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Cyt C (cyt + c)
Selected AbstractsInduced SER-Activity in Nanostructured Ag,Silica,Au Supports via Long-Range Plasmon CouplingADVANCED FUNCTIONAL MATERIALS, Issue 12 2010Jiu-Ju Feng Abstract A novel Ag,silica,Au hybrid device is developed that displays a long-range plasmon transfer of Ag to Au leading to enhanced Raman scattering of molecules largely separated from the optically excited Ag surface. A nanoscopically rough Ag surface is coated by a silica spacer of variable thickness from ,1 to 21,nm and a thin Au film of ,25,nm thickness. The outer Au surface is further functionalized by a self-assembled monolayer (SAM) for electrostatic binding of the heme protein cytochrome c (Cyt c) that serves as a Raman probe and model enzyme. High-quality surface-enhanced resonance Raman (SERR) spectra are obtained with 413,nm excitation, demonstrating that the enhancement results exclusively from excitation of Ag surface plasmons. The enhancement factor is estimated to be 2,×,104,8,×,103 for a separation of Cyt c from the Ag surface by 28,47,nm, corresponding to an attenuation of the enhancement by a factor of only 2,6 compared to Cyt c adsorbed directly on a SAM-coated Ag electrode. Upon immobilization of Cyt c on the functionalized Ag,silica,Au device, the native structure and redox properties are preserved as demonstrated by time- and potential-dependent SERR spectroscopy. [source] Calcium-induced Cytochrome c release from CNS mitochondria is associated with the permeability transition and rupture of the outer membraneJOURNAL OF NEUROCHEMISTRY, Issue 2 2002Nickolay Brustovetsky Abstract The mechanisms of Ca2+ -induced release of Cytochrome c (Cyt c) from rat brain mitochondria were examined quantitatively using a capture ELISA. In 75 or 125 mm KCl-based media 1.4 µmol Ca2+/mg protein caused depolarization and mitochondrial swelling. However, this resulted in partial Cyt c release only in 75 mm KCl. The release was inhibited by Ru360, an inhibitor of the Ca2+ uniporter, and by cyclosporin A plus ADP, a combination of mitochondrial permeability transition inhibitors. Transmission electron microscopy (TEM) revealed that Ca2+ -induced swelling caused rupture of the outer membrane only in 75 mm KCl. Koenig's polyanion, an inhibitor of mitochondrial porin (VDAC), enhanced swelling and amplified Cyt c release. Dextran T70 that is known to enhance mitochondrial contact site formation did not prevent Cyt c release. Exposure of cultured cortical neurons to 500 µm glutamate for 5 min caused Cyt c release into the cytosol 30 min after glutamate removal. MK-801 or CsA inhibited this release. Thus, the release of Cyt c from CNS mitochondria induced by Ca2+in vitro as well as in situ involved the mPT and appeared to require the rupture of the outer membrane. [source] Melatonin induces apoptotic death in LNCaP cells via p38 and JNK pathways: therapeutic implications for prostate cancerJOURNAL OF PINEAL RESEARCH, Issue 1 2009Seong Soo Joo Abstract:, Apoptosis, a form of cell death, is a fundamental process for the development and maintenance of multicellular organisms that promotes the removal of damaged, senescent or unwanted cells. Induction of cancer cell apoptosis is an important strategy of anticancer therapy. In this study, we examined if melatonin, the main secretory product of the pineal gland, inhibited the growth of prostate cancer cells (LNCaP) and promoted apoptosis via mitogen-activated protein kinases (MAPKs), which are closely associated with apoptosis and survival. Melatonin treatment significantly inhibited the growth of LNCaP cells in a dose- and time-dependent manner. It clearly induced both an early stage of apoptosis (propidium iodide,, FITC Annexin-V+) and a late apoptosis/secondary necrosis (propidium iodide+ and FITC Annexin-V+), which indicated induction of serial stages of apoptosis in cells. Moreover, melatonin markedly activated c-JUN N-terminal kinase (JNK) and p38 kinase, whereas extracellular signal-regulated kinase (ERK) was not responsive to melatonin. Treatment with MAPK inhibitors, PD98059 (ERK inhibitor), SP600125 (JNK inhibitor) and SB202190 (p38 inhibitor), confirmed that melatonin-induced apoptosis was JNK- and p38-dependent, but ERK-independent. In the presence of PD98059, caspase-3 activity increased, while levels of Bax/cytochrome c (Cyt c) and Bcl-2 decreased. These effects were opposite to those observed with SP600125 and SB202190 treatments. Together, these results strongly suggest that JNK and p38 activation directly participate in apoptosis induced by melatonin. Thus, melatonin may be of promise for anti-prostate cancer strategies. [source] Malonate induces cell death via mitochondrial potential collapse and delayed swelling through an ROS-dependent pathwayBRITISH JOURNAL OF PHARMACOLOGY, Issue 4 2005Francisco J Fernandez-Gomez 1Herein we study the effects of the mitochondrial complex II inhibitor malonate on its primary target, the mitochondrion. 