Low Levels Of Cytochrome Research Articles

Evaluation of the adaptogenic potential exerted by ginsenosides Rb1 and Rg1 against oxidative stress-mediated neurotoxicity in an in vitro neuronal model.

BackgroundGinseng (Panax sp.) is a drug with multiple pharmacological actions that has been largely used in traditional medicines for the treatment of many health problems. In the therapy of neurodegenerative disorders, it has been employed due to its capacity to strengthen mental processes by enhancing cognitive performance and psychological function. Current work aimed at evaluating the adaptogenic potential of Rb1 and Rg1 against oxidative-stress mediated degeneration in a model of nervous cells.MethodsOxidative stress and mitochondrial dysfunction were achieved by exposing SH-SY5Y cells to the mitochondrial complex I inhibitor rotenone. The cytoprotective activity of pre-treatments with ginsenosides Rb1 and Rg1 against rotenone was assessed by determining biochemical markers regarding oxidative stress (ROS scavenging, glutathione and lipid peroxidation levels, SOD activity and Nrf2 activation) and apoptosis-related alterations (mitochondrial membrane potential, calcium levels, aconitase activity and pro/antiapoptotic proteins). Their capacity to cross the blood brain barrier was also estimated.ResultsAt their optimal doses, ginsenosides Rb1 and Rg1 significantly ameliorated redox status within the cells; they reduced ROS and TBARS levels and improved the glutathione system, as well as they enhanced SOD activity and Nrf2 pathway activation. They protected neuronal cells against MMP loss, calcium homeostasis disruption and aconitase inhibition. Consequently, apoptotic cell death was attenuated by the pre-treatment with ginsenosides, as evidenced by the reduction in caspase-3 and Bax, and the increase in Bcl-2 expressions; also, lower levels of cytochrome C were found in the cytosol. Poor BBB permeation was demonstrated for both ginsenosides.ConclusionsIn conclusion, ginsenosides Rb1 and Rg1 exhibit neuroprotective potential which is achieved, at least in part, via mitochondrial protection and the plausible involvement of Nrf2 pathway activation. Our results contribute to validate the traditional use of ginseng for cognitive-enhancing purposes and provide basis to encourage further research on the potential of ginsenosides in the treatment of neurodegenerative diseases.

Bioenergetic differences between MCF-7 and T47D breast cancer cells and their regulation by oestradiol and tamoxifen

Oestrogen receptor α (ERα+) breast tumours rely on mitochondria (mt) to generate ATP. The goal of the present study was to determine how oestradiol (E2) and 4-hydroxytamoxifen (4-OHT) affect cellular bioenergetic function in MCF-7 and T47D ERα+ breast cancer cells in serum-replete compared with dextran-coated charcoal (DCC)-stripped foetal bovine serum (FBS)-containing medium ('serum-starved'). Serum-starvation reduced oxygen consumption rate (OCR), extracellular acidification rate (ECAR), ATP-linked OCR and maximum mt capacity, reflecting lower ATP demand and mt respiration. Cellular respiratory stateapparent was unchanged by serum deprivation. 4-OHT reduced OCR independent of serum status. Despite having a higher mt DNA/nuclear DNA ratio than MCF-7 cells, T47D cells have a lower OCR and ATP levels and higher proton leak. T47D express higher nuclear respiratory factor-1 (NRF-1) and NRF-1-regulated, nuclear-encoded mitochondrial transcription factor TFAM and cytochrome c, but lower levels of cytochrome c oxidase, subunit IV, isoform 1 (COX4, COX4I1). Mitochondrial reserve capacity, reflecting tolerance to cellular stress, was higher in serum-starved T47D cells and was increased by 4-OHT, but was decreased by 4-OHT in MCF-7 cells. These data demonstrate critical differences in cellular energetics and responses to 4-OHT in these two ERα+ cell lines, likely reflecting cancer cell avoidance of apoptosis.

A mutation in the human CBP4 ortholog UQCC3 impairs complex III assembly, activity and cytochrome b stability.

