Both ANT and ATPase are essential for mitochondrial permeability transition but not depolarization.

An increase in permeability of the mitochondrial inner membrane, mitochondrial permeability transition (PT), is the central event responsible for cell death and tissue damage in conditions such as stroke and heart attack. PT is caused by the cyclosporin A (CSA)-dependent calcium-induced pore, the permeability transition pore (PTP). The molecular details of PTP are incompletely understood. We utilized holographic and fluorescent microscopy to assess the contribution of ATP synthase and adenine nucleotide translocator (ANT) toward PTP. In cells lacking either ATP synthase or ANT, we observed CSA-sensitive membrane depolarization, but not high-conductance PTP. In wild-type cells, calcium-induced CSA-sensitive depolarization preceded opening of PTP, which occurred only after nearly complete mitochondrial membrane depolarization. We propose that both ATP synthase and ANT are required for high-conductance PTP but not depolarization, which presumably occurs through activation of the low-conductance PT, which has a molecular nature that is different from both complexes.

Electrophysiological properties of the mitochondrial permeability transition pores: Channel diversity and disease implication.

The mitochondrial permeability transition pore (mPTP) is a channel that, when open, is responsible for a dramatic increase in the permeability of the mitochondrial inner membrane, a process known as the mitochondrial permeability transition (mPT). mPTP activation during Ca2+ dyshomeostasis and oxidative stress disrupts normal mitochondrial function and induces cell death. mPTP opening has been implicated as a critical event in many diseases, including hypoxic injuries, neurodegeneration, and diabetes. Discoveries of recent years indicate that mPTP demonstrates very complicated behavior and regulation, and depending on specific induction or stress conditions, it can function as a high-conductance pore, a small channel, or a non-specific membrane leak. The focus of this review is to summarize the literature on the electrophysiological properties of the mPTP and to evaluate the evidence that it has multiple molecular identities. This review also provides perspective on how an electrophysiological approach can be used to quantitatively investigate the biophysical properties of the mPTP under physiological, pharmacological, pathophysiological, and disease conditions.

Comparative Analysis of the Concentrations of Ions (Na+, K+, Ca2+, and Cl‒) in the Blood of the Brown Trout Salmo trutta from Different Rivers.

The contents of ions in the blood have been compared in three ecological groups of brown trout (parr, mature, and resident) living in rives of the basins of Lake Onega and Lake Ladoga. Brown trout from the Alats-oya (the Ladoga basin, remote from two other rivers belonging to the Onega Lake) is characterized by the lowest concentrations of the Na+, Ca2+, and Cl- ions in blood. The probable effect of the ion level in blood of brown trout on selection of life strategy is discussed.

Mitochondrial permeability transition pore induction is linked to formation of the complex of ATPase C-subunit, polyhydroxybutyrate and inorganic polyphosphate.

Mitochondrial permeability transition pore (mPTP) opening allows free movement of ions and small molecules leading to mitochondrial membrane depolarization and ATP depletion that triggers cell death. A multi-protein complex of the mitochondrial ATP synthase has an essential role in mPTP. However, the molecular identity of the central 'pore' part of mPTP complex is not known. A highly purified fraction of mammalian mitochondria containing C-subunit of ATPase (C-subunit), calcium, inorganic polyphosphate (polyP) and polyhydroxybutyrate (PHB) forms ion channels with properties that resemble the native mPTP. We demonstrate here that amount of this channel-forming complex dramatically increases in intact mitochondria during mPTP activation. This increase is inhibited by both Cyclosporine A, an inhibitor of mPTP and Ruthenium Red, an inhibitor of the Mitochondrial Calcium Uniporter. Similar increases in the amount of complex formation occurs in areas of mouse brain damaged by ischemia-reperfusion injury. These findings suggest that calcium-induced mPTP is associated with de novo assembly of a channel comprising C-subunit, polyP and PHB.

Contribution of inorganic polyphosphate towards regulation of mitochondrial free calcium.

BACKGROUND: Calcium signaling plays a key role in the regulation of multiple processes in mammalian mitochondria, from cellular bioenergetics to the induction of stress-induced cell death. While the total concentration of calcium inside the mitochondria can increase by several orders of magnitude, the concentration of bioavailable free calcium in mitochondria is maintained within the micromolar range by the mitochondrial calcium buffering system. This calcium buffering system involves the participation of inorganic phosphate. However, the mechanisms of its function are not yet understood. Specifically, it is not clear how calcium-orthophosphate interactions, which normally lead to formation of insoluble precipitates, are capable to dynamically regulate free calcium concentration. Here we test the hypothesis that inorganic polyphosphate, which is a polymerized form of orthophosphate, is capable to from soluble complexes with calcium, playing a significant role in the regulation of the mitochondrial free calcium concentration. METHODS: We used confocal fluorescence microscopy to measure the relative levels of mitochondrial free calcium in cultured hepatoma cells (HepG2) with variable levels of inorganic polyphosphate (polyP). RESULTS: The depletion of polyP leads to the significantly lower levels of mitochondrial free calcium concentration under conditions of pathological calcium overload. These results are coherent with previous observations showing that inorganic polyphosphate (polyP) can inhibit calcium-phosphate precipitation and, thus, increase the amount of free calcium. CONCLUSIONS: Inorganic polyphosphate plays an important role in the regulation of mitochondrial free calcium, leading to its significant increase. GENERAL SIGNIFICANCE: Inorganic polyphosphate is a previously unrecognized integral component of the mitochondrial calcium buffering system.

