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Cyclosporine‐A (CsA) is a powerful immunosuppressive agent and hepatotoxicity results from CsA treatment. This study aimed to elucidate the effectiveness of tyrosine kinase inhibitor nilotinib against CsA‐induced hepatotoxicity and the underlying molecular mechanisms. Male Sprague‐Dawley rats were allocated into four groups and received drugs for 28 days as follows: Control group: received vehicle, Nilotinib group: received nilotinib (20 mg/kg orally), CsA group: received CsA by subcutaneous injection (20 mg/kg daily), CsA‐nilotinib: received nilotinib and CsA. Serum lactate dehydrogenase (LDH), liver function biomarkers, hepatic levels of oxidative stress biomarkers, nuclear factor erythroid‐2 like‐2 (Nrf2), total antioxidant capacity (TAC), interleukin‐2 (IL‐2), IL‐1β, IL‐6, and cytochrome‐C were assessed. Additionally, the protein levels and mRNA expression of Bcl2 associated X protein (Bax), caspase‐3, nuclear factor‐κB (NF‐κB), hemoxygenase‐1 (HO‐1) were measured. Moreover, liver tissues were assessed histopathologically using hematoxylin–eosin and Masson trichrome stain. Nilotinib treatment decreased serum LDH, alanine aminotransferase, aspartate aminotransferase, and γ‐glutamyltransferase (γ‐GT), hepatic malondialdehyde, and cytochrome‐C. It also increased superoxide dismutase, reduced glutathione, glutathione reductase, glutathione peroxidase, glutathione‐S‐transferase (GST), TAC, and Nrf2 compared to CsA‐injected rats. In addition, nilotinib decreased NF‐κB, IL‐1β, IL‐6, Bax, and caspase‐3, while elevated IL‐2 and immunoexpression of HO‐1. Additionally, mRNA expression of Bax and caspase‐3 was elevated and that of HO‐1 and inhibitory protein κB‐α was reduced in the nilotinib‐treated group. Moreover, nilotinib significantly attenuated CsA‐induced histopathological alterations. Nilotinib may have a promising role as a hepato‐protective through its antiapoptotic, antioxidant, and anti‐inflammatory effects. Research Highlights: Nilotinib has an ameliorative effect against Cyclosporine‐A‐induced liver injury.Nilotinib decreased serum LDH and ALT, AST, and γ‐GT.Nilotinib increased GSH, SOD, GR, GPx, GST, TAC, Nrf2, and HO‐1 levelsNilotinib decreased hepatic MDA, NF‐κB, IL‐1β, IL‐6, Bax, and caspase‐3 levels.Nilotinib attenuated Cyclosporine‐A‐induced histopathological alterations.Nilotinib raised Bax and caspase‐3 mRNA, lowered HO‐1 and NFκBia mRNA expression. [ABSTRACT FROM AUTHOR]

Introduction. Worldwide, nanoparticles especially gold-nanoparticles (Au-NPs) are widely used in medicine, cancer treatment and cosmetic industry. They are easily conjugated with different biomedical and biological agents and effortlessly absorbed with few side effects. The pars distalis of the pituitary gland is considered as the maestro of the endocrine peripheral glands since it secrets trophic hormones that controls their functions. 5-10% of the non-granular pars distalis cells are folliculo-stellate cells (FSCs) that support the granular cells' functions. The aim of the study was to explore the histological and the biochemical effects of repeated exposure to Au-NPs on the pars distalis in adult male albino rats with highlighting the impact on FSCs. Material and methods. Thirty-six adult male albino rats were divided equally into control group and Au-NPs group (received 40 μg/kg/day of 11 ± 2 nm spherical Au-NPs orally for 2 weeks). Then, rats were euthanized and deposition of Au-NPs in pars distalis was investigated. Biochemical investigations and histological studies including hematoxylin and eosin staining, periodic acid Schiff's reaction, immunohistochemistry (IHC) for S-100, connexin 43 (Cx43) and Cytochrome-C (Cyt-C) as well as electron-microscopic and morphometric studies were carried out. Results. The Au-NPs group demonstrated structural disorganization in the pars distalis, inflammation, congestion and increased extracellular PAS-positive colloid deposition due to the accumulation of Au-NPs. A significant increase in the immunoreactivity of S-100, Cx43 and Cyt-c, along with a significant increase in TNF-a, MDA, and bFGF content in the pituitary homogenates, was noted as compared to the control group. Ultrastructurally, degenerative changes were observed in the secretory cells. FSCs showed proliferation and increased phagocytic activity. Conclusions. Repetitive exposure of adult male albino rats to Au-NPs prompted the accumulation of these nanoparticles in the pars distalis that was accompanied by cellular degeneration and dysfunction of the secretory cell and proliferation of FSCs. Thus, monitoring of the pars distalis hormonal levels might be useful for early detection of some hazardous effects possibly associated with the use of gold-nanoparticles. [ABSTRACT FROM AUTHOR]

