Citalopram, an antipsychotic agent, induces G1/G0 phase cell cycle arrest and promotes apoptosis in human laryngeal carcinoma HEP-2 cells.

Human laryngeal squamous carcinoma (LSCC) is a common malignant tumor in the head and neck. Despite the recently developed therapies for the treatment of LSCC, patients' overall survival rate still did not enhance remarkably; this highlights the need to formulate alternative strategies to develop novel treatments. The antitumor effects of antidepressant drugs such as citalopram have been reported on several cancer cells; however, they have yet to be investigated against LSCC. The current study was directed to explore the possible antitumor effects of citalopram on human laryngeal carcinoma cell lines (HEP-2). HEP-2 cells were cultured and treated with different doses of citalopram (50-400 µM) for 24, 48, and 72 h. The effects of citalopram on the viability of cancer cells were determined by the MTT assay. In addition, apoptosis and cell cycle analysis were performed by flow cytometry. Moreover, evaluation of the expression of proapoptotic and apoptotic proteins, such as cytochrome c, cleaved caspases 3 and 9, Bcl-2, and BAX, was performed by western blotting analysis. Our results revealed that citalopram significantly suppressed the proliferation of HEP-2 cells through the upregulation of p21 expression, resulting in the subsequent arrest of the cell cycle at the G0/G1 phase. Furthermore, citalopram treatment-induced HEP-2 cell apoptosis; this was indicated by the significant increase of cytochrome c, cleaved caspases 3 and 9, and BAX protein expression. On the contrary, Bcl-2 protein expression was significantly downregulated following treatment with citalopram. The ultrastructure studies were in accordance with the protein expression findings and showed clear signs of apoptosis with ring chromatin condensation upon treatment with citalopram. These findings suggest that citalopram's anti-tumor activities on HEP-2 cells entailed stimulation of the intrinsic apoptotic pathway, which was mediated via Bcl-2 suppression.

Origin of Enantioselectivity in Engineered Cytochrome c-Catalyzed Carbon-Radical FePP Hydrolysis Revealed Using QM/MM (ABEEM Polarizable Force Field) and MD Simulations.

The origin of highly efficient asymmetric aminohydroxylation of styrene catalyzed by engineered cytochrome c is investigated by the developed Atom-Bond Electronegativity Equalization Method polarizable force field (ABEEM PFF), which is a combined outcome of electronic and steric effects. Model molecules were used to establish the charge parameters of the ABEEM PFF, for which the bond-stretching and angle-bending parameters were obtained by using a combination of modified Seminario and scan methods. The interactions between carbon-radical Fe-porphyrin (FePP) and waters are simulated by molecular dynamics, which shows a clear preference for the pre-R over the pre-S. This preference is attributed to the hydrogen-bond between the mutated 100S and 101P residues as well as van der Waals interactions, enforcing a specific conformation of the carbon-radical FePP complex within the binding pocket. Meanwhile, the hydrogen-bond between water and the nitrogen atom in the active intermediate dictates the stereochemical outcome. Quantum mechanics/molecular mechanics (QM/MM (ABEEM PFF)) and free-energy perturbation calculations elucidate that the 3RTS is characterized by sandwich-like structure among adjacent amino acid residues, which exhibits greater stability than crowed arrangement in 3STS and enables the R enantiomer to form more favorably. Thus, this study provides mechanistic insight into the catalytic reaction of hemoproteins.

Attenuating mitochondrial dysfunction and morphological disruption with PT320 delays dopamine degeneration in MitoPark mice.

