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Synthetic Strategies For Tailoring The Physicochemical And Magnetic Properties Of Hydrophobic Magnetic Ionic Liquids, Omprakash Nacham, Kevin D. Clark, Honglian Yu, Jared L. Anderson
Synthetic Strategies For Tailoring The Physicochemical And Magnetic Properties Of Hydrophobic Magnetic Ionic Liquids, Omprakash Nacham, Kevin D. Clark, Honglian Yu, Jared L. Anderson
Jared L. Anderson
Magnetic ionic liquids (MILs) are a subclass of ionic liquids (ILs) containing paramagnetic components and are readily manipulated by an external magnetic field. Due to their hydrophilic nature, very few applications of MILs in aqueous systems have been reported. In this study, three general classes of hydrophobic MILs including monocationic, symmetrical/unsymmetrical dicationic, and symmetrical/unsymmetrical tricationic MILs were synthesized and characterized. By tuning the structure of the MIL, various physicochemical properties including water solubility, magnetic susceptibility, and melting point were regulated. MILs synthesized with the benzimidazolium cation were shown to exhibit lower water solubility (0.1% (w/v)) when compared to those containing …
Extraction Of Dna By Magnetic Ionic Liquids: Tunable Solvents For Rapid And Selective Dna Analysis, Kevin D. Clark, Omprakash Nacham, Honglian Yu, Tianhao Li, Melissa M. Yamsek, Donald R. Ronning, Jared L. Anderson
Extraction Of Dna By Magnetic Ionic Liquids: Tunable Solvents For Rapid And Selective Dna Analysis, Kevin D. Clark, Omprakash Nacham, Honglian Yu, Tianhao Li, Melissa M. Yamsek, Donald R. Ronning, Jared L. Anderson
Jared L. Anderson
DNA extraction represents a significant bottleneck in nucleic acid analysis. In this study, hydrophobic magnetic ionic liquids (MILs) were synthesized and employed as solvents for the rapid and efficient extraction of DNA from aqueous solution. The DNA-enriched microdroplets were manipulated by application of a magnetic field. The three MILs examined in this study exhibited unique DNA extraction capabilities when applied toward a variety of DNA samples and matrices. High extraction efficiencies were obtained for smaller single-stranded and double-stranded DNA using the benzyltrioctylammonium bromotrichloroferrate(III) ([(C8)3BnN+][FeCl3Br–]) MIL, while the dicationic 1,12-di(3-hexadecylbenzimidazolium)dodecane bis[(trifluoromethyl)sulfonyl]imide bromotrichloroferrate(III) ([(C16BnIM)2C122+][NTf2–, FeCl3Br–]) MIL produced higher extraction efficiencies for larger …
Repeated Stressors In Adulthood Increase The Rate Of Biological Ageing, Michaela Hau, Mark F. Haussmann, Timothy J. Greives, Christa Matlack, David Costantini, Michael Quetting, James S. Adelman, Ana C. Miranda, Jesko Partecke
Repeated Stressors In Adulthood Increase The Rate Of Biological Ageing, Michaela Hau, Mark F. Haussmann, Timothy J. Greives, Christa Matlack, David Costantini, Michael Quetting, James S. Adelman, Ana C. Miranda, Jesko Partecke
James S. Adelman
Individuals of the same age can differ substantially in the degree to which they have accumulated tissue damage, akin to bodily wear and tear, from past experiences. This accumulated tissue damage reflects the individual’s biological age and may better predict physiological and behavioural performance than the individual‘s chronological age. However, at present it remains unclear how to reliably assess biological age in individual wild vertebrates. We exposed hand-raised adult Eurasian blackbirds (Turdus merula) to a combination of repeated immune and disturbance stressors for over one year to determine the effects of chronic stress on potential biomarkers of biological ageing including …
