Inhibition by purine nucleotides of the release of reactive oxygen species from muscle mitochondria: indication for a function of uncoupling proteins as superoxide anion transporters

Release of reactive oxygen species (ROS), measured as the sum of hydrogen peroxide (H₂O₂) and superoxide anion radical (), from respiring rat heart and skeletal muscle mitochondria was significantly decreased by millimolar concentrations of GTP or GDP. Attempts to differentiate between the two forms of ROS showed that the release of rather than that of H₂O₂ was affected. Meanwhile, intramitochondrial ROS accumulation, measured by inactivation of aconitase, increased. These results suggest that guanine nucleotides inhibit the release of from mitochondria. As these nucleotides are known inhibitors of uncoupling proteins (UCPs), it is proposed that UCPs may function as carriers of , thus enabling its removal from the matrix compartment.     

Mitochondrial permeability transition involves dissociation of F1FO ATP synthase dimers and C‐ring conformation

The impact of the mitochondrial permeability transition (MPT) on cellular physiology is well characterized. In contrast, the composition and mode of action of the permeability transition pore complex (PTPC), the supramolecular entity that initiates MPT, remain to be elucidated. Specifically, the precise contribution of the mitochondrial F₁F_O ATP synthase (or subunits thereof) to MPT is a matter of debate. We demonstrate that F₁F_O ATP synthase dimers dissociate as the PTPC opens upon MPT induction. Stabilizing F₁F_O ATP synthase dimers by genetic approaches inhibits PTPC opening and MPT. Specific mutations in the F₁F_O ATP synthase c subunit that alter C‐ring conformation sensitize cells to MPT induction, which can be reverted by stabilizing F₁F_O ATP synthase dimers. Destabilizing F₁F_O ATP synthase dimers fails to trigger PTPC opening in the presence of mutants of the c subunit that inhibit MPT. The current study does not provide direct evidence that the C‐ring is the long‐sought pore‐forming subunit of the PTPC, but reveals that PTPC opening requires the dissociation of F₁F_O ATP synthase dimers and involves the C‐ring.     

Astronomically controlled deep-sea life in the Late Cretaceous reconstructed from ultra-high-resolution inoceramid shell archives

The periodicity of the mutual position of celestial bodies in the Earth-Moon-Sun system is crucial to the functioning of life on Earth. Biological rhythms affect most of the processes inside organisms, and some can be recorded in skeletal remains, allowing one to reconstruct the cycles that occur in nature deep in time. In the present study, we have used ultra-high-resolution elemental ratio scans of Mg/Ca, Sr/Ca and Mn/Ca from the fossil, ca. 70 Ma old inoceramid bivalve Inoceramus (Platyceramus) salisburgensis from deep aphotic water and identified a clear regularity of repetition of the geochemical signal every of ~0.006 mm. We estimate that the shell accretion rate is on average ~0.4 cm of shell thickness per lunar year. Visible light–dark lamination, interpreted as a seasonal signal corresponding to the semilunar-related cycle, gives a rough shell age estimate and growth rate for this large bivalve species supported by a dual feeding strategy. We recognize a biological clock that follows either a semilunar (model A) or a tidal (model B) cycle. This cycle of tidal dominance seems to fit better considering the biological behaviour of I. (P.) salisburgensis, including the estimated age and growth rate of the studied specimens. We interpret that the major control in such deep-sea environment, well below the photic zone and storm wave base, was due to barotropic tidal forces, thus changing the water pressure.     

Putative Structural and Functional Coupling of the Mitochondrial BKCa Channel to the Respiratory Chain

Potassium channels have been found in the inner mitochondrial membranes of various cells. These channels regulate the mitochondrial membrane potential, the matrix volume and respiration. The activation of these channels is cytoprotective. In our study, the single-channel activity of a large-conductance Ca²⁺-regulated potassium channel (mitoBK_Ca channel) was measured by patch-clamping mitoplasts isolated from the human astrocytoma (glioblastoma) U-87 MG cell line. A potassium-selective current was recorded with a mean conductance of 290 pS in symmetrical 150 mM KCl solution. The channel was activated by Ca²⁺ at micromolar concentrations and by the potassium channel opener NS1619. The channel was inhibited by paxilline and iberiotoxin, known inhibitors of BK_Ca channels. Western blot analysis, immuno-gold electron microscopy, high-resolution immunofluorescence assays and polymerase chain reaction demonstrated the presence of the BK_Ca channel β4 subunit in the inner mitochondrial membrane of the human astrocytoma cells. We showed that substrates of the respiratory chain, such as NADH, succinate, and glutamate/malate, decrease the activity of the channel at positive voltages. This effect was abolished by rotenone, antimycin and cyanide, inhibitors of the respiratory chain. The putative interaction of the β4 subunit of mitoBK_Ca with cytochrome c oxidase was demonstrated using blue native electrophoresis. Our findings indicate possible structural and functional coupling of the mitoBK_Ca channel with the mitochondrial respiratory chain in human astrocytoma U-87 MG cells.     

