Reactive Oxygen Species In Cells Research Articles

Antioxidant activity of sulfated Porphyra yezoensis polysaccharides and their regulating effect on calcium oxalate crystal growth

The nucleation, growth and aggregation of calcium oxalate (CaOx) crystals and the oxidative damage of renal tubular epithelial cells are the key factors to induce kidney stones. In this study, degraded Porphyra yezoensis polysaccharide (PYP0) with 14.14% sulfate group (–OSO3−) content was modified via the sulfur trioxide–pyridine method to obtain three kinds of sulfated P. yezoensis polysaccharides (PYPs), namely, PYPS1, PYPS2, and PYPS3, with –OSO3− group contents of 17.11%, 20.28%, and 27.14% respectively. Fourier transform infrared spectroscopy, 1H NMR, and 13C NMR analyses showed that the –OSO3− groups replaced the hydroxyl groups at the C2, C4, and C6 positions on (1 → 3)-linked β-D-galactose, the basic structural skeleton unit of PYP0. The antioxidant activity of the PYPSs increased after sulfation, and their scavenging capacity for OH and DPPH free radicals was enhanced with the increase in their –OSO3− group content. Calcium oxalate (CaOx) crystal growth experiments showed that sulfated PYPs promoted the conversion of the thermodynamically stable and sharp CaOx monohydrate (COM) crystals into the thermodynamically unstable and round CaOx dihydrate crystals. With the increase in the –OSO3− group content of the polysaccharides, the concentration of soluble Ca2+ ions in the supernatant increased and the amount of CaOx precipitate decreased. PYPs were nontoxic to human kidney proximal tubular epithelial cells (HK-2) and could protect HK-2 from oxidative damage caused by nano-COM and reduce the level of reactive oxygen species in cells. PYPS3, which had the highest degree of sulfation, had the best protective capability. The results of this work showed that sulfation improved the biological activity of PYPs. This study could provide inspiration for the development of new drugs for the prevention and treatment of kidney stones.

Multifunctional Tannic Acid-Alendronate Nanocomplexes with Antioxidant, Anti-Inflammatory, and Osteogenic Potency.

In the current study, we fabricated tannic acid-alendronate (TA-ALN) nanocomplexes (NPXs) via self-assembly. These TA-ALNs were characterized by dynamic light scattering, zeta potential, transmission electron microscopy, and FT-IR spectroscopy. The TA-ALNs were evaluated for antioxidant, anti-inflammatory, and osteogenesis-accelerating abilities in osteoblast-like cells (MC3T3-E1 cells). All TA-ALNs displayed nano-sized beads that were circular in form. Treatment with TA-ALN (1:0.1) efficiently removed reactive oxygen species in cells and protected osteoblast-like cells from toxic hydrogen peroxide conditions. Moreover, TA-ALN (1:0.1) could markedly decrease the mRNA levels of pro-inflammatory mediators in lipopolysaccharide-stimulated cells. Furthermore, cells treated with TA-ALN (1:1) exhibited not only significantly greater alkaline phosphatase activity and calcium collection, but also outstandingly higher mRNA levels of osteogenesis-related elements such as collagen type I and osteocalcin. These outcomes indicate that the prepared TA-ALNs are excellent for antioxidant, anti-inflammatory, and osteogenic acceleration. Accordingly, TA-ALN can be used latently for bone renovation and regeneration in people with bone fractures, diseases, or disorders.

Ecotoxicological effects of micronized car tire wear particles and their heavy metals on the earthworm (Eisenia fetida) in soil

