Three-dimensional (3D) printing technology offers attractive possibilities for many fields. In electrochemistry, 3D printing technology has been used to fabricate customized 3D-printed electrodes as a platform to develop bio/sensing, energy generation and storage devices. Here, we use a 3D-printed graphene/polylactic (PLA) electrode made by additive manufacturing technology and immobilize horseradish peroxidase (HRP) to create a direct electron transfer enzyme-based biosensors for hydrogen peroxide detection. Gold nanoparticles are included in the system to confirm and facilitate heterogeneous electron transfer. This work opens a new direction for the fabrication of third-generation electrochemical biosensors using 3D printing technology, with implications for applications in the environmental and biomedical fields.
Biological effects of high fluence low-power (HFLP) lasers have been reported for some time, yet the molecular mechanisms procuring cellular responses remain obscure. A better understanding of the effects of HFLP lasers on living cells will be instrumental for the development of new experimental and therapeutic strategies. Therefore, we investigated sub-cellular mechanisms involved in the laser interaction with human hepatic cell lines. We show that mitochondria serve as sub-cellular "sensor" and "effector" of laser light non-specific interactions with cells. We demonstrated that despite blue and red laser irradiation results in similar apoptotic death, cellular signaling and kinetic of biochemical responses are distinct. Based on our data, we concluded that blue laser irradiation inhibited cytochrome c oxidase activity in electron transport chain of mitochondria. Contrary, red laser triggered cytochrome c oxidase excessive activation. Moreover, we showed that Bcl-2 protein inhibited laser-induced toxicity by stabilizing mitochondria membrane potential. Thus, cells that either overexpress or have elevated levels of Bcl-2 are protected from laser-induced cytotoxicity. Our findings reveal the mechanism how HFLP laser irradiation interfere with cell homeostasis and underscore that such laser irradiation permits remote control of mitochondrial function in the absence of chemical or biological agents.
- MeSH
- Apoptosis radiation effects MeSH
- Hep G2 Cells MeSH
- Phototherapy * MeSH
- Low-Level Light Therapy * MeSH
- Humans MeSH
- Membrane Potential, Mitochondrial genetics radiation effects MeSH
- Mitochondrial Membranes metabolism radiation effects MeSH
- Mitochondria genetics radiation effects MeSH
- Oxidation-Reduction radiation effects MeSH
- Reactive Oxygen Species metabolism MeSH
- Gene Expression Regulation radiation effects MeSH
- Electron Transport Complex IV genetics MeSH
- Electron Transport genetics radiation effects MeSH
- Cell Survival genetics radiation effects MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Dihydroorotate dehydrogenase (DHODH) is an enzyme of the de novo pyrimidine synthesis pathway that provides nucleotides for RNA/DNA synthesis essential for proliferation. In mammalian cells, DHODH is localized in mitochondria, linked to the respiratory chain via the coenzyme Q pool. Here we discuss the role of DHODH in the oxidative phosphorylation system and in the initiation and progression of cancer. We summarize recent findings on DHODH biology, the progress made in the development of new, specific inhibitors of DHODH intended for cancer therapy, and the mechanistic insights into the consequences of DHODH inhibition.
