Dependent Enzyme Research Articles

A SIRT7-dependent acetylation switch regulates early B cell differentiation and lineage commitment through Pax5.

B lymphopoiesis is orchestrated by lineage-specific transcription factors. In B cell progenitors, lineage commitment is mediated by Pax5, which is commonly mutated in B cell acute lymphoblastic leukemia. Despite its essential role in immunity, the mechanisms regulating Pax5 function remain largely unknown. Here, we found that the NAD+-dependent enzyme SIRT7 coordinates B cell development through deacetylation of Pax5 at K198, which promotes Pax5 protein stability and transcriptional activity. Neither Pax5K198 deacetylated nor acetylated mimics rescued B cell differentiation in Pax5-/- pro-B cells, suggesting that B cell development requires Pax5 dynamic deacetylation. The Pax5K198 deacetylation mimic restored lineage commitment in Pax5-/- pro-B cells and B cell differentiation in Sirt7-/- pro-B cells, suggesting the uncoupling of differentiation from lineage commitment. The SIRT7-Pax5 interplay was conserved in B cell acute lymphoblastic leukemia, where SIRT7 expression correlated with good prognosis. Our findings reveal a crucial mechanism for B lymphopoiesis and highlight the relevance of sirtuins in immune function.

Enhanced SIRT1 Activity by Galangin Mitigates UVB-Induced Senescence in Dermal Fibroblasts via p53 Acetylation Regulation and Activation.

Human skin aging, a complex process influenced by intrinsic aging and extrinsic photoaging, is marked by the accumulation of reactive oxygen species (ROS) that cause DNA damage, impaired dermal fibroblast function, and wrinkle formation. External stressors, such as ultraviolet (UV) radiation, can trigger cellular senescence. Sirtuin-1 (SIRT1), an NAD+-dependent enzyme in the sirtuin family, plays a crucial role in deacetylating p53, thereby inhibiting its nuclear translocation and reducing skin senescence. Galangin, a flavonoid found in honey and Alpinia officinarum root, has antioxidant and anti-inflammatory properties. This study investigates the protective mechanism of galangin against UVB-induced senescence in human dermal fibroblasts (HDFs) by examining its effects on SIRT1 and its target, acetylated-p53. An in vitro model of UVB-induced senescence using HDFs and an in vivo model using nude mice were employed to assess the dermal protective effects of galangin. The results demonstrate that while UVB exposure does not decrease SIRT1 protein levels, it impairs its enzymatic function. However, galangin treatment counteracts these adverse effects. Additionally, UVB exposure significantly reduces cell viability and upregulates senescence markers like p16, p21, and p53 nuclear transactivation. An increase in senescence-associated β-galactosidase (SA-β-gal) positive cells was observed in UVB-exposed dermal fibroblasts. Galangin treatment mitigates UVB-induced cellular senescence by enhancing SIRT1-mediated p53 deacetylation, thereby inhibiting nuclear translocation and reducing dermal senescence. These findings suggest that galangin is a promising agent for alleviating UVB-induced skin aging and could be a potential component in antiaging cosmetic formulations.

Identification of a novel NADPH generation reaction in the pentose phosphate pathway in Escherichia coli using mBFP.

