Changes In Cyt Research Articles

Electrochemical analysis of gold nanoparticles multifunctionalised with Cytochrome c and a zinc Porphyrin

Cytochrome c (Cyt c), known for its functional redox capabilities, plays a pivotal role in biological processes such as the electron transport chain and apoptosis. However, understanding how different conjugation strategies impact its structural and redox characteristics is limited. To fill this gap, we investigated the effects of conjugating Cyt c and a zinc(II) porphyrin (Zn Porph) to gold nanoparticles (AuNPs). We used circular dichroism (CD) spectroscopy to detect structural conformational changes in Cyt c upon conjugation and time-of-flight secondary ion mass spectrometry (ToF-SIMS) to identify protein orientation. Cyt c was predicted to have different orientations depending on the size of AuNPs and methods used to conjugate the protein, it was hypothesised that the orientation of Cyt c may influence the redox properties of the protein. The electrochemical properties of Cyt c were assessed using cyclic voltammetry (CV) and differential pulse voltammetry (DPV). We used DPV-based to determine the heterogeneous rate constant (k0). The results show a lower k0for conjugated Cyt c than free Cyt c, likely due to structural changes in the protein. The spatial orientation of Cyt c had minimal influence on k0, while ligand density and AuNP size had an effect. The k0 value of Zn Porph did not decrease on conjugation. Despite these changes, Cyt c and Zn Porph maintained their electrochemical capabilities after conjugation.

Echinacoside inhibits PASMCs calcium overload to prevent hypoxic pulmonary artery remodeling by regulating TRPC1/4/6 and calmodulin.

Research indicates that hypoxic pulmonary hypertension (HPH) potentially stimulates the sympathetic nervous system, which may increase norepinephrine (NE) release and cause excessive Ca2+ influx into pulmonary artery smooth muscle cells (PASMCs), leading to calcium overload and abnormal PASMC proliferation, factors closely associated with pulmonary vascular remodeling (PVR). This study investigates the potential mechanisms underlying echinacoside (ECH) treatment in HPH. In the in vitro experiment, NE-induced PASMCs were used to simulate HPH-induced PASMCs' calcium overload and abnormal proliferation. Postincubation with ECH, [Ca2+]cyt changes were detected using Fluo-4 AM. Flow cytometry was employed to ascertain ECH's inhibitory effect on PASMCs proliferation. For in vivo experiments, rats were exposed to a hypoxic and low-pressure oxygen environment to establish the HPH model. Post-ECH treatment, hematoxylin and eosin (HE) staining was conducted to assess PVR, and western blot analysis was used to examine protein expression in the lung tissues of the different groups. ECH was observed to inhibit [Ca2+]cyt increase in NE-induced PASMCs in a concentration-dependent manner, effectively reducing abnormal cell proliferation. It also reduced the expression of Transient receptor potential channel (TRPC) 1 (TRPC1), TRPC4, TRPC6, and calmodulin in PASMCs. In vivo studies demonstrated that ECH lowered the expression of these proteins in lung tissues of HPH rats, significantly decreased mean pulmonary artery pressure, and mitigated PVR.

Effect of pH on the conformational structure of cytochrome c and subsequent enzymatic cross-linking catalyzed by laccase

BackgroundThe aim of the present study was to investigate the effect of substrate conformational structure changes on the laccase-induced protein cross-linking. The effects of laccase amount, pH, and ferulic acid (FA) on the enzymatic cross-linking of substrate, Cyt C, were determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. High-performance size exclusion chromatography, laser particle size analysis and isothermal titration calorimetry (ITC) were also applied to investigate the cross-linking product and enthalpy changes. Structural changes of Cyt C at different pH values were analyzed by ultraviolet–visible (UV–vis), fluorescence, and circular dichroism (CD) measurements. ResultsComplete cross-linking, partial cross-linking, minute cross-linking, and no cross-linking occurred at pH 2.0, 4.0, 6.0, and 8.0, respectively. ITC analysis demonstrated that the enzymatic cross-linking of Cyt C was an endothermic process. The UV–vis, fluorescence, and CD measurements exhibited that the tertiary structure of Cyt C was disrupted, and part of the α-helical polypeptide region unfolded at pH 2.0. The structural flexibilities decreased, and the tertiary structure of Cyt C became increasingly compact with the increase in pH values from 4.0 to 8.0. The gradual changes in the structure of Cyt C at different pH values were in accordance with the cross-linking results of Cyt C catalyzed by laccase. ConclusionsThe results demonstrated that minute structure changes of substrate had a remarkable effect on the laccase-induced cross-linking. The findings promote the understanding of the substrate requirement of laccase in protein cross-linking and are instructive for the modulation of laccase-induced protein cross-linking.How to cite: Li D-X, Qi Z-Y, Liu J-Y, et al. Effect of pH on the conformational structure of cytochrome c and subsequent enzymatic cross-linking catalyzed by laccase. Electron J Biotechnol 2022;60. https://doi.org/10.1016/j.ejbt.2022.07.002.

