RESUMO
Membrane-bound proteins can play a role in the binding of anionic gold nanoparticles (AuNPs) to model bilayers; however, the mechanism for this binding remains unresolved. In this work, we determine the relative orientation of the peripheral membrane protein cytochrome c in binding to a mercaptopropionic acid-functionalized AuNP (MPA-AuNP). As this is nonrigid binding, traditional methods involving crystallographic or rigid molecular docking techniques are ineffective at resolving the question. Instead, we have implemented a computational assay technique using a cross-correlation of a small ensemble of 200 ns long molecular dynamics trajectories to identify a preferred nonrigid binding orientation or pose of cytochrome c on MPA-AuNPs. We have also employed a mass spectrometry-based footprinting method that enables the characterization of the stable protein corona that forms at long time-scales in solution but remains in a dynamic state. Through the combination of these computational and experimental primary results, we have established a consensus result establishing the identity of the exposed regions of cytochrome c in proximity to MPA-AuNPs and its complementary pose(s) with amino-acid specificity. Moreover, the tandem use of the two methods can be applied broadly to determine the accessibility of membrane-binding sites for peripheral membrane proteins upon adsorption to AuNPs or to determine the exposed amino-acid residues of the hard corona that drive the acquisition of dynamic soft coronas. We anticipate that the combined use of simulation and experimental methods to characterize biomolecule-nanoparticle interactions, as demonstrated here, will become increasingly necessary as the complexity of such target systems grows.
Assuntos
Grupo dos Citocromos c/química , Nanopartículas Metálicas/química , Simulação de Acoplamento Molecular , Ácido 3-Mercaptopropiônico/química , Animais , Ânions/química , Grupo dos Citocromos c/metabolismo , Ouro/química , Cavalos , Ligantes , Lisina/química , Ligação ProteicaRESUMO
Müller cells, the supporting cells of the retina, play a key role in responding to retinal stress by releasing chemokines, including CCL2, to recruit microglia and macrophages (MG/MΦ) into the damaged retina. Photobiomodulation (PBM) with 670 nm light has been shown to reduce inflammation in models of retinal degeneration. In this study, we aimed to investigate whether 670 nm light had an effect on Müller cell-initiated inflammation under retinal photo-oxidative damage (PD) in vivo and in vitro. Sprague-Dawley rats were pre-treated with 670 nm light (9J/cm2) once daily over 5 days prior to PD. The expression of inflammatory genes including CCL2 and IL-1ß was analysed in retinas. In vitro, primary Müller cells dissociated from neonatal rat retinas were co-cultured with 661W photoreceptor cells. Co-cultures were exposed to PD, followed by 670 nm light treatment to the Müller cells only, and Müller cell stress and inflammation were assessed. Primary MG/MΦ were incubated with supernatant from the co-cultures, and collected for analysis of inflammatory activation. To further understand the mechanism of 670 nm light, the expression of COX5a and mitochondrial membrane potential (ΔΨm) were measured in Müller cells. Following PD, 670 nm light-treated Müller cells had a reduced inflammatory activation, with lower levels of CCL2, IL-1ß and IL-6. Supernatant from 670 nm light-treated co-cultures reduced activation of primary MG/MΦ, and lowered the expression of pro-inflammatory cytokines, compared to untreated PD controls. Additionally, 670 nm light-treated Müller cells had an increased expression of COX5a and an elevated ΔΨm following PD, suggesting that retrograde signaling plays a role in the effects of 670 nm light on Müller cell gene expression. Our data indicates that 670 nm light reduces Müller cell-mediated retinal inflammation, and offers a potential cellular mechanism for 670 nm light therapy in regulating inflammation associated with retinal degenerations.
