Analysis of Mitochondrial Respiratory Chain Complexes in Cultured Human Cells using Blue Native Polyacrylamide Gel Electrophoresis and Immunoblotting

Mitochondrial respiration is performed by oxidative phosphorylation (OXPHOS) complexes within mitochondria. Internal and environmental factors can perturb the assembly and stability of OXPHOS complexes. This protocol describes the analysis of mitochondrial respiratory chain complexes by blue native polyacrylamide gel electrophoresis (BN-PAGE) in application to cultured human cells. First, mitochondria are extracted from the cells using digitonin, then using lauryl maltoside, the intact OXPHOS complexes are isolated from the mitochondrial membranes. The OXPHOS complexes are then resolved by gradient gel electrophoresis in the presence of the negatively charged dye, Coomassie blue, which prevents protein aggregation and ensures electrophoretic mobility of protein complexes towards the cathode. Finally, the OXPHOS complexes are detected by standard immunoblotting. Thus, BN-PAGE is a convenient and inexpensive technique that can be used to evaluate the assembly of entire OXPHOS complexes, in contrast to the basic SDS-PAGE allowing the study of only individual OXPHOS complex subunits.Peer reviewe

Chitosan Technology from Crustacean Shells of the Northern Seas

Technological schemes for the production of chitin and chitosan from the crustaceans of the Barents Sea have been developed. We used shells of king crab (Paralithodes camtschaticus) and snow crab (Chionoecetes opilio) as chitin-containing raw materials, which are waste from the processing of crabs and contain 5.5 and 4.9 wt.% chitin, respectively. Technological schemes are developed taking into account the chemical composition of the used raw materials containing a large amount of residual protein (up to 26 wt.% in the king crab shell) and mineral substances (up to 17 wt.% in the snow crab shell). A chemical method for chitin production has been used. The technological scheme includes the stages of the first deproteinization, demineralization, the second deproteinization and depigmentation of the raw materials using chemical reagents - acids, alkalis, etc. The deacetylation reaction in an alkaline medium was used as the main method for chitosan production from chitin. Technological solutions have been found to significantly reduce the consumption of alkali, to form a circuit of alkaline solutions. This leads to the reduction of pollution of wastewater generated during the production of chitin and chitosan. The resulting polysaccharide chitosan has a degree of deacetylation of 80–85%. Such a product is considered as a valuable ingredient for high-quality functional foods

To involvement the conformation of the adenine nucleotide translocase in opening the Tl+-induced permeability transition pore in Ca2+-loaded rat liver mitochondria

The conformation of adenine nucleotide translocase (ANT) has a profound impact in opening the mitochondrial permeability transition pore (MPTP) in the inner membrane. Fixing the ANT in 'c' conformation by phenylarsine oxide (PAO), tert-butylhydroperoxide (tBHP), and carboxyatractyloside as well as the interaction of 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) with mitochondrial thiols markedly attenuated the ability of ADP to inhibit the MPTP opening. We earlier found (Korotkov and Saris, 2011) that calcium load of rat liver mitochondria in medium containing TINO3 and KNO3 stimulated the Tl+-induced MPTP opening in the inner mitochondrial membrane. The MPTP opening as well as followed increase in swelling, a drop in membrane potential (Delta Psi(mito)), and a decrease in state 3, state 4, and 2,4-dinitrophenol-uncoupled respiration were visibly enhanced in the presence of PAO, tBHP, DIDS, and carboxyatractyloside. However, these effects were markedly inhibited by ADP and membrane-penetrant hydrophobic thiol reagent, N-ethylmaleimide (NEM) which fix the ANT in 'm' conformation. Cyclosporine A additionally potentiated these effects of ADP and NEM. Our data suggest that conformational changes of the ANT may be directly involved in the opening of the Tl+-induced MPTP in the inner membrane of Ca2+-loaded rat liver mitochondria. Using the Tl+-induced MPTP model is discussed in terms finding new transition pore inhibitors and inducers among different chemical and natural compounds. (C) 2016 Elsevier Ltd. All rights reserved.Peer reviewe

2-Hydroxy­imino-N′-[1-(2-pyrid­yl)ethyl­idene]propanohydrazide

The title compound, C10H12N4O2, features an intra­molecular N—H⋯N hydrogen bond formed between the imine NH and oxime N atoms. The oxime group and the amide C=O bond are anti to each other. In the crystal, mol­ecules are connected by O—H⋯O hydrogen bonds into supra­molecular zigzag chains along the c axis

MATHEMATICAL MODELING OF DIFFUSION PROCESSES OF MASS TRANSFER OF «FREE CALCIUM HYDROXIDE» DURING CORROSION OF CEMENT CONCRETES

The paper presents a mathematical model of mass transfer in the processes of corrosion of the first type of cement concrete at the level of phenomenological equations for a closed reservoir-liquid system. A step-by-step transition to the recording of the boundary value mass conduction problem in dimensionless coordinates is shown. The solutions of the boundary value mass conduction problem for the region of large and small values of Fourier numbers are obtained

ALS and Parkinson's disease genes CHCHD10 and CHCHD2 modify synaptic transcriptomes in human iPSC-derived motor neurons

