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1
Allen, John F. "Why chloroplasts and mitochondria retain their own genomes and genetic systems: Colocation for redox regulation of gene expression." Proceedings of the National Academy of Sciences 112, no. 33 (May 18, 2015): 10231–38. http://dx.doi.org/10.1073/pnas.1500012112.
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Chloroplasts and mitochondria are subcellular bioenergetic organelles with their own genomes and genetic systems. DNA replication and transmission to daughter organelles produces cytoplasmic inheritance of characters associated with primary events in photosynthesis and respiration. The prokaryotic ancestors of chloroplasts and mitochondria were endosymbionts whose genes became copied to the genomes of their cellular hosts. These copies gave rise to nuclear chromosomal genes that encode cytosolic proteins and precursor proteins that are synthesized in the cytosol for import into the organelle into which the endosymbiont evolved. What accounts for the retention of genes for the complete synthesis within chloroplasts and mitochondria of a tiny minority of their protein subunits? One hypothesis is that expression of genes for protein subunits of energy-transducing enzymes must respond to physical environmental change by means of a direct and unconditional regulatory control—control exerted by change in the redox state of the corresponding gene product. This hypothesis proposes that, to preserve function, an entire redox regulatory system has to be retained within its original membrane-bound compartment. Colocation of gene and gene product for redox regulation of gene expression (CoRR) is a hypothesis in agreement with the results of a variety of experiments designed to test it and which seem to have no other satisfactory explanation. Here, I review evidence relating to CoRR and discuss its development, conclusions, and implications. This overview also identifies predictions concerning the results of experiments that may yet prove the hypothesis to be incorrect.
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McTernan, Patrick M., Paige S. Katz, Constance Porretta, David A. Welsh, and Robert W. Siggins. "A Novel FACS-Based Workflow for Simultaneous Assessment of RedOx Status, Cellular Phenotype, and Mitochondrial Genome Stability." BioChem 1, no. 1 (April 2, 2021): 1–18. http://dx.doi.org/10.3390/biochem1010001.
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Intracellular reduction-oxidation (RedOx) status mediates a myriad of critical biological processes. Importantly, RedOx status regulates the differentiation of hematopoietic stem and progenitor cells (HSPCs), mesenchymal stromal cells (MSCs) and maturation of CD8+ T Lymphocytes. In most cells, mitochondria are the greatest contributors of intracellular reactive oxygen species (ROS). Excess ROS leads to mitochondrial DNA (mtDNA) damage and protein depletion. We have developed a fluorescence-activated cell sorting (FACS)-based protocol to simultaneously analyze RedOx status and mtDNA integrity. This simultaneous analysis includes measurements of ROS (reduced glutathione (GSH)), ATP5H (nuclear encoded protein), MTCO1 (mitochondrial DNA encoded protein), and cell surface markers to allow discrimination of different cell populations. Using the ratio of MTCO1 to ATP5H median fluorescence intensity (MFI), we can gain an understanding of mtDNA genomic stability, since MTCO1 levels are decreased when mtDNA becomes significantly damaged. Furthermore, this workflow can be optimized for sorting cells, using any of the above parameters, allowing for downstream quantification of mtDNA genome copies/nucleus by quantitative PCR (qPCR). This unique methodology can be used to enhance analyses of the impacts of pharmacological interventions, as well as physiological and pathophysiological processes on RedOx status along with mitochondrial dynamics in most cell types.
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Spezia, Pietro Giorgio, Andreina Baj, Francesca Drago Ferrante, Sara Boutahar, Lorenzo Azzi, Angelo Genoni, Daniela Dalla Gasperina, et al. "Detection of Torquetenovirus and Redondovirus DNA in Saliva Samples from SARS-CoV-2-Positive and -Negative Subjects." Viruses 14, no. 11 (November 9, 2022): 2482. http://dx.doi.org/10.3390/v14112482.
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Objectives: Torquetenovirus (TTV) and Redondovirus (ReDoV) are the most prevalent viruses found in the human respiratory virome in viral metagenomics studies. A large-scale epidemiological study was performed to investigate their prevalence and loads in saliva samples according to SARS-CoV-2 status. Methods: Saliva samples from 448 individuals (73% SARS-CoV-2 negative and 27% SARS-CoV-2 positive) aged 23–88 years were tested. SARS-CoV-2 and TTV were determined in saliva by specific qualitative and quantitative real-time PCRs, respectively. A sub-cohort of 377 subjects was additionally tested for the presence and load of ReDoV in saliva, and a different sub-cohort of 120 subjects for which paired saliva and plasma samples were available was tested for TTV and ReDoV viremia at the same timepoints as saliva. Results: TTV in saliva was 72% prevalent in the entire cohort, at a mean DNA load of 4.6 log copies/mL, with no difference regardless of SARS-CoV-2 status. ReDoV was found in saliva from 61% of the entire cohort and was more prevalent in the SARS-CoV-2-negative subgroup (65% vs. 52%, respectively). In saliva, the total mean load of ReDoV was very similar to the one of TTV, with a value of 4.4 log copies/mL. The mean viral loads in subjects infected with a single virus, namely, those infected with TTV or ReDoV alone, was lower than in dually infected samples, and Tukey’s multiple-comparison test showed that ReDoV single-infected samples resulted in the only true outlier (p = 0.004). Differently from TTV, ReDoV was not detected in any blood samples. Conclusions: This study establishes the prevalence and mean value of TTV and ReDoV in saliva samples and demonstrates the existence of differences between these two components of the human virome.
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Carlisle, Ricarda, Carol Ann Rhoads, Tak Yee Aw, and Lynn Harrison. "Endothelial cells maintain a reduced redox environment even as mitochondrial function declines." American Journal of Physiology-Cell Physiology 283, no. 6 (December 1, 2002): C1675—C1686. http://dx.doi.org/10.1152/ajpcell.00092.2002.
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Human umbilical vein endothelial cells (HUVECs) are an endothelial model of replicative senescence. Oxidative stress, possibly due to dysfunctional mitochondria, is believed to play a key role in replicative senescence and atherosclerosis, an age-related vascular disease. In this study, we determined the effect of cell division on genomic instability, mitochondrial function, and redox status in HUVECs that were able to replicate for ∼60 cumulative population doublings (CPD). After 20 CPD, the nuclear genome deteriorated and the protein content of the cell population increased. This indicated an increase in cell size, which was accompanied by an increase in oxygen consumption, ATP production, and mitochondrial genome copy number and ∼10% increase in mitochondrial mass. The antioxidant capacity increased, as seen by an increase in reduced glutathione, glutathione peroxidase, GSSG reductase, and glucose-6-phosphate dehydrogenase. However, by CPD 52, the latter two enzymes decreased, as well as the ratio of mitochondrial-to-nuclear genome copies, the mitochondrial mass, and the oxygen consumption per milligram of protein. Our results signify that HUVECs maintain a highly reducing (GSH) environment as they replicate despite genomic instability and loss of mitochondrial function.
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Kim, Yeo, Lim, Song, Chun, and Kim. "APEX1 Expression as a Potential Diagnostic Biomarker of Clear Cell Renal Cell Carcinoma and Hepatobiliary Carcinomas." Journal of Clinical Medicine 8, no. 8 (August 1, 2019): 1151. http://dx.doi.org/10.3390/jcm8081151.
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Apurinic/apyrimidinic endonuclease 1/redox effector factor 1 (APEX1) has been known to play key roles in DNA repair, the regulation of diverse transcriptional activity, and cellular responses to redox activity. This study aimed to examine serum APEX1 (s-APEX1) expression as a possible screening biomarker for clear cell renal cell carcinoma (ccRCC), hepatocellular carcinoma (HCC), and proximal and distal cholangiocarcinoma (CC). A total of 216 frozen serum samples were collected from 39 healthy control cases, 32 patients with ≥58 copies/mL of hepatitis B viral DNA (HBV DNA (+)), 40 ccRCC cases, 59 HCC cases, and 46 CC cases. The serum samples were examined for s-APEX1 concentration by enzyme-linked immunosorbent assay. The association of APEX1 expression with clinicopathological characteristics was also studied by immunohistochemical staining in 106 ccRCC, 131 HCC, and 32 intrahepatic CC cases. The median s-APEX1 concentrations of the HCC, CC, ccRCC, healthy control, and HBV DNA (+) groups were 0.294, 0.710, 0.474, 0.038, and 2.384 ng/mL, respectively (p < 0.001). Univariate and multivariate analyses revealed that increased cytoplasmic APEX1 expression led to a shorter disease-free survival period in HCC and CC cases. We suggest that the s-APEX1 level could be a potential diagnostic biomarker of ccRCC, HCC, and CC. Additionally, cytoplasmic APEX1 expression in cancer cells could be used to predict relapses in patients with HCC or CC.
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Lin, Xueju, David Kennedy, Aaron Peacock, James McKinley, Charles T. Resch, James Fredrickson, and Allan Konopka. "Distribution of Microbial Biomass and Potential for Anaerobic Respiration in Hanford Site 300 Area Subsurface Sediment." Applied and Environmental Microbiology 78, no. 3 (December 2, 2011): 759–67. http://dx.doi.org/10.1128/aem.07404-11.
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ABSTRACTSubsurface sediments were recovered from a 52-m-deep borehole cored in the 300 Area of the Hanford Site in southeastern Washington State to assess the potential for biogeochemical transformation of radionuclide contaminants. Microbial analyses were made on 17 sediment samples traversing multiple geological units: the oxic coarse-grained Hanford formation (9 to 17.4 m), the oxic fine-grained upper Ringold formation (17.7 to 18.1 m), and the reduced Ringold formation (18.3 to 52 m). Microbial biomass (measured as phospholipid fatty acids) ranged from 7 to 974 pmols per g in discrete samples, with the highest numbers found in the Hanford formation. On average, strata below 17.4 m had 13-fold less biomass than those from shallower strata. ThenosZgene that encodes nitrous oxide reductase (measured by quantitative real-time PCR) had an abundance of 5 to 17 relative to that of total 16S rRNA genes below 18.3 m and <5 above 18.1 m. MostnosZsequences were affiliated withOchrobactrum anthropi(97 sequence similarity) or had a nearest neighbor ofAchromobacter xylosoxidans(90 similarity). Passive multilevel sampling of groundwater geochemistry demonstrated a redox gradient in the 1.5-m region between the Hanford-Ringold formation contact and the Ringold oxic-anoxic interface. Within this zone, copies of thedsrAgene andGeobacteraceaehad the highest relative abundance. The majority ofdsrAgenes detected near the interface were related toDesulfotomaculumspp. These analyses indicate that the region just below the contact between the Hanford and Ringold formations is a zone of active biogeochemical redox cycling.
