Cellular Cytochrome Research Articles

Copper Modulates Mitochondrial Oxidative Phosphorylation to Enhance Dermal Papilla Cells Proliferation in Rex Rabbits.

Copper (Cu) is an important coenzyme factor in cell signaling, such as cytochrome c oxidase (Complex IV). Metabolism plays an important role in regulating the fate of mammalian cells. The aim of this study is to experimentally investigate the effect of copper on cell metabolism in the dermal papilla cells of the Rex rabbit. In this study, Cu promoted proliferation of dermal papilla cells (p = 0.0008) while also increasing levels of cellular CIII, CIV, Complex IV and ATP. Moreover, fifty metabolites that were significantly different between Cu and controls were identified as potential biomarkers of Cu stimulation. Copper-stimulated cells had altered levels of arachidonic acid derivatives, S-glutamic acid, and citric acid, which were primarily linked to two different pathways: arachidonic acid metabolism (p < 0.0001) and alanine, aspartate and glutamate metabolism (p = 0.0003). The addition of Cu can increase the proliferation of Rex rabbit dermal papilla cells. Increased levels of ubiquinol-cytochrome c reductase complex core protein 2 (CIII) and cytochrome c oxidase subunit 1 (CIV) were associated with the increased levels of cellular cytochrome c oxidase (Complex IV) and adenosine triphosphate (ATP). In a word, copper promotes cell proliferation by maintaining the function of the cellular mitochondrial electron transport chain (ETC) pathway.

Discovery of a selective and covalent small-molecule inhibitor of BFL-1 protein that induces robust apoptosis in cancer cells

Induction of apoptosis by the FDA-approved drug Venetoclax in cancer cells mainly derives from blocking the interactions between BCL-2 and BH3-only proteins. Anti-apoptotic BFL-1, a homolog of BCL-2, also competitively binds to the BH3-only proteins and is responsible for Venetoclax-induced drug resistance. Compared to BCL-2, small-molecule inhibitors of BFL-1 are relatively underexplored. In order to tackle this issue, in-house compound library was screened and a hit compound was identified and optimized to obtain 12 (ZH97) functioning as a covalent and selective inhibitor of BFL-1. 12 modifies BFL-1 at the C55 residue, blocks BFL-1/BID interaction in vitro, promotes cellular cytochrome c release from mitochondria, and induced apoptosis in BFL-1 overexpressing cancer cells. Mechanistic studies show that 12 inhibited BFL-1/PUMA interaction in cell lysate and is effective in cancer cells that harboring high expression level of BFL-1. In summary, blockade of BFL-1/BH3-only proteins interactions with a covalent small-molecule inhibitor induced apoptosis and elicited antitumor activity. Thus, our study demonstrates an appealing strategy for selective modulation of cellular BFL-1 for cancer therapy.

Multifunctional Cellular Targeting, Molecular Delivery, and Imaging by Integrated Mesoporous-Silica with Optical Nanocrescent Antenna: MONA.

Multifunctional nanoprobes have attracted significant attention in a wide range of disciplines such as nanomedicine, precision medicine, and cancer diagnosis and treatment. However, integrating multifunctional ability in a nanoscale structure to precisely target, image, and deliver with cellular spatial/temporal resolution is still challenging in cellulo applications. This is because the development of such high-precision resolution needs to be carried out without labeling, photobleaching, and structurally segregating live cells. In this study, we present an integrated nanostructure of a mesoporous-silica nanosphere with an optical nanocrescent antenna (MONA) for multifunctional cellular targeting, drug delivery, and molecular imaging with spatiotemporal resolution. MONA comprises a systematically constructed Au nanocrescent (AuNC) antenna as a nanosensor and optical switch on a mesoporous-silica nanosphere as a cargo to molecular delivery. MONA made of antiepithelial cell adhesion molecules (anti-EpCAM)-conjugated AuNC facilitates the specific targeting of breast cancer cells, resulting in a highly focused photothermal gradient that functions as a molecular emitter. This light-driven molecular, doxorubicin (DOX) delivery function allows rapid apoptosis of breast cancer cells. Since MONA permits the tracking of quantum biological electron-transfer processes, in addition to its role as an on-demand optical switch, it enables the monitoring of the dynamic behavior of cellular cytochrome c pivoting cell apoptosis in response to the DOX delivery. Owing to the integrated functions of molecular actuation and direct sensing at the precisely targeted spot afforded by MONA, we anticipate that this multifunctional optical nanoantenna structure will have an impact in the fields of nanomedicine, cancer theranostics, and basic life sciences.

