering cabbage; however, its specific mechanism remains relatively unknown. In this study, the application of external 6-BA delayed leaf senescence in Chinese flowering cabbage, showing that 6-BA effectively prevented the decrease in the maximum quantum yield (Fv/Fm) and overall chlorophyll content and suppressed the expression of the senescence-associated gene BrSAG12 over a 7-day period of storage. Moreover, treatment with 6-BA decreased the respiratory rate, NAD(H) content, the activities of hexose phosphate isomerase (PHI), succinate dehydrogenase (SDH), cytochrome c oxidase (CCO), and ascorbic acid oxidase (AAO) using enzyme-linked immunosorbent assay, and the transcriptional abundance of related genes by real-time quantitative polymerase chain reaction. Furthermore, 6-BA also increased the activity and expression levels of glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphate gluconate dehydrogenase (6-PGDH). The group treated with 6-BA retained elevated levels of NADP (H), ATP, total ATPase, and nicotinamide adenine dinucleotide kinase (NADK) activity, as well as the expression of respiratory enzymes. Molecular docking indicated that 6-BA hinders the glycolysis pathway (EMP), tricarboxylic acid cycle (TCA), and cytochrome pathway (CCP), and sustains elevated levels of the pentose phosphate pathway (PPP) through interactions with the PHI, SDH, 6-PGDH, G6PDH, CCO, and AAO proteins, consequently delaying postharvest leaf senescence in Chinese flowering cabbage.

The Effects of Mixed Foliar Nutrients of Calcium and Magnesium on the Major Bypass Respiratory Pathways in the Pulp of ‘Feizixiao’ Litchi

During the period of ‘Feizixiao’ litchi fruit pericarp’s full coloring, there is a phenomenon of “sugar withdrawal” in the pulp, and the mixed foliar nutrients of calcium and magnesium (Ca+Mg) can effectively overcome this phenomenon. One of the reasons for this may be that it is related to the influence of the mixed nutrients of Ca+Mg on the bypass respiratory pathways of the pulp. The major fruit quality indicators, the rates of cytochrome and cyanide-resistant respiratory pathways (CP and AP) in the pulp and the activities of their key enzymes, were observed continuously in 2021 and 2022, and the deferentially expressed genes (DEGs) related to the two bypass respiratory pathways in the pulp were screened by RNA-seq analysis, with a qPCR of the random genes performed to verify the results. Ca+Mg treatment kept the content of the total soluble sugar in the pulp stable and higher than that the control in the ripening stage; Ca+Mg treatment increased the activities of electron-transferring enzymes in the electron transport chain, such as NADH dehydrogenase (ND), succinate dehydrogenase (SDH), cytochrome bc1 complex, and cytochrome c (Cyt c) through up-regulating their gene expression. In terms of the rate-limiting enzymes in the pulp, Ca+Mg treatment increased the activity of cytochrome oxidase (COX) in the CP pathway by up-regulating the expression of COX genes, then increased the CP respiratory rate and inhibited the CP respiratory rate decrease; meanwhile, it also inhibited the activity of AOX (alternate oxidase) in the pulp in the AP pathway by down-regulating the expression of AOX genes, then inhibited the increase in the AP respiration rate. The qPCR validation of randomly selected DEGs showed a significant unitary linear correlation between their expression levels and the results of the RNA-seq analysis. Therefore, one of the physiological mechanisms on the mixed foliar nutrients of Ca and Mg overcoming the phenomenon of “sugar withdrawal” in the ‘Feizixiao’ litchi pulp could be to promote CP and to inhibit AP, and then to delay the ripening and senescence of the pulp.

Alternative Oxidase: From Molecule and Function to Future Inhibitors.