Interplay Of Hydrogen Bonds And N→Π* Interactions In Proteins, Gail J. Bartlett, Robert W. Newberry, Brett Vanveller, Ronald T. Raines, Derek N. Woolfson
Interplay Of Hydrogen Bonds And N→Π* Interactions In Proteins, Gail J. Bartlett, Robert W. Newberry, Brett Vanveller, Ronald T. Raines, Derek N. Woolfson
Brett VanVeller
Protein structures are stabilized by multiple weak interactions, including the hydrophobic effect, hydrogen bonds, electrostatic effects, and van der Waals interactions. Among these interactions, the hydrogen bond is distinct in having its origins in electron delocalization. Recently, another type of electron delocalization, the n→π* interaction between carbonyl groups, has been shown to play a role in stabilizing protein structure. Here we examine the interplay between hydrogen bonding and n→π* interactions. To address this issue, we used data available from high-resolution protein crystal structures to interrogate asparagine side-chain oxygen atoms that are both acceptors of a hydrogen bond and donors of …
Detection Of Boronic Acids Through Excited-State Intramolecular Proton-Transfer Fluorescence, Matthew R. Aronoff, Brett Vanveller, Ronald T. Raines
Detection Of Boronic Acids Through Excited-State Intramolecular Proton-Transfer Fluorescence, Matthew R. Aronoff, Brett Vanveller, Ronald T. Raines
Brett VanVeller
Boronic acids are versatile reagents for the chemical synthesis of organic molecules. They and other boron-containing compounds can be detected readily by the interruption of the excited-state intramolecular proton transfer (ESIPT) of 10-hydroxybenzo[h]quinolone. This method is highly sensitive and selective, and useful for monitoring synthetic reactions and detecting boron-containing compounds on a solid support.
A Divalent Protecting Group For Benzoxaboroles, Brett Vanveller, Matthew R. Aronoff, Ronald T. Raines
A Divalent Protecting Group For Benzoxaboroles, Brett Vanveller, Matthew R. Aronoff, Ronald T. Raines
Brett VanVeller
1-Dimethylamino-8-methylaminonaphthalene is put forth as a protecting group for benzoxaboroles. The ensuing complex is fluorescent, charge-neutral, highly stable under basic conditions, stable to anhydrous acid, and readily cleavable in aqueous acid to return the free benzoxaborole.
N→Π* Interactions Of Amides And Thioamides: Implications For Protein Stability, Robert W. Newberry, Brett Vanveller, Ilia A. Guzei, Ronald T. Raines
N→Π* Interactions Of Amides And Thioamides: Implications For Protein Stability, Robert W. Newberry, Brett Vanveller, Ilia A. Guzei, Ronald T. Raines
Brett VanVeller
Carbonyl–carbonyl interactions between adjacent backbone amides have been implicated in the conformational stability of proteins. By combining experimental and computational approaches, we show that relevant amidic carbonyl groups associate through an n→π* donor–acceptor interaction with an energy of at least 0.27 kcal/mol. The n→π* interaction between two thioamides is 3-fold stronger than between two oxoamides due to increased overlap and reduced energy difference between the donor and acceptor orbitals. This result suggests that backbone thioamide incorporation could stabilize protein structures. Finally, we demonstrate that intimate carbonyl interactions are described more completely as donor–acceptor orbital interactions rather than dipole–dipole interactions.
