Isolation of glyoxalase II from bovine liver mitochondria

Bovine liver mitochondria contain about 10% of the total glyoxalase II activity in the homogenate. Electrophoresis and isoelectric focussing of either crude mitochondrial extract or the purified mitochondrial glyoxalase II resolved the enzyme activity into five forms (pl 6.3, 6.7, 7.1, 7.7, and 7.9). Since bovine liver cytosol contains a single form of glyoxalase II (pl 7.5), at least four forms are exclusively mitochondrial with no counterpart in the cytosol. The relative molecular mass of mitochondrial glyoxalase II is about 23-24 kDa, similar to the cytosolic form. The kinetic constants obtained using S-D-lactoyl, S-acetyl-, S-acetoacetyl-, and S-succinyl-glutathione as substrates are similar to those reported for glyoxalase II from rat liver mitochondria. S-D-Lactoyl- and S-acetoacetyl-glutathione are the best substrates. S-Acetylglutathione is the poorest substrate with respect to both Vmax and Km values.     

Intramitochondrial proteolytic activity of rat liver]

Highly purified mitochondria and lysosomes are isolated from rat liver homogenate. pH optimum of proteolytic activity with respect to proteins of own structures and to mitochondrial structural protein is investigated. The purification of mitochondria from lysosomes is found to be accompanied by the change of proteolytic activity pH optimum from 5.0 to 6.0 in coarse and purified mitochondria respectively. Comparative study of structural protein hydrolysis products with enzyme preparations from purified mitochondria and lysosomes has revealed differences in the spectrum of the reaction products. The data obtained suggest a presence of a proteolytic enzyme in rat liver mitochondria.     

THE SARCOTUBULAR SYSTEM OF FROG SKELETAL MUSCLE : A Morphological and Biochemical Study

In the frog skeletal muscle cell a well defined and highly organized system of tubular elements is located in the sarcoplasm between the myofibrils. The sarcoplasmic component is called the sarcotubular system. By means of differential centrifugation it has been possible to isolate from the frog muscle homogenate a fraction composed of small vesicles, tubules, and particles. This fraction is without cytochrome oxidase activity, which is localized in the mitochondrial membranes. This indicates that the structural components of this fraction do not derive from the mitochondrial fragmentation, but probably from the sarcotubular system. This fraction, called sarcotubular fraction, has a Mg⁺⁺-stimulated ATPase activity which differs from that of muscle mitochondria in that it is 3 to 4 times higher on the protein basis as compared with the mitochondrial ATPase, and is inhibited by Ca⁺⁺ and by deoxycholate like the Kielley and Meyerhof ATPase. We therefore conclude that the "granules" of the Kielley and Meyerhof ATPase, which were shown to have a relaxing effect, are fragments of the sarcotubular system. The isolated sarcotubular fraction has a high RNA content and demonstrable activity in incorporating labeled amino acids, even in the absence of added supernatant.     

Effect of tumor growth and preliminary irradiation on the state of DNA degradation processes in the liver cell nuclei

This study is devoted to the analysis of DNase activity and DNA fragmentation level in liver cells nuclei of rats with transplanted Guerin's carcinoma on irradiation background. Was shown, that in an organism of previously irradiated rat with tumor the dominance of neoplasm development over the consequences of the irradiation is observed on the initial stages of the experiment. The alignment of gamma-irradiation influencing already takes place from the first stages after Guerin's carcinoma transplantation, which is testified by the decrease of DNase activity of liver cells nuclei and the decrease of a degree of nuclear DNA fragmentation. On the stationary stages of tumor growth the general action of the factors causes differences in the parameters of the investigated animal groups. It shows that together with the constant reduced level of DNase activity the processes of DNA fragmentation strengthen and their intensity reaches maximum during this period. The nature of the fragmented DNA accumulation is similar to the irradiated organism on the first days after the radiation factor removal. Thus, the radiation-induced signal in the organism with tumor is leveled under the conditions of active neoplasm growth; nevertheless, the consequents of its operating do not disappear, and only move away in time.     