BACKGROUND: Mitochondria are essential organelles involved in cellular energy production. Changes in mitochondrial function can lead to dysfunction and cell death in aging and age-related disorders. Recent research suggests that mitochondrial dysfunction is closely linked to neurodegenerative diseases. Glucagon-like peptide-1 receptor (GLP-1R) agonist has gained interest as a potential treatment for Parkinson's disease (PD). However, the exact mechanisms responsible for the therapeutic effects of GLP-1R-related agonists are not yet fully understood. METHODS: In this study, we explores the effects of early treatment with PT320, a sustained release formulation of the GLP-1R agonist Exenatide, on mitochondrial functions and morphology in a progressive PD mouse model, the MitoPark (MP) mouse. RESULTS: Our findings demonstrate that administration of a clinically translatable dose of PT320 ameliorates the reduction in tyrosine hydroxylase expression, lowers reactive oxygen species (ROS) levels, and inhibits mitochondrial cytochrome c release during nigrostriatal dopaminergic denervation in MP mice. PT320 treatment significantly preserved mitochondrial function and morphology but did not influence the reduction in mitochondria numbers during PD progression in MP mice. Genetic analysis indicated that the cytoprotective effect of PT320 is attributed to a reduction in the expression of mitochondrial fission protein 1 (Fis1) and an increase in the expression of optic atrophy type 1 (Opa1), which is known to play a role in maintaining mitochondrial homeostasis and decreasing cytochrome c release through remodeling of the cristae. CONCLUSION: Our findings suggest that the early administration of PT320 shows potential as a neuroprotective treatment for PD, as it can preserve mitochondrial function. Through enhancing mitochondrial health by regulating Opa1 and Fis1, PT320 presents a new neuroprotective therapy in PD.

Pulsed Direct Current Arc-Induced Nanoelectrospray Ionization Mass Spectrometry.

This study explores the innovative field of pulsed direct current arc-induced nanoelectrospray ionization mass spectrometry (DCAI-nano-ESI-MS), which utilizes a low-temperature direct current (DC) arc to induce ESI during MS analyses. By employing a 15 kV output voltage, the DCAI-nano-ESI source effectively identifies various biological molecules, including angiotensin II, bradykinin, cytochrome C, and soybean lecithin, showcasing impressive analyte signals and facilitating multicharge MS in positive- and negative-ion modes. Notably, results show that the oxidation of fatty acids using a DC arc produces [M + O - H]- ions, which aid in identifying the location of C═C bonds in unsaturated fatty acids and distinguishing between isomers based on diagnostic ions observed during collision-induced dissociation tandem MS. This study presents an approach for identifying the sn-1 and sn-2 positions in phosphatidylcholine using phosphatidylcholine and nitrate adduct ions, accurately determining phosphatidylcholine molecular configurations via the Paternò-Büchi reaction. With all the advantages above, DCAI-nano-ESI holds significant promise for future analytical and bioanalytical applications.

Influence of flavonoids from Sedum aizoon L. on mitochondrial function of Rhizopus nigricans in strawberry.

Rhizopus nigricans (R. nigricans), one of the fungi that grows the fastest, is frequently discovered in postharvest fruits, it's the main pathogen of strawberry root rot. Flavonoids in Sedum aizoon L. (FSAL) is a kind of green and safe natural substance extracted from Sedum aizoon L. which has antifungal activity. In this study, the minimum inhibitory concentration (MIC) of FSAL on R. nigricans and cell apoptosis tests were studied to explore the inhibitory effect of FSAL on R. nigricans. The effects of FSAL on mitochondria of R. nigricans were investigated through the changes of mitochondrial permeability transition pore(mPTP), mitochondrial membrane potential(MMP), Ca2+ content, H2O2 content, cytochrome c (Cyt c) content, the related enzyme activity and related genes of mitochondria. The results showed that the MIC of FSAL on R. nigricans was 1.800 mg/mL, with the addition of FSAL (1.800 mg/mL), the mPTP openness of R. nigricans increased and the MMP reduced. Resulting in an increase in Ca2+ content, accumulation of H2O2 content and decrease of Cyt c content, the activity of related enzymes was inhibited and related genes were up-regulated (VDAC1, ANT) or down-regulated (SDHA, NOX2). This suggests that FSAL may achieve the inhibitory effect of fungi by damaging mitochondria, thereby realizing the postharvest freshness preservation of strawberries. This lays the foundation for the development of a new plant-derived antimicrobial agent.