- MeSH
- Enzyme Inhibitors therapeutic use MeSH
- Humans MeSH
- Mitochondria genetics metabolism MeSH
- Neoplasms genetics pathology MeSH
- Oxidative Phosphorylation * MeSH
- Oxidoreductases Acting on CH-CH Group Donors antagonists & inhibitors genetics MeSH
- Cell Proliferation drug effects MeSH
- Electron Transport genetics MeSH
- Ubiquinone analogs & derivatives genetics MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
Interquinone QA- → QB electron-transfer (ET) in isolated photosystem II reaction centers (PSII-RC) is protein-gated. The temperature-dependent gating frequency "k" is described by the Eyring equation till levelling off at T ≥ 240 °K. Although central to photosynthesis, the gating mechanism has not been resolved and due to experimental limitations, could not be explored in vivo. Here we mimic the temperature dependency of "k" by enlarging VD1-208, the volume of a single residue at the crossing point of the D1 and D2 PSII-RC subunits in Synechocystis 6803 whole cells. By controlling the interactions of the D1/D2 subunits, VD1-208 (or 1/T) determines the frequency of attaining an ET-active conformation. Decelerated ET, impaired photosynthesis, D1 repair rate and overall cell physiology upon increasing VD1-208 to above 130 Å3, rationalize the >99% conservation of small residues at D1-208 and its homologous motif in non-oxygenic bacteria. The experimental means and resolved mechanism are relevant for numerous transmembrane protein-gated reactions.
- MeSH
- Cell Respiration genetics MeSH
- Electrons MeSH
- Photosynthesis genetics MeSH
- Photosystem II Protein Complex chemistry genetics MeSH
- Kinetics MeSH
- Light MeSH
- Light-Harvesting Protein Complexes chemistry genetics MeSH
- Synechocystis chemistry genetics MeSH
- Electron Transport genetics MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Trypanosomatids are a very diverse group composed of monoxenous and dixenous parasites belonging to the excavate class Kinetoplastea. Here we studied the respiration of five monoxenous species (Blechomonas ayalai, Herpetomonas muscarum, H. samuelpessoai, Leptomonas pyrrhocoris and Sergeia podlipaevi) introduced into culture, each representing a novel yet globally distributed and/or species-rich clade, and compare them with well-studied flagellates Trypanosoma brucei, Phytomonas serpens, Crithidia fasciculata and Leishmania tarentolae. Differences in structure and activities of respiratory chain complexes, respiration and other biochemical parameters recorded under laboratory conditions reveal their substantial diversity, likely a reflection of different host environments. Phylogenetic relationships of the analysed trypanosomatids do not correlate with their biochemical parameters, with the differences within clades by far exceeding those among clades. As the S. podlipaevi canonical respiratory chain complexes have very low activities, we believe that its mitochondrion is utilised for purposes other than oxidative phosphorylation. Hence, the single reticulated mitochondrion of diverse trypanosomatids seems to retain multipotency, with the capacity to activate its individual components based on the host environment.
- MeSH
- Phylogeny MeSH
- Leishmania genetics metabolism MeSH
- Mitochondria genetics physiology MeSH
- Oxidative Phosphorylation MeSH
- Electron Transport genetics physiology MeSH
- Trypanosoma brucei brucei genetics metabolism MeSH
- Trypanosomatina genetics metabolism MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Early onset mitochondrial encephalo-cardiomyopathy due to isolated deficiency of ATP synthase is frequently caused by mutations in TMEM70 gene encoding enzyme-specific ancillary factor. Diminished ATP synthase results in low ATP production, elevated mitochondrial membrane potential and increased ROS production. To test whether the patient cells may react to metabolic disbalance by changes in oxidative phosphorylation system, we performed a quantitative analysis of respiratory chain complexes and intramitochondrial proteases involved in their turnover. SDS- and BN-PAGE Western blot analysis of fibroblasts from 10 patients with TMEM70 317-2A>G homozygous mutation showed a significant 82-89% decrease of ATP synthase and 50-162% increase of respiratory chain complex IV and 22-53% increase of complex III. The content of Lon protease, paraplegin and prohibitins 1 and 2 was not significantly changed. Whole genome expression profiling revealed a generalized upregulation of transcriptional activity, but did not show any consistent changes in mRNA levels of structural subunits, specific assembly factors of respiratory chain complexes, or in regulatory genes of mitochondrial biogenesis which would parallel the protein data. The mtDNA content in patient cells was also not changed. The results indicate involvement of posttranscriptional events in the adaptive regulation of mitochondrial biogenesis that allows for the compensatory increase of respiratory chain complexes III and IV in response to deficiency of ATP synthase.
