Biomedical subjects
Publications and source records attributed to G Wagner.
Cloacal malformations are rare, complex inhibitional anomalies of early embryogenesis. We report a patient with a cloacal malformation in which a septate vagina and a rectal fistula emptied through a common orifice onto an exstrophic bladder plate. Additional anomalies included an omphalocele and malformations of the upper urinary tract and the lower extremities, skeleton, and vertebral column.
PURPOSE: The purpose of this study was to determine whether exercise mediates the psychological and nutritional effects of testosterone therapy in men with symptomatic HIV illness, low serum testosterone, and clinical symptoms of hypogonadism. METHODS: A 12-wk open trial of biweekly intramuscular testosterone injections was conducted, with 54 men completing the trial and exercise assessments. Most (71%) men were diagnosed with AIDS; 41% had a CD4 < 50. One-third of the men were diagnosed with major depression, and nearly half had some evidence of wasting. Twenty-nine men (54%) engaged in exercise (predominantly resistance training) during the trial. Exercisers did not differ from nonexercisers on any measure of psychological well being or nutritional status at baseline. RESULTS: After 12 wk of testosterone treatment, those who exercised showed significant improvement in mood (Hamilton Rating Scale for Depression; HAM-D) and overall distress (Brief Symptom Inventory; BSI) (P < 0.000 for both), as well as a significant increase in body cell mass (P < 0.01) and lean body mass (mean increase of 2.6 kg; P < 0.000) as measured by bioelectric impedance analysis. In contrast, nonexercisers showed improvement on the HAM-D (P < 0.000), but not the BSI or measures of nutritional status. CONCLUSION: These findings indicate that exercise may be an important adjunct to testosterone therapy in the treatment of psychological distress and wasting symptoms in men with symptomatic HIV illness.
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The incidence of Plasmodium falciparum infection has been followed in a birth cohort of 71 infants in southern Ghana, an area of perennial malaria transmission. Parasite DNA detection established the presence of a high rate of infection in newborns (13.6%), a low level of infection from two to 26 weeks (1.5-9.7%) and a steadily increasing parasite rate from 26 weeks of age. The median age to first infection was 42 weeks. Five cases of fever (temperature > or = 37.5 degrees C) and parasite density greater than 1,000 parasites/microl of blood, all in children more than 18 weeks of age, were considered possible cases of clinical malaria. The risk of infection was almost three times higher in the wet season than in the dry season and increased significantly from the age of 18 weeks. The level of malaria-specific IgG at birth was positively correlated with risk of infection in children 6-12 months of age, indicating that maternally derived anti-malarial IgG is correlated with exposure to malaria infection. There was no association between malaria-specific IgG at birth and risk of infection in children 0-6 months of age. However, infants do appear to possess mechanisms to limit parasite growth and a role for maternal antibody cannot be ruled out.
Anacystis nidulans flavodoxin, an electron-transfer protein containing a flavin mononucleotide (FMN) molecule as its prosthetic group, has a redox potential for the oxidized/semiquinone equilibrium close to that of free flavin. Whereas the redox potential for the semiquinone/hydroquinone equilibrium is more negative. To gain an understanding of the contribution of mobility to redox potential modulation, we studied the backbone mobility of the oxidized A. nidulans flavodoxin at pH 6.6, 303 K by 15N NMR relaxation measurements. The spin-lattice relaxation rate constants (RN(Nz)=1/T1), spin-spin relaxation rate constants (RN(Nx,y)=1/T2) and 1H-15N nuclear Overhauser effects (NOE) were obtained for 143 of the 166 protonated backbone 15N nuclei and for the FMN N3 nucleus without ambiguity. The 15N T1, T2 and NOE data were analyzed by reduced spectral density mapping, and the so-called model-free approach. In contrast to most other proteins studied with 15N relaxation experiments, we found an almost complete absence of internal mobility. The overall correlation time of this 169-residue flavodoxin (>19 kDa) is significantly shorter (7.4 to 7.8 ns) than that of other proteins of this size, suggesting that the absence of internal mobility is correlated with faster overall rotational diffusion. The uniformity of the motional parameters along the backbone is in strong contrast to the crystallographic B-factors, which vary significantly along the sequence in this and other flavodoxins. The NMR relaxation parameters are primarily sensitive to rotational diffusive motions of the N-H bond vectors, while the crystallographic B-factors would be sensitive to translational internal motions as well. However, the large B-factors in this protein may originate from crystal packing and crystal lattice disorder. The relatively fast overall tumbling results in sharp NMR resonances. Hence, much larger proteins with such favorable dynamic behavior could be excellent candidates for studies by NMR.
