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G Wagner

Publications and source records attributed to G Wagner.

At least 181 records · Page 10Linked to original sources
PubMed1997

Structure and mobility of the PUT3 dimer.

The solution structure and backbone dynamics of the transcriptional activator PUT3 (31-100) has been characterized using NMR spectroscopy. PUT3 (31-100) contains three distinct domains: a cysteine zinc cluster, linker, and dimerization domain. The cysteine zinc cluster of PUT3 closely resembles the solution structure of GAL4, while the dimerization domain forms a long coiled-coil similar to that observed in the crystal structures of GAL4 and PPR1. However, the residues at the N-terminal end of the coiled-coil behave very differently in each of these proteins. A comparison of the structural elements within this region provides a model for the DNA binding specificity of these proteins. Furthermore, we have characterized the dynamics of PUT3 to find that the zinc cluster and dimerization domains have very diverse dynamics in solution. The dimerization domain behaves as a large protein, while the peripheral cysteine zinc clusters have dynamic properties similar to small proteins.

Amino Acid Sequence↗
PubMed1997

Synchronous primary neuroendocrine carcinoma and adenocarcinoma in Barrett's esophagus.

We report a case of a synchronous primary small-cell carcinoma and adenocarcinoma of the esophagus with ectopic gastrin and calcitonin production. The double primary carcinomas appeared to arise from dysplastic Barrett's mucosa in a 43-year-old woman with a long history of reflux esophagitis. She is one of the few reported long-term survivors treated by surgical resection and combination chemotherapy. The histopathology and treatment of this rare tumor are briefly discussed.

Adenocarcinoma↗
PubMed1997

Clinical grading of spinal osteoporosis: quality of life components and spinal deformity in women with chronic low back pain and women with vertebral osteoporosis.

Clinical consequences of osteoporotic vertebral fractures, such as back pain, functional limitations, and impairment of mood, are often cited as justification for prevention and therapy. But these symptoms are poorly characterized, and a clinical grading system is not available. The aim of this study was to compare clinical measures for spinal deformation and quality of life components between patients with osteoporosis and patients with chronic low back pain (CLBP) and to determine the relationship between spinal deformation and quality of life components. A total of 130 female patients (63 osteoporotic patients, 65 +/- 7.9 years, and 77 CLBP patients, 56 +/- 6.5 years) had a standardized interview on quality of life components (pain, activities of daily life, mood) and clinical measures of spinal deformation (height reduction [HR], distance from occiput to wall [DOW], and distance from iliac crest to ribs [DIR]). Spinal X-rays were reviewed in all patients for the evidence of vertebral fractures. In osteoporotic patients, vertebral deformity was quantified by the spine deformity index (SDI) on X-rays. It was assessed whether subgroups could be identified by a combination of indices for spinal deformation (SDI, HR, DOW) using a cluster analysis. Back pain was a major complaint in both groups, without differences in pain intensity and frequency. Impairment of general well being and mood was found in about one-third of the patients in both groups. Independent of age, the disability score was significantly higher in patients with osteoporosis than in patients with CLBP. Both groups differed with respect to clinical measures of spinal deformity (HR, DOW, DIR). Among osteoporotic patients, parameters of quality of life were not linearly related to the degree of radiologically assessed vertebral deformity, but osteoporotic patients with two or more vertebral fractures tended to have more functional limitations than those with only one fracture. There was, however, a significant linear relationship between components of quality of life (disability score, pain) and clinical measures of spinal deformation (HR, DOW, DIR). The osteoporotic patients were subdivided into three clusters. The first group was characterized by low spinal deformation (decreases SDI, decreases HR, decreases DOW) and little impairment of quality of life. The second group had significantly greater spinal deformation (increases SDI, increases HR, increases DOW) and significantly more pain and functional limitations. The third group was characterized by increased kyphosis, mainly caused by nonskeletal dysfunction (decreases SDI, decreases HR, increases DOW), but pain and functional limitations were impaired to the same degree as in the second group with severe skeletal spinal deformation. We conclude that with respect to quality of life components, functional limitation is the most specific to spinal osteoporosis and is related to clinical measures of spinal deformation. Furthermore, spinal deformation and the clinical course of osteoporosis appears to be insufficiently reflected by radiological indices of vertebral deformity (such as SDI) alone. For grading the disease and for therapeutical concepts, radiological measures and clinical evaluation should be considered in combination.