[Cytological State of Gonads and Level of Thyroid and Sex Steroid Hormones in Black Sea Trout Salmo trutta labrax Pall].

Cytological state of the gonads and hormonal state of hatchery Black Sea trout before differentiation into resident and anadromous forms (parr) at an age of 15 months have been examined. It has been shown that the hormonal changes associated with the choice of life strategy in the Black Sea trout females and males are pronounced to different degrees. As compared with the resident and anadromous individuals; the female parr display a low rate of oogenesis and similar hormonal status, while characteristic of the male parr are an intermediate rate of spermatogenesis, a low level of thyroid hormones and estradiol, and a medium testosterone level. As has been found, the undifferentiated Black Sea trout individuals predominantly develop into the resident form.

Synergistic neuroprotection by epicatechin and quercetin: Activation of convergent mitochondrial signaling pathways.

In view of evidence that increased consumption of epicatechin (E) and quercetin (Q) may reduce the risk of stroke, we have measured the effects of combining E and Q on mitochondrial function and neuronal survival following oxygen-glucose deprivation (OGD). Relative to mouse cortical neuron cultures pretreated (24h) with either E or Q (0.1-10µM), E+Q synergistically attenuated OGD-induced neuronal cell death. E, Q and E+Q (0.3µM) increased spare respiratory capacity but only E+Q (0.3µM) preserved this crucial parameter of neuronal mitochondrial function after OGD. These improvements were accompanied by corresponding increases in cyclic AMP response element binding protein (CREB) phosphorylation and the expression of CREB-target genes that promote neuronal survival (Bcl-2) and mitochondrial biogenesis (PGC-1α). Consistent with these findings, E+Q (0.1 and 1.0µM) elevated mitochondrial gene expression (MT-ND2 and MT-ATP6) to a greater extent than E or Q after OGD. Q (0.3-3.0µM), but not E (3.0µM), elevated cytosolic calcium (Ca(2+)) spikes and the mitochondrial membrane potential. Conversely, E and E+Q (0.1 and 0.3µM), but not Q (0.1 and 0.3µM), activated protein kinase B (Akt). Nitric oxide synthase (NOS) inhibition with L-N(G)-nitroarginine methyl ester (1.0µM) blocked neuroprotection by E (0.3µM) or Q (1.0µM). Oral administration of E+Q (75mg/kg; once daily for 5days) reduced hypoxic-ischemic brain injury. These findings suggest E and Q activate Akt- and Ca(2+)-mediated signaling pathways that converge on NOS and CREB resulting in synergistic improvements in neuronal mitochondrial performance which confer profound protection against ischemic injury.

[Peculiarities of diagnosis and surgical policy in elderly patients with pathological tortuosity of the internal carotid artery].

The study was aimed at optimizing surgical treatment management of elderly patients presenting with pathological tortuosity of the internal carotid artery (ICA). We examined a total of 94 patients with unilateral haemodynamically significant tortuosity of the ICA. Depending on the age, the patients were subdivided into two groups: Group One comprising forty-six (49%) 50-to-60-year-old patients and Group Two consisting of 48 (51%) patients above 60 years (from 61 to 84 years). 37% of patients had were found to have pathological tortuosity combined with haemodynamically significant ICA stenosis. In these patients linear blood velocity in the zone of the largest deformation was by 15% less than in patients with isolated tortuosity (p<0.05). All patients underwent reconstructive operations of carotid arteries with a good clinical effect. A combination of stenosis and pathological tortuosity of the ICA was treated by eversion carotid endarterectomy with lowering down and reimplantation of the artery into the previous ostium; microaneurysms present in the ICA wall were managed by resection of the artery's portion with autovenous prosthetic repair or bringing down the artery into the previous ostium. In 77% of patients above 60 years the operation was carried out under regional anaesthesia. According to the findings of duplex scanning, rectilinearity of the ICA after surgery was restored in 100% of cases, blood flow was of major type, with no turbulence registered. It was demonstrated that surgical management of elderly patients with pathological tortuosity is an effective method of prevention of ischaemic stroke. The complications rate in patients presenting with combined atherosclerotic lesions of the ICA and its pathological tortuosity, should adequate surgical policy be employed, falls within the framework of the accepted standards and does not depend on the type of ICA lesion.

