Absence of Low-Energy Shape Coexistence in

The ^{80}Ge structure was investigated in a high-statistics ß-decay experiment of ^{80}Ga using the GRIFFIN spectrometer at TRIUMF-ISAC through γ, ß-e, e-γ, and γ-γ spectroscopy. No evidence was found for the recently reported 0_{2}^{+} 639-keV level suggested as evidence for low-energy shape coexistence in ^{80}Ge. Large-scale shell model calculations performed in ^{78,80,82}Ge place the 0_{2}^{+} level in ^{80}Ge at 2 MeV. The new experimental evidence combined with shell model predictions indicate that low-energy shape coexistence is not present in ^{80}Ge.

Non-interpretive radiology: an Irish perspective.

AIM: To describe and quantify the range of non-interpretive tasks engaged in by consultant radiologists in Ireland today. MATERIALS AND METHODS: A multiple-choice electronic survey was circulated to over 200 Irish consultant radiologists and results were analysed. RESULTS: Responses were received from approximately 40% of the 267 full-time equivalent consultants in Ireland at the time of the survey. There was a wide sub-specialty mix, and responses from both clinical directors and those without designated administrative responsibility. Overall, the three most time-consuming activities were reported to be multidisciplinary meetings, vetting, and informal consultations. Non-interpretive tasks were estimated to account for 35% of the working week, with higher figures (up to 60%) for clinical directors. CONCLUSION: Consultant radiologists in Ireland spend a significant proportion of their time engaged in non-interpretive radiology; acknowledgement and scheduling of non-interpretive tasks will need to be supported by appropriate workforce planning. Non-interpretive skills will also need to be addressed during training to adequately prepare trainees for the reality of the workplace.

Mitochondrial ROS Metabolism: 10 Years Later.

The role of mitochondria in oxidative stress is well recognized, but many questions are still to be answered. This article is intended to update our comprehensive review in 2005 by highlighting the progress in understanding of mitochondrial reactive oxygen species (ROS) metabolism over the past 10 years. We review the recently identified or re-appraised sources of ROS generation in mitochondria, such as p66(shc) protein, succinate dehydrogenase, and recently discovered properties of the mitochondrial antioxidant system. We also reflect upon some controversies, disputes, and misconceptions that confound the field.

Bnip3 impairs mitochondrial bioenergetics and stimulates mitochondrial turnover.

Bnip3 (Bcl-2/adenovirus E1B 19-kDa-interacting protein 3) is a mitochondrial BH3-only protein that contributes to cell death through activation of the mitochondrial pathway of apoptosis. Bnip3 is also known to induce autophagy, but the functional role of autophagy is unclear. In this study, we investigated the relationship between mitochondrial dysfunction and upregulation of autophagy in response to Bnip3 in cells lacking Bax and Bak. We found that Bnip3 induced mitochondrial autophagy in the absence of mitochondrial membrane permeabilization and Bax/Bak. Also, co-immunoprecipitation experiments showed that Bnip3 interacted with the autophagy protein LC3 (microtubule-associated protein light chain 3). Although Bax-/Bak-deficient cells were resistant to Bnip3-mediated cell death, inhibition of mitochondrial autophagy induced necrotic cell death. When investigating why these mitochondria had to be removed by autophagy, we discovered that Bnip3 reduced both nuclear- and mitochondria-encoded proteins involved in oxidative phosphorylation. Interestingly, Bnip3 had no effect on other mitochondrial proteins, such as Tom20 and MnSOD, or actin and tubulin in the cytosol. Bnip3 did not seem to reduce transcription or translation of these proteins. However, we found that Bnip3 caused an increase in mitochondrial protease activity, suggesting that Bnip3 might promote degradation of proteins in the mitochondria. Thus, Bnip3-mediated impairment of mitochondrial respiration induces mitochondrial turnover by activating mitochondrial autophagy.