Assuntos
Células Ependimogliais/efeitos da radiação , Macrófagos/efeitos da radiação , Microglia/efeitos da radiação , Degeneração Retiniana/radioterapia , Animais , Quimiocinas/metabolismo , Grupo dos Citocromos c/metabolismo , Modelos Animais de Doenças , Células Ependimogliais/fisiologia , Interleucinas/metabolismo , Potencial da Membrana Mitocondrial/efeitos da radiação , Estresse Oxidativo/efeitos da radiação , Ratos , Ratos Sprague-Dawley , Degeneração Retiniana/metabolismoRESUMO
Stroke is a common neurodegenerative disease in the wide world, and mitochondrial defects underlie the pathogenesis of ischemia, especially during reperfusion. Picroside II, the principal active component of Picrorhiza, is a traditional Chinese medicine. Our previous study demonstrated that the best therapeutic dose and time window were injection of picroside II at a dose of 10-20 mg/kg body weight following cerebral ischemia by 1.5-2.0 h. In this paper, the neuroprotective effect and the mechanism of picroside II were investigated, as well as its involvement in antioxidant and mitochondria cytochrome C (CytC) signal pathway following ischemia reperfusion (I/R) injury in rats. After 24 h of cerebral I/R, the neurobehavioral function was measured by modified neurological severity score test; the content of reactive oxygen species in brain tissue was measured by enzyme-linked immunosorbent assay; the cerebral infarction volume was detected by TTC staining; the morphology of brain tissue was observed by hematoxylin-eosin; the apoptotic cells were counted by terminal deoxynucleotidyl transferase dUTP nick end labeling assay; the ultrastructure of the cortical brain tissues was observation by transmission electron microscopy; the expressions of CytC and Caspase-3 were determined by immunohistochemical assay and Western blot. The results indicated that picroside II could scavenge ROS contents, decrease the cerebral infarction volume and apoptotic cells, protect the structure of mitochondria, down-regulate the expression of CytC and Caspase-3 in cerebral I/R rats. It can be concluded that picroside II exerts a neuroprotective effect by inhibiting the mitochondria CytC signal pathway following ischemia reperfusion injury in rats.
Assuntos
Antioxidantes/farmacologia , Isquemia Encefálica/tratamento farmacológico , Cinamatos/farmacologia , Grupo dos Citocromos c/metabolismo , Glucosídeos Iridoides/farmacologia , Mitocôndrias/metabolismo , Fármacos Neuroprotetores/farmacologia , Traumatismo por Reperfusão/tratamento farmacológico , Animais , Apoptose , Encéfalo/irrigação sanguínea , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Isquemia Encefálica/metabolismo , Masculino , Mitocôndrias/efeitos dos fármacos , Ratos , Ratos Wistar , Traumatismo por Reperfusão/metabolismo , Transdução de SinaisRESUMO
In this issue of Molecular Cell, Barrow et al. (2016) use two complementary approaches-one an assessment of a chemical library, and the other a genome-wide CRISPR screen-that both identify bromodomain-containing protein 4 (Brd4) as a therapeutic target for mtDNA diseases affecting complex I.
Assuntos
Benzodiazepinas/farmacologia , Grupo dos Citocromos c/genética , Complexo I de Transporte de Elétrons/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Proteínas de Ciclo Celular , Fusão Celular , Linhagem Celular , Grupo dos Citocromos c/metabolismo , Complexo I de Transporte de Elétrons/deficiência , Complexo IV da Cadeia de Transporte de Elétrons , Regulação da Expressão Gênica , Ensaios de Triagem em Larga Escala , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Doenças Mitocondriais/tratamento farmacológico , Doenças Mitocondriais/genética , Doenças Mitocondriais/metabolismo , Doenças Mitocondriais/patologia , Proteínas Mitocondriais/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Transdução de Sinais , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismoRESUMO
Mitochondrial diseases comprise a heterogeneous group of genetically inherited disorders that cause failures in energetic and metabolic function. Boosting residual oxidative phosphorylation (OXPHOS) activity can partially correct these failures. Herein, using a high-throughput chemical screen, we identified the bromodomain inhibitor I-BET 525762A as one of the top hits that increases COX5a protein levels in complex I (CI) mutant cybrid cells. In parallel, bromodomain-containing protein 4 (BRD4), a target of I-BET 525762A, was identified using a genome-wide CRISPR screen to search for genes whose loss of function rescues death of CI-impaired cybrids grown under conditions requiring OXPHOS activity for survival. We show that I-BET525762A or loss of BRD4 remodeled the mitochondrial proteome to increase the levels and activity of OXPHOS protein complexes, leading to rescue of the bioenergetic defects and cell death caused by mutations or chemical inhibition of CI. These studies show that BRD4 inhibition may have therapeutic implications for the treatment of mitochondrial diseases.