Mechanisms underlying Clostridium pasteurianum's metabolic shift when grown with Geobacter sulfurreducens.

Recently, a study showed that glycerol fermentation by Clostridium pasteurianum could be metabolically redirected when the electroactive bacterium Geobacter sulfurreducens was added in the culture. It was assumed that this metabolic shift of the fermentative species resulted from an interspecies electron transfer. The aim of this study was to find out the mechanisms used for this interaction and how they affect the metabolism of C. pasteurianum. To get insights into the mechanisms involved, several coculture setups and RNA sequencing with differential expression analysis were performed. As a result, a putative interaction model was proposed: G. sulfurreducens produces cobamide molecules that possibly modify C. pasteurianum metabolic pathway at the key enzyme glycerol dehydratase, and affect its vanadium nitrogenase expression. In addition, the results suggested that G. sulfurreducens' electrons could enter C. pasteurianum through its transmembrane flavin-bound polyferredoxin and cellular cytochrome b5-rubredoxin interplay, putatively reinforcing the metabolic shift. Unravelling the mechanisms behind the interaction between fermentative and electroactive bacteria helps to better understand the role of bacterial interactions in fermentation setups. KEY POINTS: • C. pasteurianum-G. sulfurreducens interaction inducing a metabolic shift is mediated • C. pasteurianum's metabolic shift in coculture might be induced by cobamides • Electrons possibly enter C. pasteurianum through a multiflavin polyferredoxin.

Ferrimicrobium acidiphilum Exchanges Electrons With a Platinum Electrode via a Cytochrome With Reduced Absorbance Maxima at 448 and 605 nm.

Ferrimicrobium acidiphilum is a Gram-positive member of the Actinobacteria phylum that can respire aerobically or anaerobically with soluble Fe(II) or Fe(III), respectively, in sulfuric acid at pH 1.5. Cyclic voltammetry measurements using intact F. acidiphilum at pH 1.5 produced fully reversible voltammograms that were highly reproducible. The maximum current observed with the anodic peak was considerably less than was the maximum current observed with the cathodic peak. This difference was attributed to the competition between the platinum electrode and the soluble oxygen for the available electrons that were introduced by the cathodic wave into this facultative aerobic organism. The standard reduction potential of the intact organism was determined to be 786 mV vs. the standard hydrogen electrode, slightly more positive than that of 735 mV that was determined for soluble iron at pH 1.5 using the same apparatus. Chronocoulometry measurements conducted at different cell densities revealed that the intact organism remained in close proximity to the working electrode during the measurement, whereas soluble ionic iron did not. When the cyclic voltammetry of intact F. acidiphilum was monitored using an integrating cavity absorption meter, the only small changes in absorbance that were detected were consistent with the participation of a cellular cytochrome with reduced absorbance peaks at 448 and 605 nm. The cytochrome that participated in the exchange of electrons between the intact organism and extracellular solid electrodes like platinum was the same cytochrome whose oxidation was previously shown to be rate-limiting when the organism respired aerobically on extracellular soluble iron.

Effect of polyaromatic hydrocarbons on cellular cytochrome P450 1A induction

Effect of polyaromatic hydrocarbons on cellular cytochrome P450 1A induction

Vitamin D3 and calcium cosupplementation alleviates cadmium hepatotoxicity in the rat: Enhanced antioxidative and anti‐inflammatory actions by remodeling cellular calcium pathways