NADPH is a redox cofactor that drives the anabolic reactions. Although major NADPH generation reactions have been identified in Escherichia coli, some minor reactions have not been identified. In the present study, we explored novel NADPH generation reactions by monitoring the fluorescence dynamics after the addition of carbon sources to starved cells, using a metagenome-derived blue fluorescent protein (mBFP) as an intracellular NADPH reporter. Perturbation analyses were performed on a glucose-6-phosphate isomerase (PGI) deletion strain and its parental strain. Interestingly, mBFP fluorescence increased not only in the parental strain but also in the ΔPGI strain after the addition of xylose. Because the ΔPGI strain cannot metabolize xylose through the oxidative pentose phosphate pathway, this suggests that an unexpected NADPH generation reaction contributes to an increase in fluorescence. To unravel this mystery, we deleted the NADPH generation enzymes including transhydrogenase, isocitrate dehydrogenase, NADP+-dependent malic enzyme, glucose-6-phosphate dehydrogenase (G6PDH), and 6-phosphogluconate dehydrogenase (6PGDH) in the ΔPGI strain, and revealed that G6PDH and 6PGDH contribute to an increase in fluorescence under xylose conditions. In vitro assays using purified enzymes showed that G6PDH can produce NADPH using erythrose-4-phosphate (E4P) as a substitute for glucose-6-phosphate. Because the Km (0.65 mM) for E4P was much higher than the reported intracellular E4P concentrations in E. coli, little E4P must be metabolized through this bypass in the parental strain. However, the flux would increase when E4P accumulates in the cells owing to genetic modifications. This finding provides a metabolic engineering strategy for generating NADPH to produce useful compounds using xylose as a carbon source.IMPORTANCEBecause NADPH is consumed during the synthesis of various useful compounds, enhancing NADPH regeneration is highly desirable in metabolic engineering. In this study, we explored novel NADPH generation reactions in Escherichia coli using a fluorescent NADPH reporter and found that glucose-6-phosphate dehydrogenase can produce NADPH using erythrose-4-phosphate as a substrate under xylose conditions. Xylose is an abundant sugar in nature and is an attractive carbon source for bioproduction. Therefore, this finding contributes to novel pathway engineering strategies using a xylose carbon source in E. coli to produce useful compounds that consume NADPH for their synthesis.

Molecular characterization and expression pattern analysis of Sirtuin family genes in the eastern honeybee, Apis cerana

Sirtuin proteins are a family of evolutionarily conserved NAD+-dependent deacetylase enzymes which play crucial roles in regulating insect metabolism, maintaining genome stability, and lifespan. However, there is no report on the genome-wide analysis of the Sirtuin gene family in the eastern honeybee, Apis cerana. Here, 6 Sirtuin genes (AcSirt1, AcSirt2, AcSirt4 ~ AcSirt7) were obtained in the A. cerana genome, which were located on 6 different chromosomes and were all single-copy genes. Gene structure analysis revealed that these 6 genes were all multi-exon genes. The Sirtuin family genes contained 9 conserved motifs and 5 conserved domains, which exhibited typical Sirtuin protein structural characteristics. The phylogenetic analysis of the Sirtuin family genes indicates that A. cerana lacked the Sirt3 gene, and the 6 Sirtuin encoding genes could be clustered into 5 groups and 6 branches, showing high homology with the Apis mellifera and the Apis dorsata. The results of the tissue and period expression profiles by qRT-PCR indicated that the 6 Sirtuin family genes showed transcriptional activity in different tissues of worker bees, and the gene expression level reached the peak in the 4-day-old larvae and pupa stages. It was speculated that Sirtuin genes expression level was affected by fasting behavior, caloric restriction and organ differentiation during different developmental stages of A. cerana. Meanwhile, the differential expression patterns of Sirtuin genes in various tissues further demonstrated the diversity and complexity of their functions, providing a basis for target selection in future studies on the functionality of Sirtuin genes.

Key structural role of a conserved cis-proline revealed by the P285S variant of soybean serine hydroxymethyltransferase 8.

The enzyme serine hydroxymethyltransferase (SHMT) plays a key role in folate metabolism and is conserved in all kingdoms of life. SHMT is a pyridoxal 5'-phosphate (PLP) - dependent enzyme that catalyzes the conversion of L-serine and (6S)-tetrahydrofolate to glycine and 5,10-methylene tetrahydrofolate. Crystal structures of multiple members of the SHMT family have shown that the enzyme has a single conserved cis proline, which is located near the active site. Here, we have characterized a Pro to Ser amino acid variant (P285S) that affects this conserved cis proline in soybean SHMT8. P285S was identified as one of a set of mutations that affect the resistance of soybean to the agricultural pathogen soybean cyst nematode. We find that replacement of Pro285 by serine eliminates PLP-mediated catalytic activity of SHMT8, reduces folate binding, decreases enzyme stability, and affects the dimer-tetramer ratio of the enzyme in solution. Crystal structures at 1.9 - 2.2 Å resolution reveal a local reordering of the polypeptide chain that extends an a-helix and shifts a turn region into the active site. This results in a dramatically perturbed PLP-binding pose, where the ring of the cofactor is flipped by ~180° with concomitant loss of conserved enzyme-PLP interactions. A nearby region of the polypeptide becomes disordered, evidenced by missing electron density for ~10 residues. These structural perturbations are consistent with the loss of enzyme activity and folate binding and underscore the important role of the Pro285 cis-peptide in SHMT structure and function.

