Mitochondrial Malate Dehydrogenase Research Articles

Insights into the Cardioprotective Effects of Pyridoxine Treatment in Diabetic Rats: A Study on Cardiac Oxidative Stress, Cardiometabolic Status, and Cardiovascular Biomarkers.

The aims of this study were to examine the effects of pyridoxine administration on the activities of cardiac antioxidant stress enzymes superoxide dismutase (SOD) and catalase (CAT) and enzyme indicators of cardiometabolic status, lactate and malate dehydrogenase (LDH, MDH), as well as LDH and MDH isoforms' distribution in the cardiac tissue of healthy and diabetic Wistar male rats. Experimental animals were divided into five groups: C1-control (0.9% sodium chloride-NaCl-1 mL/kg, intraperitoneally (i.p.), 1 day); C2-second control (0.9% NaCl 1 mL/kg, i.p., 28 days); DM-diabetes mellitus (streptozotocin 100 mg/kg in 0.9% NaCl, i.p., 1 day); P-pyridoxine (7 mg/kg, i.p., 28 days); and DM + P-diabetes mellitus and pyridoxine (streptozotocin 100 mg/kg, i.p., 1 day and pyridoxine 7 mg/kg, i.p., 28 days). Pyridoxine treatment reduced CAT and MDH activity in diabetic rats. In diabetic rats, the administration of pyridoxine increased LDH1 and decreased LDH4 isoform activities, as well as decreased peroxisomal MDH and increased mitochondrial MDH activities. Our findings highlight the positive effects of pyridoxine administration on the complex interplay between oxidative stress, antioxidant enzymes, and metabolic changes in diabetic cardiomyopathy.

BomMDH1 regulates malate-mediated oxidative stress in tobacco BY-2 suspension cells

Programmed cell death (PCD) is a genetically regulated process of cell suicide essential for plant development. The ‘malate valve’ is a mechanism that ensures redox balance across different subcellular compartments. In broccoli, the BomMDH1 gene encodes malate dehydrogenase in mitochondria, a critical enzyme in the ‘malate circulation’ pathway. This study investigates the functional role of BomMDH1 in malate (MA)-induced apoptosis in bright yellow-2 (BY-2) suspension cells. Findings revealed that transgenic cells overexpressing BomMDH1 showed enhanced viability under MA-induced oxidative stress compared to wild-type (WT) cells. Overexpression of BomMDH1 also reduced levels of reactive oxygen species (ROS), hydrogen peroxide (H2O2), and malondialdehyde (MDA), while increasing the expression of antioxidant enzyme genes such as NtAPX, NtAOX1a, NtSOD, and NtMDHAR. Additionally, treatment with salicylhydroxamic acid (SHAM), a characteristic inhibitor of mitochondrial respiration, further improved the anti-apoptotic activity of BY-2 cells. Overall, these results highlighted the function of the BomMDH1 gene and the potential of SHAM treatment in mitigating oxidative stress in BY-2 suspension cells.

Phosphorylation of mammalian cytosolic and mitochondrial malate dehydrogenase: insights into regulation.

Malate dehydrogenase (MDH) is a key enzyme in mammalian metabolic pathways in cytosolic and mitochondrial compartments. Regulation of MDH through phosphorylation remains an underexplored area. In this review we consolidate evidence supporting the potential role of phosphorylation in modulating the function of mammalian MDH. Parallels are drawn with the phosphorylation of lactate dehydrogenase, a homologous enzyme, to reveal its regulatory significance and to suggest a similar regulatory strategy for MDH. Comprehensive mining of phosphorylation databases, provides substantial experimental (primarily mass spectrometry) evidence of MDH phosphorylation in mammalian cells. Experimentally identified phosphorylation sites are overlaid with MDH's functional domains, offering perspective on how these modifications could influence enzyme activity. Preliminary results are presented from phosphomimetic mutations (serine/threonine residues changed to aspartate) generated in recombinant MDH proteins serving as a proof of concept for the regulatory impact of phosphorylation. We also examine and highlight several approaches to probe the structural and cellular impact of phosphorylation. This review highlights the need to explore the dynamic nature of MDH phosphorylation and calls for identifying the responsible kinases and the physiological conditions underpinning this modification. The synthesis of current evidence and experimental data aims to provide insights for future research on understanding MDH regulation, offering new avenues for therapeutic interventions in metabolic disorders and cancer.