Akt mediates mitochondrial protection in cardiomyocytes through phosphorylation of mitochondrial hexokinase-II.

Akt activation supports survival of cardiomyocytes against ischemia/reperfusion, which induces cell death through opening of the mitochondrial permeability transition pore (PT-pore). Mitochondrial depolarization induced by treatment of cardiomyocytes with H(2)O(2) is prevented by activation of Akt with leukemia inhibitory factor (LIF). This protective effect is observed even when cardiomyocytes treated with LIF are permeabilized and mitochondrial depolarization is elicited by elevating Ca(2+). Cell fractionation studies demonstrate that LIF treatment increases both total and phosphorylated Akt in the mitochondrial fraction. Furthermore, the association of Akt with HK-II is increased by LIF. HK-II contains consensus sequences for phosphorylation by Akt and LIF treatment induces PI3K- and Akt-dependent HK-II phosphorylation. Addition of recombinant kinase-active Akt to isolated adult mouse heart mitochondria stimulates phosphorylation of HK-II and concomitantly inhibits the ability of Ca(2+) to induce cytochrome c release. This protection is prevented when HK-II is dissociated from mitochondria by incubation with glucose 6-phosphate or HK-II-dissociating peptide. Finally LIF increases HK-II association with mitochondria and dissociation of HK-II from mitochondria attenuates the protective effect of LIF on H(2)O(2)-induced mitochondrial depolarization in cardiomyocytes. We conclude that Akt has a direct effect at the level of the mitochondrion, which is mediated via phosphorylation of HK-II and results in protection of mitochondria against oxidant or Ca(2+)-stimulated PT-pore opening.

Mitochondria frozen with trehalose retain a number of biological functions and preserve outer membrane integrity.

In apoptosis, Bcl-2-family proteins regulate the barrier function of the mitochondrial outer membrane (MOM), controlling the release of proapoptotic proteins from the intermembrane space into the cytoplasm. This process can be studied in vitro with freshly isolated mouse liver mitochondria. Unfortunately, mitochondria frozen/thawed in standard sucrose-mannitol buffers become leaky and useless for apoptosis research. However, here we show that mitochondria frozen in buffer containing the sugar, trehalose, maintained MOM integrity and responsiveness to Bcl-2-family proteins, much like fresh mitochondria. Trehalose also preserved ultrastructure, as well as biological functions such as ATP synthesis, calcium-induced swelling, transmembrane potential, and the import and processing of protein precursors. However, bioenergetic function was somewhat reduced. Thus, trehalose-frozen mitochondria retained most of the biological features of mitochondria including MOM integrity. Although not ideal for studies involving bioenergetics, this method will facilitate research on apoptosis and other mitochondrial functions that rely on an intact MOM.

Mitochondria in neurodegeneration: bioenergetic function in cell life and death.

The biochemical pathways to cell death in chronic and acute forms of neurodegeneration are poorly understood, limiting the ability to develop effective therapeutic approaches. As details of the apoptotic and necrotic pathways have been revealed, an appreciation for the decisive role that mitochondria play in life-death decisions for the cell has grown. As a result, the need has arisen to reevaluate the significance to cell viability of mitochondrial Ca2+ sequestration, reactive oxygen species generation, and the membrane permeability transition. This review provides basic information on these mitochondrial functions as they relate to control over cell death.

Bcl-2 and Ca(2+)-mediated mitochondrial dysfunction in neural cell death.

Although altered Ca2+ homoeostasis is believed to be a primary cause of death for many cell types in response to toxic insults, the specific Ca(2+)-stimulated event responsible for directing cells down the death pathway has remained elusive. Recent publications support the hypothesis that mitochondrial Ca2+ sequestration is the critical event in induction of excitotoxic neuronal death. If similar pathways are involved in the induction of Ca(2+)-induced necrotic and apoptotic death, then agents that mimic the action of the anti-apoptotic protein Bcl-2 should be particularly useful. Our previous results provide evidence that Bcl-2 increases the maximal capacity of mitochondria to accumulate Ca2+ while providing resistance to Ca(2+)-induced respiratory damage. In addition, we have found that Bcl-2 can block Ca(2+)-ionophore-induced delayed cell death. These data predict that in response to a challenging mitochondrial Ca2+ load, Bcl-2-containing mitochondria would be capable of continuing bioenergetic function, potentially avoiding a catastrophic death signalling event.