The programmed cell death (PCD) occurs when the targeted cells have fulfilled their task or under conditions as oxidative stress generated by ROS species. Thus, plants have to deal with the singlet oxygen 1O2 produced in chloroplasts. 1O2 is unlikely to act as a primary retrograde signal owing to its high reactivity and short half-life. In addition to its high toxicity, the 1O2 generated under an excess or low excitation energy might also act as a highly versatile signal triggering chloroplast-to-nucleus retrograde signaling (ChNRS) and nuclear reprogramming or cell death. Molecular and biochemical studies with the flu mutant, which accumulates protochlorophyllide in the dark, demonstrated that chloroplastic 1O2-driven EXECUTER-1 (EX1) and EX2 proteins are involved in the 1O2-dependent response. Both EX1 and EX2 are necessary for full suppression of 1O2-induced gene expression. That is, EXECUTER proteolysis via the ATP-dependent zinc protease (FtsH) is an integral part of 1O2-triggered retrograde signaling. The existence of at least two independent ChNRS involving EX1 and β-cyclocitral, and dihydroactinidiolide and OXI1, respectively, seem clear. Besides, this update also focuses on plant PCD and its relation with mitochondrial cytochrome-c (Cytc) release to cytosol. Changes in the dynamics and morphology of mitochondria were shown during the onset of cell death. The mitochondrial damage and translocation of Cytc may be one of the major causes of PCD triggering. Together, this current overview illustrates the complexity of the cellular response to oxidative stress development. A puzzle with the majority of its pieces still not placed. [ABSTRACT FROM AUTHOR]

Rapid, efficient, and robust quantitative analyses of dynamic apoptotic events are essential in a high‐throughput screening workflow. Currently used methods have several bottlenecks, specifically, limitations in available fluorophores for downstream assays and misinterpretation of statistical image data analysis. In this study, we developed cytochrome‐C (Cyt‐C) and caspase‐3/‐8 reporter cell lines using lung (PC9) and breast (T47D) cancer cells, and characterized them from the response to apoptotic stimuli. In these two reporter cell lines, the spatial fluorescent signal translocation patterns served as reporters of activations of apoptotic events, such as Cyt‐C release and caspase‐3/‐8 activation. We also developed a vision‐based, tunable, automated algorithm in MATLAB to implement the robust and accurate analysis of signal translocation in single or multiple cells. Construction of the reporter cell lines allows live monitoring of apoptotic events without the need for any other dyes or fixatives. Our algorithmic implementation forgoes the use of simple image statistics for more robust analytics. Our optimized algorithm can achieve a precision greater than 90% and a sensitivity higher than 85%. Combining our automated algorithm with reporter cells bearing a single‐color dye/fluorophore, we expect our approach to become an integral component in the high‐throughput drug screening workflow. [ABSTRACT FROM AUTHOR]

Proteins fold via a number of intermediates that help them to attain their unique native 3D structure. These intermediates can be trapped under extreme conditions of pH, temperature and chemical denaturants. Similar states can also be achieved by other processes like chemical modification, site directed mutagenesis (or point mutation) and cleavage of covalent bonds of natural proteins under physiological conditions usually taken as dilute buffer (near neutral pH) and 25 °C. Structural characterization of molten globules is hampered due to (i) their transient nature, (ii) very low population at equilibrium, and (iii) prone to aggregation at high concentration. Furthermore, the dynamic nature of these folding intermediates makes them unsuitable for X-ray diffraction. Hence, understanding their structures at the atomic level is often a challenge. However, characterization of these intermediates at the atomic level is possible by NMR, which could possibly unravel new details of the protein folding process. We have previously shown that the L94G mutant of horse cytochrome-c displays characteristics of the molten globule (MG) state at pH 6.0 and 25 °C. As a first step towards characterizing this MG state at the atomic level by NMR, we report its complete backbone, side chain and heme chemical shift assignments. [ABSTRACT FROM AUTHOR]

