Nano-grids of Yeast Cytochrome C

One innovative thought in biomolecular electronics is the exploitation of electron transfer proteins. Using nature's self assembly techniques, proteins can build highly organized edifices with retained functional activity, and they can serve as platforms for biosensors. In this research work, Yeast Cytochrome C (YCC) is immobilized with a help of a linker molecule, 3-Mercaptopropyltrimethoxysilane (3-MPTS) on a hydroxylated surface of a silicon substrate. Atomic Force Microscopy (AFM) is used for characterization. AFM data shows immobilization of one YCC molecule in between eight grids that are formed by the linker molecules. 3-MPTS monolayers are organized in grids that are 1.2 nm apart. Immobilization of 3-MPTS was optimized using a concentration of 5 mM in a completely dehydrated state for 30 minutes. The functionally active grids of YCC can now be incorporated with Cytochrome C oxidase on a Platinum electrode surface for transfer of electrons in development of biosensors, such as nitrate sensor, that are small in size, cheaper, and easier to manufacture than the top-down approach of fabrication of molecular biodevices

Influence de l'inflammation sur le métabolisme catalysé par les cytochromes P450 chez le rat : approches in vitro et implications sur la pharmacocinétique d'agonistes delta

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Rôle du système endocrinien de la vitamine D³ sur l'induction de cytochromes P450 hépatiques impliquées dans la détoxication-cytoprotection

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Influence de l'inflammation sur le métabolisme catalysé par les cytochromes P450 chez le rat : approches in vitro et implications sur la pharmacocinétique d'agonistes delta

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Rôle du système endocrinien de la vitamine D³ sur l'induction de cytochromes P450 hépatiques impliquées dans la détoxication-cytoprotection

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Enhancement of the Cytotoxic Effect of Anticancer Agent by Cytochrome c Functionalised Hybrid Nanoparticles in Hepatocellular Cancer Cells

Treatment of hepatocellular cancer with chemotherapeutic agents has limited successin clinical practice and their efficient IC50 concentration would require extremely highdoses of drug administration which could not be tolerated due to systemic side effects.In order to potentiate the efficacy of anticancer agents we explored the potentialof co-treatment with pro-apoptotic Cytochrome c which activates the apoptoticpathway downstream of p53 that is frequently mutated in cancer. To this end weused hybrid iron oxide-gold nanoparticles as a drug delivery system to facilitate theinternalisation of Cytochrome c into cultured HepG2 hepatocellular carcinoma cells.Our results showed that Cytochrome c can be easily conjugated to the gold shell ofthe nanoparticles which are readily taken up by the cells. We used Cytochrome cin concentration (0.2μgmL-1) below the threshold required to induce apoptosis onits own. When the conjugate was administered to cells treated by doxorubicin, itsignificantly reduced its IC50 concentration from 9μgmL-1 to 3.5μgmL-1 as detectedby cell viability assay, and the efficiency of doxorubicin on decreasing viability ofHepG2 cells was significantly enhanced in the lower concentration range between0.01μgmL-1 to 5μgmL-1. The results demonstrate the potential of the application oftherapeutic proteins in activating the apoptotic pathway to complement conventionalchemotherapy to increase its efficacy. The application of hybrid iron oxide-goldnanoparticles can also augment the specificity of drug targeting and could serve as amodel drug delivery system for pro-apoptotic protein targeting and delivery.

Niveles de expresión de citocromos P450 (CYP6AA7, CYP4C52v1, CYP6BY3, CYP9J34, CYP9M10, CYP9J40 y CYP9AL1) determinados por RT-qPCR en larvas de Culex quinquefasciatus expuestas a diversos insecticidas.

En el presente trabajo, se describen los niveles de expresión de siete transcritos de Culex quinquefasciatus que codifican para citocromos P450, estos medidos por qPCR en larvas expuestas a dos concentraciones del insecticida permetrina (0.51 y 0.71 mg) comparadas con un grupo control sin exponer, las cuales fueron colectadas en distintas localidades del noreste de México. Debido a que los genes analizados no se encontraban reportados, fue necesario diseñar oligonucleótidos utilizando secuencias trazas producto de la secuenciación del genoma del mosquito antes mencionado. Con los oligonucleótidos diseñados y utilizando DNA complementario, sintetizado a partir de RNA total de las larvas fueron amplificados por PCR los mensajeros, se procedió a clonar cada uno de estos genes en nueve poblaciones analizadas, posteriormente fueron secuenciados y éstas fueron comparadas para deducir los porcentajes de similitud. Se diseñaron sondas taqman en segmentos conservados, y utilizando el DNA complementario previamente sintetizado fueron deducidos los niveles de expresión de cada uno de los genes analizados bajo las condiciones citadas anteriormente para las poblaciones. Con el trabajo realizado, reportamos las secuencias nucleótidicos y aminoacídicas de cada uno de los genes analizados de cada una de las poblaciones estudiadas y sus niveles de expresión. Concluimos que los genes analizados son una herramienta potente para ahondar en el estatus de resistencia que han desarrollado las poblaciones silvestres de Culex quinquefasciatus en localidades del noreste de México.