Study of DNA-binding proteins in mitochondria of rat liver gamma-irradiation

Acid-soluble proteins were isolated from the liver mitochondria of control and irradiated (8 Gy) rats. By means of electrophoresis in 15% polyacrylamide gel, these proteins were separated into more than 20 polypeptides of molecular masses between 10 and 120 kDa. The irradiation of rats with a dose of 8 Gy led to changes in the polypeptide content of mitochondrial acid-soluble proteins in the postradiation period. It was found that the liver acid-soluble proteins of control and irradiated rats were able to form nucleoproteid complexes with DNA at the physiological NaCl concentration. It was shown that along with mitochondrial acid-soluble proteins, proteases were also released, their activity increased in the presence of DNA. Twenty four hours after irradiation of rats with 8 Gy, the activity of proteases cleaving mitochondrial acid-soluble proteins decreased. Probably, the acid-soluble proteins and DNA-activated proteases of mitochondria are involved in the regulation of the structural organization and functional activity of mitochondrial DNA.     

Dynamic Adaptation of Liver Mitochondria to Chronic Alcohol Feeding in Mice: BIOGENESIS,REMODELING, AND FUNCTIONAL ALTERATIONS*

Liver mitochondria undergo dynamic alterations following chronic alcohol feeding to mice. Intragastric alcohol feeding to mice resulted in 1) increased state III respiration (109% compared with control) in isolated liver mitochondria, probably due to increased levels of complexes I, IV, and V being incorporated into the respiratory chain; 2) increased mitochondrial NAD⁺ and NADH levels (∼2-fold), with no change in the redox status; 3) alteration in mitochondrial morphology, with increased numbers of elongated mitochondria; and 4) enhanced mitochondrial biogenesis in the liver, which corresponded with an up-regulation of PGC-1α (peroxisome proliferator-activated receptor γ coactivator-1α). Oral alcohol feeding to mice, which is associated with less liver injury and steatosis, slightly enhanced respiration in isolated liver mitochondria (30.8% compared with control), lower than the striking increase caused by intragastric alcohol feeding. Mitochondrial respiration increased with both oral and intragastric alcohol feeding despite extensive N-acetylation of mitochondrial proteins. The alcohol-induced mitochondrial alterations are probably an adaptive response to enhance alcohol metabolism in the liver. Isolated liver mitochondria from alcohol-treated mice had a greater rate of acetaldehyde metabolism and respiration when treated with acetaldehyde than control. Aldehyde dehydrogenase-2 levels were unaltered in response to alcohol, suggesting that the greater acetaldehyde metabolism by isolated mitochondria from alcohol-treated mice was due to increased mitochondrial respiration that regenerated NAD⁺, the rate-limiting substrate in alcohol/acetaldehyde metabolism. Overall, our work suggests that mitochondrial plasticity in the liver may be an important adaptive response to the metabolic stress caused by alcohol intake and could potentially play a role in many other vital functions performed by the liver.     

Ultrastructural changes of bovine pulmonary artery endothelial cells irradiated in vitro with a 137Cs source

Bovine pulmonary artery endothelial cells in culture were evaluated by phase-contrast and electron microscopy at various times after being irradiated with ¹³⁷Cs in vitro. Cells irradiated prior to reaching confluency showed vacuolization and increased numbers of lysosomes beginning at 48 hr after irradiation with 300–500 rad and at 24 hr after irradiation with 1500–5000 rad. After 7 days the morphological changes appeared to be reversible for cells receiving the lower doses, but were progressive for higher doses of radiation. The same qualitative changes, with a delayed onset, were observed for cells irradiated at confluency. An observed decrease in the endoplasmic reticulum and polysomes occurred only late in the course of radiation injury. There was no observable structural alteration of mitochondria even when there was evidence of otherwise marked cytoplasmic injury. We conclude that structural changes of the lysosomes constitute an early phase of injury by irradiation of the endothelial cell in culture, while decreases in endoplasmic reticulum and polysomcs occur relatively late. The mitochondrial structure of the endothelial cell appears to be relatively resistant to radiation. All morphological changes occur subsequent to impaired transport of α-aminoisobutyric acid, which is observed within 6 hr as previously reported (Kwock et al., 1982).     

