Internal Mitochondrial Membrane Research Articles

Endocrinological features and epileptic encephalopathy in COX deficiency due to SCO1 mutations: case series and review of literature.

Cytochrome C oxidase (COX) is the fourth component of the respiratory chain and is located within the internal membrane of mitochondria. COX deficiency causes an inherited mitochondrial disease with significant genetic and phenotypic heterogeneity. Four clinical subtypes have been identified, each with distinct phenotypes and genetic variants. Mitochondrial complex IV deficiency nuclear type 4 (MC4DN4) is a form of COX deficiency associated with pathogenic variants in the SCO1 gene. We describe three patients with MC4DN4 with developmental and epileptic encephalopathy (DEE), hypopituitarism, and SCO1 pathogenic variants. These patients' phenotypes considerably differ from previously reported MC4DN4 phenotypes as they associate DEE with progressive hypopituitarism and survival beyond the first months after birth. Pituitary deficiency in these patients progressively worsened and mainly involved growth hormone secretion and thyroid function. Our findings expand knowledge of phenotypic variability in MC4DN4 and suggest that SCO1 is a candidate gene for genetic hypopituitarism and DEE. Our paper describes three patients affected by MC4DN4 with hypopituitarism and developmental and epileptic encephalopathy (DEE), two features that have never been associated with this condition. In addition, we reviewed the clinical features of all previous cases of MC4DN4 to give the other clinicians a wide picture of the clinical phenotype of this genetic disease. We hope that the publication of our data may help others to identify this disease and consider the chance to analyze the SCO1 gene in cases of DEE associated with pituitary dysfunction. Our article contributes to expanding the spectrum of genetic hypopituitarism and proposes a model to explain an association between this condition, mitochondrial anomalies, and neurodevelopmental defects.

Tissue-specific differences in Ca2+ sensitivity of the mitochondrial permeability transition pore (PTP). Experiments in male rat liver and heart.

Permeability transition pore (PTP) opening dissipates ion and electron gradients across the internal mitochondrial membrane (IMM), including excess Ca2+ in the mitochondrial matrix. After opening, immediate PTP closure must follow to prevent outer membrane disruption, loss of cytochrome c, and eventual apoptosis. Flickering, defined as the rapid alternative opening/closing of PTP, has been reported in heart, which undergoes frequent, large variations in Ca2+. In contrast, in tissues that undergo depolarization events less often, such as the liver, PTP would not need to be as dynamic and thus these tissues would not be as resistant to stress. To evaluate this idea, it was decided to follow the reversibility of the permeability transition (PT) in isolated murine mitochondria from two different tissues: the very dynamic heart, and the liver, which suffers depolarizations less frequently. It was observed that in heart mitochondria PT remained reversible for longer periods and at higher Ca2+ loads than in liver mitochondria. In all cases, Ca2+ uptake was inhibited by ruthenium red and PT was delayed by Cyclosporine A. Characterization of this phenomenon included measuring the rate of oxygen consumption, organelle swelling and Ca2+ uptake and retention. Results strongly suggest that there are tissue-specific differences in PTP physiology, as it resists many more Ca2+ additions before opening in a highly active organ such as the heart than in an organ that seldom suffers Ca2+ loading, such as the liver.

How mitochondrial cristae illuminate the important role of oxygen during eukaryogenesis.

Inner membranes of mitochondria are extensively folded, forming cristae. The observed overall correlation between efficient eukaryotic ATP generation and the area of internal mitochondrial inner membranes both in unicellular organisms and metazoan tissues seems to explain why they evolved. However, the crucial use of molecular oxygen (O2) as final acceptor of the electron transport chain is still not sufficiently appreciated. O2 was an essential prerequisite for cristae development during early eukaryogenesis and could be the factor allowing cristae retention upon loss of mitochondrial ATP generation. Here I analyze illuminating bacterial and unicellular eukaryotic examples. I also discuss formative influences of intracellular O2 consumption on the evolution of the last eukaryotic common ancestor (LECA). These considerations bring about an explanation for the many genes coming from other organisms than the archaeon and bacterium merging at the start of eukaryogenesis.