Soil fungal communities varied across aspects of restored grassland in former mining areas of the Qinghai-Tibet Plateau.

To determine whether different aspects lead to a heterogeneous distribution of soil fungi, we investigated artificially established alpine grasslands in the Muli mining area in the Qinghai-Tibet Plateau. Employing high-throughput sequencing techniques, we analyzed the composition, diversity, and function of soil fungal communities across various aspects (flat, East-facing, South-facing, West-facing, North-facing). We also examined their relationships with environmental factors. Soil fungal communities of restored alpine grasslands differed significantly across aspects in terms of the dominant phyla, classes and species level. Compared with No aspect, the Shannon index of fungi respectively decreased by 2.99%, 19.32%, 19.37% and 10.56% for East aspect, South aspect, West aspect and North aspect, respectively, and the Chao1 index of fungi respectively decreased by-2.44%, 35.50%, 42.15% and 3.21%, respectively. A total of 22 different types of fungi were identified in the study area. Predictive analysis, based on PICRUSt2, indicated that the primary functions of the fungal communities across different aspects were aerobic respiration I (cytochrome c) and aerobic respiration II (cytochrome c). Among the environmental variables, total phosphorus (P) and total nitrogen (N) were the principal factors influencing the fungal community composition.In conclusion, aspect plays a significant role in shaping the composition of fungal communities and also affects their overall diversity.

Phosphorylations and Acetylations of Cytochrome c Control Mitochondrial Respiration, Mitochondrial Membrane Potential, Energy, ROS, and Apoptosis.

Cytochrome c (Cytc) has both life-sustaining and cellular death-related functions, depending on subcellular localization. Within mitochondria, Cytc acts as a single electron carrier as part of the electron transport chain (ETC). When released into the cytosol after cellular insult, Cytc triggers the assembly of the apoptosome, committing the cell to intrinsic apoptosis. Due to these dual natures, Cytc requires strong regulation by the cell, including post-translational modifications, such as phosphorylation and acetylation. Six phosphorylation sites and three acetylation sites have been detected on Cytc in vivo. Phosphorylations at T28, S47, Y48, T49, T58, and Y97 tend to be present under basal conditions in a tissue-specific manner. In contrast, the acetylations at K8, K39, and K53 tend to be present in specific pathophysiological conditions. All of the phosphorylation sites and two of the three acetylation sites partially inhibit respiration, which we propose serves to maintain an optimal, intermediate mitochondrial membrane potential (ΔΨm) to minimize reactive oxygen species (ROS) production. Cytc phosphorylations are lost during ischemia, which drives ETC hyperactivity and ΔΨm hyperpolarization, resulting in exponential ROS production thus causing reperfusion injury following ischemia. One of the acetylation sites, K39, shows a unique behavior in that it is gained during ischemia, stimulating respiration while blocking apoptosis, demonstrating that skeletal muscle, which is particularly resilient to ischemia-reperfusion injury compared to other organs, possesses a different metabolic strategy to handle ischemic stress. The regulation of Cytc by these post-translational modifications underscores the importance of Cytc for the ETC, ΔΨm, ROS production, apoptosis, and the cell as a whole.

One-Step Affinity Purification of Leucine-Rich α2-Glycoproteins from Snake Sera and Characterization of Their Phospholipase A2-Inhibitory Activities as ß-Type Phospholipase A2 Inhibitors.