2Malonate induces mitochondrial potential collapse, mitochondrial swelling, cytochrome c (Cyt c) release and depletes glutathione (GSH) and nicotinamide adenine dinucleotide coenzyme (NAD(P)H) stores in brain-isolated mitochondria. 3Although, mitochondrial potential collapse was almost immediate after malonate addition, mitochondrial swelling was not evident before 15 min of drug presence. This latter effect was blocked by cyclosporin A (CSA), Ruthenium Red (RR), magnesium, catalase, GSH and vitamin E. 4Malonate added to SH-SY5Y cell cultures produced a marked loss of cell viability together with the release of Cyt c and depletion of GSH and NAD(P)H concentrations. All these effects were not apparent in SH-SY5Y cells overexpressing Bcl-xL. 5When GSH concentrations were lowered with buthionine sulphoximine, cytoprotection afforded by Bcl-xL overexpression was not evident anymore. 6Taken together, all these data suggest that malonate causes a rapid mitochondrial potential collapse and reactive oxygen species production that overwhelms mitochondrial antioxidant capacity and leads to mitochondrial swelling. Further permeability transition pore opening and the subsequent release of proapoptotic factors such as Cyt c could therefore be, at least in part, responsible for malonate-induced toxicity. British Journal of Pharmacology (2005) 144, 528,537. doi:10.1038/sj.bjp.0706069 [source] 3242: MsrA repair of Cytochrome c and ,-crystallin links chaperone and oxidative repair pathways with mitochondrial protection and apoptotic control in lens cells.ACTA OPHTHALMOLOGICA, Issue 2010M KANTOROW Purpose Methionine sulfoxide reductase A (MsrA) is essential for lens defense against oxidative stress and cataract formation through its ability to repair oxidized protein methionine (PMSO). Here, we examined ,-crystallin and cytochrome c (Cyt c) as potential targets for MsrA-repair in the lens. Methods The oxidized methionine content of ,-crystallin and Cyt c was evaluated from the lenses of MsrA knockout and w+ mice. The activities of ,,crystallin and Cyt c were evaluated upon methionine oxidation. The ability of MsrA to repair these proteins and restore their respective chaperone and electron transport activities was determined. All three proteins were localized in lens cells and their abilities to form complexes in vitro and in vivo evaluated. Results Cyt c was oxidized at met 80 and ,,-crystallin at met 68 in MsrA-knockout relative to w+ mice. Methionine oxidization of 138 of ,A-crystallin and 68 of ,B-crystallin resulted in loss of ,-crystallin chaperone function while oxidation of met 80 resulted in loss of Cyt c electron transport function. ,-crystallin protected Cyt c against methioinine oxidation. MsrA repaired and restored the functions of all three proteins which co-localized and formed complexes in lens mitochondria. Conclusion MsrA is likely required for repair of ,-crystallin and Cyt c in lens cells suggesting that it maintains and regulates the activities of ,-crystallin and Cyt c in lens mitochondria where all three proteins were localized. Loss of MsrA-repair could contribute to cataract formation through loss of chaperone function, loss of oxidative phosphorylation and/or increased Cyt c mediated apoptosis. [source] The effect of GHRH antagonists on human glioblastomas and their mechanism of action,INTERNATIONAL JOURNAL OF CANCER, Issue 10 2010Eva Pozsgai Abstract The effects of new growth hormone-releasing hormone (GHRH) antagonists JMR-132 and MIA-602 and their mechanism of action were investigated on 2 human glioblastoma cell lines, DBTRG-05 and U-87MG, in vitro and in vivo. GHRH receptors and their main splice variant, SV1 were found on both cell lines. After treatment with JMR-132 or MIA-602, the cell viability decreased significantly. A major decrease in the levels of phospho-Akt, phospho-GSK3, and phosho-ERK 1/2 was detected at 5 and 10 min following treatment with the GHRH antagonists, whereas elevated levels of phospho-p38 were observed at 24 hr. The expression of caspase-3 and poly(ADP-ribose) (PARP), as the downstream executioners of apoptosis were found to be significantly elevated after treatment. Following treatment of the glioblastoma cells with GHRH antagonists, nuclear translocation of apoptosis inducing factor (AIF) and Endonuclease G (Endo G) and the mitochondrial release of cytochrome c (cyt c) were detected, indicating that the cells were undergoing apoptosis. In cells treated with GHRH antagonists, the collapse of the mitochondrial membrane potential was shown with fluorescence microscopy and JC-1 membrane potential sensitive dye. There were no significant differences between results obtained in DBTRG-05 or U-87MG cell lines. After