In the respiratory chain of the majority of aerobic organisms, the enzyme alternative oxidase (AOX) functions as the terminal oxidase and has important roles in maintaining metabolic and signaling homeostasis in mitochondria. AOX endows the respiratory system with flexibility in the coupling among the carbon metabolism pathway, electron transport chain (ETC) activity, and ATP turnover. AOX allows electrons to bypass the main cytochrome pathway to restrict the generation of reactive oxygen species (ROS). The inhibition of AOX leads to oxidative damage and contributes to the loss of adaptability and viability in some pathogenic organisms. Although AOXs have recently been identified in several organisms, crystal structures and major functions still need to be explored. Recent work on the trypanosome alternative oxidase has provided a crystal structure of an AOX protein, which contributes to the structure-activity relationship of the inhibitors of AOX. Here, we review the current knowledge on the development, structure, and properties of AOXs, as well as their roles and mechanisms in plants, animals, algae, protists, fungi, and bacteria, with a special emphasis on the development of AOX inhibitors, which will improve the understanding of respiratory regulation in many organisms and provide references for subsequent studies of AOX-targeted inhibitors.

Reaction pathways, proton transfer, and proton pumping in ba3 class cytochrome c oxidase: perspectives from DFT quantum chemistry and molecular dynamics.

After drawing comparisons between the reaction pathways of cytochrome c oxidase (CcO, Complex 4) and the preceding complex cytochrome bc1 (Complex 3), both being proton pumping complexes along the electron transport chain, we provide an analysis of the reaction pathways in bacterial ba3 class CcO, comparing spectroscopic results and kinetics observations with results from DFT calculations. For an important arc of the catalytic cycle in CcO, we can trace the energy pathways for the chemical protons and show how these pathways drive proton pumping of the vectorial protons. We then explore the proton loading network above the Fe heme a3-CuB catalytic center, showing how protons are loaded in and then released by combining DFT-based reaction energies with molecular dynamics simulations over states of that cycle. We also propose some additional reaction pathways for the chemical and vector protons based on our recent work with spectroscopic support.

Particulate Air Pollution and Blood Pressure: Signaling by the Arachidonate Metabolism.

Short-term exposure to ambient particulate matter (PM) can raise blood pressure, but the underlying mechanisms are unclear. We explored whether arachidonate metabolites serve as biological intermediates in PM-associated prohypertensive changes. This panel study recruited 110 adults aged 50 to 65 years living in Beijing, China. The participants' blood pressure, arterial stiffness, and cardiac and endothelial function were measured up to 7 times. The serum concentrations of arachidonate metabolites were quantified by targeted lipidomics. Ambient concentrations of fine PM (PM2.5), black carbon, and accumulation mode particles were continuously monitored at a station and their associations with the health indicators were evaluated. Interquartile range increases in 25 to 96-hour-lag exposure to PM2.5, black carbon, and accumulation mode particles were associated with significant increases in systolic blood pressure (brachial: 0.8-3.2 mm Hg; central: 0.7-2.8 mm Hg) and diastolic blood pressure (brachial, 0.5-1.5 mm Hg; central, 0.5-1.6 mm Hg). At least 1 pollutant was associated with increases in augmentation pressure and heart rate and decreases in reactive hyperemia index and ejection time. The serum concentrations of arachidonate were significantly increased by 3.3% to 14.6% in association with PM exposure, which mediated 9% of the PM-associated increases in blood pressure. The levels of eicosanoids from the cytochrome P450, cyclooxygenase, and lipoxygenase pathways changed with PM exposure, and those from the cytochrome pathway significantly mediated the association between PM exposure and blood pressure. Short-term exposure to particulate air pollution was associated with a prohypertensive change in adults, which was in part mediated by alteration of arachidonate metabolism.