Ca(2+)-mediated mitochondrial dysfunction and the protective effects of Bcl-2.

Mitochondrial Ca2+ sequestration likely contributes to cell death in excitotoxicity and ischemia reperfusion injury, and may also be involved in chronic forms of neurodegeneration in which a compromise in bioenergetic function alters cellular Ca2+ homeostasis. Bcl-2 overexpression is known to protect against Ca(2+)-mediated death; the mechanism of protection remains unresolved. Our data of the ability of Bcl-2 to potentiate mitochondrial Ca2+ uptake capacity and resistance to Ca(2+)-induced damage is discussed in light of current information on apoptotic signaling pathways.

Bcl-2 potentiates the maximal calcium uptake capacity of neural cell mitochondria.

Expression of the human protooncogene bcl-2 protects neural cells from death induced by many forms of stress, including conditions that greatly elevate intracellular Ca2+. Considering that Bcl-2 is partially localized to mitochondrial membranes and that excessive mitochondrial Ca2+ uptake can impair electron transport and oxidative phosphorylation, the present study tested the hypothesis that mitochondria from Bcl-2-expressing cells have a higher capacity for energy-dependent Ca2+ uptake and a greater resistance to Ca(2+)-induced respiratory injury than mitochondria from cells that do not express this protein. The overexpression of bcl-2 enhanced the mitochondrial Ca2+ uptake capacity using either digitonin-permeabilized GT1-7 neural cells or isolated GT1-7 mitochondria by 1.7 and 3.9 fold, respectively, when glutamate and malate were used as respiratory substrates. This difference was less apparent when respiration was driven by the oxidation of succinate in the presence of the respiratory complex I inhibitor rotenone. Mitochondria from Bcl-2 expressors were also much more resistant to inhibition of NADH-dependent respiration caused by sequestration of large Ca2+ loads. The enhanced ability of mitochondria within Bcl-2-expressing cells to sequester large quantities of Ca2+ without undergoing profound respiratory impairment provides a plausible mechanism by which Bcl-2 inhibits certain forms of delayed cell death, including neuronal death associated with ischemia and excitotoxicity.

Shift of the cellular oxidation-reduction potential in neural cells expressing Bcl-2.

Expression of the protooncogene bcl-2 inhibits both apoptotic and in some cases necrotic cell death in many cell types, including neural cells, and in response to a wide variety of inducers. The mechanism by which the Bcl-2 protein acts to prevent cell death remains elusive. One mechanism by which Bcl-2 has been proposed to act is by decreasing the net cellular generation of reactive oxygen species. To evaluate this proposal, we measured activities of antioxidant enzymes as well as levels of glutathione and pyridine nucleotides in control and bcl-2 transfectants in two different neural cell lines-rat pheochromocytoma PC12 and the hypothalamic GnRH cell line GT1-7. Both neural cell lines overexpressing bcl-2 had elevated total glutathione levels when compared with control transfectants. The ratios of oxidized glutathione to total glutathione in PC12 and GT1-7 cells overexpressing bcl-2 were significantly reduced. In addition, the NAD+/NADH ratio of bcl-2-expressing PC12 and GT1-7 cells was two- to threefold less than that of control cell lines. GT1-7 cells overexpressing bcl-2 had the same level of glutathione peroxidase, catalase, superoxide dismutase, and glutathione reductase activities as control cells. PC12 cells overexpressing bcl-2 had a twofold increase in superoxide dismutase and catalase activity when compared with matched control transfected cells. The levels of glutathione peroxidase and glutathione reductase in PC12 cells overexpressing bcl-2 were similar to those of control cells. These results indicate that the overexpression of bcl-2 shifts the cellular redox potential to a more reduced state, without consistently affecting the major cellular antioxidant enzymes.