As one of the most common and persistent emerging human-made pollutants, microplastics (MPs) have become a global environmental problem. Although many studies have demonstrated the impact of MPs on aquatic organisms, the potential effects on terrestrial fauna are relatively unknown, even though soil is a major sink for plastic pollution. In this study, earthworms (Eisenia fetida) were exposed to MPs from tires (TMPs) in artificial soil at different TMP concentrations (0, 1, 5, 10, and 20%, w/w), at different TMP size fractions (2 mm–350 μm; 350 μm–50 μm; 50 μm–25 μm; and <25 μm), and time periods (14 and 28 days). The purpose was to evaluate the impact of TMPs on the bioaccumulation of heavy metals (Zn, Cd, and Pb) and levels of oxidative stress in the earthworms. Fluorescence imaging of TMPs stained with Nile red dye clearly showed that the worms had ingested the TMPs, with smaller particles preferentially consumed. The ingested TMPs had altered the surface morphology, degradation that increased surface area and uptake of heavy metals into the organisms. Earthworms exposed to TMPs at the higher concentrations showed clear signs of oxidative stress, with significantly (p < 0.05) increased catalase and peroxidase activity and lipid peroxidation levels, and reduced activity of superoxide dismutase and glutathione S-transferase. SOD, POD, and CAT are important cell protection enzymes that prevent excessive levels of reactive oxygen species in cells. MDA represents the total scavenging ability of free radicals in the tissue. SOD decreases and MDA accumulates rapidly. GST has a detoxification effect. Overall, this study adds to the growing body of evidence that TMPs are harmful to certain terrestrial organisms.

EkFLS overexpression promotes flavonoid accumulation and abiotic stress tolerance in plant.

Flavonoids with great medicinal value play an important role in plant individual growth and stress resistance. Flavonol synthetase (FLS) is one of the key enzymes to synthesize flavonoids. However, the role of the FLS gene in flavonoid accumulation and tolerance to abiotic stresses, as well as its mechanism has not yet been investigated systematically in plants. The aim of this research is to evaluate the effect of FLS overexpression on the accumulation of active ingredients and stress resistance in Euphorbia kansui Liou. The results showed that when the EkFLS gene was overexpressed in Arabidopsis thaliana, the accumulation of flavonoids was improved. In addition, when the wild-type and EkFLS overexpressed Arabidopsis plants were treated with ABA and MeJA, compared with WT Arabidopsis, EkFLS overexpressed Arabidopsis promoted stomatal aperture to influence photosynthesis of the plants, which in turn can promote stress resistance. Meanwhile, under MeJA, NaCl, and PEG treatment, EkFLS overexpressed in Arabidopsis induced higher accumulation of flavonoids, which significantly enhanced peroxidase (POD) and superoxide dismutase (SOD) activities that can scavenge reactive oxygen species in cells to protect the plant. These results indicated that EkFLS overexpression is strongly correlated to the increase of flavonoid synthesis and therefore the tolerance to abiotic stresses in plants, providing a theoretical basis for further improving the quality of medicinal plants and their resistance to abiotic stresses simultaneously.

Are mitochondria the main contributor of reactive oxygen species in cells?

Physiologists often assume that mitochondria are the main producers of reactive oxygen species (ROS) in cells. Consequently, in biomedicine, mitochondria are considered as important targets for therapeutic treatments, and in evolutionary biology, they are considered as mediators of life-history tradeoffs. Surprisingly, data supporting such an assumption are lacking, at least partially due to the technical difficulties in accurately measuring the level of ROS produced by different subcellular compartments in intact cells. In this Commentary, we first review three potential reasons underlying the misassumption of mitochondrial dominance in the production of cellular ROS. We then introduce some other major sites/enzymes responsible for cellular ROS production. With the use of a recently developed cell-based assay, we further discuss the contribution of mitochondria to the total rate of ROS release in cell lines and primary cells of different species. In these cells, the contribution of mitochondria varies between cell types but mitochondria are never the main source of cellular ROS. This indicates that although mitochondria are one of the significant sources of cellular ROS, they are not necessarily the main contributor under normal conditions. Intriguingly, similar findings were also observed in cells under a variety of stressors, life-history strategies and pathological stages, in which the rates of cellular ROS production were significantly enhanced. Finally, we make recommendations for designing future studies. We hope this paper will encourage investigators to carefully consider non-mitochondrial sources of cellular ROS in their study systems or models.

Phyto-Courier, a Silicon Particle-Based Nano-biostimulant: Evidence from Cannabis sativa Exposed to Salinity.

