Biomedical subjects
G Wagner
Publications and source records attributed to G Wagner.
Solution structure of the CIDE-N domain of CIDE-B and a model for CIDE-N/CIDE-N interactions in the DNA fragmentation pathway of apoptosis.
Apoptotic DNA fragmentation and chromatin condensation are mediated by the caspase-activated DFF40/ CAD nuclease, which is chaperoned and inhibited by DFF45/ICAD. CIDE proteins share a homologous regulatory CIDE-N domain with DFF40/CAD and DFF45/ ICAD. Here we report the solution structure of CIDE-N of human CIDE-B. We show that the CIDE-N of CIDE-B interacts with CIDE-N domains of both DFF40 and DFF45. The binding epitopes are similar and map to a highly charged bipolar surface region of CIDE-B. Furthermore, we demonstrate that the CIDE-N of CIDE-B regulates enzymatic activity of the DFF40/ DFF45 complex in vitro. Based on these results and mutagenesis data, we propose a model for the CIDE-N/ CIDE-N complex and discuss the role of this novel bipolar interaction in mediating downstream events of apoptosis.
The crystal structure of a T cell receptor in complex with peptide and MHC class II.
The crystal structure of a complex involving the D10 T cell receptor (TCR), 16-residue foreign peptide antigen, and the I-Ak self major histocompatibility complex (MHC) class II molecule is reported at 3.2 angstrom resolution. The D10 TCR is oriented in an orthogonal mode relative to its peptide-MHC (pMHC) ligand, necessitated by the amino-terminal extension of peptide residues projecting from the MHC class II antigen-binding groove as part of a mini beta sheet. Consequently, the disposition of D10 complementarity-determining region loops is altered relative to that of most pMHCI-specific TCRs; the latter TCRs assume a diagonal orientation, although with substantial variability. Peptide recognition, which involves P-1 to P8 residues, is dominated by the Valpha domain, which also binds to the class II MHC beta1 helix. That docking is limited to one segment of MHC-bound peptide offers an explanation for epitope recognition and altered peptide ligand effects, suggests a structural basis for alloreactivity, and illustrates how bacterial superantigens can span the TCR-pMHCII surface.
Solution structure of Apaf-1 CARD and its interaction with caspase-9 CARD: a structural basis for specific adaptor/caspase interaction.
Direct recruitment and activation of caspase-9 by Apaf-1 through the homophilic CARD/CARD (Caspase Recruitment Domain) interaction is critical for the activation of caspases downstream of mitochondrial damage in apoptosis. Here we report the solution structure of the Apaf-1 CARD domain and its surface of interaction with caspase-9 CARD. Apaf-1 CARD consists of six tightly packed amphipathic alpha-helices and is topologically similar to the RAIDD CARD, with the exception of a kink observed in the middle of the N-terminal helix. By using chemical shift perturbation data, the homophilic interaction was mapped to the acidic surface of Apaf-1 CARD centered around helices 2 and 3. Interestingly, a significant portion of the chemically perturbed residues are hydrophobic, indicating that in addition to the electrostatic interactions predicted previously, hydrophobic interaction is also an important driving force underlying the CARD/CARD interaction. On the basis of the identified functional residues of Apaf-1 CARD and the surface charge complementarity, we propose a model of CARD/CARD interaction between Apaf-1 and caspase-9.
Screening of heavy metal pollution in Bulgaria using Populus nigra 'Italica'.
The pollution with As, Cd, Co, Cr, Cu, Fe, Mn, Pb, Sb and Zn on the territory of Bulgaria has been evaluated using standardized samples of leaves of Populus nigra 'Italica' collected during the last 10 years. Background values for the respective elements are established in the leaves of the plant. The comparisons of urban and industrial pollution to some other European countries indicates similar or lower concentrations. Using cluster analysis of the analytical data the territory of Bulgaria is divided into six regions with different pollution levels.
The Cap-binding protein eIF4E promotes folding of a functional domain of yeast translation initiation factor eIF4G1.
The association of eucaryotic translation initiation factor eIF4G with the cap-binding protein eIF4E establishes a critical link between the mRNA and the ribosome during translation initiation. This association requires a conserved seven amino acid peptide within eIF4G that binds to eIF4E. Here we report that a 98-amino acid fragment of S. cerevisiae eIF4G1 that contains this eIF4E binding peptide undergoes an unfolded to folded transition upon binding to eIF4E. The folding of the eIF4G1 domain was evidenced by the eIF4E-dependent changes in its protease sensitivity and (1)H-(15)N HSQC NMR spectrum. Analysis of a series of charge-to-alanine mutations throughout the essential 55.4-kDa core of yeast eIF4G1 also revealed substitutions within this 98-amino acid region that led to reduced eIF4E binding in vivo and in vitro. These data suggest that the association of yeast eIF4E with eIF4G1 leads to the formation of a structured domain within eIF4G1 that could serve as a specific site for interactions with other components of the translational apparatus. They also suggest that the stability of the native eIF4E-eIF4G complex is determined by amino acid residues outside of the conserved seven-residue consensus sequence.