Mammalian target of differentiation-inducing factor-1 is mitochondrial malate dehydrogenase for activation of AMP-activated protein kinase and induction of mitochondrial fission

AimsDifferentiation-inducing factor-1 (DIF-1) is a polyketide produced by Dictyostelium discoideum that inhibits growth and migration, while promoting the differentiation of Dictyostelium stalk cells through unknown mechanisms. DIF-1 localizes in stalk mitochondria. In addition to its effect on Dictyostelium, DIF-1 also inhibits growth and migration, and induces mitochondrial fission followed by mitophagy in mammalian cells, at least in part by activating AMP-activated protein kinase (AMPK). In a previous study, we found that DIF-1 binds to mitochondrial malate dehydrogenase (MDH2) and inhibits its activity in HeLa cells. In the present study, we investigated whether MDH2 serves as a pharmacological target of DIF-1 in mammalian cells. Main methodsTo examine the enzymatic activity of MDH, mitochondrial morphology, and molecular mechanisms of DIF-1 action, we conducted an MDH reverse reaction assay, immunofluorescence staining, western blotting, and RNA interference using mammalian cells such as human umbilical vein endothelial cells, human cervical cancer cells, mouse endothelial cells, and mouse breast cancer cells. Key findingsDIF-1 inhibited mitochondrial but not cytoplasmic MDH activity. Similar to DIF-1, LW6, an authentic MDH2 inhibitor, induced phosphorylation of AMPK, resulting in the phosphorylation of acetyl-CoA carboxylase (ACC) and the dephosphorylation of p70 S6 kinase with approximately the same potency. DIF-1 and LW6 induced mitochondrial fission. Furthermore, MDH2 knockdown using siRNA reproduced the DIF-1 action on the AMPK signaling and mitochondrial morphology. Conversely, an AMPK inhibitor prevented DIF-1-induced mitochondrial fission. SignificanceWe propose that MDH2 is a mammalian target of DIF-1 for the activation of AMPK and induction of mitochondrial fission.

Insights into the Mechanism of Catalytic Activity of Plasmodium Parasite Malate-Quinone Oxidoreductase.

Plasmodium malate-quinone oxidoreductase (MQO) is a membrane flavoprotein catalyzing the oxidation of malate to oxaloacetate and the reduction of quinone to quinol. Recently, using a yeast expression system, we demonstrated that MQO, expressed in place of mitochondrial malate dehydrogenase (MDH), contributes to the TCA cycle and the electron transport chain in mitochondria, making MQO attractive as a promising drug target in Plasmodium malaria parasites, which lack mitochondrial MDH. However, there is little information on the structure of MQO and its catalytic mechanism, information that will be required to develop novel drugs. Here, we investigated the catalytic site of P. falciparum MQO (PfMQO) using our yeast expression system. We generated a model structure for PfMQO with the AI tool AlphaFold and used protein footprinting by acetylation with acetic anhydride to analyze the surface topology of the model, confirming the computational prediction to be reasonably accurate. Moreover, a putative catalytic site, which includes a possible flavin-binding site, was identified by this combination of protein footprinting and structural prediction model. This active site was analyzed by site-directed mutagenesis. By measuring enzyme activity and protein expression levels in the PfMQO mutants, we showed that several residues at the active site are essential for enzyme function. In addition, a single substitution mutation near the catalytic site resulted in enhanced sensitivity to ferulenol, an inhibitor of PfMQO that competes with malate for binding to the enzyme. This strongly supports the notion that the substrate binds to the proposed catalytic site. Then, the location of the catalytic site was demonstrated by structural comparison with a homologous enzyme. Finally, we used our results to propose a mechanism for the catalytic activity of MQO by reference to the mechanism of action of structurally or functionally homologous enzymes.