Mitochondrial intermembrane space proteins CHCHD2 and CHCHD10 have roles in motor neuron diseases such as amyotrophic lateral sclerosis, spinal muscular atrophy and axonal neuropathy and in Parkinson's disease. They form a complex of unknown function. Here we address the importance of these two proteins in human motor neurons. We show that gene edited human induced pluripotent stem cells (iPSC) lacking either CHCHD2 or CHCHD10 are viable and can be differentiated into functional motor neurons that fire spontaneous and evoked action potentials. Mitochondria in knockout iPSC and motor neurons sustain ultrastructure but show increased proton leakage and respiration, and reciprocal compensatory increases in CHCHD2 or CHCHD10. Knockout motor neurons have largely overlapping transcriptome profiles compared to isogenic control line, in particular for synaptic gene expression. Our results show that the absence of either CHCHD2 or CHCHD10 alters mitochondrial respiration in human motor neurons, inducing similar compensatory responses. Thus, pathogenic mechanisms may involve loss of synaptic function resulting from defective energy metabolism.Peer reviewe

Truncated HSPB1 causes axonal neuropathy and impairs tolerance to unfolded protein stress

Peer reviewe

Redox regulation of GRPEL2 nucleotide exchange factor for mitochondrial HSP70 chaperone

Mitochondria are central organelles to cellular metabolism. Their function relies largely on nuclear-encoded proteins that must be imported from the cytosol, and thus the protein import pathways are important for the maintenance of mitochondrial proteostasis. Mitochondrial HSP70 (mtHsp70) is a key component in facilitating the translocation of proteins through the inner membrane into the mitochondrial matrix. Its protein folding cycle is regulated by the nucleotide-exchange factor GrpE, which triggers the release of folded proteins by ATP rebinding. Vertebrates have two mitochondrial GrpE paralogs, GRPEL1 and 2, but without clearly defined roles. Using BioID proximity labeling to identify potential binding partners of the GRPELs in the mitochondrial matrix, we obtained results supporting a model where both GRPELs regulate mtHsp70 as homodimers. We show that GRPEL2 is not essential in human cultured cells, and its absence does not prevent mitochondrial protein import. Instead we find that GRPEL2 is redox regulated in oxidative stress. In the presence of hydrogen peroxide, GRPEL2 forms dimers through intermolecular disulfide bonds in which Cys87 is the thiol switch. We propose that the dimerization of GRPEL2 may activate the folding machinery responsible for protein import into mitochondrial matrix or enhance the chaperone activity of mtHSP70, thus protecting mitochondrial proteostasis in oxidative stress.Peer reviewe

A patient with pontocerebellar hypoplasia type 6 : Novel RARS2 mutations, comparison to previously published patients and clinical distinction from PEHO syndrome

Pontocerebellar hypoplasia type 6 (PCH6) is a rare infantile-onset progressive encephalopathy caused by biallelic mutations in RARS2 that encodes the mitochondrial arginine-tRNA synthetase enzyme (mtArgRS). The clinical presentation overlaps that of PEHO syndrome (Progressive Encephalopathy with edema, Hypsarrhythmia and Optic atrophy). The proband presented with severe intellectual disability, epilepsy with varying seizure types, optic atrophy, axial hypotonia, acquired microcephaly, dysmorphic features and progressive cerebral and cerebellar atrophy and delayed myelination on MRI. The presentation had resemblance to PEHO syndrome but sequencing of ZNHIT3 did not identify pathogenic variants. Subsequent whole genome sequencing revealed novel compound heterozygous variants in RARS2, a missense variant affecting a highly conserved amino acid and a frameshift variant with consequent degradation of the transcript resulting in decreased mtArgRS protein level confirming the diagnosis of PCH6. Features distinguishing the proband's phenotype from PEHO syndrome were later appearance of hypotonia and elevated lactate levels in blood and cerebrospinal fluid. On MRI the proband presented with more severe supratentorial atrophy and lesser degree of abnormal myelination than PEHO syndrome patients. The study highlights the challenges in clinical diagnosis of patients with neonatal and early infantile encephalopathies with overlapping clinical features and brain MRI findings.Peer reviewe

Aspects of Serological Diagnostics of Listeriosis (Literature Review)

The review presents data on the antigenic structure and the current classification of epidemically significant serovariants of Listeria. Description of species-specific properties of serovariants of Listeria, which may be common for two or more species, and common antigens with staphylococci and typhoid and paratyphoid bacteria, are given. It has been shown that only the antigenic scheme of Listeria monocytogenes is of practical interest for medical microbiology. Importance of serotyping in the epidemiological analysis to determine the source of infections and ways of its spreading has been determined. Differences in the designation of serovariants in the diagnosis of listeriosis in medical practice are observed. High level of adaptive properties of Listeria, its ability to reproduce in an abiotic environment, including food, susceptibility of immunodeficient individuals, prevalence of food pathway of infection pose a significant danger of increased sickness rate with listeriosis. Serological diagnostics of Listeria has not been developed in detail, and the existing serological methods are aimed at identifying specific antibodies to listeria. Advantages of the serological method include: quick results and the possibility to study any biological material. Currently available serological methods have a number of disadvantages: low reliability of results and low specificity of the study. The most promising method for identification of a serological group of cultures, according to the world classification, is the multiplex PCR method, based on the correlation between the serogroup of an isolate and the presence of specific open reading frames in its genome