The SKN-1 transcription factor specifies early embryonic cell fates in Caenorhabditis elegans. SKN-1 binds DNA at high affinity as a monomer, by means of a basic region like those of basic-leucine zipper (bZIP) proteins, which bind DNA only as dimers. We have investigated how the SKN-1 DNA-binding domain (the Skn domain) promotes stable binding of a basic region monomer to DNA. A flexible arm at the Skn domain amino terminus binds in the minor groove, but a support segment adjacent to the carboxy-terminal basic region can independently stabilize basic region-DNA binding. Off DNA, the basic region and arm are unfolded and, surprisingly, the support segment forms a molten globule of four alpha-helices. On binding DNA, the Skn domain adopts a tertiary structure in which the basic region helix extends directly from a support segment alpha-helix, which is required for binding. The remainder of the support segment anchors this uninterrupted helix on DNA, but leaves the basic region exposed in the major groove. This is similar to how the bZIP basic region extends from the leucine zipper, indicating that positioning and cooperative stability provided by helix extension are conserved mechanisms that promote binding of basic regions to DNA.
Proteins of the rho subfamily of ras GTPases have been shown to be crucial components of pathways leading to cell growth and the establishment of cell polarity and mobility. Presented here is the solution structure of one such protein, Cdc42Hs, which provides insight into the structural basis for specificity of interactions between this protein and its effector and regulatory proteins. Standard heteronuclear NMR methods were used to assign the protein, and approximately 2100 distance and dihedral angle constraints were used to calculate a set of 20 structures using a combination of distance geometry and simulated annealing refinement. These structures show overall similarity to those of other GTP-binding proteins, with some exceptions. The regions corresponding to switch I and switch II in H-ras are disordered, and no evidence was found for an alpha-helix in switch II. The 13-residue insertion, which is only present in rho-subtype proteins and has been shown to be an important mediator of binding of regulatory and target proteins, forms a compact structure containing a short helix lying adjacent to the beta4-alpha3 loop. The insert forms one edge of a "switch surface" and, unexpectedly, does not change conformation upon activation of the protein by the exchange of GTP analogs for GDP. These studies indicate the insert region forms a stable invariant "footrest" for docking of regulatory and effector proteins.
Two chronic haemodialyzed patients with digitalis intoxication are reported. One of them took digoxin 0.25 mg three times daily for an unknown period and the other took digitoxin 0.1 mg twice daily for two weeks. The symptoms of intoxication were mainly concealed by uremic syndrome. The diagnosis was established by noticed sinus bradycardia, first- and second-degree atrioventricular block in ECG and the determination of sera levels of glycosides (serum digoxin concentration was 7.36 ng/ml, serum digitoxin concentration was 46.5 ng/ml) in both cases. Considering the probable long elimination period of digitalis and the potentially life-threatening situation the patients were given digoxin-specific antibody (Fab) fragments with potassium replacement therapy. The symptoms disappeared within a few hours after therapy, side effects and rebound toxicity did not develop. In connection with these cases the aim of this report is to publish a method which can reverse the life-threatening digitalis intoxication in patients suffering from renal failure as well. As to the above method, the authors have not found any similar case reports in the Hungarian medical literature.
The antitumor compound PT523 [N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L- ornithine] was found to have an inhibition constant (K(i)) of 0.35 +/- 0.10 pM against human dihydrofolate reductase (hDHFR), 15-fold lower than that of the classical antifolate drug methotrexate (MTX). The structure of PT523 bound to hDHFR and hDHFR-NADPH was investigated using multinuclear NMR techniques. NMR data indicate that the binary complex has two distinct conformations in solution which are in slow exchange and that the addition of NADPH stabilizes the ternary complex in a single bound state. Comparison of resonance assignments in the PT523 and MTX ternary complexes revealed that substantial protein chemical shift differences are limited to small regions of hDHFR tertiary structure. A restrained molecular dynamics and energy minimization protocol was performed for the hDHFR-PT523-NADPH complex, using 185 NOE restraints (33 intermolecular) to define the ligand-binding region. The positions of the pteridine and pABA rings of PT523 and the nicotinamide and ribose rings of NADPH are well defined in the solution structures (RMSD = 0.59 A) and are consistent with previously determined structures of DHFR complexes. The N(delta)-hemiphthaloyl-L-ornithine group of PT523 is less well defined, and the calculated model structures suggest the hemiphthaloyl ring may adopt more than one conformation in solution. Contacts between the hemiphthaloyl ring and hDHFR, which are not possible in the hDHFR-MTX-NADPH complex, may explain the greater inhibition potency of PT523.