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Jun, Daniel, Adrian Grzedowski, J. Thomas Beatty, and Dan Bizzotto. "Photosynthetic Reaction Centres Assembled on a Gold Electrode and the Photocurrent - Potential Response." ECS Meeting Abstracts MA2022-01, no. 45 (July 7, 2022): 1892. http://dx.doi.org/10.1149/ma2022-01451892mtgabs.
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The photosynthetic reaction centre (RC) from Rhodobacter sphaeroides has been studied for use in biohybrid solar cells. Much of the previous work has focussed on improving photocurrent generation by loading the electrode surface with many copies of the protein resulting in multilayers. The primary disadvantage with this approach is the random orientation of proteins, with some supposedly oriented properly. We used RCs with Cys for covalent attachment to a gold electrode and for proper orientation. Areas of bare electrode surface and RCs bound non-specifically (i.e. not bound via the Cys) were competitively displaced by an insulating, non-redox layer of mercaptohexanol (MCH). The adsorbed monolayer of RCs was imaged using atomic force microscopy to detail the distribution of RCs on the gold surface for surfaces prepared with different RC deposition concentrations. Photocurrents were measured for all RC modified surfaces using a LED modulation method which enabled measurement of photocurrent in the presence of large faradaic currents from the sacrificial reactant (hydroquinone) at a variety of applied potentials.[1] The photocurrents generated from a monolayer composed of RCs and MCH resulted in consistent photocurrent currents. which enabled modeling of the photocurrent generation using the Marcus-Hush-Chidsey theory to extract a reorganization energy for this process. Multilayers of adsorbed RCs were distinctly different and revealed that the local environment in which the RCs are embedded significantly influenced photocurrent generation. [1] Jun, D.; Beatty, J. T.; Bizzotto, D. Highly Sensitive Method to Isolate Photocurrent Signals From Large Background Redox Currents on Protein‐Modified Electrodes. ChemElectroChem 2019, 6 (11), 2870–2875.
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Huang, S. T., S. S. Tzean, B. Y. Tsai, and H. J. Hsieh. "Cloning and heterologous expression of a novel ligninolytic peroxidase gene from poroid brown-rot fungus Antrodia cinnamomea." Microbiology 155, no. 2 (February 1, 2009): 424–33. http://dx.doi.org/10.1099/mic.0.022459-0.
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A novel ligninolytic peroxidase gene (ACLnP) was cloned and characterized from a poroid brown-rot fungus, Antrodia cinnamomea. The genomic DNA of the fungus harboured two copies of ACLnP, with a length of 2111 bp, interlaced with 12 introns, while the full-length cDNA was 1183 bp, with a 66 bp signal peptide and an ORF of 990 bp. The three-dimensional molecular structure model was comparable to that of the versatile peroxidase of Pleurotus eryngii. ACLnP was cloned into vector pQE31, successfully expressed in Escherichia coli strain M15 under the control of the T5 promoter and produced a non-glycosylated protein of about 38 kDa, pI 5.42. The native and recombinant ACLnP was capable of oxidizing the redox mediator veratryl alcohol, and also decolorized bromophenol blue and 2,6-dimethoxyphenol dyes, implicating a functional extracellular peroxidase activity. The significance of discovering a functional ACLnP gene in A. cinnamomea in terms of wood degradation and colonization capacity in its unique niche is discussed.
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Cramm, R., A. Büsch, and K. Strube. "NO-dependent transcriptional activation of gene expression in Ralstonia eutropha H16." Biochemical Society Transactions 34, no. 1 (January 20, 2006): 182–84. http://dx.doi.org/10.1042/bst0340182.
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The σ54-dependent transcriptional regulator NorR of Ralstonia eutropha H16 activates gene expression in response to nitric oxide (NO). The N-terminal domain of NorR is thought to be involved in signal perception. A C112S exchange within this domain abolished promoter activation by the mutated protein, indicating that Cys112 is essential for the signalling mechanism of NorR. The DNA region recognized by NorR contains three copies of a conserved element termed the NorR-box. Alteration of bases within any of the NorR-boxes resulted in a significant decrease in promoter activation. Therefore all three boxes have to be recognized by NorR to activate its target promoter. NorR controls expression of an operon that encodes a redox-active non-haem-iron protein NorA and an NO reductase NorB. NorA exerts a negative effect on signal-dependent promoter activation by NorR. Optical spectroscopy of purified NorA indicates that the reduced protein can react with NO to form a ferrous nitrosyl adduct. Hence, NO binding by NorA opens up the possibility that NorA and NorR compete for NO in the cytoplasm.
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Allen, John F. "Why Chloroplasts and Mitochondria Contain Genomes." Comparative and Functional Genomics 4, no. 1 (2003): 31–36. http://dx.doi.org/10.1002/cfg.245.
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Chloroplasts and mitochondria originated as bacterial symbionts. The larger, host cells acquired genetic information from their prokaryotic guests by lateral gene transfer. The prokaryotically-derived genes of the eukaryotic cell nucleus now function to encode the great majority of chloroplast and mitochondrial proteins, as well as many proteins of the nucleus and cytosol. Genes are copied and moved between cellular compartments with relative ease, and there is no established obstacle to successful import of any protein precursor from the cytosol. Yet chloroplasts and mitochondria have not abdicated all genes and gene expression to the nucleus and to cytosolic translation. What, then, do chloroplast- and mitochondrially-encoded proteins have in common that confers a selective advantage on the cytoplasmic location of their genes? The proposal advanced here is that co-location of chloroplast and mitochondrial genes with their gene products is required for rapid and direct regulatory coupling. Redox control of gene expression is suggested as the common feature of those chloroplast and mitochondrial proteins that are encodedin situ. Recent evidence is consistent with this hypothesis, and its underlying assumptions and predictions are described.
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Heathcote, Peter, Michael R. Jones, and Paul K. Fyfe. "Type I photosynthetic reaction centres: structure and function." Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences 358, no. 1429 (January 29, 2003): 231–43. http://dx.doi.org/10.1098/rstb.2002.1178.
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We review recent advances in the study of the photosystem I reaction centre, following the determination of a spectacular 2.5 Å resolution crystal structure for this complex of Synechococcus elongatus . Photosystem I is proving different to type II reaction centres in structure and organization, and the mechanism of transmembrane electron transfer, and is providing insights into the control of function in reaction centres that operate at very low redox potentials. The photosystem I complex of oxygenic organisms has a counterpart in non–oxygenic bacteria, the strictly anaerobic phototrophic green sulphur bacteria and heliobacteria. The most distinctive feature of these type I reaction centres is that they contain two copies of a large core polypeptide (i.e. a homodimer), rather than a heterodimeric arrangement of two related, but different, polypeptides as in the photosystem I complex. To compare the structural organization of the two forms of type I reaction centre, we have modelled the structure of the central region of the reaction centre from green sulphur bacteria, using sequence alignments and the structural coordinates of the S. elongatus Photosystem I complex. The outcome of these modelling studies is described, concentrating on regions of the type I reaction centre where important structure–function relationships have been demonstrated or inferred.
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Allen, John F. "The function of genomes in bioenergetic organelles." Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences 358, no. 1429 (January 29, 2003): 19–38. http://dx.doi.org/10.1098/rstb.2002.1191.
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Mitochondria and chloroplasts are energy-transducing organelles of the cytoplasm of eukaryotic cells. They originated as bacterial symbionts whose host cells acquired respiration from the precursor of the mitochondrion, and oxygenic photosynthesis from the precursor of the chloroplast. The host cells also acquired genetic information from their symbionts, eventually incorporating much of it into their own genomes. Genes of the eukaryotic cell nucleus now encode most mitochondrial and chloroplast proteins. Genes are copied and moved between cellular compartments with relative ease, and there is no obvious obstacle to successful import of any protein precursor from the cytosol. So why are any genes at all retained in cytoplasmic organelles? One proposal is that these small but functional genomes provide a location for genes that is close to, and in the same compartment as, their gene products. This co-location facilitates rapid and direct regulatory coupling. Redox control of synthesis de novo is put forward as the common property of those proteins that must be encoded and synthesized within mitochondria and chloroplasts. This testable hypothesis is termed CORR, for co -location for r edox r egulation. Principles, predictions and consequences of CORR are examined in the context of competing hypotheses and current evidence.
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Ma, Ke, Ralf Conrad, and Yahai Lu. "Responses of MethanogenmcrAGenes and Their Transcripts to an Alternate Dry/Wet Cycle of Paddy Field Soil." Applied and Environmental Microbiology 78, no. 2 (November 18, 2011): 445–54. http://dx.doi.org/10.1128/aem.06934-11.