Aged↗
PubMed1997

An account of NMR in structural biology.

With the ability to determine atomic resolution structures of biological macromolecules in semi-physiological conditions, nuclear magnetic resonance spectroscopy (NMR) has become an eminent tool in structural biology. NMR provides a means for studying critical biological phenomena including protein structure, dynamics and folding as well as a practical approach to drug design.

Crystallography, X-Ray↗
PubMed1996

Development and validation of an indirect enzyme immunoassay for detection of antibody to Anaplasma marginale in bovine sera.

An indirect enzyme immunoassay (IELISA) for detection of bovine antibody activity to Anaplasma marginale was developed. This assay used a crude antigen prepared from erythrocytes of infected calves, immobilized in a polystyrene matrix and a mouse monoclonal antibody to bovine IgG1, conjugated with horseradish peroxidase. Negative sera (n = 1842) were tested and the diagnostic specificity was 98.4 +/- 0.6% before retesting 29 positive samples. After retesting, eight samples remained positive and the specificity was calculated to be 99.6 +/- 0.3%. The diagnostic sensitivity, using 831 serum samples collected from naturally or experimentally infected cattle in Argentina, 370 from Mexico and 525 sera from experimentally vaccinated or infected cattle from Texas, was 87.3 +/- 1.6%.

Agglutination Tests↗
PubMed1996

Backbone dynamics, amide hydrogen exchange, and resonance assignments of the DNA methylphosphotriester repair domain of Escherichia coli Ada using NMR.

The 10kDa amino-terminal fragment of Escherichia coli Ada protein (N-Ada10) repairs methyl phosphotriesters in DNA and possesses a tightly bound zinc ion. The complete resonance assignments of this protein domain have been obtained using multidimensional homonuclear and heteronuclear NMR experiments. The assignments served to study the internal mobility of this protein domain via 15N relaxation experiments. This involved the measurement of longitudinal and transverse 15N relaxation rates, as well as the amide proton solvent exchange rates. Relaxation rates in the rotating frame, R1 rho, of 15N nuclei were measured at different spin-lock field strengths, leading to the detection of two slow conformational exchange processes at Gly-25 and Gln-73. For the latter, which is next to the active site of this protein domain, the characteristic time of this process was found to be around 60 microseconds. The other relaxation experiments unveiled some regions of fast internal motions, faster than the overall correlation time. These motions were found in the N- and C- terminal tails, in segment 33-35 which forms the turn between beta-strands S1 and S2, and residues 47-52 located in a long loop preceding strand S3. The latter loop belongs to the potential DNA binding surface of N-Ada10. While the structure from residue 18 to residue 26 appears not well defined in the calculated structure, the relaxation experiments do not indicate higher mobility for this region. Residues at the N-terminal portion, including the first helix, the sequentially adjacent loop, and part of the second helix, exhibit internal motions close to the time scale of the overall rotational correlation time. This appears to be related to the fact that the first helix has no hydrogen bonds or salt bridges to the rest of the protein and is stabilized only by the involvement of some of its side chains in a hydrophobic core consisting of the side chains of two phenylalanines, a tryptophan, a leucine, and a valine. The four cysteines which bind the zinc show motions on different time scales ranging from microseconds to picoseconds. Thus the motions in the immediate region around the bound zinc of the DNA methyl phosphotriester repair domain are of relatively small amplitude but take place over a wide time range. On the other hand, high mobility is found in the turn connecting S1 and S2 and in the loop preceding S3, regions of the potential DNA binding surface.

Bacterial Proteins↗
PubMed1996

A 39 amino acid fragment of the cell cycle regulator p21 is sufficient to bind PCNA and partially inhibit DNA replication in vivo.