Bcl-2 protects neural cells from cyanide/aglycemia-induced lipid oxidation, mitochondrial injury, and loss of viability.

The protooncogene bcl-2 rescues cells from a wide variety of insults. Recent evidence suggests that the mechanism of action of Bcl-2 involves antioxidant activity. The involvement of free radicals in ischemia/reperfusion injury to neural cells has led us to investigate the effect of Bcl-2 in a model of delayed neural cell death. We have examined the survival of control and bcl-2 transfectants of a hypothalamic tumor cell line, GT1-7, exposed to potassium cyanide in the absence of glucose (chemical hypoxia/aglycemia). After 30 min of treatment, no loss of viability was evident in control or bcl-2 transfectants; however, Bcl-2-expressing cells were protected from delayed cell death measured following 24-72 h of reoxygenation. Under these conditions, the rate and extent of ATP depletion in response to treatment with cyanide in the absence of glucose and the rate of recovery of ATP during reenergization were similar in control and Bcl-2-expressing cells. Bcl-2-expressing cells were protected from oxidative damage resulting from this treatment, as indicated by significantly lower levels of oxidized lipids. Mitochondrial respiration in control but not Bcl-2-expressing cells was compromised immediately following hypoxic treatment. These results indicate that Bcl-2 can protect neural cells from delayed death resulting from chemical hypoxia and reenergization, and may do so by an antioxidant mechanism. The results thereby provide evidence that Bcl-2 or a Bcl-2 mimetic has potential therapeutic application in the treatment of neuropathologies involving oxidative stress, including focal and global cerebral ischemia.

Tissue inhibitor of metalloproteinases-2 inhibits bFGF-induced human microvascular endothelial cell proliferation.

Tissue inhibitor of metalloproteinase-2 (TIMP-2), a protease inhibitor that binds to the latent and active forms of 72 kDa type IV collagenase (gelatinase A), was found to inhibit the in vitro proliferation of human microvascular endothelial (HME) cells stimulated with bFGF and 5% serum. The maximal inhibitory effect of TIMP-2 on incorporation of 3H-thymidine was evident 24 hours after bFGF stimulation of these cells and ranged between 45 and 60%. The half-maximal effective concentration of TIMP-2 was 107 +/- 12 nM (S.D.). In contrast, TIMP-1 was not found to slow the growth of HME cells. The inhibition of cell proliferation observed with TIMP-2 was not mimicked by addition to the culture medium of BB94, a general matrix metalloproteinase inhibitor, nor antibodies to the 72 kDa type IV collagenase. In addition to growth, two other cell functions associated with the angiogenic process were tested for sensitivity to TIMP-2. Cell adhesion to tissue culture plastic was slightly stimulated by TIMP-2, and cell migration was inhibited with short-term exposure to TIMP-2, but neither process was affected by longer-term exposure. The ability of TIMP-2 to inhibit cultured endothelial cell proliferation independent of protease inhibitory activity suggests that TIMP-2 may have additional actions which may limit neovascularization associated with solid tumor growth and metastasis in vivo.

Type IV collagenase(s) and TIMPs modulate endothelial cell morphogenesis in vitro.