Mitochondrial Sensitivity To Submaximal ADP Concentrations Following Bed Rest: A Double Exponential Model Analysis

We recently demonstrated that a 10-day exposure to inactivity/microgravity (bed rest, BR) determined impairments of peak oxygen uptake (V̇O2), peak cardiac output, peripheral and microvascular function, whereas maximal ADP-stimulated mitochondrial respiration ex vivo in isolated permeabilized fibers and muscle V̇O2 off-kinetics in vivo were unaffected. PURPOSE: To evaluate mitochondrial sensitivity to submaximal [ADP]. METHODS: Isolated permeabilized vastus lateralis fibers were analyzed by high-resolution respirometry, before (PRE) and after (POST) a 10-day BR in 10 young males. 11 submaximal titrations of ADP (from 12.5 to 10,000 μM) were utilized to assess complex I + II-linked ADP sensitivity which was analyzed by both a Michaelis-Menten (MM) kinetics equation, with the calculation of the apparent Km by standard methods, and by applying a double-exponential function, with the calculation of [ADP] corresponding to 50% of the maximal mitochondrial respiration ([ADP] at 50% JO2max). RESULTS: The double-exponential function provided a better fitting of the data according to AIC model selection. The sum of squared residuals was substantially lower with the double-exponential vs. MM fitting (411 ± 419 and 1209 ± 495 [pmol/s/mg]2, respectively). R2 was higher with the double-exponential vs. the MM fitting (0.97 ± 0.04 and 0.90 ± 0.05, respectively). Analysis of residuals showed that the fitting by MM markedly overestimated JO2 from 1000 to 2000 μM, and underestimated JO2 from 6000 to 10,000 μM. The apparent Km for MM was lower than the [ADP] at 50% JO2max, both in PRE (843 ± 706 and 1270 ± 1015 μM, respectively) and in POST (375 ± 173 and 409 ± 398 μM, respectively). Only for the [ADP] at 50% JO2max the difference between POST vs. PRE was statistically significant (P = 0.046). CONCLUSIONS: The double-exponential function provided a better fit of experimental data. It might also give insights into cellular mechanisms regulating mitochondrial respiration, possibly related to the transport of nucleotides and phosphate across external and internal mitochondrial membranes, ATP synthase activity and respiratory chain activity. The enhanced mitochondrial sensitivity to submaximal [ADP] after BR could represent an attempt to maintain metabolic homeostasis in the presence of impairments upstream of mitochondria.

Bone Marrow Mononuclear Cells Restore Normal Mitochondrial Ca2+ Handling and Ca2+-Induced Depolarization of the Internal Mitochondrial Membrane by Inhibiting the Permeability Transition Pore After Ischemia/Reperfusion

Acute kidney injury due to ischemia followed by reperfusion (IR) is a severe clinical condition with high death rates. IR affects the proximal tubule segments due to their predominantly oxidative metabolism and profoundly altered mitochondrial functions. We previously described the impact of IR on oxygen consumption, the generation of membrane potential (ΔΨ), and formation of reactive oxygen species, together with inflammatory and structural alterations. We also demonstrated the benefits of bone marrow mononuclear cells (BMMC) administration in these alterations. The objective of the present study has been to investigate the effect of IR and the influence of BMMC on the mechanisms of Ca2+ handling in mitochondria of the proximal tubule cells. IR inhibited the rapid accumulation of Ca2+ (Ca2+ green fluorescence assays) and induced the opening of the cyclosporine A-sensitive permeability transition pore (PTP), alterations prevented by BMMC. IR accelerated Ca2+-induced decrease of ΔΨ (Safranin O fluorescence assays), as evidenced by decreased requirement for Ca2+ load and t1/2 for complete depolarization. Addition of BMMC and ADP recovered the normal depolarization profile, suggesting that stabilization of the adenine nucleotide translocase (ANT) in a conformation that inhibits PTP opening offers a partial defense mechanism against IR injury. Moreover, as ANT forms a complex with the voltage-dependent anion channel (VDAC) in the outer mitochondrial membrane, it is possible that this complex is also a target for IR injury—thus favoring Ca2+ release, as well as the supramolecular structure that BMMC protects. These beneficial effects are accompanied by a stimulus of the citric acid cycle—which feed the mitochondrial complexes with the electrons removed from different substrates—as the result of accentuated stimulus of citrate synthase activity by BMMC.