[Content of Thyroid and Sex Steroid Hormones in Young-of-the-Year of Black Sea Trout Salmo trutta labrax from Two Spatial Groups for Different Duration of Starvation].

The content of thyroid and sex steroid hormones is determined in fish-farm juveniles of Black Sea trout 5.5 months old, from the bottom and pelagic spatial groups differing in the probability of future selection of the resident or anadromous life strategies, respectively. Differences in the concentration of the aforementioned hormones are found in young-of-the-year corresponding to those in the migratory and resident forms of yearlings of trout. In the juveniles from the pelagic group at the age 0+, the level of triiodothyronine, thyroxine, and testosterone is higher than in specimens from the bottom group. Prolonged starvation results in a higher content of triiodthyronine, thyroxine, and testosterone in the blood of juveniles from both spatial groups. The concentration of estradiol-17ß increases in pelagic specimens and decreases in bottom specimens.

[Diurnal dynamics of thyroid and sex steroid hormones in the blood of yearlings of the resident form of Black Sea trout Salmo trutta labrax].

The diurnal dynamics of the content of thyroid and sex steroid hormones is investigated in the blood of the resident form of Black Sea trout in summer. The maximums and minimums of concentration of the investigated hormones do not coincide over 24 h, except for the decrease in the level of T3 and testosterone before dawn. The dynamics of the investigated hormones is controlled to a high extent by the sex of fish in the morning and in the daytime.

[Postspawning survival in lacustrine sock-eyed salmon Oncorhynchus nerka Walb].

The state of gonads, age, structure of scales, and size of specimens of the resident lacustrine form of sock-eyed salmon--kokanee Onchorhynchus nerka--are analyzed. In stocked, previously fishless, lakes, there are specimens that have survived spawning and have remained active for a year or several years. No evidence was found of the possibility of repeated spawning. Thus, such fish do not belong to the spawning stock of the population, and their ecological function is not clear.

[Nibentan in arresting acute atrial fibrillation: risk or benefit?].

AIM: To study efficacy and safety of using a domestic anti-arrhythmic drug nibentan for arresting acute atrial fibrillation. MATERIAL AND METHODS: A total of 210 patients received nibentan in a dose 0.125 mg/kg dissolved in 20.0 ml of 0.9% sodium chloride solution by intravenous slow jet 3-min injection. In no response, the injection was repeated 20 min later. Nibentan injection was accompanied with continuous ECG monitoring, measurements of QT interval, arterial pressure. The criterion of efficacy was atrial fibrillation conversion into the sinus rhythm within 24 hours after injection of a total nibentan dose. RESULTS: Nibentan in a dose 0.125 mg/kg demonstrated high efficacy (91.9%) and sufficient safety (incidence of polymorphic ventricular tachycardia 2.4%) including patients taking other antiarrhythmic drugs. A basic marker of an electrophysiological effect of nibentan on the myocardium is a dynamic change in the interval QT which may serve a predictor of sinus rhythm reestablishment (in QT > or =480 ms) and of a risk to develop proarrhythmogenic complications (in QT > or =640 ms).

Radiation sterilisation of doxorubicin bound to poly(butyl cyanoacrylate) nanoparticles.

Doxorubicin-loaded poly(butyl cyanoacrylate) (PBCA) nanoparticles were prepared by anionic polymerisation under non-aseptic conditions. The feasibility of sterilisation of this formulation using either gamma-irradiation or electron beam irradiation was investigated. The irradiation doses ranged from 10 to 35 kGy. Bacillus pumilus was used as the official test microorganism. The bioburden of the untreated formulation was found to be 100 CFU/g. Microbiological monitoring revealed that at this level of the bioburden the irradiation dose of 15 kGy was sufficient for sterilisation of the nanoparticles. The formulation showed excellent stability with both types of irradiation in the investigated dose range. The irradiation did not influence the physicochemical parameters of the drug-loaded and empty nanoparticles, such as the mean particle size, polydispersity, and aggregation stability. The molecular weights of the PBCA polymer as well as the polydispersity indices (M(w)/M(n)) remained nearly unchanged. The drug substance was stable to radiolysis. Additionally, the presence of irradiation-induced radicals was evaluated by ESR spectroscopy after storage of the particles at ambient temperature. The paramagnetic species found in the formulation were mainly produced by irradiation of mannitol and dextran used as excipients.

A novel, high conductance channel of mitochondria linked to apoptosis in mammalian cells and Bax expression in yeast.

