TIP47 protects mitochondrial membrane integrity and inhibits oxidative-stress-induced cell death

We found that overexpression of tail interacting protein of 47 kDa (TIP47), but not its truncated form (t-TIP47) protected NIH3T3 cells from hydrogen-peroxide-induced cell death, prevented the hydrogen-peroxide-induced mitochondrial depolarization determined by 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazolylcarbocyanine iodide (JC1), while suppression of TIP47 in HeLa cells facilitated oxidative-stress-induced cell death. TIP47 was located to the cytoplasm of untreated cells, but some was associated to mitochondria in oxidative stress. Recombinant TIP47, but not t-TIP47 increased the mitochondrial membrane potential (Δψ), and partially prevented Ca²⁺ induced depolarization. It is assumed that TIP47 can bind to mitochondria in oxidative stress, and inhibit mitochondria mediated cell death by protecting mitochondrial membrane integrity.     

Biochemistry and physiology of mitochondrial ion channels involved in cardioprotection

Over the past decades there has been considerable progress in understanding the multifunctional roles of mitochondrial ion channels in metabolism, energy transduction, ion transport, signaling, and cell death. Recent data have suggested that some of these channels function under physiological condition, and others may be activated in response to pathological insults and play a key role in cytoprotection. This review outlines our current understanding of the molecular identity and pathophysiological roles of the mitochondrial ion channels in the heart with particular emphasis on cardioprotection against ischemia/reperfusion injury, and future research on mitochondrial ion channels.     

Differential Involvement of Intracellular Ca2+ in 1-Methyl-4-phenylpyridinium- or 6-Hydroxydopamine-Induced Cell Viability Loss in PC12 Cells

1-Methyl-4-phenylpyridinium (MPP⁺) or 6-hydroxydopamine (6-OHDA) caused a nuclear damage, the mitochondrial membrane permeability changes, leading to the cytochrome c release and caspase-3 activation, the formation of reactive oxygen species and the depletion of GSH in PC12 cells. Nicardipine (a calcium channel blocker), EGTA (an extracellular calcium chelator), BAPTA-AM (a cell permeable calcium chelator) and calmodulin antagonists (W-7 and calmidazolium) attenuated the MPP⁺-induced mitochondrial damage and cell death. In contrast, the compounds did not reduce the toxicity of 6-OHDA. Treatment with MPP⁺ or 6-OHDA evoked the elevation of intracellular Ca²⁺ levels. Unlike cell injury, addition of nicardipine, BAPTA-AM and calmodulin antagonists prevented the elevation of intracellular Ca²⁺ levels due to both toxins. The results show that the MPP⁺-induced formation of the mitochondrial permeability transition seems to be mediated by elevation of intracellular Ca²⁺ levels and calmodulin action. In contrast, the 6-OHDA-induced cell death seems to be mediated by Ca²⁺-independent manner.     

Expression analysis of LacZ gene placed in the locus of Cnot7 exhibits its activity in osteoblasts in vivo and in mineralized nodules in vitro

CCR4-NOT complex 7 (Cnot7) was identified as a regulator of gene expression in yeast and evolutionally conserved in mammals. Cnot7 deficient male mice exhibit abnormality in spermatogenesis. As these mice contained construct to express LacZ, we followed the expression patterning in these animals. LacZ was expressed in osteoblasts located in the primary spongiosa in adult mice. Cellular analysis indicated that LacZ is expressed in osteoblasts but not in osteoclasts. In the mineralized nodules formed in the culture of bone marrow cells obtained from Cnot7 +/- mice, LacZ expression was mainly observed in the cells forming mineralized nodules but not in un-mineralized area scattered around the periphery of the nodules. LacZ blue positive cells were gradually depositing minerals along its time course of the in vitro mineralization assay. Cnot7 expression was enhanced by the treatment with BMP. These data suggest that Cnot7 is expressed in osteoblasts and is associated with mineralization.     