Alteration in the Lipid Profile and the Desaturases Activity in Patients With Severe Pneumonia by SARS-CoV-2.

The kidnapping of the lipid metabolism of the host’s cells by severe acute respiratory syndrome (SARS-CoV-2) allows the virus to transform the cells into optimal machines for its assembly and replication. Here we evaluated changes in the fatty acid (FA) profile and the participation of the activity of the desaturases, in plasma of patients with severe pneumonia by SARS-CoV-2. We found that SARS-CoV-2 alters the FA metabolism in the cells of the host. Changes are characterized by variations in the desaturases that lead to a decrease in total fatty acid (TFA), phospholipids (PL) and non-esterified fatty acids (NEFAs). These alterations include a decrease in palmitic and stearic acids (p ≤ 0.009) which could be used for the formation of the viral membranes and for the reparation of the host’s own membrane. There is also an increase in oleic acid (OA; p = 0.001) which could modulate the inflammatory process, the cytokine release, apoptosis, necrosis, oxidative stress (OS). An increase in linoleic acid (LA) in TFA (p = 0.03) and a decreased in PL (p = 0.001) was also present. They result from damage of the internal mitochondrial membrane. The arachidonic acid (AA) percentage was elevated (p = 0.02) in the TFA and this can be participated in the inflammatory process. EPA was decreased (p = 0.001) and this may decrease of pro-resolving mediators with increase in the inflammatory process. The total of NEFAs (p = 0.03), PL (p = 0.001), cholesterol, HDL and LDL were decreased, and triglycerides were increased in plasma of the COVID-19 patients. Therefore, SARS-CoV-2 alters the FA metabolism, the changes are characterized by alterations in the desaturases that lead to variations in the TFA, PL, and NEFAs profiles. These changes may favor the replication of the virus but, at the same time, they are part of the defense system provided by the host cell metabolism in its eagerness to repair damage caused by the virus to cell membranes.

Three-dimensional imaging of mitochondrial cristae complexity using cryo-soft X-ray tomography

Mitochondria are dynamic organelles that change morphology to adapt to cellular energetic demands under both physiological and stress conditions. Cardiomyopathies and neuronal disorders are associated with structure-related dysfunction in mitochondria, but three-dimensional characterizations of the organelles are still lacking. In this study, we combined high-resolution imaging and 3D electron density information provided by cryo-soft X-ray tomography to characterize mitochondria cristae morphology isolated from murine. Using the linear attenuation coefficient, the mitochondria were identified (0.247 ± 0.04 µm−1) presenting average dimensions of 0.90 ± 0.20 µm in length and 0.63 ± 0.12 µm in width. The internal mitochondria structure was successfully identified by reaching up the limit of spatial resolution of 35 nm. The internal mitochondrial membranes invagination (cristae) complexity was calculated by the mitochondrial complexity index (MCI) providing quantitative and morphological information of mitochondria larger than 0.90 mm in length. The segmentation to visualize the cristae invaginations into the mitochondrial matrix was possible in mitochondria with MCI ≥ 7. Altogether, we demonstrated that the MCI is a valuable quantitative morphological parameter to evaluate cristae modelling and can be applied to compare healthy and disease state associated to mitochondria morphology.

Study on the Characteristics of ATP-Sensitive Potassium Channels in Neonatal Rat Hypoxic-Ischemic Encephalopathy