Global warming and sea level rise are serious threats to agriculture. The negative effects caused by severe salinity include discoloration and reduced surface of the leaves, as well as wilting due to an impaired uptake of water from the soil by roots. Nanotechnology is emerging as a valuable ally in agriculture: several studies have indeed already proven the role of silicon nanoparticles in ameliorating the conditions of plants subjected to (a) biotic stressors. Here, we introduce the concept of phyto-courier: hydrolyzable nanoparticles of porous silicon, stabilized with the nonreducing saccharide trehalose and containing different combinations of lipids and/or amino acids, were used as vehicle for the delivery of the bioactive compound quercetin to the leaves of salt-stressed hemp (Cannabis sativa L., Santhica 27). Hemp was used as a representative model of an economically important crop with multiple uses. Quercetin is an antioxidant known to scavenge reactive oxygen species in cells. Four different silicon-based formulations were administered via spraying in order to investigate their ability to improve the plant's stress response, thereby acting as nano-biostimulants. We show that two formulations proved to be effective at decreasing stress symptoms by modulating the amount of soluble sugars and the expression of genes that are markers of stress-response in hemp. The study proves the suitability of the phyto-courier technology for agricultural applications aimed at crop protection.

Ferroptosis Inducers Are a Novel Therapeutic Approach for Advanced Prostate Cancer.

Ferroptosis is a type of programmed cell death induced by the accumulation of lipid peroxidation and lipid reactive oxygen species in cells. It has been recently demonstrated that cancer cells are vulnerable to ferroptosis inducers (FIN). However, the therapeutic potential of FINs in prostate cancer in preclinical settings has not been explored. In this study, we demonstrate that mediators of ferroptosis, solute carrier family 7 member 11, SLC3A2, and glutathione peroxidase, are expressed in treatment-resistant prostate cancer. We further demonstrate that treatment-resistant prostate cancer cells are sensitive to two FINs, erastin and RSL3. Treatment with erastin and RSL3 led to a significant decrease in prostate cancer cell growth and migration in vitro and significantly delayed the tumor growth of treatment-resistant prostate cancer in vivo, with no measurable side effects. Combination of erastin or RSL3 with standard-of-care second-generation antiandrogens for advanced prostate cancer halted prostate cancer cell growth and migration in vitro and tumor growth in vivo. These results demonstrate the potential of erastin or RSL3 independently and in combination with standard-of-care second-generation antiandrogens as novel therapeutic strategies for advanced prostate cancer. SIGNIFICANCE: These findings reveal that induction of ferroptosis is a new therapeutic strategy for advanced prostate cancer as a monotherapy and in combination with second-generation antiandrogens.

Dynamic in vivo protein carbonyl biosensor for measuring oxidative stress

AbstractOxidative stress is intimately linked to cellular energy balance and occurs when there is an imbalance between production and accumulation of reactive oxygen species in cells and tissues and the ability of a biological system to keep in a redox steady state. While protein carbonyls are an easily detectable marker of oxygen metabolism and oxidative stress, all methods to date have required significant sample preparation and do not provide the ability to measure dynamic changes. To overcome this problem, we develop the first method for measuring in vivo concentration changes of protein carbonyls using a minimally invasive optical fibre biosensor. We use this biosensor to measure redox balance and oxidative stress within living systems by measuring dynamic in vivo concentration changes of protein carbonyls.

Gold/Silver Hybrid Nanoparticles with Enduring Inhibition of Coronavirus Multiplication through Multisite Mechanisms.

As a large enveloped RNA virus, coronavirus is of considerable medical and veterinary significance, and anticoronavirus treatment is challenging due to its biodiversity and rapid variability. In this study, Au@Ag nanorods (Au@AgNRs) were successfully synthesized by coating AuNRs with silver and were shown for the first time to have activity against the replication of porcine epidemic diarrhea virus (PEDV). Viral titer analysis demonstrated that Au@AgNRs could inhibit PEDV infection by 4 orders of magnitude at 12 h post-infection, which was verified by viral protein expression analysis. The potential mechanism of action showed that Au@AgNRs could inhibit the entry of PEDV and decrease the mitochondrial membrane potential and caspase-3 activity. Additionally, we demonstrated that a large amount of virus proliferation can cause the generation of reactive oxygen species in cells, and the released Ag+ and exposed AuNRs by Au@AgNRs after the stimulation of reactive oxygen species has superior antiviral activity to ensure long-term inhibition of the PEDV replication cycle. The integrated results support that Au@AgNRs can serve as a potential therapeutic strategy to prevent the replication of coronavirus.

