Competitive oxidation of key pentose phosphate pathway enzymes modulates the fate of intermediates and NAPDH production.

The oxidative phase of the pentose phosphate pathway (PPP) involving the enzymes glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconolactonase (6PGL), and 6-phosphogluconate dehydrogenase (6PGDH), is critical to NADPH generation within cells, with these enzymes catalyzing the conversion of glucose-6-phosphate (G6P) into ribulose-5-phosphate (Ribu5-P). We have previously studied peroxyl radical (ROO•) mediated oxidative inactivation of E. coli G6PDH, 6PGL, and 6PGDH. However, these data were obtained from experiments where each enzyme was independently exposed to ROO•, a condition not reflecting biological reality. In this work we investigated how NADPH production is modulated when these enzymes are jointly exposed to ROO•. Enzyme mixtures (1:1:1 ratio) were exposed to ROO• produced from thermolysis of 100 mM 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH). NADPH was quantified at 340 nm, and protein oxidation analyzed by liquid chromatography with mass spectrometric detection (LC-MS). The data obtained were rationalized using a mathematical model. The mixture of non-oxidized enzymes, G6P and NADP+ generated ∼175 µM NADPH. Computational simulations showed a constant decrease of G6P associated with NADPH formation, consistent with experimental data. When the enzyme mixture was exposed to AAPH (3 h, 37 °C), lower levels of NADPH were detected (∼100 µM) which also fitted with computational simulations. LC-MS analyses indicated modifications at Tyr, Trp, and Met residues but at lower concentrations than detected for the isolated enzymes. Quantification of NADPH generation showed that the pathway activity was not altered during the initial stages of the oxidations, consistent with a buffering role of G6PDH towards inactivation of the oxidative phase of the pathway.

The extremophilic Andean isolate Acinetobacter sp. Ver3 expresses two ferredoxin-NADP+ reductase isoforms with different catalytic properties.

Ferredoxin/flavodoxin-NADPH reductases (FPRs) catalyze the reversible electron transfer between NADPH and ferredoxin/flavodoxin. The Acinetobacter sp. Ver3 isolated from high-altitude Andean lakes contains two isoenzymes, FPR1ver3 and FPR2ver3. Absorption spectra of these FPRs revealed typical features of flavoproteins, consistent with the use of FAD as a prosthetic group. Spectral differences indicate distinct electronic arrangements for the flavin in each enzyme. Steady-state kinetic measurements show that the enzymes display catalytic efficiencies in the order of 1-6 µm-1·s-1, although FPR1ver3 exhibited higher kcat values compared to FPR2ver3. When flavodoxinver3 was used as a substrate, both reductases exhibited dissimilar behavior. Moreover, only FPR1ver3 is induced by oxidative stimuli, indicating that the polyextremophile Ver3 has evolved diverse strategies to cope with oxidative environments.

Fragment library screening by X-ray crystallography and binding site analysis on thioredoxin glutathione reductase of Schistosoma mansoni.

Schistosomiasis is caused by parasites of the genus Schistosoma, which infect more than 200 million people. Praziquantel (PZQ) has been the main drug for controlling schistosomiasis for over four decades, but despite that it is ineffective against juvenile worms and size and taste issues with its pharmaceutical forms impose challenges for treating school-aged children. It is also important to note that PZQ resistant strains can be generated in laboratory conditions and observed in the field, hence its extensive use in mass drug administration programs raises concerns about resistance, highlighting the need to search for new schistosomicidal drugs. Schistosomes survival relies on the redox enzyme thioredoxin glutathione reductase (TGR), a validated target for the development of new anti-schistosomal drugs. Here we report a high-throughput fragment screening campaign of 768 compounds against S. mansoni TGR (SmTGR) using X-ray crystallography. We observed 49 binding events involving 35 distinct molecular fragments which were found to be distributed across 16 binding sites. Most sites are described for the first time within SmTGR, a noteworthy exception being the "doorstop pocket" near the NADPH binding site. We have compared results from hotspots and pocket druggability analysis of SmTGR with the experimental binding sites found in this work, with our results indicating only limited coincidence between experimental and computational results. Finally, we discuss that binding sites at the doorstop/NADPH binding site and in the SmTGR dimer interface, should be prioritized for developing SmTGR inhibitors as new antischistosomal drugs.