Changes of malic enzyme activity in the developing rat brain are due to both the increase of mitochondrial protein content and the increase of specific activity.

1. The pattern of NADP-linked malic enzyme activity estimated in the whole brain homogenate did not parallel that found in liver of developing rat. 2. Studies on intracellular distribution of malic enzyme in brain showed that the mitochondrial enzyme increased about three-fold between 10th and 40th day of life. Thereafter, a slow gradual increase to the adult level was observed. 3. The extramitochondrial malic enzyme from brain, like the liver enzyme, increased at the time of weaning, although to a lesser extent. At day 5 the brain malic enzyme was equally distributed between mitochondria and cytosol. 4. During the postnatal development, the contribution of the mitochondrial malic enzyme in the total activity was increasing, reaching the value approx. 80% at day 150 after birth. 5. The increase with age of the malic enzyme specific activity was observed in both synaptosomal and non-synaptosomal mitochondria, the changes in the last fraction being more pronounced. 6. The activity of citrate synthase developed markedly between 10-40 postnatal days, increasing about five-fold, while the specific activity of the enzyme did change neither in the synaptosomal nor in non-synaptosomal mitochondria at this period. 7. We conclude that the changes in malic enzyme activity in the developing rat brain are mainly due both to the increase of mitochondrial protein content and to the increase of specific activity of the mitochondrial malic enzyme.     

The functional size of ferrochelatase determined in situ by radiation inactivation.

Ferrochelatase (EC 4.99.1.1) catalyzes the final step of heme biosynthesis, the insertion of iron(II) into protoporphyrin. It is an integral protein of the inner mitochondrial membrane. The functional size of bovine hepatic ferrochelatase has been studied in situ using radiation inactivation analysis. The functional unit required for enzymic activity in intact mitochondria was found to have a mass of 82 +/- 13 kDa. In contrast, the structural unit (evaluated in immunoblots following sodium dodecyl sulfate-polyacrylamide gel electrophoresis) has a mass of 40 +/- 10 kDa. Similar results were obtained when irradiation was performed on sodium cholate-solubilized mitochondria. The presence or absence of dithiothreitol during irradiation had no effect on target sizes obtained from either intact or solubilized mitochondria. Pairwise comparison of the functional and structural target sizes from each set of irradiated samples yielded a ratio of 2.0 +/- 0.4. Previous studies using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography have shown that a Mr 40,000 peptide is associated with ferrochelatase activity. This study shows that the functional size of bovine ferrochelatase is approximately 80 kDa; the data are most consistent with a model for active ferrochelatase composed of two structural subunits of about 40 kDa each.     

Radiation-induced signaling results in mitochondrial impairment in mouse heart at 4 weeks after exposure to X-rays