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ABSTRACTIntermittent drainage can substantially reduce methane emission from rice fields, but the microbial mechanisms remain poorly understood. In the present study, we determined the rates of methane production and emission, the dynamics of ferric iron and sulfate, and the abundance of methanogenmcrAgenes (encoding the alpha subunit of methyl coenzyme M reductase) and their transcripts in response to alternate dry/wet cycles in paddy field soil. We found that intermittent drainage did not affect the growth of rice plants but significantly reduced the rates of both methane production and emission. The dry/wet cycles also resulted in shifts of soil redox conditions, increasing the concentrations of ferric iron and sulfate in the soil. Quantitative PCR analysis revealed that bothmcrAgene copies andmcrAtranscripts significantly decreased after dry/wet alternation compared to continuous flooding. Correlation and regression analyses showed that the abundance ofmcrAgenes and transcripts positively correlated with methane production potential and soil water content and negatively correlated with the concentrations of ferric iron and sulfate in the soil. However, the transcription ofmcrAgenes was reduced to a greater extent than the abundance ofmcrAgenes, resulting in very lowmcrAtranscript/gene ratios after intermittent drainage. Furthermore, terminal restriction fragment length polymorphism analysis revealed that the composition of methanogenic community remained stable under dry/wet cycles, whereas that of metabolically active methanogens strongly changed. Collectively, our study demonstrated a stronger effect of intermittent drainage on the abundance ofmcrAtranscripts than ofmcrAgenes in rice field soil.
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Elizareva, E. N., A. A. Fakhertdinova, A. N. Elizaryev, D. A. Tarakanov, and N. V. Kudashkina. "Geoecological Assessment of Phytoremediation Features When Using Fallopia Sachalinensis in Conditions of Anthropogenic Heavy Metal Pollution of Soils of the Bashkir Trans-Urals." Bulletin of Irkutsk State University. Series Earth Sciences 42 (2022): 41–57. http://dx.doi.org/10.26516/2073-3402.2022.42.41.
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The relevance of this work is related to solving the problem of soil pollution with heavy metals by applying Fallopia Sachalinensis and obtaining benefits in the recovery process. The aim of the work is to develop an algorithm for geoecological assessment of phytoremediation features when using plants, taking into account the conditions of anthropogenic soil pollution with heavy metals, as well as physiological characteristics and species-specific reactions under abiotic and biotic stress. For the quantitative determination of flavonoids in Fallopia Sachalinensis, a spectrophotometric method was used in the work on a Shimadzu – UV 1800 instrument using a complexing additive – 5% aluminum chloride solution, to determine the content of tannins, the redox titration method was used, in order to detect amino acids, qualitative reactions were carried out with aqueous extraction for three copies of each sample. Quantitative determination of amino acids in the studied samples is carried out on the AAA-339 amino acid analyzer (Czechoslovakia). Approbation of the algorithm on Fallopia Sachalinensis under conditions of anthropogenic contamination of soils of the Bashkir Trans-Urals with heavy metals showed that the plant has an indicator potential, but excludes the production of natural biologically active substances from biomass. At the same time, Fallopia Sachalinensis is resistant to soil pollution with heavy metals and has an increased resistance to industrial pollution, and the possibility of using it as an indicator of soil pollution has also been determined
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Yu, J. H., and L. Dai. "RETRACTED: Strain rate and interfacial property effects of random fibre cementitious composites." Journal of Strain Analysis for Engineering Design 44, no. 6 (August 1, 2009): 417–25. http://dx.doi.org/10.1243/03093247jsa513.
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Yu, J. H. and Dai, L. Strain rate and interfacial property effects of random fibre cementitious composites. Journal of Strain Analysis for Engineering Design, 2009, 44: 417–425. DOI: 10.1234/03093247JSA513. This article has been retracted at the request of the Editor and the Publishers. Reason for Retraction: The results of a plagiarism investigation showed that the authors of this work (Yu, J. H., and Dai, L) are in breach of the agreement that they signed in their Licence to Publish Form for JSA513, having copied substantial portions of material from the following: Yang, E. H. Designing added functions in engineered cementitious composites. Ph.D. Dissertation, University of Michigan, 2008. Yang, E. H. and Li, V. C. Rate Dependencies in Engineered Cementitious Composites. Proceedings of International RILEM workshop on HPFRCC in structural applications, Honolulu, Hawaii. Published by RILEM SARL, pp. 83–92, 2005. Redon, C., V. C. Li, C. Wu, H. Hoshiro, T. Saito, and Ogawa, A. Measuring and Modifying Interface Properties of PVA Fibers in ECC Matrix. ASCE J. Materials in Civil Engineering, 13 (6): 399–406, 2001. We apologize that this was not detected during the peer review process in 2009.
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Wang, Rui-Yu, Vivian R. Ruvolo, Teresa McQueen, Wendy D. Schober, Steven Kornblau, Marina Konopleva, and Michael Andreeff. "Overexpression of Apoptosis-Inducing Factor (AIF) and Pro-Survival Function in AML." Blood 108, no. 11 (November 16, 2006): 4395. http://dx.doi.org/10.1182/blood.v108.11.4395.4395.
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Abstract Apoptosis inducing factor (AIF) is a bifunctional flavoprotein with NADH oxidase activity with a vital function in bioenergetics and redox metabolism while also inducing apoptosis in cancer cells. The gene is localized on chromosome human X (q25–26). AIF is synthesized in the cytosol as a 67KDa precursor molecule and then imported into the intermembrane space of mitochondria. In response to pro-apoptotic stimuli, the change of mitochondrial membrane potential (MMP) results in release of mature AIF (57KDa) to the cytoplasm and translocation into the nuclei where AIF can induce caspase-independent large scale DNA fragmentation (~ 50 Kbp) and chromatin condensation. The mechanism of induction of cell death by AIF remains poorly understood. We have previously reported that AIF induced cell death is both caspase-dependent and independent and related to mitochondrial membrane depolarization in leukemic cells treated with chemotherapeutic agents. In this study, we investigate the expression level and function of AIF and its relationship to Bcl-2 and Bcl-2 family members in leukemia cells; and the expression levels of AIF in AML patient’s samples. To study the function of AIF, we introduced AIF si-RNA into K562 CML cell line using lentiviral vectors and established permanent AIF knockdown cell lines. Western blot analysis showed that the expression levels of XIAP, MCL-1, Bcl-xl and suvivin were increased while the AIF expression level was reduced. Cell growth was diminished two-fold (siAIF-clone 2, cell doubling time ~52-hr) to four-fold (siAIF-clone 1, cell doubling time ~99-hr) in AIF knockdown cells compared to the parental cells (24-hr). This suggests that AIF is supporting cell growth. Next, AIF knockdown and scrambled siRNA control cells were treated with Ara-C (10μM) or STI571 (Gleeevec) (2 μM). The frequency of apoptotic cells was determined by flow cytometry (table). 24hr 48hr Control Ara-C STI571 Control Ara-C STI-571 Control 17.04 30.56 30.23 20.21 48.48 76.12 siAIF-1 3.09 3.36 17.03 7.5 10.12 44.92 siAIF-2 6.77 6.64 7.73 11.95 10.73 41.85 AIF-siRNA in K562 cells showed increased sensitivity to Ara-C and STI-571 (Gleevec). The results support again a pro-survival function of AIF in K562 cells. To examine the expression levels of AIF in primary AML patients, 148 samples (newly diagnosed n=32, relapsed n=27, refractory n=18, CR n=5, and normal-donors n=31) were analyzed by western blot and 40 samples of newly diagnosed AML were analyzed by RT-PCR. Results show that in comparison with normal CD34+ cells, AIF mRNA levels in newly diagnosed AML patients were significantly higher (95.0 copies AIF per hundred copies ABL1 in AML, 57.4 copies AIF per hundred copies in NBM CD34+, p = 0.02). The AIF/actin protein ratios in AML (0.43±0.05) was increased compared with normal BM-CD34+ (0.12±0.02) (p=0.01). Comparison of AIF expression levels in normal CD34+ cells vs. AML with good (0.5±0.05), bad (0.34±0.05), and intermediate (0.39±0.06) cytogenetics respectively, were significantly different (p<0.002). The transcription levels of AIF also showed significant differences (p<0.04) in patients with different cytogenetics abnormalities. Taken together, we conclude that AIF transcript and protein levels are significantly higher in AML compared to normal CD34 cells. AIF in K562 cells functions as pro-survival factor for drug sensitivity. These results question the previously assumed predominant pro-apoptotic function of AIF.
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Zanini, Giada, Valentina Selleri, Mara Malerba, Kateryna Solodka, Giorgia Sinigaglia, Milena Nasi, Anna Vittoria Mattioli, and Marcello Pinti. "The Role of Lonp1 on Mitochondrial Functions during Cardiovascular and Muscular Diseases." Antioxidants 12, no. 3 (February 28, 2023): 598. http://dx.doi.org/10.3390/antiox12030598.
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The mitochondrial protease Lonp1 is a multifunctional enzyme that regulates crucial mitochondrial functions, including the degradation of oxidized proteins, folding of imported proteins and maintenance the correct number of copies of mitochondrial DNA. A series of recent studies has put Lonp1 at the center of the stage in the homeostasis of cardiomyocytes and muscle skeletal cells. During heart development, Lonp1 allows the metabolic shift from anaerobic glycolysis to mitochondrial oxidative phosphorylation. Knock out of Lonp1 arrests heart development and determines cardiomyocyte apoptosis. In adults, Lonp1 acts as a cardioprotective protein, as its upregulation mitigates cardiac injury by preventing the oxidative damage of proteins and lipids, and by preserving mitochondrial redox balance. In skeletal muscle, Lonp1 is crucial for cell development, as it mediates the activation of PINK1/Parkin pathway needed for proper myoblast differentiation. Skeletal muscle-specific ablation of Lonp1 in mice causes reduced muscle fiber size and strength due to the accumulation of mitochondrial-retained protein in muscle. Lonp1 expression and activity decline with age in different tissues, including skeletal muscle, and are associated with a functional decline and structural impairment of muscle fibers. Aerobic exercise increases unfolded protein response markers including Lonp1 in the skeletal muscle of aged animals and is associated with muscle functional recovery. Finally, mutations of Lonp1 cause a syndrome named CODAS (Cerebral, Ocular, Dental, Auricular, and Skeletal anomalies) characterized by the impaired development of multiple organs and tissues, including myocytes. CODAS patients show hypotonia and ptosis, indicative of skeletal muscle reduced performance. Overall, this body of observations points Lonp1 as a crucial regulator of mitochondrial functions in the heart and in skeletal muscle.