Glycerol as substrate and NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase enable higher production of 3-hydroxypropionic acid through the ß-alanine pathway in E. coli.

Microbial engineering is a promising way to produce3-HP using biorenewable substrates such as glycerol. However, theglycerol pathway to obtain 3-HPrequires vitamin B-12, which hinders its economic viability. The present work showed that 3-HP can be efficiently produced from glycerol through the ß-alanine pathway. To develop a cell factory for this purpose, glycerol was evaluated as a substrate and showed more than two-fold improved 3-HP production compared to glucose. Next, the reducing power was modulated by overexpression of an NADP+ -dependent glyceraldehyde-3-phosphate dehydrogenase coupled with CRISPR-based repression of the endogenous gapA gene, resulting in a 91 % increase in 3-HP titer. Finally, the toxicity of 3-HP accumulation was addressed by overexpressing a putative exporter (YohJK). Fed-batch cultivation of the final strain yielded 72.2 g/L of 3-HP and a productivity of 1.64 g/L/h, which are the best results for the ß-alanine pathway and are similar to those found for other pathways.

NERNST: a genetically-encoded ratiometric non-destructive sensing tool to estimate NADP(H) redox status in bacterial, plant and animal systems.

NADP(H) is a central metabolic hub providing reducing equivalents to multiple biosynthetic, regulatory and antioxidative pathways in all living organisms. While biosensors are available to determine NADP+ or NADPH levels in vivo, no probe exists to estimate the NADP(H) redox status, a determinant of the cell energy availability. We describe herein the design and characterization of a genetically-encoded ratiometric biosensor, termed NERNST, able to interact with NADP(H) and estimate ENADP(H). NERNST consists of a redox-sensitive green fluorescent protein (roGFP2) fused to an NADPH-thioredoxin reductase C module which selectively monitors NADP(H) redox states via oxido-reduction of the roGFP2 moiety. NERNST is functional in bacterial, plant and animal cells, and organelles such as chloroplasts and mitochondria. Using NERNST, we monitor NADP(H) dynamics during bacterial growth, environmental stresses in plants, metabolic challenges to mammalian cells, and wounding in zebrafish. NERNST estimates the NADP(H) redox poise in living organisms, with various potential applications in biochemical, biotechnological and biomedical research.

Plant NADPH-dependent thioredoxin reductases are crucial for the metabolism of sink leaves and plant acclimation to elevated CO2.

Plants contain three NADPH-thioredoxin reductases (NTR) located in the cytosol/mitochondria (NTRA/B) and the plastid (NTRC) with important metabolic functions. However, mutants deficient in all NTRs remained to be investigated. Here, we generated and characterised the triple Arabidopsis ntrabc mutant alongside with ntrc single and ntrab double mutants under different environmental conditions. Both ntrc and ntrabc mutants showed reduced growth and substantial metabolic alterations, especially in sink leaves and under high CO2 (HC), as compared to the wild type. However, ntrabc showed higher effective quantum yield of PSII under both constant and fluctuating light conditions, altered redox states of NADH/NAD+ and glutathione (GSH/GSSG) and lower potential quantum yield of PSII in sink leaves in ambient but not high CO2 concentrations, as compared to ntrc, suggesting a functional interaction between chloroplastic and extra-chloroplastic NTRs in photosynthesis regulation depending on leaf development and environmental conditions. Our results unveil a previously unknown role of the NTR system in regulating sink leaf metabolism and plant acclimation to HC, while it is not affecting full plant development, indicating that the lack of the NTR system can be compensated, at least to some extent, by other redox mechanisms.

Lighting the light reactions of photosynthesis by means of redox-responsive genetically encoded biosensors for photosynthetic intermediates.