BACKROUND: Radiation therapy treatment of breast cancer, Hodgkin's disease or childhood cancers expose the heart to high local radiation doses, causing an increased risk of cardiovascular disease in the survivors decades after the treatment. The mechanisms that underlie the radiation damage remain poorly understood so far. Previous data show that impairment of mitochondrial oxidative metabolism is directly linked to the development of cardiovascular disease. METHODOLOGY/PRINCIPAL FINDINGS: In this study, the radiation-induced in vivo effects on cardiac mitochondrial proteome and function were investigated. C57BL/6N mice were exposed to local irradiation of the heart with doses of 0.2 Gy or 2 Gy (X-ray, 200 kV) at the age of eight weeks, the control mice were sham-irradiated. After four weeks the cardiac mitochondria were isolated and tested for proteomic and functional alterations. Two complementary proteomics approaches using both peptide and protein quantification strategies showed radiation-induced deregulation of 25 proteins in total. Three main biological categories were affected: the oxidative phophorylation, the pyruvate metabolism, and the cytoskeletal structure. The mitochondria exposed to high-dose irradiation showed functional impairment reflected as partial deactivation of Complex I (32%) and Complex III (11%), decreased succinate-driven respiratory capacity (13%), increased level of reactive oxygen species and enhanced oxidation of mitochondrial proteins. The changes in the pyruvate metabolism and structural proteins were seen with both low and high radiation doses. CONCLUSION/SIGNIFICANCE: This is the first study showing the biological alterations in the murine heart mitochondria several weeks after the exposure to low- and high-dose of ionizing radiation. Our results show that doses, equivalent to a single dose in radiotherapy, cause long-lasting changes in mitochondrial oxidative metabolism and mitochondria-associated cytoskeleton. This prompts us to propose that these first pathological changes lead to an increased risk of cardiovascular disease after radiation exposure.     

DNA SYNTHESIS IN THE OOPLASM OF DROSOPHILA MELANOGASTER

Tritiated thymidine was injected into 2-day-old Drosophila melanogaster females, and tissue sections were prepared from the ovary for radioautography with both the light and electron microscopes. Besides the expected incorporation of H³-thymidine into nuclei of nurse cells and follicle cells, there was a relatively high level of incorporation of label into ooplasmic DNA. The highest level of incorporation occurred at stage 12. At the same time, the 15 nurse cell nuclei also incorporate thymidine in spite of the fact that they are breaking down and degenerating. The label in the ooplasm is not removed by extraction with DNase (although this removes nuclear label) unless extraction is preceded by a treatment with protease. Electron microscopic radioautography revealed that 36% of the silver grains resulting from decay of H³-thymidine are found over mitochondria, with a further 28% being located within 0.25 µ of these organelles. The remaining 36% of the silver grains was not found to be associated with any organelles, and it probably represents synthesis in the cytoplasm by the "storage DNA" characteristic of many eggs. It is suggested that one mechanism acting throughout the egg chamber is responsible for the synchronous synthesis of DNA in the degenerating nurse cells, in the mitochondria of the egg, and in the "storage DNA" of the ooplasm.     

Differential long-term effects of d-chloramphenicol on the biogenesis of mitochondria in normal and regenerating rat liver

1. Normal and partially hepatectomized rats (150g) were injected daily with d-chloramphenicol (20mg) for a period of 4 weeks, in order to investigate whether defective mitochondria could be induced in vivo in higher organisms as in yeast, and to measure the degree of inhibition of the mitochondrial function thus obtained. 2. The antibiotic did not affect growth and increased the amount of liver protein without changing the mitochondrial yield. 3. The respiration of isolated mitochondria from regenerated liver (regeneration completed) with succinate, α-oxo-glutarate, isocitrate and malate, was decreased in the chloramphenicol-treated rats, whereas in normal liver the antibiotic increased the mitochondrial oxygen consumption with succinate and did not significantly change the respiration with other substrates. 4. Mitochondrial cytochromes and respiratory enzymes were also decreased in amount in regenerated liver from the treated rats and enhanced in normal liver. 5. The protein specific radioactivities of most mitochondrial and microsomal subfractions, 30min after an injection of [¹⁴C]leucine, were decreased in regenerated liver under the action of chloramphenicol. Conversely, the incorporation of [¹⁴C]leucine into proteins of most subfractions in incubations of liver slices was enhanced in the case of normal rats treated with the antibiotic. 6. It is concluded that in regenerated liver chloramphenicol induces functionally defective mitochondria by inhibiting their biogenesis, whereas in normal liver the stimulation of respiration and protein synthesis is probably a secondary detoxication response.     