A pyridine-Si-rhodamine-based near-infrared fluorescent probe for visualizing reactive oxygen species in living cells.

A lysosomal-targeted near infrared (NIR) fluorescent probe for reactive oxygen species (ROS) was developed with highly sensitive ability. The different responding activity toward H2O2, OH, and HClO were investigated. Meanwhile, the probe has been successfully applied in detecting and imaging reactive oxygen species both in cells and in vivo.

Pro-Oxidant Activity of an ALS-Linked SOD1 Mutant in Zn-Deficient Form.

Cu, Zn superoxide dismutase (SOD1) is a representative antioxidant enzyme that catalyzes dismutation of reactive oxygen species in cells. However, (E,E)-SOD1 mutants in which both copper and zinc ions were deleted exhibit pro-oxidant activity, contrary to their antioxidant nature, at physiological temperatures, following denaturation and subsequent recombination of Cu2+. This oxidative property is likely related to the pathogenesis of amyotrophic lateral sclerosis (ALS); however, the mechanism by which Cu2+ re-binds to the denatured (E,E)-SOD1 has not been elucidated, since the concentration of free copper ions in cells is almost zero. In this study, we prepared the (Cu,E) form in which only a zinc ion was deleted using ALS-linked mutant H43R (His43→Arg) and found that (Cu,E)-H43R showed an increase in the pro-oxidant activity even at physiological temperature. The increase in the pro-oxidant activity of (Cu,E)-H43R was also observed in solution mimicking intracellular environment and at high temperature. These results suggest that the zinc-deficient (Cu,E) form can contribute to oxidative stress in cells, and that the formation of (E,E)-SOD1 together with the subsequent Cu2+ rebinding is not necessary for the acquisition of the pro-oxidant activity.

Single-molecule enzymatic reaction dynamics and mechanisms of GPX3 and TRXh9 from Arabidopsis thaliana.

Glutathione peroxidases (GPXs) regulate the levels of reactive oxygen species in cells and tissues. During the redox cycling, the plant GPX is regenerated by thioredoxins (TRXs) as reductant rather than glutathione as the electron donor. However, the direct experimental observation on the interaction dynamics between GPXs and TRXs has not been reported, and the redox mechanism is unclear. In this work, the protein interactions between oxidized AtGPX3 and reduced AtTRXh9 have been studied using single-molecule fluorescence resonance energy transfer (smFRET). The obtained results indicate there are four processes in these two protein interaction, including biological recognition, binding, intermediate and unbinding state. Two enzymatic reaction intermediate states have been identified in the dissociation of AtGPX3-AtTRXh9 complex from binding to unbinding state, suggesting two types of interaction pathways and intermediate complexes. In particular, the dynamical study reveals that the redox reaction between oxidized AtGPX3 and reduced AtTRXh9 is realized through the forming and breaking of disulfide bonds via the active sites of Cys4 and Cys57 in AtTRXh9. These findings are of significant for deep understanding the redox reaction and mechanism between GPXs and TRXs enzymes, and studying other protein dynamics at single-molecule level.