Assuntos
Benzodiazepinas/farmacologia , Grupo dos Citocromos c/genética , Complexo I de Transporte de Elétrons/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Proteínas de Ciclo Celular , Fusão Celular , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Grupo dos Citocromos c/metabolismo , Complexo I de Transporte de Elétrons/deficiência , Complexo IV da Cadeia de Transporte de Elétrons , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Ensaios de Triagem em Larga Escala , Humanos , Metaboloma , Metabolômica , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Doenças Mitocondriais/tratamento farmacológico , Doenças Mitocondriais/genética , Doenças Mitocondriais/metabolismo , Doenças Mitocondriais/patologia , Proteínas Mitocondriais/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Transdução de Sinais , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismoRESUMO
Studies with pure cultures of dissimilatory metal-reducing microorganisms have demonstrated that outer-surface c-type cytochromes are important electron transfer agents for the reduction of metals, but previous environmental proteomic studies have typically not recovered cytochrome sequences from subsurface environments in which metal reduction is important. Gel-separation, heme-staining and mass spectrometry of proteins in groundwater from in situ uranium bioremediation experiments identified a putative c-type cytochrome, designated Geobacter subsurface c-type cytochrome A (GscA), encoded within the genome of strain M18, a Geobacter isolate previously recovered from the site. Homologs of GscA were identified in the genomes of other Geobacter isolates in the phylogenetic cluster known as subsurface clade 1, which predominates in a diversity of Fe(III)-reducing subsurface environments. Most of the gscA sequences recovered from groundwater genomic DNA clustered in a tight phylogenetic group closely related to strain M18. GscA was most abundant in groundwater samples in which Geobacter sp. predominated. Expression of gscA in a strain of Geobacter sulfurreducens that lacked the gene for the c-type cytochrome OmcS, thought to facilitate electron transfer from conductive pili to Fe(III) oxide, restored the capacity for Fe(III) oxide reduction. Atomic force microscopy provided evidence that GscA was associated with the pili. These results demonstrate that a c-type cytochrome with an apparent function similar to that of OmcS is abundant when Geobacter sp. are abundant in the subsurface, providing insight into the mechanisms for the growth of subsurface Geobacter sp. on Fe(III) oxide and suggesting an approach for functional analysis of other Geobacter proteins found in the subsurface.
Assuntos
Grupo dos Citocromos c/metabolismo , Geobacter/metabolismo , Urânio/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biodegradação Ambiental , Transporte de Elétrons , Compostos Férricos/metabolismo , Geobacter/classificação , Geobacter/genética , Geobacter/isolamento & purificação , Água Subterrânea/microbiologia , Dados de Sequência Molecular , Oxirredução , Filogenia , Proteômica , Alinhamento de SequênciaRESUMO
The in situ stimulation of Fe(III) oxide reduction in the subsurface stimulates the growth of Geobacter spp. and the precipitation of U(VI) from groundwater. As with Fe(III) oxide reduction, the reduction of uranium by Geobacter spp. requires the expression of their conductive pili. The pili bind the soluble uranium and catalyse its extracellular reductive precipitation along the pili filaments as a mononuclear U(IV) complexed by carbon-containing ligands. Although most of the uranium is immobilized by the pili, some uranium deposits are also observed in discreet regions of the outer membrane, consistent with the participation of redox-active foci, presumably c-type cytochromes, in the extracellular reduction of uranium. It is unlikely that cytochromes released from the outer membrane could associate with the pili and contribute to the catalysis, because scanning tunnelling microscopy spectroscopy did not reveal any haem-specific electronic features in the pili, but, rather, showed topographic and electronic features intrinsic to the pilus shaft. Pili not only enhance the rate and extent of uranium reduction per cell, but also prevent the uranium from traversing the outer membrane and mineralizing the cell envelope. As a result, pili expression preserves the essential respiratory activities of the cell envelope and the cell's viability. Hence the results support a model in which the conductive pili function as the primary mechanism for the reduction of uranium and cellular protection in Geobacter spp.
Assuntos
Fímbrias Bacterianas/metabolismo , Geobacter/metabolismo , Urânio/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas da Membrana Bacteriana Externa/fisiologia , Precipitação Química , Grupo dos Citocromos c/metabolismo , Grupo dos Citocromos c/fisiologia , Transporte de Elétrons , Compostos Férricos/metabolismo , Fímbrias Bacterianas/ultraestrutura , Geobacter/ultraestrutura , Heme/metabolismo , Viabilidade Microbiana , Oxirredução , Periplasma/metabolismo , Periplasma/ultraestrutura , Urânio/químicaRESUMO
In this work we identified the gene for the tetrathionate-forming thiosulfate dehydrogenase (TsdA) from the purple sulfur bacterium Allochromatium vinosum by sequence analysis and reverse genetics. The recombinant protein produced in Escherichia coli is a periplasmic, monomeric 25.8 kDa dihaem cytochrome c with an enzyme activity optimum at pH 4. UV-visible and electron paramagnetic resonance spectroscopy indicate methionine (strictly conserved M(222) or M(236)) and cysteine (C(123) ) as probable sixth distal axial ligands of the two haem irons in TsdA. These results place TsdA in the group of c-type cytochromes with an unusual axial histidine-cysteine coordination of the haem iron. These proteins appear to play a pivotal role in sulfur-based energy metabolism. Exchange of C(123) to glycine rendered thiosulfate dehydrogenase inactive, proving the importance of this residue for catalysis. TsdA homologues are present in α-, ß-, δ-, γ- and ε-Proteobacteria. Three of these were produced in E. coli and exhibited the expected enzymatic activity. The widespread occurrence of tsdA agrees with reports of tetrathionate formation not only by specialized sulfur oxidizers but also by many chemoorganoheterotrophs that use thiosulfate as a supplemental but not as the sole energy source.