Cytosolic RNA binding of the mitochondrial TCA cycle enzyme malate dehydrogenase.

Several enzymes of intermediary metabolism have been identified to bind RNA in cells, with potential consequences for the bound RNAs and/or the enzyme. In this study, we investigate the RNA-binding activity of the mitochondrial enzyme malate dehydrogenase 2 (MDH2), which functions in the tricarboxylic acid (TCA) cycle and the malate-aspartate shuttle. We confirmed in cellulo RNA binding of MDH2 using orthogonal biochemical assays and performed enhanced cross-linking and immunoprecipitation (eCLIP) to identify the cellular RNAs associated with endogenous MDH2. Surprisingly, MDH2 preferentially binds cytosolic over mitochondrial RNAs, although the latter are abundant in the milieu of the mature protein. Subcellular fractionation followed by RNA-binding assays revealed that MDH2-RNA interactions occur predominantly outside of mitochondria. We also found that a cytosolically retained N-terminal deletion mutant of MDH2 is competent to bind RNA, indicating that mitochondrial targeting is dispensable for MDH2-RNA interactions. MDH2 RNA binding increased when cellular NAD+ levels (MDH2's cofactor) were pharmacologically diminished, suggesting that the metabolic state of cells affects RNA binding. Taken together, our data implicate an as yet unidentified function of MDH2-binding RNA in the cytosol.

Activity-based protein profiling technology reveals malate dehydrogenase as the target protein of cinnamaldehyde against Aspergillus niger

Cinnamaldehyde displays strong antifungal activity against fungi such as Aspergillus niger, but its precise molecular mechanisms of antifungal action remain inadequately understood. In this investigation, we applied chemoproteomics and bioinformatic analysis to unveil the target proteins of cinnamaldehyde in Aspergillus niger cells. Additionally, our study encompassed the examination of cinnamaldehyde's effects on cell membranes, mitochondrial malate dehydrogenase activity, and intracellular ATP levels in Aspergillus niger cells. Our findings suggest that malate dehydrogenase could potentially serve as an inhibitory target of cinnamaldehyde in Aspergillus niger cells. By disrupting the activity of malate dehydrogenase, cinnamaldehyde interferes with the mitochondrial tricarboxylic acid (TCA) cycle, leading to a significant decrease in intracellular ATP levels. Following treatment with cinnamaldehyde at a concentration of 1 MIC, the inhibition rate of MDH activity was 74.90 %, accompanied by an 84.5 % decrease in intracellular ATP content. Furthermore, cinnamaldehyde disrupts cell membrane integrity, resulting in the release of cellular contents and subsequent cell demise. This study endeavors to unveil the molecular-level antifungal mechanism of cinnamaldehyde via a chemoproteomics approach, thereby offering valuable insights for further development and utilization of cinnamaldehyde in preventing and mitigating food spoilage.

De Novo Synthesis of Chrysin in Saccharomyces cerevisiae.

Chrysin, a flavonoid, has been found to have been widely used in the health food field. But at present, chrysin production is hindered by the low availability of precursors and the lack of catalytic enzymes with high activity. Therefore, ZmPAL was initially screened to synthesize trans-cinnamic acid with high catalytic activity and specificity. To enhance the supply of precursors, the shikimic acid and chorismic acid pathway genes were overexpressed. Besides, the expression of the intracellular and mitochondrial carbon metabolism genes CIT, MAC1/3, CTP1, YHM2, RtME, and MDH was enhanced to increase the intracellular acetyl-CoA content. Chrysin was synthesized through a novel gene combination of ScCPR-EbFNSI-1 and PcFNSI. Finally, de novo synthesis of chrysin was achieved, reaching 41.9 mg/L, which is the highest reported concentration to date. In summary, we identified efficient enzymes for chrysin production and increased it by regulating acetyl-CoA metabolism in mitochondria and the cytoplasm, laying a foundation for future large-scale production.