During apoptosis, proapoptotic factors are released from mitochondria by as yet undefined mechanisms. Patch-clamping of mitochondria and proteoliposomes formed from mitochondrial outer membranes of mammalian (FL5.12) cells has uncovered a novel ion channel whose activity correlates with onset of apoptosis. The pore diameter inferred from the largest conductance state of this channel is approximately 4 nm, sufficient to allow diffusion of cytochrome c and even larger proteins. The activity of the channel is affected by Bcl-2 family proteins in a manner consistent with their pro- or antiapoptotic properties. Thus, the channel activity correlates with presence of proapoptotic Bax in the mitochondrial outer membrane and is absent in mitochondria from cells overexpressing antiapoptotic Bcl-2. Also, a similar channel activity is found in mitochondrial outer membranes of yeast expressing human Bax. These findings implicate this channel, named mitochondrial apoptosis-induced channel, as a candidate for the outer-membrane pore through which cytochrome c and possibly other factors exit mitochondria during apoptosis.

Palmitic and stearic acids bind Ca2+ with high affinity and form nonspecific channels in black-lipid membranes. Possible relation to Ca2+-activated mitochondrial pores.

A mitochondrial hydrophobic component that forms Ca2+-induced nonspecific ion channels in black-lipid membranes (Mironova et al., 1997) has been purified and its nature elucidated. It consists of long-chain saturated fatty acids--mainly palmitic and stearic. These fatty acids, similar to the mitochondrial hydrophobic component, bind Ca2+ with high affinity in comparison with unsaturated fatty acids, saturated fatty acids with shorter aliphatic chains, phospholipids, and other lipids. Ca2+-binding is inhibited by Mg2+ but not by K+. For palmitic acid, the Kd for Ca2+ was 5 microM at pH 8.5 and 15 microM at pH 7.5, with the Bmax of 0.48 +/- 0.08 mmol/g. This corresponds to one Ca2+ ion for eight palmitic acid molecules. The data of IR spectroscopy confirm that Ca2+ does not form ionic bonds with palmitic and stearic acids under hydrophobic conditions. It has been found that in the presence of Ca2+, palmitic and stearic acids, but not unsaturated FFA induce a nonspecific permeability in black-lipid membranes. Addition of Ca2+ in order to induce the permeability transition, increases the extractable amount of palmitic and stearic acids, the effect being prevented by a phospholipase A2 inhibitor. The possible involvement of palmitic and stearic acids in the mitochondrial nonspecific permeability is discussed.

Calcium-binding properties of the mitochondrial channel-forming hydrophobic component.

A hydrophobic, low-molecular weight component extracted from mitochondria forms a Ca2+-activated ion channel in black-lipid membranes (Mironova et al., 1997). At pH 8.3-8.5, the component has a high-affinity binding site for Ca2+ with a Kd of 8 x 10(-6) M, while at pH 7.5 this Kd was decreased to 9 x 10(-5) M. Bmax for the Ca2+-binding site did not change significantly with pH. In the range studied, 0.2 +/- 0.06 mmol Ca2+/g component were bound or one calcium ion to eight molecules of the component. The Ca2+ binding was strongly decreased by 50-100 mM Na+, but not by K+. Treatment of mitochondria with CaCl2 prior to ethanolic extraction resulted in a high level of Ca2+-binding capacity of the partially purified component. Cyclosporin A, a specific inhibitor of the mitochondrial permeability transition, when added to the mitochondrial suspension, decreased the Ca2+-binding activity of the purified extract severalfold. The calcium-binding capability of the partially purified component correlates with its calcium-channel activity. This indicates that the channel-forming component might be involved in the permeability transition that stimulates its formation.

[Respiratory insufficiency in adults with diphtheric polyneuropathy]. / Dykhatel'naia nedostatochnost' pri difteriinoi polineiropatii u vzroslykh.

Twenty-five adult patients with grave diphtheric polyneuropathy after toxic diphtheria were followed up. Two symptom complexes of neurologic disorders leading to the development of peripheral respiratory failure of different severity were distinguished: 1) predominating glossopharyngeal paralysis and 2) combination of glossopharyngeal paralysis with grave generalized sensorimotor polyneuropathy (with pareses and paralyses of the respiratory muscles). The major quantitative parameters of pulmonary functions associated with various manifestations of respiratory failure are characterized. The significance of information on the type of dysfunction of vocal cords is emphasized. A high efficacy of a complex of respiratory reanimation used in the treatment of patients with peripheral respiratory failure is demonstrated.

Investigation of five cases of vesicular enteroviral stomatitis with exanthema induced by coxsackie A5 virus.

Five cases of vesicular enteroviral stomatitis with exanthema or hand-foot-mouth disease are described. The diagnosis was based on the clinical aspect, the Coxsackie A5 virus isolation from pathological samples, and confirmed by serological tests.