Measuring Markers Of Liver Function Using A Micropatterned Paper Device Designed For Blood From A Fingerstick, Sarah J. Vella, Patrick Beattie, Rebecca Cademartiri, Anna Laromaine, Andres W. Martinez, Scott T. Phillips, Katherine A. Mirica, George M. Whitesides
Measuring Markers Of Liver Function Using A Micropatterned Paper Device Designed For Blood From A Fingerstick, Sarah J. Vella, Patrick Beattie, Rebecca Cademartiri, Anna Laromaine, Andres W. Martinez, Scott T. Phillips, Katherine A. Mirica, George M. Whitesides
Rebecca Cademartiri
This paper describes a paper-based microfluidic device that measures two enzymatic markers of liver function (alkaline phosphatase, ALP, and aspartate aminotransferase, AST) and total serum protein. A device consists of four components: (i) a top plastic sheet, (ii) a filter membrane, (iii) a patterned paper chip containing the reagents necessary for analysis, and (iv) a bottom plastic sheet. The device performs both the sample preparation (separating blood plasma from erythrocytes) and the assays; it also enables both qualitative and quantitative analysis of data. The data obtained from the paper-microfluidic devices show standard deviations in calibration runs and “spiked” standards that …
Nmr Detection And Characterization Of Sialylated Glycoproteins And Cell Surface Polysaccharides, Adam Barb, Darón I. Freedberg, Marcos D. Battistel, James H. Prestegard
Nmr Detection And Characterization Of Sialylated Glycoproteins And Cell Surface Polysaccharides, Adam Barb, Darón I. Freedberg, Marcos D. Battistel, James H. Prestegard
Adam Barb
Few solution NMR pulse sequences exist that are explicitly designed to characterize carbohydrates (glycans). This is despite the essential role carbohydrate motifs play in cell–cell communication, microbial pathogenesis, autoimmune disease progression and cancer metastasis, and despite that fact that glycans, often shed to extra-cellular fluids, can be diagnostic of disease. Here we present a suite of two dimensional coherence experiments to measure three different correlations (H3–C2, H3–C1, and C1–C2) on sialic acids, a group of nine-carbon carbohydrates found on eukaryotic cell surfaces that often play a key role in disease processes. The chemical shifts of the H3, C2, and C1 …
Structures Of Domains I And Iv From Ybbr Are Representative Of A Widely Distributed Protein Family, Adam Barb, John R. Cort, Jayaraman Seetharaman, Scott Lew, Hsiau-Wei Lee, Thomas Acton, Rong Xiao, Michael A. Kenndy, Liang Tong, Gaetano T. Montelione, James H. Prestegard
Structures Of Domains I And Iv From Ybbr Are Representative Of A Widely Distributed Protein Family, Adam Barb, John R. Cort, Jayaraman Seetharaman, Scott Lew, Hsiau-Wei Lee, Thomas Acton, Rong Xiao, Michael A. Kenndy, Liang Tong, Gaetano T. Montelione, James H. Prestegard
Adam Barb
YbbR domains are widespread throughout Eubacteria and are expressed as monomeric units, linked in tandem repeats or cotranslated with other domains. Although the precise role of these domains remains undefined, the location of the multiple YbbR domain-encoding ybbR gene in the Bacillus subtilis glmM operon and its previous identification as a substrate for a surfactin-type phosphopantetheinyl transferase suggests a role in cell growth, division, and virulence. To further characterize the YbbR domains, structures of two of the four domains (I and IV) from the YbbR-like protein of Desulfitobacterium hafniense Y51 were solved by solution nuclear magnetic resonance and X-ray crystallography. …
Protection Of Retinal Cells From Ischemia By A Novel Gap Junction Inhibitor, Satyabrata Das, Dingo Lin, Snehalata Jena, Aibin Shi, Srinivas Battina, Duy H. Hua, Rachel A. Allbaugh, Dolores J. Takemoto
Protection Of Retinal Cells From Ischemia By A Novel Gap Junction Inhibitor, Satyabrata Das, Dingo Lin, Snehalata Jena, Aibin Shi, Srinivas Battina, Duy H. Hua, Rachel A. Allbaugh, Dolores J. Takemoto
Rachel A. Allbaugh