Assuntos
Chromatiaceae/enzimologia , Chromatiaceae/genética , Oxirredutases/genética , Oxirredutases/metabolismo , Sequência de Aminoácidos , Grupo dos Citocromos c/química , Grupo dos Citocromos c/genética , Grupo dos Citocromos c/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Oxirredução , Oxirredutases/química , Proteínas Recombinantes/genética , Alinhamento de Sequência , Deleção de SequênciaRESUMO
UndA(HRCR-6) was identified from the metal-reducing bacterium Shewanella sp. strain HRCR-6. Both in vivo and in vitro characterization results indicate that UndA(HRCR-6) is an outer membrane endecaheme c-type cytochrome and probably has a key functional role in the extracellular reduction of iron [Fe(III)] oxides and uranium [U(VI)] by Shewanella sp. HRCR-6.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Grupo dos Citocromos c/metabolismo , Shewanella/enzimologia , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/análise , Sequência de Bases , Biodegradação Ambiental , Grupo dos Citocromos c/análise , Grupo dos Citocromos c/genética , Compostos Férricos/metabolismo , Oxirredução , Filogenia , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Shewanella/genética , Urânio/metabolismoRESUMO
Survivin, an important member of inhibitor-of-apoptosis (IAP) family, can be up-regulated by various pro-apoptotic stimuli, such as UV, photodynamic therapy (PDT) and cisplatin. High fluence low-power laser irradiation (HF-LPLI) is a newly discovered pro-apoptotic stimulator. The anti-apoptotic mechanism of survivin during HF-LPLI-induced apoptosis is still not investigated. Here, we report that HF-LPLI up-regulates survivin activity through reactive oxygen species (ROS)/cdc25c protein phosphatase (cdc25c)/cyclin-dependent kinase (CDK1) signaling pathway in human lung adenocarcinoma cells (ASTC-a-1). The up-regulation of survivin activity can reduce HF-LPLI-induced apoptosis, while down-regulation of the activity can promote the apoptosis. In addition, activated survivin delays mitochondrial depolarization, cytochrome c release, caspase-9 and Bax activation, all of which are typical pro-apoptotic events of cell apoptosis induced by HF-LPLI. On the basis of the present studies, we conclude that survivin can mediate self-protection during tumor cell apoptosis caused by HF-LPLI.
Assuntos
Terapia com Luz de Baixa Intensidade/métodos , Proteínas Associadas aos Microtúbulos/metabolismo , Transdução de Sinais/efeitos da radiação , Apoptose/efeitos da radiação , Proteína Quinase CDC2/metabolismo , Inibidores de Caspase , Caspases/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos da radiação , Grupo dos Citocromos c/antagonistas & inibidores , Grupo dos Citocromos c/metabolismo , Humanos , Proteínas Inibidoras de Apoptose , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/radioterapia , Fosforilação , Espécies Reativas de Oxigênio/metabolismo , Survivina , Transfecção , Regulação para Cima , Proteína X Associada a bcl-2/metabolismo , Fosfatases cdc25/metabolismoRESUMO
AIM OF THE STUDY: Cinobufacini (Huachansu), an aqueous extract from the skin and parotid venom glands of Bufo bufo gargarizans Cantor, is a traditional Chinese medicine widely used in clinical cancer therapy in China. The present study sought to investigate the possible signaling pathway implicated in cinobufacini-induced apoptosis in the hepatocellular carcinoma cell lines HepG(2) and Bel-7402. MATERIALS AND METHODS: The effects of cinobufacini on cell proliferation of HepG(2) and Bel-7402 cells were evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assays. Cell apoptosis was detected by Hoechst 33258 staining and flow cytometry analysis. The mitochondrial membrane potential (Deltapsim) and caspase-9 and -3 activity were detected using MitoCapture reagent staining and colorimetric assays, respectively. The expression of apoptosis-related proteins and release of cytochrome c were assessed by Western blot analysis. RESULTS: Cinobufacini significantly inhibited cell proliferation of both cell lines in a dose- and time-dependent manner. Marked changes in apoptotic morphology and apoptosis rates were clearly observed after cinobufacini treatment. The protein expression of Bax increased whereas that of Bcl-2 decreased, leading to an increase in the Bax/Bcl-2 ratio. Subsequently, cinobufacini disrupted the mitochondrial membrane potential (Deltapsim) and resulted in the release of cytochrome c, activation of both caspase-9 and -3, and cleavage of poly (ADP-ribose) polymerase (PARP). CONCLUSION: The present study indicated that cinobufacini can induce apoptosis of HepG(2) and Bel-7402 cells through a mitochondria-mediated apoptosis pathway.
