Unbiased quantification of oxidative modifications of cysteine thiols by double labeling approach using isotope amino acids and affinity tags

2009 ◽  
Vol 189 ◽  
pp. S122
Author(s):  
Jun Adachi ◽  
Keishi Kihara ◽  
Tomonari Matsuda
Keyword(s):  
Amino Acids ◽  
Double Labeling ◽  
Affinity Tags ◽  
Oxidative Modifications ◽  
Labeling Approach ◽  
Cysteine Thiols

scholarly journals COLLAGEN METABOLISM IN THE NORMAL AND LATHYRITIC CHICK

1964 ◽  
Vol 119 (2) ◽  
pp. 275-289 ◽  
Author(s):  
Marvin L. Tanzer ◽  
Jerome Gross
Keyword(s):  
Amino Acids ◽  
Chick Embryo ◽  
Collagen Synthesis ◽  
Fibril Formation ◽  
Collagen Metabolism ◽  
Bone Collagen ◽  
Double Labeling

1. Radioisotope incorporation studies of normal and lathyritic chick embryo bone collagen do not demonstrate any interference by lathyrism with collagen synthesis or fibril formation. 2. The results indicate that a portion of the extractable collagen from lathyritic chick embryo bone represents newly synthesized protein. Evidence from a double labeling experiment and from analysis of isotope flow between the extractable and non-extractable pools suggests the extractable lathyritic collagen is heterogeneous. We propose that the lathyritic process affects collagen in all states of aggregation, probably in varying degree. 3. Puromycin, administered intravenously, reduces the amount of extractable collagen in both normal and lathyritic chick embryo bone, and diminishes the incorporation of labeled proline into collagen. 4. Marked fluctuations in incorporation of labeled amino acids into chick embryo bone collagen suggests the occurrence of wide fluctuations in metabolism of this protein.

scholarly journals RIBONUCLEOPROTEIN PARTICLES IN THE AMPHIBIAN OOCYTE NUCLEUS

1968 ◽  
Vol 36 (3) ◽  
pp. 421-432 ◽  
Author(s):  
M. Elizabeth Rogers
Keyword(s):  
Amino Acids ◽  
Ribosomal Subunit ◽  
Oocyte Nucleus ◽  
Double Labeling ◽  
Immature Oocytes ◽  
Amphibian Oocyte ◽  
Ribonucleoprotein Particles ◽  
40S Ribosomal Subunit ◽  
Nuclear Rna ◽  
Cytoplasmic Ribosomes

Studies of the sedimentation properties of RNP1 material from the nucleus of the amphibian oocyte have indicated (1) that there are few, if any, 78S ribosomes in the nucleus, (2) that there are smaller particles sedimenting at 50-55S and 30S, and (3) that the larger of these is the precursor of the 60S subunit of the cytoplasmic ribosomes. Although the nature of the 30S material is not completely clear, it probably includes precursor particles to the 40S ribosomal subunit. Heavy (50-55S) particles are predominant in immature oocytes of Triturus viridescens, whereas in immature oocytes of Triturus and Amblystoma mexicanum they are reduced greatly in amount, but are still detectable. Double-labeling studies of RNA and protein reveal that both types of particle incorporate uridine-3H, but that the 50-55S material of immature oocytes does not incorporate 14C-labeled amino acids. However, other evidence exists that favors the RNP nature of this material. Sedimentation analyses after SDS extraction show that 50-55S particles contain 40 and 30S RNA, whereas 30S particles contain 20S RNA. These types of RNA represent at least 80% of all the extractable nuclear RNA. The 50-55S particles are probably heterogeneous, including both particles containing mostly 40S RNA and particles containing only 30S RNA.

scholarly journals Relationship of glutamate and aspartate to the periaqueductal gray-raphe magnus projection: analysis using immunocytochemistry and microdialysis.