Retinal cells which become ischemic will pass apoptotic signal to adjacent cells, resulting in the spread of damage. This occurs through open gap junctions. A class of novel drugs, based on primaquine (PQ), was tested for binding to connexin 43 using simulated docking studies. A novel drug has been synthesized and tested for inhibition of gap junction activity using R28 neuro-retinal cells in culture. Four drugs were initially compared to mefloquine, a known gap junction inhibitor. The drug with optimal inhibitory activity, PQ1, was tested for inhibition and was found to inhibit dye transfer by 70% at 10 μM. Retinal …
Mechanism Of High-Mobility Group Protein B Enhancement Of Progesterone Receptor Sequence-Specific Dna Binding, James S. Adelman, Sarah C. Roemer, Mair E.A. Churchill, Dean P. Edwards
Mechanism Of High-Mobility Group Protein B Enhancement Of Progesterone Receptor Sequence-Specific Dna Binding, James S. Adelman, Sarah C. Roemer, Mair E.A. Churchill, Dean P. Edwards
James S. Adelman
The DNA-binding domain (DBD) of progesterone receptor (PR) is bipartite containing a zinc module core that interacts with progesterone response elements (PRE), and a short flexible carboxyl terminal extension (CTE) that interacts with the minor groove flanking the PRE. The chromosomal high-mobility group B proteins (HMGB), defined as DNA architectural proteins capable of bending DNA, also function as auxiliary factors that increase the DNA-binding affinity of PR and other steroid receptors by mechanisms that are not well defined. Here we show that the CTE of PR contains a specific binding site for HMGB that is required for stimulation of PR-PRE …
Surface Modification Of Cetyltrimethylammonium Bromide-Capped Gold Nanorods To Make Molecular Probes, Chenxu Yu, Leo Varghese, Joseph Irudayaraj
Surface Modification Of Cetyltrimethylammonium Bromide-Capped Gold Nanorods To Make Molecular Probes, Chenxu Yu, Leo Varghese, Joseph Irudayaraj
Chenxu Yu
A chemical procedure to replace the cetyltrimethylammonium bromide (CTAB) cap on gold nanorods (GNRs) fabricated through seed-mediated growth with organothiol compounds [3-animo-5-mercapto-1,2,4-triazole (AMTAZ) and 11-mercaptoundecaonic acid (MUDA)] was developed to reduce the cytotoxity of GNRs and facilitate further biofunctionalization. Compared to phosphatidylcholine (PC) modification, our procedure yields stable GNRs that are biocompatible and suitable for whole-cell studies. The PC-, AMTAZ-, and MUDA-activated GNRs all showed low cytotoxicity. By choosing different organothiols, net positive or negative charges could be created on the nanorod surface, for different applications. Gold nanorod molecular probes (GNrMPs) were fabricated by subsequent attachment of antibodies to the …
Identity Profiling Of Cell Surface Markers By Multiplex Gold Nanorod Probes, Chenxu Yu, Harikrishna Nakshatri, Joseph Irudayaraj
Identity Profiling Of Cell Surface Markers By Multiplex Gold Nanorod Probes, Chenxu Yu, Harikrishna Nakshatri, Joseph Irudayaraj
Chenxu Yu
Gold nanorod molecular probes (GNrMPs) were designed and fabricated for multiplex identification of cell surface markers in HBECs. Cells were probed directly using dark field microscopy integrated with a spectral imager for simultaneous detection of up to three surface markers. The immunophenotype composition of these cell lines indicative of their metastasis potential was assessed using the GNrMPs. The technique has the potential to become an important tool for diagnosis and prognosis of breast and other cancers.
Multiplex Biosensor Using Gold Nanorods, Chenxu Yu, Joseph Irudayaraj
Multiplex Biosensor Using Gold Nanorods, Chenxu Yu, Joseph Irudayaraj
Chenxu Yu
Gold nanorods (GNRs) with different aspect ratios were fabricated through seed-mediated growth and surface activation by alkanethiols for the attachment of antibodies to yield gold nanorod molecular probes (GNrMPs). Multiplex sensing was demonstrated by the distinct response of the plasmon spectra of the GNrMPs to binding events of three targets (goat anti-human IgG1 Fab, rabbit anti-mouse IgG1 Fab, rabbit anti-sheep IgG (H+L)). Plasmonic sensors are highly specific and sensitive and can be used to monitor refractive index changes caused by molecular interactions in their immediate vicinity with potential to achieve single-particle biosensing. This technique can play a key role in …
