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Theoretical analyses predict A20 regulates period of NF-kB oscillation
2009-11-04
The nuclear-cytoplasmic shuttling of NF-kB is characterized by damped oscillations of the nuclear concentration with a time period of around 1-2 hours. The NF-kB network contains several feedback loops modulating the overall response of NF-kB activity. While IkBa is known to drive and IkBe is known to dampen the oscillations, the precise role of A20 negative feedback remains to be elucidated. Here we propose a model of the NF-kB system focusing on three negative feedback loops (IkBa, IkBe and A20) which capture the experimentally observed responses in wild-type and knockout cells. We find that A20, like IkBe, efficiently dampens the oscillations albeit through a distinct mechanism. In addition, however, we have discovered a new functional role of A20 by which it controls the oscillation period of nuclear NF-kB. The design based on three nested feedback loops allows independent control of period and amplitude decay in the oscillatory response. Based on these results we predict that adjusting the expression level of A20, e.g. by siRNA, the period can be changed by up to a factor 2.
{sup 13}C relaxation in an RNA hairpin
1994-12-01
This initial survey of {sup 13}C relaxation in the {triangle}TAR RNA element has generated a number of interesting results that should prove generally useful for future studies. The most readily comparable study in the literature monitored {sup 13}C relaxation of the methyl groups from unusual bases in tRNA{sup Phe}. The study, which used T{sub 1} and NOE data only, reported order parameters for the methyl group axis that ranged between 0.51 and 0.97-a range similar to that observed here. However, they reported a breakdown of the standard order parameter analysis at higher (118-MHz {sup 13}C) frequencies, which should serve to emphasize the need for a thorough exploration of suitable motional models.
1994-12-01
The current emphasis in biological NMR work is on determining structures of biological macromolecules in solution. This emphasis is appropriate because NMR is the only technique capable of providing high-resolution structures that are comparable to those of x-ray crystallography for molecules in solution. This structural knowledge is immensely valuable and is needed in many areas of investigation. However, as valuable as such structural knowledge is, it never provides all the answers; a structure often reveals more questions than answers.
The potential for energy conservation in the United States
1993-12-31
The period of high oil prices between 1973 and 1985 was traumatic in the United States, as it was also in the rest of the world. It was also instructive in showing the kinds of adaptation that could occur rapidly in a very large industrialized economy. During the period, energy use remained essentially constant while the economy continued to grow. The efficiency of energy use, as indicated by the ratio of energy consumption to gross domestic product, increased by 24 percent. Since 1985 there has been little further improvement in energy efficiency. Can this kind of improvement in efficiency be repeated, and if so, what can make it happen? A number of energy analysts have recently made projections for the next 20 years. The projections all indicate steady increases of about 1 percent per year in the level of energy use. Since these projections assumed that gross domestic product will increase by about 2.3 percent per year, the implication is that energy efficiency is expected to increase slowly during the next two decades.
Synthesis and NMR of {sup 15}N-labeled DNA fragments
1994-12-01
DNA fragments labeled with {sup 15}N at the ring nitrogens and at the exocyclic amino groups can be used to obtain novel insight into interactions such as base pairing, hydration, drug binding, and protein binding. A number of synthetic routes to {sup 15}N-labeled pyrimidine nucleosides, purines, and purine nucleosides have been reported. Moreover, many of these labeled bases or monomers have been incorporated into nucleic acids, either by chemical synthesis or by biosynthetic procedures. The focus of this chapter will be on the preparation of {sup 15}N-labeled purine 2{prime}-deoxynucleosides, their incorporation into DNA fragments by chemical synthesis, and the results of NMR studies using these labeled DNA fragments.
1994-12-01
Complete understanding of a protein`s function and mechanism of action can only be achieved with a knowledge of its three-dimensional structure at atomic resolution. At present, there are two methods available for determining such structures. The first method, which has been established for many years, is x-ray diffraction of protein single crystals. The second method has blossomed only in the last 5 years and is based on the application of nuclear magnetic resonance (NMR) spectroscopy to proteins in solution. This review paper describes three- and four-dimensional NMR methods applied to protein structure determination and was adapted from Clore and Gronenborn. The review focuses on the underlying principals and practice of multidimensional NMR and the structural information obtained.