1990 ◽  
Vol 38 (12) ◽  
pp. 1755-1765 ◽  
Author(s):  
A J Beitz
Keyword(s):  
Amino Acids ◽  
Excitatory Amino Acid ◽  
Strong Support ◽  
Periaqueductal Gray ◽  
Microdialysis Probe ◽  
Double Labeling ◽  
Homocysteic Acid ◽  
Raphe Magnus ◽  
Projection Analysis ◽  
Relationship Of

This study tested the hypothesis that the excitatory amino acid transmitters glutamate and/or aspartate are associated with the periaqueductal gray (PAG)-raphe magnus (NRM) projection. Retrograde neuroanatomical tracing procedures utilizing the tracers WGA-HRP or D-[3H]-aspartate were combined with immunocytochemical localization of glutamate or aspartate to determine if glutamate and/or aspartate immunostained neurons projected to the NRM. Both glutamate- and aspartate-immunoreactive cells in the PAG were found to project to the NRM. Double labeling immunocytochemichemical procedures indicated that glutamate and aspartate are co-localized in many PAG neurons, suggesting the following possibilities: (a) one of these two amino acids may serve as a precursor to the other; (b) both amino acids may be co-released from the same PAG neuron; or (c) both amino acids are present in high levels in the perikarya for metabolic purposes. At the EM level, both glutamate- and aspartate-immunoreactive terminals were identified in the NRM, strengthening the concept that both amino acids participate in synaptic transmission in this medullary nucleus. To determine if glutamate and aspartate are in fact released from PAG-NRM axons, the PAG was stimulated chemically with homocysteic acid (HCA) and amino acids were collected from the NRM using a microdialysis probe. Microinjection of HCA, but not vehicle, into the PAG resulted in the release of both glutamate and aspartate in the nucleus raphe magnus. These data suggest that both glutamate and aspartate are released from PAG fibers terminating in the NRM and provide strong support for the hypothesis that excitatory amino acids play a neurotransmitter role in the PAG-NRM pathway.

scholarly journals Introduction to approaches and tools for the evaluation of protein cysteine oxidation

2020 ◽  
Vol 64 (1) ◽  
pp. 1-17 ◽  
Author(s):  
Leslie B. Poole ◽  
Cristina M. Furdui ◽  
S. Bruce King
Keyword(s):  
Reactive Oxygen Species ◽  
Best Practices ◽  
Reactive Oxygen ◽  
Cellular Proteins ◽  
Oxygen Species ◽  
Cysteine Oxidation ◽  
Oxidative Modifications ◽  
Cysteine Thiols ◽  
The Way

Abstract Oxidative modifications of cysteine thiols in cellular proteins are pivotal to the way signal-stimulated reactive oxygen species are sensed and elicit appropriate or sometimes pathological responses, but the dynamic and often transitory nature of these modifications offer a challenge to the investigator trying to identify such sites and the responses they elicit. A number of reagents and workflows have been developed to identify proteins undergoing oxidation and to query the timing, extent and location of such modifications, as described in this minireview. While no approach is perfect to capture all the redox information in a functioning cell, best practices described herein can enable considerable insights into the “redox world” of cells and organisms.

scholarly journals Affinity-Tagged Miniprion Derivatives Spontaneously Adopt Protease-Resistant Conformations

2000 ◽  
Vol 74 (24) ◽  
pp. 11928-11934 ◽  
Author(s):  
Surachai Supattapone ◽  
Hoang-Oanh B. Nguyen ◽  
Tamaki Muramoto ◽  
Fred E. Cohen ◽  
Stephen J. DeArmond ◽  
...  
Keyword(s):  
Amino Acids ◽  
Therapeutic Efficacy ◽  
Cultured Cells ◽  
Neuroblastoma Cells ◽  
New Disease ◽  
Wild Type ◽  
Affinity Tag ◽  
Affinity Tags ◽  
Protease Resistance ◽  
Protease Digestion