Islamic Fasting during Ramadan will Negatively Impact on Pandemic of COVID-19 (SARS-CoV-2)

The aim of this review is to prove Islamic Fasting during COVID-19 pandemic has negative effect on the spreading of the virus. Basically Water makes up about 60% of one’s body weight. Fasting in Islamic religion is to stop drinking and eating any kinds of food/drinks (Water). even a little amount is forbidden during this month (Ramadan) and the duration is almost 29-30 days. In this review, we have mentioned the impact of fasting in causing Organ/Cellular inflammation, dehydration, dampen cellular/body metabolism which reduces producing enough ATP by mitochondrion through citric acid cycle (CAC) which causes body and organ weakness, dizziness, tiredness and reduction in immune system functioning of the body and increasing the amounts of Reactive Oxygen Species in cells that increasing the possibility of contamination the fasted individual to several important diseases. Infectious diseases including microbial/viral diseases, high abnormal heart rate as a result of falling blood volume which put high pressure on heart and worsen heart disease. The humidity and temperature of the environment in spring time is also help spreading viruses including SARS-CoV-2. Based on evidences in this article, Islamic Fasting during the pandemic of COVID-19 will increase the rate of contaminated people.

Islamic Fasting During Ramadan will Negatively Impact on Pandemic of Covid-19 (Sars-Cov-2).

Abstract The aim of this review is to prove Islamic Fasting during COVID-19 pandemic has negative effect on the spreading of the virus. Basically Water makes up about 60% of one’s body weight. Fasting in Islamic religion is to stop drinking and eating any kinds of food/drinks (Water). even a little amount is forbidden during this month (Ramadan) and the duration is almost 29-30 days. In this review, we have mentioned the impact of fasting in causing Organ/Cellular inflammation, dehydration, dampen cellular/body metabolism which reduces producing enough ATP by mitochondrion through citric acid cycle (CAC) which causes body and organ weakness, dizziness, tiredness and reduction in immune system functioning of the body and increasing the amounts of Reactive Oxygen Species in cells that increasing the possibility of contamination the fasted individual to several important diseases. Infectious diseases including microbial/viral diseases, high abnormal heart rate as a result of falling blood volume which put high pressure on heart and worsen heart disease. The humidity and temperature of the environment in spring time is also help spreading viruses including SARS-CoV-2. Based on evidences in this article, Islamic Fasting during the pandemic of COVID-19 will increase the rate of contaminated people.

Enhancing Enzyme-like Activities of Prussian Blue Analog Nanocages by Molybdenum Doping: Toward Cytoprotecting and Online Optical Hydrogen Sulfide Monitoring.

Artificial nanozymes have been designed to solve the problems of high cost and poor stability involving natural enzymes in analytical applications. Nevertheless, the catalytic efficiency of the nanozyme still needs to be improved so that it can meet the stability and sensitivity requirements of continuous biological detection. We presented an effective tailoring strategy to enhance the enzyme-like activities of Prussian-blue-analog-based nanozymes. Molybdenum-polysulfide-deposited nickel-iron bimetal Prussian-blue-analog-based hollow nanocages (Nanocages) with peroxidase-, catalase-, and laccase-mimicking activities were synthesized. The doping of molybdenum successfully tailored the size, morphology, composition, and complex structure of the Nanocage, and the peroxidase- and laccase-mimicking activities of the Nanocage nanozyme were enhanced by over 37 and 27 times, respectively, compared with pristine Prussian blue analogs. Moreover, in environments of harsh pH, high temperature, and high salt concentration, Nanocages exhibited much higher stability than natural enzymes. The peroxidase- and catalase-mimicking activities were applied to eliminate reactive oxygen species in cells, whereas the laccase-like activity of Nanocages was integrated with an online sensing platform for in vivo and continuous optical hydrogen sulfide monitoring in the brains of living rats. Our findings may provide possibilities for advancing the design strategy of highly active nanozymes as well as nanozyme-based in vivo detection methods and will offer unique opportunities for their involvement in bioanalytical chemistry.

Exposure to Maghemite Nanoparticles Induces Epigenetic Alterations in Human Submandibular Gland Cells.