Multi-walled carbon nanotubes (MWCNTs) were incorporated into the active layer of mesoporous TiO2 films resulting in MWCNTs-TiO2 nanocomposites with improved electrical conductivity. These MWCNTs-TiO2 nanocomposite films were prepared by a direct mixing method and the “doctor blade” technique. The films were sintered at various annealing temperatures (300, 350, 400, and 450 °C) in order to examine the effect of annealing temperature to the morphology and electrochemical activity of the films. The presence of anatase TiO2 and MWCNTs has been confirmed by X-ray diffraction (XRD), field emission scanning electron microscopy (SEM), transmission electron microscopy (TEM), and Raman spectroscopy, while conductivity and electrochemical properties of the nanocomposite MWCNΤs-TiO2 films were examined via cyclic voltammetry (CV) and spectroelectrochemistry. After successful protein immobilization (Cyt-c), the electrochemical and spectroelectrochemical behavior of these hybrid electrodes (Cyt-c/MWCNTs-TiO2) was examined in detail and particularly the effect of MWCNTs on the interfacial electron transfer between the film electrode and the adsorbed protein molecules.Schematic illustration of MWCNTs-TiO2 nanocomposite films used for Cyt-c immobilization. [ABSTRACT FROM AUTHOR]

The present work is to evaluate the neurotoxicity induced by pyrethroid insecticide 'Deltamethrin' at 0.32 mg/kg/day in two main regions of the Wistar rat brain (hippocampus and striatum) and the protective effects of Quercetin at 10 mg/kg/day on this toxicity after 90 days of exposure. The assay of brain parameters showed that Deltamethrin caused a significant increase of mitochondrial metabolite level (proteins, lipids, and carbohydrates) and enzyme activity (glutathione S-transferase and superoxide dismutase); a decreased amount of mitochondrial glutathione level and catalase and glutathione peroxidase activities; and an increase of malondialdehyde (MDA) acid levels of the two regions. Furthermore, mitochondrial functional testing in the brains of treated rats exhibited a significant increase in permeability followed by a mitochondrial swelling. Instead, a statistically significant decrease in mitochondrial respiration (O consumption) was recorded in the striatum and hippocampus. Our study showed that the pesticide caused a significant increase of the cytochrome c amount correlated with activation of neuronal apoptosis mechanisms by the significant increase of caspase-3 of hippocampus and striatum. In particular, the results of behavioral tests (open field, classic maze tests of sucrose, and Morris water maze) have significant changes, namely bad behavior of the treated rats, affecting the level of anxiety, learning, and memory, and general motor activity has mainly been shown in treated rats. In addition, the histological cuts clearly confirm cerebral necrosis in the hippocampus and the striatum caused by the pesticide. They allow us to consider the necrotic areas, black spots, reduction, and denaturation of these brain regions in the treated rats. On the other hand, we have studied the protective effects against the neurotoxicity of Deltamethrin (DLM). In this context, after the gavage of Quercetin at the dose of 10 mg/kg/day, we have noticed an improvement in the entire parameters: mitochondrial enzyme, metabolic, histological, and behavioral parameters. This confirmed the improvement of preventive and curative effect of Quercetin against free radicals induced by the DLM. [ABSTRACT FROM AUTHOR]

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The Nilgai or Blue Bull (Boselaphus tragocamelus) is the largest Asian antelope which is endemic to Nepal, Pakistan and Indian regions, where these animals are found in packs. Due to rampant hunting, deforestation and habitat degradation, this vulnerable specie is near threatened in many parts of the world. There is need of conservation efforts both at genomic and geographic level. The present study was designed for genomic characterization of Boselaphus tragocamelus in Pakistan by using phylogenetic analysis. Samples from Boselaphus tragocamelus were collected from different parks, zoos and natural habitats. DNA was extracted by standard inorganic extraction method. Primer3 software was used for primer designing targeting Mitochondrial specie identification markers such as d-loop, cytochrome-b and cytochrome-c. After amplification, PCR product was sequenced. Bioinformatics tools were applied for identification of polymorphic loci. Allelic frequency of each locus was calculated. Multidimensional scaling plot illustrated low level of generic diversity among individuals. Phylogenetic analysis revealed conserved neighbouring pattern among different individuals as they shared common ancestry. This is the first report on genomic characterization of Nilgai from Pakistan. The information of selected species of deer is prerequisite for designing effective strategy in future effective practices for conservation. However further genomic investigations should be carried out at a larger scale. [ABSTRACT FROM AUTHOR]