Glutathione transferase (GST)-catalysed S-nitrosoglutathione (GSNO) formation from alkyl nitrites was determined with the homodimers 1-1, 2-2, 3-3, and 4-4 isolated from rat liver. The 4-4 isoform showed a high specificity for the alkyl nitrites. Total GST activities were studied in homogenates from different organs. The liver showed highest GST activity both with amyl nitrite and with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate, the activity ratio of amyl nitrite over CDNB being 3.8. In lung and heart, these ratios were 6.2 and 5.7, respectively, indicating a selectivity of these organs for alkyl nitrite metabolism and GSNO formation.
Transcription factors of the NFAT family regulate the production of effector proteins that coordinate the immune response. The immunosuppressive drugs FK506 and cyclosporin A (CsA) act by blocking a Ca2+-mediated signalling pathway leading to NFAT. Although FK506 and CsA have enabled human organs to be transplanted routinely, the toxic side-effects of these drugs limit their usage. This toxicity might be absent in antagonists that target NFAT directly. As a first step in the structure-based search for NFAT antagonists, we now report the identification and solution structure of a 20K domain of NFATc (NFATc-DBD) that is both necessary and sufficient to bind DNA and activate transcription cooperatively. Although the overall fold of the NFATc DNA-binding domain is related to that of NF-kappaB p50 (refs 2, 3), the two proteins use significantly different strategies for DNA recognition. On the basis of these results, we present a model for the cooperative complex formed between NFAT and the mitogenic transcription factor AP-1 on the interleukin-2 enhancer.
We have used 15N NMR relaxation experiments to probe, for the glycosylated human CD2 adhesion domain, the overall molecular motion, as well as very fast nanosecond-picosecond (ns-ps) and slow millisecond-microsecond (ms-microsecond) internal motions. Using a novel analysis method that considers all residues, we obtained a correlation time for the overall motion of 9.5 +/- 0.3 ns. Surprisingly, we found a large contiguous patch of residues in the counterreceptor (CD58) binding site of human CD2 exhibiting slow conformational exchange motions (ms-microsecond). On the other hand, almost none of the residues of the CD58 binding side display fast (ns-ps) internal motions of amplitudes larger than what is seen for well-ordered regions of the structure. Residues close to the N-glycosylation site, and the first N-acetylglucosamine of the high mannose glycan are as rigid as the protein core. Residues conserved in the immunoglobulin superfamily V-set domain are generally very rigid.
Villin 14T is the amino terminal actin monomer binding domain from the actin-severing and bundling protein villin. Its structure has been determined in solution using heteronuclear multidimensional nuclear magnetic resonance (NMR) spectroscopy (Markus MA, Nakayama T, Matsudaira P, Wagner G. 1994. Solution structure of villin 14T, a domain conserved among actin-severing proteins. Protein Science 3:70-81). An additional nuclear Overhauser effect (NOE) spectroscopy data set, acquired using improved gradient techniques, and further detailed analysis of existing data sets, produced an additional 601 NOE restraints for structure calculations. The overall fold does not change significantly with the additional NOE restraints but the definition of the structure is improved, as judged by smaller deviations among an ensemble of calculated structures that adequately satisfy the NMR restraints. Some of the side chains, especially those in the hydrophobic core of the domain, are much more defined. This improvement in the detail of the solution structure of villin 14T makes it interesting to compare the structure with the crystal structure of gelsolin segment 1, which shares 58% sequence identity with villin 14T, in an effort to gain insight into villin 14T's weaker affinity for actin monomers. Villin 14T has smaller side chains at several positions that make hydrophobic contacts with actin in the context of gelsolin segment 1. The structure is also compared with the structure of the related actin-severing domain, severin domain 2.