Arabidopsis guard cell CO2/HCO3\u2212 response mutant screening by an aequorin-based calcium imaging system

BackgroundThe increase in atmospheric CO2 is causing a number of changes in plant growth such as increases in leaf area and number, branching, plant size and biomass, and growth rate. Despite the importance of stomatal responses to CO2, little is known about the genetic and molecular mechanisms that mediate stomatal development and movement in response to CO2 levels. Deciphering the mechanisms that sense changes in CO2 and/or HCO3− concentration is critical for unraveling the role of CO2 in stomatal development movement. In Arabidopsis, CO2-induced stomatal closure is strongly Ca2+-dependent. To further dissect this signaling pathway and identify new components in the CO2 response pathway, we recorded [Ca2+]cyt changes in mutagenized Arabidopsis leaves and screened for mutants with abnormal guard cell behavior in response to CO2/HCO3−.ResultsWe observed that 1 mM HCO3− induces [Ca2+]cys transient changes in guard cells and stomatal closure both in light and darkness. The changes in [Ca2+]cys induced by HCO3− could be detected by an aequorin-based calcium imaging system. Using this system, we identified a number of Arabidopsis mutants defective in both [Ca2+]cyt changes and the stomatal response to CO2/HCO3−.ConclusionsWe provide a sensitive method for isolating stomatal CO2/HCO3− response genes that function early in stomatal closure and that have a role in regulating [Ca2+]cyt. This method will be helpful in elucidating the Ca2+-dependent regulation of guard cell behavior in response to CO2/HCO3−.

(246) Determining the Role of Presynaptic Ca2+ Signaling in the Spinal Cord in Pain Sensitization

Chronic neuropathic pain affects approximately 10% of the population and only a minority of patients experience satisfactory relief of their pain with currently available pharmaceutics. As most previous reports focused primarily on the post-synaptic signaling of the first sensory synapse via patch-clamp recordings, we aim to address a gap in the current knowledge about the potential role of presynaptic Ca2+ signaling in central sensitization following neuropathic injury. Utilizing a novel ex vivo intact spinal cord preparation, a mouse line that expresses GCaMP specifically in the sensory neurons, and multiphoton microscopy to image presynaptic Ca2+ signaling, we are able to evaluate presynaptic Ca2+ signaling in the spinal cord isolated from mice that underwent either spared nerve injury (SNI) or sham surgery. Behavioral testing was used to confirm development of mechanical allodynia in SNI (but not sham) mice prior to presynaptic Ca2+ imaging. Presynaptic [Ca2+]cyt changes were induced by dorsal root stimulation, bradykinin, and capsaicin. The effects of modulatory neurotransmitters, such as dopamine, were also tested for their ability to regulate [Ca2+]cyt in central processes of sensory afferent neurons. Preliminary results quantifying frequency and duration of presynaptic [Ca2+]cyt transients indicate that neuropathic injury alters basal Ca2+ signaling and responsiveness to sensory modulators.

Review on cytochrome C

Cytochrome c (Cyt c) is an essential mitochondrial protein which takes part in the electron transport system and apoptosis. Cyt c also scavenges reactive oxygen species (ROS) and oxidizes cardiolipin during apoptosis. The recent finding that cyt c is phosphorylated in vivo for the important role of cyt c in the regulation of making life and death decisions. An apoptotic sequence of events takes place which involves changes in cyt c phosphorylation, increased ROS through increased mitochondrial membrane potentials, the oxidation of cardiolipin by cyt c, and its release from the mitochondria. Cyt c regulation in respiration and cellular death is discussed about human disease which includes neurodegenerative and cardiovascular diseases, cancer, and sepsis.

