Elmeer K.,Biotechnology Center
Genetics and molecular research : GMR | Year: 2012
The massive destruction and deterioration of the habitat of Oryx leucoryx and illegal hunting have decimated Oryx populations significantly, and now these animals are almost extinct in the wild. Molecular analyses can significantly contribute to captive breeding and reintroduction strategies for the conservation of this endangered animal. A representative 32 identical sequences used for species identification through BOLD and GenBank/NCBI showed maximum homology 96.06% with O. dammah, which is a species of Oryx from Northern Africa, the next closest species 94.33% was O. gazella, the African antelope. DNA barcode sequences of the mitochondrial cytochrome C oxidase (COI) gene were determined for O. leucoryx; identification through BOLD could only recognize the genus correctly, whereas the species could not be identified. This was due to a lack of sequence data for O. leucoryx on BOLD. Similarly, BLAST analysis of the NCBI data base also revealed no COI sequence data for the genus Oryx.
Loaiza R.,University of Michigan |
Powers P.P.,Biotechnology Center |
Noujaim S.,University of Michigan |
Ackerman M.J.,University of Wisconsin - Madison |
And 3 more authors.
Circulation Research | Year: 2013
Rationale: Most cardiac ryanodine receptor (RyR2) mutations associated with catecholaminergic polymorphic ventricular tachycardia (CPVT) are postulated to cause a distinctive form of Ca release dysfunction. Considering the spread distribution of CPVT mutations, we hypothesized that dysfunctional heterogeneity also was feasible. Objective: To determine the molecular and cellular mechanisms by which a novel RyR2-V2475F mutation associated with CPVT in humans triggers Ca-dependent arrhythmias in whole hearts and intact mice. Methods and Results: Recombinant channels harboring CPVT-linked RyR2 mutations were functionally characterized using tritiated ryanodine binding and single-channel recordings. Homologous recombination was used to generate a knock-in mouse bearing the RyR2-V2475F mutation. Ventricular myocytes from mice heterozygous for the mutation (RyR2-V2475F) and their wild-type littermates were Ca-imaged by confocal microscopy under conditions that mimic stress. The propensity of wild-type and RyR2-V2475F mice to have development of arrhythmias was tested at the whole heart level and in intact animals. Recombinant RyR2-V2475F channels displayed increased cytosolic Ca activation, abnormal protein kinase A phosphorylation, and increased activation by luminal Ca. The RyR2-V2475F mutation appears embryonic-lethal in homozygous mice, but heterozygous mice have no alterations at baseline. Spontaneous Ca release events were more frequent and had shorter latency in isoproterenol-stimulated cardiomyocytes from RyR2-V2475F hearts, but their threshold was unchanged with respect to wild-type. Adrenergically triggered tachyarrhythmias were more frequent in RyR2-V2475F mice. Conclusions: The mutation RyR2-V2475F is phenotypically strong among other CPVT mutations and produces heterogeneous mechanisms of RyR2 dysfunction. In living mice, this mutation appears too severe to be harbored in all RyR2 channels but remains undetected under basal conditions if expressed at relatively low levels. β-adrenergic stimulation breaks the delicate Ca equilibrium of RyR2-V2475F hearts and triggers life-threatening arrhythmias. © 2012 American Heart Association, Inc.
Canton M.,University of Padua |
Menazza S.,University of Padua |
Sheeran F.L.,Monash University |
Polverino De Laureto P.,Biotechnology Center |
And 2 more authors.
Journal of the American College of Cardiology | Year: 2011
Objectives We investigated the incidence and contribution of the oxidation/nitrosylation of tropomyosin and actin to the contractile impairment and cardiomyocyte injury occurring in human end-stage heart failure (HF) as compared with nonfailing donor hearts. Background Although there is growing evidence that augmented intracellular accumulation of reactive oxygen/nitrogen species may play a key role in causing contractile dysfunction, there is a dearth of data regarding their contractile protein targets in human HF. Methods In left ventricular (LV) biopsies from explanted failing hearts (New York Heart Association functional class IV; HF group) and nonfailing donor hearts (NF group), carbonylation of actin and tropomyosin, disulphide cross-bridge (DCB) formation, and S-nitrosylation in tropomyosin were assessed, along with plasma troponin I and LV ejection fraction (LVEF). Results The LV biopsies from the HF group had 2.14 ± 0.23-fold and 2.31 ± 0.46-fold greater levels in actin and tropomyosin carbonylation, respectively, and 1.77 ± 0.45-fold greater levels of high-molecular-weight complexes of tropomyosin due to DCB formation, compared with the NF group. Tropomyosin also underwent S-nitrosylation that was 1.3 ± 0.15-fold higher in the HF group. Notably, actin and tropomyosin carbonylation was significantly correlated with both loss of viability indicated by plasma troponin I and contractile impairment as shown by reduced LVEF. Conclusions This study demonstrated that oxidative/ nitrosylative changes of actin and tropomyosin are largely increased in human failing hearts. Because these changes are inversely correlated to LVEF, actin and tropomyosin oxidation are likely to contribute to the contractile impairment evident in end-stage HF. © 2011 American College of Cardiology Foundation.