Snakes contain three types of phospholipase A2 (PLA2)-inhibitory proteins in their blood, PLIα, ß, and γ, which protect them from their own venom, PLA2. PLIß is the snake ortholog of leucine-rich α2 glycoprotein (LRG). Since autologous cytochrome c (Cyt c) serves as an endogenous ligand for LRG, in this study, we purified snake LRGs from various snake serum samples using Cyt c affinity chromatography. All purified snake LRGs were found to be dimers linked by disulfide bonds. Laticauda semifasciata and Naja kaouthia LRGs showed no inhibitory activity against L. semifasciata PLA2 and weak inhibitory activity against Gloydius brevicauda basic PLA2. Elaphe climacophora PLIß had weaker inhibitory activity against G. brevicauda basic PLA2 than G. brevicauda and Elaphe quadrivirgata PLIs, which are abundant in blood and known to neutralize G. brevicauda basic PLA2. Protobothrops flavoviridis LRG showed no inhibitory activity against basic venom PLA2, PL-X, or G. brevicauda basic PLA2. Binding analysis of P. flavoviridis LRG using surface plasmon resonance showed very strong binding to snake Cyt c, followed by that to horse Cyt c, weak binding to yeast Cyt c, and no binding to P. flavoviridis PL-X or BPI/II. We also deduced the amino acid sequences of L. semifasciata and P. flavoviridis LRG by means of cDNA sequencing and compared them with those of other known sequences of PLIs and LRGs. This study concluded that snake LRG can potentially inhibit basic PLA2, but, whether it actually functions as a PLA2-inhibitory protein, PLIß, depends on the snake.

Deciphering the Roles of Interfacial Amino Acids in Inter-Protein Charge Transport.

Elucidating charge transport (CT) through proteins is critical for gaining insights into ubiquitous CT chain reactions in biological systems and developing high-performance bioelectronic devices. While intra-protein CT has been extensively studied, crucial knowledge about inter-protein CT via interfacial amino acids is still absent due to the structural complexity. Herein, by loading cytochrome c (Cyt c) on well-defined peptide self-assembled monolayers to mimic the protein-protein interface, we provide a precisely controlled platform for identifying the roles of interfacial amino acids in solid-state CT via peptide-Cyt c junctions. The terminal amino acid of peptides serves as a fine-tuning factor for both the interfacial interaction between peptides and Cyt c and the immobilized Cyt c orientation, resulting in a nearly 10-fold difference in current through peptide-Cyt c junctions with varied asymmetry. This work provides a valuable platform for studying CT across proteins and contributes to the understanding of fundamental principles governing inter-protein CT.

Overcoming aggregation with laser heated nanoelectrospray mass spectrometry: thermal stability and pathways for loss of bicarbonate from carbonic anhydrase II.

Variable temperature electrospray mass spectrometry is useful for multiplexed measurements of the thermal stabilities of biomolecules, but the ionization process can be disrupted by aggregation-prone proteins/complexes that have irreversible unfolding transitions. Resistively heating solutions containing a mixture of bovine carbonic anhydrase II (BCAII), a CO2 fixing enzyme involved in many biochemical pathways, and cytochrome c leads to complete loss of carbonic anhydrase signal and a significant reduction in cytochrome c signal above ∼72 °C due to aggregation. In contrast, when the tips of borosilicate glass nanoelectrospray emitters are heated with a laser, complete thermal denaturation curves for both proteins are obtained in <1 minute. The simultaneous measurements of the melting temperature of BCAII and BCAII bound to bicarbonate reveal that the bicarbonate stabilizes the folded form of this protein by ∼6.4 °C. Moreover, the temperature dependences of different bicarbonate loss pathways are obtained. Although protein analytes are directly heated by the laser for only 140 ms, heat conduction further up the emitter leads to a total analyte heating time of ∼41 s. Pulsed laser heating experiments could reduce this time to ∼0.5 s for protein aggregation that occurs on a faster time scale. Laser heating provides a powerful method for studying the detailed mechanisms of cofactor/ligand loss with increasing temperature and promises a new tool for studying the effect of ligands, drugs, growth conditions, buffer additives, or other treatments on the stabilities of aggregation-prone biomolecules.

Dual Optical Response Strategy for the Detection of Cytochrome c Using Highly Luminescent Lanthanide-Based Nanotubular Sensor Arrays.