Although vitamin D (VD) and calcium (Ca) attenuate cadmium (Cd) metabolism, their combined antioxidant and anti-inflammatory actions against Cd toxicity have not been previously explored. Hence, this study measured the protective effects of VD ± Ca supplements against Cd hepatotoxicity. Forty adult male rats were distributed to: negative controls (NCs), positive controls (PCs), VD, Ca, and VD3 and Ca (VDC) groups. All groups, except NC, received CdCl2 in drinking water (44 mg/L) for 4 weeks individually or concurrently with intramuscular VD3 (600 IU/kg; three times per week) and/or oral Ca (100 mg/kg; five times per week). The PC group showed abnormal hepatic biochemical parameters and increase in cellular cytochrome C, caspase-9, and caspase-3 alongside the apoptotic/necrotic cell numbers by terminal deoxynucleotidyl transferase dUTP nick end labeling technique. The PC hepatic tissue also had substantially elevated pro-oxidants (malondialdehyde [MDA]/H2 O2 /protein carbonyls) and inflammatory cytokines (interleukin 1β [IL-1β]/IL-6/IL17A/tumor necrosis factor-α), whereas the anti-inflammatory (IL-10/IL-22) and antioxidants (glutathione [GSH]/GPx/catalase enzyme [CAT]) markers declined. Hypovitaminosis D, low hepatic tissue Ca, aberrant hepatic expression of VD-metabolizing enzymes (Cyp2R1/Cyp27a1/cyp24a1), receptor and binding protein alongside Ca-membrane (CaV 1.1/CaV 3.1), and store-operated (RyR1/ITPR1) channels, and Ca-binding proteins (CAM/CAMKIIA/S100A1/S100B) were observed in the PC group. Both monotherapies decreased serum, but not tissue Cd levels, restored the targeted hepatic VD/Ca molecules' expression. However, these effects were more prominent in the VD group than the Ca group. The VDC group, contrariwise, disclosed the greatest alleviations on serum and tissue Cd, inflammatory and oxidative markers, the VD/Ca molecules and tissue integrity. In conclusion, this report is the first to reveal boosted protection for cosupplementing VD and Ca against Cd hepatotoxicity that could be due to enhanced antioxidative, anti-inflammatory, and modulation of the Ca pathways.

Raman micro-spectroscopy monitoring of cytochrome c redox state in Candida utilis during cell death under low-temperature plasma-induced oxidative stress.

Oxidative stress may result in different modes of cell death, such as necrosis, apoptosis and necroptosis. Currently, researchers are still striving to develop efficient tools/methods to distinguish the cell death modes in direct and label-free ways. In this study, we attempted to employ Raman micro-spectroscopy to observe the molecular changes in Candida utilis cells under oxidative stress induced by low-temperature plasma (LTP) and explore the spectroscopic biomarkers for the modes of cell death under oxidative stress. In this research, we confirmed that LTP could impose oxidative stress on the yeast cells, and recorded the changes of Raman signals of cytochrome c in the cells under LTP oxidative stress. Subsequently, we identified the biochemical and morphological characteristic features corresponding to different modes of cell death. Interestingly, we found that LTP under certain conditions could induce oxidative stress which caused the yeast cell death mainly by means of necroptosis, which was verified by Annexin V/PI, HMGB1 location assay and immunoprecipitation assay of the RIP1/RIP3 necrosome. Correspondingly, we also showed that the LTP induced necroptosis, associated with the increase of cytoplasmic Ca2+ and mitochondrial ROS, the decrease of mitochondrial membrane potential, the release of oxidized cytochrome c from the mitochondrion to the cytoplasm, and the destruction of mitochondria in yeast cells. This work has therefore demonstrated that monitoring the redox state of cytochrome c using Raman micro-spectroscopy is very useful for distinguishing the modes of cell death and particularly may unveil the unique necroptosis process of cells under extrinsic oxidative stress.

Interface Sensitized Optical Microfiber Biosensors

Sensitivity is an important parameter to characterize the performance of sensors. Interfacial modification and functionalization is an effective strategy to improve the sensitivity of optical biosensors. This paper reports our recent development on interface sensitized fiber optic biosensors for the detection of ultra-low concentration of biological targets. We employ microfiber mode interferometer as transducer to sense the molecular binding interaction induced surrounding refractive index change. By modifying the microfiber surface to increase the assembling surface area and enhance the evanescent field energy density, ultra-low limit of detection of 1.65 × 10−15 M and 6.82 × 10−17 M, respectively, were realized for neurotransmitter γ-amino-butyric acid and cellular cytochrome c.