Buch A.,Molecular Microbial Biochemistry Laboratory |
Archana G.,Biotechnology Center |
Naresh Kumar G.,Molecular Microbial Biochemistry Laboratory
Bioresource Technology | Year: 2010
The Synechococcus elongatus PCC 6301 phosphoenolpyruvate carboxylase (ppc) gene was constitutively overexpressed in fluorescent pseudomonads, to increase the supply of oxaloacetate, a crucial anabolic precursor and an intermediate in biosynthesis of organic acids implicated in phosphate (P) solubilization. Pseudomonas fluorescens ATCC 13525, transformed with pAB3 plasmid containing the ppc gene showed a 14-fold increase in PPC activity under P-sufficiency resulting in increased carbon flow through the direct oxidative pathway and reduced metabolic overflow. Under P-limitation, contribution of the direct oxidative pathway significantly increased in P. fluorescens ATCC 13525; however, ppc overexpression enhanced glucose catabolism through intracellular phosphorylative pathway. These results correlated with gluconic, pyruvic and acetic acid levels as well as the activities of key glucose catabolic enzymes. Irrespective of the P-status, ppc overexpression improved biomass yield without altering growth rate, resulting in improved P- solubilizing abilities of P. fluorescens ATCC 13525 as well as of the wheat rhizosphere fluorescent pseudomonads isolates Fp585, P109 and Fp315. Collectively, ppc overexpression reversed the P-status dependent glucose distribution between the direct oxidative and phosphorylative pathways of glucose catabolism in P. fluorescens ATCC 13525 and presents a feasible genetic engineering approach for developing efficient P-solubilizing bacteria. © 2009 Elsevier Ltd. All rights reserved.
The studies can shed some light on the perennial question of how life arose, but they also have slightly more practical applications in the search for life in space, says senior author Eric Roden, a professor of geoscience at UW–Madison. Animals use oxygen and "reduce" it to produce water, but some bacteria use iron that is deficient in electrons, reducing it to a more electron-rich form of the element. Ironically, electron-rich forms of iron can also supply electrons in the opposite "oxidation" reaction, in which the bacteria literally "eat" the iron to get energy. Iron is the fourth-most abundant element on the planet, and because free oxygen is scarce underwater and underground, bacteria have "thought up," or evolved, a different solution: moving electrons to iron while metabolizing organic matter. These bacteria "eat organic matter like we do," says Roden. "We pass electrons from organic matter to oxygen. Some of these bacteria use iron oxide as their electron acceptor. On the flip side, some other microbes receive electrons donated by other iron compounds. In both cases, the electron transfer is essential to their energy cycles." Whether the reaction is oxidation or reduction, the ability to move an electron is essential for the bacteria to process energy to power its lifestyle. Roden has spent decades studying iron-metabolizing bacteria. "I focus on the activities and chemical processing of microorganisms in natural systems," he says. "We collect material from the environment, bring it back to the lab, and study the metabolism through a series of geochemical and microbiological measurements." The current studies focus on bacteria samples from Chocolate Pot hot spring, a relatively cool geothermal spring in Yellowstone National Park that is named for the dark, reddish-brown color of ferric oxide. Related studies deal with a culture obtained from a much less auspicious environment—a ditch in Germany. Both studies are online, in Applied and Environmental Microbiology and in Geobiology. During the studies, Roden and doctoral student Nathan Fortney and research scientist Shaomei He explored how the cultured organisms changed the oxidation state—the number of electrons—in the iron compounds. They also used an advanced genome-sequencing instrument at the UW–Madison Biotechnology Center to identify strings of DNA in the genomes. "More than 99 percent of microbial diversity cannot be obtained in pure culture," says He, meaning they cannot be grown as a single strain for analysis. "Instead of going through the long, laborious and often unsuccessful process of isolating strains, we apply genomic tools to understand how the organisms were doing what they were doing in mixed communities." The researchers found some unknown bacteria capable of iron metabolism, and also got genetic data on a unique capacity that some of them have: the ability to transport electrons in both directions across the cell's outer membrane. "Bacteria have not only evolved a metabolism that opens niches to use iron as an energy," says He, "but these new electron transport mechanisms give them a way to use forms of iron that can't be brought inside the cell." "These are fundamental studies, but these chemical transformations are at the heart of all kinds of environmental systems, related to soil, sediment, groundwater and waste water," says Roden. "For example, the Department of Energy is interested in finding a way to derive energy from organic matter through the activity of iron-metabolizing bacteria." These bacteria are also critical to the life-giving process of weathering rocks into soil. Iron-metabolizing bacteria have been known for a century, Roden says, and were actually discovered in Madison-area groundwater. "Geologists saw organisms that formed these unique structures that were visible under the light microscope. They formed stalks or sheaths, and it turned out they were used to move iron." Roden and He are geobiologists, interested in how microbes affect geology, but the significance of microbes in Earth's evolution is only now being fully appreciated, Roden says. "Eyebrows rose when we contacted the Biotech Center three or four year ago to discuss sequencing: 'Who are these people from geology, and what are they talking about?' But we stuck with it, and it's turned into a pretty cool collaboration that has allowed us to apply their excellent tools that are more typically applied to biomedical and related microbial issues." Some of the iron-metabolizing bacteria appear quite early on the tree of life, making the studies relevant to discovering the origins of life, but the findings also have implications in the search for life in space, Roden says. "Our support comes from NASA's astrobiology institute at UW–Madison. It's possible that on a rocky planet like Mars, life could rely on iron metabolism instead of oxygen. "A fundamental approach in astrobiology is to use terrestrial sites as analogs, where we look for insight into the possibilities on other worlds," Roden continues. "Some people believe that use of iron oxide as an electron acceptor could have been the first, or one of the first, forms of respiration on Earth. And there's so much iron around on the rocky planets." Explore further: Organic solids in soil may speed up bacterial breathing More information: N. W. Fortney et al. Microbial Fe(III) oxide reduction potential in Chocolate Pots hot spring, Yellowstone National Park, Geobiology (2016). DOI: 10.1111/gbi.12173