Enhanced cyanide-resistant respiration as the predominant respiratory metabolic pathway in abnormal chilling injury behavior of postharvest papaya

An increasing number of studies have demonstrated that reactive oxygen species (ROS) play a crucial role in the chilling injury (CI) of postharvest fruit and vegetables. This is not only associated with the free radical scavenging system but is also intimately connected to the electron transport chain pathway. However, it remains unclear whether ROS is associated with the electron transport chain in the abnormal chilling behavior observed in postharvest papaya. As such, this study investigated the electron transport chain of papaya fruit stored at 1 ℃ and 6 ℃ and its connection to ROS. In comparison to storage at 6 ℃, storage at 1 ℃ suppressed the expression of CpCCO, and induced the upregulation of CpAOX expression in papaya fruit. Throughout the middle and late stages of storage, there was an increase in pyruvate content and ubiquinone reduction level. The increase had two effects: it inhibited cytochrome oxidase (CCO) activity and promoted alternative oxidase (AOX) activity, leading to a blocked cytochrome pathway and an increased proportion of cyanide-resistant respiratory pathways. Consequently, the cyanide-resistant respiratory pathway supplanted the cytochrome pathway as the primary electron transport pathway. This transition effectively curbed the accumulation of ROS, thereby diminishing the damage degree of fruit cell membrane, boosting chilling tolerance, and ultimately inhibiting the CI of papaya fruit, which led to the emergence of abnormal chilling injury.

Alternaria alternata stimulates blackhead disease development of ‘Korla’ fragrant pear (Pyrus bretschneideri Rehd) by regulating energy status and respiratory metabolism

Blackhead disease of ‘Korla’ fragrant pear (Pyrus bretschneideri Rehd), caused by the fungus Alternaria alternata (A. alternata), limits postharvest pear fruit storage and transportation. Herein, we explored the effects of A. alternata infection on respiratory metabolism and energy status of pear fruit, and their correlations with disease development. Compared with control fruit, A. alternata-inoculated fruit displayed a higher respiration rate and disease index. Infection increased the gene expression and enzyme activities of phosphohexose isomerase (PGI), alternative oxidase (AOX), succinate dehydrogenase (SDH) and cytochrome C oxidase (CCO) in pear fruit, and decreased those of 6-phosphogluconate dehydrogenase (6PGDH) and glucose-6-phosphate dehydrogenase (G6PDH) in the late storage period. Infection reduced nicotinamide adenine dinucleotide kinase (NADK) activity and expression level in fruit, inhibited NADP accumulation, and increased NAD, NADH, and NADPH contents in the early storage period. Infection also lowered adenosine triphosphate (ATP), adenosine diphosphate (ADP) and energy charge (EC) levels as well as ATPase activity, and enhanced adenosine monophosphate (AMP) content and PbAtpB expression levels, resulting in decreased fruit energy status. All these results suggest that disease development may be connected to the enhancement of the Embden-Meyerhof-Parnas (EMP) pathway, the tricarboxylic acid (TCA) cycle, the cytochrome pathway (CCP), the alternative pathway (AP), the increased then decrease in the pentose phosphate pathway (PPP), and reduced energy status in pear fruit.

Abstract 1564: Novel combination of TRIP13 and Aurora kinase A inhibition demonstrated extensive DNA damage and immunogenic cell death in RB-deficient cancers