Exploring the Role and Pathophysiological Significance of Aldehyde Dehydrogenase 1B1 (ALDH1B1) in Human Lung Adenocarcinoma

Aldehyde dehydrogenases (ALDHs) constitute a diverse superfamily of NAD(P)+-dependent enzymes pivotal in oxidizing endogenous and exogenous aldehydes to carboxylic acids. Beyond metabolic roles, ALDHs participate in essential biological processes, including differentiation, embryogenesis and the DNA damage response, while also serving as markers for cancer stem cells (CSCs). Aldehyde dehydrogenase 1B1 (ALDH1B1) is a mitochondrial enzyme involved in the detoxification of lipid peroxidation by-products and metabolism of various aldehyde substrates. This study examines the potential role of ALDH1B1 in human lung adenocarcinoma and its association with the CSC phenotype. To this end, we utilized the lung adenocarcinoma cell line A549, engineered to stably express the human ALDH1B1 protein tagged with green fluorescent protein (GFP). Overexpression of ALDH1B1 led to notable changes in cell morphology, proliferation rate and clonogenic efficiency. Furthermore, ALDH1B1-overexpressing A549 cells exhibited enhanced resistance to the chemotherapeutic agents etoposide and cisplatin. Additionally, ALDH1B1 overexpression correlated with increased migratory potential and epithelial–mesenchymal transition (EMT), mediated by the upregulation of transcription factors such as SNAI2, ZEB2 and TWIST1, alongside the downregulation of E-cadherin. Moreover, Spearman’s rank correlation coefficient analysis using data from 507 publicly available lung adenocarcinoma clinical samples revealed a significant correlation between ALDH1B1 and various molecules implicated in CSC-related signaling pathways, including Wnt, Notch, hypoxia, Hedgehog, retinoic acid, Hippo, NF-κΒ, TGF-β, PI3K/PTEN-AKT and glycolysis/gluconeogenesis. These findings provide insights into the role of ALDH1B1 in lung tumor progression and its relation to the lung CSC phenotype, thereby offering potential therapeutic targets in the clinical management of lung adenocarcinoma.

Sirt2 Regulates Liver Metabolism in a Sex-Specific Manner.

Sirtuin-2 (Sirt2), an NAD+-dependent lysine deacylase enzyme, has previously been implicated as a regulator of glucose metabolism, but the specific mechanisms remain poorly defined. Here, we observed that Sirt2-/- males, but not females, have decreased body fat, moderate hypoglycemia upon fasting, and perturbed glucose handling during exercise compared to wild type controls. Conversion of injected lactate, pyruvate, and glycerol boluses into glucose via gluconeogenesis was impaired, but only in males. Primary Sirt2-/- male hepatocytes exhibited reduced glycolysis and reduced mitochondrial respiration. RNAseq and proteomics were used to interrogate the mechanisms behind this liver phenotype. Loss of Sirt2 did not lead to transcriptional dysregulation, as very few genes were altered in the transcriptome. In keeping with this, there were also negligible changes to protein abundance. Site-specific quantification of the hepatic acetylome, however, showed that 13% of all detected acetylated peptides were significantly increased in Sirt2-/- male liver versus wild type, representing putative Sirt2 target sites. Strikingly, none of these putative target sites were hyperacetylated in Sirt2-/- female liver. The target sites in the male liver were distributed across mitochondria (44%), cytoplasm (32%), nucleus (8%), and other compartments (16%). Despite the high number of putative mitochondrial Sirt2 targets, Sirt2 antigen was not detected in purified wild type liver mitochondria, suggesting that Sirt2's regulation of mitochondrial function occurs from outside the organelle. We conclude that Sirt2 regulates hepatic protein acetylation and metabolism in a sex-specific manner.