Molecular Validation Of Lpxc As An Antibacterial Drug Target In Pseudomonas Aeruginosa, Adam Barb, Khisimuzi E. Mdluli, Pamela R. Witte, Toni Kline, Alice L. Erwin, Bryce E. Mansfield, Amanda L. Mcclerren, Michael C. Pirrung, L. Nathan Tumey, Paul Warrener, Christian R. H. Raetz, C. Kenall Stover
Molecular Validation Of Lpxc As An Antibacterial Drug Target In Pseudomonas Aeruginosa, Adam Barb, Khisimuzi E. Mdluli, Pamela R. Witte, Toni Kline, Alice L. Erwin, Bryce E. Mansfield, Amanda L. Mcclerren, Michael C. Pirrung, L. Nathan Tumey, Paul Warrener, Christian R. H. Raetz, C. Kenall Stover
Adam Barb
LpxC [UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc deacetylase] is a metalloamidase that catalyzes the first committed step in the biosynthesis of the lipid A component of lipopolysaccharide. A previous study (H. R. Onishi, B. A. Pelak, L. S. Gerckens, L. L. Silver, F. M. Kahan, M. H. Chen, A. A. Patchett, S. M. Galloway, S. A. Hyland, M. S. Anderson, and C. R. H. Raetz, Science 274:980-982, 1996) identified a series of synthetic LpxC-inhibitory molecules that were bactericidal for Escherichia coli. These molecules did not inhibit the growth of Pseudomonas aeruginosa and were therefore not developed further as antibacterial drugs. The inactivity of the LpxC …
Mid-Ir Biosensor: Detection And Fingerprinting Of Pathogens On Gold Island Functionalized Chalcogenide Films, Chenxu Yu, Ashtosh Ganjoo, H. Jain, C. G. Pantano, Joseph Irudayaraj
Mid-Ir Biosensor: Detection And Fingerprinting Of Pathogens On Gold Island Functionalized Chalcogenide Films, Chenxu Yu, Ashtosh Ganjoo, H. Jain, C. G. Pantano, Joseph Irudayaraj
Chenxu Yu
Antibody (human IgG, anti-E. coli O157:H7, and anti-Salmonella) complexes on the surface of IR-transparent Ge-containing chalcogenide glass films were formed via thiol chemistry on 20-nm-thick gold islands. As a first step, the protocol was validated by monitoring fluorescently tagged targets to validate binding. FT-IR spectroscopy confirmed that the coating of the films with 20-nm gold did not have a significant effect on the propagation and penetration of IR evanescent waves through the film. The films functionalized with anti-E. coli O157:H7 and anti-Salmonella antibodies were used to detect E. coli O157:H7 and S. enteriditis through label-free IR fingerprinting. Highly selective detection …
Bifunctional Abietadiene Synthase: Mutual Structural Dependence Of The Active Sites For Protonation-Initiated And Ionization-Initiated Cyclizations, Reuben J. Peters, Ora A. Carter, Yan Zhang, Brian W. Matthews, Rodney B. Croteau
Bifunctional Abietadiene Synthase: Mutual Structural Dependence Of The Active Sites For Protonation-Initiated And Ionization-Initiated Cyclizations, Reuben J. Peters, Ora A. Carter, Yan Zhang, Brian W. Matthews, Rodney B. Croteau
Reuben J. Peters
Abietadiene synthase from grand fir catalyzes two sequential, mechanistically distinct cyclizations, of geranylgeranyl diphosphate and of copalyl diphosphate, in the formation of a mixture of abietadiene isomers as the committed step of diterpenoid resin acid biosynthesis. Each reaction is independently conducted at a separate active site residing in what were considered to be structurally distinct domains typical of terpene cyclases. Despite the presence of an unusual 250-residue N-terminal insertional element, a tandem pair of charged residues distal to the insertion was shown to form a functional part of the C-terminal active site. Because abietadiene synthase resembles the ancestral plant terpene …
Jun Dimerization Protein 2 Functions As A Progesterone Receptor N-Terminal Domain Coactivator, James S. Adelman, Suzanne E. Wardell, Viroj Boonyaratanakornkit, Ami Aronheim
Jun Dimerization Protein 2 Functions As A Progesterone Receptor N-Terminal Domain Coactivator, James S. Adelman, Suzanne E. Wardell, Viroj Boonyaratanakornkit, Ami Aronheim
James S. Adelman