Abstract Many lethal cancer types have inactivation of the retinoblastoma (Rb) tumor suppressor pathway including cancers caused by human papillomavirus (HPV), which causes >5% of all cancers worldwide. There are no therapies that uniquely target Rb-deficient cancers. Biomarker-selected, molecular targeted therapy for Rb-deficient cancers represents both an unmet need and a translational knowledge gap. To address this gap, we recently published data that support a model in which Rb-deficient cancers, having high levels of Mad2, survive owing to TRIP13-mediated inhibition of Mad2 and Aurora A-mediated mitotic progression. The combination of Aurora A inhibition (alisertib) plus TRIP13 depletion caused extensive apoptosis in Rb-deficient, but not in Rb-proficient, cancer cells. Both Aurora A and TRIP13 are involved in the cell’s response to DNA damage and apoptosis that can lead to immunogenic cell death (ICD), prompting us to investigate the mechanism underlying the synthetic lethality of TRIP13 and Aurora A in Rb-deficient cancer cells. We transfected two HPV+ cell lines with TRIP13 or control siRNA and incubated with 100nM alisertib. We also generated two stable HPV+ cell lines with TRIP13KO. In both these systems, TRIP13 KD or KO alone did not cause apoptosis. Incubation with alisertib resulted in significant cell death, that validated our previous findings. We then tested the effect of the combination on DNA damage. Alisertib caused robust increases in γH2AX, pTIF1β (KAP1, TRIM28), and DNA PKcs, measured using immunoblotting, in both TRIP13 KD and KO HPV+ cell lines. We also observed enhanced γH2AX staining using confocal microscopy. ICD leads to activation of immune cells by the dying cancer cells in the tumor microenvironment involving the release of antigenic fragments that are engulfed, processed and presented by antigen presenting cells for the induction of antigen-specific T-cell responses. Studies have shown DNA damage stimulates the cGAS/STING pathway leading to ICD. In our system, we saw that TRIP13KO and KD in HPV+ cells treated with alisertib, resulted in marked activation of the cGAS/STING pathway and release of cytochrome C and IL1α in the cell supernatant, which are markers of pyroptosis. Additionally, we also observed gasdermin-E cleavage. The combination further led to increased cancer cell secretion of GM-CSF, IL6, and IL18 that can activate macrophages, NK cells and T-cells. The selective effect of the combination in Rb-deficient cancer cells allowed us to significantly reduce the concentration of alisertib. By sparing normal cells that express Rb, this combination can potentially reduce treatment toxicity in patients with Rb-deficient cancers. The ICD induced by this combination may lead to host T-cell engagement, thereby further enhancing tumor elimination. This work can shape future clinical trials that can change current treatment regime. Citation Format: Soma Ghosh, Tuhina Mazumdar, Lacin Yapindi, Pragya Sinha, Jagannadha Sastry, Faye M. Johnson. Novel combination of TRIP13 and Aurora kinase A inhibition demonstrated extensive DNA damage and immunogenic cell death in RB-deficient cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1564.

Regulatory Effect of Exogenous γ-Aminobutyric Acid on Respiratory Rate through the γ-Aminobutyric Acid Shunt in Malus baccata (L.) Borkh. Roots under Suboptimal Low Root-Zone Temperature

Malus baccata (L.) Borkh. is one of the most widely used rootstocks in the apple-producing region of Northern China. However, in the early growing season, apple roots are often subjected to suboptimal low root-zone temperatures. The regulatory effects of exogenous γ-aminobutyric acid (GABA) on both the γ-aminobutyric acid shunt (GABA shunt) and the respiratory activity of roots under suboptimal low root-zone temperatures remain unknown. To explore the physiological basis for GABA alleviation of low-temperature stress in M. baccata Borkh. roots, the following treatments were examined: suboptimal low root-zone temperature (potted parts of the seedlings were maintained at 5 ± 0.5 °C; L); suboptimal low root-zone temperature + GABA (LG); and suboptimal low root-zone temperature + vigabatrin (VGB; LV), which is a specific active inhibitor of γ-aminobutyric acid transaminase (GABA-T). Each treatment was matched with a control (18 °C/8 °C day/night; CK) for comparison. Our results showed that the L treatment reduced the root vitality, increased malondialdehyde (MDA) content, promoted the accumulation of GABA, activated the GABA shunt, and inhibited the total root respiration rate (VTotal) by decreasing the respiratory rates of Embden–Meyerhof pathway (VEMP) and tricarboxylic acid cycle (VTCAC). The LG treatment significantly increased the content of endogenous GABA, accelerated the metabolism of the GABA shunt, enhanced root respiratory activity by increasing VTotal, VEMP, VTCAC, and increased the cytochrome pathway respiratory rate (VCP), thus alleviating the damage of low root-zone temperature stress. Meanwhile, contrasting results were observed in the LV treatment. These findings revealed that exogenous GABA improved the tolerance of apple rootstocks to suboptimal low temperatures in early spring by regulating the GABA shunt and root respiratory activity.

Chronic alcohol metabolism results in DNA repair infidelity and cell cycle-induced senescence in neurons.

