DNA microarrays based on noncovalent oligonucleotide attachment and hybridization in two dimensions

Short oligonucleotide probes have been linked to a solid support by simple electrostatic adsorption onto a positively charged surface film. Attachment was obtained by microfluidic application of unmodified oligonucleotides in distilled water onto amino-silanized glass. It has been demonstrated that an extremely stable monolayer of oligonucleotide is obtained by this method, at a density of about 10(11) molecules/mm(2), which approaches the limit expected for a two-dimensional closest-packed array. Application of oligonucleotide by adsorption is followed by capping with acetic anhydride in the vapor phase, and then capping with succinic anhydride in solution to form a surface with weak negative charge. The capping method has been successfully employed for microarray fabrication and for the analysis of single nucleotide polymorphisms in the k-ras gene. The data reveal that, subsequent to capping, the adsorptive association of oligonucleotide to the surface yields a probe layer which is capable of single nucleotide base mismatch discrimination and high apparent binding affinity.     

Role of mitochondria in hepatotoxicity of ethanol

The current understanding of the effects of alcohol intoxication on the basic mitochondrial functions has been presented. Both, the direct toxic effect of ethanol on biological membranes and various cellular systems and the toxicity of acetaldehyde and reactive oxygen species (the products of ethanol oxidation) are discussed, with emphasis on the effect of ethanol on the basic functions of mitochondria and Ca²⁺-dependent mitochondrial permeability transition. Based on the available experimental data, it is demonstrated that acute alcohol intoxication causes a global mitochondrial dysfunction in the liver, resulting in considerable disturbance of the whole cellular metabolism. Alcohol poisoning of the liver leads to a decreased ability of cells to withstand oxidative stress, to support the synthesis of vital metabolic intermediates (e.g., methyl groups), as well as to produce urea from ammonia, due to a decreased permeability of the outer membrane and impaired exchange of substrates between the cytoplasm and the mitochondrial matrix. This review emphasizes the role of porin channels of the outer mitochondrial membrane in ethanol-mediated disturbances of basic mitochondrial functions and its consequences for the entire cell metabolism in the liver.     

Ultrastructural and Functional Changes in Liver Mitochondria in a Rat Model of Type I Diabetes Mellitus

Biophysics - Abstract—The characteristics of the ultrastructure and functioning of mitochondria in the liver of Sprague Dawley rats in the experimental model of type I diabetes mellitus have...     

A permeability transition in liver mitochondria and liposomes induced by α,ω-dioic acids and Ca2+

The article examines the molecular mechanism of the Ca²⁺-dependent cyclosporin A (CsA)-insensitive permeability transition in rat liver mitochondria induced by α,ω-dioic acids. The addition of α,ω-hexadecanedioic acid (HDA) to Ca²⁺-loaded liver mitochondria was shown to induce a high-amplitude swelling of the organelles, a drop of membrane potential and the release of Ca²⁺ from the matrix, the effects being insensitive to CsA. The experiments with liposomes loaded with sulforhodamine B (SRB) revealed that, like palmitic acid (PA), HDA was able to cause permeabilization of liposomal membranes. However, the kinetics of HDA- and PA-induced release of SRB from liposomes was different, and HDA was less effective than PA in the induction of SRB release. Using the method of ultrasound interferometry, we also showed that the addition of Ca²⁺ to HDA-containing liposomes did not change the phase state of liposomal membranes—in contrast to what was observed when Ca²⁺ was added to PA-containing vesicles. It was suggested that HDA/Ca²⁺- and PA/Ca²⁺-induced permeability transition occurs by different mechanisms. Using the method of dynamic light scattering, we further revealed that the addition of Ca²⁺ to HDA-containing liposomes induced their aggregation/fusion. Apparently, these processes result in a partial release of SRB due to the formation of fusion pores. The possibility that this mechanism underlies the HDA/Ca²⁺-induced permeability transition of the mitochondrial membrane is discussed.     