Sodium/calcium selectivity of cloned calcium T-type channels

We studied the peculiarities of permeability with respect to the main extracellular cations, Na⁺ and Ca²⁺, of cloned low-threshold calcium channels (LTCCs) of three subtypes, Ca_v3.1 (α1G), Ca_v3.2 (α 1H), and Ca_v3.3 (α1I), functionally expressed in Xenopus oocytes. In a calcium-free solution containing 100 mM Na⁺ and 5 mM calcium-chelating EGTA buffer (to eliminate residual concentrations of Ca²⁺) we observed considerable integral currents possessing the kinetics of inactivation typical of LTCCs and characterized by reversion potentials of −10 ± 1, −12 ± 1, and −18 ± 2 mV, respectively, for Ca_v3.1, Ca_v3.2, and Ca_v3.3 channels. The presence of Ca²⁺ in the extracellular solution exerted an ambiguous effect on the examined currents. On the one hand, Ca²⁺ effectively blocked the current of monovalent cations through cloned LTCCs (K _d = 2, 10, and 18 μM for currents through channels Ca_v3.1, Ca_v3.2, and Ca_v3.3, respectively). On the other hand, at the concentration of 1 to 100 mM, Ca²⁺ itself functioned as a carrier of the inward current. Despite the fact that the calcium current reached the level of saturation in the presence of 5 mM Ca²⁺ in the external solution, extracellular Na⁺ influenced the permeability of these channels even in the presence of 10 mM Ca²⁺. The Ca_v3.3 channels were more permeable with respect to Na⁺ (P _Ca/P _Na ∼ 21) than Ca_v3.1 and Ca_v3.2 (P _Ca/P _Na ∼ 66). As a whole, our data indicate that cloned LTCCs form multi-ion Ca²⁺-selective pores, as these ions possess a high affinity for certain binding sites. Monovalent cations present together with Ca²⁺ in the external solution modulate the calcium permeability of these channels. Among the above-mentioned subtypes, Ca_v3.3 channels show the minimum selectivity with respect to Ca²⁺ and are most permeable for monovalent cations. Neirofiziologiya/Neurophysiology, Vol. 38, No. 3, pp. 183–192, May–June, 2006.     

Transgenic Medicago truncatula plants that accumulate proline display nitrogen-fixing activity with enhanced tolerance to osmotic stress

Legume root nodule nitrogen-fixing activity is severely affected by osmotic stress. Proline accumulation has been shown to induce tolerance to salt stress, and transgenic plants over-expressing Delta(1)-pyrroline-5-carboxylate synthetase (P5CS), which accumulates high levels of proline, display enhanced osmotolerance. Here, we transformed the model legume Medicago truncatula with the P5CS gene from Vigna aconitifolia, and nodule activity was evaluated under osmotic stress in transgenic plants that showed high proline accumulation levels. Nitrogen fixation was significantly less affected by salt treatment compared to wild-type (WT) plants. To our knowledge, this is the first time that transgenic legumes have been produced that display nitrogen-fixing activity with enhanced tolerance to osmotic stress. We studied the expression of M. truncatula proline-related endogenous genes M. truncatulaDelta(1)-pyrroline-5-carboxylate synthetase 1 (MtP5CS1), M. truncatulaDelta(1)-pyrroline-5-carboxylate synthetase 2 (MtP5CS2), M. truncatula ornithine delta-aminotransferase (MtOAT), M. truncatula proline dehydrogenase (MtProDH) and a proline transporter gene in both WT and transgenic plants. Our results indicate that proline metabolism is finely regulated in response to osmotic stress in an organ-specific manner. The transgenic model allowed us to analyse some of the biochemical and molecular mechanisms that are activated in the nodule in response to high salt conditions, and to ascertain the essential role of proline in the maintenance of nitrogen-fixing activity under osmotic stress.     

