Inactivating alternative NADH dehydrogenases: enhancing fungal bioprocesses by improving growth and biomass yield?

Debate still surrounds the physiological roles of the alternative respiratory enzymes found in many fungi and plants. It has been proposed that alternative NADH:ubiquinone oxidoreductases (NADH dehydrogenases) may protect against oxidative stress, conversely, elevated activity of these enzymes has been linked to senescence. Here we show that inhibition of these enzymes in a fungal protein expression system (Aspergillus niger) leads to significantly enhanced specific growth rate, substrate uptake, carbon dioxide evolution, higher protein content, and more efficient use of substrates. These findings are consistent with a protective role of the NADH dehydrogenases against oxidative stress, thus, when electron flow via these enzymes is blocked, flux through the main respiratory pathway rises, leading to enhanced ATP generation. We anticipate that our findings will stimulate further studies in fungal and plant cultures leading to significant improvements in these expression systems, and to deeper insights into the cellular roles of alternative respiration

Characterization of Apoptosis-Related Oxidoreductases from Neurospora crassa

The genome from Neurospora crassa presented three open reading frames homologous to the genes coding for human AIF and AMID proteins, which are flavoproteins with oxidoreductase activities implicated in caspase-independent apoptosis. To investigate the role of these proteins, namely within the mitochondrial respiratory chain, we studied their cellular localization and characterized the respective null mutant strains. Efficiency of the respiratory chain was analyzed by oxygen consumption studies and supramolecular organization of the OXPHOS system was assessed through BN-PAGE analysis in the respective null mutant strains. The results demonstrate that, unlike in mammalian systems, disruption of AIF in Neurospora does not affect either complex I assembly or function. Furthermore, the mitochondrial respiratory chain complexes of the mutant strains display a similar supramolecular organization to that observed in the wild type strain. Further characterization revealed that N. crassa AIF appears localized to both the mitochondria and the cytoplasm, whereas AMID was found exclusively in the cytoplasm. AMID2 was detected in both mitochondria and cytoplasm of the amid mutant strain, but was barely discernible in wild type extracts, suggesting overlapping functions for the two proteins

Influence of mitochondrial genome rearrangement on cucumber leaf carbon and nitrogen metabolism

The MSC16 cucumber (Cucumis sativus L.) mitochondrial mutant was used to study the effect of mitochondrial dysfunction and disturbed subcellular redox state on leaf day/night carbon and nitrogen metabolism. We have shown that the mitochondrial dysfunction in MSC16 plants had no effect on photosynthetic CO2 assimilation, but the concentration of soluble carbohydrates and starch was higher in leaves of MSC16 plants. Impaired mitochondrial respiratory chain activity was associated with the perturbation of mitochondrial TCA cycle manifested, e.g., by lowered decarboxylation rate. Mitochondrial dysfunction in MSC16 plants had different influence on leaf cell metabolism under dark or light conditions. In the dark, when the main mitochondrial function is the energy production, the altered activity of TCA cycle in mutated plants was connected with the accumulation of pyruvate and TCA cycle intermediates (citrate and 2-OG). In the light, when TCA activity is needed for synthesis of carbon skeletons required as the acceptors for NH4+ assimilation, the concentration of pyruvate and TCA intermediates was tightly coupled with nitrate metabolism. Enhanced incorporation of ammonium group into amino acids structures in mutated plants has resulted in decreased concentration of organic acids and accumulation of Glu

Mouse Bone Marrow-Derived Mesenchymal Stromal Cells Turn Activated Macrophages into a Regulatory-Like Profile

In recent years it has become clear that the therapeutic properties of bone marrow-derived mesenchymal stromal cells (MSC) are related not only to their ability to differentiate into different lineages but also to their capacity to suppress the immune response. We here studied the influence of MSC on macrophage function. Using mouse thioglycolate-elicited peritoneal macrophages (M) stimulated with LPS, we found that MSC markedly suppressed the production of the inflammatory cytokines TNF-α, IL-6, IL-12p70 and interferon-γ while increased the production of IL-10 and IL-12p40. Similar results were observed using supernatants from MSC suggesting that factor(s) constitutively released by MSC are involved. Supporting a role for PGE2 we observed that acetylsalicylic acid impaired the ability of MSC to inhibit the production of inflammatory cytokines and to stimulate the production of IL-10 by LPS-stimulated M. Moreover, we found that MSC constitutively produce PGE2 at levels able to inhibit the production of TNF-α and IL-6 by activated M. MSC also inhibited the up-regulation of CD86 and MHC class II in LPS-stimulated M impairing their ability to activate antigen-specific T CD4+ cells. On the other hand, they stimulated the uptake of apoptotic thymocytes by M. Of note, MSC turned M into cells highly susceptible to infection with the parasite Trypanosoma cruzi increasing more than 5-fold the rate of M infection. Using a model of inflammation triggered by s.c. implantation of glass cylinders, we found that MSC stimulated the recruitment of macrophages which showed a low expression of CD86 and the MHC class II molecule Iab and a high ability to produce IL-10 and IL-12p40, but not IL-12 p70. In summary, our results suggest that MSC switch M into a regulatory profile characterized by a low ability to produce inflammatory cytokines, a high ability to phagocyte apoptotic cells, and a marked increase in their susceptibility to infection by intracellular pathogens