Identification of influential proteins in the classical retinoic acid signaling pathway

BackgroundIn the classical pathway of retinoic acid (RA) mediated gene transcription, RA binds to a nuclear hormone receptor dimer composed of retinoic acid receptor (RAR) and retinoid X receptor (RXR), to induce the expression of its downstream target genes. In addition to nuclear receptors, there are other intracellular RA binding proteins such as cellular retinoic acid binding proteins (CRABP1 and CRABP2) and cytochrome P450 (CYP) enzymes, whose contributions to the RA signaling pathway have not been fully understood. The objective of this study was to compare the significance of various RA binding receptors, i.e. CRABP1, CRABP2, CYP and RAR in the RA signaling pathway. In this regard, we developed a mathematical model of the RA pathway, which is one of the few models, if not the only one, that includes all main intracellular RA binding receptors. We then performed a global sensitivity analysis (GSA) to investigate the contribution of the RA receptors to RA-induced mRNA production, when the cells were treated with a wide range of RA levels, from physiological to pharmacological concentrations.ResultsOur results show that CRABP2 and RAR are the most and the least important proteins, respectively, in controlling the model performance at physiological concentrations of RA (1–10 nM). However, at higher concentrations of RA, CYP and RAR are the most sensitive parameters of the system. Furthermore, we found that depending on the concentrations of all RA binding proteins, the rate of metabolism of RA can either change or remain constant following RA therapy. The cellular levels of CRABP1 are more important than that of CRABP2 in controlling RA metabolite formation at pharmacological conditions (RA = 0.1–1 μM). Finally, our results indicate a significant negative correlation between total mRNA production and total RA metabolite formation at pharmacological levels of RA.ConclusionsOur simulations indicate that the significance of the RA binding proteins in the RA pathway of gene expression strongly depends on intracellular concentration of RA. This study not only can explain why various cell types respond to RA therapy differently, but also can potentially help develop pharmacological methods to increase the efficacy of the drug.

Quinone and nitrofurantoin redox cycling by recombinant cytochrome b5 reductase

NADH cytochrome b5 reductase mediates electron transfer from NADH to cytochrome b5 utilizing flavin adenine dinucleotide as a redox cofactor. Reduced cytochrome b5 is an important cofactor in many metabolic reactions including cytochrome P450-mediated xenobiotic metabolism, steroid biosynthesis and fatty acid metabolism, hemoglobin reduction, and methionine and plasmalogen synthesis. Using recombinant human enzyme, we discovered that cytochrome b5 reductase mediates redox cycling of a variety of quinones generating superoxide anion, hydrogen peroxide, and, in the presence of transition metals, hydroxyl radicals. Redox cycling activity was oxygen-dependent and preferentially utilized NADH as a co-substrate; NADH was 5–10 times more active than NADPH in supporting redox cycling. Redox cycling activity was greatest for 9,10-phenanthrenequinone and 1,2-naphthoquinone, followed by 1,4-naphthoquinone and 2-methyl-1,4-naphthoquinone (menadione), nitrofurantoin and 2-hydroxyestradiol. Using menadione as the substrate, quinone redox cycling was found to inhibit reduction of cytochrome b5 by cytochrome b5 reductase, as measured by heme spectral changes in cytochrome b5. Under anaerobic conditions where redox cycling is inhibited, menadione had no effect on the reduction of cytochrome b5. Chemical redox cycling by cytochrome b5 reductase may be important in generating cytotoxic reactive oxygen species in target tissues. This activity, together with the inhibition of cytochrome b5 reduction by redox-active chemicals and consequent deficiencies in available cellular cytochrome b5, are likely to contribute to tissue injury following exposure to quinones and related redox active chemicals.

Real-Time Cellular Cytochrome C Monitoring through an Optical Microfiber: Enabled by a Silver-Decorated Graphene Nanointerface.

The translocation of cytochrome c (cyt c) from mitochondria and out of cell is an important signal of cell apoptosis. Monitoring this process extracellularly without invasion and cytotoxicity to cells is of great importance to understand certain diseases at the cellular level; however, it requires sensors with ultrahigh sensitivity and miniature size. This study reports an optical microfiber aptasensor with a silver‐decorated graphene (Ag@RGO) nanointerface for real‐time cellular cyt c monitoring. Owing to an interfacial sensitization effect coupled with the plasmonic electromagnetic enhancement of silver nanoparticles and chemical enhancement of graphene platforms, which enhances the energy density on microfiber surface obviously, the lowest limit of detection achieved is 6.82 × 10−17 m, which is approximately five orders of magnitude lower than those of existing methods. This microfiber successfully detects the ultralow concentrations of cyt c present during the initial stage of apoptosis in situ. As the microfiber functionalized by Ag@RGO nanointerface can be varied to meet any specific detection objective, this work opens up new opportunities to quantitatively monitor biological functions occurring at the cellular level.