Structure of the soluble methane monooxygenase regulatory protein B.
The soluble methane monooxygenase (sMMO; EC 1.14.13.25) from the pseudothermophile Methylococcus capsulatus (Bath) is a three-component enzyme system that catalyzes the selective oxidation of methane to methanol. We have used NMR spectroscopy to produce a highly refined structure of MMOB, the 16-kDa regulatory protein of this system. This structure has a unique and intricate fold containing seven beta-strands forming two beta-sheets oriented perpendicular to each other and bridged by three alpha-helices. The rate and efficiency of the methane hydroxylation by sMMO depend on dynamic binding interactions of the hydroxylase with the reductase and regulatory protein components during catalysis. We have monitored by NMR the binding of MMOB to the hydroxylase in the presence and absence of the reductase. The results of these studies provide structural insight into how the regulatory protein interacts with the hydroxylase.
Solution structure of the catalytic domain of GCN5 histone acetyltransferase bound to coenzyme A.
Gene transcription requires the release of inactive DNA from its packaging of histone proteins. Following the discovery of the first transcription-associated histone acetyltransferase, tetrahymena GCN5, it was shown that yeast GCN5 is recruited to the promoter and causes hyper-acetylation of histones and transcriptional activation of target genes, establishing a direct connection between histone acetylation and transcriptional activation. Many other important transcription regulators have been found to have histone acetyltransferase activity, including TAFII230/250, p300/CBP and its associated factor PCAF. Here we present the solution structure of the catalytic domain of tGCN5 (residues 47-210) in complex with coenzyme A. The structure contains two domains; the amino-terminal domain is similar to those of other GCN5-related N-acetyltransferases but the carboxy-terminal domain is not. Coenzyme A binds in a deep hydrophobic pocket between the two domains. Chemical shift changes upon titration with histone H3 peptides indicate a binding site at the domain boundary opposite to the coenzyme A site. The structural data indicate a single-step acetyl-transfer reaction mechanism catalysed by a hydrogen bond to the backbone amide group of leucine 126 and the side-chain carboxyl group of a conserved acidic residue.
Structure of a heterophilic adhesion complex between the human CD2 and CD58 (LFA-3) counterreceptors.
Interaction between CD2 and its counterreceptor, CD58 (LFA-3), on opposing cells optimizes immune recognition, facilitating contacts between helper T lymphocytes and antigen-presenting cells as well as between cytolytic effectors and target cells. Here, we report the crystal structure of the heterophilic adhesion complex between the amino-terminal domains of human CD2 and CD58. A strikingly asymmetric, orthogonal, face-to-face interaction involving the major beta sheets of the respective immunoglobulin-like domains with poor shape complementarity is revealed. In the virtual absence of hydrophobic forces, interdigitating charged amino acid side chains form hydrogen bonds and salt links at the interface (approximately 1200 A2), imparting a high degree of specificity albeit with low affinity (K(D) of approximately microM). These features explain CD2-CD58 dynamic binding, offering insights into interactions of related immunoglobulin superfamily receptors.
Management factors associated with Babesia bovis seroprevalence in cattle from eastern Yucatán, Mexico.
The effect of management on the seroprevalence of Babesia bovis was studied in 399 Bos indicus cattle 1-2 years old from 92 farms in the eastern Yucatán, México. The management factors studied were: farm-type, production system, herd size, farm size, stocking density, vector control, dipping interval, type of dipping, type of acaricide and cattle introduction to the farm. A cross-sectional study was carried out (2-stage cluster sampling). The number of serum samples was proportionally distributed according to the number of farms in the nine locations of eastern Yucatán, México (399 animals from 92 farms). Antibody activity to B. bovis was tested using an indirect ELISA. The farms with a seroprevalence < or = 75% were considered as cases and those with seroprevalence > 75% were considered as controls. The variables with p < or = 0.20 were included in fixed effects logistic regression. The seroprevalence of the zone was 73.8% (66.3-81.3%). The following risk factors were found: Stocking density (< 1 head/ha, OR = 4.04, CI (OR) = 1.20-13.62) and dipping interval (> 60 days, OR = 5.07 CI (OR) = 1.26-20.48).
Functional glycan-free adhesion domain of human cell surface receptor CD58: design, production and NMR studies.
A general strategy is presented here for producing glycan-free forms of glycoproteins without loss of function by employing apolar-to-polar mutations of surface residues in functionally irrelevant epitopes. The success of this structure-based approach was demonstrated through the expression in Escherichia coli of a soluble 11 kDa adhesion domain extracted from the heavily glycosylated 55 kDa human CD58 ectodomain. The solution structure was subsequently determined and binding to its counter-receptor CD2 studied by NMR. This mutant adhesion domain is functional as determined by several experimental methods, and the size of its binding site has been probed by chemical shift perturbations in NMR titration experiments. The new structural information supports a 'hand-shake' model of CD2-CD58 interaction involving the GFCC'C" faces of both CD2 and CD58 adhesion domains. The region responsible for binding specificity is most likely localized on the C, C' and C" strands and the C-C' and C'-C" loops on CD58.
