[43 Detection of DNA Base Mismatches Using DNA Intercalators

Kisko, and Jacqueline K. Barton Introduction The most prevalent mechanisms leading to mutations in DNA are direct mis-incorporation of bases during replication and sustained chemical damage. Under normal circumstances, the cell corrects these problems using DNA polymerase proofreading mechanisms as well as the complex repair machinery of the cell. In certain tissues that contain mismatch repair deficiencies, DNA mispairs may accumulate.15 Even in healthy cells,...

Principles

The enzyme-linked immunospot assay is based on incubating live cells, stimulated or unstimulated, in a microtiter plate in which the solid-phase bottom (nitrocellulose, PVDF, or polystyrene) is precoated with a specific monoclonal antibody (MAb), termed the capture Ab, against the antigen, for example, Trx or TrxR (Fig. 1). After an incubation period of 16 to 48 hr, as a rule, the cells are removed, and the position of the captured antigen is visualized at the bottom of the plate by 19 N. K....

Ho1

LPS induces HO-1 protein in heart and lung tissues. Total heart and lung protein was extracted from mice treated with (+) or without (-) LPS for 24 hr. Aliquots (25 xg) were subjected to Western blotting with polyclonal anti-HO-1 antiserum. before incubation with horseradish peroxidase-conjugated goat anti-rabbit serum (Amersham, Piscataway, NJ), diluted 1 4000, for 1 hr at room temperature. The blots are washed with 4 (w v) milk in TBST two times (15 min each), and then with TBST two...

Protein Structure And Function

The requirement of SIR2 for rDNA silencing. (A) Spot assay. Reporter strains containing mURA3 in the rDNA were spotted onto either SC-Ura or nonselective SC medium and allowed to grow for 3 days. Loss of silencing in the sir2 A strains is indicated by increased growth on the SC-Ura plates. (B) Colony color assay. Wild-type or sir2A strains containing MET15 in the rDNA were streaked onto rich medium containing lead nitrate. A lighter colony color and increased numbers of dark brown...

[17 Redox Properties of Vanillyl Alcohol Oxidase

H. van den Heuvel, Marco W. Fraaije, and Willem J. H. van Berkel Flavins are a ubiquitous class of redox-active coenzymes that are able to catalyze a number of different chemical reactions when bound to apoproteins. They play an important role in (de)hydrogenation and hydroxylation reactions, in oxygen activation, and in one- and two-electron transfer processes from and to redox centers.1'2 Because of their chemical versatility, flavins are involved in a wide range of biological...

[23 Redox Control of 20S Proteasome

By Bertrand Friguet, Anne-Laure Bulteau, Mariangela Conconi, Oxidative modifications of proteins have been implicated in age- and disease-related impairment of cellular functions and are known to affect protein turnover.1 3 Because the 20S proteasome has been shown to be the major actor in the degradation of oxidized protein4 and consequently to be important in the regulation of the steady state level of altered proteins in the cell, the fate of proteasome subjected to oxidative processes has...

J

Detection of BioGEE incorporation into protein, and selectivity for reactive cysteines. (A) HeLa cells were loaded with 0.5 mM BioGEE for 1 hr and then exposed to H202 (0,0.12,0.25,0.5, and 1 mM) for 15 min. A soluble protein extract was obtained as described in text and 40 g of protein from each extract was resolved by SDS-PAGE under either nonreducing or reducing conditions. The proteins were transferred to nitrocellulose and the blots were stained with Ponceau S. The blots were then...

Info

An aliquot of BioGEE prepared as described in text was solubi-lized in 0.05 (w v) TFA and separated by reversed phase HPLC with both spectrophotometric and mass spectrometric detection (model 1100 Hewlett-Packard, Palo Alto, CA), using a Vydac narrow-bore Cig column (218TP5205 Vydac, Hesperia, CA). The initial solvent was 0.05 (w v) trifluoroacetic acid and the other solvent was acetonitrile-0.05 (w v) trifluoroacetic acid. The acetonitrile gradient was...

[10 Detection and Affinity Purification of Oxidant Sensitive Proteins Using Biotinylated Glutathione Ethyl Ester

Levine, and Toren Finkel Introduction Effects of oxidative stress in biological systems have long been the subject of intense scientific scrutiny. This work has established at least an ancillary role for oxidants in an astonishingly wide range of human diseases and, more recently, a role in normal signal transduction. Many of these studies have focused on toxicological effects of highly reactive species such as peroxyl, alkoxyl, and hydroxyl radicals, which are...

[37 Transgenic Shuttle Vector Assays for Determining Genetic Differences in Oxidative B Cell Mutagenesis in Vivo

Rockwood, and Siegfried Janz Because tumorigenesis is an in vivo phenomenon that can be evaluated optimally only in in vivo studies, it has been postulated that attempts to link tumor development with mutagenesis may be most informative when mutagenesis can also be assessed in vivo. We have chosen two transgenic shuttle vector assays, A LIZ (Big Blue) and pUR288 (placZ), to explore in mice the role of oxidative B cell mutagenesis in the development of the malignant...