Metformin hydrolase is a recently evolved nickel-dependent heteromeric ureohydrolase

The anti-diabetic drug metformin is one of the most widely prescribed medicines in the world. Together with its degradation product guanylurea, it is a major pharmaceutical pollutant in wastewater treatment plants and surface waters. An operon comprising two genes of the ureohydrolase family in Pseudomonas and Aminobacter species has recently been implicated in metformin degradation. However, the corresponding proteins have not been characterized. Here we show that these genes encode a Ni2+-dependent enzyme that efficiently and specifically hydrolyzes metformin to guanylurea and dimethylamine. The active enzyme is a heteromeric complex of α- and β- subunits in which only the α-subunits contain the conserved His and Asp residues for the coordination of two Ni2+ ions in the active site. A crystal structure of metformin hydrolase reveals an α2β4 stoichiometry of the hexameric complex, which is unprecedented in the ureohydrolase family. By studying a closely related but more widely distributed enzyme, we find that the putative predecessor specifically hydrolyzes dimethylguanidine instead of metformin. Our findings establish the molecular basis for metformin hydrolysis to guanylurea as the primary pathway for metformin biodegradation and provide insight into the recent evolution of ureohydrolase family proteins in response to an anthropogenic compound.

Electrochemical glucose sensor based on a phenothiazine derivative mediator with nicotinamide adenine dinucleotide-dependent glucose dehydrogenase

The phenothiazine derivative (M2) was utilized as a redox mediator for the redox processes of the nicotinamide adenine dinucleotide (NAD+) cofactor. The composite solutions were prepared by mixing NAD+-dependent glucose dehydrogenase with NAD+, M2, poly-l-lysine, and glutaraldehyde. Glucose sensors were fabricated by casting various composite solutions onto the reduced graphene oxide-modified gold electrode and additional Nafion coating processes. The composition of electrode materials (ratio and amount of each component) and measurement conditions (applied potential, pH, and temperature) were optimized to obtain the highest sensitivity. The sensor showed a low reference effect, high selectivity toward interfering species, and long-term stability. The glucose sensor performed well when human serum was injected as the actual sample and compared with a standard solution. These findings promote the development of electrochemical sensors based on other NAD+-dependent enzymes.

Identification of Kibdelomycin and Related Biosynthetic Gene Clusters and Characterization of the C‐Branching of Amycolose

AbstractMany bacterial natural products contain C‐branched sugars, including components from the outer cell wall or antibiotically active metabolites. The enzymatic C‐branching of keto sugars leading to longer side chains (≥C2) is catalyzed by thiamine diphosphate (ThDP)‐dependent enzymes. Chiral tertiary α‐hydroxy ketones are formed in this process. The ThDP‐dependent enzymes that catalyze C‐branching reactions belong to one of three enzymatic superfamilies: decarboxylases, transketolases, and α‐ketoacid dehydrogenases 2, but branching of keto sugars has only been demonstrated for decarboxylases. In this study, we showed that an α‐ketoacid dehydrogenase is responsible for C‐branching of the deoxyketo sugar amycolose in the biosynthesis of kibdelomycin in Kibdelosporangium sp. MA7385. In addition, we characterized an amino transferase in the same biosynthetic gene cluster (BGC) that accepts a sterically demanding tertiary α‐hydroxy ketone in a downstream reaction. Subsequently, we identified approximately 400 similar BGCs in silico, suggesting that there is a large diversity of possible ThDP‐dependent enzymes catalyzing the C‐branching of keto sugars and subsequent modifications.

Structure, Function, and Activity of Small Molecule and Peptide Inhibitors of Protein Arginine Methyltransferase 1.

Protein arginine N-methyltransferases (PRMT) are a family of S-adenosyl-l-methionine (SAM)-dependent enzymes that transfer methyl-groups to the ω-N of arginyl residues in proteins. PRMTs are involved in regulating gene expression, RNA splicing, and other activities. PRMT1 is responsible for most cellular arginine methylation, and its dysregulation is involved in many cancers. Accordingly, many groups have targeted PRMT1 using small molecules and peptide inhibitors. In this Perspective, we discuss the structure and function of selected peptide and small molecule inhibitors of PRMT1. We examine inhibitors that target the substrate arginyl peptide, SAM, or both binding sites, and the type of inhibition that results. Small molecules, and peptides that are bisubstrate, and/or PRMT transition state mimic inhibitors as well as inhibitors that alkylate PRMTs will be discussed. We define a structure-activity relationship for the aromatic/heteroaromatic N-methylethylenediamine inhibitors of PRMT1 and review current progress of PRMT1 inhibitors in clinical trials.