Even though nanotechnology has revolutionized the biomedical research, a plethora of studies debate the nanoparticles safety. In order to contribute to these studies, we evaluated the cytotoxic and epigenetic effects of maghemite nanoparticles covered with citric acid on human submandibular gland cells. Objective: This work objective was to evaluate the cytotoxic effects and epigenetic alterations induced in human salivary gland cells after treatment with maghemite nanoparticles covered with citric acid. Methods: For that, human submandibular gland cells were cultured and treated with nanoparticles for 24 or 48 hours. To assess cytotoxicity we used lactate dehydrogenase, a general oxidative stress indicator assay and microscopy. Epigenetic status was detected by colorimetric assays and the results were confirmed by quantitative polymerase chain reaction. Results: No cytotoxic effects were detected on cells exposed to up 3.0 mgFemL-1 for 48 hours, although cytoplasmic vacuoles formation was detected by light microscopy analyses. An increased generation of reactive oxygen species in cells exposed to nanoparticles was evidenced and iron clusters accumulated in the cytoplasm of treated cells. Global DNA methylation and histones H3 and H4 acetylation were also altered in response to nanoparticles exposure, thus suggesting a reprogramming of the epigenome. Transcripts accumulation analyses showed that genes related to iron metabolism and oxidative stress were upregulated, while the gene related to epigenetic reprogramming presented reduced transcript accumulation after treatment. Conclusion: We concluded that maghemite nanoparticles covered with citric acid exposure provoked several biological responses without impairment of human submandibular gland cells viability. This is the first report on the epigenetic effects of maghemite nanoparticles on this cell lineage.

Blue light-triggered photochemistry and cytotoxicity of retinal

The chemical- and photo- toxicity of chromophore retinal on cells have long been debated. Although we recently showed that retinal and blue light exposure interrupt cellular signaling, a comprehensive study examining molecular underpinnings of this perturbation and its consequences to cellular fate is lacking. Here, we report molecular evidence for blue light excited-retinal induced oxidative damage of polyunsaturated lipid anchors in membrane-interacting signaling molecules and DNA damage in cells using live-cell imaging and in vitro experimentation. The incurred molecular damage irreversibly disrupted subcellular localization of these molecules, a crucial criterion for their signaling. We further show retinal accumulation in lipid-bilayers of cell membranes could enhance the lifetime of retinal in cells. Comparative response-signatures suggest that retinal triggers reactions upon photoexcitation similar to photodynamic therapy agents and generate reactive oxygen species in cells. Additionally, data also shows that exposing retinal-containing cells to sunlight induces substantial cytotoxicity. Collectively, our results explain a likely in vivo mechanism and reaction conditions under which bio-available retinal in physiological light conditions damages cells.

Exploring the antibacterial potential and unraveling the mechanism of action of non-doped and heteroatom-doped carbon nanodots

A comprehensive study of the antibacterial (bacteriostatic and bactericidal) properties of non-doped carbon nanodots (CNDs), nitrogen-doped (N-doped), and nitrogen/sulfur co-doped (N,S-doped) CNDs against Escherichia coli (a model organism) is discussed herein. The CNDs, with a size of ca. 5 nm, were found to be capable of inhibiting the growth of the bacterial biofilm but not significantly the growth of planktonic bacteria. Heteroatom doping was found to considerably improve the potential of CNDs as bactericidal agents. Further experiments were conducted to shed light on the potential mechanism of the antibacterial activity of the CNDs. The results showed that the CNDs do not interact with the cellular membrane via electrostatic forces. Following a simple metabolomic workflow, no alterations of the bacterial metabolome were observed except for the activation of the metabolism of α-linolenic acid. The CNDs neither oxidize cell membrane lipids and intracellular proteins nor elevate the concentration of reactive oxygen species in cells. Finally, the interactions of CNDs with genomic DNA and RNA revealed that CNDs are able to intercalate into their structure. The different affinities of the three kinds of CNDs for DNA/RNA account for the differences in their antibacterial activity and constitute the main mechanism via which CND activity is achieved.

Achyranthes bidentata polypeptides prevent apoptosis by inhibiting the glutamate current in cultured hippocampal neurons.