Expression and purification of active plant diphosphonucleotide phosphatase/phosphodiesterase from baculovirus-infected insect cells

We have previously purified and characterized a diphosphonucleotide phosphatase/phosphodiesterase (PPD1) from yellow lupin seeds. This report describes an efficient strategy for overexpression in baculovirus-infected Spodoptera frugiperda Sf9 cells and purification of a functional PPD1 enzyme. We tested six variants of recombinant PPD1, differing in secretion peptides, fusion tags, and promoters. The highest expression level of the active PPD1 was achieved when the native signal peptide and the C-terminal V5-6His tag were attached. This recombinant protein was secreted at very high level (18.4 mg/L) to serum-free medium. Single-step purification procedure using metal affinity chromatography resulted in the homogeneous PPD1. The overexpressed protein showed enzymatic activity comparable to the native enzyme isolated previously from plant material. We showed that PPD1, which belongs to purple acid phosphatase family, formed tetrameric structure, which is non-typical for this group of enzymes.     

Non-homologous DNA end joining repair in normal and leukemic cells depends on the substrate ends

Double-strand breaks (DSBs) are the most serious DNA damage which, if unrepaired or misrepaired, may lead to cell death, genomic instability or cancer transformation. In human cells they can be repaired mainly by non-homologous DNA end joining (NHEJ). The efficacy of NHEJ pathway was examined in normal human lymphocytes and K562 myeloid leukemic cells expressing the BCR/ABL oncogenic tyrosine kinase activity and lacking p53 tumor suppressor protein. In our studies we employed a simple and rapid in vitro DSB end joining assay based on fluorescent detection of repair products. Normal and cancer cells were able to repair DNA damage caused by restriction endonucleases, but the efficiency of the end joining was dependent on the type of cells and the structure of DNA ends. K562 cells displayed decreased NHEJ activity in comparison to normal cells for 5' complementary DNA overhang. For blunt-ended DNA there was no significant difference in end joining activity. Both kinds of cells were found about 10-fold more efficient for joining DNA substrates with compatible 5' overhangs than those with blunt ends. Our recent findings have shown that stimulation of DNA repair could be involved in the drug resistance of BCR/ABL-positive cells in anticancer therapy. For the first time the role of STI571 was investigated, a specific inhibitor of BCR/ABL oncogenic protein approved for leukemia treatment in the NHEJ pathway. Surprisingly, STI571 did not change the response of BCR/ABL-positive K562 cells in terms of NHEJ for both complementary and blunt ends. Our results suggest that the various responses of the cells to DNA damage via NHEJ can be correlated with the differences in the genetic constitution of human normal and cancer cells. However, the role of NHEJ in anticancer drug resistance in BCR/ABL-positive cells is questionable.     

Spread of alien (non-indigenous) fish species <Emphasis Type="Italic">Neogobius melanostomus</Emphasis> in the Gulf of Gdansk (south Baltic)

The round goby (Neogobius melanostomus) was first noticed in the Gulf of Gdansk in 1990. This Ponto-Caspian fish was most likely introduced to the Baltic environment from ships ballast water. During the first years of invasion, slow population growth was observed in the initially colonized regions. From 1994, round goby gradually spread in all shallow water zones of the Gulf of Gdansk. The area occupied by the population and the number of fish grew rapidly. By the end of the nineties, round goby became a dominant fish in shallow waters of the western part of the Gulf of Gdansk, where the fish used all solid substrates on the bottom, including concrete piers, big stones or even dumped waste for spawning and refuge. The presence of round goby in the Vistula Lagoon and shallow waters near Rugia (Western Baltic) was noticed for the first time in 1999. The present state, size and condition of the round goby population in the Gulf of Gdansk led us to predict a continual spread of this species into new regions of the Baltic. In addition, the population of round goby is so large that the species has started playing a part in commercial fishing in the Gulf of Gdansk.     

Mitochondrial fission in apoptosis

Mitochondria fuse and divide continuously within cells to form a dynamic network. One of the steps in apoptosis is the fragmentation of mitochondria, and recent evidence indicates that the mitochondrial fission machinery actively participates in the process of programmed cell death.