KATP channels are present on the surface of cardiac internal membranes, skeletal muscle, brain, and mitochondrial membranes. KATP channels can be activated with selective drugs including diazoxide and cromakalim that may be able to limit ischemia-induced damage or brain cell death. KATP channels have no relationship with a homogenous group, while other channels do not have the same combinations. This study was to characterize the effects of the neuro-protective KATP channel openers diazoxide and cromakalim in neonatal rats with hypoxic-ischemic encephalopathy (HIE) induced by intrauterine asphyxia. Seventy-two hours-old Sprague Dawley rat pups were subjected to intrauterine asphyxia and hypoxia for 30 min, and then randomly assigned to seven groups: Vehicle, Hypoxic-ischemia (HI), Diazoxide (Dia), Cromakalim (Cro), Diazoxide+Glibenclamide (Dia + Gli), Cromakalim+Glibenclamide (Cro + Gli), and Solvent (NaOH). Brain injury was maintained by neurologic deficit or mass injury 72 hours after HI. Moreover, gene expression of KATP channels was measured by RT-PCR 72 hours after HI. Results showed that the hippocampus and cortex had the most serious injury after HI, next highest injury was in the cerebellum, and the least amount of injury was in the thalamus and brainstem. Diazoxide and cromakalim showed a neuroprotective effect to the five brain regions tested after intrauterine asphyxia. The neuroprotective effect between diazoxide and cromakalim was not statistically significant and could be inhibited by glibenclamide. Diazoxide and cromakalim had the strongest neuroprotective effect in the hippocampus and cortex, which showed the most serious injury after intrauterine asphyxia.

Role of SNPs of CPTIA and CROT genes in the carnitine-shuttle in coronary artery disease: a case-control study

Abstract Objective Fatty acid β-oxidation defects can lead to difficulties at covering energy requirement of heart. The carnitine-shuttle is responsible for the transfering of long-chain fatty acids from the internal mitochondrial membrane. The role of genetic variants of the enzymes in the carnitine shuttle in coronary artery disease (CAD) has not been studied. Therefore, we performed a case-control study investigating the possible relation between the CPTIA-rs3019613 and CROT-rs2214930 gene variations located carnitine shuttle and CAD risk. Materials and methods Study groups were comprised of 96 CAD patients and 85 controls. CPTIA-rs3019613 G > A and CROT-rs2214930 T > C polymorphisms were determined by real-time-PCR. Results The CROT-rs2214930-CC genotype was found to be associated with decreased HDL-cholesterol (HDL-C) in controls (p = 0.029). In patients with CPTIA-rs3019613-A allele, body mass index (BMI) (p = 0.016) and BMI threshold-value (p = 0.030) were found be higher compared to those with GG-genotype, while HDL-C threshold-value (HDL-C ≤ 0.90 mmol/L) was found to be lower (p = 0.015). Regression analysis confirmed CPTIA-rs3019613-A allele has a significant relationship with decreased HDL-C (p = 0.009) in patients. Conclusion Our study indicated that the polymorphisms of the CROT and CPTIA genes related to β-oxidation of long-chain fatty acids had an important effect on serum HDL-C levels and may be a potential risk for CAD.

Study of the Mechanism of the Neuron Sensitization to the Repeated Glutamate Challenge

Exposure of cultured neurons to high concentrations of Glu leads to a strong depolarization of mitochondria, which develops synchronously with the secondary rise in the intracellular Ca2+ concentration (delayed calcium deregulation, DCD). In this study, using the primary culture of rat cerebellar neurons, we investigated the mechanism of neuronal sensitization, which manifests itself in the reduction of latent periods of DCD during repeated exposures to Glu. It was shown that the most likely cause of sensitization is the inability of mitochondria to maintain a high transmembrane potential (ΔΨm) as a result of an increase in the proton conductivity of the internal mitochondrial membrane, but not the opening of the mitochondrial permeability transition pore in the inner mitochondrial membrane. Mitochondrial dysfunction reduces the production of ATP, leading to the inability of neurons to quickly restore the concentration of Na+, ATP, and NADH in the intervals between successive Glu administrations. One of the reasons that aggravate the dysfunction of mitochondria and contribute to the sensitization of neurons to the repeated action of Glu is Ca2+ accumulated in the mitochondria during the first glutamate impact.

ПОСТКОНДИЦИОНИРОВАНИЕ КАК МЕТОД ПОВЫШЕНИЯ ВЫЖИВАЕМОСТИ ТКАНЕЙ ПРИ ИШЕМИЧЕСКОМ ПОВРЕЖДЕНИИ

The article presents the present-day data on the phenomenon of ischemic myocardial postconditioning. It has been shown, that this phenomenon consists in protection of the heart from reperfusion damage by the means of short episodes of ischemia/reperfusion performed at the early stage after prolonged ischemia. It is presented that postconditioning effect manifests in limiting the size of infarction and preserving endothelial function in the region exposed to ischemic injury. The article reports on the modern concept of role of various intracellular signal cascades in providing survival of the cell after the episode of ischemia/reperfusion. Much attention is given to changing in the state of pores localized in internal mitochondrial membrane as end links of realization of postconditioning effect. Prospects of clinical use of postconditioning are very optimistic due to the fact that application of different variants of preconditioning is limited because in most cases it is impossible to predict the time of occurrence of ischemic injury, whereas postconditioning may be used after prolonged ischemia.