1994-12-01
Carbohydrates play important roles in many key biochemical processes in living cells. For example, they are metabolized to produce energy, mediate cell-cell recognition, and play an indirect role (as constituents of DNA and RNA) in DNA replication, RNA transcription, and protein synthesis. These roles, and others of comparable biochemical significance, have been studied to varying extends with the use of stable isotopically labeled molecules, usually in conjunction with NMR spectroscopy and/or mass spectrometry. For example, carbohydrate metabolism has been monitored in vitro and in vivo with the use of isotopically labeled compounds. Molecular aspects of cell-cell recognition, mediated by cell-surface glycoproteins and glycolipids, have been probed through NMR studies of isotopically labeled oligosaccharides. More recently, the solution behavior of DNA and RNA has been examined through the use of labeled oligonucleotides. In all of these pursuits, the effort and expense to prepare labeled molecules, both of which can be substantial, are more than offset by the wealth of information derived from these studies. This information often cannot be accessed, or can be accessed only with great difficulty, using natural (unlabeled) compounds.
Selective {sup 2}H and {sup 13}C labeling in NMR analysis of solution protein structure and dynamics
1994-12-01
Preparation of samples bearing combined isotope enrichment patterns has played a central role in the recent advances in NMR analysis of proteins in solution. In particular, uniform {sup 13}C, {sup 15}N enrichment has made it possible to apply heteronuclear multidimensional correlation experiments for the mainchain assignments of proteins larger than 30 KDa. In contrast, selective labeling approaches can offer advantages in terms of the directedness of the information provided, such as chirality and residue type assignments, as well as through enhancements in resolution and sensitivity that result from editing the spectral complexity, the relaxation pathways and the scalar coupling networks. In addition, the combination of selective {sup 13}C and {sup 2}H enrichment can greatly facilitate the determination of heteronuclear relaxation behavior.
REDOR NMR of stable-isotope-labeled protein binding sites
1994-12-01
Rotational-echo, double resonance (REDOR) NMR, a new analytical spectroscopic technique for solids spinning at the magic angle, has been developed over the last 5 years. REDOR provides a direct measure of heteronuclear dipolar coupling between isolated pairs of labeled nuclei. In a solid with a {sup 13}C-{sup 15}N labeled pair, for example, the {sup 13}C rotational echoes that form each rotor period following a{sup 1}H-{sup 13}C cross-polarization transfer can be prevented from reaching full intensity by insertion of a {sup 15}N {pi} pulse each half rotor period. The REDOR difference (the difference between a {sup 13}C NMR spectrum obtained under these conditions and one obtained with no {sup 15}N {pi} pulses) has a strong dependence on the {sup 13}C-{sup 15}N dipolar coupling, and hence, the {sup 13}C-{sup 15}N internuclear distance. REDOR is described as double-resonance even though three radio frequencies (typically {sup 1}H, {sup 13}C, and {sup 15}N) are used because the protons are removed from the important evolution part of the experiment by resonant decoupling. The dephasing of magnetization in REDOR arises from a local dipolar {sup 13}C-{sup 15}N field gradient and involves no polarization transfer. REDOR has no dependence on {sup 13}C or {sup 15}N chemical-shift tensors and does not require resolution of a {sup 13}C-{sup 15}N coupling in the chemical-shift dimension.
Overview of the EPA quality system for environmental programs
1993-12-31
Formalized quality assurance program requirements for the U.S. Environmental Protection Agency (EPA) have been established for more than a decade. During this period, the environmental issues and concerns addressed by the EPA have changed. Many issues, such as ozone depletion and global climate warming, have become international concerns among the world environmental community. Other issues, such as hazardous waste cleanup and clean air, remain a focus of national environmental concerns. As the environmental issues of the 1980`s evolved, the traditional quality assurance (QA) program was transformed through the use of quality management principles into a Quality System to help managers meet the needs of the 1990`s and beyond.
New strategy for stable-isotope-aided, multidimensional NMR spectroscopy of DNA oligomers
1994-12-01
Nuclear Magnetic Resonance (NMR) is the most efficient method for determining the solution structures of biomolecules. By applying multidimensional heteronuclear NMR techniques to {sup 13}C/{sup 15}N-labeled proteins, we can determine the solution structures of proteins with molecular mass of 20 to 30kDa at an accuracy similar to that of x-ray crystallography. Improvements in NMR instrumentation and techniques as well as the development of protein engineering methods for labeling proteins have rapidly advanced multidimensional heteronuclear NMR of proteins. In contrast, multidimensional heteronuclear NMR studies of nucleic acids is less advanced because there were no efficient methods for preparing large amounts of labeled DNA/RNA oligomers. In this report, we focused on the chemical synthesis of DNA oligomers labeled at specific residue(s). RNA oligomers with specific labels, which are difficult to synthesize by the enzyme method, can be synthesized by the chemical method. The specific labels are useful for conformational analysis of larger molecules such as protein-nucleic acid complexes.