STRUCTURAL CHANGES IN ISOLATED LIVER MITOCHONDRIA OF RATS DURING ESSENTIAL FATTY ACID DEFICIENCY

Liver mitochondria isolated in 0.44 M sucrose from rats deficient in essential fatty acids (EFA) oxidized citrate, succinate, α-ketoglutarate, glutamate, and pyruvate at a faster rate than did mitochondria isolated from normal rats; however, the oxidation of malate, caprylate, and β-hydroxybutyrate was not significantly increased. The mitochondria from deficient rats exhibited an increased ATPase activity and extensive structural damage as revealed by electron microscope examination of thin sections. An increase in citrate oxidation and ATPase activity, together with some structural damage, could be demonstrated as early as the 4^th week in rats on a fat-free diet. Saturated fat in the diet did not prevent the change in mitochondrial structure but accelerated its appearance. Both the biochemical and structural defects could be reversed within three weeks after feeding deficient rats a source of EFA. In the presence of a phosphate acceptor the effect of EFA deficiency on substrate oxidation was largely eliminated. A trend toward a reduced efficiency of oxidative phosphorylation was noted in mitochondria from EFA-deficient rats, but significant uncoupling was found only in the case of citrate, β-hydroxybutyrate, and glutamate in the presence of malonate. Together with the increased ATPase activity, the uncoupling of phosphorylation could account for the poor respiratory control found with the deficient preparation. However, EFA deficiency was without effect on the respiration of liver slices, which supports the belief that the observed changes in oxidation and phosphorylation are an artifact resulting from damage sustained by the deficient mitochondria during their isolation.     

Isolated mouse liver mitochondria are devoid of glucokinase

Glucokinase is a hexokinase isoform with low affinity for glucose that has previously been identified as a cytosolic enzyme. A recent report claims that glucokinase physically associates with liver mitochondria to form a multi-protein complex that may be physiologically important in apoptotic signaling [N.N. Danial, C.F. Gramm, L. Scorrano, C.Y. Zhang, S. Krauss, A.M. Ranger, S.R. Datta, M.E. Greenberg, L.J. Licklider, B.B. Lowell, S.P. Gygi, S.J. Korsmeyer, Nature 424 (2003) 952-956]. Here, we re-examined the association of glucokinase with isolated mouse liver mitochondria. When glucokinase activity was measured by coupled enzyme assay, robust activity was present in whole liver homogenates and their 9500 g supernatants (cytosol), but activity in the purified mitochondrial fraction was below detection (<0.2% of homogenate). Furthermore, addition of 45 mM glucose in the presence of ATP did not increase mitochondrial respiration, indicating the absence of ADP formation by glucokinase or any other hexokinase isoform. Immunoblots of liver homogenates and cytosol revealed strong glucokinase bands, but no immunoreactivity was detected in mitochondria. In conclusion, mouse liver mitochondria lack measurable glucokinase. Thus, functional linkage of glucokinase to mitochondrial metabolism and apoptotic signaling is unlikely to be mediated by the physical association of glucokinase with mitochondria.     

DEVELOPMENT OF AN IMPROVED MEDIUM FOR THE ISOLATION OF LIVER MITOCHONDRIA

In view of the unsatisfactory appearance, under the electron microscope, of liver mitochondria isolated in isotonic sucrose medium, alternative media have been examined. It was found to be advantageous to replace sucrose by raffinose, and to add levan or, preferably, dextran, together with heparin in suitable concentration. With the optimal medium, the constituents of which are raffinose, versene (optional), dextran of high molecular weight, heparin, and AMP (optional), most of the mitochondria in the osmium-fixed pellet are apparently intact, and show the membranes characteristic of mitochondria as seen in cell sections. The optimal medium has no adverse effect on the activity of the several tissue enzymes which have been studied, except that Mg⁺⁺-activated ATPase is partially inhibited if the medium is present in high concentration in the assay system. Mitochondrial fractions isolated in the new medium have, in common with sucrose fractions, appreciable "free" ATPase activity, this activity being evidently a poor criterion of mitochondrial integrity. Use of the new medium does not decrease the proportion of cytoplasmic ATPase which fails to sediment with the mitochondria, but does give a mitochondrial fraction low in RNA and in acid phosphatase activity and little contaminated with microsomal material. Particles tentatively identified as "lysosomes" have been seen in certain sections.     