Assuntos
Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Adenosina Difosfato Ribose/metabolismo , Animais , Apoptose/fisiologia , Western Blotting , Bufo bufo/metabolismo , Caspase 9/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , China , Grupo dos Citocromos c/metabolismo , Citocromos c/metabolismo , Citometria de Fluxo , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neoplasias/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Transdução de Sinais , Proteína X Associada a bcl-2/metabolismoRESUMO
AIM OF THE STUDY: Cordyceps is a parasitic fungus and has long been used as a traditional Chinese medicine to treat illnesses, promote longevity, increase athletic power, and relieve exhaustion and cancer. In this study, we reveal the mechanisms underlying apoptosis induced by Cordyceps pruinosa butanol fraction (CPBF) in the human cervical adenocarcinoma cell line, HeLa. MATERIALS AND METHODS: Proliferation and apoptosis of cells were examined by MTT assay, DNA fragmentation, phosphatidyl serine distribution assay, Western blot analysis, and immunocytochemistry. To determine the association between CPBF related apoptosis and ROS, electron spin resonance (ESR) trapping experiments were used. RESULTS: CPBF inhibited proliferation and induced apoptosis in HeLa cells in a dose-dependent manner using a MTT assay, DNA fragmentation, and a phosphatidyl serine distribution assay. Western blot analysis showed that apoptosis in HeLa cells was caspase-3- and -9-dependent. Proteolytic cleavage of PARP and the release of cytochrome c from the mitochondria into the cytosol were significantly increased and the Bcl-2/Bax protein ratio was decreased. Apoptosis induced by CPBF was not prevented by various antioxidants. CONCLUSIONS: These results indicate that apoptotic effects of CPBF on HeLa cells are mediated by mitochondria-dependent death-signaling pathway independent of reactive oxygen species, suggesting that CPBF might be effective as an anti-proliferative agent for cancer.
Assuntos
Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Caspases/metabolismo , Cordyceps/metabolismo , Extratos Vegetais/farmacologia , Grupo dos Citocromos c/metabolismo , Citocromos c/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Células HeLa , Humanos , Mitocôndrias/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína X Associada a bcl-2/metabolismoRESUMO
Anaerobic, bacterial reduction of water-soluble U(VI) complexes to the poorly soluble U(IV) mineral uraninite has been intensively studied as a strategy for in situ remediation of uranium-contaminated groundwater. A novel and potentially counteracting metabolic process, anaerobic, nitrate-dependent U(IV) oxidation, has recently been described in two bacterial species (Geobacter metallireducens and Thiobacillus denitrificans), but the underlying biochemistry and genetics are completely unknown. We report here that two diheme, c-type cytochromes (putatively c(4) and c(5) cytochromes) play a major role in nitrate-dependent U(IV) oxidation by T. denitrificans. Insertion mutations in each of the two genes encoding these cytochromes resulted in a greater than 50% decrease in U(IV) oxidation activity, and complementation in trans restored activity to wild-type levels. Sucrose-density-gradient ultracentrifugation confirmed that both cytochromes are membrane-associated. Insertion mutations in genes encoding other membrane-associated, c-type cytochromes did not diminish U(IV) oxidation. This is the first report of proteins involved in anaerobic U(IV) oxidation.