The progesterone receptor (PR) contains two transcription activation function (AF) domains, constitutive AF-1 in the N terminus and AF-2 in the C terminus. AF-2 activity is mediated by a hormone-dependent interaction with a family of steroid receptor coactivators (SRCs). SRC-1 can also stimulate AF-1 activity through a secondary domain that interacts simultaneously with the primary AF-2 interaction site. Other protein interactions and mechanisms that mediate AF-1 activity are not well defined. By interaction cloning, we identified an AP-1 family member, Jun dimerization protein 2 (JDP-2), as a novel PR-interacting protein. JDP-2 was first defined as a c-Jun interacting protein that …
Mechanism Of Abietadiene Synthase Catalysis: Stereochemistry And Stabilization Of The Cryptic Pimarenyl Carbocation Intermediates, Reuben J. Peters, Matthew M. Ravn, Robert M. Coates, Rodney Croteau
Mechanism Of Abietadiene Synthase Catalysis: Stereochemistry And Stabilization Of The Cryptic Pimarenyl Carbocation Intermediates, Reuben J. Peters, Matthew M. Ravn, Robert M. Coates, Rodney Croteau
Reuben J. Peters
Abietadiene synthase (AS) catalyzes the complex cyclization-rearrangement of (E,E,E)-geranylgeranyl diphosphate (8, GGPP) to a mixture of abietadiene (1a), double bond isomers 2a-4a and pimaradienes 5a-7a as a key step in the biosynthesis of the abietane resin acid constituents (1b-4b) of conifer oleoresin. The reaction proceeds at two active sites by way of the intermediate, copalyl diphosphate (9). In the second site, a putative tricyclic pimaradiene or pimarenyl(+) carbocation intermediate of undefined C13 stereochemistry and annular double bond position is formed. Three 8-oxy-17-nor analogues of 9 (17 and 19a,b) and three isomeric 15,16-bisnorpimarenyl-N-methylamines (26a-c) were synthesized and evaluated as alternative substrates …
Abietadiene Synthase Catalysis: Conserved Residues Involved In Protonation-Initiated Cyclization Of Geranylgeranyl Diphosphate To (+)-Copalyl Diphosphate, Reuben J. Peters, Rodney B. Croteau
Abietadiene Synthase Catalysis: Conserved Residues Involved In Protonation-Initiated Cyclization Of Geranylgeranyl Diphosphate To (+)-Copalyl Diphosphate, Reuben J. Peters, Rodney B. Croteau
Reuben J. Peters
Abietadiene synthase catalyzes two sequential, mechanistically distinct cyclization reactions in the formation of a mixture of abietadiene double bond isomers as the committed step in resin acid biosynthesis. Each reaction is carried out at a separate active site residing in a structurally distinct domain, and the reactions are kinetically separable. The first cyclization reaction is initiated by protonation of the terminal double bond of the universal diterpene precursor, geranylgeranyl diphosphate. The pH dependence of the overall reaction is consistent with an acid-base catalytic mechanism, and a divalent metal ion plays a role in this reaction probably by binding the diphosphate …
Phylogeographic Analysis Of The Threatened And Endangered Superconglutinate-Producing Mussels Of The Genus Lampsilis (Bivalvia: Unionidae), Kevin J. Roe, Charles Lydeard, Paul D. Hartfield
Phylogeographic Analysis Of The Threatened And Endangered Superconglutinate-Producing Mussels Of The Genus Lampsilis (Bivalvia: Unionidae), Kevin J. Roe, Charles Lydeard, Paul D. Hartfield
Kevin J. Roe
Several species of freshwater unionid mussels in the genus Lampsilis exhibit a remarkable reproductive strategy. Female mussels of these species enclose their larvae in a minnow-like lure, called a ‘superconglutinate’, to attract piscivorous fishes. When a fish attempts to ingest the superconglutinate the lure ruptures and the larvae are released to parasitize the fish. Of the four species of mussel which exhibit this strategy and are endemic to the Gulf Coast drainages of the southeastern United States, three are protected under the Endangered Species Act, and one is recognized as imperilled. Phylogenetic analysis of nucleotide sequences of the mitochondrial 16S …