Here, we demonstrate a label-free dual optical response strategy for the detection of cytochrome c (Cyt c) with ultrahigh sensitivity using highly luminescent lanthanides containing inorganic-organic hybrid nanotubular sensor arrays. These sensor arrays are formed by the sequential incorporation of the photosensitizers 2,3-dihydroxynaphthalene (DHN) or 1,10-phenanthroline (Phen), and trivalent lanthanide terbium ions (Tb3+) into sodium lithocholate (NaLC) nanotube templates. Our sensing platform relies on the detection and quantification of Cyt c in solution by providing dual photoluminescence quenching responses from the nanotubular hybrid arrays in the presence of Cyt c. The large quenching of the sensitized Tb3+ emission within the DHN/Phen-Tb3+-NaLC nanotubular sensor arrays caused by the strong binding of the photosensitizers to Cyt c provides an important signal response for the selective detection of Cyt c. This long-lived lanthanide emission response-based sensing strategy can be highly advantageous for the detection of Cyt c in a cellular environment eliminating background fluorescence and scattering signals through time-gated measurements. The DHN containing nanotubular sensor arrays (DHN-NaLC and DHN-Tb3+-NaLC) provide an additional quenching response characterized by a unique spectral valley splitting with quantized quenching dip on the DHN fluorescence emission. This spectral quenching dip resulting from efficient FRET between the protein bound DHN photosensitizer and the heme group of Cyt c serves as an important means for specific detection and quantification of Cyt c in the concentration range of 0-30 µM with a low detection limit of around 20 nM.

Exploring the role of ghrelin and des-acyl ghrelin in chemotherapy-induced nausea and vomiting.

Ghrelin and its mimetics have been shown to reduce cisplatin-induced emesis in preclinical studies using ferrets and shrews. This study investigated the effectiveness of ghrelin and des-acyl ghrelin (DAG) in antagonizing cisplatin-induced emesis and physiological changes indicative of nausea in Suncus murinus. Animals implanted with radiotelemetry devices were administered ghrelin (0.2, 1.0, and 5.0 µg/day), DAG (0.2, 1.0, and 5.0 µg/day), or saline (14 µL/day) intracerebroventricularly 4 days before and 3 days after treatment with cisplatin (30 mg/kg). At the end, the anti-apoptotic potentials of ghrelin and DAG were assessed by measuring Bax expression and cytochrome C activity. Neurotransmitter changes in the brain were evaluated using liquid chromatography-mass spectrometry analysis. Ghrelin and DAG reduced cisplatin-induced emesis in the delayed (24-72 h) but not the acute phase (0-24 h) of emesis. Ghrelin also partially reversed the inhibitory effects of cisplatin on food intake without affecting gastrointestinal myoelectrical activity or causing hypothermia; however, ghrelin or DAG did not prevent these effects. Ghrelin and DAG could attenuate the cisplatin-induced upregulation of Bax and cytochrome C in the ileum. Cisplatin dysregulated neurotransmitter levels in the frontal cortex, amygdala, thalamus, hypothalamus, and brainstem, and this was partially restored by low doses of ghrelin and DAG. Our findings suggest that ghrelin and DAG exhibit protective effects against cisplatin-induced delayed emesis. The underlying antiemetic mechanism may involve GHSR and/or unspecified pathways that modulate the neurotransmitters involved in emesis control in the brain and an action to attenuate apoptosis in the gastrointestinal tract.

Iron-mediated DAMO-anammox process: Revealing the mechanism of electron transfer.