Glutamate-induced excitotoxicity plays a critical role in the neurological impairment caused by middle cerebral artery occlusion. Achyranthes bidentata polypeptides have been shown to protect against neurological functional damage caused by middle cerebral artery occlusion, but the underlying neuroprotective mechanisms and the relationship to glutamate-induced excitotoxicity remain unclear. Therefore, in the current study, we investigated the protective effects of Achyranthes bidentata polypeptides against glutamate-induced excitotoxicity in cultured hippocampal neurons. Hippocampal neurons were treated with Mg2+-free extracellular solution containing glutamate (300 µM) for 3 hours as a model of glutamate-mediated excitotoxicity (glutamate group). In the normal group, hippocampal neurons were incubated in Mg2+-free extracellular solution. In the Achyranthes bidentata polypeptide group, hippocampal neurons were incubated in Mg2+-free extracellular solution containing glutamate (300 µM) and Achyranthes bidentata polypeptide at different concentrations. At 24 hours after exposure to the agents, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and Hoechst 33258 staining were used to assess neuronal viability and nuclear morphology, respectively. Caspase-3 expression and activity were evaluated using western blot assay and colorimetric enzymatic assay, respectively. At various time points after glutamate treatment, reactive oxygen species in cells were detected by H2DCF-DA, and mitochondrial membrane potential was detected by rhodamine 123 staining. To examine the effect of Achyranthes bidentata polypeptides on glutamate receptors, electrophysiological recording was used to measure the glutamate-induced inward current in cultured hippocampal neurons. Achyranthes bidentata polypeptide decreased the percentage of apoptotic cells and reduced the changes in caspase-3 expression and activity induced by glutamate. In addition, Achyranthes bidentata polypeptide attenuated the amplitude of the glutamate-induced current. Furthermore, the glutamate-induced increase in intracellular reactive oxygen species and reduction in mitochondrial membrane potential were attenuated by Achyranthes bidentata polypeptide treatment. These findings collectively suggest that Achyranthes bidentata polypeptides exert a neuroprotective effect in cultured hippocampal neurons by suppressing the overactivation of glutamate receptors and inhibiting the caspase-3-dependent mitochondrial apoptotic pathway. All animal studies were approved by the Animal Care and Use Committee, Nantong University, China (approval No. 20120216-001) on February 16, 2012.

Overexpression of mitochondrial histidyl-tRNA synthetase restores mitochondrial dysfunction caused by a deafness-associated tRNAHis mutation.

The deafness-associated m.12201T>C mutation affects the A5-U68 base-pairing within the acceptor stem of mitochondrial tRNAHis The primary defect in this mutation is an alteration in tRNAHis aminoacylation. Here, we further investigate the molecular mechanism of the deafness-associated tRNAHis 12201T>C mutation and test whether the overexpression of the human mitochondrial histidyl-tRNA synthetase gene (HARS2) in cytoplasmic hybrid (cybrid) cells carrying the m.12201T>C mutation reverses mitochondrial dysfunctions. Using molecular dynamics simulations, we demonstrate that the m.12201T>C mutation perturbs the tRNAHis structure and function, supported by decreased melting temperature, conformational changes, and instability of mutated tRNA. We show that the m.12201T>C mutation-induced alteration of aminoacylation tRNAHis causes mitochondrial translational defects and respiratory deficiency. We found that the transfer of HARS2 into the cybrids carrying the m.12201T>C mutation raises the levels of aminoacylated tRNAHis from 56.3 to 75.0% but does not change the aminoacylation of other tRNAs. Strikingly, HARS2 overexpression increased the steady-state levels of tRNAHis and of noncognate tRNAs, including tRNAAla, tRNAGln, tRNAGlu, tRNALeu(UUR), tRNALys, and tRNAMet, in cells bearing the m.12201T>C mutation. This improved tRNA metabolism elevated the efficiency of mitochondrial translation, activities of oxidative phosphorylation complexes, and respiration capacity. Furthermore, HARS2 overexpression markedly increased mitochondrial ATP levels and membrane potential and reduced production of reactive oxygen species in cells carrying the m.12201T>C mutation. These results indicate that HARS2 overexpression corrects the mitochondrial dysfunction caused by the tRNAHis mutation. These findings provide critical insights into the pathophysiology of mitochondrial disease and represent a step toward improved therapeutic interventions for mitochondrial disorders.