Selectivity and Reactivity of ZrIV and CeIV Substituted Keggin Type Polyoxometalates Toward Cytochrome c in Surfactant Solutions.

In this paper we investigate the effect of three different types of surfactants, on the hydrolysis of Cytochrome c (Cyt c), a predominantly α helical protein containing a heme group, promoted by [Ce(α PW11O39)2]10- (CeK) and [Zr(α PW11O39)2]10- (ZrK) polyoxometalates. In the presence of SDS, Zw3 12, or CHAPS surfactants, which are commonly used for solubilizing hydrophobic proteins, the specificity of CeK or ZrK toward hydrolysis of Cyt c does not change. However, the hydrolysis rate of Cyt c by CeK was increased in the presence of SDS, but decreased in the presence of CHAPS, and was nearly inhibited in the presence of Zw3 12. The Circular dichroism and Tryptophan fluorescence spectroscopy have shown that the structural changes in Cyt c caused by surfactants are similar to those caused by POMs, hence the same specificity in the absence or presence of surfactants was observed. The results also indicate that for Cyt c hydrolysis to occur, large unfolding of the protein is needed in order to accommodate the POMs. While SDS readily unfolds Cyt c, the protein remains largely folded in the presence of CHAPS and Zw3 12. Addition of POMs to Cyt c solutions in CHAPS results in unfolding of the structure allowing the interaction with POMs to occur and results in protein hydrolysis. Zw3 12, however, locks Cyt c in a conformation that resists unfolding upon addition of POM, and therefore results in nearly complete inhibition of protein hydrolysis.

Polyethylene glycol promotes autoxidation of cytochrome c

Cytochrome c (Cyt c) was rapidly oxidized by molecular oxygen in the presence, but not absence of PEG. The redox potential of heme c was determined by the potentiometric titration to be +236 ± 3 mV in the absence of PEG, which was negatively shifted to +200 ± 4 mV in the presence of PEG. The underlying the rapid oxidation was explored by examining the structural changes in Cyt c in the presence of PEG using UV–visible absorption, circular dichroism, resonance Raman, and fluorescence spectroscopies. These spectroscopic analyses suggested that heme oxidation was induced by a modest tertiary structural change accompanied by a slight shift in the heme position (<1.0 Å) rather than by partial denaturation, as is observed in the presence of cardiolipin. The near-infrared spectra showed that PEG induced dehydration from Cyt c, which triggered heme displacement. The primary dehydration site was estimated to be around surface-exposed hydrophobic residues near the heme center: Ile81 and Val83. These findings and our previous studies, which showed that hydrated water molecules around Ile81 and Val83 are expelled when Cyt c forms a complex with CcO, proposed that dehydration of these residues is functionally significant to electron transfer from Cyt c to CcO.

Assessment of cytosolic free calcium changes during ceramide-induced cell death in MDA-MB-231 breast cancer cells expressing the calcium sensor GCaMP6m

Alterations in Ca2+ signaling can regulate key cancer hallmarks such as proliferation, invasiveness and resistance to cell death. Changes in the regulation of intracellular Ca2+ and specific components of Ca2+ influx are a feature of several cancers and/or cancer subtypes, including the basal-like breast cancer subtype, which has a poor prognosis. The development of genetically encoded calcium indicators, such as GCaMP6, represents an opportunity to measure changes in intracellular free Ca2+ during processes relevant to breast cancer progression that occur over long periods (e.g. hours), such as cell death. This study describes the development of a MDA-MB-231 breast cancer cell line stably expressing GCaMP6m. The cell line retained the key features of this aggressive basal-like breast cancer cell line. Using this model, we defined alterations in relative cytosolic free Ca2+ ([Ca2+]CYT) when the cells were treated with C2-ceramide. Cell death was measured simultaneously via assessment of propidium iodide permeability. Treatment with ceramide produced delayed and heterogeneous sustained increases in [Ca2+]CYT. Where cell death occurred, [Ca2+]CYT increases preceded cell death. The sustained increases in [Ca2+]CYT were not related to the rapid morphological changes induced by ceramide. Silencing of the plasma membrane Ca2+ ATPase isoform 1 (PMCA1) was associated with an augmentation in ceramide-induced increases in [Ca2+]CYT and also cell death. This work demonstrates the utility of GCaMP6 Ca2+ indicators for investigating [Ca2+]CYT changes in breast cancer cells during events relevant to tumor progression, which occur over hours rather than minutes.