First Cases of Dominant Optic Atrophy in Saudi Arabia

Fifty to 60% of patients with dominant optic atrophy (DOA) have mutations of the OPA1 gene, which encodes dynamin-related GTPase, a protein of the internal mitochondrial membrane. To date, more than 200 OPA1 mutations in the OPA1 gene have been described. However, DOA is genetically heterogeneous with certain families linked to other chromosomal loci, that is, OPA3, OPA4, OPA5, and OPA7. This study describes a clinical series of 40 patients from Saudi Arabia with a positive DOA phenotype (i.e., decreased visual acuity during the first 2 decades of life, temporal or global optic disc pallor, and absence of other neurological or ophthalmological diseases that could explain the optic neuropathy) who underwent molecular genetic testing for OPA1 (and, in some cases, for OPA3). This study describes for the first time 4 OPA1 mutations in DOA patients from Saudi Arabia, including 2 novel OPA1 mutations in 2 different patients. The question remains whether certain patients in Saudi Arabia with a clearly defined DOA phenotype may be due to mutations in chromosomal loci other than OPA1 and OPA3. It is likely that genetic alterations associated with different loci will be discovered in the future.

Mitochondrial Transmembrane Potential (ψm) Assay Using TMRM

During cellular respiration, nutrients are oxidized to generate energy through a mechanism called oxidative phosphorylation, which occurs in the mitochondria. During oxidative phosphorylation, the gradual degradation of molecules through the TCA cycle releases electrons from the covalent bonds that are broken. These electrons are captured by NAD+ through its reduction into NADH. Finally, NADH transports the electrons to the complexes of the electron chain in the internal membrane of mitochondria. These complexes use the energy released by the electrons to pump protons into the intermembrane space, generating an electrochemical gradient across the internal membrane of mitochondria, which provides energy for the ATP-synthase complex, ultimately producing adenosine triphosphate (ATP). We assessed the mitochondrial membrane potential (ψm) using tetramethylrhodamine methyl ester (TMRM), a cell-permeant, cationic, red fluorescent dye. To measure specifically the mitochondrial membrane potential (ψm) we quantified the fluorescence intensity before and after applying FCCP, a mitochondrial electron chain uncoupler. The difference of intensity before and after applying FCCP corresponds specifically to the mitochondrial membrane potential. We analyzed mitochondrial membrane potential (ψm) by cytofluorimetry. The ratio between the total level of signal and the signal generated after uncoupling provided a normalized value for the difference in cell size. Furthermore, to normalize for the different size of cells that we were analyzing we have analyzed TMRM in live imaging using IN Cell Analyzer, so that the level of mitochondrial membrane potential could be detected per unit of mitochondrial membrane area measured. Thus, our protocol can also be used to compare the mitochondrial membrane potential of cells that are different in size.

L-carnitine in cardiology: reality and perspectives

Carnitine is a substance close to vitamins B, which participates in the mitochondrial energy synthesis. L-carnitine controls the rate of the long-chain fatty acid oxidation and acts as a specific co-factor facilitating their transport via the internal mitochondrial membrane. It also participates in the elimination of long-chain fatty acid excess from mitochondria and cytoplasm. These effects prevent cytotoxicity. This review presents the results of the studies which comply with the standards of evidence-based medicine. It has been shown that in patients with acute myocardial infarction (AMI), L-carnitine therapy was associated with a reduction in myocardial necrosis and an improved clinical course of AMI (end-point incidence in the main and control groups was 15,6% and 26%, respectively). In the first 5 days of L-carnitine therapy, there was a significant reduction in mortality in the main group of AMI patients. In patients with stable angina, L-carnitine reduces the risk of all-cause mortality, repeat AMI, and ventricular arrhythmias. In patients with intermittent claudication, L-carnitine significantly increases the pain-free walking distance. This medication is well tolerated, with no registered major adverse effects.