In silico Evolutionary Insights into Prokaryotic and Eukaryotic Malate Dehydrogenases (MDH) Support the Archaebacterial Origin of Eukaryotes

Malate Dehydrogenase (MDH) stands as a pivotal enzyme crucial for cellular energy metabolism, orchestrating the conversion of malate to oxaloacetate in both prokaryotic and eukaryotic organisms. This study delves into the evolutionary trajectories of MDH1 (cytoplasmic) and MDH2 (mitochondrial), offering substantial evidence supporting the archaebacterial origin of eukaryotes. The dataset spans nine groups, encompassing human MDH1 and MDH2, mammalian MDH1 and MDH2, amphibian MDH2, arthropod MDH2, amoeba MDH1, archaea, and bacteria. Protein BLAST analysis revealed significant sequence homology in mammalian MDH1, particularly among primates, underlining a close evolutionary connection. Conversely, lower eukaryotes, including amoeba, arthropods, and amphibians, exhibited marked divergence from human MDH1 and MDH2. Phylogenetic analysis unveils distinct clusters for MDH1 and MDH2, accentuating significant genetic diversity between mitochondrial and cytoplasmic MDH enzymes. Prokaryotic MDH sequences cluster with human mitochondrial MDH2, while MDH1 forms a separate cluster. Staphylococcus MDH aligns with archaeal MDH, emphasizing the diversity within bacterial MDH evolution. Protein variability analysis indicates noteworthy divergence of human MDH1 from prokaryotic MDH, while MDH2 displays comparatively lower divergence. Pairwise evolutionary divergence analysis sheds light on complex relationships among MDH protein sequences. Human MDH1 shows close evolutionary ties to mammalian MDH1, whereas MDH2 exhibits a unique pattern, aligning closely with mammalian MDH2, arthropods, and amphibians. Furthermore, MDH2 demonstrates closer proximity to bacterial MDH, supporting a bacterial origin of mitochondrial MDH. In contrast, MDH1 displays less divergence to archaeal MDH than its bacterial counterpart, endorsing an archaeal origin for cytoplasmic MDH. In conclusion, this study provides compelling support for the archaebacterial origin of eukaryotes, suggesting a bacterial endosymbiont within an archaeal host that evolved into mitochondria. It contributes valuable insights into MDH evolution, unraveling the intricate relationships and unique adaptations shaping the evolutionary history of eukaryotic cells.
 Bangladesh J Microbiol, Volume 40, Number 1, June 2023, pp 15-24

Abstract 1317 Characterization of cytoplasmic, mitochondrial, and peroxisomal malate dehydrogenase via missense single nucleotide mutations and homologous recombination mutations in the budding yeast Saccharomyces cerevisiae

Abstract 1317 Characterization of cytoplasmic, mitochondrial, and peroxisomal malate dehydrogenase via missense single nucleotide mutations and homologous recombination mutations in the budding yeast Saccharomyces cerevisiae

Abstract 2011 The structural and functional effects of phosphorylation on human mitochondrial malate dehydrogenase

Abstract 2011 The structural and functional effects of phosphorylation on human mitochondrial malate dehydrogenase

Induced pluripotent stem cell-derived hepatocytes reveal TCA cycle disruption and the potential basis for triheptanoin treatment for malate dehydrogenase 2 deficiency