Acid-enhanced conformation changes of yeast cytochrome c coated onto gold nanoparticles, a FT-IR spectroscopic analysis

Under conditions with or without linker molecules, the effects of acidic pH on the conformation of yeast iso-1-cytochrome c coated onto gold nanoparticles (AuNPs) in correlation with color changes of a Cyt c-coated AuNPs solution/suspension were examined by Fourier transform infrared (FT-IR) spectroscopy and correlated to color change. The results of detailed secondary structural analysis revealed that although the color changes coincide with acid-induced conformational changes in Cyt c coated onto AuNPs, the pH-related conformational unfolding of Cyt c coated onto AuNPs differed dramatically from that of its counterpart in solution. For Cyt c free in solution, the acid-induced unfolding did not occur until the pH was below 3.0, whereas for Cyt c coated onto AuNPs via C102 coordination near the C-terminal, a partial unfolding was observed even at near neutral pH which continuously intensified as pH decreased. Insertion of a short alkanethiol (3-mercaptoproprionic acid, 3-MPA) molecule between Cyt c and AuNP, which changes the interaction mode from a thiol coordination between Cyt c and AuNP to an electrostatic interaction between Cyt c and 3-MPA, which stabilized the conformation of Cyt c significantly, but did not prevent the acid-induced aggregation of Cyt c-3MPA-AuNPs.

Simulation insight into the cytochrome c adsorption on graphene and graphene oxide surfaces

Abstract Graphene-based materials might serve as an ideal platform for the regulation and promotion of metalloprotein electron transfer (ET); however, the underpinning mechanism at the molecular level has not yet been fully revealed. The orientation of cytochrome c (Cyt c) on surfaces is vital for ET. In this work, the orientation of Cyt c on graphene and graphene oxide (GO) surfaces, as well as the dominant driving forces, the conformational change and the ET pathways were investigated by molecular dynamics simulations. The results show that Cyt c is adsorbed onto the GO surface mainly through lysine residues; whereas hydrophobic interaction contributes to the Cyt c adsorption on graphene surface. There is no significant conformational change of Cyt c upon adsorption. The heme plane of Cyt c tends to be horizontally oriented and far away from the graphene surface, which is not conducive to ET. On the GO surface, the heme plane is slightly deviated from the normal direction to the surface and the axial ligand Met80 is much closer to the surface, which facilitates the ET. These findings shed some light on the ET mechanism of Cyt c on graphene-based materials and provide guidance for the development of bionic electronic devices.

Probing the conformational changes and peroxidase-like activity of cytochrome c upon interaction with iron nanoparticles

Herein, the interaction of iron nanoparticle (Fe-NP) with cytochrome c (Cyt c) was investigated, and a range of techniques such as dynamic light scattering (DLS), zeta potential measurements, static and synchronous fluorescence spectroscopy, near and far circular dichroism (CD) spectroscopy, and ultraviolet–visible (UV–vis) spectroscopy were used to analyze the interaction between Cyt c and Fe-NP. DLS and zeta potential measurements showed that the values of hydrodynamic radius and charge distribution of Fe-NP are 83.95 ± 3.7 nm and 4.5 ± .8 mV, respectively. The fluorescence spectroscopy results demonstrated that the binding of Fe-NP with Cyt c is mediated by hydrogen bonds and van der Waals interactions. Also Fe-NP induced conformational changes in Cyt c and reduced the melting temperature value of Cyt c from 79.18 to 71.33°C. CD experiments of interaction between Fe-NP and Cyt c revealed that the secondary structure of Cyt c with the dominant α-helix structures remained unchanged whereas the tertiary structure and heme position of Cyt c are subjected to remarkable changes. Absorption spectroscopy at 695 nm revealed that Fe-NP considerably disrupt the Fe…S(Met80) bond. In addition, the UV–vis experiment showed the peroxidase-like activity of Cyt c upon interaction with Fe-NP. Hence, the data indicate the Fe-NP results in unfolding of Cyt c and subsequent peroxidase-like activity of denatured species. It was concluded that a comprehensive study of the interaction of Fe-NP with biological system is a crucial step for their potential application as intracellular delivery carriers and medicinal agents.