Ner protein of phage Mu: Assignments using {sup 13}C/{sup 15}N-labeled protein
1994-12-01
The Ner protein is a small (74-amino acid) DNA-binding protein that regulates a switch between the lysogenic and lytic stages of phage Mu. It inhibits expression of the C repressor gene and down-regulates its own expression. Two-dimensional NMR experiments on uniformly {sup 15}N-labeled protein provided most of the backbone and some of the sidechain proton assignments. The secondary structure determination using two-dimensional NOESY experiments showed that Ner consists of five {alpha}-helices. However, because most of the sidechain protons could not be assigned, the full structure was not determined. Using uniformly {sup 13}C/{sup 15}N-labeled Ner and a set of three-dimensional experiments, we were able to assign all of the backbone and 98% of the sidechain protons. In particular, the CBCANH and CBCA(CO)NH experiments were used to sequentially assign the C{alpha} and C{beta} resonances; the HCCH-CTOCSY and HCCH-COSY were used to assign sidechain carbon and proton resonances.
NMR studies of isotopically labeled RNA
1994-12-01
In summary, the ability to generate NMR quantities of {sup 15}N and {sup 13}C-labeled RNAs has led to the development of heteronuclear multi-dimensional NMR techniques for simplifying the resonance assignment and structure determination of RNAs. These methods for synthesizing isotopically labeled RNAs are only several years old, and thus there are still relatively few applications of heteronuclear multi-dimensional NMR techniques to RNA. However, given the critical role that RNAs play in cellular function, one can expect to see an increasing number of NMR structural studies of biologically active RNAs.
Mammography accreditation program
1993-12-31
In the mid-1980`s, the movement toward the use of dedicated mammography equipment provided significant improvement in breast cancer detection. However, several studies demonstrated that this change was not sufficient to ensure optimal image quality at a low radiation dose. In particular, the 1985 Nationwide Evaluation of X-ray Trends identified the wide variations in image quality and radiation dose, even from dedicated units. During this time period, the American Cancer Society (ACS) launched its Breast Cancer Awareness Screening Campaign. However, there were concerns about the ability of radiology to respond to the increased demand for optimal screening examinations that would result from the ACS program. To respond to these concerns, the ACS and the American College of Radiology (ACR) established a joint committee on mammography screening in 1986. After much discussion, it was decided to use the ACR Diagnostic Practice Accreditation Program as a model for the development of a mammography accreditation program. However, some constraints were required in order to make the program meet the needs of the ACS. This voluntary, peer review program had to be timely and cost effective. It was determined that the best way to address these needs would be to conduct the program by mail. Finally, by placing emphasis on the educational nature of the program, it would provide an even greater opportunity for improving mammographic quality. The result of this effort was that, almost six years ago, in May 1987, the pilot study for the ACR Mammography Accreditation Program (MAP) began, and in August of that year, the first applications were received. In November 1987, the first 3-year accreditation certificates were awarded.
Magnetic resonance studies of isotopically labeled paramagnetic proteins: (2FE-2S) ferredoxins
1994-12-01
Recent developments in NMR spectroscopy, especially multidimensional, multinuclear NMR techniques, have made NMR the most versatile tool available for studying protein structure and function in solution. Unlike diamagnetic proteins, paramagnetic proteins contain centers with unpaired electrons. These unpaired electrons interact with magnetic nuclei either through chemical bonds by a contact mechanism or through space by a pseudocontact mechanism. Such interactions make the acquisition and analysis of NMR spectra of paramagnetic proteins more challenging than those of diamagnetic proteins. Some NMR signals from paramagnetic proteins are shifted outside the chemical shift region characteristic of diamagnetic proteins; these {open_quotes}hyperfine-shifted{close_quotes} resonances originate from nuclei that interact with unpaired electrons from the paramagnetic center. The large chemical shift dispersion in spectra of paramagnetic proteins makes it difficult to excite the entire spectral window and leads to distortions in the baseline. Interactions with paramagnetic centers shorten T{sub 1} and T{sub 2} relaxation times of nuclei; the consequences are line broadening and lower spectral sensitivity. Scalar (through bond) and dipolar (through space) interactions between pairs of nuclei are what give rise to crosspeak signals in multi-dimensional NMR spectra of small diamagnetic proteins. When such interactions involve a nucleus that is strongly relaxed by interaction with a paramagnetic center, specialized methods may be needed for its detection or it may be completely undetectable by present nD NMR methods.
1994-12-01
This paper deals with compounds that are chiral-at least in part, due to isotope substitution-and their use in tracing the steric course of enzyme reaction in vitro and in vivo. There are other applications of isotopically chiral compounds (for example, in analyzing the steric course of nonenzymatic reactions and in probing the conformation of biomolecules) that are important but they will not be discussed in this context.