treatment with MIA-602 and JMR-132, the reduction rate in the growth of DBTRG-05 glioblastoma, xenografted into nude mice, was significant and tumor doubling time was also significantly extended when compared with controls. Our study demonstrates that GHRH antagonists induce apoptosis through key proapoptotic pathways and shows the efficacy of MIA-602 for experimental treatment of glioblastoma. [source] A modified Ising model for the thermodynamic properties of local and global protein folding,unfolding observed by circular dichroism and small-angle X-ray scatteringJOURNAL OF APPLIED CRYSTALLOGRAPHY, Issue 2007Ying-Jen Shiu Based on the mean-field approximation, we have applied a modified Ising model to describe general protein unfolding behavior at thermodynamic equilibrium with the free energy contributed by the subgroup units (amino acids or peptide bonds) of the protein. With the thermodynamic properties of the protein, this model can associate the stepwise change of an unfolding fraction ratio profile with the local and global conformation unfolding. Taking cytochrome c (cyt c) as a model protein, we have observed, using small-angle X-ray scattering and circular dichroism (CD), the global and local structure changes for the protein in three kinds of denaturant environments: acid, urea and guanidine hydrochloride. The small-angle X-ray scattering and CD results are mapped to the unfolding fractions as a function of the pH value or denaturant concentration, from which we have extracted local and global unfolding free energies of cyt c in different denaturant environments using a modified Ising model. Based on the characteristics of the thermodynamic properties deduced from the local and global protein folding,unfolding, we discuss the thermodynamic stabilities of the protein in the three denaturant environments, and the possible correlation between the global conformation change of the protein and the local unfolding activities of the S,Fe bond in the Met80-heme and the ,-helices. [source] Loss of E-cadherin mediated cell,cell adhesion as an early trigger of apoptosis induced by photodynamic treatmentJOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2005Sergio Galaz Photodynamic treatment with different photosensitizers (PSs) can result in the specific induction of apoptosis in many cell types. It is commonly accepted that this apoptotic response depends on the mitochondrial accumulation of the PS. Accumulation in other cellular organelles, such as lysosomes or the Golgi complex, and subsequent photodamage resulting in an apoptotic process has been also described. However, the role played by cell adhesion in apoptosis induced in epithelial cells after photodynamic treatment is not well characterized. Here, we have used a murine keratinocyte line, showing a strong dependence on E-cadherin for cell,cell adhesion and survival, to analyze the relevance of this adhesion complex in the context of zinc(II)-phthalocyanine (ZnPc) photodynamic treatment. We report that under apoptotic conditions, ZnPc phototreatment induces a rapid disorganization of the E-cadherin mediated cell,cell adhesion, which largely preceded both the detachment of cells from the substrate, via ,-1 integrins and the induction of apoptotic mitochondrial markers. Therefore, the alteration in E-cadherin, ,- and ,-catenins adhesion proteins preceded the release of cytochrome c (cyt c) from mitochondria to the cytosol and the activation of caspase 3. In addition, blocking E-cadherin function with a specific antibody (Decma-1) induced apoptosis in this cell system. These results strongly suggest that the E-cadherin adhesion complex could be the primary target of ZnPc phototreatment, and that loss of E-cadherin mediated cell adhesion after early photodamage triggers an apoptotic response. © 2005 Wiley-Liss, Inc. [source] NO-induced neuroprotection in ischemic preconditioning stimulates mitochondrial Mn-SOD activity and expression via RAS/ERK1/2 pathwayJOURNAL OF NEUROCHEMISTRY, Issue 4 2007A. Scorziello Abstract To identify the transductional mechanisms responsible for the neuroprotective effect of nitric oxide (NO) during ischemic preconditioning (IPC), we investigated the effects of this gaseous mediator on mitochondrial Mn-superoxide dismutase (Mn-SOD) expression and activity. In addition, the possible involvement of Ras/extracellular-regulated kinase (ERK) ERK1/2 pathway in preserving cortical neurons exposed to oxygen and glucose deprivation (OGD) followed by reoxygenation was also examined. Ischemic preconditioning was obtained by exposing neurons to a 30-min sublethal OGD (95% N2 and 5% CO2). Then, after a 24-h interval, neurons were exposed to 3 h of OGD followed by 24 h of reoxygenation (OGD/Rx). Our results revealed that IPC reduced cytochrome c (cyt c) release into the cytosol, improved mitochondrial function, and