The nitrate denitrifying anaerobic methane oxidation-anaerobic ammonia oxidation (DAMO-anammox) can accomplish nitrogen removal and methane (CH4) reduction. This process greatly contributes to carbon emission mitigation and carbon neutrality. In this study, we investigated the electron transfer process of functional microorganisms in the iron-mediated DAMO-anammox system. Fe3+ could be bound to several functional groups (-CH3, COO-, -CH) in extracellular polymeric substance (EPS), and the functional groups bound were different at different iron concentration. Fe3+ underwent reduction reactions to produce Fe2+. Most Fe3+ and Fe2+ react with microorganisms and formed chelates with EPS. Three-dimensional fluorescence spectra showed that Fe3+ affected the secretion of tyrosine and tryptophan, which were essential for cytochrome synthesis. The presence of Fe3+ accelerated c-type cytochrome-mediated extracellular electron transfer (EET), and when more Fe3+ existed, the more cytochrome C expressed. DAMO archaea (M. nitroreducens) in the system exhibited a high positive correlation with the functional genes (resa and ccda) for cytochrome c synthesis. Some denitrifying microorganisms showed positive correlations with the abundance of riboflavin. This finding showed that riboflavin secreted by functional microorganisms acted as an electron shuttle. In addition, DAMO archaea were positively correlated with the hair synthesis gene pily1, which indicated that direct interspecies electron transfer (DIET) may exist in the iron-mediated DAMO-anammox system.

Diverse functions of cytochrome c in cell death and disease.

The redox-active protein cytochrome c is a highly positively charged hemoglobin that regulates cell fate decisions of life and death. Under normal physiological conditions, cytochrome c is localized in the mitochondrial intermembrane space, and its distribution can extend to the cytosol, nucleus, and extracellular space under specific pathological or stress-induced conditions. In the mitochondria, cytochrome c acts as an electron carrier in the electron transport chain, facilitating adenosine triphosphate synthesis, regulating cardiolipin peroxidation, and influencing reactive oxygen species dynamics. Upon cellular stress, it can be released into the cytosol, where it interacts with apoptotic peptidase activator 1 (APAF1) to form the apoptosome, initiating caspase-dependent apoptotic cell death. Additionally, following exposure to pro-apoptotic compounds, cytochrome c contributes to the survival of drug-tolerant persister cells. When translocated to the nucleus, it can induce chromatin condensation and disrupt nucleosome assembly. Upon its release into the extracellular space, cytochrome c may act as an immune mediator during cell death processes, highlighting its multifaceted role in cellular biology. In this review, we explore the diverse structural and functional aspects of cytochrome c in physiological and pathological responses. We summarize how posttranslational modifications of cytochrome c (e.g., phosphorylation, acetylation, tyrosine nitration, and oxidation), binding proteins (e.g., HIGD1A, CHCHD2, ITPR1, and nucleophosmin), and mutations (e.g., G41S, Y48H, and A51V) affect its function. Furthermore, we provide an overview of the latest advanced technologies utilized for detecting cytochrome c, along with potential therapeutic approaches related to this protein. These strategies hold tremendous promise in personalized health care, presenting opportunities for targeted interventions in a wide range of conditions, including neurodegenerative disorders, cardiovascular diseases, and cancer.

Cardiolipin Membranes Promote Cytochrome c Transformation of Polycyclic Aromatic Hydrocarbons and Their In Vivo Metabolites.

The catalytic properties of cytochrome c (Cc) have captured great interest in respect to mitochondrial physiology and apoptosis, and hold potential for novel enzymatic bioremediation systems. Nevertheless, its contribution to the metabolism of environmental toxicants remains unstudied. Human exposure to polycyclic aromatic hydrocarbons (PAHs) has been associated with impactful diseases, and animal models have unveiled concerning signs of PAHs' toxicity to mitochondria. In this work, a series of eight PAHs with ionization potentials between 7.2 and 8.1 eV were used to challenge the catalytic ability of Cc and to evaluate the effect of vesicles containing cardiolipin mimicking mitochondrial membranes activating the peroxidase activity of Cc. With moderate levels of H2O2 and at pH 7.0, Cc catalyzed the oxidation of toxic PAHs, such as benzo[a]pyrene, anthracene, and benzo[a]anthracene, and the cardiolipin-containing membranes clearly increased the PAH conversions. Our results also demonstrate for the first time that Cc and Cc-cardiolipin complexes efficiently transformed the PAH metabolites 2-hydroxynaphthalene and 1-hydroxypyrene. In comparison to horseradish peroxidase, Cc was shown to reach more potent oxidizing states and react with PAHs with ionization potentials up to 7.70 eV, including pyrene and acenaphthene. Spectral assays indicated that anthracene binds to Cc, and docking simulations proposed possible binding sites positioning anthracene for oxidation. The results give support to the participation of Cc in the metabolism of PAHs, especially in mitochondria, and encourage further investigation of the molecular interaction between PAHs and Cc.