Chronic binge-like drinking is a risk factor for age-related dementia, however, the lasting and irreversible effect of alcohol on the brain remains elusive. Transcriptomic changes in brain cortices revealed pro-ageing hallmarks upon chronic ethanol exposure and these changes predominantly occur in neurons. The changes are attributed to a prioritized ethyl alcohol oxidation in these cells via the NADPH-dependent cytochrome pathway. This hijacks the folate metabolism of the 1-carbon network which supports the pathway choice of DNA repair via the non-cell cycle-dependent mismatch repair networks. The lost-in-function of such results in the de-inactivation of the less preferred cell cycle-dependent homologous recombination (HR) repair, forcing these post-mitotic cells to re-engage in a cell cycle-like process. However, mature neurons are post-mitotic. Therefore, instead of successfully completing a full round of cell cycle which is necessary for the completion of HR-mediated repair; these cells are arrested at checkpoints. The resulting persistence of repair intermediates induces and promotes the nuclear accumulation of p21 and cyclin B-a trigger for permanent cell cycle exits and irreversible senescence response. Supplementation of bioactive 5-methyl tetrahydrofolate simultaneously at times with ethyl alcohol exposure supports the fidelity of the 1-carbon network and hence the activity of the mismatch repair. This prevents aberrant and irreversible cell cycle re-entry and senescence events of neurons. Together, our findings offer a direct connection between binge-drinking behaviour and its irreversible impact on the brain, which makes it a potential risk factor for dementia.

Photosynthesis, Respiration, and Thermal Energy Dissipation in Leaves of Two Phenotypes of Plantago media L. under Environmental Conditions

The ability to maintain the balance between the absorbed light energy and energy used in photosynthesis is a key factor of plant adaptation to variable environmental conditions. In this work, diurnal variations in photosynthesis, respiration, thermal energy dissipation, and the activity of the antioxidant system were studied in hoary plantain (Plantago media L.) growing on an open slope (sun plants) and under natural shading in the herbage (shade plants). The highest leaves net photosynthetic rate (Pn) was observed early in the morning and amounted to 2.6 and 9.2 μmol CO2/m2 s in shade and sun plants, respectively. In the daytime, the Pn values of sun plants decreased significantly (threefold) along with the decrease in stomatal conductance; changes of both parameters developed concurrently with the increase in insolation and air temperature. The Pn changes in leaves of shade plants were less pronounced and weakly dependent on stomatal conductance. The leaves of shade plants contained comparatively high amounts of soluble carbohydrates, whereas the sun plant leaves accumulated larger amounts of starch. In the daytime, nonphotochemical quenching (NPQ) of chlorophyll a fluorescence in photosystem II of sun plant leaves could be as large as 2.6 rel. units, which was four- to fivefold higher than NPQ in shade plants. In leaves of sun plants in the morning and evening hours, the ratio of cytochrome pathway (CP) and the alternative (AP) respiratory pathways was approximately 1.0, whereas this ratio decreased to 0.4 during the day, synchronously with an increase in NPQ. The CP/AP ratio in shade plant leaves remained constant throughout the diurnal cycle and equaled 1.4, indicating a comparatively high energy efficiency of respiration in shaded plants growing under the grass canopy. The leaves of sun plants featured an increased content of superoxide anion radical and hydrogen peroxide as well as the elevated activity of antioxidant enzymes (superoxide dismutase, guaiacol peroxidase) that control the accumulation of reactive oxygen species. The results emphasize the importance of coordinated changes in energy-dissipating processes and the activity of the antioxidant system for maintaining the energy and redox balance in phototrophic tissues during long-term adaptation of plants to environmental conditions, excessive insolation in particular.