decreased free radical production. Moreover, it induced an increase in nNOS expression and NO production and promoted ERK1/2 activation. These effects were paralleled by an increase in Mn-SOD expression and activity that persisted throughout the following OGD phase. When the neurons were treated with L-NAME, a well known NOS inhibitor, the increase in Mn-SOD expression occurring during IPC was reduced and, as a result, IPC-induced neuroprotection was prevented. Similarly, when ERK1/2 was inhibited by its selective inhibitor PD98059, the increase in Mn-SOD expression observed during IPC was almost completely abolished. As a result, its neuroprotective effect on cellular survival was thwarted. The present findings indicate that during IPC the increase in Mn-SOD expression and activity are paralleled by NO production. This suggests that NO neuroprotective role occurs through the stimulation of Mn-SOD expression and activity. In particular, NO via Ras activation stimulates downstream ERK1/2 cascade. This pathway, in turn, post-transcriptionally activates Mn-SOD expression and activity, thus promoting neuroprotection during preconditioning. [source] Hypoxia-Induced Apoptotic Cell Death is Prevented by Oestradiol Via Oestrogen Receptors in the Developing Central Nervous SystemJOURNAL OF NEUROENDOCRINOLOGY, Issue 3 2008V. M. Pozo Devoto The neuroprotective effects of oestrogens have been demonstrated against a variety of insults, including excitotoxicity, oxidative stress and cerebral ischemia under certain conditions. However, the molecular mechanisms underlying oestrogen neuroprotection are still unclear. We aimed to determine whether 17,-oestradiol (E2) administration post-hypoxia (p-hx) was neuroprotective and whether these actions were mediated through oestrogen receptors (ER). For this purpose, 12-embyonic day-old chickens were subjected to acute hypoxia [8% (O2), 60 min], followed by different reoxygenation periods. To test the neuroprotective effect of E2 and its mechanism, embryos were injected 30 min after the end of hypoxia with E2 alone or with ICI 182 780, a competitive antagonist of ER. Cytochrome c (cyt c) release, an indicator of mitochondrial apoptotic pathway, was measured by western blot in optic lobe cytosolic extracts. DNA fragmentation by TUNEL fluorescence and caspase-3 fragmentation by immunofluorescence were detected on optic lobe sections. Acute hypoxia produces a significant increase in cyt c release from mitochondria at 4 h p-hx, followed by an increase in TUNEL positive cells 2 h later (6 h p-hx). Administration of E2 (0.5 mg/egg) produced a significant decrease in cytosolic cyt c levels at 4 h p-hx, in casapse-3 activation and in TUNEL positive cells at 6 h p-hx compared to vehicle treated embryos. In the E2 -ICI 182 780 treated embryos, cyt c release, caspase-3 fragmentation and TUNEL positive cells were similar to the hypoxic embryos, thus suggesting the requirement of an E2,ER interaction for E2 mediated neuroprotective effects. In conclusion, E2 prevents hypoxia-induced cyt c release and posterior cell death and these effects are mediated by oestrogen receptors. [source] Mitochondria-targeted disruptors and inhibitors of cytochrome c/cardiolipin peroxidase complexes: A new strategy in anti-apoptotic drug discoveryMOLECULAR NUTRITION & FOOD RESEARCH (FORMERLY NAHRUNG/FOOD), Issue 1 2009Valerian E. Kagan Abstract Thre critical role of mitochondria in programmed cell death leads to the design of mitochondriotropic agents as a strategy in regulating apoptosis. For anticancer therapy, stimulation of proapoptotic mitochondrial events in tumor cells and their suppression in surrounding normal cells represents a promising paradigm for new therapies. Different approaches targeting regulation of components of mitochondrial antioxidant system such as Mn-SOD demonstrated significant antitumor efficiency, particularly in combination therapy. This review is focused on a newly discovered early stage of mitochondria-dependent apoptosis , oxidative lipid signaling involving a mitochondria-specific phospholipid cardiolipin (CL). Cytochrome c (cyt c) acts as a CL-specific peroxidase very early in apoptosis. At this stage, the hostile events are still secluded within the mitochondria and do not reach the cytosolic targets. CL oxidation process is required for the release of pro-apoptotic factors into the cytosol. Manipulation of cyt c interactions with CL, inhibition of peroxidase activity, and prevention of CL peroxidation are prime targets for the discovery of anti-apoptotic drugs acting before the "point-of-no-return" in the fulfillment of the cell death program. Therefore, mitochondria-targeted disruptors and inhibitors of cyt c/CL peroxidase complexes and suppression of CL peroxidation represent new strategies in anti-apoptotic drug discovery. [source] Weak interactions and