The molecular mechanisms underlying stress-induced depression have not been fully outlined. Hence, the current study aimed at testing the link between behavioral changes in chronic mild stress (CMS) model and changes in hippocampal energy metabolism and the role of paroxetine (PAROX) in ameliorating these changes. Male Wistar rats were divided into three groups: vehicle control, CMS-exposed rats, and CMS-exposed rats receiving PAROX (10 mg/kg/day intraperitoneally). Sucrose preference, open-field, and forced swimming tests were carried out. Corticosterone (CORT) was measured in serum, while adenosine triphosphate and its metabolites, cytosolic cytochrome-c (Cyt-c), caspase-3 (Casp-3), as well as nitric oxide metabolites (NOx) were measured in hippocampal tissue homogenates. CMS-exposed rats showed a decrease in sucrose preference as well as body weight compared to control, which was reversed by PAROX. The latter further ameliorated the CMS-induced elevation of CORT in serum (91.71±1.77 ng/mL vs 124.5±4.44 ng/mL, P<0.001) as well as the changes in adenosine triphosphate/adenosine diphosphate (3.76±0.02 nmol/mg protein vs 1.07±0.01 nmol/mg protein, P<0.001). Furthermore, PAROX reduced the expression of Cyt-c and Casp-3, as well as restoring NOx levels. This study highlights the role of PAROX in reversing depressive behavior associated with stress-induced apoptosis and changes in hippocampal energy metabolism in the CMS model of depression. [ABSTRACT FROM AUTHOR]

Objective : In the present study we analyzed neuroprotective and antiapoptotic effect of the difumarate salt S-15176, as an anti-ischemic, an antioxidant and a stabilizer of mitochondrial membrane in secondary damage following spinal cord injury (SCI) in a rat model. Methods : Three groups were performed with 30 Wistar rats; control (1), trauma (2), and a trauma+S-15176 (10 mg/kg i.p., dimethyl sulfoxide) treatment (3). SCI was performed at the thoracic level using the weight-drop technique. Spinal cord tissues were collected following intracardiac perfusion in 3rd and 7th days of posttrauma. Hematoxylin and eosin staining for histopatology, terminal deoxynucleotidyl transferase dUTP nick end labeling assay for apoptotic cells and immunohistochemistry for proapoptotic cytochrome-c, Bax and caspase 9 were performed to all groups. Functional recovery test were applied to each group in 3rd and 7th days following SCI. Results : In trauma group, edematous regions, diffuse hemorrhage, necrosis, leukocyte infiltration and severe degeneration in motor neurons were observed prominently in gray matter. The number of apoptotic cells was significantly higher (p<0.05) than control group. In the S-15176-treated groups, apoptotic cell number in 3rd and 7th days (p<0.001), also cytochrome-c (p<0.001), Bax (p<0.001) and caspase 9 immunoreactive cells (p<0.001) were significantly decreased in number compared to trauma groups. Hemorrhage and edema in the focal areas were also noticed in gray matter of treatment groups. Results of the locomotor test were significantly increased in treatment group (p<0.05) when compared to trauma groups. Conclusion : We suggest that difumarate salt S-15176 prevents mitochondrial pathways of apoptosis and protects spinal cord from secondary injury and helps to preserve motor function following SCI in rats. [ABSTRACT FROM AUTHOR]

Objectives To investigate the effect of tibolone and raloxifene on the serum apoptotic markers soluble Fas (sFas), soluble Fas ligand (sFasL) and cytochrome-c (cyt-c) in postmenopausal women. Methods A total of 89 healthy postmenopausal women, attending the University Menopause Clinic, were randomly allocated to tibolone ( n =30), raloxifene ( n =29) or no treatment ( n =30). Serum apoptotic markers sFas, sFasL and cyt-c were measured at baseline and at 6 months. Results Serum sFasL decreased significantly in women receiving tibolone (baseline: 53.8±28.3 pg/ml, 6 months: 40.45±19.2 pg/ml, p =0.001), whilst sFas levels did not significantly change in this group. Serum sFas or sFasL did not change either in the raloxifene group or in the control group. Serum cyt-c concentrations were under the detection limit of the assay in all women assessed. Conclusions Tibolone use resulted in a significant decrease in serum sFasL, but not in serum sFas. Raloxifene had no effect on either sFas or sFasL. These results may indicate that tibolone use is associated with a decrease in receptor-mediated apoptosis. [ABSTRACT FROM AUTHOR]