Developmentally regulated mitochondrial biogenesis and cell death competence in maize pollen

BackgroundCytoplasmic male sterility (CMS) is a maternally inherited failure to produce functional pollen that most commonly results from expression of novel, chimeric mitochondrial genes. In Zea mays, cytoplasmic male sterility type S (CMS-S) is characterized by the collapse of immature, bi-cellular pollen. Molecular and cellular features of developing CMS-S and normal (N) cytoplasm pollen were compared to determine the role of mitochondria in these differing developmental fates.ResultsTerminal deoxynucleotidyl transferase dUTP nick end labeling revealed both chromatin and nuclear fragmentation in the collapsed CMS-S pollen, demonstrating a programmed cell death (PCD) event sharing morphological features with mitochondria-signaled apoptosis in animals. Maize plants expressing mitochondria-targeted green fluorescent protein (GFP) demonstrated dynamic changes in mitochondrial morphology and association with actin filaments through the course of N-cytoplasm pollen development, whereas mitochondrial targeting of GFP was lost and actin filaments were disorganized in developing CMS-S pollen. Immunoblotting revealed significant developmental regulation of mitochondrial biogenesis in both CMS-S and N mito-types. Nuclear and mitochondrial genome encoded components of the cytochrome respiratory pathway and ATP synthase were of low abundance at the microspore stage, but microspores accumulated abundant nuclear-encoded alternative oxidase (AOX). Cytochrome pathway and ATP synthase components accumulated whereas AOX levels declined during the maturation of N bi-cellular pollen. Increased abundance of cytochrome pathway components and declining AOX also characterized collapsed CMS-S pollen. The accumulation and robust RNA editing of mitochondrial transcripts implicated translational or post-translational control for the developmentally regulated accumulation of mitochondria-encoded proteins in both mito-types.ConclusionsCMS-S pollen collapse is a PCD event coincident with developmentally programmed mitochondrial events including the accumulation of mitochondrial respiratory proteins and declining protection against mitochondrial generation of reactive oxygen species.

High Expression of ALTERNATIVE OXIDASE 2 in Latent Axillary Buds Suggests Its Key Role in Quiescence Maintenance in Rosebush.

Most vegetative axes remain quiescent as dormant axillary buds until metabolic and hormonal signals, driven by environmental changes, trigger bud outgrowth. While the resumption of growth activity is well documented, the establishment and maintenance of quiescence is comparatively poorly understood, despite its major importance in the adaptation of plants to the seasonal cycle or in the establishment of their shape. Here, using the rosebush Rosa hybrida 'Radrazz' as a plant model, we highlighted that the quiescent state was the consequence of an internal and active energy control of buds, under the influence of hormonal factors previously identified in the bud outgrowth process. We found that the quiescent state in the non-growing vegetative axis of dormant axillary buds displayed a low energy state along with a high expression of the ALTERNATIVE OXIDASE 2 (AOX2) and the accumulation of the corresponding protein. Conversely, AOX2 expression and protein amount strongly decreased during bud burst as energy status shifted to a high state, allowing growth. Since AOX2 can deviate electrons from the cytochrome pathway in the mitochondrial respiratory chain, it could drastically reduce the formation of ATP, which would result in a low energy status unfavorable for growth activities. We provide evidence that the presence/absence of AOX2 in quiescent/growing vegetative axes of buds was under hormonal control and thus may constitute the mechanistic basis of both quiescence and sink strength manifestation, two important aspects of budbreak.

Exploration of shared TF-miRNA‒mRNA and mRNA-RBP-pseudogene networks in type 2 diabetes mellitus and breast cancer.

Type 2 diabetes mellitus (T2DM) has been confirmed to be closely associated with breast cancer (BC). However, the shared mechanisms between these diseases remain unclear. By comparing different datasets, we identified shared differentially expressed (DE) RNAs in T2DM and BC, including 427 mRNAs and 6 miRNAs from the GEO(Gene Expression Omnibus) database. We used databases to predict interactions to construct two critical networks. The transcription factor (TF)-miRNA‒mRNA network contained 236 TFs, while the RNA binding protein (RBP)-pseudogene-mRNA network showed that the pseudogene S-phase kinase associated protein 1 pseudogene 1 (SKP1P1) might play a key role in regulating gene expression. The shared mRNAs between T2DM and BC were enriched in cytochrome (CYP) pathways, and further analysis of CPEB1 and COLEC12 expression in cell lines, single cells and other cancers showed that they were strongly correlated with the survival and prognosis of patients with BC. This result suggested that patients with T2DM presenting the downregulation of CPEB1 and COLEC12 might have a higher risk of developing BC. Overall, our work revealed that high expression of CYPs in patients with T2DM might be a susceptibility factor for BC and identified novel gene candidates and immune features that are promising targets for immunotherapy in patients with BC.