Situating the phosphonated calixarene-cytochrome C association by molecular dynamics simulations.

Protein-calixarenes binding plays an increasingly central role in many applications, spanning from molecular recognition to drug delivery strategies and protein inhibition. These ligands obey a specific bio-supramolecular chemistry, which can be revealed by computational approaches, such as molecular dynamics simulations. In this paper, we rely on all-atom, explicit-solvent molecular dynamics simulations to capture the electrostatically driven association of a phosphonated calix-[4]-arene with cytochome-C, which critically relies on surface-exposed paired lysines. Beyond two binding sites identified in direct agreement with the x-ray structure, the association has a larger structural impact on the protein dynamics. Then, our simulations allow a direct comparison to analogous calixarenes, namely, sulfonato, similarly reported as "molecular glue." Our work can contribute to a robust in silico predictive tool to assess binding sites for any given protein of interest for crystallization, with the specificity of a macromolecular cage whose endo/exo orientation plays a role in the binding.

Cyclizine induces cytotoxicity and apoptosis in macrophages through the extrinsic and intrinsic apoptotic pathways.

Cyclizine, an over-the-counter and prescription antihistamine, finds widespread application in the prevention and treatment of motion sickness, encompassing symptoms such as nausea, vomiting, dizziness, along with its effectiveness in managing vertigo. However, the overuse or misuse of cyclizine may lead to hallucinations, confusion, tachycardia, and hypertension. The molecular mechanisms underlying cyclizine-induced cytotoxicity and apoptosis remain unclear. During the 24 h incubation duration, RAW264.7 macrophages were exposed to different concentrations of cyclizine. Cytotoxicity was assessed through the lactate dehydrogenase assay. Flow cytometry employing annexin V-fluorescein isothiocyanate and propidium iodide was utilized to evaluate apoptosis and necrosis. Caspase activity and mitochondrial dysfunction were evaluated through a fluorogenic substrate assay and JC-1 dye, respectively. Flow cytometry employing fluorogenic antibodies was utilized to evaluate the release of cytochrome c and expression of death receptor, including tumor necrosis factor-α receptor and Fas receptor. Western blotting was utilized to evaluate the expression of the Bcl2 and Bad apoptotic regulatory proteins. The findings unveiled from the present study demonstrated that cyclizine exerted a concentration-dependent effect on RAW264.7 macrophages, leading to the induction of cytotoxicity, apoptosis, and necrosis. This compound further activated the intrinsic apoptotic pathway by inducing mitochondrial dysfunction, Bcl2/Bad exchange expression, cytochrome c liberation, and activation of caspases contained caspase 3, 8, and 9. Moreover, the activation of the extrinsic apoptotic pathway was observed as cyclizine induced the upregulation of death receptors and increased caspase activities. Based on our investigations, it can be inferred that cyclizine prompts cytotoxicity and apoptosis in RAW264.7 macrophages in a concentration-dependent manner by triggering both the intrinsic and extrinsic apoptotic pathways.

Protective role of chlorogenic acid in preserving cytochrome-c stability against HFIP-induced molten globule state at physiological pH.