Ziram as a dithiocarbamate fungicide is widely used throughout the world in agriculture. We previously found that ziram significantly inhibited cytotoxic T lymphocyte activity in a dose-dependent manner. To explore the mechanism of this inhibition, we investigated ziram-induced apoptosis in human T lymphocytes. Jurkat T cells were treated with ziram at 0.031-1 μM for 2-24 h. Freshly isolated primary human T cells were treated with ziram at 0.0625-1 μM for 15 and 24 h. Apoptosis was determined by FITC-Annexin V/PI staining and the TUNEL assay. To explore the mechanism of apoptosis, intracellular levels of active caspases 3, 3/7, 8, and 9 and pan-caspase and mitochondrial cytochrome-c release were determined by flow cytometry. Disruption to mitochondrial transmembrane potential was determined with a MitoLight Apoptosis Detection Kit. We found that ziram induced apoptosis in a time- and dose-dependent manner in both Jurkat cells and primary human T cells. The primary human T cells were more sensitive to ziram than the Jurkat cell line. Ziram induced increases in active caspases 3, 3/7, 8, and 9 and pan-caspase in a dose-dependent manner, and a caspase-3 inhibitor, Z-DEVD-FMK, partially but significantly inhibited the apoptosis. Moreover, a general caspase inhibitor, Z-VAD-FMK, significantly and almost completely blocked the apoptosis. Ziram also disrupted mitochondrial transmembrane potential and caused mitochondrial cytochrome-c release. These findings indicate that ziram can induce apoptosis in human T cells, and the apoptosis is mediated by both the caspase-cascade and the mitochondria/cytochrome-c pathways. [ABSTRACT FROM AUTHOR]

Acute respiratory distress syndrome (ARDS) induced by lipopolysaccharide (LPS) is known to be associated with pulmonary cytokine production resulting in infiltration of neutrophils and pulmonary injury. The study presented here is focused on the changes in cell death regulating proteins during lung injury and studied the relationship between the LPS challenged ARDS and the subsequent role of surfactant therapy against ARDS. Apoptotic features, such as induction of DNA fragmentation ladders in lung tissue, expression of cytoplasmic proteins (Bcl-2, Bax), mitochondrial proteins (cytochrome c, Apaf-1) and activation of caspases were detected following LPS exposure. Instillation of the surfactant derived from the natural source as well as synthetic surfactant prevents LPS induced apoptosis. These results suggest that the mechanism of induction of apoptosis by LPS depends on mitochondrial dysfunction and the anti-apoptotic effects of surfactant treatment conferred protection in the rat model during lung injury mediated via suppression of mitochondrial intrinsic pathway. [ABSTRACT FROM AUTHOR]

Unilamellar liposomes, prepared from synthetic lipid mixture of DMPC and DMPG either by sonication or extrusion, were used to entrap water soluble and water insoluble molecules to investigate the efficacy of encapsulation by different liposome preparation methods. In the case of entrapment of hydrophilic protein cytochrome-C, the solutions were subjected to a series of ultrafiltration steps to eliminate any free protein outside the vesicles. It was observed that the protein could be encapsulated by the vesicles only if cholesterol was present in the bilayer. The release of cytochrome-C was observed spectrophotometrically upon vesicle-breakdown. The amount of protein encapsulated depended on the method of preparation and was found to be 10 times greater in extruded liposomes compared to those produced by sonication. Hydrophobic Vitamin E, on the other hand, could be encapsulated in the liposome bilayer, independently of the presence of cholesterol and the method of preparation. These fundamental results can be used to develop more efficient drug encapsulations and to have better understanding about their release. [ABSTRACT FROM AUTHOR]

A number of species of Gram-negative bacteria can use insoluble minerals of Fe(lll) and Mn(IV) as extracellular respiratory electron acceptors. In some species of Shewanella, deca-heme electron transfer proteins lie at the extracellular face of the outer membrane (OM). where they can interact with insoluble substrates. To reduce extracellular substrates, these redox proteins must be charged by the inner membrane/periplasmic electron transfer system. Here, we present a spectro-potentiometric characterization of a trans-OM icosa-heme complex, MtrCAB, and demonstrate its capacity to move electrons across a lipid bilayer after incorporation into proteoliposomes. We also show that a stable MtrAB subcomplex can assemble in the absence of MtrC; an MtrBC subcomplex is not assembled in the absence of MtrA; and MtrA is only associated to the membrane in cells when MtrB is present. We propose a model for the modular organization of the MtrCAB complex in which MtrC is an extracellular element that mediates electron transfer to extracellular substrates and MtrB is a trans-OM spanning β-barrel protein that serves as a sheath, within which MtrA and MtrC exchange electrons. We have identified the MtrAB module in a range of bacterial phyla, suggesting that it is widely used in electron exchange with the extracellular environment. [ABSTRACT FROM AUTHOR]