ABSTRACT An abridged PrP molecule of 106 amino acids designated PrP106 can form infectious miniprions in transgenic (Tg) mice (29). Addition of six-histidine (His6) affinity tags to selective sites within PrP106 resulted unexpectedly in new PrP proteins that spontaneously adopted protease-resistant conformations when expressed in neuroblastoma cells and Tg mice. Acquisition of protease resistance depended on the length, charge, and placement of the affinity tag. Introduction of the disease-linked mutation E200K into the sequence of PrP106(140/6His) increased the recovery of protease-resistant PrP fivefold, whereas introduction of the mutations C213A and Δ214–220 did not affect the recovery of protease-resistant PrP. Treatment of cultured cells expressing affinity-tagged PrP106 mutants with polypropyleneimine dendrimer rendered these proteins sensitive to protease digestion in a manner similar to wild-type PrPSc. We conclude that certain affinity-tagged PrP106 proteins spontaneously fold into conformations partially resembling, yet distinct from, wild-type PrPSc. These proteins might be useful tools in the identification of new disease-causing mutations as well as for screening compounds for therapeutic efficacy.

Ribosomal Structure and Functional Sites Mapped by Immune Electron Microscopy

1980 ◽  
Vol 38 ◽  
pp. 546-547
Author(s):  
James A. Lake
Keyword(s):  
Binding Sites ◽  
Ribosomal Proteins ◽  
Ribosomal Subunit ◽  
Double Labeling ◽  
Functional Sites ◽  
Initiation Factors ◽  
Functional Regions ◽  
Igg Binding ◽  
Labeling Approach ◽  
Trna Binding

Ribosomal protein locations have been mapped on the ribosomal surface by using antibodies directed against specific ribosomal proteins. Relative orientations of the large and the small subunits in the monomeric ribosome have been determined using a double labeling approach; and functional regions such as the codon-anticodon interaction site, the tRNA binding sites and the peptidyl transferase have been suggested from the locations of ribosomal proteins. The small ribosomal subunit (see Figure 1) is divided by a constriction into a one-third, or head, and a two-thirds, or base, region (1). A platform, located on the base, forms a cleft between it and the upper one-third of the subunit. The cleft, the upper one-third and the platform are thought to comprise the binding sites for mRNA and for initiation factors IFl, IF2 and IF3. The locations of some of the proteins involved in tRNA binding that have been mapped in collaboration with Dr. L. Kahan (Department of Physiological Chemistry, University of Wisconsin) are summarized in Figure 1. In addition, some proteins such as S4 are not accessible for IgG binding (2). Protein S4 is being mapped using reconstituted subunits lacking S5 and S12 (3). The binding sites for proteins Si, S11, S12 and S18 (not shown) are located on the platform and suggest that the site of the codon-anticodon interaction is also there.

scholarly journals Intracisternally Injected L-Proline Activates Hypothalamic Supraoptic, but Not Paraventricular, Vasopressin-Expressing Neurons in Conscious Rats

2011 ◽  
Vol 2011 ◽  
pp. 1-8 ◽  
Author(s):  
Yumi Takemoto
Keyword(s):  
Blood Pressure ◽  
Supraoptic Nucleus ◽  
Excitatory Amino Acid ◽  
Brain Regions ◽  
Double Labeling ◽  
Amino Acid Receptors ◽  
Freely Moving Rats ◽  
Artificial Cerebrospinal Fluid ◽  
Freely Moving ◽  
Labeling Approach

When injected into specific rat brain regions, the neurotransmitter candidate L-proline produces various cardiovascular changes through ionotropic excitatory amino acid receptors. The present study used an immunohistochemical double-labeling approach to determine whether intracisternally injected L-proline in freely moving rats, which increases blood pressure, activates hypothalamic vasopressin-expressing neurons and ventral medullary tyrosine-hydroxylase- (TH-) containing neurons. Following injection of L-proline, the number of activated hypothalamic neurons that coexpressed vasopressin and c-Fos was much greater in the supraoptic nucleus (SON) than in the paraventricular nucleus (PVN) of rats with increased blood pressure. The number of activated TH-containing neurons was significantly greater following L-proline treatment than following control injections of artificial cerebrospinal fluid (ACSF). These results clearly demonstrate that intracisternally injected L-proline activates hypothalamic supraoptic, but not paraventricular, vasopressin-expressing neurons and medullary TH-containing (A1/C1) neurons in freely moving rats.

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