Mitochondrial malate dehydrogenase 2 (MDH2) is crucial to cellular energy generation through direct participation in the tricarboxylic acid (TCA) cycle and the malate aspartate shuttle (MAS). Inherited MDH2 deficiency is an ultra-rare metabolic disease caused by bi-allelic pathogenic variants in the MDH2 gene, resulting in early-onset encephalopathy, psychomotor delay, muscular hypotonia and frequent seizures. Currently, there is no cure for this devastating disease. We recently reported symptomatic improvement of a three-year-old girl with MDH2 deficiency following treatment with the triglyceride triheptanoin. Here, we aimed to better characterize this disease and improve our understanding of the potential utility of triheptanoin treatment. Using fibroblasts derived from this patient, we generated induced pluripotent stem cells (hiPSCs) and differentiated them into hepatocytes (hiPSC-Heps). Characterization of patient-derived hiPSCs and hiPSC-Heps revealed significantly reduced MDH2 protein expression. Untargeted proteotyping of hiPSC-Heps revealed global dysregulation of mitochondrial proteins, including upregulation of TCA cycle and fatty acid oxidation enzymes. Metabolomic profiling confirmed TCA cycle and MAS dysregulation, and demonstrated normalization of malate, fumarate and aspartate following treatment with the triheptanoin components glycerol and heptanoate. Taken together, our results provide the first patient-derived hiPSC-Hep-based model of MDH2 deficiency, confirm altered TCA cycle function, and provide further evidence for the implementation of triheptanoin therapy for this ultra-rare disease. SynopsisThis study reveals altered expression of mitochondrial pathways including the tricarboxylic acid cycle and changes in metabolite profiles in malate dehydrogenase 2 deficiency and provides the molecular basis for triheptanoin treatment in this ultra-rare disease.

Dictyostelium Differentiation-Inducing Factor 1 Promotes Glucose Uptake via Direct Inhibition of Mitochondrial Malate Dehydrogenase in Mouse 3T3-L1 Cells.

Differentiation-inducing factor 1 (DIF-1), found in Dictyostelium discoideum, has antiproliferative and glucose-uptake-promoting activities in mammalian cells. DIF-1 is a potential lead for the development of antitumor and/or antiobesity/antidiabetes drugs, but the mechanisms underlying its actions have not been fully elucidated. In this study, we searched for target molecules of DIF-1 that mediate the actions of DIF-1 in mammalian cells by identifying DIF-1-binding proteins in human cervical cancer HeLa cells and mouse 3T3-L1 fibroblast cells using affinity chromatography and liquid chromatography-tandem mass spectrometry and found mitochondrial malate dehydrogenase (MDH2) to be a DIF-1-binding protein in both cell lines. Since DIF-1 has been shown to directly inhibit MDH2 activity, we compared the effects of DIF-1 and the MDH2 inhibitor LW6 on the growth of HeLa and 3T3-L1 cells and on glucose uptake in confluent 3T3-L1 cells in vitro. In both HeLa and 3T3-L1 cells, DIF-1 at 10-40 μM dose-dependently suppressed growth, whereas LW6 at 20 μM, but not at 2-10 μM, significantly suppressed growth in these cells. In confluent 3T3-L1 cells, DIF-1 at 10-40 μM significantly promoted glucose uptake, with the strongest effect at 20 μM DIF-1, whereas LW6 at 2-20 μM significantly promoted glucose uptake, with the strongest effect at 10 μM LW6. Western blot analyses showed that LW6 (10 μM) and DIF-1 (20 μM) phosphorylated and, thus, activated AMP kinase in 3T3-L1 cells. Our results suggest that MDH2 inhibition can suppress cell growth and promote glucose uptake in the cells, but appears to promote glucose uptake more strongly than it suppresses cell growth. Thus, DIF-1 may promote glucose uptake, at least in part, via direct inhibition of MDH2 and a subsequent activation of AMP kinase in 3T3-L1 cells.

SUMOylation Modulates Reactive Oxygen Species (ROS) Levels and Acts as a Protective Mechanism in the Type 2 Model of Diabetic Peripheral Neuropathy.