Long-distance electron transport in individual, living cable bacteria

Electron transport within living cells is essential for energy conservation in all respiring and photosynthetic organisms. While a few bacteria transport electrons over micrometer distances to their surroundings, filaments of cable bacteria are hypothesized to conduct electric currents over centimeter distances. We used resonance Raman microscopy to analyze cytochrome redox states in living cable bacteria. Cable-bacteria filaments were placed in microscope chambers with sulfide as electron source and oxygen as electron sink at opposite ends. Along individual filaments a gradient in cytochrome redox potential was detected, which immediately broke down upon removal of oxygen or laser cutting of the filaments. Without access to oxygen, a rapid shift toward more reduced cytochromes was observed, as electrons were no longer drained from the filament but accumulated in the cellular cytochromes. These results provide direct evidence for long-distance electron transport in living multicellular bacteria.

Membrane Transporters for Bilirubin and Its Conjugates: A Systematic Review.

Background: Bilirubin is a highly-hydrophobic tetrapyrrole which binds to plasma albumin. It is conjugated in the liver to glucuronic acid, and the water-soluble glucuronides are excreted in urine and bile. The membrane transporters of bilirubin diglucuronide are well-known. Still undefined are however the transporters performing the uptake of bilirubin from the blood into the liver, a process known to be fast and not rate-limited. The biological importance of this process may be appraised by considering that in normal adults 200–300 mg of bilirubin are produced daily, as a result of the physiologic turnover of hemoglobin and cellular cytochromes. Nevertheless, research in this field has yielded controversial and contradicting results. We have undertaken a systematic review of the literature, believing in its utility to improve the existing knowledge and promote further advancements.Methods: We have sourced the PubMed database until 30 June 2017 by applying 5 sequential searches. Screening and eligibility criteria were applied to retain research articles reporting results obtained by using bilirubin molecules in membrane transport assays in vitro or by assessing serum bilirubin levels in in vivo experiments.Results: We have identified 311 articles, retaining 44, reporting data on experimental models having 6 incremental increases of complexity (isolated proteins, membrane vesicles, cells, organ fragments, in vivo rodents, and human studies), demonstrating the function of 19 membrane transporters, encoded by either SLCO or ABC genes. Three other bilirubin transporters have no gene, though one, i.e., bilitranslocase, is annotated in the Transporter Classification Database.Conclusions: This is the first review that has systematically examined the membrane transporters for bilirubin and its conjugates. Paradoxically, the remarkable advancements in the field of membrane transport of bilirubin have pointed to the elusive mechanism(s) enabling bilirubin to diffuse into the liver as if no cellular boundary existed.

3D cell clusters combined with a bioreactor system to enhance the drug metabolism activities of C3A hepatoma cell lines.

Since clinical drugs need to be approved for their liver metabolism efficiency before commercialization, a powerful in vitro drug-screening platform is imperative and indispensable for the clinical medicine and pharmaceutical industries. An essential issue in the development of drug screening platforms is choosing cell candidates that mimic and perform cell/tissue functions of normal hepatic tissues in vivo. In this study, we developed a self-designed bioreactor system to provide and mimic an appropriate environment for systematic cell expansion, micro-tissue formation, and increased cellular cytochrome P450 (CYP) enzymatic activities. Since CYP3A4 is the most plentiful and crucial enzyme in drug metabolism among liver CYP superfamily members, we demonstrated that micro-tissue formation under three-dimensional dynamic conditions could enhance cellular CYP3A4 enzymatic activity, maintain cell viability, and preserve adhesive abilities. Furthermore, Ca-alginate scaffolds used in this study can be completely removed by a non-toxic chelating reagent (EDTA solution), and the functional micro-tissues can be collected by slow-speed centrifugation. In conclusion, these micro-tissues are advantageous and show great potential in in vitro drug metabolizing assays.