Recent and future changes in the combination of annual temperature and precipitation throughout China

ABSTRACTClimate involves different combinations of temperature and precipitation, and each year's combination of factors can be assigned a climatic year type (CYT; e.g. Warm‐Humid). Describing the changes in the CYT provides more information than describing the temperature or precipitation data alone. In this study, we defined nine CYTs using the probability density function of annual temperature and precipitation. Recent and future spatiotemporal changes in CYT were analysed using 507‐station observational data and projected data obtained from the CMIP5 multi‐model ensemble under RCP2.6, RCP4.5, and RCP8.5 scenarios. China was divided into six subregions to analyse the spatiotemporal changes. Obvious differences in spatial patterns among the various CYTs reflect the climate regime throughout China. The warmth‐associated CYTs (Warm‐Humid, Warm‐Dry, and Warm‐Normal) mainly occur in West China (e.g. Southwest China). The cold‐associated CYTs (Cold‐Humid, Cold‐Dry, and Cold‐Normal) dominate at high latitudes and high altitudes (e.g. Northeast China and the Tibetan Plateau). The climate in China changed from cold to warm in the last half‐century, accompanying the transformation of Cold‐Humid, Cold‐Dry, and Cold‐Normal before the early 1990s to Warm‐Humid, Warm‐Dry, and Warm‐Normal from the early 1990s onward. In the 21st century, the projected CYTs are mainly Warm‐Humid, Warm‐Dry, and Warm‐Normal in China. Warm‐Humid dominates in West China, North China, and Northeast China. Warm‐Dry is mainly projected in the Yellow River Valley and South China. High‐frequency Warm‐Normal is projected in the Yellow River Valley. Warm‐Humid is projected to increase whereas Warm‐Dry and Warm‐Normal are projected to decrease from 2015 to 2099. All three CYTs are projected to exhibit larger changes in trends under stronger versus weaker RCPs (RCP8.5 &gt; RCP4.5 &gt; RCP2.6). Compared with temperature or precipitation data alone, CYTs provide more complete information on climate change and more accurately characterize regional differences in climate throughout China.

Chemical glycosylation of cytochrome c improves physical and chemical protein stability.

BackgroundCytochrome c (Cyt c) is an apoptosis-initiating protein when released into the cytoplasm of eukaryotic cells and therefore a possible cancer drug candidate. Although proteins have been increasingly important as pharmaceutical agents, their chemical and physical instability during production, storage, and delivery remains a problem. Chemical glycosylation has been devised as a method to increase protein stability and thus enhance their long-lasting bioavailability.ResultsThree different molecular weight glycans (lactose and two dextrans with 1 kD and 10 kD) were chemically coupled to surface exposed Cyt c lysine (Lys) residues using succinimidyl chemistry via amide bonds. Five neo-glycoconjugates were synthesized, Lac4-Cyt-c, Lac9-Cyt-c, Dex5(10kD)-Cyt-c, Dex8(10kD)-Cyt-c, and Dex3(1kD)-Cyt-c. Subsequently, we investigated glycoconjugate structure, activity, and stability. Circular dichroism (CD) spectra demonstrated that Cyt c glycosylation did not cause significant changes to the secondary structure, while high glycosylation levels caused some minor tertiary structure perturbations. Functionality of the Cyt c glycoconjugates was determined by performing cell-free caspase 3 and caspase 9 induction assays and by measuring the peroxidase-like pseudo enzyme activity. The glycoconjugates showed ≥94% residual enzyme activity and 86 ± 3 to 95 ± 1% relative caspase 3 activation compared to non-modified Cyt c. Caspase 9 activation by the glycoconjugates was with 92 ± 7% to 96 ± 4% within the error the same as the caspase 3 activation. There were no major changes in Cyt c activity upon glycosylation. Incubation of Dex3(1 kD)-Cyt c with mercaptoethanol caused significant loss in the tertiary structure and a drop in caspase 3 and 9 activation to only 24 ± 8% and 26 ± 6%, respectively. This demonstrates that tertiary structure intactness of Cyt c was essential for apoptosis induction. Furthermore, glycosylation protected Cyt c from detrimental effects by some stresses (i.e., elevated temperature and humidity) and from proteolytic degradation. In addition, non-modified Cyt c was more susceptible to denaturation by a water-organic solvent interface than its glycoconjugates, important for the formulation in polymers.ConclusionThe results demonstrate that chemical glycosylation is a potentially valuable method to increase Cyt c stability during formulation and storage and potentially during its application after administration.