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Sowinska, Marta, Maja Morawiak, Marta Bochyńska-Czyż, Andrzej Lipkowski, Elżbieta Ziemińska, Barbara Zabłocka, and Zofia Urbanczyk-Lipkowska. "Molecular Antioxidant Properties and In Vitro Cell Toxicity of the p-Aminobenzoic Acid (PABA) Functionalized Peptide Dendrimers." Biomolecules 9, no. 3 (March 5, 2019): 89. http://dx.doi.org/10.3390/biom9030089.
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Background: Exposure to ozone level and ultraviolet (UV) radiation is one of the major concerns in the context of public health. Numerous studies confirmed that abundant free radicals initiate undesired processes, e.g. carcinogenesis, cells degeneration, etc. Therefore, the design of redox-active molecules with novel structures, containing radical quenchers molecules with novel structures, and understanding their chemistry and biology, might be one of the prospective solutions. Methods: We designed a group of peptide dendrimers carrying multiple copies of p-aminobenzoic acid (PABA) and evaluated their molecular antioxidant properties in 1,1’-diphenyl-2-picrylhydrazyl (DPPH) and 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) tests. Cytotoxicity against human melanoma and fibroblast cells as well as against primary cerebral granule cells (CGC) alone and challenged by neurotoxic sodium glutamate and production of reactive oxygen species (ROS) in presence of dendrimers were measured. Results: PABA-terminated dendrimers express enhanced radical and radical cation scavenging properties in relation to PABA alone. In cellular tests, the dendrimers at 100 M fully suppress and between 20–100 M reduce proliferation of the human melanoma cell line. In concentration 20 M dendrimers generate small amount of the reactive oxygen species (<25%) but even in their presence human fibroblast and mouse cerebellar granule cells remain intact Moreover, dendrimers at 0.2–20 µM concentration (except one) increased the percentage of viable fibroblasts and CGC cells treated with 100 M glutamate. Conclusions: Designed PABA-functionalized peptide dendrimers might be a potential source of new antioxidants with cationic and neutral radicals scavenging potency and/or new compounds with marked selectivity against human melanoma cell or glutamate-stressed CGC neurons. The scavenging level of dendrimers depends strongly on the chemical structure of dendrimer and the presence of other groups that may be prompted into radical form. The present studies found different biological properties for dendrimers constructed from the same chemical fragments but the differing structure of the dendrimer tree provides once again evidence that the structure of dendrimer can have a significant impact on drug–target interactions.
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Summerfield, Tina C., Sowmya Nagarajan, and Louis A. Sherman. "Gene expression under low-oxygen conditions in the cyanobacterium Synechocystis sp. PCC 6803 demonstrates Hik31-dependent and -independent responses." Microbiology 157, no. 2 (February 1, 2011): 301–12. http://dx.doi.org/10.1099/mic.0.041053-0.
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We have investigated the response of the cyanobacterium Synechocystis sp. PCC 6803 during growth at very low O2 concentration (bubbled with 99.9 % N2/0.1 % CO2). Significant transcriptional changes upon low-O2 incubation included upregulation of a cluster of genes that contained psbA1 and an operon that includes a gene encoding the two-component regulatory histidine kinase, Hik31. This regulatory cluster is of particular interest, since there are virtually identical copies on both the chromosome and plasmid pSYSX. We used a knockout mutant lacking the chromosomal copy of hik31 and studied differential transcription during the aerobic–low-O2 transition in this ΔHik31 strain and the wild-type. We observed two distinct responses to this transition, one Hik31 dependent, the other Hik31 independent. The Hik31-independent responses included the psbA1 induction and genes involved in chlorophyll biosynthesis. In addition, there were changes in a number of genes that may be involved in assembling or stabilizing photosystem (PS)II, and the hox operon and the LexA-like protein (Sll1626) were upregulated during low-O2 growth. This family of responses mostly focused on PSII and overall redox control. There was also a large set of genes that responded differently in the absence of the chromosomal Hik31. In the vast majority of these cases, Hik31 functioned as a repressor and transcription was enhanced when Hik31 was deleted. Genes in this category encoded both core and peripheral proteins for PSI and PSII, the main phycobilisome proteins, chaperones, the ATP synthase cluster and virtually all of the ribosomal proteins. These findings, coupled with the fact that ΔHik31 grew better than the wild-type under low-O2 conditions, suggested that Hik31 helps to regulate growth and overall cellular homeostasis. We detected changes in the transcription of other regulatory genes that may compensate for the loss of Hik31. We conclude that Hik31 regulates an important series of genes that relate to energy production and growth and that help to determine how Synechocystis responds to changes in O2 conditions.
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Rossi, Edmund A., William J. McBride, Diane L. Nordstrom, Preeti Trisal, Thomas M. Cardillo, David M. Goldenberg, and Chien-Hsing Chang. "A New Class of Dimeric α-Interferons (IFN-α) Made by the Dock and Lock (DNL) Method for Improved Cancer Therapy." Blood 110, no. 11 (November 16, 2007): 1396. http://dx.doi.org/10.1182/blood.v110.11.1396.1396.
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Abstract Background: IFN-α2 is indicated for the therapy of hairy cell leukemia, chronic myelogenous leukemia, follicular lymphoma, and malignant melanoma. As is the case for most cytokines, the pharmacokinetic (PK) properties of IFN-α2 are critical for dosing and efficacy. In vivo, the protein is quickly degraded, diffuses widely throughout the body, and has a rapid rate of renal clearance. Pegylation of IFN-α2 significantly increases the serum half-life and reduces renal clearance, thus enhancing its efficacy. However, established pegylation of IFN-α2 results in a mixture of positional isomers and reduced in vitro activity, as known for PEGASYS (Roche) and PEG-INTRON (Schering-Plough). Methods: To improve the PK properties and the potency, the DNL method (Rossi et al. Proc. Natl. Acad. Sci. USA, 2006,103:6841) was used to generate novel agents having two copies of IFN-α2b conjugated to polyethylene glycol (PEG). Results: A fusion protein (DDD2-IFN-α2b) composed of IFN-α2b with a dimerization-and-docking domain (DDD2) and a six-His tag was expressed both in myeloma cells and in E. coli. Two PEG-based modules, each composed of a fluorescent molecule, an anchor domain (AD) and either a 20-kDa PEG (IMP362) or a 30-kDa PEG (IMP413), were synthesized. Combining DDD2-IFN-α2b and IMP362 or IMP413 under redox conditions resulted in the desirable DNL conjugates consisting of two copies of IFN-α2b and one PEG linked site-specifically via the DDD and AD interaction. The cytotoxic activity of DDD2-IFN-α2b on Daudi lymphoma cells was similar to that of commercially available recombinant IFN-α2 (rhIFN-α2). The purity and identity of the two DNL conjugates (α2b-413 and α2b-362) were demonstrated by SDS-PAGE, immunoblotting, and fluorescence imaging. Both also showed potent cytotoxic activity on Daudi cells in vitro and superior PK properties to PEG-INTRON. For example, the mean blood residence times for α2b-362 (10.3 h) and α2b-413 (21.7 h) were significantly longer than those of rhIFN-α2 (0.7 h) and PEG-INTRON (5.1 h). Initial studies in mice bearing Daudi xenografts showed a significant therapeutic advantage over PEG-INTRON for both α2b-362 and α2b-413. Animals given 14,000 IU of PEG-INTRON had a median survival (MS) of 32 days compared to 21 days for saline control, whereas those receiving α2b-362 at 14,000 IU, 7,000 IU and 3,500 IU resulted in MS of 45, 41 and 32 days, respectively. More remarkably, α2b-413 was the most effective, achieving MS of 46, >53, and >53 days with 3,500 IU, 7,000 IU and 14,000 IU, respectively, all statistically significant improvements (P< 0.0028) compared to PEG-INTRON given at each equivalent activity. Conclusions: The DNL method provides a novel pegylation strategy for generating a dimeric IFN-α2b that is linked site-specifically to a single PEG at the predetermined location. Since the resulting conjugates exhibit improved PK and efficacy in a Burkitt lymphoma model, they may represent a new class of interferons for use in cancer and infectious disease therapy.
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Dumbrava, Ecaterina Elena, Amit Mahipal, Xin Gao, Geoffrey Shapiro, Jason S. Starr, Parminder Singh, Muhammad Furqan, et al. "Phase 1/2 study of eprenetapopt (APR-246) in combination with pembrolizumab in patients with solid tumor malignancies." Journal of Clinical Oncology 39, no. 15_suppl (May 20, 2021): TPS3161. http://dx.doi.org/10.1200/jco.2021.39.15_suppl.tps3161.
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TPS3161 Background: The p53 pathway has been implicated in antitumor immunity, including antigen presentation and T-cell proliferation. Loss of p53 function can increase resistance to immunotherapy across many tumor types. Eprenetapopt (eprenet) is a small molecule that stabilizes the folded structure of p53, resulting in activation of mutant p53 and stabilization of wild-type (WT) p53. It also targets the cellular redox homeostasis, resulting in induction of apoptosis in tumor cells. In vivo, mice carrying supernumerary copies of the TP53 gene harbor a pro-inflammatory tumor microenvironment, an effect recapitulated in TP53 normal-copy mice treated with eprenetapopt. Combining eprenetapopt and anti-PD1 or anti-CTLA4 therapy resulted in enhanced tumor growth inhibition and improved survival in TP53 WT mice inoculated with B16 melanoma and MC38 colon adenocarcinoma cells . Based on these results, we hypothesized that eprenet-induced p53 stabilization may augment response to immunotherapy. To test this hypothesis, we are conducting a phase 1b/2 study of eprenet in combination with pembrolizumab (eprenet+pembro) in pts with solid tumors. Methods: The primary objectives are to determine the maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D) and to assess the safety and tolerability of eprenet+pembro in pts with advanced solid tumors. The secondary objectives are to estimate the anti-tumor activity and to describe the pharmacokinetics of the combination. Exploratory objectives include assessing predictive and pharmacodynamic markers of response. The study includes a safety lead-in with a 3+3 dose de-escalation design for pts with advanced solid tumors with known tumor TP53 mutation status ( TP53 WT is acceptable) (max 18 pts), followed by expansion cohorts in pts with NSCLC, gastric/GEJ and urothelial cancer (max 100 pts). In expansion, pts with urothelial and gastric cancers must be naïve to anti-PD-1/ L1 therapy. Eprenet is given IV once daily on Days 1–4 while pembro is administered on Day 3 of each 21-day cycle. The RP2D of eprenet+pembro is considered the dose at which ≤ 1 of 6 pts in a cohort has a dose-limiting toxicity (DLT). Primary endpoints are occurrence of DLTs, adverse events (AEs) and serious AEs with eprenet+pembro. Key secondary endpoints are best objective response, progression free survival and overall survival. Exploratory endpoints include gene mutations by next generation sequencing (including TP53), mRNA expression, multiplex immunohistochemistry and transcriptomics, multiplex flow cytometry on peripheral blood mononuclear cells and cytokines in serum. Continuous monitoring of toxicity will be conducted. The trial opened in May 2020 and is actively enrolling patients. Clinical trial information: NCT04383938.