Oxygenic photosynthesis involves light and dark phases. In the light phase, photosynthetic electron transport provides reducing power and energy to support the carbon assimilation process. It also contributes signals to defensive, repair, and metabolic pathways critical for plant growth and survival. The redox state of components of the photosynthetic machinery and associated routes determines the extent and direction of plant responses to environmental and developmental stimuli, and therefore, their space- and time-resolved detection in planta becomes critical to understand and engineer plant metabolism. Until recently, studies in living systems have been hampered by the inadequacy of disruptive analytical methods. Genetically encoded indicators based on fluorescent proteins provide new opportunities to illuminate these important issues. We summarize here information about available biosensors designed to monitor the levels and redox state of various components of the light reactions, including NADP(H), glutathione, thioredoxin, and reactive oxygen species. Comparatively few probes have been used in plants, and their application to chloroplasts poses still additional challenges. We discuss advantages and limitations of biosensors based on different principles and propose rationales for the design of novel probes to estimate the NADP(H) and ferredoxin/flavodoxin redox poise, as examples of the exciting questions that could be addressed by further development of these tools. Genetically encoded fluorescent biosensors are remarkable tools to monitor the levels and/or redox state of components of the photosynthetic light reactions and accessory pathways. Reducing equivalents generated at the photosynthetic electron transport chain in the form of NADPH and reduced ferredoxin (FD) are used in central metabolism, regulation, and detoxification of reactive oxygen species (ROS). Redox components of these pathways whose levels and/or redox status have been imaged in plants using biosensors are highlighted in green (NADPH, glutathione, H2O2, thioredoxins). Analytes with available biosensors not tried in plants are shown in pink (NADP+). Finally, redox shuttles with no existing biosensors are circled in light blue. APX, ASC peroxidase; ASC, ascorbate; DHA, dehydroascorbate; DHAR, DHA reductase; FNR, FD-NADP+ reductase; FTR, FD-TRX reductase; GPX, glutathione peroxidase; GR, glutathione reductase; GSH, reduced glutathione; GSSG, oxidized glutathione; MDA, monodehydroascorbate; MDAR, MDA reductase; NTRC, NADPH-TRX reductase C; OAA, oxaloacetate; PRX, peroxiredoxin; PSI, photosystem I; PSII: photosystem II; SOD, superoxide dismutase; TRX, thioredoxin.

NAADP-Evoked Ca2+ Signaling Leads to Mutant Huntingtin Aggregation and Autophagy Impairment in Murine Astrocytes.

Huntington's disease (HD) is a progressive neurodegenerative disease characterized by mutations in the huntingtin gene (mHtt), causing an unstable repeat of the CAG trinucleotide, leading to abnormal long repeats of polyglutamine (poly-Q) in the N-terminal region of the huntingtin, which form abnormal conformations and aggregates. Alterations in Ca2+ signaling are involved in HD models and the accumulation of mutated huntingtin interferes with Ca2+ homeostasis. Lysosomes are intracellular Ca2+ storages that participate in endocytic and lysosomal degradation processes, including autophagy. Nicotinic acid adenine dinucleotide phosphate (NAADP) is an intracellular second messenger that promotes Ca2+ release from the endo-lysosomal system via Two-Pore Channels (TPCs) activation. Herein, we show the impact of lysosomal Ca2+ signals on mHtt aggregation and autophagy blockade in murine astrocytes overexpressing mHtt-Q74. We observed that mHtt-Q74 overexpression causes an increase in NAADP-evoked Ca2+ signals and mHtt aggregation, which was inhibited in the presence of Ned-19, a TPC antagonist, or BAPTA-AM, a Ca2+ chelator. Additionally, TPC2 silencing revert the mHtt aggregation. Furthermore, mHtt has been shown co-localized with TPC2 which may contribute to its effects on lysosomal homeostasis. Moreover, NAADP-mediated autophagy was also blocked since its function is dependent on lysosomal functionality. Taken together, our data show that increased levels of cytosolic Ca2+ mediated by NAADP causes mHtt aggregation. Additionally, mHtt co-localizes with the lysosomes, where it possibly affects organelle functions and impairs autophagy.

Metabolomic Insights of Biosurfactant Activity from Bacillus niabensis against Planktonic Cells and Biofilm of Pseudomonas stutzeri Involved in Marine Biofouling.