Toxicity of Copper on Isolated Liver Mitochondria: Impairment at Complexes I,II, and IV Leads to Increased ROS Production

Oxidative damage has been implicated in disorders associated with abnormal copper metabolism and also Cu²⁺ overloading states. Besides, mitochondria are one of the most important targets for Cu²⁺, an essential redox transition metal, induced hepatotoxicity. In this study, we aimed to investigate the mitochondrial toxicity mechanisms on isolated rat liver mitochondria. Rat liver mitochondria in both in vivo and in vitro experiments were obtained by differential ultracentrifugation and the isolated liver mitochondria were then incubated with different concentrations of Cu²⁺. Our results showed that Cu²⁺ induced a concentration and time-dependent rise in mitochondrial ROS formation, lipid peroxidation, and mitochondrial membrane potential collapse before mitochondrial swelling ensued. Increased disturbance in oxidative phosphorylation was also shown by decreased ATP concentration and decreased ATP/ADP ratio in Cu²⁺-treated isolated mitochondria. In addition, collapse of mitochondrial membrane potential (MMP), mitochondrial swelling, and release of cytochrome c following of Cu²⁺ treatment were well inhibited by pretreatment of mitochondria with CsA and BHT. Our results showed that Cu²⁺ could interact with respiratory complexes (I, II, and IV). This suggests that Cu²⁺-induced liver toxicity is the result of metal’s disruptive effect on liver hepatocyte mitochondrial respiratory chain that is the obvious cause of Cu²⁺-induced ROS formation, lipid peroxidation, mitochondrial membrane potential decline, and cytochrome c expulsion which start cell death signaling.     

Alterations of neuronal nuclear matrix and chromatin structure after irradiation under aerobic and anoxic conditions

This study was undertaken to determine if structural alterations of the bulk chromatin and the amount of protein associated with the nuclear matrix in cerebellar neurons depend on radiation dose and a cell's state of oxygenation. After irradiation with 2.5 to 25.0 Gy under both aerobic and anoxic conditions, the sensitivity of the neuronal chromatin to m. nuclease digestion increase linearly with dose up to about 5 Gy, beyond which there was no further increase. The same increase in accessibility of chromatin to micrococcal nuclease digestion was observed when neuronal nuclei were irradiated at 4 degrees C. Neuronal nuclei were stained with propidium iodide (PI) for DNA and with fluorescein isothiocyanate (FITC) for protein, both before and after complete digestion with DNase I, and analyzed by flow cytometry. There was no change in either the PI (P greater than 0.4) or the FITC (P greater than 0.9) fluorescence of undigested nuclei after irradiation. For the DNase I digested nuclei, the PI fluorescence was unchanged after irradiation (P greater than 0.4), but the FITC fluorescence increased significantly (P less than 0.02). This increase in the FITC fluorescence was linear with dose up to about 5 Gy, beyond which there was no further increase. The flow cytometry results from DNase I digested nuclei were identical for neurons irradiated under aerobic or anoxic conditions, indicating that this phenomenon is oxygen independent. This increase in FITC fluorescence after irradiation was inhibited at ice-cold temperatures and probably reflects an increase in protein content at the nuclear matrix that requires metabolism. This may explain our previously observed resistance of nuclear matrix-associated DNA to digestion by DNase I. This protein increase at the nuclear matrix appears to follow "saturation" kinetics identical to that previously reported for repair of DNA strand breaks in cerebellar neurons. However, the exact molecular nature of this process and its role in DNA repair or cell survival remains to be determined.     