Assuntos
Grupo dos Citocromos c/metabolismo , Oxirredução , Thiobacillus/metabolismo , Urânio/metabolismo , Anaerobiose , Biodegradação Ambiental , Nitratos/metabolismo , UltracentrifugaçãoRESUMO
The antioxidant activity of C.oil in cerebral stroke has been reported earlier. We have attempted here to clarify the mechanisms underlying the neuroprotection against experimental cerebral ischemia by Curcuma oil (C.oil), isolated from the rhizomes of Curcuma longa. C.oil (250 mg/kg i.p.) was given 30 min before focal ischemia in rats caused by occlusion of the middle cerebral artery (1h of occlusion, 24h of reflow). Ischemia, leads to elevation in [Ca(2+)] this sets into motion a cascades of ischemic injury which was attenuated by C.oil. C.oil reduced post-ischemic brain neutrophil infiltration in the ischemic area, controlled tissue NOx levels and the neuronal levels of nitric oxide, peroxynitrite and reactive oxygen species when measured after 24h of reflow. Double immunofluorescence staining analysis and Western immunoblot analysis with C.oil treatment showed that the expression of nitric oxide synthase (NOS) isoforms were decreased significantly compared to the untreated ischemia group. Ischemia is associated with increased in TUNEL (TdT-mediated dUTP nick-end labeling) positive cells in brain sections indicating DNA fragmentation. The C.oil treated group showed a significant decrease in numbers of apoptotic cells compared to the untreated ischemia group, as seen in the flowcytometric analysis of the neurons. Results of immunohistochemistry and Western immunoblot indicate that C.oil suppressed the elevated protein level of Bax, and aided mitochondrial translocation and activation of Bcl-2 by altered mitochondrial membrane potential. It also inhibits the cytosolic release of apoptogenic molecules like cytochrome c, inhibits the activation of caspase-3 and the expression of p53 ultimately inhibiting apoptosis. Our observations suggest that high levels of NO generated by NOS isoforms are partially responsible for exacerbating the neuronal damage induced by MCAo by intraluminal filament.
Assuntos
Apoptose/efeitos dos fármacos , Isquemia Encefálica/tratamento farmacológico , Curcuma/química , Neurônios/efeitos dos fármacos , Óxido Nítrico/metabolismo , Óleos Voláteis/uso terapêutico , Animais , Western Blotting , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Infarto Cerebral/tratamento farmacológico , Infarto Cerebral/metabolismo , Infarto Cerebral/patologia , Grupo dos Citocromos c/metabolismo , Fragmentação do DNA , Citometria de Fluxo , Regulação da Expressão Gênica , Marcação In Situ das Extremidades Cortadas , Masculino , Mitocôndrias/enzimologia , Mitocôndrias/patologia , Neurônios/metabolismo , Neurônios/patologia , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Óleos Voláteis/química , Óleos Voláteis/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
BACKGROUND: In order to study the mechanism of U(VI) reduction, the effect of deleting c-type cytochrome genes on the capacity of Geobacter sulfurreducens to reduce U(VI) with acetate serving as the electron donor was investigated. RESULTS: The ability of several c-type cytochrome deficient mutants to reduce U(VI) was lower than that of the wild type strain. Elimination of two confirmed outer membrane cytochromes and two putative outer membrane cytochromes significantly decreased (ca. 50-60%) the ability of G. sulfurreducens to reduce U(VI). Involvement in U(VI) reduction did not appear to be a general property of outer membrane cytochromes, as elimination of two other confirmed outer membrane cytochromes, OmcB and OmcC, had very little impact on U(VI) reduction. Among the periplasmic cytochromes, only MacA, proposed to transfer electrons from the inner membrane to the periplasm, appeared to play a significant role in U(VI) reduction. A subpopulation of both wild type and U(VI) reduction-impaired cells, 24-30%, accumulated amorphous uranium in the periplasm. Comparison of uranium-accumulating cells demonstrated a similar amount of periplasmic uranium accumulation in U(VI) reduction-impaired and wild type G. sulfurreducens. Assessment of the ability of the various suspensions to reduce Fe(III) revealed no correlation between the impact of cytochrome deletion on U(VI) reduction and reduction of Fe(III) hydroxide and chelated Fe(III). CONCLUSION: This study indicates that c-type cytochromes are involved in U(VI) reduction by Geobacter sulfurreducens. The data provide new evidence for extracellular uranium reduction by G. sulfurreducens but do not rule out the possibility of periplasmic uranium reduction. Occurrence of U(VI) reduction at the cell surface is supported by the significant impact of elimination of outer membrane cytochromes on U(VI) reduction and the lack of correlation between periplasmic uranium accumulation and the capacity for uranium reduction. Periplasmic uranium accumulation may reflect the ability of uranium to penetrate the outer membrane rather than the occurrence of enzymatic U(VI) reduction. Elimination of cytochromes rarely had a similar impact on both Fe(III) and U(VI) reduction, suggesting that there are differences in the routes of electron transfer to U(VI) and Fe(III). Further studies are required to clarify the pathways leading to U(VI) reduction in G. sulfurreducens.