Biotechnological Perspective of Reactive Oxygen Species (ROS)-Mediated Stress Tolerance in Plants

All environmental cues lead to develop secondary stress conditions like osmotic and oxidative stress conditions that reduces average crop yields by more than 50% every year. The univalent reduction of molecular oxygen (O2) in metabolic reactions consequently produces superoxide anions (O2•−) and other reactive oxygen species (ROS) ubiquitously in all compartments of the cell that disturbs redox potential and causes threat to cellular organelles. The production of ROS further increases under stress conditions and especially in combination with high light intensity. Plants have evolved different strategies to minimize the accumulation of excess ROS like avoidance mechanisms such as physiological adaptation, efficient photosystems such as C4 or CAM metabolism and scavenging mechanisms through production of antioxidants and antioxidative enzymes. Ascorbate-glutathione pathway plays an important role in detoxifying excess ROS in plant cells, which includes superoxide dismutase (SOD) and ascorbate peroxidase (APX) in detoxifying O2•−radical and hydrogen peroxide (H2O2) respectively, monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) involved in recycling of reduced substrates such as ascorbate and glutathione. Efficient ROS management is one of the strategies used by tolerant plants to survive and perform cellular activities under stress conditions. The present chapter describes different sites of ROS generation and and their consequences under abiotic stress conditions and also described the approaches to overcome oxidative stress through genomics and genetic engineering

Effects of Anacetrapib in Patients with Atherosclerotic Vascular Disease

BACKGROUND: Patients with atherosclerotic vascular disease remain at high risk for cardiovascular events despite effective statin-based treatment of low-density lipoprotein (LDL) cholesterol levels. The inhibition of cholesteryl ester transfer protein (CETP) by anacetrapib reduces LDL cholesterol levels and increases high-density lipoprotein (HDL) cholesterol levels. However, trials of other CETP inhibitors have shown neutral or adverse effects on cardiovascular outcomes. METHODS: We conducted a randomized, double-blind, placebo-controlled trial involving 30,449 adults with atherosclerotic vascular disease who were receiving intensive atorvastatin therapy and who had a mean LDL cholesterol level of 61 mg per deciliter (1.58 mmol per liter), a mean non-HDL cholesterol level of 92 mg per deciliter (2.38 mmol per liter), and a mean HDL cholesterol level of 40 mg per deciliter (1.03 mmol per liter). The patients were assigned to receive either 100 mg of anacetrapib once daily (15,225 patients) or matching placebo (15,224 patients). The primary outcome was the first major coronary event, a composite of coronary death, myocardial infarction, or coronary revascularization. RESULTS: During the median follow-up period of 4.1 years, the primary outcome occurred in significantly fewer patients in the anacetrapib group than in the placebo group (1640 of 15,225 patients [10.8%] vs. 1803 of 15,224 patients [11.8%]; rate ratio, 0.91; 95% confidence interval, 0.85 to 0.97; P=0.004). The relative difference in risk was similar across multiple prespecified subgroups. At the trial midpoint, the mean level of HDL cholesterol was higher by 43 mg per deciliter (1.12 mmol per liter) in the anacetrapib group than in the placebo group (a relative difference of 104%), and the mean level of non-HDL cholesterol was lower by 17 mg per deciliter (0.44 mmol per liter), a relative difference of -18%. There were no significant between-group differences in the risk of death, cancer, or other serious adverse events. CONCLUSIONS: Among patients with atherosclerotic vascular disease who were receiving intensive statin therapy, the use of anacetrapib resulted in a lower incidence of major coronary events than the use of placebo. (Funded by Merck and others; Current Controlled Trials number, ISRCTN48678192 ; ClinicalTrials.gov number, NCT01252953 ; and EudraCT number, 2010-023467-18 .)