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Rossi, Edmund A., Eric Chan, Pankaj Gupta, David M. Goldenberg, and Chien-Hsing Chang. "A Novel Ribonuclease-Based Immunotoxin Comprising Quadruple Ranpirnase (Rap) Site-Specifically Conjugated to An Anti-CD22 IgG Showing Potent Anti-Lymphoma Activity." Blood 114, no. 22 (November 20, 2009): 3721. http://dx.doi.org/10.1182/blood.v114.22.3721.3721.
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Abstract Abstract 3721 Poster Board III-657 Background Rap is a single-chain ribonuclease of 104 amino acids originally isolated from the oocytes of Rana pipiens. Rap exhibits cytostatic and cytotoxic effects on a variety of tumor cell lines in vitro, as well as antitumor activity in vivo. The amphibian ribonuclease enters cells via receptor-mediated endocytosis and once internalized into the cytosol, selectively degrades tRNA, resulting in inhibition of protein synthesis and induction of apoptosis. Rap is in advanced Phase IIIb clinical trials against malignant mesothelioma, with reversible dose-limiting renal toxicity and no reported immunogenicity. We have previously shown that effective therapy of human lymphoma xenografts could be achieved with a recombinant fusion protein comprising Rap and a rapidly internalizing anti-CD74 humanized IgG4 antibody (Chang et al., Blood, 2005; 106: 4308-14). In this study, we applied the Dock-and-Lock (DNL) method to generate another novel class of immunotoxins, each of which contains four copies of Rap site-specifically linked to a bivalent IgG. Methods The DNL platform technology exploits a pair of distinct protein domains involved in the natural binding of cAMP-dependent protein kinase (PKA) and A-kinase anchoring proteins (AKAPs). These domains serve as linkers for site-specific conjugation of 2 types of modules, one containing the dimerization and docking domain (DDD) of PKA and the other containing the anchoring domain (AD) of an interactive AKAP. We have combined a recombinant Rap-DDD module with a recombinant IgG-AD module derived from the internalizing anti-CD22 humanized antibody, epratuzumab, to generate 22-Rap, in which a dimer of Rap is covalently tethered to the c-terminus of each heavy chain of epratuzumab. The Rap-DDD module was expressed in E. coli as inclusion bodies, which were purified by immobilized metal affinity chromatography and refolded. The epratuzumab-AD module was expressed in myeloma cells and purified from the culture supernatant using Protein A affinity chromatography. Results 22-Rap was prepared by mixing the epratuzumab-AD module with the Rap-DDD module under mild redox conditions and purified to near homogeneity in a single step using Protein A affinity chromatography. Size exclusion HPLC revealed the presence of a single peak of a retention time consistent with the molecular size of ∼230 kDa, indicative of an IgG and four Rap groups. In vitro transcription/translation assays showed that both the Rap-DDD2 module and 22-Rap retained the activity of Rap. Results of in vitro cytotoxicity assays using Daudi Burkitt lymphoma cells demonstrated that 22-Rap was highly cytotoxic, resulting in nearly 100% cell killing at 1 nM, with an IC50 <50 pM. In comparison, recombinant Rap at 100 nM, either alone or in combination with epratuzumab, resulted in only 50% inhibition of Daudi proliferation. Thus, 22-Rap is about 3 logs more potent than free Rap against Daudi cells. Conclusion The DNL method provides a modular approach to efficiently tether multiple cytotoxins onto a targeting antibody, resulting in novel immunotoxins that are expected to show higher in vivo potency due to improved pharmacokinetics and targeting specificity. Disclosures: Rossi: Immunomedics, Inc: Employment. Chan:Immunomedics, Inc: Employment. Gupta:Immunomedics Inc.: Employment. Goldenberg:Immunomedics Inc.: Consultancy, Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Chang:Immunomedics Inc.: Employment.
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Rossi, Edmund A., Chien-Hsing Chang, Thomas M. Cardillo, Diane L. Nordstrom, and David M. Goldenberg. "A Novel Anti-CD20 IgG-Interferon-α2b Fusion Protein with Potent in Vitro and in Vivo Anti-Lymphoma Activity Made by the Dock-and-Lock (DNL) Method." Blood 112, no. 11 (November 16, 2008): 1590. http://dx.doi.org/10.1182/blood.v112.11.1590.1590.
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Abstract BACKGROUND: Interferon-α2b (IFN-α2b) is active alone and in combination with other agents in the therapy of a variety of cancers, including hairy cell leukemia, chronic myelocytic leukemia, follicular lymphoma, and malignant melanoma. As for most cytokines, the pharmacokinetics are a major factor affecting schedule and efficacy. The protein is rapidly degraded, diffuses widely throughout the body, and has a rapid rate of renal clearance. Commercially available IFNs that are pegylated, such as PEG-INTRON and PEGASYS, have increased serum half-life and reduced renal clearance, which augment their biological activity. For therapy of lymphoma and other cancers, fusing IFN-α2 to tumor-targeting antibodies could increase serum half-life and target the IFN-α2 to the tumor, conceivably allowing less frequent and lower dosing with improved therapeutic efficacy and reduced side effects. METHODS: The modular DNL method exploits a pair of distinct protein domains involved in the natural binding between protein kinase A (PKA) and A-kinase anchoring proteins (AKAP), whereby the dimerization-and-docking domain (DDD) of PKA and the anchoring domain (AD) of an interactive AKAP are each fused to a biological entity, resulting in respective DDD- and AD-modules that are readily combined to quantitatively generate stably-tethered structures of defined composition with retained bioactivity. We have selectively combined recombinant DDD-modules comprising IFN-α2b with recombinant AD-modules derived from the anti-CD20 humanized mAb, veltuzumab, and other humanized mAbs to generate complexes comprising four copies of IFN-α2b site-specifically linked to the bivalent IgG. RESULTS: The IgG-AD2 and IFN-α2b-DDD2 modules were expressed in separate myeloma cell cultures and purified from culture broths by Protein A and IMAC, respectively. Combining an IgG-AD2 module with slightly more than 2 molar equivalents of the cytokine-DDD2 module under mild redox conditions resulted in the formation of a covalent complex comprising one IgG and 4 IFN-α2b via the docking of each of the two AD2 domains on IgG with a dimer of IFN-α2b-DDD2, and subsequent formation of disulfide bonds (locking) between DDD2 and AD2. The 255-kDa conjugates, which were purified by Protein A, were readily detected by size-exclusion HPLC and non-reducing SDS-PAGE, and retained the biological functions of IFN-α2b in vitro. The IgG-IFN-α2b constructs exhibited potent anti-viral activity in vitro, with specific activities approaching that of recombinant human IFN-α2b. Additionally, the constructs all showed highly potent in vitro cytotoxicity against Burkitt lymphoma cell lines. Notably, the CD20-targeted IFN-α2b construct (20-2b) was 30-fold more potent than a control, non-targeting IgG-IFN-α2b. The enhanced cytotoxicity of 20-2b was not reproduced when non-targeting IgG-IFN-α2b was used in combination with veltuzumab, suggesting that IFN-α2b must be physically linked to achieve maximal potency. IgG-IFN-α2b fusion proteins, including 20-2b, induced significantly more potent ADCC compared to their parental MAbs. The targeting properties of 20-2b were comparable to veltuzumab, and its serum half-life was significantly longer than PEG-INTRON and PEGASYS. In the human Daudi xenograft model, 20-2b showed superior anti-tumor efficacy compared to both veltuzumab and other IgG-IFN-α2 agents. The median survival time (MST) for mice treated with single-dose 170 ng 20-2b was 101.5 days, whereas those treated with an equivalent dose of veltuzumab and untreated mice survived 39 and 28 days, respectively (P<0.0005). Lower 20-2b doses of 80, 17 and 8 ng resulted in MST of 97.5, 56.5 and 48 days, respectively, with the lowest dose still significantly better than the highest dose of veltuzumab (P=0.0434). Using a single 170-ng dose, a CD22-targeting IFN-α2b (22-2b) also increased MST significantly to 47 days (P =0.0119), while a non-targeting IgG-IFN-α2b (734-2b) did not. CONCLUSIONS: The DNL method provided an IFN-α2-targeting mAb fusion protein that showed improved anti-tumor efficacy over the mAb by itself, based on improved pharmacokinetics, ADCC, and tumor targeting, as well as reduced systemic toxicity. Thus, DNL provides a modular approach to efficiently tether cytokines to targeting antibodies, resulting in higher in vivo potency than the original cytokines or mAbs.
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Nordstrom, Diane L., Edmund A. Rossi, David M. Goldenberg, and Chien-Hsing Chang. "First Bispecific Antibody Immunocytokine (Anti-CD20/HLA-DR-Interferon-á2b) Is Highly Toxic for Human Lymphoma Cells in Vitro." Blood 114, no. 22 (November 20, 2009): 1695. http://dx.doi.org/10.1182/blood.v114.22.1695.1695.