Hydrogen Sulfide Detection Using a Gold Nanoparticle/Metalloprotein Based Probe

We present a simple method for direct detection of hydrogen sulfide (H2S) in an aqueous solution. This method represents a novel biosensor based on metalloprotein cytochrome c (cyt c) with the localized surface plasmon resonance of gold nanoparticles (AuNPs). For this purpose, we develop a new approach based on attaching chemically-modified cyt c onto AuNPs. Here, by reacting H2S with protein heme center, its conformation changes in the locality of the heme moiety. The conformational changes occurring in the protein alter the spectral characteristics by changing the dielectric properties of AuNPs. The conformational changes of cyt c induced by the H2S interaction are characterized by the UV-visible absorption spectroscopy and the circular dichroism technique. The limit of the detection and sensitivity of the AuNPs/cyt c biosensor are evaluated by using UV-visible spectroscopy. According to the experiments, it is revealed that H2S can be detected at a concentration of 4.0 μM (1.3 ppb) by the fabricated AuNPs/cyt c biosensor. In addition, the sensor retains activity and gives reproducible results after storage in 4°C for 60 d. This simple and cost-effective sensing platform provides a rapid and convenient detection for H2S at concentrations far below the hazardous limit.

Single-Walled Carbon Nanotubes Alter Cytochrome c Electron Transfer and Modulate Mitochondrial Function

Single-walled carbon nanotubes (SWCNTs) are broadly used for various biomedical applications such as drug delivery, in vivo imaging, and cancer photothermal therapy due to their unique physiochemical properties. However, once they enter the cells, the effects of SWCNTs on the intracellular organelles and macromolecules are not comprehensively understood. Cytochrome c (Cyt c), as a key component of the electron transport chain in mitochondria, plays an essential role in cellular energy consumption, growth, and differentiation. In this study, we found the mitochondrial membrane potential and mitochondrial oxygen uptake were greatly decreased in human epithelial KB cells treated with SWCNTs, which accompanies the reduction of Cyt c. SWCNTs deoxidized Cyt c in a pH-dependent manner, as evidenced by the appearance of a 550 nm characteristic absorption peak, the intensity of which increased as the pH increased. Circular dichroism measurement confirmed the pH-dependent conformational change, which facilitated closer association of SWCNTs with the heme pocket of Cyt c and thus expedited the reduction of Cyt c. The electron transfer of Cyt c is also disturbed by SWCNTs, as measured with electron spin resonance spectroscopy. In conclusion, the redox activity of Cyt c was affected by SWCNTs treatment due to attenuated electron transfer and conformational change of Cyt c, which consequently changed mitochondrial respiration of SWCNTs-treated cells. This work is significant to SWCNTs research because it provides a novel understanding of SWCNTs' disruption of mitochondria function and has important implications for biomedical applications of SWCNTs.

Mechanisms for the Direct Electron Transfer of Cytochrome c Induced by Multi-Walled Carbon Nanotubes

Multi-walled carbon nanotube (MWCNT)-modified electrodes can promote the direct electron transfer (DET) of cytochrome c (Cyt c). There are several possible mechanisms that explain the DET of Cyt c. In this study, several experimental methods, including Fourier transform infrared spectroscopy, circular dichroism, ultraviolet-visible absorption spectroscopy, and electron paramagnetic resonance spectroscopy were utilized to investigate the conformational changes of Cyt c induced by MWCNTs. The DET mechanism was demonstrated at various nano-levels: secondary structure, spatial orientation, and spin state. In the presence of MWCNTs, the secondary structure of Cyt c changes, which exposes the active site, then, the orientation of the heme is optimized, revolving the exposed active center to the optimum spatial orientation for DET; and finally, a transition of spin states is induced, providing relatively high energy and a more open microenvironment for electron transfer. These changes at different nano-levels are closely connected and form a complex process that promotes the electron transfer of Cyt c.