Uranium is a naturally occurring radioactive material present everywhere in the environment. It is toxic because of its chemical or radioactive properties. Uranium enters environment mainly from mines and industry and cause threat to human health by accumulating in lungs as a result of inhalation. In our previous study, we have shown the effectiveness of antioxidant system response to the oxidative stress induced by uranyl acetate (UA) in rat lung epithelial (LE) cells. As part of our continuing studies; here, we investigated the mechanism underlying when LE cells are exposed to different concentration of UA. Oxidative stress may lead to apoptotic signaling pathways. LE cells treated with 0.25, 0.5 and 1 mM of UA results in dose and time-dependent increase in activity of both caspases-3 and -8. Increase in the concentration of cytochrome-c oxidase in cytosol was seen in LE cells treated with 1 mM UA as a result of mitochondria membrane permeability. The cytochrome-c leakage may trigger the apoptotic pathway. TUNEL assay performed in LE cells treated with 1 mM of UA showed significant incorporation of dNTPs in the nucleus after 24 h. In the presence of the caspase inhibitors, we observed the significant decrease in the activity of caspases-8 and -3 in 0.5 and 1 mM UA-treated LE cells. [ABSTRACT FROM AUTHOR]

Individual cells within a population undergo apoptosis at distinct, apparently random time points. By analyzing cellular mitotic history, we identified that sibling HeLa cell pairs, in contrast to random cell pairs, underwent apoptosis synchronously. This allowed us to use high-speed cellular imaging to investigate mitochondrial outer membrane permeabilization (MOMP), a highly coordinated, rapid process during apoptosis, at a temporal resolution approximately 100 times higher than possible previously. We obtained new functional and mechanistic insight into the process of MOMP: We were able to determine the kinetics of pore formation in the outer mitochondrial membrane from the initiation phase of cytochrome-c-GFP redistribution, and showed differential pore formation kinetics in response to intrinsic or extrinsic apoptotic stimuli (staurosporine, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)). We also detected that the onset of mitochondrial permeabilization frequently proceeded as a wave through the cytosol, and that the frequency of wave occurrence in response to TRAIL was reduced by inhibition of protein kinase CK2. Computational analysis by a partial differential equation model suggested that the spread of permeabilization signals could sufficiently be explained by diffusion–adsorption velocities of locally generated permeabilization inducers. Taken together, our study yielded the first comprehensive analysis of clonal cell-to-cell variability in apoptosis execution and allowed to visualize and explain the dynamics of MOMP in cells undergoing apoptosis.Cell Death and Differentiation (2009) 16, 613–623; doi:10.1038/cdd.2008.187; published online 9 January 2009 [ABSTRACT FROM AUTHOR]

In bacterial empyema the pleural mesothelium is constantly exposed to microorganisms. Staphylococcus aureus ( S. aureus) is one of the most frequent pathogens associated with empyema. In an earlier study we demonstrated that S. aureus induced barrier dysfunction in pleural mesothelial cell monolayers. In the present study we report that S. aureus activates the early response genes c -fos and c -jun and activator protein-1 (AP-1), and induces proapoptosis genes Bad and Bak in primary mouse pleural mesothelial cells (PMCs). Our data indicate that in PMCs S. aureus induces apoptosis in a time- and multiplicity of infection (MOI)-dependent manner. Staphylococcus aureus induced Bcl 2, Bcl-X L, c- fos, c -jun, and AP-1 expression in PMCs during the initial phase of infection . In S. aureus-infected PMCs, Bad and Bak gene expression was increased and correlated with DNA fragmentation and cytochrome-c release. Bcl 2 and Bcl-X L gene expression was significantly lower in S. aureus-infected PMCs than in uninfected PMCs 12 h postinfection. We conclude that at the initial stage of infection S. aureus modulates expression of early response genes c -fos and c- jun, and in the late phase of infection S. aureus induces expression of proapoptotic genes Bak and Bad in PMCs. Silencing AP-1 significantly inhibited S. aureus-induced Bak and Bad expression in PMCs. The upregulation of early response genes during the early phase of infection may contribute to the activation of proapoptotic genes Bak and Bad and release of cytochrome-c, caspase-3 thereby resulting in apoptosis in PMCs. [ABSTRACT FROM AUTHOR]