[3 Enzyme Linked Immunospot Assay for Detection of Thioredoxin and Thioredoxin Reductase Secretion from Cells

By Bita Sahaf, Anita S derberg, Christina Ekerfelt, Staffan Paulie, and Anders Rosen Oxidative stress response was determined in this study by enzyme-linked immunospot (ELISpot) assays for thioredoxin (Trx) and Trx reductase (TrxR). On exposure to oxidative stress, cells can launch a variety of defense mechanisms, including release of antioxidant proteins. The Trx system, consisting of Trx, TrxR, and NADPH, constitutes one of these cellular defense systems for maintenance of a healthy...

[12 Lysozyme Osmotic Shock Methods for Localization of Periplasmic Redox Proteins in Bacteria

Davidson and Dapeng Sun The cell envelope of gram-negative bacteria is composed of a cytoplasmic membrane (plasma membrane), a murein peptidoglycan layer, and an outer membrane that is linked by lipoproteins to the murein layer. The compartment between the cytoplasmic and outer membrane is called the periplasm, or periplasmic space.1 Several proteins are specifically localized in the periplasm. These have long been known to include binding proteins for nutrients, and hydrolytic and...

[4 Determination of Redox Properties of Protein Disulfide Bonds by Radiolytic Methods

Disulfide dithiol redox systems are involved in the regulation of cell growth and proliferation,1'2 in human cancer development,3,4 and in the development of postirradiation effects.5 Protein sulfur functions are essential in numerous events related to oxidative stress. In the regulation of all events involving free radicals, such as oxidative stress and irradiation by ionizing radiation, one-electron redox processes take place and sulfur free radicals are created. Their properties play a key...

[5 Crystal Structures of Oxidized and Reduced Forms of NADH Peroxidase

Modulations of protein function by oxidation-reduction reactions through co-factors such as FAD, iron-sulfur centers, heme prosthetic groups, and redox-active disulfides are well known. However, only more recently have cysteine-sulfenic acid (Cys-SOH) derivatives been recognized as novel moieties (cofactors) functioning in enzyme catalysis and redox regulation. A major reason for the delayed recognition of the functional importance of this moiety in proteins is that sulfenic acids are difficult...

Gskg

_ 3JK1GEP* _ BllgycddxaaB < kcgmtiphkkw-lr a w lv 1 of just the bacterial Sir2p species is included as well. Note that the bacterial species have much shorter N and C termini and are more conserved as a group. Interestingly, the region surrounding the GAGISTS motif does have some weak similarity to a region of bacterial 6-phosphogluconate dehydrogenases, an NAD+-binding protein.16 This enzyme uses NADP+ or NAD+ as a cofactor in its activity and one-half of its binding site is similar to...

Water Saturated 1-butanol Recipe For Chicken

Fig. 4. (A) SDS-polyacrylamide gel of Sir2-Af2 with 10-fold dilutions showing more than 99 purity. (B) SDS-polyacrylamide gel of TmSir2p with 10-fold dilutions showing approximately 99 purity. Arrowheads indicate position of 30 kDa marker. Fig. 4. (A) SDS-polyacrylamide gel of Sir2-Af2 with 10-fold dilutions showing more than 99 purity. (B) SDS-polyacrylamide gel of TmSir2p with 10-fold dilutions showing approximately 99 purity. Arrowheads indicate position of 30 kDa marker. Basic Protocol for...

[26 SIR2 Family of NADDependent Protein Deacetylases

Smith, Jose Avalos, Ivana Celic, Shabazz Muhammad, Cynthia Wolberger, and Jef D. Boeke Nicotinamide adenine dinucleotide (NAD+) is a key cofactor in numerous metabolic pathways, including glycolysis and the citric acid cycle. The reduction of NAD+ in these reactions produces NADH, which subsequently acts as the key electron carrier in the oxidative phosphorylation process of ATP generation. Oxidative phosphorylation oxidizes NADH to regenerate NAD+. The NAD+ NADH and NADP+ NADPH...

[13 Alterations in Membrane Cholesterol That Affect Structure and Function of Caveolae

Anderson A widely acknowledged function of cellular membrane is to promote biological reactions by creating a two-dimensional space that facilitates interactions among resident molecules. Less well appreciated is that cell membranes are subdivided into specific domains that compartmentalize a variety of essential activities. Three examples of membrane domains are clathrin-coated pits, focal adhesion sites, and caveolae. Each requires specific cellular...

Edited by Lawrence C Kuo and Jules A Shafer

Neoglycoconjugates Part A Edited by Y. C. Lee and Reiko T. Lee Volume 243. Inorganic Microbial Sulfur Metabolism Edited by Harry D. Peck, Jr., and Jean LeGall Volume 244. Proteolytic Enzymes Serine and Cysteine Peptidases Edited by Alan J. Barrett Volume 245. Extracellular Matrix Components Edited by E. Ruoslahti and E. Engvall Volume 246. Biochemical Spectroscopy Edited by Kenneth Sauer Volume 247. Neoglycoconjugates Part B Biomedical Applications Edited by Y. C. Lee and reiko T....