Endocrine Responses To Chronic Androstenedione Intake In 30- To 56-Year-Old Men, Gregory A. Brown, Matthew D. Vukovich, Emily R. Martini, Marian L. Kohut, Warren D. Franke, David A. Jackson, Douglas S. King
Endocrine Responses To Chronic Androstenedione Intake In 30- To 56-Year-Old Men, Gregory A. Brown, Matthew D. Vukovich, Emily R. Martini, Marian L. Kohut, Warren D. Franke, David A. Jackson, Douglas S. King
Warren D Franke
In young men, chronic ingestion of 100 mg androstenedione (ASD), three times per day, does not increase serum total testosterone but does increase serum estrogen and ASD concentrations. We investigated the effects of ASD ingestion in healthy 30- to 56-yr-old men. In a double-blind, randomly assigned manner, subjects consumed 100 mg ASD three times daily (n 5 28), or placebo (n 5 27) for 28 days. Serum ASD , dihydrotestosterone (DHT), free and total testosterone, estradiol, prostate-specific antigen (PSA), and lipid concentrations were measured at week 0 and each week throughout the supplementation period. Serum total testosterone and PSA concentrations …
Development Of An Enzyme-Linked Immunosorbent Assay (Elisa) For Quantification Of Skeletal Muscle Calpastatin, M. E. Doumit, Steven M. Lonergan, J. R. Arbona, J. Killefer, M. Koohmaraie
Development Of An Enzyme-Linked Immunosorbent Assay (Elisa) For Quantification Of Skeletal Muscle Calpastatin, M. E. Doumit, Steven M. Lonergan, J. R. Arbona, J. Killefer, M. Koohmaraie
Steven M. Lonergan
An indirect antibody ELISA was developed for rapid and sensitive quantification of skeletal muscle calpastatin. Polyclonal antibodies were raised in rabbits against recombinant calpastatin, corresponding to domains 2, 3, and 4 of bovine skeletal muscle calpastatin. Western blot analysis revealed that these antibodies specifically recognize an immunoreactive calpastatin protein of approximately 130 kDa in prerigor skeletal muscle extracts. The intensity of the immunoreactive bands corresponds qualitatively with assayable calpastatin activity. For ELISA development, optimum dilutions of sample, primary anti-calpastatin antibody, and peroxidase-conjugated secondary antibody were determined by titration. A dilution optimum for coating of Immulonâ 4 (Dynatech) plates was observed …
Light-Dependent And Tissue-Specific Expression Of The H-Protein Of The Glycine Decarboxylase Complex, R. Srinivasan, David J. Oliver
Light-Dependent And Tissue-Specific Expression Of The H-Protein Of The Glycine Decarboxylase Complex, R. Srinivasan, David J. Oliver
David J. Oliver
Glycine decarboxylase is a mitochondrial enzyme complex, which is the site of photorespiratory CO2 and NH3 release. Although the proteins that constitute the complex are located within the mitochondria, because of their intimate association with photosynthesis their expression is controlled by light. Comparisons of the kinetics of mRNA accumulation between the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and the H-protein of glycine decarboxylase during the greening of etiolated Arabidopsis thaliana suggest that their expression is controlled in parallel. A genomic clone for the H-protein (gdcH) was isolated from Arabidopsis and sequenced. The upstream region from -856 to +62 was fused to …
The Phosphate Transporter From Pea Mitochondria (Isolation And Characterization In Proteolipid Vesicles), Cecilia A. Mcintosh, David J. Oliver
The Phosphate Transporter From Pea Mitochondria (Isolation And Characterization In Proteolipid Vesicles), Cecilia A. Mcintosh, David J. Oliver
David J. Oliver
The phosphate transporter from mitochondria will exchange matrix phosphate for cytosolic phosphate and facilitate either phosphate/proton symport or phosphate/hydroxyl ion antiport. The phosphate transported into the matrix by this carrier is either used for ATP synthesis or exchanges back out to the cytosol on the dicarboxylate transporter, permitting entry of malate and succinate into the matrix. The phosphate transporter was solubilized from etiolated pea (Pisum sativum L. cv Alaska) mitochondrial membranes with Triton X-114, purified approximately 500-fold by hydroxylapatite chromatography, and reconstituted into azolectin vesicles that were preloaded with 0.1 or 10 mM phosphate. Phosphate transport was measured as the …