Gentamicin side-effects on cochlear structures and function are well known, but not the detailed intracellular molecular mechanisms which lead to aminoglycoside induced ototoxicity. Hair cell death occurs by apoptosis, by the activation of enzymatic cascades known to be involved in the programmed cell death, or by the release of cytochrome-c from the damaged mitochondria. In this paper we have investigated the active role of minocycline, a second-generation tetracycline, and of MDL 28170, a selective calpain inhibitor, in the protection of hair cells from GM damage in in vitro organospecific cultures of the organ of Corti. We used cultures from neonatal (P3) rat cochlea, treated with different dosages of GM, alone, or with the two protector drugs. We have observed a dose-dependent OHC and IHC loss. The GM damage was reduced after treatment with both drugs. These results were also supported by the Disk Diffusion Susceptibility Test (Kirby-Bauer Method), in which we used drug treated organs of Corti. The data suggest that minocycline, previously reported to inhibit the release of cytochrome-c, and MDL 28170, prevent GM induced programmed cell death pathway in cochlear HC. [ABSTRACT FROM AUTHOR]

The retention data of the components of the peptide mixture obtained by the enzymatic digestion of cytochrome-c were measured under different gradient conditions, using different organic modifiers (acetonitrile, methanol, and tetrahydrofuran) in a 0.1% ( v/v) aqueous solution of trifluoroacetic acid. The influences of the nature of the organic modifier, of the gradient steepness, and of the column length on the retention and the resolution of these peptides were studied. Peak capacity and retention factor calculations for gradient separations were helpful in determining a desired gradient for this separation. All three organic modifiers are shown to cause reversal orders of some peaks in the cytochrome-c digest separation. [ABSTRACT FROM AUTHOR]

Bis(ethylenediamine) copper(II) complex was encapsulated in NaY, KL, Naβ and NaZSM-5 zeolite. The redox properties of metal complexes in neat and encapsulated state were studied by cyclic voltammetry. The redox potential of metal complex is altered towards negative value upon encapsulation in various zeolites. This may be due to axial interaction with various zeolite matrix. The redox properties of biological systems are also altered (cytochrome-C, and Vitamin-B12) when they are immobilized on NaAlMCM-41 materials. The copper ethylenediamine complex in constrained environment shows higher activity for the oxidation of dimethyl sulfide compared to that of neat complex. [ABSTRACT FROM AUTHOR]

A novel reaction of cytochrome-c from the horse heart with the enzyme glucose oxidase from Aspergillus niger (EC 1.1.3.4), in acidic media is described. Glucose oxidase is able to induce a rapid, profound and irreversible physico-chemical change in cytochrome-c, under anaerobic conditions and in the presence of glucose. The initial rate of reaction is almost independent of the concentration of enzyme and glucose. The striking feature of this reaction is the fact that the reaction proceeds efficiently even below a concentration of 10 nM enzyme. [ABSTRACT FROM AUTHOR]

Cytochrome c (Cyt-c), a small mitochondrial electron transport heme protein, has been employed in bioelectrochemical and therapeutic applications. However, its potential as both a biosensor and anticancer drug is significantly impaired due to poor long-term and thermal stability. To overcome these drawbacks, we developed a site-specific PEGylation protocol for Cyt-c. The PEG derivative used was a 5 kDa mPEG-NHS, and a site-directed PEGylation at the lysine amino-acids was performed. The effects of the pH of the reaction media, molar ratio (Cyt-c:mPEG-NHS) and reaction time were evaluated. The best conditions were defined as pH 7, 1:25 Cyt-c:mPEG-NHS and 15 min reaction time, resulting in PEGylation yield of 45% for Cyt-c-PEG-4 and 34% for Cyt-c-PEG-8 (PEGylated cytochrome c with 4 and 8 PEG molecules, respectively). Circular dichroism spectra demonstrated that PEGylation did not cause significant changes to the secondary and tertiary structures of the Cyt-c. The long-term stability of native and PEGylated Cyt-c forms was also investigated in terms of peroxidative activity. The results demonstrated that both Cyt-c-PEG-4 and Cyt-c-PEG-8 were more stable, presenting higher half-life than unPEGylated protein. In particular, Cyt-c-PEG-8 presented great potential for biomedical applications, since it retained 30–40% more residual activity than Cyt-c over 60-days of storage, at both studied temperatures of 4 °C and 25 °C. [ABSTRACT FROM AUTHOR]