In marine environments, biofilm can cause negative impacts, including the biofouling process. In the search for new non-toxic formulations that inhibit biofilm, biosurfactants (BS) produced by the genus Bacillus have demonstrated considerable potential. To elucidate the changes that BS from B. niabensis promote in growth inhibition and biofilm formation, this research performed a nuclear magnetic resonance (NMR) metabolomic profile analysis to compare the metabolic differences between planktonic cells and biofilms of Pseudomonas stutzeri, a pioneer fouling bacteria. The multivariate analysis showed a clear separation between groups with a higher concentration of metabolites in the biofilm than in planktonic cells of P. stutzeri. When planktonic and biofilm stages were treated with BS, some differences were found among them. In planktonic cells, the addition of BS had a minor effect on growth inhibition, but at a metabolic level, NADP+, trehalose, acetone, glucose, and betaine were up-regulated in response to osmotic stress. When the biofilm was treated with the BS, a clear inhibition was observed and metabolites such as glucose, acetic acid, histidine, lactic acid, phenylalanine, uracil, and NADP+ were also up-regulated, while trehalose and histamine were down-regulated in response to the antibacterial effect of the BS.

Reactive oxygen species-dependent-NLRP3 inflammasome activation in human neutrophils induced by l-amino acid oxidase derived from Calloselasma rhodostoma venom.

l-Amino acid oxidase isolated from Calloselasma rhodostoma (Cr-LAAO) snake venom is a potent stimulus for neutrophil activation and production of inflammatory mediators, contributing to local inflammatory effects in victims of envenoming. Cr-LAAO triggered the activation of nicotinamide adenine dinucleotide phosphatase (NADPH) oxidase complex and protein kinase C (PKC)-α signaling protein for reactive oxygen species (ROS) production. This study aims to evaluate the ROS participation in the NLRP3 inflammasome complex activation in human neutrophil. Human neutrophils were isolated and stimulated for 1 or 2 h with RPMI (negative control), LPS (1 µg/mL, positive control) or Cr-LAAO (50 µg/mL). The neutrophil transcriptome was examined using the microarray technique, and RT-qPCR for confirmation of gene expression. Immunofluorescence assays for NLRP3, caspase-1, IL-1ß and GSDMD proteins was performed by Western blot in the presence and/or absence of Apocynin, an inhibitor of NADPH oxidase. IL-1ß release was also detected in the presence and/or absence of NLRP3, caspase-1 and NADPH oxidase inhibitors. Results showed that Cr-LAAO upregulated the expression of genes that participate in the NADPH oxidase complex formation and inflammasome assembly. NLRP3 was activated and accumulated in the cytosol forming punctas, indicating its activation. Gasdermin D was not cleaved but lactate dehydrogenase was released. Furthermore, ROS inhibition decreased the expression of NLRP3 inflammasome complex proteins, as observed by protein expression in the presence and/or absence of apocynin, an NADPH oxidase inhibitor. IL-1ß was also released, and pharmacological inhibition of NLRP3, caspase-1, and ROS reduced the amount of released cytokine. This is the first report demonstrating the activation of the NLRP3 inflammasome complex via ROS generation by Cr-LAAO, which may lead to the development of local inflammatory effects observed in snakebite victims.

Biochemical regulatory processes in the control of oxidants and antioxidants production in the brain of rats with iron and copper chronic overloads.

Iron [Fe(II)] and copper [Cu(II)] overloads in rat brain are associated with oxidative stress and damage. The purpose of this research is to study whether brain antioxidant enzymes are involved in the control of intracellular redox homeostasis in the brain of rats male Sprague-Dawley rats (80-90 g) that received drinking water supplemented with either 1.0 g/L of ferrous chloride (n = 24) or 0.5 g/L cupric sulfate (n = 24) for 42 days. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx) and glutathione transferase (GT) activities in brain were determined by spectrophotometric methods and NO production by the content of nitrite concentration in the organ. Chronic treatment with Fe(II) and Cu(II) led to a significant decrease of nitrite content and SOD activity in brain. Activity of NADPH oxidase increased with Cu(II) treatment. Concerning Fe(II), catalase and GT activities increased in brain after 28 and 4 days of treatment, respectively. In the case of Cu(II), catalase activity decreased whereas GT activity increased after 2 and 14 days, respectively. The regulation of redox homeostasis in brain involves changes of the activity of these enzymes to control the steady state of oxidant species related to redox signaling pathways upon Cu and Fe overload. NO may serve to detoxify cells from superoxide anion and hydrogen peroxide with the concomitant formation of peroxynitrite. However, the latest is a powerful oxidant which leads to oxidative modifications of biomolecules. These results suggest a common pathway to oxidative stress and damage in brain for Cu(II) and Fe(II).