The cell cycle regulator p21 interacts with and inhibits the DNA replication and repair factor proliferating cell nuclear antigen (PCNA). We have defined a 39 amino acid fragment of p21 which is sufficient to bind PCNA with high affinity (Kd 10-20 nM). This peptide can inhibit DNA replication in vitro and microinjection of a GST fusion protein containing this domain inhibited S phase in vivo. Despite its high affinity for PCNA, the free 39 amino acid peptide does not have a well-defined structure, as judged from circular dichroism and nuclear magnetic resonance measurements, suggesting an induced fit mechanism for the PCNA-p21 interaction. The association of the small peptide with PCNA was thermolabile, suggesting that portions of p21 adjoining the minimal region of contact stabilize the interaction. In addition, a domain containing 67 amino acids from the N-terminus of PCNA was defined as both necessary and sufficient for binding to p21.

Amino Acid Sequence↗
PubMed1996

Internal mobility in the partially folded DNA binding and dimerization domains of GAL4: NMR analysis of the N-H spectral density functions.

The DNA binding domain (residues 1--65) of the yeast transcriptional activator GAL4 is only partially folded. While residues 10-41, the DNA recognition domain, form a well-defined structure in the free protein, the whole polypeptide folds up and dimerizes upon binding DNA. In order to describe the mobility of the protein, we have characterized the frequency spectrum of the motions of N-H bond vectors of GAL4(1-65) using a reduced spectral density mapping approach (an approximation of the full spectral density mapping technique) [Peng, J. W., & Wagner, G. (1992a) J. Magn. Reson. 98, 308-332; Peng. J. W., & Wagner, G. (1992b) Biochemistry 31, 8571-8586]. 15N spin-lattice relaxation [Rn(Nz)], spin-spin relaxation [Rn(Nx,y)], cross-relaxation [RN(Hz-->Nz)], two-spin order [RNH(2HzNz)], and antiphase [RNH(2HzNx,y)] rates were determined for 52 of the 65 backbone amide groups at 10 degrees C and ph 6.5 at 11.74 T. Calculations of the spectral density functions using a reduced set of RN(Nz),RN(Nx,y),RN(Hz-->Nz), and RNH(2HzNz) gave excellent agreement with those calculated using all six sets. The reduced method has the added advantage that the errant behavior seen at high field values is circumvented. A linear correlation was found between J(omega N) and J(0) with a limited and clearly defined range of J(0) values which defines the range of rates for internal motions in GAL4(1-65). It appears that all residues experience a combination of two movements: one of the overall tumbling (correlation time, 8.65 ns) and the other of fast internal fluctuations of the structure. The respective weights of these contributions vary with the primary sequence and faithfully mirror the secondary and tertiary elements of the protein. The position on the correlation line of J(omega N) versus J(0) indicates the amount of angular averaging relative to the overall motion of the protein. A spectral density function for internal motions can be described.

Amino Acid Sequence↗
PubMed1996

Local mobility within villin 14T probed via heteronuclear relaxation measurements and a reduced spectral density mapping.

Villin 14T, a representative domain from the actin severing and bundling protein villin, binds calcium ions and actin monomers. To begin to understand the contributions of mobility to the villin-calcium and villin-actin interactions, relaxation rates for magnetization involving the amide nitrogens and protons have been measured for 15N-labeled villin 14T in solution. Although we have measured the complete set of rates required for a full spectral density map, difficulties in the accurate measurement of relaxation rates for antiphase coherence and two-spin order led us to consider a reduced mapping formalism. From the reduced spectral density map, a model-free analysis, or directly from the measured Nx,y relaxation rates, local variations in mobility along the backbone of villin 14T have been revealed. Fast motions are evident not only at the amino and carboxyl termini but also in the turn between strands beta 4 and beta 5 of the central beta-sheet and in the turn between helix alpha 3 and strand beta 7. Slower motions are suggested for the turn between strands beta 2 and beta 3. Motions on the microsecond to millisecond time scale have been probed directly by examining the dependence of the proton transverse relaxation rate on the spin-locking field strength. Leu11 shows a strong dependence on field strength, implying conformational exchange with a time constant of 125 +/- 69 microseconds. The backbone at the actin-binding interface appears to be rather rigid.

Actins↗