Effect of herbal formulation on glimepiride pharmacokinetics and pharmacodynamics in nicotinamide-streptozotocin-induced diabetic rats

BackgroundTraditional medicinal herbs are widely consumed in developing countries to treat diabetes as they are perceived to be safer, less expensive, and have fewer side effects as compared to the conventional medicines. Diabecon (DB), Himalaya Herbal Healthcare, India is herbal over-the-counter formulation which contains several herbs that are reported in the traditional texts for the treatment of diabetes. The majority of these herbs have been investigated and found to interfere with the cytochrome pathway. The most common oral antihyperglycemic drug used today in clinical practice is Glimepiride (GP).The CYP2C9 enzyme is mainly responsible for the metabolism of GP. Herein we hypothesize that the co-administration of GP with DB may result in possible Herb–Drug Interactions (HDIs) as DB has the potential to significantly inhibit the CYP2C9 enzyme. ObjectiveIn the current study, the pharmacokinetic and pharmacodynamic interactions of GP (0.82 mg/kg) with DB (110.95 mg/kg) was investigated in diabetes induced (Nicotinamide-STZ) rats by co-administering both drugs orally for 21 days. Materials and methodsFor the study of the HDI, Bioanalytical RP-HPLC/PDA method for quantifying GP in plasma of rats was developed and validated as per US-FDA guidelines. In vivo pharmacokinetic and pharmacodynamic parameters were studied on day 1 and day 21 post administration. ResultsThe RP-HPLC/PDA method was successfully employed for quantification of GP in the PK studies. The co-administration of GP and DB in diabetic rats resulted in beneficial pharmacodynamic interactions, but there were no notable changes in the pharmacokinetic parameters of GP. ConclusionThis current investigation in an animal model suggests that co-administration of GP and DB may have significant therapeutic benefits in the treatment of diabetes; however, additional research, randomized clinical trials or case studies in humans, is needed.

A yeast suppressor screen links Coa4 to the mitochondrial copper delivery pathway for cytochrome c oxidase.

Cytochrome c oxidase (CcO) is a multimeric copper-containing enzyme of the mitochondrial respiratory chain that powers cellular energy production. The two core subunits of cytochrome c oxidase, Cox1 and Cox2, harbor the catalytic CuB and CuA sites, respectively. Biogenesis of each copper site occurs separately and requires multiple proteins that constitute the mitochondrial copper delivery pathway. Currently, the identity of all the members of the pathway is not known, though several evolutionarily conserved twin CX9C motif-containing proteins have been implicated in this process. Here, we performed a targeted yeast suppressor screen that placed Coa4, a twin CX9C motif-containing protein, in the copper delivery pathway to the Cox1 subunit. Specifically, we show that overexpression of Cox11, a copper metallochaperone required for the formation of CuB site, can restore Cox1 abundance, cytochrome c oxidase assembly, and mitochondrial respiration in coa4Δ cells. This rescue is dependent on the copper-coordinating cysteines of Cox11. The abundance of Coa4 and Cox11 in mitochondria is reciprocally regulated, further linking Coa4 to the CuB site biogenesis. Additionally, we find that coa4Δ cells have reduced levels of copper and exogenous copper supplementation can partially ameliorate its respiratory-deficient phenotype, a finding that connects Coa4 to cellular copper homeostasis. Finally, we demonstrate that human COA4 can replace the function of yeast Coa4 indicating its evolutionarily conserved role. Our work provides genetic evidences for the role of Coa4 in the copper delivery pathway to the CuB site of cytochrome c oxidase.