This paper introduces a novel computational method for estimating relaxation rates among pairs of spin orders. This method simultaneously estimates all the auto- and cross-relaxation rates from the same measurements, and avoids the ill-conditioning problems associated with multiexponential fits. The method models the relaxation dynamics by a system of linear differential equations, and assumes that measurements of the spin orders have been made at an equally spaced sequence of time points. It computes a nonlinear least-squares fit of the exponential of the rate matrix at the shortest time point to these measurements. Preliminary estimates of the exponential matrix and initial spin orders from which to start the computations are obtained by solving simpler linear-least-squares problems. The performance of the method on simulated 2 x 2 test problems indicates that when measurements at eight or more equally spaced times spanning the maximum and inflection points of the build-up curves are available, the relative errors in the rates are usually less than the relative errors in the measurements. The method is further demonstrated by applying it to the problem of determining the cross correlation-induced cross-relaxation rates between the in-phase and antiphase coherence of the amide groups in the 15N-labeled protein oxidized flavodoxin. Finally, the possibility of extending the method to other kinds of relaxation measurements and larger spin systems is discussed.
Maximum-entropy spectrum reconstruction derives much of its power from its nonlinearity. This nonlinearity causes difficulties in several contexts, however, including computation of multidimensional spectra and quantification of reconstructed spectra. We describe two methods for avoiding these difficulties: a "Constant-lambda" algorithm for performing row-wise reconstructions, which uses a fixed weighting of the entropy and the experimental constraint in the objective function, and in situ error analysis, for calibrating the nonlinearity. These methods are applied to data from quantitative J-correlation and relaxation experiments.
OBJECTIVES: To develop a brief, reliable, self-administered measure of erectile function that is cross-culturally valid and psychometrically sound, with the sensitivity and specificity for detecting treatment-related changes in patients with erectile dysfunction. METHODS: Relevant domains of sexual function across various cultures were identified via a literature search of existing questionnaires and interviews of male patients with erectile dysfunction and of their partners. An initial questionnaire was administered to patients with erectile dysfunction, with results reviewed by an international panel of experts. Following linguistic validation in 10 languages, the final 15-item questionnaire, the international index of Erectile Function (IIEF), was examined for sensitivity, specificity, reliability (internal consistency and test-retest repeatability), and construct (concurrent, convergent, and discriminant) validity. RESULTS: A principal components analysis identified five factors (that is, erectile function, orgasmic function, sexual desire, intercourse satisfaction, and overall satisfaction) with eigenvalues greater than 1.0. A high degree of internal consistency was observed for each of the five domains and for the total scale (Cronbach's alpha values of 0.73 and higher and 0.91 and higher, respectively) in the populations studied. Test-retest repeatability correlation coefficients for the five domain scores were highly significant. The IIEF demonstrated adequate construct validity, and all five domains showed a high degree of sensitivity and specificity to the effects of treatment. Significant (P values = 0.0001) changes between baseline and post-treatment scores were observed across all five domains in the treatment responder cohort, but not in the treatment nonresponder cohort. CONCLUSIONS: The IIEF addresses the relevant domains of male sexual function (that is, erectile function, orgasmic function, sexual desire, intercourse satisfaction, and overall satisfaction), is psychometrically sound, and has been linguistically validated in 10 languages. This questionnaire is readily self-administered in research or clinical settings. The IIEF demonstrates the sensitivity and specificity for detecting treatment-related changes in patients with erectile dysfunction.
We have refined the solution structure of cadmium-bound GAL4 and present its 15N and 1H NMR assignments. The root-mean-square (rms) deviation to the average structure was 0.4 +/- 0.05 A for backbone atoms, and 0.9 +/- 0.1 A for all heavy atoms. The three-bond heteronuclear 3J(113Cd,1H) coupling constants were found to disobey a Karplus-type relationship, which was attributable to the unusual constraints imposed by the bimetal-thiolate cluster in GAL4. We conclude that the structural parameters that correlate to 3J(113Cd,1H) are complex.
eIF4E, the mRNA cap binding protein, is a master switch that controls eukaryotic translation. To be active, it must bind eIF4G and form the eIF4F complex, which also contains eIF4A. Translation is downregulated by association of eIF4E with 4E-BP, which occupies the eIF4G binding site. Signalling events acting on 4E-BP cause it to dissociate from eIF4E, and eIF4E is then free to bind eIF4G to form the active eIF4F complex. We have solved the structure of the yeast eIF4E/m7Gpp complex in a CHAPS micelle. We determined the position of the second nucleotide in a complex with m7GpppA, and identified the 4E-BP binding site. eIF4E has a curved eight-stranded antiparallel beta-sheet, decorated with three helices on the convex face and three smaller helices inserted in connecting loops. The m7G of the cap is intercalated into a stack of tryptophans in the concave face. The 4E-BP binding site is located in a region encompassing one edge of the beta-sheet, the adjacent helix a2 and several regions of non-regular secondary structure. It is adjacent to, but does not overlap the cap-binding site.