Diabetic peripheral neuropathy (DPN) is the prevalent type of peripheral neuropathy; it primarily impacts extremity nerves. Its multifaceted nature makes the molecular mechanisms of diabetic neuropathy intricate and incompletely elucidated. Several types of post-translational modifications (PTMs) have been implicated in the development and progression of DPN, including phosphorylation, glycation, acetylation and SUMOylation. SUMOylation involves the covalent attachment of small ubiquitin-like modifier (SUMO) proteins to target proteins, and it plays a role in various cellular processes, including protein localization, stability, and function. While the specific relationship between high blood glucose and SUMOylation is not extensively studied, recent evidence implies its involvement in the development of DPN in type 1 diabetes. In this study, we investigated the impact of SUMOylation on the onset and progression of DPN in a type 2 diabetes model using genetically modified mutant mice lacking SUMOylation, specifically in peripheral sensory neurons (SNS-Ubc9-/-). Behavioural measurement for evoked pain, morphological analyses of nerve fibre loss in the epidermis, measurement of reactive oxygen species (ROS) levels, and antioxidant molecules were analysed over several months in SUMOylation-deficient and control mice. Our longitudinal analysis at 30 weeks post-high-fat diet revealed that SNS-Ubc9-/- mice exhibited earlier and more pronounced thermal and mechanical sensation loss and accelerated intraepidermal nerve fibre loss compared to control mice. Mechanistically, these changes are associated with increased levels of ROS both in sensory neuronal soma and in peripheral axonal nerve endings in SNS-Ubc9-/- mice. In addition, we observed compromised detoxifying potential, impaired respiratory chain complexes, and reduced levels of protective lipids in sensory neurons upon deletion of SUMOylation in diabetic mice. Importantly, we also identified mitochondrial malate dehydrogenase (MDH2) as a SUMOylation target, the activity of which is negatively regulated by SUMOylation. Our results indicate that SUMOylation is an essential neuroprotective mechanism in sensory neurons in type 2 diabetes, the deletion of which causes oxidative stress and an impaired respiratory chain, resulting in energy depletion and subsequent damage to sensory neurons.

Effects of OsAOX1a Deficiency on Mitochondrial Metabolism at Critical Node of Seed Viability in Rice.

Mitochondrial alternative oxidase 1a (AOX1a) plays an extremely important role in the critical node of seed viability during storage. However, the regulatory mechanism is still poorly understood. The aim of this study was to identify the regulatory mechanisms by comparing OsAOX1a-RNAi and wild-type (WT) rice seed during artificial aging treatment. Weight gain and time for the seed germination percentage decreased to 50% (P50) in OsAOX1a-RNAi rice seed, indicating possible impairment in seed development and storability. Compared to WT seeds at 100%, 90%, 80%, and 70% germination, the NADH- and succinate-dependent O2 consumption, the activity of mitochondrial malate dehydrogenase, and ATP contents all decreased in the OsAOX1a-RNAi seeds, indicating that mitochondrial status in the OsAOX1a-RNAi seeds after imbibition was weaker than in the WT seeds. In addition, the reduction in the abundance of Complex I subunits showed that the capacity of the mitochondrial electron transfer chain was significantly inhibited in the OsAOX1a-RNAi seeds at the critical node of seed viability. The results indicate that ATP production was impaired in the OsAOX1a-RNAi seeds during aging. Therefore, we conclude that mitochondrial metabolism and alternative pathways were severely inhibited in the OsAOX1a-RNAi seeds at critical node of viability, which could accelerate the collapse of seed viability. The precise regulatory mechanism of the alternative pathway at the critical node of viability needs to be further analyzed. This finding might provide the basis for developing monitoring and warning indicators when seed viability declines to the critical node during storage.

Plasmodium Parasite Malate-Quinone Oxidoreductase Functionally Complements a Yeast Deletion Mutant of Mitochondrial Malate Dehydrogenase.