Chronic hypoxia leads to a glycolytic phenotype and suppressed HIF-2 signaling in PC12 cells

BackgroundAlong with other regulators of cell metabolism, hypoxia-inducible factors HIF-1 and HIF-2 differentially regulate cell adaptation to hypoxia. Switches in HIF-1/HIF-2 signaling in chronic hypoxia have not been fully investigated. MethodsProliferation, viability, apoptosis, neuronal and bioenergetic markers, mitochondrial function, respiration, glycolysis, HIF signalling, responses to O2 and glucose deprivation (OGD) were examined using tumor PC12 and SH-SY5Y cells continuously grown at 3% O2. ResultsHypoxic PC12 cells (H-cells) exhibit reduced proliferation and histone H4 acetylation, NGF-independent differentiation, activation of AMPK, inhibition of Akt, altered mitochondria and response to NGF. Cellular cytochrome c is increased with no effect on apoptosis. Reduction in respiration has minor effect on cellular ATP which is maintained through activated uptake (GLUT1) and utilization (HK2, PFK2) of glucose. H-cells exhibit resistance to OGD linked to increased glycogen stores. HIF-2alpha protein is decreased without changes in mRNA. Unlike HIF-1alpha, HIF-2alpha is not stabilized pharmacologically or by O2 deprivation. Capacity for HIF-2alpha stabilization is partly restored when H-cells are cultured at normoxia. In low-respiring SH-SY5Y cells cultured under the same conditions HIF-2alpha stabilization and energy budget are not affected. ConclusionsIn chronically hypoxic PC12 cells glycolytic energy budget, increased energy preservation and low susceptibility to OGD are observed. HIF-2alpha no longer orchestrates adaptive responses to anoxia. General significanceDemonstrated switch in HIF-1/HIF-2 signaling upon chronic hypoxia can facilitate cell survival in energy crisis, by regulating balance between energy saving and decrease in proliferation, on one hand and active cell growth and tumor expansion, on the other.

Modulation of Serines 17 and 24 in the LC3-interacting Region of Bnip3 Determines Pro-survival Mitophagy versus Apoptosis

BH3-only proteins integrate apoptosis and autophagy pathways, yet regulation and functional consequences of pathway cross-talk are not fully resolved. The BH3-only protein Bnip3 is an autophagy receptor that signals autophagic degradation of mitochondria (mitophagy) via interaction of its LC3-interacting region (LIR) with Atg8 proteins. Here we report that phosphorylation of serine residues 17 and 24 flanking the Bnip3 LIR promotes binding to specific Atg8 members LC3B and GATE-16. Using quantitative multispectral image-based flow cytometry, we demonstrate that enhancing Bnip3-Atg8 interactions via phosphorylation-mimicked LIR mutations increased mitochondrial sequestration, lysosomal delivery, and degradation. Importantly, mitochondria were targeted by mitophagy prior to cytochrome c release, resulting in reduced cellular cytochrome c release capacity. Intriguingly, pro-survival Bcl-x(L) positively regulated Bnip3 binding to LC3B, sequestration, and mitochondrial autophagy, further supporting an anti-apoptotic role for Bnip3-induced mitophagy. The ensemble of these results demonstrates that the phosphorylation state of the Bnip3 LIR signals either the induction of apoptosis or pro-survival mitophagy.

In situ Spectroscopy on Intact Leptospirillum ferrooxidans Reveals that Reduced Cytochrome 579 is an Obligatory Intermediate in the Aerobic Iron Respiratory Chain.

Electron transfer reactions among colored cytochromes in intact bacterial cells were monitored using an integrating cavity absorption meter that permitted the acquisition of accurate absorbance data in suspensions of cells that scatter light. The aerobic iron respiratory chain of Leptospirillum ferrooxidans was dominated by the redox status of an abundant cellular cytochrome that had an absorbance peak at 579 nm in the reduced state. Intracellular cytochrome 579 was reduced within the time that it took to mix a suspension of the bacteria with soluble ferrous iron at pH 1.7. Steady state turnover experiments were conducted where the initial concentrations of ferrous iron were less than or equal to that of the oxygen concentration. Under these conditions, the initial absorbance spectrum of the bacterium observed under air-oxidized conditions was always regenerated from that of the bacterium observed in the presence of Fe(II). The kinetics of aerobic respiration on soluble iron by intact L. ferrooxidans conformed to the Michaelis–Menten formalism, where the reduced intracellular cytochrome 579 represented the Michaelis complex whose subsequent oxidation appeared to be the rate-limiting step in the overall aerobic respiratory process. The velocity of formation of ferric iron at any time point was directly proportional to the concentration of the reduced cytochrome 579. Further, the integral over time of the concentration of the reduced cytochrome was directly proportional to the total concentration of ferrous iron in each reaction mixture. These kinetic data obtained using whole cells were consistent with the hypothesis that reduced cytochrome 579 is an obligatory steady state intermediate in the iron respiratory chain of this bacterium. The capability of conducting visible spectroscopy in suspensions of intact cells comprises a powerful post-reductionist means to study cellular respiration in situ under physiological conditions for the organism.