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Abstract Abstract 1695 Poster Board I-721 Background IFN-á2 is indicated for the therapy of a variety of hematopoietic tumors. As with most cytokines, the short serum half-life and severe side effects of IFN-á2 are major factors affecting its dosing schedule and efficacy. Fusion or conjugation of IFN-á2 to a tumor-targeting IgG has the potential to enhance in vivo potency due to increased tumor localization and more favorable pharmacokinetics. We have recently demonstrated that 20-2b, a monospecific immunocytokine generated by the dock-and-lock (DNL) method to comprise tetrameric IFN-á2b covalently linked to veltuzumab, a humanized anti-CD20 mAb, exhibited very potent anti-tumor activity in vitro and in human lymphoma xenografts (Rossi et al., Blood, in press). However, lymphomas and leukemias that express little or no CD20 are expected to be resistant to therapy with 20-2b. HLA-DR is expressed on many hematopoietic tumors and some solid cancers. A bispecific immunocytokine that could target IFN-á to both CD20 and HLA-DR might be a more effective therapeutic against a wide variety of hematopoietic malignancies, including those that express CD20, HLA-DR, or both. Since each component of the multifunctional complex (veltuzumab, anti-HLA-DR F(ab)2, and IFN-á2b) has anti-tumor activity independently, we evaluated if the bispecific immunocytokine can potentially be even more potent than the monospecific immunocytokine, 20-2b. Methods One strategy of the modular DNL method is to fuse either the dimerization-and-docking domain (DDD) derived from protein kinase A, or the anchoring domain (AD) of a cognate A-kinase anchoring protein, to a biological entity, resulting in respective DDD- and AD-modules that are readily combined to quantitatively generate stably-tethered structures of defined composition with retained bioactivity. We have selectively combined recombinant DDD-modules of both IFN-á2b and anti-HLA-DR Fab (derived from humanized L243) together with a recombinant AD-module of anti-CD20 IgG (veltuzumab) to generate the first bispecific antibody-based immunocytokine, designated 20-C2-2b, which comprises two copies of IFN-á2b and a stabilized F(ab)2 of hL243 site-specifically linked to veltuzumab. Results Each of the three modules, veltuzumab-AD, hL243-Fab-DDD, and IFN-á2b-DDD, was produced recombinantly in separate myeloma cell cultures. Combining equimolar amounts of the three modules under mild redox conditions resulted in the formation of 20-C2-2b, which was purified by sequential chromatographic processes involving Protein A, IMAC and anion exchange chromatography to remove potential side-products such as 20-2b and 20-C2 (the hexavalent bispecific antibody comprising veltuzumab and four Fabs of hL243). Size-exclusion HPLC analysis indicated a major peak of a retention time consistent with a ∼310 kDa protein. Reducing SDS-PAGE of 20-C2-2b revealed the presence of all three constituents. The complex was immunoreactive with an anti-IFN-á2b, an anti-idiotype to hL243, as well as an anti-idiotype to veltuzumab, and showed increased binding to Raji lymphoma cells compared to either veltuzumab or hL243. More importantly, 20-C2-2b was found to be extremely cytotoxic to Daudi, an IFNá-sensitive Burkitt lymphoma cell line, having an IC50 = 0.035 pM, which was 100,000-fold more potent than hL243 IgG, 100-fold more potent than a combination of veltuzumab, hL243 IgG and a structural analog comprising an irrelevant IgG and IFN-á2b, and 5-fold more potent than 20-2b. In the same assay, we have also determined that 20-C2-2b was about 2-fold more potent than C2-2b, which comprises hL243 IgG linked to four molecules of IFN-á2b. Conclusions The DNL method provides a modular approach to enable the creation of novel multifunctional complexes. Based on our experience with 20-2b, the bispecific immunocytokine 20-C2-2b is expected to have greater in vivo potency than IFN-á due to improved pharmacokinetics and endowed targeting specificity, and may potentially be useful for therapy of a variety of hematopoietic tumors that express either CD20 or HLA-DR. Disclosures Nordstrom: Immunomedics, Inc.: Employment. Rossi:Immunomedics, Inc: Employment. Goldenberg:Immunomedics Inc.: Consultancy, Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Chang:Immunomedics Inc.: Employment.
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"Application of copper oxide-based nanomaterials in electrochemical energy‐storage devices." Journal of NanoScience Technology, May 17, 2022. http://dx.doi.org/10.52319/j.nanoscitec.2022.11.
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Copper oxide is established as an important compound in technology due to its semi-conducting nature and high chemical stability as well as economic benefits. These features have made it a good candidate in energy storage applications. Further, extensive attention has been paid to advancement of supercapacitors- a complementary device between battery and conventional capacitors – due to their unique features such as high power, long cycle life, and environmentally friendly nature. In addition, copper oxide has sparked interest in preparation of applicable positive electrodes which can be used in preparation of supercapacitors. Meanwhile, copper oxide is mixed with polarized liquids and polymers easily, and it is has relative stable chemical and physical properties. Electrochemical features of copper oxide depend on the morphology where proper architectural design of electrode materials can be optimized in these devices. In this review, copper oxide synthesis and its redox mechanism as cathode materials will be explored as well as the application of various copper oxide compounds in preparation of high-performance supercapacitors.
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Loll, Bernhard, Matthias Broser, Peter B. Kós, Jan Kern, Jacek Biesiadka, Imre Vass, Wolfram Saenger, and Athina Zouni. "Modeling of variant copies of subunit D1 in the structure of photosystem II from Thermosynechococcus elongatus." Biological Chemistry 389, no. 5 (May 1, 2008). http://dx.doi.org/10.1515/bc.2008.058.
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Abstract In the cyanobacterium Thermosynechococcus elongatus BP-1, living in hot springs, the light environment directly regulates expression of genes that encode key components of the photosynthetic multi-subunit protein-pigment complex photosystem II (PSII). Light is not only essential as an energy source to power photosynthesis, but leads to formation of aggressive radicals which induce severe damage of protein subunits and organic cofactors. Photosynthetic organisms develop several protection mechanisms against this photo-damage, such as the differential expression of genes coding for the reaction center subunit D1 in PSII. Testing the expression of the three different genes (psbAI, psbAII, psbAIII) coding for D1 in T. elongatus under culture conditions used for preparing the material used in crystallization of PSII showed that under these conditions only subunit PsbA1 is present. However, exposure to high-light intensity induced partial replacement of PsbA1 with PsbA3. Modeling of the variant amino acids of the three different D1 copies in the 3.0 Å resolution crystal structure of PSII revealed that most of them are in the direct vicinity to redox-active cofactors of the electron transfer chain. Possible structural and mechanistic consequences for electron transfer are discussed.
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Patil, Varsha, and Vikas Jain. "Understanding Metabolic Remodeling in Mycobacterium smegmatis to Overcome Energy Exigency and Reductive Stress Under Energy-Compromised State." Frontiers in Microbiology 12 (September 1, 2021). http://dx.doi.org/10.3389/fmicb.2021.722229.
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Mycobacteria such as Mycobacterium tuberculosis, the causative agent of tuberculosis that annually kills several million people worldwide, and Mycobacterium smegmatis, the non-pathogenic fast-growing mycobacteria, require oxidative phosphorylation to meet their energy requirements. We have previously shown that deletion of one of the two copies of atpD gene that codes for the ATP synthase β-subunit establishes an energy-compromised state in M. smegmatis. Here we report that upon such deletion, a major routing of electron flux occurs through the less energy-efficient complexes of its respiratory chain. ΔatpD bacterium also shows an increased reduced state which is further confirmed by the overexpression of WhiB3, a major redox sensor. We show a substantial modulation of the biosynthesis of cell wall associated lipids and triacylglycerol (TAG). An accumulation of TAG-containing lipid bodies is further confirmed by using 14C oleate incorporation. Interestingly, the mutant also shows an overexpression of TAG-degrading lipase genes, and the intracellular lipolytic enzymes mediate TAG hydrolysis for their utilization as energy source. We believe that our in vitro energy-depleted model will allow us to explore the critical link between energy metabolism, redox homeostasis, and lipid biosynthesis during ATP-depleted state, which will enhance our understanding of the bacterial adaptation, and will allow us to identify novel drug targets to counter mycobacterial infections.
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Sanyal, Ravi Prakash, Vishal Prashar, Narendra Jawali, Ramanjulu Sunkar, Hari Sharan Misra, and Ajay Saini. "Molecular and Biochemical Analysis of Duplicated Cytosolic CuZn Superoxide Dismutases of Rice and in silico Analysis in Plants." Frontiers in Plant Science 13 (May 30, 2022). http://dx.doi.org/10.3389/fpls.2022.864330.
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Superoxide dismutases (SODs, EC 1.15.1.1) are ubiquitous antioxidant metalloenzymes important for oxidative stress tolerance and cellular redox environment. Multiple factors have contributed toward the origin and diversity of SOD isoforms among different organisms. In plants, the genome duplication events, responsible for the generation of multiple gene copies/gene families, have also contributed toward the SOD diversity. However, the importance of such molecular events on the characteristics of SODs has not been studied well. This study investigated the effects of divergence on important characteristics of two block-duplicated rice cytosolic CuZn SODs (OsCSD1, OsCSD4), along with in silico assessment of similar events in other plants. The analysis revealed heterogeneity in gene length, regulatory regions, untranslated regions (UTRs), and coding regions of two OsCSDs. An inconsistency in the database-predicted OsCSD1 gene structure was also identified and validated experimentally. Transcript analysis showed differences in the basal levels and stress responsiveness of OsCSD1 and OsCSD4, and indicated the presence of two transcription start sites in the OsCSD1. At the amino acid level, the two OsCSDs showed differences at 18 sites; however, both exist as a homodimer, displaying typical CuZn SOD characteristics, and enhancing the oxidative stress tolerance of Escherichia coli cells. However, OsCSD4 showed higher specific activity as well as stability. The comparison of the two OsCSDs with reported thermostable CSDs from other plants identified regions likely to be associated with stability, while the homology modeling and superposition highlighted structural differences. The two OsCSDs displayed heteromeric interaction capability and forms an enzymatically active heterodimer (OsCSD1:OsCSD4) on co-expression, which may have significance as both are cytosolic. In silico analysis of 74 plant genomes revealed the prevalence of block duplications for multiple CSD copies (mostly cytosolic). The divergence and clustering analysis of CSDs suggested the possibility of an ancestral duplication event in monocots. Conserved SOD features indicating retention of SOD function among CSD duplicates were evident in few monocots and dicots. In most other species, the CSD copies lacked critical features and may not harbor SOD function; however, other feature-associated functions or novel functions might be present. These aspects of divergent CSD copies encoding co-localized CSDs may have implications in plant SOD functions in the cytosol and other organelles.