Assuntos
Grupo dos Citocromos c/metabolismo , Geobacter/metabolismo , Urânio/metabolismo , Biodegradação Ambiental , Grupo dos Citocromos c/genética , Compostos Férricos/química , Compostos Férricos/metabolismo , Geobacter/genética , Geobacter/ultraestrutura , Microscopia Eletrônica de Transmissão , Mutação , Oxirredução , Periplasma/metabolismo , Periplasma/ultraestrutura , Urânio/químicaRESUMO
Modern approaches for bioremediation of radionuclide contaminated environments are based on the ability of microorganisms to effectively catalyze changes in the oxidation states of metals that in turn influence their solubility. Although microbial metal reduction has been identified as an effective means for immobilizing highly-soluble uranium(VI) complexes in situ, the biomolecular mechanisms of U(VI) reduction are not well understood. Here, we show that c-type cytochromes of a dissimilatory metal-reducing bacterium, Shewanella oneidensis MR-1, are essential for the reduction of U(VI) and formation of extracellular UO(2) nanoparticles. In particular, the outer membrane (OM) decaheme cytochrome MtrC (metal reduction), previously implicated in Mn(IV) and Fe(III) reduction, directly transferred electrons to U(VI). Additionally, deletions of mtrC and/or omcA significantly affected the in vivo U(VI) reduction rate relative to wild-type MR-1. Similar to the wild-type, the mutants accumulated UO(2) nanoparticles extracellularly to high densities in association with an extracellular polymeric substance (EPS). In wild-type cells, this UO(2)-EPS matrix exhibited glycocalyx-like properties and contained multiple elements of the OM, polysaccharide, and heme-containing proteins. Using a novel combination of methods including synchrotron-based X-ray fluorescence microscopy and high-resolution immune-electron microscopy, we demonstrate a close association of the extracellular UO(2) nanoparticles with MtrC and OmcA (outer membrane cytochrome). This is the first study to our knowledge to directly localize the OM-associated cytochromes with EPS, which contains biogenic UO(2) nanoparticles. In the environment, such association of UO(2) nanoparticles with biopolymers may exert a strong influence on subsequent behavior including susceptibility to oxidation by O(2) or transport in soils and sediments.
Assuntos
Grupo dos Citocromos c/metabolismo , Shewanella/metabolismo , Compostos de Urânio/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Biodegradação Ambiental , Glicocálix/química , Ferro/metabolismo , Nanopartículas Metálicas/química , Oxirredução , Fósforo/metabolismo , Polissacarídeos Bacterianos/metabolismo , Distribuição Tecidual , Urânio/farmacocinética , Compostos de Urânio/farmacocinéticaRESUMO
"Reduced minus oxidized" difference extinction coefficients Deltavarepsilon in the alpha-bands of Cyt b559 and Cyt c550 were determined by using functionally and structurally well-characterized PS II core complexes from the thermophilic cyanobacterium Thermosynechococcus elongatus. Values of 25.1+/-1.0 mM(-1) cm(-1) and 27.0+/-1.0 mM(-1) cm(-1) were obtained for Cyt b559 and Cyt c550, respectively. Anaerobic redox titrations covering the wide range from -250 up to +450 mV revealed that the heme groups of both Cyt b559 and Cyt c550 exhibit homogenous redox properties in the sample preparation used, with E(m) values at pH 6.5 of 244+/-11 mV and -94+/-21 mV, respectively. No HP form of Cyt b559 could be detected. Experiments performed on PS II membrane fragments of higher plants where the content of the high potential form of Cyt b559 was varied by special treatments (pH, heat) have shown that the alpha-band extinction of Cyt b559 does not depend on the redox form of the heme group. Based on the results of this study the Cyt b559/PSII stoichiometry is inferred to be 1:1 not only in thermophilic cyanobacteria as known from the crystal structure but also in PSII of plants. Possible interrelationships between the structure of the Q(B) site and the microenvironment of the heme group of Cyt b559 are discussed.
Assuntos
Beta vulgaris/enzimologia , Grupo dos Citocromos b/metabolismo , Grupo dos Citocromos c/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Synechococcus/enzimologia , Anaerobiose , Grupo dos Citocromos b/química , Grupo dos Citocromos c/química , Oxirredução , Complexo de Proteína do Fotossistema II/química , Espectrofotometria/métodosRESUMO
Physalis species is a popular folk medicine used for treating cancer, leukemia, hepatitis and other diseases. Studies have shown that the ethanol extract of Physalis peruviana (EEPP) inhibits growth and induces apoptotic death of human Hep G2 cells in culture, whereas proliferation of the mouse BALB/C normal liver cells was not affected. In this study, we performed detailed studies to define the molecular mechanism of EEPP-induced apoptosis in Hep G2 cells. The results further confirmed that EEPP inhibited cell proliferation in a dose- and time-dependent manner. At 50 microg/ml, EEPP significantly increased the accumulation of the sub-G1 peak (hypoploid) and the portion of apoptotic annexin V positive cells. EEPP was found to trigger apoptosis through the release of cytochrome c, Smac/DIABLO and Omi/HtrA2 from mitochondria to cytosol and consequently resulted in caspase-3 activation. Pre-treatment with a general caspase inhibitor (z-VAD-fmk) prevented cytochrome c release. After 48 h of EEPP treatment, the apoptosis of Hep G2 cells was found to associate with an elevated p53, and CD95 and CD95L proteins expression. Furthermore, a marked down-regulation of the expression of the Bcl-2, Bcl-XL and XIAP, and up-regulation of the Bax and Bad proteins were noted. Taken together, the present results suggest that EEPP-induced Hep G2 cell apoptosis was possibly mediated through the CD95/CD95L system and the mitochondrial signaling transduction pathway.