- MeSH
- Fibroblasts metabolism pathology MeSH
- Humans MeSH
- Membrane Proteins genetics MeSH
- RNA, Messenger genetics metabolism MeSH
- DNA, Mitochondrial metabolism MeSH
- Mitochondrial Proteins genetics MeSH
- Mitochondrial Proton-Translocating ATPases deficiency metabolism MeSH
- Mitochondria enzymology genetics MeSH
- Mutation genetics MeSH
- Oxidative Phosphorylation MeSH
- Peptide Hydrolases metabolism MeSH
- Electron Transport Complex III metabolism MeSH
- Electron Transport Complex IV metabolism MeSH
- Gene Expression Profiling MeSH
- Electron Transport genetics MeSH
- Up-Regulation * MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The impact of point mutations in mitochondrial tRNA genes on the amount and stability of respiratory chain complexes and ATP synthase (OXPHOS) has been broadly characterized in cultured skin fibroblasts, skeletal muscle samples, and mitochondrial cybrids. However, less is known about how these mutations affect other tissues, especially the brain. We have compared OXPHOS protein deficiency patterns in skeletal muscle mitochondria of patients with Leigh (8363G>A), MERRF (8344A>G), and MELAS (3243A>G) syndromes. Both mutations that affect mt-tRNA(Lys) (8363G>A, 8344A>G) resulted in severe combined deficiency of complexes I and IV, compared to an isolated severe defect of complex I in the 3243A>G sample (mt-tRNA(LeuUUR). Furthermore, we compared obtained patterns with those found in the heart, frontal cortex, and liver of 8363G>A and 3243A>G patients. In the frontal cortex mitochondria of both patients, the patterns of OXPHOS deficiencies differed substantially from those observed in other tissues, and this difference was particularly striking for ATP synthase. Surprisingly, in the frontal cortex of the 3243A>G patient, whose ATP synthase level was below the detection limit, the assembly of complex IV, as inferred from 2D-PAGE immunoblotting, appeared to be hindered by some factor other than the availability of mtDNA-encoded subunits.
- MeSH
- Electrophoresis, Gel, Two-Dimensional MeSH
- Child MeSH
- Fatal Outcome MeSH
- Financing, Organized MeSH
- Immunoblotting MeSH
- Kinetics MeSH
- Muscle Fibers, Skeletal enzymology pathology MeSH
- Muscle, Skeletal enzymology pathology MeSH
- Humans MeSH
- Mitochondrial Proton-Translocating ATPases metabolism MeSH
- Mitochondria enzymology genetics MeSH
- Adolescent MeSH
- Brain enzymology MeSH
- Mutation genetics MeSH
- Infant, Newborn MeSH
- Organ Specificity MeSH
- Oxidative Phosphorylation MeSH
- Protein Subunits metabolism MeSH
- RNA, Transfer, Lys genetics MeSH
- Oxygen Consumption MeSH
- Electron Transport genetics MeSH
- Check Tag
- Child MeSH
- Humans MeSH
- Adolescent MeSH
- Male MeSH
- Infant, Newborn MeSH
- Female MeSH
- Publication type
- Case Reports MeSH
- Comparative Study MeSH
- MeSH
- Antioxidants metabolism MeSH
- Arteriosclerosis etiology pathology MeSH
- Research Support as Topic MeSH
- Glutathione physiology metabolism therapeutic use MeSH
- Glutathione Peroxidase physiology MeSH
- Insulin Resistance MeSH
- Oxidative Stress genetics MeSH
- Electron Transport genetics MeSH
- Publication type
- Review MeSH
- MeSH
- Cytochromes c physiology MeSH
- Oxygen metabolism MeSH
- Mitochondria enzymology MeSH
- Protons MeSH
- Electron Transport Complex IV analysis physiology chemistry MeSH
- Electron Transport genetics MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Review MeSH