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Alcaide, Fernando, Rocío Trastoy, Raquel Moure, Mónica González-Bardanca, Antón Ambroa, María López, Inés Bleriot, et al. "Multiplex Real-Time PCR-shortTUB Assay for Detection of the Mycobacterium tuberculosis Complex in Smear-Negative Clinical Samples with Low Mycobacterial Loads." Journal of Clinical Microbiology 57, no. 8 (June 12, 2019). http://dx.doi.org/10.1128/jcm.00733-19.
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ABSTRACT Tuberculosis (TB) remains a major health problem worldwide. Control of TB requires rapid, accurate diagnosis of active disease. However, extrapulmonary TB is very difficult to diagnose because the clinical specimens have very low bacterial loads. Several molecular methods involving direct detection of the Mycobacterium tuberculosis complex (MTBC) have emerged in recent years. Real-time PCR amplification simultaneously combines the amplification and detection of candidate sequences by using fluorescent probes (mainly TaqMan or Molecular Beacons) in automated systems. The multiplex real-time PCR-short assay is performed using locked nucleic acid (LNA) probes (length, 8 to 9 nucleotides) in combination with CodUNG to detect multiple pathogens in clinical samples. In this study, we evaluated the performance of this novel multiplex assay for detecting the MTBC in comparison with that of the conventional culture-based method. The multiplex real-time PCR-shortTUB assay targets two genes, whiB3 (redox-responsive transcriptional regulator) and pstS1 (phosphate-specific transporter), yielding limits of detection (LOD) of 10 copies and 100 copies, respectively, and amplification efficiencies of 92% and 99.7%, respectively. A total of 94 extrapulmonary samples and pulmonary samples with low mycobacterial loads (all smear negative; 75 MTBC culture positive) were analyzed using the test, yielding an overall sensitivity of 88% and a specificity of 95%. For pleural fluid and tissues/biopsy specimens, the sensitivity was 83% and 85%, respectively. In summary, this technique could be implemented in routine clinical microbiology testing to reduce the overall turnaround time for MTBC detection and may therefore be a useful tool for the diagnosis of extrapulmonary tuberculosis and diagnosis using pulmonary samples with low mycobacterial loads.
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Haja, Dominik K., and Michael W. W. Adams. "pH Homeostasis and Sodium Ion Pumping by Multiple Resistance and pH Antiporters in Pyrococcus furiosus." Frontiers in Microbiology 12 (August 16, 2021). http://dx.doi.org/10.3389/fmicb.2021.712104.
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Multiple Resistance and pH (Mrp) antiporters are seven-subunit complexes that couple transport of ions across the membrane in response to a proton motive force (PMF) and have various physiological roles, including sodium ion sensing and pH homeostasis. The hyperthermophilic archaeon Pyrococcus furiosus contains three copies of Mrp encoding genes in its genome. Two are found as integral components of two respiratory complexes, membrane bound hydrogenase (MBH) and the membrane bound sulfane sulfur reductase (MBS) that couple redox activity to sodium translocation, while the third copy is a stand-alone Mrp. Sequence alignments show that this Mrp does not contain an energy-input (PMF) module but contains all other predicted functional Mrp domains. The P. furiosus Mrp deletion strain exhibits no significant changes in optimal pH or sodium ion concentration for growth but is more sensitive to medium acidification during growth. Cell suspension hydrogen gas production assays using the deletion strain show that this Mrp uses sodium as the coupling ion. Mrp likely maintains cytoplasmic pH by exchanging protons inside the cell for extracellular sodium ions. Deletion of the MBH sodium-translocating module demonstrates that hydrogen gas production is uncoupled from ion pumping and provides insights into the evolution of this Mrp-containing respiratory complex.
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Sui, Yu-fei, Tabea Schütze, Li-ming Ouyang, Hongzhong Lu, Peng Liu, Xianzun Xiao, Jie Qi, Ying-Ping Zhuang, and Vera Meyer. "Engineering cofactor metabolism for improved protein and glucoamylase production in Aspergillus niger." Microbial Cell Factories 19, no. 1 (October 23, 2020). http://dx.doi.org/10.1186/s12934-020-01450-w.
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Abstract Background Nicotinamide adenine dinucleotide phosphate (NADPH) is an important cofactor ensuring intracellular redox balance, anabolism and cell growth in all living systems. Our recent multi-omics analyses of glucoamylase (GlaA) biosynthesis in the filamentous fungal cell factory Aspergillus niger indicated that low availability of NADPH might be a limiting factor for GlaA overproduction. Results We thus employed the Design-Build-Test-Learn cycle for metabolic engineering to identify and prioritize effective cofactor engineering strategies for GlaA overproduction. Based on available metabolomics and 13C metabolic flux analysis data, we individually overexpressed seven predicted genes encoding NADPH generation enzymes under the control of the Tet-on gene switch in two A. niger recipient strains, one carrying a single and one carrying seven glaA gene copies, respectively, to test their individual effects on GlaA and total protein overproduction. Both strains were selected to understand if a strong pull towards glaA biosynthesis (seven gene copies) mandates a higher NADPH supply compared to the native condition (one gene copy). Detailed analysis of all 14 strains cultivated in shake flask cultures uncovered that overexpression of the gsdA gene (glucose 6-phosphate dehydrogenase), gndA gene (6-phosphogluconate dehydrogenase) and maeA gene (NADP-dependent malic enzyme) supported GlaA production on a subtle (10%) but significant level in the background strain carrying seven glaA gene copies. We thus performed maltose-limited chemostat cultures combining metabolome analysis for these three isolates to characterize metabolic-level fluctuations caused by cofactor engineering. In these cultures, overexpression of either the gndA or maeA gene increased the intracellular NADPH pool by 45% and 66%, and the yield of GlaA by 65% and 30%, respectively. In contrast, overexpression of the gsdA gene had a negative effect on both total protein and glucoamylase production. Conclusions This data suggests for the first time that increased NADPH availability can indeed underpin protein and especially GlaA production in strains where a strong pull towards GlaA biosynthesis exists. This data also indicates that the highest impact on GlaA production can be engineered on a genetic level by increasing the flux through the pentose phosphate pathway (gndA gene) followed by engineering the flux through the reverse TCA cycle (maeA gene). We thus propose that NADPH cofactor engineering is indeed a valid strategy for metabolic engineering of A. niger to improve GlaA production, a strategy which is certainly also applicable to the rational design of other microbial cell factories.
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Pérez Castro, Sherlynette, Mikayla A. Borton, Kathleen Regan, Isabella Hrabe de Angelis, Kelly C. Wrighton, Andreas P. Teske, Marc Strous, and S. Emil Ruff. "Degradation of biological macromolecules supports uncultured microbial populations in Guaymas Basin hydrothermal sediments." ISME Journal, June 10, 2021. http://dx.doi.org/10.1038/s41396-021-01026-5.
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AbstractHydrothermal sediments contain large numbers of uncultured heterotrophic microbial lineages. Here, we amended Guaymas Basin sediments with proteins, polysaccharides, nucleic acids or lipids under different redox conditions and cultivated heterotrophic thermophiles with the genomic potential for macromolecule degradation. We reconstructed 20 metagenome-assembled genomes (MAGs) of uncultured lineages affiliating with known archaeal and bacterial phyla, including endospore-forming Bacilli and candidate phylum Marinisomatota. One Marinisomatota MAG had 35 different glycoside hydrolases often in multiple copies, seven extracellular CAZymes, six polysaccharide lyases, and multiple sugar transporters. This population has the potential to degrade a broad spectrum of polysaccharides including chitin, cellulose, pectin, alginate, chondroitin, and carrageenan. We also describe thermophiles affiliating with the genera Thermosyntropha, Thermovirga, and Kosmotoga with the capability to make a living on nucleic acids, lipids, or multiple macromolecule classes, respectively. Several populations seemed to lack extracellular enzyme machinery and thus likely scavenged oligo- or monomers (e.g., MAGs affiliating with Archaeoglobus) or metabolic products like hydrogen (e.g., MAGs affiliating with Thermodesulfobacterium or Desulforudaceae). The growth of methanogens or the production of methane was not observed in any condition, indicating that the tested macromolecules are not degraded into substrates for methanogenesis in hydrothermal sediments. We provide new insights into the niches, and genomes of microorganisms that actively degrade abundant necromass macromolecules under oxic, sulfate-reducing, and fermentative thermophilic conditions. These findings improve our understanding of the carbon flow across trophic levels and indicate how primary produced biomass sustains complex and productive ecosystems.
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Ortiz-Merino, Raúl A., Nurzhan Kuanyshev, Kevin P. Byrne, Javier A. Varela, John P. Morrissey, Danilo Porro, Kenneth H. Wolfe, and Paola Branduardi. "Transcriptional Response to Lactic Acid Stress in the Hybrid Yeast Zygosaccharomyces parabailii." Applied and Environmental Microbiology 84, no. 5 (December 21, 2017). http://dx.doi.org/10.1128/aem.02294-17.