The effect of cytochrome-C, hemoglobin, and myoglobin on photo-osmosis through liquid membrane bilayers generated by bacteriorhodopsin (BR) has been studied. The magnitude of photo-osmotic velocity was found to be much greater when BR was combined with any one of the three pigments than that of BR alone. This has been because of the exclusion of protons and electrons in the illuminated compartment by the action of light, where one acts as the acceptor for the others. The rate of light-induced volume flux of the combined system depends on temperature, intensity, and wavelength of incident light, and the nature and concentration of electron donors and acceptors. [ABSTRACT FROM AUTHOR]

Monte Carlo simulation has commonly been used in phylogenetic studies to test different tree-reconstruction methods, and consequently, its application for testing evolutionary models can be considered as a natural extension of this usage. Repetitive simulation of a given evolutionary process, under the restrictions imposed by the model to be tested, along a determinate tree topology allow the estimate of probability distributions for the desired parameters. Next, the phylogenetic tree can be reconstructed again without the constraints of the model, and the parameter of interest, derived from this tree, can be compared to the corresponding probability distribution derived from the restricted, simulated trees. As an example we have used Monte Carlo simulation to test the constancy of evolutionary rates in a set of cytochrome-c protein sequences. [ABSTRACT FROM AUTHOR]

Bio-electrochemical systems such as microbial fuel cells and microbial electrosynthesis cells depend on efficient electron transfer between the microorganisms and the electrodes. Understanding the mechanisms and dynamics of the electron transfer is important in order to design more efficient reactors, as well as modifying microorganisms for enhanced electricity production. Geobacter are well known for their ability to form thick biofilms and transfer electrons to the surfaces of electrodes. Currently, there are not many "on-line" systems for monitoring the activity of the biofilm and the electron transfer process without harming the biofilm. Raman microscopy was shown to be capable of providing biochemical information, i.e., the redox state of C-type cytochromes, which is integral to external electron transfer, without harming the biofilm. In the current study, a custom 3D printed flow-through cuvette was used in order to analyze the oxidation state of the C-type cytochromes of suspended cultures of three Geobacter sulfurreducens strains (PCA, KN400 and ΔpilA). It was found that the oxidation state is a good indicator of the metabolic state of the cells. Furthermore, an anaerobic fluidic system enabling in situ Raman measurements was designed and applied successfully to monitor and characterize G. sulfurreducens biofilms during electricity generation, for both a wild strain, PCA, and a mutant, ΔS. The cytochrome redox state, monitored by the Raman peak areas, could be modulated by applying different poise voltages to the electrodes. This also correlated with the modulation of current transferred from the cytochromes to the electrode. The Raman peak area changed in a predictable and reversible manner, indicating that the system could be used for analyzing the oxidation state of the proteins responsible for the electron transfer process and the kinetics thereof in-situ. [ABSTRACT FROM AUTHOR]

It is unclear whether whey protein, popular with many sportspeople due to its high calorific value and because it increases body mass, has adverse effects on the male reproductive system. The aim of this study was to investigate the effects of whey protein on apoptotic signaling molecules and testosterone release in the testis. Thirty male Wistar albino rats were divided into three groups, a control group, a group receiving short-term whey protein (5 days, 252 g/kg) (WS) and a group receiving long-term whey protein (4 weeks, 252 g/kg) (WL). Immunohistochemical techniques were applied to P-53, Bax, Bcl-2 and cytochrome-c signaling molecules and testosterone in extracted testis tissue. In the control group, seminiferous tubules P53 reaction was observed in some primary spermatocytes (PSs) and Sertoli cells (SCs), while in the WS group, expression ranging from moderate to strong was determined in most PSs and SCs. In the WL group, strong immune reaction was observed in both PSs and SCs. No Bax immunoreaction was encountered in any group. Bcl-2 expression was moderate in control group PSs but weaker in the other two groups. Cytochromec expression in the control group was weak and granular in form in secondary spermatocytes (SSs), while expression in the WS and WL groups was again observed only in SSs and in strongly granular form. Testosterone immunoreaction exhibited strong release in control group Leydig cells, while expression was moderate in WS and WL. Whey protein use, and particularly long-term use, causes damage via the P-53 signaling molecule in SSs, which are of vital importance for sperm nourishment and support in the seminiferous tubules. It also causes excessive expression of the cytochrome-c molecule in SSs through the mitochondrial pathway. Both long-and short-term use can lead to some degree of decrease in testosterone release from Leydig cells. [ABSTRACT FROM AUTHOR]