Bioenergetic parameters of lamprey and frog liver mitochondria during metabolic depression and activity

The objective of this study is to elucidate the role of mitochondria in reversible metabolic depression of hepatocytes of the Baltic lamprey (Lampetra fluviatilis) taking place in the last year of its life cycle and to compare their main bioenergetic parameters with those of the frog (Rana temporaria) and the white outbred mouse (Mus musculus). Using isolated mitochondria as a model, we have revealed significant seasonal variations in the main bioenergetic parameters of the lamprey liver. These changes indicate that the metabolic depression is mediated by prolonged reversible alterations of mitochondrial functions, which manifest in low activity of the mitochondrial respiratory chain, low oxidative phosphorylation, low content of mitochondrial adenine nucleotides, high level of reduced mitochondrial pyridine nucleotides and leaky mitochondrial membranes observed in winter. The enhanced ion membrane permeability of winter lamprey liver mitochondria is found to be sensitive to EGTA and to cyclosporine A in combination with ADP and Mg²⁺ and is likely mediated opening the mitochondrial permeability transition pore in its low conductance state. The sharp activation of oxidation and phosphorylation in the lamprey liver mitochondria followed by spawning and death of the animal is observed in spring. The possible causes of the phenomenon and the differences obtained between lamprey, frog and mouse are under discussion.     

Long-term effects of low-dose mouse liver irradiation involve ultrastructural and biochemical changes in hepatocytes that depend on lipid metabolism

The aim of the study was to investigate long-term effects of radiation on the (ultra)structure and function of the liver in mice. The experiments were conducted on wild-type C57BL/6J and apolipoprotein E knock-out (ApoE^?/?) male mice which received a single dose (2 or 8 Gy) of X-rays to the heart with simultaneous exposure of liver to low doses (no more than 30 and 120 mGy, respectively). Livers were collected for analysis 60 weeks after irradiation and used for morphological, ultrastructural, and biochemical studies. The results show increased damage to mitochondrial ultrastructure and lipid deposition in hepatocytes of irradiated animals as compared to non-irradiated controls. Stronger radiation-related effects were noted in ApoE^?/? mice than wild-type animals. In contrast, radiation-related changes in the activity of lysosomal hydrolases, including acid phosphatase, β-glucuronidase, N-acetyl-β-d-hexosaminidase, β-galactosidase, and α-glucosidase, were observed in wild type but not in ApoE-deficient mice, which together with ultrastructural picture suggests a higher activity of autophagy in ApoE-proficient animals. Irradiation caused a reduction of plasma markers of liver damage in wild-type mice, while an increased level of hepatic lipase was observed in plasma of ApoE-deficient mice, which collectively indicates a higher resistance of hepatocytes from ApoE-proficient animals to radiation-mediated damage. In conclusion, liver dysfunctions were observed as late effects of irradiation with an apparent association with malfunction of lipid metabolism.     

Bax is present as a high molecular weight oligomer/complex in the mitochondrial membrane of apoptotic cells

Bax is a Bcl-2 family protein with proapoptotic activity, which has been shown to trigger cytochrome c release from mitochondria both in vitro and in vivo. In control HeLa cells, Bax is present in the cytosol and weakly associated with mitochondria as a monomer with an apparent molecular mass of 20,000 Da. After treatment of the HeLa cells with the apoptosis inducer staurosporine or UV irradiation, Bax associated with mitochondria is present as two large molecular weight oligomers/complexes of 96,000 and 260,000 Da, which are integrated into the mitochondrial membrane. Bcl-2 prevents Bax oligomerization and insertion into the mitochondrial membrane. The outer mitochondrial membrane protein voltage-dependent anion channel and the inner mitochondrial membrane protein adenosine nucleotide translocator do not coelute with the large molecular weight Bax oligomers/complexes on gel filtration. Bax oligomerization appears to be required for its proapoptotic activity, and the Bax oligomer/complex might constitute the structural entirety of the cytochrome c-conducting channel in the outer mitochondrial membrane.