Exogenous Proline Alleviated Low Temperature Stress in Maize Embryos by Optimizing Seed Germination, Inner Proline Metabolism, Respiratory Metabolism and a Hormone Regulation Mechanism

Proline (Pro) is not only an important osmotic adjustment substance, but it also plays an important role in regulating plant abiotic stress resistance. The maize varieties, Xinxin 2 (low temperature insensitive) and Damin 3307 (low temperature sensitive), were chosen as materials, setting a normal temperature for germination (22 °C/10 °C, 9d), low temperature germination (4 °C/4 °C, 5d) and normal temperature recovery (22 °C/10 °C, 4d), combined with a proline (15 mmol·L−1) soaking treatment, to reveal the seed germination and regulation mechanism in maize embryos. The results showed that proline significantly improved the germination potential, germination rate, germination index and vigor index of seeds under low temperature stress, increasing the length of the coleoptile and radicle, increasing the dry and fresh weight of young buds (coleoptile + radicle), and increasing the activity of α-amylase. Proline enhanced the activities of Δ1-pyrroline-5-carboxylic acid synthetase (P5CS) and ornithine aminotransferase (OAT) in maize embryos under low temperature stress, enhanced the proline synthesis pathways, and further enhanced proline accumulation. Proline induced the activity of proline dehydrogenase (ProDH) in the early stage of low temperature stress and stress relief. Under low temperature stress, the activities of hexokinase (HXK), phosphofructokinase (PFK), pyruvate kinase (PK), isocitrate dehydrogenase (IDH), succinate dehydrogenase (SDH), malate dehydrogenase (MDH) and glucose-6-phosphate dehydrogenase (G-6-PDH) and glucose-6-phosphate dehydrogenase (G-6-PDH and 6-P-GDH) in maize embryos were decreased, resulting in a decrease in the glycolysis (EMP) pathway, tricarboxylic acid (TCA) cycle and pentose phosphate pathway (PPP). Proline alleviated the inhibition of key enzyme activities of the EMP pathway, TCA cycle and PPP of maize embryos under low temperature stress, increased the activities of cytochrome oxidase (COX) and alternative oxidase (AOX), increased the ATP content, alleviated the inhibition of low temperature stress on main cytochrome pathway activity (ρVcyt), while further increasing the total respiratory activity (Vt) and the actual operational activity of the alternative pathway (ρValt) during seed germination at the initial stage of low temperature stress, as well as improving the inhibition of the Vt and ρValt in the middle and late stages of low temperature stress. Under low temperature stress, the content of abscisic acid (ABA) increased significantly, while gibberellin (GA), auxin (IAA) and zeatin nucleoside (ZR) decreased significantly. Proline alleviated the decrease in IAA, ZR and GA contents in maize embryos under low temperature stress, reduced the increase in the ABA content, and reduced the inhibition of low temperature on seed germination.

Experimental Setup for Investigation of Acute Mitochondrial Oxygen Sensing in Primary Cells.

The ability to sense and respond to acute changes in oxygen is essential for the viability of cells and organisms. To study molecular mechanisms of acute oxygen sensing, we established a setup for the adjustment of acute hypoxic conditions in cultured cells, exemplified here for the use of primary pulmonary arterial smooth muscle cells (PASMCs). The mitochondrial electron transport chain (ETC) is the main consumer of oxygen but recently also emerged as essential oxygen sensor suggesting that the ETC itself adapts its electron flux to oxygen availability. To test this assumption and to experimentally manipulate electron flux through the ETC, we used alternative oxidase (AOX), which bypasses the cytochrome pathway of the ETC when blocked. The described combination of our experimental setup and AOX allowed us in previous publications unprecedented insights into the role of the ETC in cellular oxygen sensing and cellular response mechanisms in living cells. Against this background, we here describe and discuss this method in detail, which will allow transfer to other cell types and research questions.