Assuntos
Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Neoplasias Hepáticas/tratamento farmacológico , Glicoproteínas de Membrana/metabolismo , Proteínas Mitocondriais/metabolismo , Physalis/química , Receptor fas/metabolismo , Ciclo Celular/efeitos dos fármacos , Grupo dos Citocromos c/metabolismo , Proteína Ligante Fas , Humanos , Transdução de Sinais , Células Tumorais CultivadasRESUMO
Cytochrome c(3) of Desulfovibrio desulfuricans strain G20 is an electron carrier for uranium (VI) reduction. When D. desulfuricans G20 was grown in medium containing a non-lethal concentration of uranyl acetate (1 mM), the rate at which the cells reduced U(VI) was decreased compared to cells grown in the absence of uranium. Western analysis did not detect cytochrome c(3) in periplasmic extracts from cells grown in the presence of uranium. The expression of this predominant tetraheme cytochrome was not detectably altered by uranium during growth of the cells as monitored through a translational fusion of the gene encoding cytochrome c(3) ( cycA) to lacZ. Instead, cytochrome c(3) protein was found tightly associated with insoluble U(IV), uraninite, after the periplasmic contents of cells were harvested by a pH shift. The association of cytochrome c(3) with U(IV) was interpreted to be non-specific, since pure cytochrome c(3) adsorbed to other insoluble metal oxides, including cupric oxide (CuO), ferric oxide (Fe(2)O(3)), and commercially available U(IV) oxide.
Assuntos
Grupo dos Citocromos c/metabolismo , Desulfovibrio desulfuricans/metabolismo , Urânio/metabolismo , Adsorção , Fusão Gênica Artificial , Cobre/química , Grupo dos Citocromos c/genética , Compostos Férricos/química , Regulação Bacteriana da Expressão Gênica , Genes Reporter , Óperon Lac/genética , Óperon Lac/fisiologia , Oxirredução , Periplasma/química , Proteínas Periplásmicas/análise , RNA Bacteriano/análise , RNA Mensageiro/análise , Transcrição Gênica , Compostos de Urânio/química , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
Kinetic parameters and the role of cytochrome c(3) in sulfate, Fe(III), and U(VI) reduction were investigated in Desulfovibrio vulgaris Hildenborough. While sulfate reduction followed Michaelis-Menten kinetics (K(m) = 220 micro M), loss of Fe(III) and U(VI) was first-order at all concentrations tested. Initial reduction rates of all electron acceptors were similar for cells grown with H(2) and sulfate, while cultures grown using lactate and sulfate had similar rates of metal loss but lower sulfate reduction activities. The similarities in metal, but not sulfate, reduction with H(2) and lactate suggest divergent pathways. Respiration assays and reduced minus oxidized spectra were carried out to determine c-type cytochrome involvement in electron acceptor reduction. c-type cytochrome oxidation was immediate with Fe(III) and U(VI) in the presence of H(2), lactate, or pyruvate. Sulfidogenesis occurred with all three electron donors and effectively oxidized the c-type cytochrome in lactate- or pyruvate-reduced, but not H(2)-reduced cells. Correspondingly, electron acceptor competition assays with lactate or pyruvate as electron donors showed that Fe(III) inhibited U(VI) reduction, and U(VI) inhibited sulfate loss. However, sulfate reduction was slowed but not halted when H(2) was the electron donor in the presence of Fe(III) or U(VI). U(VI) loss was still impeded by Fe(III) when H(2) was used. Hence, we propose a modified pathway for the reduction of sulfate, Fe(III), and U(VI) which helps explain why these bacteria cannot grow using these metals. We further propose that cytochrome c(3) is an electron carrier involved in lactate and pyruvate oxidation and is the reductase for alternate electron acceptors with higher redox potentials than sulfate.