The emergence of drug-resistant variants of malaria-causing Plasmodium parasites is a life-threatening problem worldwide. Investigation of the physiological function of individual parasite proteins is a prerequisite for a deeper understanding of the metabolic pathways required for parasite survival and therefore a requirement for the development of novel antimalarials. A Plasmodium membrane protein, malate-quinone oxidoreductase (MQO), is thought to contribute to the tricarboxylic acid (TCA) cycle and the electron transport chain (ETC) and is an antimalarial drug target. However, there is little information on its expression and function. Here, we investigated the function of Plasmodium falciparum MQO (PfMQO) in mitochondria using a yeast heterologous expression system. Using a yeast deletion mutant of mitochondrial malate dehydrogenase (MDH1), which is expected to be functionally similar to MQO, as a background strain, we successfully constructed PfMQO-expressing yeast. We confirmed that expression of PfMQO complemented the growth defect of the MDH1 deletion, indicating that PfMQO can adopt the metabolic role of MDH1 in energy transduction for growth in the recombinant yeast. Analysis of cell fractions confirmed that PfMQO was expressed and enriched in yeast mitochondria. By measuring MQO activity, we also confirmed that PfMQO expressed in yeast mitochondria was active. Measurement of oxygen consumption rates showed that mitochondrial respiration was driven by the TCA cycle through PfMQO. In addition, we found that MQO activity was enhanced when intact mitochondria were sonicated, indicating that the malate binding site of PfMQO is located facing the mitochondrial matrix. IMPORTANCE We constructed a model organism to study the physiological role and function of P. falciparum malate-quinone oxidoreductase (PfMQO) in a yeast expression system. PfMQO is actively expressed in yeast mitochondria and functions in place of yeast mitochondrial malate dehydrogenase, which catalyzes the oxidation of malate to oxaloacetate in the TCA cycle. The catalytic site for the oxidation of malate in PfMQO, which is a membrane-bound protein, faces into the mitochondrial matrix, not the mitochondrial inner membrane space. Our findings clearly show that PfMQO is a TCA cycle enzyme and is coupled with the ETC via ubiquinone reduction.

Clozapine-induced Myocarditis: Pathophysiologic Mechanisms and Implications for Therapeutic Approaches.

Clozapine, a superior treatment for treatment-resistant schizophrenia can cause potentially life-threatening myocarditis and dilated cardiomyopathy. While the occurrence of this condition is well known, its molecular mechanisms are unclear and may be multifactorial. Putative mechanisms warrant an in-depth review not only from the perspective of toxicity but also for understanding the molecular mechanisms of the adverse cardiac effects of clozapine and the development of novel therapeutic approaches. Clozapine-induced cardiac toxicity encompasses a diverse set of pathways, including (i) immune modulation and proinflammatory processes encompassing an IgEmediated (type I hypersensitivity) response and perhaps a cytokine release syndrome (ii) catecholaminergic activation (iii) induction of free radicals and oxidative stress (iv) activation of cardiomyocyte cell death pathways, including apoptosis, ischemia through impairment in coronary blood flow via changes in endothelial production of NO and vasoconstriction induced by norepinephrine as well as other factors released from cardiac mast cells. (v) In addition, an extensive examination of the effects of clozapine on non-cardiac cellular proteins demonstrates that clozapine can impair enzymes involved in cellular metabolism, such as pyruvate kinase, mitochondrial malate dehydrogenase, and other proteins, including α-enolase, triosephosphate isomerase and cofilin, which might explain clozapine-induced reductions in myocardial energy generation for cell viability as well as contractile function. Pharmacologic antagonism of these cellular protein effects may lead to the development of strategies to antagonize the cardiac damage induced by clozapine.

The neuroprotective effect of betanin nanoparticles on brain ischemia–reperfusion injury

BackgroundBetanin is an antioxidant and anti-inflammatory compound that has attracted the attention of researchers in a variety of diseases in recent years. Therefore, In the present study the effect of pretreatment of betanin nanoparticles (NB) in cerebral ischemia rats was investigated. MethodsOral administration of NB started five days before bilateral common carotid artery occlusion (BCCAO) induction and continued for three days thereafter in rats. The neurological disorder scores (NDS), brain swelling and edema were evaluated. After preparing brain homogenate, the intensity of oxidative stress, the activities of antioxidant enzymes, GSH content, the activities of mitochondrial enzymes SDH, MDH and LDH were studied. Also, the expressions of IL-1β, IL-6, TNF-α cytokines were measured. The expression levels of NF-ĸB and pSTAT3 genes and proteins and ERK and TFEB proteins were also studied. ResultsNB showed strong anti-inflammatory and antioxidant effects in cerebral I/R rats and improved mitochondrial and antioxidant enzymes activities. Decreases in NDS, swelling and edema of the brain were also observed in the cerebral I/R rats receiving NB. Also, administration of NB decreased the expressions of inflammatory cytokines and upregulated the expressions of NF-KB and pSTAT3 genes and proteins and overexpressed ERK and TEFB proteins. ConclusionNB has neuroprotective effects in cerebral I/R condition due to its antioxidant and anti-inflammation properties, which can be considered as a suitable treatment option in stroke conditions.