Polyphenolic Extracts fromOlea europea L.Protect Against Cytokine-Induced β-Cell Damage Through Maintenance of Redox Homeostasis

Various pancreatic β-cell stressors, including cytokines, are known to induce oxidative stress, resulting in apoptotic/necrotic cell death and inhibition of insulin secretion. Traditionally, olive leaves or fruits are used for treating diabetes, but the cellular mechanism(s) of their effects are not known. We examined the effects of Olea europea L. (olive) leaf and fruit extracts and their component oleuropein on cytokine-induced β-cell toxicity. INS-1, an insulin-producing β-cell line, was preincubated with or without increasing concentrations of olive leaf or fruit extract or oleuropein for 24 hr followed by exposure to a cytokine cocktail containing 0.15 ng/mL interleukin-1β (IL-1β), 1 ng/mL interferon-γ (IFN-γ), and 1 ng/mL tumor necrosis factor-α (TNF-α) for 6 hr. The cytotoxicity was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) testing. Apoptosis was quantified by detecting acridine orange/ethidium bromide-stained condensed nuclei under a fluorescent microscope. The cells exposed to cytokines had a higher apoptotic rate, a decreased viability (MTT), and an increased caspase 3/7 activity. Both extracts and oleuropein partially increased the proportion of living cells and improved the viability of cells after cytokines. The protective effects of extracts on live cell viability were mediated through the suppression of caspase 3/7 activity. Oleuropein did not decrease the amount of both apoptotic and necrotic cells, whereas extracts significantly protected cells against cytokine-induced death. Cytokines led to an increase in reactive oxygen species (ROS) generation and inhibited glutathione level, superoxide dismutase activity, and insulin secretion in INS-1. Insulin secretion was almost completely protected by leaf extract, but was partially affected by fruit extract or oleuropein. Neither cytokines nor olive derivatives had a significant effect on cellular cytochrome c release and catalase activity. Moreover, the cells incubated with each extract or oleuropein showed a significant reduction in cytokine-induced ROS production and ameliorated abnormal antioxidant defense. The molecular mechanism by which olive polyphenols inhibit cytokine-mediated β-cell toxicity appears to be involving the maintenance of redox homeostasis.

Human Prion Protein Mutants with Deleted and Inserted Octarepeats Undergo Different Pathways to Trigger Cell Apoptosis

Octarepeats region sequence is one of the most important characteristics of PrP topology. To explore the mechanism of deleted and inserted octarepeats mutants PrP-caused apoptosis, wild-type PrP (PrP-PG5), and PrP with deleted octarepeats (PrP-PG0) and with four (PrP-PG9) and seven (PrP-PG12) extra octarepeats were transiently induced into SH-SY5Y cell. The results indicated PrP-PG9 and PrP-PG12 mainly retained in fraction of cytoplasm, while PrP-PG5 and PrP-PG0 presented both in cell membrane and cytoplasm. Cells expressing PrP-PG9 and PrP-PG12 were sensitive to endoplasmic reticulum (ER) stimuli, tunicamycin, and brefeldin A. ER-stress-related proteins, Grp94, XBP1, TRAF2, and CHOP, were significantly increased in cells expressing PrP-PG9 and PrP-PG12, while Grp78 increased markedly 12 h and pro-caspase-12 decreased sharply 20 h post-transfection. It indicates that expressions of PrP mutants with inserted octarepeats cause ER stress and lead to cell apoptosis lately. Meanwhile, cellular Cytochrome C increased and Bcl-2 decreased obviously in cells expressing PrP-PG0, indicating triggering a mitochondrial-related apoptosis. These data highlight that PrP mutants in region of octarepeats may undergo different pathways to trigger cell apoptosis, in which PrPs with inserted octarepeats via ER stress and PrP mutant without octarepeats via mitochondrial-related pathway.