Effects of isocoumarins isolated from Paepalanthus bromelioides on mitochondria: uncoupling, and induction/inhibition of mitochondrial permeability transition

Isolated mitochondria may undergo uncoupling, and in presence of Ca(2+) at different conditions, a mitochondrial permeability transition (MPT) linked to protein thiol oxidation, and demonstrated by CsA-sensitive mitochondrial swelling; these processes may cause cell death either by necrosis or by apoptosis. Isocoumarins isolated from the Brazilian plant Paepalanthus bromelioides (Eriocaulaceae) paepalantine (9,10-dihydroxy-5,7-dimethoxy-1H-naptho(2,3c)pyran-1-one), 8,8'-paepalantine dimer, and vioxanthin were assayed at 1-50 microM on isolated rat liver mitochondria, for respiration, MPT, protein thiol oxidation, and interaction with the mitochondrial membrane using 1,6-diphenyl-1,3,5-hexatriene (DPH). The isocoumarins did not significantly affect state 3 respiration of succinate-energized mitochondria; they did however, stimulate 4 respiration, indicating mitochondrial uncoupling. Induction of MPT and protein thiol oxidation were assessed in succinate-energized mitochondria exposed to 10 microM Ca(2+); inhibition of these processes was assessed in non-energized organelles in the presence of 300 microM t-butyl hydroperoxide plus 500 microM Ca(2+). Only paepalantine was an effective MPT/protein thiol oxidation inducer, also releasing cytochrome c from mitochondria; the protein thiol oxidation, unlike mitochondrial swelling, was neither inhibited by CsA nor dependent on the presence of Ca(2+). Vioxanthin was an effective inhibitor of MPT/protein thiol oxidation. All isocoumarins inserted deeply into the mitochondrial membrane, but only paepalantine dimer and vioxantin decreased the membrane's fluidity. A direct reaction with mitochondrial membrane protein thiols, involving an oxidation of these groups, is proposed to account for MPT induction by paepalantine, while a restriction of oxidation of these same thiol groups imposed by the decrease of membrane fluidity, is proposed to account for MPT inhibition by vioxanthin.     

Influence of inhibitors of cytoskeleton proteins on water exchange of wheat roots under the after-action of water stress

The dynamics of water molecular state and transport in winter wheat (Triticum aestivum L.) of roots different resistance cultivars was studied by a biophysical method, Nuclear Magnetic Resonance (NMR), and a physiological method, Water-Holding Capacity (WHC). The effective coefficient of water self-diffusion (D(eff)), spin-spin relaxation times (T(2)) and WHC were measured after structural modification of cytoskeleton by colchicine and cytochalasin B after the action of water stress. New information about molecular mechanisms of water state and water transport regulation determined by the influence of dynamic cytoskeleton structure has been obtained. This is very important for the development of a fundamental theory of water exchange in plants, and for the ways of its optimization under conditions of environmental stress.     

Toxicity of depleted uranium on isolated rat kidney mitochondria

Background

Kidney is known as the most sensitive target organ for depleted uranium (DU) toxicity in comparison to other organs. Although the oxidative stress and mitochondrial damage induced by DU has been well investigated, the precise mechanism of DU-induced nephrotoxicity has not been thoroughly recognized yet.

Methods

Kidney mitochondria were obtained using differential centrifugation from Wistar rats and mitochondrial toxicity endpoints were then determined in both in vivo and in vitro uranyl acetate (UA) exposure cases.

Results

Single injection of UA (0, 0.5, 1 and 2 mg/kg, i.p.) caused a significant increase in blood urea nitrogen and creatinine levels. Isolated mitochondria from the UA-treated rat kidney showed a marked elevation in oxidative stress accompanied by mitochondrial membrane potential (MMP) collapse as compared to control group. Incubation of isolated kidney mitochondria with UA (50, 100 and 200 μM) manifested that UA can disrupt the electron transfer chain at complex II and III that leads to induction of reactive oxygen species (ROS) formation, lipid peroxidation, and glutathione oxidation. Disturbances in oxidative phosphorylation were also demonstrated through decreased ATP concentration and ATP/ADP ratio in UA-treated mitochondria. In addition, UA induced a significant damage in mitochondrial outer membrane. Moreover, MMP collapse, mitochondrial swelling and cytochrome c release were observed following the UA treatment in isolated mitochondria.

General significance

Both our in vivo and in vitro results showed that UA-induced nephrotoxicity is linked to the impairment of electron transfer chain especially at complex II and III which leads to subsequent oxidative stress.