Potential Therapeutic Interventions Targeting NAD+ Metabolism for ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting both upper and lower motor neurons. While there have been many potential factors implicated for ALS development, such as oxidative stress and mitochondrial dysfunction, no exact mechanism has been determined at this time. Nicotinamide adenine dinucleotide (NAD+) is one of the most abundant metabolites in mammalian cells and is crucial for a broad range of cellular functions from DNA repair to energy homeostasis. NAD+ can be synthesized from three different intracellular pathways, but it is the NAD+ salvage pathway that generates the largest proportion of NAD+. Impaired NAD+ homeostasis has been connected to aging and neurodegenerative disease-related dysfunctions. In ALS mice, NAD+ homeostasis is potentially disrupted prior to the appearance of physical symptoms and is significantly reduced in the nervous system at the end stage. Treatments targeting NAD+ metabolism, either by administering NAD+ precursor metabolites or small molecules that alter NAD+-dependent enzyme activity, have shown strong beneficial effects in ALS disease models. Here, we review the therapeutic interventions targeting NAD+ metabolism for ALS and their effects on the most prominent pathological aspects of ALS in animal and cell models.

NAD+ Metabolism and Mitochondrial Activity in the Aged Oocyte: Focus on the Effects of NAMPT Stimulation.

The ovary experiences an age-dependent decline starting during the fourth decade of life. Ovarian aging is the predominant factor driving female reproductive aging. Modern trend to postpone childbearing age contributes to reduced fertility and natality worldwide. Recently, the beneficial role of NAD+ precursors on the maintenance of oocyte competence and female fertility affected by aging has emerged. Nevertheless, age-related changes in NAD+ regulatory network have not been investigated so far. In this context, our goal was to investigate changes induced by the aging process in the expression level of genes participating in NAD+ biosynthetic and NAD+ consuming pathways and in the cellular bioenergetics in the mouse oocyte. From Ingenuity Pathway Analysis (IPA) it emerged that aging caused the downregulation of all cellular pathways for NAD+ synthesis (Kynurenine pathway, Preiss-Handler pathway and NAD+ salvage pathway) and deeply influenced the activity of NAD+-dependent enzymes, i.e. PARPs and SIRTs, with effects on many cellular functions including compromised ROS detoxification. Considering that NAMPT, the rate-limiting enzyme of NAD+ salvage pathway, was deregulated, aged oocytes were matured in the presence of P7C3, NAMPT activator. P7C3 improved spindle assembly and mitochondrial bioenergetics and reduced mitochondrial proton leak. Moreover, P7C3 influenced gene expression of NAD+ regulatory network, with Sirt1 as the central node of IPA-interfered target gene network. Finally, P7C3 effectively counteracted oocyte alterations induced by exposure to oxidative stress. Our study contributes to establish effective NAD+ boosting interventions to alleviate the effects of advanced maternal age on fertility and explore their potential in redox-related fertility disorders.

Integrated multi-omics unveil the impact of H-phosphinic analogs of glutamate and α-ketoglutarate on Escherichia coli metabolism

Desmethylphosphinothricin (L-Glu-γ-PH) is the H-phosphinic analogue of glutamate with carbon-phosphorus-hydrogen (C-P-H) bonds. In L-Glu-γ-PH the phosphinic group acts as a bioisostere of glutamate γ-carboxyl group allowing the molecule to be a substrate of Escherichia coli glutamate decarboxylase, a pyridoxal 5’-phosphate-dependent α-decarboxylase. In addition, the L-Glu-γ-PH decarboxylation product, GABA-PH, is further metabolized by bacterial GABA-transaminase, another pyridoxal 5’-phosphate-dependent enzyme, and succinic semialdehyde dehydrogenase, a NADP+-dependent enzyme. The product of these consecutive reactions, the so-called GABA shunt, is succinate-PH, the H-phosphinic analogue of succinate, a tricarboxylic acid cycle intermediate. Notably, L-Glu-γ-PH displays an antibacterial activity in the same concentration range of well-established antibiotics in E. coli. The dipeptide L-Leu-Glu-γ-PH was shown to display an even higher efficacy, likely as a consequence of an improved penetration into the bacteria.Herein, with the aim of further understanding the intracellular effects of L-Glu-γ-PH, 1H NMR-based metabolomics and LC-MS-based shotgun proteomics were used. This study included also the keto-derivative of L-Glu-γ-PH, α-ketoglutarate-γ-PH (α-KG-γ-PH), which also exhibits antimicrobial activity. L-Glu-γ-PH and α-KG-γ-PH are found to similarly impact the bacterial metabolism, though the overall effect of α-KG-γ-PH is more pervasive. Notably α-KG-γ-PH is converted intracellularly into L-Glu-γ-PH, but the opposite was not found. In general, both molecules impact the pathways where aspartate, glutamate and glutamine are used as precursors for the biosynthesis of related metabolites, activate the acid stress response and deprive cells of nitrogen.This work highlights the multi-target drug potential of L-Glu-γ-PH and α-KG-γ-PH and paves the way for their exploitation as antimicrobials.