Mitochondrial lipid pore in the mechanism of glutamate-induced calcium deregulation of brain neurons

The work examines the mechanism of central nerve cell death upon stimulation of brain NMDA receptors with the stimulatory mediator glutamate. A prolonged stimulation of neurons with glutamate is known to result in the disorder of Ca²⁺ homeostasis and severe mitochondrial depolarization followed by cell death. It has been shown that the overload of mitochondria with Sr²⁺ leads to the release of the cation, medium alkalization, decrease of membrane potential and mitochondrial swelling, indicating a nonspecific permeabilization of the mitochondrial membrane. The permeabilization, in our opinion, is caused by the activation of Ca²⁺/Sr²⁺-dependent phospholipase A₂ (PLA₂), resulting in the formation of free palmitic and stearic acids in the mitochondrial membrane. These fatty acids bind Ca²⁺ with high affinity and the process of binding is accompanied by the formation of a transient lipid pore—a phenomenon demonstrated earlier on both artificial and mitochondrial membranes. The inhibitors of PLA₂ have been shown to suppress permeabilization of mitochondrial membranes. In the culture of granular cerebellum neurons, the PLA₂ inhibitors prolonged the lag of the delayed Sr²⁺ deregulation and membrane depolarization. On the basis of data obtained on isolated mitochondria and neurons we suppose that the initial stages of glutamate-induced Ca²⁺ deregulation of neurons are underlain by the opening of lipid pores in brain mitochondria.     

A permeability transition in liposomes induced by the formation of Ca/palmitic acid complexes

Formation of palmitic acid/Ca²⁺ (PA/Ca²⁺) complexes was suggested to play a key role in the non-classical permeability transition in mitochondria (NCPT), which seems to be involved in the PA-induced apoptosis of cardiomyocytes. Our previous studies of complexation of free fatty acids (FFA) with Ca²⁺ showed that long-chain (C:16-C:22) saturated FFA had an affinity to Ca²⁺, which was much higher than that of other FFA and lipids. The formation of FFA/Ca²⁺ complexes in the black-lipid membrane (BLM) was demonstrated to induce a nonspecific ion permeability of the membrane. In the present work, we have found that binding of Ca²⁺ to PA incorporated into the membrane of sulforhodamine B (SRB)-loaded liposomes results in an instant release of a part of SRB, with the quantity of SRB released depending on the concentration of PA and Ca²⁺. The pH-optimum of this phenomenon, similar to that of PA/Ca²⁺ complexation, is in the alkaline range. The same picture of SRB release has been revealed for stearic, but not for linoleic acid. Along with Ca²⁺, some other bivalent cations (Ba²⁺, Sr²⁺, Mn²⁺, Ni²⁺, Co²⁺) also induce SRB release upon binding to PA-containing liposomes, while Mg²⁺ turns out to be relatively ineffective. As revealed by fluorescence correlation spectroscopy, the apparent size of liposomes does not alter after the addition of PA, Ca²⁺ or their combination. So it has been supposed that the cause of SRB release from liposomes is the formation of lipid pores. The effect of FFA/Ca²⁺-induced permeabilization of liposomal membranes has several analogies with NCPT, suggesting that both these phenomena are of similar nature.     

Interaction of Phospholipase A of the E. coli Outer Membrane with the Inhibitors of Eucaryotic Phospholipases A2 and Their Effect on the Ca2+-Induced Permeabilization of the Bacterial Membrane

Phospholipase A of the bacterial outer membrane (OMPLA) is a β-barrel membrane protein which is activated under various stress conditions. The current study examines interaction of inhibitors of eucaryotic phospholipases A₂—palmitoyl trifluoromethyl ketone (PACOCF₃) and aristolochic acid (AA)—with OMPLA and considers a possible involvement of the enzyme in the Ca²⁺-dependent permeabilization of the outer membrane of Escherichia coli. Using the method of molecular docking, it has been predicted that PACOCF₃ and AA bind to OMPLA at the same site and with the same affinity as the OMPLA inhibitors, hexadecanesulfonylfluoride and bromophenacyl bromide, and the substrate of the enzyme palmitoyl oleoyl phosphatidylethanolamine. It has also been shown that PACOCF₃, AA, and bromophenacyl bromide inhibit the Ca²⁺-induced temperature-dependent changes in the permeability of the bacterial membrane for the fluorescent probe propidium iodide and suppressed the transformation of E. coli cells with plasmid DNA induced by Ca²⁺ and heat shock. The cell viability was not affected by the eucaryotic phospholipases A₂ inhibitors. The study discusses a possible involvement of OMPLA in the mechanisms of bacterial transmembrane transport based on the permeabilization of the bacterial outer membrane.     