Glucose-6-phosphate dehydrogenase immobilized onto magnetic beads (G6PDH-Mb) as a generator system for production of NADPH: Development and application in metabolism studies.

Reduced nicotinamide adenine dinucleotide phosphate (NADPH) participates in several anabolic and catabolic pathways, being essential in numerous biochemical reactions involving energy release. Most of these reactions require a high amount of NADPH, which can be expensive from an industry point of view. Thus, biotechnology industries developed a great interest in NADPH production. Currently, there are different ways to obtain NADPH in situ, however, the most common is by enzymatic reactions, known as generator systems. Although this approach can be beneficial in terms of cost, the major drawback is the impossibility of reusing the enzyme. To overcome this, enzyme immobilization is a proven alternative. Herein, we report the use of glucose-6-phosphate dehydrogenase immobilized onto magnetic beads (G6PDH-Mb) through glutaraldehyde coupling to produce high amounts of NADPH. The G6PDH-Mbs were kinetically characterized showing a sigmoidal curve. Besides, the stability was evaluated, and their reuse was demonstrated for a period superior to 40 days. The G6PDH-Mb was used to in situ production of the NADPH metabolism experiments, using human liver microsome solutions and either albendazole or fiscalin B as model targets. The production of in vitro metabolites from albendazole and fiscalin B was evaluated by comparing the use of NADPH generated in situ with those obtained by commercial NADPH. Moreover, the activity of the G6PDH-Mb was monitored after using it for five consecutive albendazole metabolism reactions, with only a minor decrease in the enzyme activity (3.58 ± 1.67%) after the fifth time of use. The higher concentration obtained when using the designed G6PDH-Mb generator system demonstrated proof of the concept and its applicability.

The Possible Role of Glucose-6-Phosphate Dehydrogenase in the SARS-CoV-2 Infection.

Glucose-6-phosphate dehydrogenase (G6PD) is the second rate-limiting enzyme of the pentose phosphate pathway. This enzyme is present in the cytoplasm of all mammalian cells, and its activity is essential for an adequate functioning of the antioxidant system and for the response of innate immunity. It is responsible for the production of nicotinamide adenine dinucleotide phosphate (NADPH), the first redox equivalent, in the pentose phosphate pathway. Viral infections such as SARS-CoV-2 may induce the Warburg effect with an increase in anaerobic glycolysis and production of lactate. This condition ensures the success of viral replication and production of the virion. Therefore, the activity of G6PD may be increased in COVID-19 patients raising the level of the NADPH, which is needed for the enzymatic and non-enzymatic antioxidant systems that counteract the oxidative stress caused by the cytokine storm. G6PD deficiency affects approximately 350-400 million people worldwide; therefore, it is one of the most prevalent diseases related to enzymatic deficiency worldwide. In G6PD-deficient patients exposed to SARS-CoV-2, the amount of NADPH is reduced, increasing the susceptibility for viral infection. There is loss of the redox homeostasis in them, resulting in severe pneumonia and fatal outcomes.

Structural Determinants of Sugar Alcohol Biosynthesis in Plants: The Crystal Structures of Mannose-6-Phosphate and Aldose-6-Phosphate Reductases.