Chemical Cytometry Quantitates Superoxide Levels in the Mitochondrial Matrix of Single Myoblasts

Triphenylphosphonium hydroethidine (TPP-HE) is a membrane-permeable probe that reacts with superoxide and forms hydroxytriphenylphosphonium ethidium (OH-TPP-E(+)), a fluorescent product that has been previously used in qualitative measurements of superoxide production. In order to develop quantitative methods to measure superoxide, it is necessary to take into consideration the principles that drive TPP-HE accumulation into various subcellular compartments. In the mitochondria matrix, TPP-HE accumulation depends on the mitochondrial membrane potential, which varies from cell to cell. Here we address this issue by including rhodamine 123 (R123) as an internal mitochondrial membrane potential calibrant in chemical cytometry experiments. After loading with TPP-HE and R123, a single cell is lysed within a separation capillary and its contents are separated and detected by micellar electrokinetic capillary chromatography with laser-induced fluorescence detection (MEKC-LIF). Using theoretical arguments, we show that the ratio [OH-TPP-E(+)]/[R123] is adequate to obtain a relative quantitation of mitochondrial matrix superoxide levels for each analyzed cell. We applied this method to single skeletal muscle myoblasts and determined that the steady state superoxide levels in the mitochondrial matrix is approximately (0.29 +/- 0.10) x 10(-12) M. The development of this quantitative method is a critical step toward establishing the importance of reactive oxygen species in biological systems, including those relevant to aging and disease.

Mouse brain expression patterns of Spg7, Afg3l1, and Afg3l2 transcripts, encoding for the mitochondrial m-AAA protease

BackgroundThe m-AAA (ATPases Associated with a variety of cellular Activities) is an evolutionary conserved metalloprotease complex located in the internal mitochondrial membrane. In the mouse, it is a hetero-oligomer variably formed by the Spg7, Afg3l1, and Afg3l2 encoded proteins, or a homo-oligomer formed by either Afg3l1 or Afg3l2. In humans, AFG3L2 and SPG7 genes are conserved, whereas AFG3L1 became a pseudogene. Both AFG3L2 and SPG7 are involved in a neurodegenerative disease, namely the autosomal dominant spinocerebellar ataxia SCA28 and a recessive form of spastic paraplegia, respectively.ResultsUsing quantitative RT-PCR, we measured the expression levels of Spg7, Afg3l1, and Afg3l2 in the mouse brain. In all regions Afg3l2 is the most abundant transcript, followed by Spg7, and Afg3l1, with a ratio of approximately 5:3:1 in whole-brain mRNA. Using in-situ hybridization, we showed that Spg7, Afg3l1 and Afg3l2 have a similar cellular pattern of expression, with high levels in mitral cells, Purkinje cells, deep cerebellar nuclei cells, neocortical and hippocampal pyramidal neurons, and brainstem motor neurons. However, in some neuronal types, differences in the level of expression of these genes were present, suggesting distinct degrees of contribution of their proteins.ConclusionsNeurons involved in SCA28 and hereditary spastic paraplegia display high levels of expression, but similar or even higher expression is also present in other types of neurons, not involved in these diseases, suggesting that the selective cell sensitivity should be attributed to other, still unknown, mechanisms.

New data on biochemical mechanism of programmed senescence of organisms and antioxidant defense of mitochondria