molecular recognition in systems involving electron transfer proteinsTHE CHEMICAL RECORD, Issue 4 2001Shun Hirota Abstract Electrostatic interactions and other weak interactions between amino acid side chains on protein surfaces play important roles in molecular recognition, and the mechanism of their intermolecular interactions has gained much interest. We established that charged peptides are useful for investigating the molecular recognition character of proteins and their molecular interaction induced structural changes. Positively charged lysine peptides competitively inhibited electron transfer from reduced cytochrome f (cyt f) or cytochrome c (cyt c) to oxidized plastocyanin (PC), due to neutralization of the negatively charged site of PC by formation of PC,lysine peptide complexes. Lysine peptides also inhibited electron transfer from cyt c to cytochrome c peroxidase. Likewise, negatively charged aspartic acid peptides interacted with the positively charged sites of cyt f and cyt c, and competitively inhibited electron transfer from reduced cyt f or cyt c to oxidized PC and from [Fe(CN)6]4, to oxidized cyt c. Changes in the geometry and a shift to a higher redox potential of the active site Cu of PC on oligolysine binding were detected by spectroscopic and electrochemical measurements, owing to the absence of absorption in the visible region for lysine peptides. Structural and redox potential changes were also observed for cyt f and cyt c by interaction with aspartic acid peptides. ©2001 John Wiley & Sons, Inc. and The Japan Chemical Journal Forum Chem Rec 1:290,299, 2001 [source] Characterization of the polyanion-induced molten globule-like state of cytochrome cBIOPOLYMERS, Issue 2 2007Erik Sedlák Abstract Cytochrome c (cyt c) undergoes a poly(vinylsulphate) (PVS)-induced transition at slightly acidic pH into a molten globule-like state that resembles the effect that negatively charged membrane surfaces have on this protein. In this work, the thermodynamic properties of the molten globule-like state of cyt c in complex with PVS are studied using differential scanning calorimetry, circular dichroism, fluorescence, and absorbance spectroscopy. The temperature-induced transition of the molten globule-like state of cyt c in the complex with PVS is characterized by a significantly lower calorimetric enthalpy than in the "typical" molten globule state of cyt c, i.e. free protein at pH 2.0 in high ionic strength. Moreover, the thermally-denatured state of cyt c in the complex at pH < 6 contains nearly 50% of the native secondary structure. The dependence of the transition temperature on the pH indicates a role for histidine residues in the destabilization of the cyt c structure in the PVS complex and in stabilization of the denatured state with the residual secondary structure. A comparison of the effects of small anions and polyanions demonstrates the importance of cooperativity among the anions in the destabilization of cyt c. Predictably, other hydrophilic flexible polyanions such as heparin, polyglutamate, and polyadenylate also have a destabilizing effect on the structure of cyt c. However, a correlation between the properties of the polyanions and their effect on the protein stability is still unclear. © 2007 Wiley Periodicals, Inc. Biopolymers 86: 119,126, 2007. This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com [source] A Colloidal Au Monolayer Modulates the Conformation and Orientation of a Protein at the Electrode/Solution InterfaceCHEMPHYSCHEM, Issue 8 2005Xiue Jiang Abstract The orientation and conformation of adsorbed cytochrome c (cyt c) at the interface between an electrode modified with colloidal Au and a solution were studied by electrochemical, spectroscopic, and spectroelectrochemical techniques. The results indicate that the colloidal Au monolayer formed via preformation of an organic self-assembled monolayer (SAM) can increase the electronic coupling between the SAM and cyt c in the same manner as bifunctional molecular bridges, one functional group of which is bound to the electrode surface while the other interacts with the protein surface. The approach of cyt c to the modified electrode/solution interface can be assisted by strong interactions of the intrinsic charge of colloidal particles with cyt c, while the heme pocket remains almost unchanged due to the screening effect of the negatively charged field created by the intrinsic charge. The conformational changes of cyt c induced by its adsorption at a bare glassy carbon electrode/solution interface and the effect of the electric field on the ligation state of the heme can be avoided at the colloidal-Au-modified electrode/solution interface. Finally, a possible model for the adsorption orientation of cyt c at the colloidal-Au-modified electrode/solution interface is proposed. [source] | ||||||||||||||||