Complex III (cytochrome bc1) is a protein complex of the mitochondrial inner membrane that transfers electrons from ubiquinol to cytochrome c. Its assembly requires the coordinated expression of mitochondrial-encoded cytochrome b and nuclear-encoded subunits and assembly factors. Complex III deficiency is a severe multisystem disorder caused by mutations in subunit genes or assembly factors. Sequence-profile-based orthology predicts C11orf83, hereafter named UQCC3, to be the ortholog of the fungal complex III assembly factor CBP4. We describe a homozygous c.59T>A missense mutation in UQCC3 from a consanguineous patient diagnosed with isolated complex III deficiency, displaying lactic acidosis, hypoglycemia, hypotonia and delayed development without dysmorphic features. Patient fibroblasts have reduced complex III activity and lower levels of the holocomplex and its subunits than controls. They have no detectable UQCC3 protein and have lower levels of cytochrome b protein. Furthermore, in patient cells, cytochrome b is absent from a high-molecular-weight complex III. UQCC3 is reduced in cells depleted for the complex III assembly factors UQCC1 and UQCC2. Conversely, absence of UQCC3 in patient cells does not affect UQCC1 and UQCC2. This suggests that UQCC3 functions in the complex III assembly pathway downstream of UQCC1 and UQCC2 and is consistent with what is known about the function of Cbp4 and of the fungal orthologs of UQCC1 and UQCC2, Cbp3 and Cbp6. We conclude that UQCC3 functions in complex III assembly and that the c.59T>A mutation has a causal role in complex III deficiency.

Peculiarities of cell death mechanisms in neutrophils.

Analyses of neutrophil death mechanisms have revealed many similarities with other cell types; however, a few important molecular features make these cells unique executors of cell death mechanisms. For instance, in order to fight invading pathogens, neutrophils possess a potent machinery to produce reactive oxygen species (ROS), the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Evidence is emerging that these ROS are crucial in the execution of most neutrophil cell death mechanisms. Likewise, neutrophils exhibit many diverse granules that are packed with cytotoxic mediators. Of those, cathepsins were recently shown to activate pro-apoptotic B-cell lymphoma-2 (Bcl-2) family members and caspases, thus acting on apoptosis regulators. Moreover, neutrophils have few mitochondria, which hardly participate in ATP synthesis, as neutrophils gain energy from glycolysis. In spite of relatively low levels of cytochrome c in these cells, the mitochondrial death pathway is functional. In addition to these pecularities defining neutrophil death pathways, neutrophils are terminally differentiated cells, hence they do not divide but undergo apoptosis shortly after maturation. The initial trigger of this spontaneous apoptosis remains to be determined, but may result from low transcription and translation activities in mature neutrophils. Due to the unique biological characteristics of neutrophils, pharmacological intervention of inflammation has revealed unexpected and sometimes disappointing results when neutrophils were among the prime target cells during therapy. In this study, we review the current and emerging models of neutrophil cell death mechanisms with a focus on neutrophil peculiarities.

Compartmentalization and regulation of mitochondrial function by methionine sulfoxide reductases in yeast.

Elevated levels of reactive oxygen species can damage proteins. Sulfur-containing amino acid residues, cysteine and methionine, are particularly susceptible to such damage. Various enzymes evolved to protect proteins or repair oxidized residues, including methionine sulfoxide reductases MsrA and MsrB, which reduce methionine (S)-sulfoxide (Met-SO) and methionine (R)-sulfoxide (Met-RO) residues, respectively, back to methionine. Here, we show that MsrA and MsrB are involved in the regulation of mitochondrial function. Saccharomyces cerevisiae mutant cells lacking MsrA, MsrB, or both proteins had normal levels of mitochondria but lower levels of cytochrome c and fewer respiration-competent mitochondria. The growth of single MsrA or MsrB mutants on respiratory carbon sources was inhibited, and that of the double mutant was severely compromised, indicating impairment of mitochondrial function. Although MsrA and MsrB are thought to have similar roles in oxidative protein repair each targeting a diastereomer of methionine sulfoxide, their deletion resulted in different phenotypes. GFP fusions of MsrA and MsrB showed different localization patterns and primarily localized to cytoplasm and mitochondria, respectively. This finding agreed with compartment-specific enrichment of MsrA and MsrB activities. These results show that oxidative stress contributes to mitochondrial dysfunction through oxidation of methionine residues in proteins located in different cellular compartments.

Variable response of selected cuproproteins in rat choroid plexus and cerebellum following perinatal copper deficiency

Recent immunohistochemical characterization of the copper transport protein, Ctr1, reported enriched levels in mouse choroid plexus, and enhancement by copper deficiency. To extend and confirm this, experiments were conducted with Holtzman rats. Following perinatal copper deficiency there was an 80% reduction in brain copper of 24-27 day old copper-deficient (Cu-) rat pups compared to copper-adequate (Cu+) controls. Choroid plexus immunoblot analysis with rabbit anti-hCtr1 demonstrated a 50% higher Ctr1 protein expression in Cu-samples. However, levels of copper chaperone for superoxide dismutase (CCS) were unchanged, suggesting that Ctr1 buffers the choroid plexus against copper deficiency, since CCS normally is much higher in Cu-tissues. There were 13% lower levels of cytochrome c oxidase subunit IV (COX IV) detected in Cuchoroid plexus. In contrast, in cerebellum of Cu-rats CCS was 2-fold higher and COXIV 1.7-fold lower than Cu+ rats consistent with severe copper deficiency. Brain mitochondria from Cu-rats had severe reductions in COXIV content and CCO activity and modest but significant elevations in CCS and reductions in Cu, Zn-superoxide dismutase. COXIV may be a more sensitive marker for copper deficiency than CCS and may prove useful to assess copper status.