Numerous neurodegenerative disorders are characterized by protein misfolding and aggregation. The mechanism of protein aggregation is intricate, and it is very challenging to study at cellular level. Inhibition of protein aggregation by interfering with its pathway is one of the ways to prevent neurodegenerative diseases. In the present work, we have evaluated the protective effect of a polyphenol compound chlorogenic acid (CGA) on the native and molten globule state of horse heart cytochrome c (cyt c). A molten globule state of this heme protein was achieved in the presence of fluorinated alcohol 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) at physiological pH, as studied by UV-Vis absorption, circular dichroism, intrinsic and ANS fluorescence. We found that at 50 % (v/v) HFIP, the native cyt c transformed into a molten globule state. The same techniques were also used to analyze the protective effect of CGA on the molten globule state of cyt c, and the results show that the CGA prevented the molten globular state and retained the protein close to the native state at 1:1 protein:CGA sub molar ratio. Molecular dynamics study also revealed that CGA retains the stability of cyt c in HFIP medium by preserving it in an intermediate state close to native conformation.

The structure of the diheme cytochrome c4 from Neisseria gonorrhoeae reveals multiple contributors to tuning reduction potentials.

Cytochrome c4 (c4) is a diheme protein implicated as an electron donor to cbb3 oxidases in multiple pathogenic bacteria. Despite its prevalence, understanding of how specific structural features of c4 optimize its function is lacking. The human pathogen Neisseria gonorrhoeae (Ng) thrives in low oxygen environments owing to the activity of its cbb3 oxidase. Herein, we report characterization of Ng c4. Spectroelectrochemistry experiments of the wild-type (WT) protein have shown that the two Met/His-ligated hemes differ in potentials by ∼100 mV, and studies of the two His/His-ligated variants provided unambiguous assignment of heme A from the N-terminal domain of the protein as the high-potential heme. The crystal structure of the WT protein at 2.45 Å resolution has revealed that the two hemes differ in their solvent accessibility. In particular, interactions made by residues His57 and Ser59 in Loop1 near the axial ligand Met63 contribute to the tight enclosure of heme A, working together with the surface charge, to raise the reduction potential of the heme iron in this domain. The structure reveals a prominent positively-charged patch, which encompasses surfaces of both domains. In contrast to prior findings with c4 from Pseudomonas stutzeri, the interdomain interface of Ng c4 contributes minimally to the values of the heme iron potentials in the two domains. Analyses of the heme solvent accessibility, interface properties, and surface charges offer insights into the interplay of these structural elements in tuning redox properties of c4 and other multiheme proteins.

Significantly Improved Cold Preservation of Rat Hind Limb Vascularized Composite Allografts Using the New PrC-210 Free Radical Scavenger.

Vascularized composite allotransplantation (VCA) represents a promising reconstructive solution primarily conducted to improve quality of life. However, tissue damage caused by cold-ischemia (CI) storage prior to transplant represents a major factor limiting widespread application. This study investigates the addition of the novel free radical scavenger PrC-210 to UW Organ Preservation Solution (UW Solution) to suppress CI-induced skeletal muscle injury in a rat hind limb amputation model. Lewis rats received systemic perfusion of UW solution +/- PrC-210 (0 mM control, 10 mM, 20 mM, 30 mM, or 40 mM), followed by bilateral transfemoral amputation. Limbs were stored in 40 mL of the same perfusate at 4 °C for 48 h. Muscle punch biopsies were taken at set times over the 48 h cold-storage period and analyzed for caspase-3,7 activity, cytochrome C levels, and qualitative histology. A single 15 s perfusion of PrC-210-containing UW Solution conferred a dose-dependent reduction in CI-induced muscle cell death over 48 h. In the presence of PrC-210, muscle cell mitochondrial cytochrome C release was equivalent to 0 h controls, with profound reductions in the caspase-3,7 apoptotic marker that correlated with limb histology. PrC-210 conferred complete prevention of ROS-induced mitochondrial lysis in vitro, as measured by cytochrome C release. We conclude that the addition of 30 mM PrC210 to UW Solution conferred the most consistent reduction in CI limb damage, and it warrants further investigation for clinical application in the VCA setting.