It has been proposed that proteases are important in endothelial cell behavior. We examined the contribution of the gelatinase/type IV collagenase system in an in vitro model of endothelial differentiation. Human umbilical vein endothelial cells rapidly align and form networks of tubes when cultured on a basement membrane preparation, Matrigel. Zymograms of culture supernates demonstrate a 72-kD and a 92-kD gelatinase activity; the cells produce most of the 72-kD gelatinase, whereas the 92-kD activity is derived entirely from the Matrigel. Addition of antibodies against type IV gelatinase/collagenase decreases the area of the tube network. Both tissue inhibitors of metalloproteinases, TIMP-1 and TIMP-2, similarly decrease tube formation when added to cultures. Conversely, exogenous recombinant 72-kD gelatinase increases tube-forming activity. The effects of the anti-gelatinase antibodies and the TIMPs are not additive. Inhibition by either antibodies or TIMPs is greatest when they are added at culture initiation, suggesting that the protease activity is important in the early steps of morphogenesis. However, culture of the cells on Matrigel does not increase early expression of mRNA for the 72-kD gelatinase. Expression of message for the enzyme actually decreases during the course of the assay, while transcription of mRNAs for TIMPs increases, further supporting the concept that collagenases facilitate an early event in tube formation. These data demonstrate that gelatinase/type IV collagenase activity is important in endothelial cell morphogenesis on Matrigel, and suggest a role for collagenases in formation of new capillaries in vivo.

Submicromolar Ca2+ regulates phosphorylating respiration by normal rat liver and AS-30D hepatoma mitochondria by different mechanisms.

The stimulation of 2-oxoglutarate and NAD(+)-isocitrate dehydrogenase by Ca2+ in mitochondria from normal tissues has been proposed to mediate partially the activation of oxidative energy metabolism elicited by physiological elevations in cytosolic Ca2+. This mode of regulation may also occur in tumor cells in which several aspects of mitochondrial metabolism are known to be altered. This study provides a comparison of the stimulation by submicromolar concentrations of Ca2+ on the rates of ATP-generating (state 3) respiration under physiologically realistic conditions by mitochondria isolated from normal rat liver and from highly malignant rat AS-30D ascites hepatoma cells. The K0.5 for activation of glutamate-dependent state 3 respiration by Ca2+ in the presence of ATP at 37 degrees C was determined to be 0.70 +/- 0.05 (S.E.) microM for hepatoma mitochondria and 0.90 +/- 0.03 microM for rat liver mitochondria. This activation was also reflected by a Ca2(+)-induced shift in the oxidation-reduction state of hepatoma mitochondrial pyridine nucleotides to a more reduced level and Ca2+ stimulation of 14CO2 production from [1-14C]glutamate. Whereas the Ca2+ sensitivity of state 3 respiration by hepatoma mitochondria can be explained by the activation of 2-oxoglutarate and possibly NAD(+)-isocitrate dehydrogenases, the Ca2+ sensitivity of liver mitochondrial respiration appears to be predominantly mediated by activation of electron flow through ubiquinone and Complex III of the electron transport chain, as indicated by the specificity of the effects of Ca2+ on respiration with different oxidizable substrates. Although rat liver and hepatoma mitochondria employ different modes of Ca2(+)-activated ATP generation, these results support the hypothesis that changes in cytosolic Ca2+ play a significant role in the potentiation of energy production in tumor, as well as normal tissue.

Calcium sensitive isocitrate and 2-oxoglutarate dehydrogenase activities in rat liver and AS-30D hepatoma mitochondria.

NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase in extracts of mitochondria from the highly malignant AS-30D rat hepatoma cell line demonstrate Ca2+ sensitivities and affinities for substrates similar to those of normal liver mitochondria. However, the maximal activities of NAD+- and NADP+-dependent isocitrate dehydrogenase were found to be 8 and 3.5 fold higher in hepatoma mitochondrial extracts than those of liver mitochondria, whereas maximal activities of succinate and 2-oxoglutarate dehydrogenases were similar in the two tissues. At pyridine nucleotide concentrations giving the lowest physiological NADH/NAD+ ratio, NAD+-isocitrate dehydrogenase activity in hepatoma mitochondrial extracts was completely inhibited at subsaturating concentrations of Ca2+, substrate, and NAD+, in contrast to rat liver mitochondrial extracts which retained significant activity.