SERR spectroelectrochemical study of cytochrome cd1 nitrite reductase co-immobilized with physiological redox partner cytochrome c552 on biocompatible metal electrodes

© 2015 Silveira et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

c-Type cytochrome biogenesis can occur via a natural Ccm system lacking CcmH, CcmG, and the heme-binding histidine of CcmE

The Ccm cytochrome c maturation System I catalyzes covalent attachment of heme to apocytochromes c in many bacterial species and some mitochondria. A covalent, but transient, bond between heme and a conserved histidine in CcmE along with an interaction between CcmH and the apocytochrome have been previously indicated as core aspects of the Ccm system. Here, we show that in the Ccm system from Desulfovibrio desulfuricans, no CcmH is required, and the holo-CcmE covalent bond occurs via a cysteine residue. These observations call for reconsideration of the accepted models of System I-mediated c-type cytochrome biogenesis. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

Fatty Acid Synthase Inhibitors Induce Apoptosis In Non-tumorigenic Melan-a Cells Associated With Inhibition Of Mitochondrial Respiration.

The metabolic enzyme fatty acid synthase (FASN) is responsible for the endogenous synthesis of palmitate, a saturated long-chain fatty acid. In contrast to most normal tissues, a variety of human cancers overexpress FASN. One such cancer is cutaneous melanoma, in which the level of FASN expression is associated with tumor invasion and poor prognosis. We previously reported that two FASN inhibitors, cerulenin and orlistat, induce apoptosis in B16-F10 mouse melanoma cells via the intrinsic apoptosis pathway. Here, we investigated the effects of these inhibitors on non-tumorigenic melan-a cells. Cerulenin and orlistat treatments were found to induce apoptosis and decrease cell proliferation, in addition to inducing the release of mitochondrial cytochrome c and activating caspases-9 and -3. Transfection with FASN siRNA did not result in apoptosis. Mass spectrometry analysis demonstrated that treatment with the FASN inhibitors did not alter either the mitochondrial free fatty acid content or composition. This result suggests that cerulenin- and orlistat-induced apoptosis events are independent of FASN inhibition. Analysis of the energy-linked functions of melan-a mitochondria demonstrated the inhibition of respiration, followed by a significant decrease in mitochondrial membrane potential (ΔΨm) and the stimulation of superoxide anion generation. The inhibition of NADH-linked substrate oxidation was approximately 40% and 61% for cerulenin and orlistat treatments, respectively, and the inhibition of succinate oxidation was approximately 46% and 52%, respectively. In contrast, no significant inhibition occurred when respiration was supported by the complex IV substrate N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD). The protection conferred by the free radical scavenger N-acetyl-cysteine indicates that the FASN inhibitors induced apoptosis through an oxidative stress-associated mechanism. In combination, the present results demonstrate that cerulenin and orlistat induce apoptosis in non-tumorigenic cells via mitochondrial dysfunction, independent of FASN inhibition.

Multitarget Effects Of Quercetin In Leukemia.

This study proposes to investigate quercetin antitumor efficacy in vitro and in vivo, using the P39 cell line as a model. The experimental design comprised leukemic cells or xenografts of P39 cells, treated in vitro or in vivo, respectively, with quercetin; apoptosis, cell-cycle and autophagy activation were then evaluated. Quercetin caused pronounced apoptosis in P39 leukemia cells, followed by Bcl-2, Bcl-xL, Mcl-1 downregulation, Bax upregulation, and mitochondrial translocation, triggering cytochrome c release and caspases activation. Quercetin also induced the expression of FasL protein. Furthermore, our results demonstrated an antioxidant activity of quercetin. Quercetin treatment resulted in an increased cell arrest in G1 phase of the cell cycle, with pronounced decrease in CDK2, CDK6, cyclin D, cyclin E, and cyclin A proteins, decreased Rb phosphorylation and increased p21 and p27 expression. Quercetin induced autophagosome formation in the P39 cell line. Autophagy inhibition induced by quercetin with chloroquine triggered apoptosis but did not alter quercetin modulation in the G1 phase. P39 cell treatment with a combination of quercetin and selective inhibitors of ERK1/2 and/or JNK (PD184352 or SP600125, respectively), significantly decreased cells in G1 phase, this treatment, however, did not change the apoptotic cell number. Furthermore, in vivo administration of quercetin significantly reduced tumor volume in P39 xenografts and confirmed in vitro results regarding apoptosis, autophagy, and cell-cycle arrest. The antitumor activity of quercetin both in vitro and in vivo revealed in this study, point to quercetin as an attractive antitumor agent for hematologic malignancies.