Sugar alcohols are major photosynthetic products in plant species from the Apiaceae and Plantaginaceae families. Mannose-6-phosphate reductase (Man6PRase) and aldose-6-phosphate reductase (Ald6PRase) are key enzymes for synthesizing mannitol and glucitol in celery (Apium graveolens) and peach (Prunus persica), respectively. In this work, we report the first crystal structures of dimeric plant aldo/keto reductases (AKRs), celery Man6PRase (solved in the presence of mannonic acid and NADP+) and peach Ald6PRase (obtained in the apo form). Both structures displayed the typical TIM barrel folding commonly observed in proteins from the AKR superfamily. Analysis of the Man6PRase holo form showed that residues putatively involved in the catalytic mechanism are located close to the nicotinamide ring of NADP+, where the hydride transfer to the sugar phosphate should take place. Additionally, we found that Lys48 is important for the binding of the sugar phosphate. Interestingly, the Man6PRase K48A mutant had a lower catalytic efficiency with mannose-6-phosphate but a higher catalytic efficiency with mannose than the wild type. Overall, our work sheds light on the structure-function relationships of important enzymes to synthesize sugar alcohols in plants.

Redox imbalance induces remodeling of glucose metabolism in Rhipicephalus microplus embryonic cell line.

Carbohydrate metabolism not only functions in supplying cellular energy but also has an important role in maintaining physiological homeostasis and in preventing oxidative damage caused by reactive oxygen species. Previously, we showed that arthropod embryonic cell lines have high tolerance to H2O2 exposure. Here, we describe that Rhipicephalus microplus tick embryonic cell line (BME26) employs an adaptive glucose metabolism mechanism that confers tolerance to hydrogen peroxide at concentrations too high for other organisms. This adaptive mechanism sustained by glucose metabolism remodeling promotes cell survival and redox balance in BME26 cell line after millimolar H2O2 exposure. The present work shows that this tick cell line could tolerate high H2O2 concentrations by initiating a carbohydrate-related adaptive response. We demonstrate that gluconeogenesis was induced as a compensation strategy that involved, among other molecules, the metabolic enzymes NADP-ICDH, G6PDH, and PEPCK. We also found that this phenomenon was coupled to glycogen accumulation and glucose uptake, supporting the pentose phosphate pathway to sustain NADPH production and leading to cell survival and proliferation. Our findings suggest that the described response is not atypical, being also observed in cancer cells, which highlights the importance of this model to all proliferative cells. We propose that these results will be useful in generating basic biological information to support the development of new strategies for disease treatment and parasite control.

Identification, analysis, and modeling of the YUCCA protein family genome-wide in Coffea canephora.

Auxin is involved in almost every aspect of plant growth and development, from embryogenesis to senescence. Indole-3-acetic acid (IAA) is the main known natural auxin that is synthesized by enzymes tryptophan aminotransferase of arabidopsis (TAA) and YUCCA (YUC) of the flavin-containing monooxygenases family (FMO) from one of the tryptophan-dependent pathways. Genome-wide identification and comprehensive analysis of the YUC-protein family have been conducted in Coffea canephora in the present study. A total of 10 members CcYUC gene family were identified in C. canephora. Phylogenetic analysis revealed that the CcYUC protein family is evolutionarily conserved, and they consist of four groups. In contrast, bioinformatic analysis predicted a hydrophobic transmembrane helix (TMH) for one CcYUC (YUC10) member only. Isoelectric point (pI), molecular mass (Ms), signal peptide, subcellular localization, and phosphorylation sites were predicted for CcYUC proteins. YUC enzymes require the prosthetic group flavin adenine dinucleotide (FAD) and the cofactor nicotinamide adenine dinucleotide phosphate (NADPH) for their enzymatic activity. Therefore, we include the molecular docking for CcYUC2-FAD-NADPH-IPyA and yucasin, which is a specific inhibitor for YUC activity. The docking results showed FAD and NADPH binding at the big and small domain sites, respectively, in CcYUC2. IPyA binds very close to FAD along the big domain, and yucasin competes for the same site as IPA, blocking IAA production. Furthermore, in silico point mutations affect the stability of the CcYUC2-4 proteins.

Mitochondrial NAD(P)+ Transhydrogenase: From Molecular Features to Physiology and Disease.