Activation and inhibition of sirtuins: From bench to bedside.

The sirtuin family comprises seven NAD+-dependent enzymes which catalyze protein lysine deacylation and mono ADP-ribosylation. Sirtuins act as central regulators of genomic stability and gene expression and control key processes, including energetic metabolism, cell cycle, differentiation, apoptosis, and aging. As a result, all sirtuins play critical roles in cellular homeostasis and organism wellness, and their dysregulation has been linked to metabolic, cardiovascular, and neurological diseases. Furthermore, sirtuins have shown dichotomous roles in cancer, acting as context-dependent tumor suppressors or promoters. Given their central role in different cellular processes, sirtuins have attracted increasing research interest aimed at developing both activators and inhibitors. Indeed, sirtuin modulation may have therapeutic effects in many age-related diseases, including diabetes, cardiovascular and neurodegenerative disorders, and cancer. Moreover, isoform selective modulators may increase our knowledge of sirtuin biology and aid to develop better therapies. Through this review, we provide critical insights into sirtuin pharmacology and illustrate their enzymatic activities and biological functions. Furthermore, we outline the most relevant sirtuin modulators in terms of their modes of action, structure-activity relationships, pharmacological effects, and clinical applications.

The INSIGHT platform: Enhancing NAD(P)-dependent specificity prediction for co-factor specificity engineering

Enzyme specificity towards cofactors like NAD(P)H is crucial for applications in bioremediation and eco-friendly chemical synthesis. Despite their role in converting pollutants and creating sustainable products, predicting enzyme specificity faces challenges due to sparse data and inadequate models. To bridge this gap, we developed the cutting-edge INSIGHT platform to enhance the prediction of coenzyme specificity in NAD(P)-dependent enzymes. INSIGHT integrates extensive data from principal bioinformatics resources, concentrating on both NADH and NADPH specificities, and utilizes advanced protein language models to refine the predictions. This integration not only strengthens computational predictions but also meets the practical demands of high-throughput screening and optimization. Experimental validation confirms INSIGHT's effectiveness, boosting our ability to engineer enzymes for efficient, sustainable industrial and environmental processes. This work advances the practical use of computational tools in enzyme research, addressing industrial needs and offering scalable solutions for environmental challenges.

Thiamine (Vitamin B1) Promoted Organic Transformations: A Recent Updates.

Due to the growing significance of sustainable and environmentally friendly organic transformations, there has been increasing interest in utilizing vitamins as catalysts owing to their green nature, biocompatibility, and ease of preparation. Among these, Vitamin B1, also known as thiamine stands out for its nonflammable, water-soluble, inexpensive, and non-toxic characteristics. This review summarized recent developments on the catalytic application of Vitamin B1 in organic transformations, particularly in facilitating C-C and C-X (N, O, S) bond formations, thus demonstrating its efficacy in synthesizing complex molecules. Vitamin B1 exhibits versatility in these reactions, functioning as both an organocatalyst as well as a co-catalyst or ligand with other metal catalysts. The review also delves into the application of thiamine diphosphate-dependent enzymes as catalysts in organic reactions, drawing inspiration from natural enzymatic processes. Additionally, the mechanistic intricacies of thiamine-catalyzed reactions and the roles of co-catalysts or additives are thoroughly examined, providing insights into reaction pathways and facilitating informed catalyst design strategies.