Isotope labeling for NMR studies of macromolecular structure and interactions
1994-12-01
Implementation of biosynthetic methods for uniform or specific isotope labeling of proteins, coupled with the recent development of powerful heteronuclear multidimensional NMR methods, has led to a dramatic increase in the size and complexity of macromolecular systems that are now amenable to NMR structural analysis. In recent years, a new technology has emerged that combines uniform {sup 13}C, {sup 15}N labeling with heteronuclear multidimensional NMR methods to allow NMR structural studies of systems approaching 25 to 30 kDa in molecular weight. In addition, with the introduction of specific {sup 13}C and {sup 15}N labels into ligands, meaningful NMR studies of complexes of even higher molecular weight have become feasible. These advances usher in a new era in which the earlier, rather stringent molecular weight limitations have been greatly surpassed and NMR can begin to address many central biological problems that involve macromolecular structure, dynamics, and interactions.
1993-12-31
For more than 20 years, the American Association of Physicists in Medicine (AAPM) has operated an accreditation program for secondary standards laboratories that calibrate radiation measuring instruments. Except for one short period, that program has been able to provide the facilities to satisfy the national need for accurate calibrations of such instruments. That exception, in 1981, due to the combination of the U.S. Nuclear Regulatory Commission (NRC) requiring instrument calibrations by users of cobalt-60 teletherapy units and the withdrawal of one of the three laboratories accredited at that time. However, after successful operation as a Task Group of the Radiation Therapy Committee (RTC) of the AAPM for two decades, a reorganization of this structure is now under serious consideration by the administration of the AAPM.
1994-12-01
To understand the details of macromolecular function, high-resolution structural and dynamic detail is essential. The polypeptide fold of the gramicidin channel has been effectively modeled for the past 20 years, yet the functional changes in conductance and channel lifetime associated with amino acid substitutions cannot be predicted. To accomplish this goal, high-resolution electrostatic modeling and the precise orientation of all dipoles are required. Furthermore, an enhanced knowledge of the complex molecular environment of this membrane-bound peptide is needed. An aqueous environment is relatively uniform and achiral. The membrane environment is very heterogenous and chiral. A knowledge of the interactions, specific and nonspecific, between peptide and lipid will aid in developing a better understanding of this environment. To accomplish this goal, it is necessary to study the peptide in an extended lipid bilayer, rather than in a vesicular or micellar form. These latter environments are likely to possess increased dynamics, increased water penetration, and distorted interactions between the polypeptide and membrane surface. To perform NMR studies on bilayer bound peptides, solid state NMR methods are required, and for specific site information, isotopic labels are incorporated using solid phase peptide synthesis.
Complex DNA structures and structures of DNA complexes
1994-12-01
Complex DNA structures (for example, triplexes, quadruplexes, junctions) and DNA-ligand complexes are more difficult to study by NMR than standard DNA duplexes are because they have high molecular weights, show nonstandard or distorted local conformations, and exhibit large resonance linewidths and severe {sup 1}H spectral overlap. These systems also tend to have limited solubility and may require specialized solution conditions to maintain favorable spectral characteristics, which adds to the spectroscopic difficulties. Furthermore, with more atoms in the system, both assignment and structure calculation become more challenging. In this article, we focus on demonstrating the current status of NMR studies of such systems and the limitations to further progress; we also indicate in what ways isotopic enrichment can be useful.
1997-01-01
A 27 kt water volume is investigated as a target for a long baseline neutrino beam from CERN to Gran Sasso. Charged secondaries from the neutrino interactions produce Cherenkov photons in water which are imaged as rings by a spherical mirror. The photon detector elements are 14 400 photomultipliers (PM`s) of 127 mm diameter or 3600 HPD`s of 250 mm diameter with single photon sensitivity. A coincidence signal of about 300 pixel elements in time with the SPS beam starts readout in bins of 1 ns over a period of 128 ns. Momentum, direction, and velocity of hadrons and mucons are determined from the width, center, and radius of the rings, respectively. Momentum is measured if multiple scattering dominates the ring width, as is the case for most of the particles of interest. Momentum, direction, and velocity of hadrons and muons are determined from the width, center, and radius of the rings, respectively. Momentum is measured if multiple scattering dominates the ring width, as is the case for most of the particles of interest. Momentum resolutions of 1-10%, mass resolutions of 5-50 MeV, and direction resolutions of < 1 mrad are achievable. Thresholds in water for muons, pions, kaons, and protons are 0.12, 0.16, 0.55, and 1.05 GeV/c, respectively. Electrons and gammas can be measured with energy resolution {sigma}{sub E}/E{approx}8.5%/{radical}E(GeV) and with direction resolution {approx} 1 mrad. The detector can be sited either inside a Gran Sasso tunnel or above ground because it is directional and the SPS beam is pulsed; thus the rejection of cosmic ray background is excellent.
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