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ABSTRACT Lactic acid has a wide range of applications starting from its undissociated form, and its production using cell factories requires stress-tolerant microbial hosts. The interspecies hybrid yeast Zygosaccharomyces parabailii has great potential to be exploited as a novel host for lactic acid production, due to high organic acid tolerance at low pH and a fermentative metabolism with a high growth rate. Here we used mRNA sequencing (RNA-seq) to analyze Z. parabailii 's transcriptional response to lactic acid added exogenously, and we explore the biological mechanisms involved in tolerance. Z. parabailii contains two homeologous copies of most genes. Under lactic acid stress, the two genes in each homeolog pair tend to diverge in expression to a significantly greater extent than under control conditions, indicating that stress tolerance is facilitated by interactions between the two gene sets in the hybrid. Lactic acid induces downregulation of genes related to cell wall and plasma membrane functions, possibly altering the rate of diffusion of lactic acid into cells. Genes related to iron transport and redox processes were upregulated, suggesting an important role for respiratory functions and oxidative stress defense. We found differences in the expression profiles of genes putatively regulated by Haa1 and Aft1/Aft2, previously described as lactic acid responsive in Saccharomyces cerevisiae . Furthermore, formate dehydrogenase ( FDH ) genes form a lactic acid-responsive gene family that has been specifically amplified in Z. parabailii in comparison to other closely related species. Our study provides a useful starting point for the engineering of Z. parabailii as a host for lactic acid production. IMPORTANCE Hybrid yeasts are important in biotechnology because of their tolerance to harsh industrial conditions. The molecular mechanisms of tolerance can be studied by analyzing differential gene expression under conditions of interest and relating gene expression patterns to protein functions. However, hybrid organisms present a challenge to the standard use of mRNA sequencing (RNA-seq) to study transcriptional responses to stress, because their genomes contain two similar copies of almost every gene. Here we used stringent mapping methods and a high-quality genome sequence to study the transcriptional response to lactic acid stress in Zygosaccharomyces parabailii ATCC 60483, a natural interspecies hybrid yeast that contains two complete subgenomes that are approximately 7% divergent in sequence. Beyond the insights we gained into lactic acid tolerance in this study, the methods we developed will be broadly applicable to other yeast hybrid strains.
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De Vega, Jose J., Abel Teshome, Manfred Klaas, Jim Grant, John Finnan, and Susanne Barth. "Physiological and transcriptional response to drought stress among bioenergy grass Miscanthus species." Biotechnology for Biofuels 14, no. 1 (March 6, 2021). http://dx.doi.org/10.1186/s13068-021-01915-z.
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Abstract Background Miscanthus is a commercial lignocellulosic biomass crop owing to its high biomass productivity, resilience and photosynthetic capacity at low temperature. These qualities make Miscanthus a particularly good candidate for temperate marginal land, where yields can be limited by insufficient or excessive water supply. Differences in response to water stress have been observed among Miscanthus species, which correlated to origin. In this study, we compared the physiological and molecular responses among Miscanthus species under excessive (flooded) and insufficient (drought) water supply in glasshouse conditions. Results A significant biomass loss was observed under drought conditions in all genotypes. M. x giganteus showed a lower reduction in biomass yield under drought conditions compared to the control than the other species. Under flooded conditions, biomass yield was as good as or better than control conditions in all species. 4389 of the 67,789 genes (6.4%) in the reference genome were differentially expressed during drought among four Miscanthus genotypes from different species. We observed the same biological processes were regulated across Miscanthus species during drought stress despite the DEGs being not similar. Upregulated differentially expressed genes were significantly involved in sucrose and starch metabolism, redox, and water and glycerol homeostasis and channel activity. Multiple copies of the starch metabolic enzymes BAM and waxy GBSS-I were strongly up-regulated in drought stress in all Miscanthus genotypes, and 12 aquaporins (PIP1, PIP2 and NIP2) were also up-regulated in drought stress across genotypes. Conclusions Different phenotypic responses were observed during drought stress among Miscanthus genotypes from different species, supporting differences in genetic adaption. The low number of DEGs and higher biomass yield in flooded conditions supported Miscanthus use in flooded land. The molecular processes regulated during drought were shared among Miscanthus species and consistent with functional categories known to be critical during drought stress in model organisms. However, differences in the regulated genes, likely associated with ploidy and heterosis, highlighted the value of exploring its diversity for breeding.
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Akob, Denise M., Shaun M. Baesman, John M. Sutton, Janna L. Fierst, Adam C. Mumford, Yesha Shrestha, Amisha T. Poret-Peterson, et al. "Detection of Diazotrophy in the Acetylene-Fermenting Anaerobe Pelobacter sp. Strain SFB93." Applied and Environmental Microbiology 83, no. 17 (June 30, 2017). http://dx.doi.org/10.1128/aem.01198-17.
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ABSTRACT Acetylene (C2H2) is a trace constituent of the present Earth's oxidizing atmosphere, reflecting a mixture of terrestrial and marine emissions from anthropogenic, biomass-burning, and unidentified biogenic sources. Fermentation of acetylene was serendipitously discovered during C2H2 block assays of N2O reductase, and Pelobacter acetylenicus was shown to grow on C2H2 via acetylene hydratase (AH). AH is a W-containing, catabolic, low-redox-potential enzyme that, unlike nitrogenase (N2ase), is specific for acetylene. Acetylene fermentation is a rare metabolic process that is well characterized only in P. acetylenicus DSM3246 and DSM3247 and Pelobacter sp. strain SFB93. To better understand the genetic controls for AH activity, we sequenced the genomes of the three acetylene-fermenting Pelobacter strains. Genome assembly and annotation produced three novel genomes containing gene sequences for AH, with two copies being present in SFB93. In addition, gene sequences for all five compulsory genes for iron-molybdenum N2ase were also present in the three genomes, indicating the cooccurrence of two acetylene transformation pathways. Nitrogen fixation growth assays showed that DSM3426 could ferment acetylene in the absence of ammonium, but no ethylene was produced. However, SFB93 degraded acetylene and, in the absence of ammonium, produced ethylene, indicating an active N2ase. Diazotrophic growth was observed under N2 but not in experimental controls incubated under argon. SFB93 exhibits acetylene fermentation and nitrogen fixation, the only known biochemical mechanisms for acetylene transformation. Our results indicate complex interactions between N2ase and AH and suggest novel evolutionary pathways for these relic enzymes from early Earth to modern days. IMPORTANCE Here we show that a single Pelobacter strain can grow via acetylene fermentation and carry out nitrogen fixation, using the only two enzymes known to transform acetylene. These findings provide new insights into acetylene transformations and adaptations for nutrient (C and N) and energy acquisition by microorganisms. Enhanced understanding of acetylene transformations (i.e., extent, occurrence, and rates) in modern environments is important for the use of acetylene as a potential biomarker for extraterrestrial life and for degradation of anthropogenic contaminants.
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Calabresi, Steven G. "Does Institutional Design Make a Difference? / O Desenho Institucional faz Diferença?" Revista Direito, Estado e Sociedade, no. 45 (March 3, 2015). http://dx.doi.org/10.17808/des.45.436.
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Abstract:The goal of essay is to identify two key features of U.S. constitutional design integral to the success of U.S. federal and presidential separation of powers, but which are not widely known and are therefore not widely copied when newly emerging democracies around the world choose to write a constitution. First, the focus is on the fact that American federalism is characterized by a much larger number of state entities than exist in most federal regimes and on the fact that state boundary lines are drawn pretty arbitrarily and cross-cut regional and ethnic cleavages. In Part II, the focus is on five features of the U.S. system of presidential separation of powers which make out presidents much weaker than the presidents of other countries with presidential systems.Keywords: Institutional Design; Federalism; Separation of Powers; Presidentialism. Resumo:O objetivo deste artigo é identificar as duas principais características do desenho constitucional dos Estados Unidos que são essenciais para o sucesso da sua separação dos poderes nos planos federativo e presidencialista, mas que não são amplamente conhecidas e, portanto, não são copiadas por democracias emergentes ao redor do mundo no momento em que formulam suas constituições. Primeiro, o foco do artigo se concentra no fato de que o federalismo americano se caracteriza por um número de entidades estatais muito maior do que se nota na maior parte dos demais regimes federativos, assim como no fato de que as linhas divisoras dos estados são desenhadas, de certa forma, de maneira arbitrária, ignorando diferenças regionais e étnicas. Na parte II, o foco se mantém nas cinco características do sistema de separação de poderes dos Estados Unidos que tornam o presidente muito mais frágil do que os presidentes de outros países com sistemas presidencialistas.
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Banerjee, Richik. "Locating an efficacy of the humane time in Ray’s Agantuk: a travel beyond the object." Rupkatha Journal on Interdisciplinary Studies in Humanities 13, no. 4 (December 11, 2021). http://dx.doi.org/10.21659/rupkatha.v13n4.45.
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The ontology of time and space has always been a subject of materialist prospectus bearing a halo effect of ‘modernity’ and ‘progress’. The enquiry into the sign of modern is a mechanical category of production where substantial copies of ‘progress’ have religiously been equated with a break from the past. This breaking away from the centre (soul) is, of course, associated with a desire for the non-native design. Simultaneously, the past becomes historicized as primitive dangers while the present/‘modern’ morphs into a non-past spectacular diffusion. Satyajit Ray reloads his artillery of the cerebral one last time in his masterpiece titled, Agantuk (The Stranger), where he pits the idea of a spectral past having an agency to redo the class binary against the totalitarian time(s) in a modern urban space which prides itself on the abuse of power-as-civility. Ray introduces a nuclear family of three (a married couple and their son) where the protagonist, Manmohan Mitra, returns as an archived data in the body of a forgotten relative. His entry into the house ruptures the canny knots of the ‘home’ where the director exposes limits of the modernized time. This paper tries to analyze how Ray uses the motif of ‘travel’ in its cinematic cloth to critique the ingestion of global progress as nothing but an accumulation of fallen spectacles that commodify both a subject who is consuming the object-in-time (progress) and also the object that is all the time getting alienated from its own subjective merit. Mitra becomes the mouthpiece of the director for conveying the paradoxes of time-as-capital in the burgeoning of speculative modernity.