Pharmacodynamic profiling in three patients with molybdenum cofactor deficiency type A reveals prolonged biological effects after withdrawal of cyclic pyranopterin monophosphate

Molybdenum cofactor deficiency type A has successfully been treated in a small number of children with daily intravenous administration of cyclic pyranopterin monophosphate. Pharmacodynamic data for this novel treatment have not been published and alternative dosing intervals have not been explored. We monitored pharmacodynamic biomarkers of sulfite oxidase and xanthine oxidoreductase activity in three patients with MoCD-A for a period of 2 to 9 months after discontinuation of cPMP substitution. We found that the clinical and metabolic effects were sustained for longer than expected, over 7 days at least. Our data implicate a biological half-life of the molybdenum cofactor dependent enzyme activities of approximately 3 days and suggest the possibility that less frequent than once daily dosing intervals could be a safe alternative to current practice.

Enhanced Late INa Induces Intracellular Ion Disturbances and Automatic Activity in the Guinea Pig Pulmonary Vein Cardiomyocytes.

The effects of enhanced late INa, a persistent component of the Na+ channel current, on the intracellular ion dynamics and the automaticity of the pulmonary vein cardiomyocytes were studied with fluorescent microscopy. Anemonia viridis toxin II (ATX- II), an enhancer of late INa, caused increases in the basal Na+ and Ca2+ concentrations, increases in the number of Ca2+ sparks and Ca2+ waves, and the generation of repetitive Ca2+ transients. These phenomena were inhibited by eleclazine, a blocker of the late INa; SEA0400, an inhibitor of the Na+/Ca2+ exchanger (NCX); H89, a protein kinase A (PKA) inhibitor; and KN-93, a Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor. These results suggest that enhancement of late INa in the pulmonary vein cardiomyocytes causes disturbance of the intracellular ion environment through activation of the NCX and Ca2+-dependent enzymes. Such mechanisms are probably involved in the ectopic electrical activity of the pulmonary vein myocardium.

Oxidative Stress in Cerebral Ischemia/Reperfusion Injury

Oxidative stress in cerebral ischemia/reperfusion injury (CIRI) involves reactive oxygen and nitrogen species (ROS and RNS). Despite efficient antioxidant pathways in the brain, hypoxia triggers the production of oxygen free radicals and downregulates ATP, which leads to oxidative stress. Sources of free radicals during CIRI include Ca<sup>2+</sup>-dependent enzymes, phospholipid degradation and mitochondrial enlargement. Upon reperfusion, the abrupt increase of oxygen triggers a massive radical production via enzymes like xantin oxidase (XO), phospholipase A2 (PLA2) and oxide synthases (OS). These enzymes play an essential role in neuronal damage by excitotoxicity, lipoperoxidation, nitrosylation, inflammation and programmed cell death (PCD). Endothelial nitric oxide synthase (eNOS) decreases as compared to neuronal nitric oxide synthase (nNOS). This is associated with neuronal damage, endothelial inflammation, apoptosis and oxidative stress. Strategies promoting activation of eNOS while inhibiting nNOS could offer neuroprotective benefits in CIRI. Understanding and targeting these pathways could mitigate brain damage in ischemia/reperfusion events. Clinically, tissue plasminogen activator (t-PA) has been shown to restore cerebral blood flow. However, serious side effects have been described, including hemorrhagic transformation. Different treatments are currently under investigation to avoid I/R injury. Baicalin has been reported as a potential agent that could improve t-PA adverse effects, which have to do with peroxynitrite synthesis and matrix metalloproteinase (MMP) expression. In this review, CIRI and interventions in oxidative stress are addressed. Special attention is paid to efficient antioxidant mechanisms in the brain and the production of free radicals, especially nNOS-derived nitric oxide (NO). The primary purpose is to describe accessible radical pathways with the activity of Ca<sup>2+</sup>-dependent oxidative enzymes, leading to membrane phospholipids and mitochondrial breakdown. <strong>Key</strong><strong>w</strong><strong>ords</strong>Oxidative stress; cerebral ischemia/reperfusion; nitric oxide; reactive oxygen species; nitric oxide synthase