Oxidative phosphorylation and ion transport in the mitochondria of two strains of rats varying in their resistance to stress and hypoxia

The role of mitochondria in the inherited or ontogenetically acquired reactions of organism to stress is not studied enough. In the present work, we examined the functional state of the coupled respiratory chain, potassium and calcium transport and rate of hydrogen peroxide production on two rat lines: August and Wistar—which possess different resistance to emotional stress and hypoxia. It was established that the respiration rate and efficiency of oxidative phosphorylation were higher in August rats than in Wistar ones. In August rats, the rate of potassium transport and ATP-dependent mitochondrial swelling as well as the concentration of the ion in the mitochondrial matrix were almost twice as higher comparatively to those parameters in Wistar rats. The rate of H₂O₂ production was found to be decreased in the mitochondria of August rats. It was also demonstrated that the two rat lines differed by their resistance to the opening of the palmitate/Ca²⁺-induced pore and by their ability to retain calcium within mitochondria. The paper discusses the involvement of the mitochondrial ATP-dependent potassium channel in the adaptation of animals to adverse effects.     

A permeability transition in liposomes induced by the formation of Ca2+/palmitic acid complexes

Formation of palmitic acid/Ca(2+) (PA/Ca(2+)) complexes was suggested to play a key role in the non-classical permeability transition in mitochondria (NCPT), which seems to be involved in the PA-induced apoptosis of cardiomyocytes. Our previous studies of complexation of free fatty acids (FFA) with Ca(2+) showed that long-chain (C:16-C:22) saturated FFA had an affinity to Ca(2+), which was much higher than that of other FFA and lipids. The formation of FFA/Ca(2+) complexes in the black-lipid membrane (BLM) was demonstrated to induce a nonspecific ion permeability of the membrane. In the present work, we have found that binding of Ca(2+) to PA incorporated into the membrane of sulforhodamine B (SRB)-loaded liposomes results in an instant release of a part of SRB, with the quantity of SRB released depending on the concentration of PA and Ca(2+). The pH-optimum of this phenomenon, similar to that of PA/Ca(2+) complexation, is in the alkaline range. The same picture of SRB release has been revealed for stearic, but not for linoleic acid. Along with Ca(2+), some other bivalent cations (Ba(2+), Sr(2+), Mn(2+), Ni(2+), Co(2+)) also induce SRB release upon binding to PA-containing liposomes, while Mg(2+) turns out to be relatively ineffective. As revealed by fluorescence correlation spectroscopy, the apparent size of liposomes does not alter after the addition of PA, Ca(2+) or their combination. So it has been supposed that the cause of SRB release from liposomes is the formation of lipid pores. The effect of FFA/Ca(2+)-induced permeabilization of liposomal membranes has several analogies with NCPT, suggesting that both these phenomena are of similar nature.     

The effect of DS16570511, a new inhibitor of mitochondrial calcium uniporter,on calcium homeostasis,metabolism, and functional state of cultured cortical neurons and isolated brain mitochondria

BackgroundDisorders of mitochondrial Ca²⁺ homeostasis play a key role in the glutamate excitotoxicity of brain neurons. DS16570511 (DS) is a new penetrating inhibitor of mitochondrial Ca²⁺ uniporter complex (MCUC). The paper examines the effects of DS on the cultivated cortical neurons and isolated mitochondria of the rat brain.MethodsThe functions of neurons and mitochondria were examined using fluorescence microscopy, XF24 microplate-based сell respirometry, ion-selective microelectrodes, spectrophotometry, and polarographic technique.ResultsAt the doses of 30 and 45 μM, DS reliably slowed down the onset of glutamate-induced delayed calcium deregulation of neurons and suppressed their death. 30 μM DS caused hyperpolarization of mitochondria of resting neurons, and 45 μM DS temporarily depolarized neuronal mitochondria. It was also demonstrated that 30–60 μM DS stimulated cellular respiration. DS was shown to suppress Ca²⁺ uptake by isolated brain mitochondria. In addition, DS inhibited ADP-stimulated mitochondrial respiration and ADP-induced decrease in the mitochondrial membrane potential. It was found that DS inhibited the activity of complex II of the respiratory chain. In the presence of Ca²⁺, high DS concentrations caused a collapse of the mitochondrial membrane potential.ConclusionsThe data obtained indicate that, in addition to the inhibition of MCUC, DS affects the main energy-transducing functions of mitochondria.General significanceThe using DS as a tool for studying MCUC and its functional role in neuronal cells should be done with care, bearing in mind multiple effects of DS, a proper evaluation of which would require multivariate analysis.