Isolation And Characterization Of The Tricarboxylate Transporter From Pea Mitochondria, Cecilia A. Mcintosh, David J. Oliver
Isolation And Characterization Of The Tricarboxylate Transporter From Pea Mitochondria, Cecilia A. Mcintosh, David J. Oliver
David J. Oliver
The tricarboxylate transporter was solubilized from pea (Pisum sativum) mitochondria with Triton X-114, partially purified over a hydroxylapatite column, and reconstituted in phospholipid vesicles. The proteoliposomes exchanged external [14C]citrate for internal citrate or malate but not for preloaded D,L-isocitrate. Similarly, although external malate, succinate, and citrate competed with [14C]citrate in the exchange reaction, D,L-isocitrate and phosphoenolpyruvate did not. This tricarboxylate transporter differed from the equivalent activity from animal tissues in that it did not transport isocitrate and phosphoenolpyruvate. In addition, tricarboxylate transport in isolated plant mitochondria, as well as that measured with the partially purified and reconstituted transporter, was less …
Nad+-Linked Isocitrate Dehydrogenase: Isolation, Purification, And Characterization Of The Protein From Pea Mitochondria, Cecilia A. Mcintosh, David J. Oliver
Nad+-Linked Isocitrate Dehydrogenase: Isolation, Purification, And Characterization Of The Protein From Pea Mitochondria, Cecilia A. Mcintosh, David J. Oliver
David J. Oliver
The NAD+-dependent isocitrate dehydrogenase from etiolated pea (Pisum sativum L.) mitochondria was purified more than 200-fold by dye-ligand binding on Matrix Gel Blue A and gel filtration on Superose 6. The enzyme was stabilized during purification by the inclusion of 20% glycerol. In crude matrix extracts, the enzyme activity eluted from Superose 6 with apparent molecular masses of 1400 ± 200, 690 ± 90, and 300 ± 50 kD. During subsequent purification steps the larger molecular mass species disappeared and an additional peak at 94 ± 16 kD was evident. The monomer for the enzyme was tentatively identified at 47 …
H-Protein Of The Glycine Decarboxylase Multienzyme Complex: Complementary Dna Encoding The Protein From Arabidopsis Thaliana, David J. Oliver
H-Protein Of The Glycine Decarboxylase Multienzyme Complex: Complementary Dna Encoding The Protein From Arabidopsis Thaliana, David J. Oliver
David J. Oliver
The glycine decarboxylase multienzyme complex is composed of four component enzymes, the 100-kD P-protein (a pyridoxal 5-phosphate-dependent amino acid decarboxylase), the 14-kD H-protein (a lipoamide-containing carrier protein), the 45-kD T-protein (a tetrahydrofolate transferase), and the 59-kD L-protein (lipoamide dehydrogenase) (5). The Iipoamide cofactor of the H-protein carries reaction intermediates between the active sites of the other three component proteins. These proteins form a large molecular weight complex (4) within the matrix ofleaf mitochondria where, together with the enzyme serine hydroxymethyltransferase, it catalyzes the photorespiratory conversion of two molecules of glycine to one each of serine, NH3, and CO2 with the …
Lignin Distribution In Thermomechanically Pulped Wood Fibers Via X-Ray Microanalysis, Douglas Stokke, Xuerong Wang
Lignin Distribution In Thermomechanically Pulped Wood Fibers Via X-Ray Microanalysis, Douglas Stokke, Xuerong Wang
Douglas D. Stokke
The three major framework organic constituents of the secondary xylem (wood) of trees are cellulose, hemicelluloses, and lignin. Lignin imparts a number of important physical properties to the woody cell wall. In addition, lignin serves as the "cement" which binds cells together, and is the material which must be softened, altered, or removed during pulping processes. The distribution of lignin within pulp cell walls is of importance with respect to use of the pulp fibers. In this study, x-ray analysis was used to investigate lignin distribution in thermomechanically pulped (i.e., pulped with heat and pressure, with no chemical treatment) aspen …