Antifungal mechanisms of volatile organic compounds produced by Pseudomonas fluorescens ZX as biological fumigants against Botrytis cinerea

To explore the antifungal mechanisms of volatile organic compounds (VOCs) produced by Pseudomonas fluorescens ZX against Botrytis cinerea, biochemical analyses and transcriptomic techniques were employed in this work. The results showed that P. fluorescens ZX-producing VOCs can increase the cell membrane permeability of B. cinerea and disrupt cell membrane integrity, resulting in leakage of the pathogen’s cellular contents, inhibition of ergosterol biosynthesis (about 76%), and an increase in malondialdehyde (MDA) content. Additionally, for B. cinerea respiration, P. fluorescens ZX-producing VOCs (1 × 109 CFU /mL) significantly inhibited the activities of ATPase (55.7%), malate dehydrogenase (MDH) (33.1%), and succinate dehydrogenase (SDH) (57.9%), seriously interfering with energy metabolism and causing accumulation of reactive oxygen species (ROS). Furthermore, transcriptome analysis of B. cinerea following exposure to VOCs revealed 4590 differentially expressed genes (DEGs) (1388 upregulated, 3202 downregulated). Through GO analysis, these DEGs were determined to be enriched in intrinsic components of membrane, integral components of membrane, and membrane parts, while KEGG analysis indicated that they were enriched in many amino acid metabolism pathways. Significantly, the DEGs related to ergosterol biosynthesis, ATPase, mitochondrial respiratory chain, malate dehydrogenase, and cell membrane showed down-regulation, corroborating the biochemical analyses. Taken together, these results suggest that the antifungal activity of P. fluorescens ZX-producing VOCs against B. cinerea occurs primary mechanisms: causing significant damage to the cell membrane, negatively affecting respiration, and interfering with amino acid metabolism.

Agomelatine, Ketamine and Vortioxetine Attenuate Energy Cell Metabolism-In Vitro Study.

This determination of the mitochondrial effect of pharmacologically different antidepressants (agomelatine, ketamine and vortioxetine) was evaluated and quantified in vitro in pig brain-isolated mitochondria. We measured the activity of mitochondrial complexes, citrate synthase, malate dehydrogenase and monoamine oxidase, and the mitochondrial respiratory rate. Total hydrogen peroxide production and ATP production were assayed. The most potent inhibitor of all mitochondrial complexes and complex I-linked respiration was vortioxetine. Agomelatine and ketamine inhibited only complex IV activity. None of the drugs affected complex II-linked respiration, citrate synthase or malate dehydrogenase activity. Hydrogen peroxide production was mildly increased by agomelatine, which might contribute to increased oxidative damage and adverse effects at high drug concentrations. Vortioxetine significantly reduced hydrogen peroxide concentrations, which might suggest antioxidant mechanism activation. All tested antidepressants were partial MAO-A inhibitors, which might contribute to their antidepressant effect. We observed vortioxetine-induced MAO-B inhibition, which might be linked to decreased hydrogen peroxide formation and contribute to its procognitive and neuroprotective effects. Mitochondrial dysfunction could be linked to the adverse effects of vortioxetine, as vortioxetine is the most potent inhibitor of mitochondrial complexes and complex I-linked respiration. Clarifying the molecular interaction between drugs and mitochondria is important to fully understand their mechanism of action and the connection between their mechanisms and their therapeutic and/or adverse effects.