Copper deficiency alters isomyosin types and levels of laminin, fibronectin and cytochrome c oxidase subunits from rat hearts

The relative amounts of carddac proteins such as laminin, fibronectin, cytochrome c oxidase, and isomyosin types were studied by gel electrophoresis and Western blotting in control and copper-deficient Sprague-Dawley rats of both sexes fed their respective diets from weanling for 3 weeks. Isomyosin types appeared to shift from V 1 to greater levels of V 3 in copper deficient rats for both genders. Male copper deficient rats had increased cardiac levels of fibronectin, decreased laminin levels, cardiac hypertrophy and anemia. Both male and female rats fed copper-deficient diet had lower levels of cytochrome c oxidase (CCO) subunit IV, and low liver copper, and high heart-to-body weight ratios compared with their respective controls.

The Effect of Carbon Dioxide as a Preslaughter Stunning Method for Turkeys

The objective of the present research was to determine the effects of CO2 as an immobilization agent on turkey meat quality. In the study, two concentrations of CO2 were used, 40 and 60%; electrical stunning served as the control. As compared with electrical stunning, there was a significant (P<.05) reduction in severity of struggle during the lag stage of immobilization with both concentrations of CO2; during the lag phase of CO2 immobilization the bird shows no sign of stress. Hemorrhagic scores for the thigh muscle were significantly lower (P<.05) for the 40 and 60% CO2 groups when compared with the electrical stunning treatment. Total heme pigments and hemoglobin concentrations were significantly lower in the groups immobilized by CO2 (P<.05) than in the control group. However, control birds exhibited significantly (P<.05) lower levels of cytochrome c when compared with the groups immobilized by CO2. Postslaughter pH of birds immobilized with CO2 was significantly (P<.05) lower than that of the control groups. There was no significant difference (P<.05) in water holding capacity between treatment groups. Shear force was significantly lower (P<.05) for the 40% CO2 treatment group, when compared with other immobilization treatments. These results indicate that CO2 immobilization may have potential application as a humane stunning technique.

Control of proteoliposomal cytochrome c oxidase: the partial reactions

The steady-state spectroscopic behaviour and the turnover of cytochrome c oxidase incorporated into proteoliposomes have been investigated as functions of membrane potential and pH gradient. The respiration rate is almost linearly dependent on [cytochrome c2+] at high flux, but while the cytochrome a redox state is always dependent on the [cytochrome c2+] steady state, it reaches a maximum reduction level less than 100% in each case. The maximal aerobic steady-state reduction level of cytochrome a is highest in the presence of valinomycin and lowest in the presence of nigericin. The proportion of [cytochrome c2+] required to achieve 50% of maximal reduction of cytochrome a varies with the added ionophores; the apparent redox potential of cytochrome a is most positive in the fully decontrolled system (plus valinomycin and nigericin). At low levels of cytochrome a reduction, the rate of respiration is no longer a linear function of [cytochrome c2+], but is dependent upon the redox state of both cytochromes a and c. That is, proteoliposomal oxidase does not follow Smith-Conrad kinetics at low cytochrome c reduction levels, especially in the controlled states. The control of cytochrome oxidase turnover by delta pH and by delta psi can be explained either by an allosteric model or by a model with reversed electron transfer between the binuclear centre and cytochrome a. Other evidence suggests that the reversed electron transfer model may be the correct one.

The kinetics of photoinactivation and long-term photosensitivity of pigmented and nonpigmented strains of the smut fungusUstilago violacea

White, pink, pumpkin, and yellow strains ofUstilago violacea containing high and low levels of cytochrome c and various carotenes were subjected to high light intensities to characterize the kinetics of photoinactivation. Additionally, these strains were grown at two light intensities to characterize their long-term resistance to photoinactivation. The orange strain 2.D37291S and yellow strain 1.C2y had the highest carotene contents and were the most light resistant in the kinetics and growth experiments. The pink strains 2.C425, AB278a-1, and 1.C421 accumulated cytochrome c as well as carotenes. These strains were slightly more photosensitive than the yellow or orange strains in the kinetics experiment but were much more sensitive in the growth experiment. The phytoene-containing white strain 2.C419, which contains a small amount of cytochrome c, had a high level of resistance to light in the kinetics and growth experiments. The carotene-less strains were most sensitive in the kinetics experiment but not in the growth experiment. The overall photosensitivity ofU. violacea strains is related to the carotene and cytochrome c contents.