Much evidence has recently been reported suggesting that reactive oxygen species (ROS) produced in mitochondria play a crucial role in the programmed senescence of organisms. In particular, it has been shown that antioxidants addressed to mitochondria slow down the appearance of symptoms of senescence and development of senile diseases and increase the median lifespan of various organisms from fungi to mammals. At the biochemical level, the mechanism of action of such rechargeable antioxidants as plastoquinonyldecyltriphenyl phosphonium (SkQ1) includes, in particular, prevention of oxidation of mitochondrial cardiolipin by ROS. The hormone melatonin also exhibits a number of such effects, and decrease in its level with age could explain the weakening of antioxidant protection upon aging. According to Moosmann et al., there exists a natural mechanism of antioxidant protection that, like SkQ1, is localized in the internal mitochondrial membrane and is rechargeable. It involves methionine residues in the surface regions of proteins encoded by mitochondrial DNA. It appears that in organisms with high respiratory metabolism the genetic code in the mitochondrial system of protein biosynthesis has changed. In these organisms (including some yeasts, insects, crustaceans, and vertebrates), the AUA codon codes for methionine rather than isoleucine, as in the case of synthesis of proteins encoded either in the nucleus or in mitochondria of organisms with lower rates of metabolism (other yeast species, sponges, and echinoderms). Methionine quenches ROS, being converted to methionine sulfoxide, which is re-reduced to the initial methionine by NADPH.

Role of uncoupling proteins in obesity

The discovery of a protein of the internal mitochondrial membrane of the brown adipocytes, the UCP1, marked an important advance in the understanding of the thermogenic process, as well as of the working of the brown adipose tissue. This protein is only of importance in the newly born and small animals, however the later discovery of proteins that were analogues of UCP1 (UCP2, widely distributed, and UCP3, mainly present in the muscle) with a similar functioning and also present in human tissue, created new perspectives and scientific goals. These proteins uncouple the respiratory chain of the oxidative phosphorylation, thus dissipating energy in the form of heat without producing ATP, by means of a mechanism that is still the subject of debate. From the studies of regulation that have been made, it emerges that their activity is modified when facing different physiological and nutritional stimuli, with greater activity observed in situations where an increase of energy expenditure is required. The studies carried on humans seem to corroborate the results obtained in experiments on animals, and action can thus be proposed on the activity, or the quantity, of these proteins in humans, as a means for fighting overweightedness and obesity. However, there is still an evident need to complete and improve the existing information on the importance of these proton transporting proteins in humans.

Proteomic analysis of heart mitochondria from Bos taurus: I. Application of proteomic methods to identification of transmembrane domains of proteins of the internal mitochondrial membrane

This study is part of a large-scale investigation of the proteome of mitochondria from the heart muscle of Bos taurus. We developed a special approach to simplification of the protein mixture by separation of mitochondrial fractions with stable protein compositions. At the first stage of this approach, we isolated and purified internal mitochondrial membranes. The protein composition of this fraction was analyzed by the following proteomic methods: enzymatic or/and chemical cleavage of the proteins, chromatographic fractionation of the complex mixture of the resulting peptides, mass-spectrometric identification of these peptides, and a search for proteins in databases of amino acid sequences. We reliably identified 147 unique proteins with the use of the SwissProt database. The subcellular location and functions of these proteins were analyzed. Approaches to studies of transmembrane domains of integral membrane proteins of the internal mitochondrial membrane were proposed on the basis of proteomic methods of analysis. Considerable coincidence of the experimental data with the results of determination of the 3D structures of the proteins by X-ray analysis was shown.

Nickel toxicity in the hindgut of an isopod Porcellio scaber (Oniscidea)

Studies on toxic effects of nickel were conducted in a chronic experiment on four groups of woodlice Porcellio scaber kept for 24 weeks on dry maple leaves containing Ni 0.1 (control), 8 (Ni1), 75 (Ni2) and 270 (Ni3) μg g −1 dry weight. Micro-PIXE elemental mapping complemented by proton backscattering was used to study relations between Ni and other elements in the hindgut. Data processed with GeoPIXE II demonstrated the highest amount of Ni in the hindgut of (Ni2) woodlice followed by the highest levels of Ca, Mn and Fe. Inverse relationship was observed between hindgut concentration of Zn and the Ni levels in the diet. Transmission electron microscopy (TEM) was used for checking possible relations between the ultrastructure of the hindgut epithelial cells and nickel tissue burden. In animals from all the “nickel groups” a series of ultrastructural alterations (disturbances in apical and basal membrane labyrinth and presence of lysosome-like structures with electron-dense material in the cytoplasm) was identified when compared with control woodlice. Large mitochondria with distinctly visible electron-dense precipitations inside them were present in the cells of animals from groups (Ni1) and (Ni3). In addition, a disturbed internal mitochondrial membrane system was observed in the cells of the (Ni2) woodlice group.