Improvement Of The Physical Performance Is Associated With Activation Of No/pgc-1α/mttfa Signaling Pathway And Increased Protein Expressions Of Electron Transport Chain In Gastrocnemius Muscle From Rats Supplemented With L-arginine.

To examine the influence of l-arginine supplementation in combination with physical training on mitochondrial biomarkers from gastrocnemius muscle and its relationship with physical performance. Male Wistar rats were divided into four groups: control sedentary (SD), sedentary supplemented with l-arginine (SDLA), trained (TR) and trained supplemented with l-arginine (TRLA). Supplementation of l-arginine was administered by gavage (62.5mg/ml/day/rat). Physical training consisted of 60min/day, 5days/week, 0% grade, speed of 1.2km/h. The study lasted 8weeks. Skeletal muscle mitochondrial enriched fraction as well as cytoplasmic fractions were obtained for Western blotting and biochemical analyses. Protein expressions of transcriptor coactivator (PGC-1α), transcriptor factors (mtTFA), ATP synthase subunit c, cytochrome oxidase (COXIV), constitutive nitric oxide synthases (eNOS and nNOS), Cu/Zn-superoxide dismutase (SOD) and manganese-SOD (Mn-SOD) were evaluated. We also assessed in plasma: lipid profile, glycemia and malondialdehyde (MDA) levels. The nitrite/nitrate (NOx(-)) levels were measured in both plasma and cytosol fraction of the gastrocnemius muscle. 8-week l-arginine supplementation associated with physical training was effective in promoting greater tolerance to exercise that was accompanied by up-regulation of the protein expressions of mtTFA, PGC-1α, ATP synthase subunit c, COXIV, Cu/Zn-SOD and Mn-SOD. The upstream pathway was associated with improvement of NO bioavailability, but not in NO production since no changes in nNOS or eNOS protein expressions were observed. This combination would be an alternative approach for preventing cardiometabolic diseases given that in overt diseases a profound impairment in the physical performance of the patients is observed.

Nek5 Interacts With Mitochondrial Proteins And Interferes Negatively In Mitochondrial Mediated Cell Death And Respiration.

Mitochondria are involved in energy supply, signaling, cell death and cellular differentiation and have been implicated in several human diseases. Neks (NIMA-related kinases) represent a family of mammal protein kinases that play essential roles in cell-cycle progression, but other functions have recently been related. A yeast two-hybrid (Y2H) screen was performed to identify and characterize Nek5 interaction partners and the mitochondrial proteins Cox11, MTX-2 and BCLAF1 were retrieved. Apoptosis assay showed protective effects of stable hNek5 expression from Hek293-T's cell death after thapsigargin treatment (2μM). Nek5 silenced cells as well as cells expressing a kinase dead version of Nek5, displayed an increase in ROS formation after 4h of thapsigargin treatment. Mitochondrial respiratory chain activity was found decreased upon stable hNek5expression. Cells silenced for hNek5 on the other hand presented 1.7 fold increased basal rates of respiration, especially at the electrons transfer steps from TMPD to cytochrome c and at the complex II. In conclusion, our data suggest for the first time mitochondrial localization and functions for Nek5 and its participation in cell death and cell respiration regulation. Stable expression of hNek5 in Hek293T cells resulted in enhanced cell viability, decreased cell death and drug resistance, while depletion of hNek5by shRNA overcame cancer cell drug resistance and induced apoptosis in vitro. Stable expression of hNek5 also inhibits thapsigargin promoted apoptosis and the respiratory chain complex IV in HEK293T cells.

Thaptomys Thomas 1915 (Rodentia, Sigmodontinae, Akodontini) with karyotypes 2n = 50, FN = 48, and 2n = 52, FN = 52: two monophyletic lineages recovered by molecular phylogeny

A novel karyotype with 2n = 50, FN = 48, was described for specimens of Thaptomys collected at Una, State of Bahia, Brazil, which are morphologically indistinguishable from Thaptomys nigrita, 2n = 52, FN = 52, found in other localities. It was hence proposed that the 2n = 50 karyotype could belong to a distinct species, cryptic of Thaptomys nigrita, once chromosomal rearrangements observed, along with the geographic distance, might represent a reproductive barrier between both forms. Phylogenetic analyses using maximum parsimony and maximum likelihood based on partial cytochrome b sequences with 1077 bp were performed, attempting to establish the relationships among the individuals with distinct karyotypes along the geographic distribution of the genus; the sample comprised 18 karyotyped specimens of Thaptomys, encompassing 15 haplotypes, from eight different localities of the Atlantic Rainforest. The intra-generic relationships corroborated the distinct diploid numbers, once both phylogenetic reconstructions recovered two monophyletic lineages, a northeastern clade grouping the 2n = 50 and a southeastern clade with three subclades, grouping the 2n = 52 karyotype. The sequence divergence observed between their individuals ranged from 1.9% to 3.5%.