Photosensitivity of pigmented and nonpigmented strains ofUstilago violacea

White, pink, pumpkin, orange, and yellow strains ofUstilago violacea containing high and low levels of cytochrome c and various carotenes were subjected to high light intensities in the presence and absence of the endogenous photosensitizer toluidine blue (TB). The white strain 15.10 and the cytochrome-c-accumulating pink strain 1.C429 were completely lacking carotenes and were the most sensitive to visible light under all conditions. The phytoene-containing white strain 2.C419, the carotene-containing pink strains AB278a-1, 1.C421, and 2.C425, and pumpkin strain 2.C415 were more resistant to photodestruction in the presence and absence of TB than strains 15.10 and 1.C429. Consistently, the most light resistant strains were the high carotene, low cytochrome-c-accumulating orange, and yellow strains. The photosensitivity of strains ofU. violacea was related to the level of carotene, cytochrome c, the presence or absence of TB, and the age of the culture.

Ultraviolet light sensitivity of carotene-and cytochrome-c-accumulating strains of the smut fungusUstilago violacea

White, pink, orange, and yellow strains ofUstilago violacea containing high and low levels of cytochrome c and various carotenes were exposed to ultraviolet light. The survival curves for all strains were of exponential decay form, but the carotene-accumulating strains were generally more resistant to UV than those strains with no carotenes at all. The UV exposure time leading to 90% loss in viability, LD90, was quantitatively related to the carotene content in the form of a power function, where LD90 increased as the fifth root of the total carotene content per cell. We also determined that the ratio of total carotenes to total cytochrome c per cell was quantitatively related to the rate of viability loss during UV exposure.

Isolation and characterization of subcellular membranes from canine stomach smooth muscle.

Subcellular membrane fractions were isolated from the circular muscle of the corpus of canine stomach by differential and isopycnic sucrose density gradient centrifugation. Differential centrifugation gave a mitochondrial fraction enriched (fourfold) in cytochrome c oxidase and a microsomal fraction enriched (fourfold) in 5'-nucleotidase and NADPH-cytochrome c reductase over postnuclear supernatant. On the basis of a study using continuous gradient, a discontinuous sucrose density gradient was prepared to yield F1 to F5 fractions. The F3 fraction at the interface of 18-32% (w/w) sucrose was maximally enriched (13-fold) in 5'-nucleotidase. The fraction contained very low levels of cytochrome c oxidase but did contain NADPH-cytochrome c reductase (eightfold enrichment). The F4 fraction, at the interface of 32-40% (w/w) sucrose, was maximally enriched in NADPH-cytochrome c reductase (12-fold) and cytochrome c oxidase (6-fold). The distribution of the azide-insensitive. ATP-dependent Ca2+ uptake correlated very well with that of 5'-nucleotidase but less well with NADPH-cytochrome c reductase and not at all with cytochrome c oxidase. Sodium azide and ruthenium red inhibited the ATP-dependent Ca2+ uptake by the mitochondrial fraction and postnuclear supernatant, but not by the F3 fraction. ATP-dependent Ca2+ uptake by the F3 fraction was inhibited by calcium ionophores A23187 and ionomycin, but not by the sodium ionophore, monensin. These results are consistent with the hypothesis that the plasma membrane plays a major role ih regulating intracellular Ca2+ concentration in canine corpus circular muscle.

Biogenesis of mitochondria: The effects of membrane unsaturated fatty acid content on the activity and assembly of the yeast mitochondrial protein-synthesizing system

The steady-state unsaturated fatty acid content of yeast mitochondria was controlled by growing cells of an unsaturated fatty acid auxotroph under derepressed conditions in glucose-limited chemostat cultures. Variations in mitochondrial unsaturated fatty acid content influenced the activity and assembly of the mitochondrial protein-synthesizing system. The temperature of discontinuity of Arrhenius plots of in vitro mitochondrial protein synthetic activity increased from below 6 ° to 24 °C as the proportion of membrane unsaturated fatty acid was lowered from 75 to 44%. Arrhenius plots of the inner mitochondrial membrane functions ATPase and NADH oxidase gave similar results. When the proportion of membrane unsaturated fatty acid was reduced below about 30%, no in vitro mitochondrial protein synthetic activity could be detected. These mitochondria lacked cytochrome aa 3, had low levels of cytochrome b, and the ATPase was insensitive to oligomycin inhibition, indicating that mitochondrial protein synthesis was also inactive in vivo. No mitochondrial ribosomal subunits could be detected in mitochondria containing 28% or less of their fatty acids unsaturated. Although mitochondrial transcription occurred, the RNA fractions isolated were all of low molecular weight, and intact mitochondrial ribosomal RNA could not be detected.