Significance: Proton-translocating NAD(P)+ transhydrogenase, also known as nicotinamide nucleotide transhydrogenase (NNT), catalyzes a reversible reaction coupling the protonmotive force across the inner mitochondrial membrane and hydride (H-, a proton plus two electrons) transfer between the mitochondrial pools of NAD(H) and NADP(H). The forward NNT reaction is a source of NADPH in the mitochondrial matrix, fueling antioxidant and biosynthetic pathways with reductive potential. Despite the greater emphasis given to the net forward reaction, the reverse NNT reaction that oxidizes NADPH also occurs in physiological and pathological conditions. Recent Advances: NNT (dys)function has been linked to various metabolic pathways and disease phenotypes. Most of these findings have been based on spontaneous loss-of-function Nnt mutations found in the C57BL/6J mouse strain (NntC57BL/6J mutation) and disease-causing Nnt mutations in humans. The present review focuses on recent advances based on the mouse NntC57BL/6J mutation. Critical Issues: Most studies associating NNT function with disease phenotypes have been based on comparisons between different strains of inbred mice (with or without the NntC57BL/6J mutation), which creates uncertainties over the actual contribution of NNT in the context of other potential genetic modifiers. Future Directions: Future research might contribute to understanding the role of NNT in pathological conditions and elucidate how NNT regulates physiological signaling through its forward and reverse reactions. The importance of NNT in redox balance and tumor cell proliferation makes it a potential target of new therapeutic strategies for oxidative-stress-mediated diseases and cancer. Antioxid. Redox Signal. 36, 864-884.

Evolution of a key enzyme of aerobic metabolism reveals Proterozoic functional subunit duplication events and an ancient origin of animals.

The biological toolkits for aerobic respiration were critical for the rise and diversification of early animals. Aerobic life forms generate ATP through the oxidation of organic molecules in a process known as Krebs' Cycle, where the enzyme isocitrate dehydrogenase (IDH) regulates the cycle's turnover rate. Evolutionary reconstructions and molecular dating of proteins related to oxidative metabolism, such as IDH, can therefore provide an estimate of when the diversification of major taxa occurred, and their coevolution with the oxidative state of oceans and atmosphere. To establish the evolutionary history and divergence time of NAD-dependent IDH, we examined transcriptomic data from 195 eukaryotes (mostly animals). We demonstrate that two duplication events occurred in the evolutionary history of NAD-IDH, one in the ancestor of eukaryotes approximately at 1967 Ma, and another at 1629 Ma, both in the Paleoproterozoic Era. Moreover, NAD-IDH regulatory subunits ß and γ are exclusive to metazoans, arising in the Mesoproterozoic. Our results therefore support the concept of an ''earlier-than-Tonian'' diversification of eukaryotes and the pre-Cryogenian emergence of a metazoan IDH enzyme.

Nitrergic neurons of the forepaw representation in the rat somatosensory and motor cortices: A quantitative study.

Nitrergic neurons (NNs) are inhibitory neurons capable of releasing nitric oxide (NO) that are labeled with nicotinamide adenine dinucleotide phosphate diaphorase histochemistry. The rat primary somatosensory (S1) and motor (M1) cortices are a favorable model to investigate NN populations by comparing their morphology, since these areas share the border of forepaw representation. The distribution of the Type I NN of the forepaw representation in the S1 and M1 cortices of the rat in different laminar compartments and the morphological parameters related to the cell body and dendritic arborization were measured and compared. We observed that the neuronal density in the S1 (130 NN/mm3 ) was higher than the neuronal density in the M1 (119 NN/mm3 ). Most NN neurons were multipolar (S1 with 58%; M1 with 69%), and a minority of the NN neurons were horizontal (S1 with 6%; M1 with 12%). NN found in S1 had a higher verticality index than NN found in M1, and no significant differences were observed for the other morphological parameters. We also demonstrated significant differences in most of the morphological parameters of the NN between different cortical compartments of S1 and M1. Our results indicate that the NN of the forepaw in S1 and M1 corresponds to a neuronal population, where the functionality is independent of the different types of sensory and motor processing. However, the morphological differences found between the cortical compartments of S1 and M1, as well as the higher density of NNs found in S1, indicate that the release of NO varies between the areas.