Anionic lipid-induced conformational changes in human phagocyte flavocytochrome b precede assembly and activation of the NADPH oxidase complex

Phagocyte NADPH oxidases generate superoxide at high rates in defense against infectious agents, a process regulated by second messenger anionic lipids using incompletely understood mechanisms. We reconstituted the catalytic core of the human neutrophil NADPH oxidase, flavocytochrome b (Cyt b) in 99% phosphatidylcholine vesicles in order to correlate anionic lipid-dependent conformational changes in membrane-bound Cyt b and oxidase activity. The anionic lipid 10:0 phosphatidic acid (10:0 PA) specifically induced conformational changes in Cyt b as measured by a combination of fluorescence resonance energy transfer methods and size exclusion chromatography. The fluorescence lifetime of a complex between Cyt b and Cascade Blue-derivatized anti-p22phox antibody (CCB-CS9), increased after exposure to 10:PA by ∼50% of the change observed when the complex is dissociated, indicating a structural rearrangement of p22phox and/or the Cyt b heme prosthetic groups. Half of the quenching relaxation occurred at 10:0 PA concentrations permissive to less than 10% full NADPH oxidase activity, but saturated near the saturation in activity in a matched cell-free oxidase assay. We conclude that anionic lipids modulate the conformation of Cyt b in the membrane and suggest they may serve to modulate the structure of Cyt b as a control mechanism for the NADPH oxidase.

The changes of mitochondrial cytochrome c and GABAergic neuron in neuropathic pain model

BackgroundRole of cytochrome c (Cyt c) is an apoptogenic agent under certain conditions. The mitochondrial permeability transition pore (MPTP) plays an important role in cell death since it opens, leading to mitochondrial swelling and release of Cyt c, which initiates apoptosis. By inhibiting the opening of MPTP, cyclosporine A (CSA) may contribute to maintaining mitochondrial homeostasis. We investigate the effects of the partial sciatic nerve injury (PSNI)-induced neuropathic pain model on mitochondrial Cyt c release and the effects of CSA on neuroprotection by mitochondrial stabilizing activity in PSNI rats.MethodsRats were assigned to two groups that received different operations (Group P; PSNI operation, Group S; sham operation). The changes of cyt c and GABAergic neuron were evaluated in the spinal cord tissue. After which, PSNI rats randomly received CSA (Group C) or saline (Group S), and the changes of mechanical thresholds with Cyt c and GABAergic neuron were checked.ResultsPSNI in rats increased the release of cytosolic Cyt c. However, GABAergic cells were not decreased in the spinal cord level on the ipsilateral side to the PSNI. The second experiment reveal a reduction in Cyt c release, using CSA in PSNI model. Rats receiving CSA were afforded the antiallodynia without decrease of GABAergic cell.ConclusionsThe Cyt c probably contributes to nerve dysfunction after PSNI. PSNI induced neuropathic pain was profoundly linked to mitochondrial stabilization. Thus, the potent neuroprotector, CSA, might produce antiallodynia through its capability to inhibit the opening of MPTP.

Diamond nanotextured surfaces for enhanced protein redox activity

AbstractA nanotextured diamond electrode was utilized to achieve optimized electrochemical redox‐interactions of cytochrome c (cyt c) with the diamond transducer. The nanotextured surface was realized using a top–down technology where diamond nanoparticles are used as etching masks. Macroscopic characterizations of the OH‐terminated surface using cyclic voltammetry reveal adsorption‐controlled cyt c/electrode interaction. The adsorption is assigned to the electrostatic attraction force of positively charged cyt c parts with the slightly negatively charged diamond surface in pH 7 buffer. Atomic force microscopy experiments in physiologic buffer solutions on flat diamond surface confirms the adsorption of cyt c on OH‐terminated diamond surfaces without structural changes of cyt c.