Structure and interactions of the translation initiation factor eIF1.
eIF1 is a universally conserved translation factor that is necessary for scanning and involved in initiation site selection. We have determined the solution structure of human eIF1 with an N-terminal His tag using NMR spectroscopy. Residues 29-113 of the native sequence form a tightly packed domain with two alpha-helices on one side of a five-stranded parallel and antiparallel beta-sheet. The fold is new but similar to that of several ribosomal proteins and RNA-binding domains. A likely binding site is indicated by yeast mutations and conserved residues located together on the surface. No interaction with recombinant eIF5 or the initiation site RNA GCCACAAUGGCA was detected by NMR, but GST pull-down experiments show that eIF1 binds specifically to the p110 subunit of eIF3. This interaction explains how eIF1 is recruited to the 40S ribosomal subunit.
Endometrial cancer after combined hormone replacement therapy.
OBJECTIVE: It has been suggested that exposure to relatively high levels of unopposed estrogen is a risk factor for endometrial cancer. Combined therapy of estrogen with cyclic progestagen was therefore highly recommended for menopausal women with an intact uterus. METHODS: The cases of two postmenopausal women who developed endometrial cancer after taking continuous sequential HRT for 15 months are reported. Both were without bleeding for more than 2 years and presented with a normal vaginal ultrasound. They had severe menopausal symptoms and asked for HRT. RESULTS: After 15 months irregular bleeding occurred and a hysterectomy was performed. The pathohistological finding in both cases was endometrial cancer. As we measured the serum estradiol levels 4 h after tablet ingestion supraphysiologic values ranging between 418 and 442 pg/ml were found. CONCLUSION: Our report strengthens the evidence that supraphysiologic estradiol levels despite combination with cyclic progestagen therapy, increase the risk of endometrial cancer.
Solution structure of BID, an intracellular amplifier of apoptotic signaling.
We report the solution structure of BID, an intracellular cross-talk agent that can amplify FAS/TNF apoptotic signal through the mitochondria death pathway after Caspase 8 cleavage. BID contains eight alpha helices where two central hydrophobic helices are surrounded by six amphipathic ones. The fold resembles poreforming bacterial toxins and shows similarity to BCL-XL although sequence homology to BCL-XL is limited to the 16-residue BH3 domain. Furthermore, we modeled a complex of BCL-XL and BID by aligning the BID and BAK BH3 motifs in the known BCL-XL-BAK BH3 complex. Additionally, we show that the overall structure of BID is preserved after cleavage by Caspase 8. We propose that BID has both BH3 domain-dependent and -independent modes of action in inducing mitochondrial damage.
Relative contributions of a single-admission 12-lead electrocardiogram and early 24-hour continuous electrocardiographic monitoring for early risk stratification in patients with unstable coronary artery disease.
Patients with unstable coronary syndromes are a heterogeneous group with varying degrees of ischemia and prognosis. The present study compares the prognostic value of a standard electrocardiogram (ECG) obtained at admission to the hospital with the information from 24-hour continuous electrocardiographic monitoring obtained immediately after admission. The admission ECGs and 24 hours of vectorcardiographic (VCG) monitoring from 308 patients admitted with unstable coronary artery disease were analyzed centrally regarding standard electrocardiographic ST-T changes, ST-vector magnitude (ST-VM), and ST change vector magnitude episodes. End points were death, acute myocardial infarction, and refractory angina pectoris within a 30-day follow-up period. ST-VM episodes (> or = 50 microV for > or = 1 minute) during VCG monitoring was the only independent predictor of death or acute myocardial infarction by multivariate analysis. ST-VM episodes during vectorcardiography was associated with a relative risk of 12.7 for having a cardiac event, hypertension was associated with a relative risk of 1.7, and ST depression on the admission ECG was associated with a relative risk of 5.7. Patients with ST depression at admission had an event rate (death or acute myocardial infarction) of 17% at 30-day follow-up. Patients without ST depression could further be risk stratified by 24 hours of VCG monitoring into a subgroup with ST-VM episodes at similar (8%) risk and a subgroup without ST-VM episodes at low (1%) risk (p = 0.00005). Continuous VCG monitoring provides important information for evaluating patients with unstable coronary artery disease. It is recommended that patients not initially estimated at high risk based on the admission ECG are referred for 24 hours of VCG monitoring for further risk stratification.
[Erectile dysfunction. An update].
Although around 10% of men aged 40 to 70 years have complete erectile dysfunction, only a few seek medical help. As erectile dysfunction is frequently associated with a number of systemic illnesses and surgical treatments, a wide range of doctors should be aware of the condition in their patients. Current effective treatments include psychosexual counselling, vacuum erection devices, intracavernosal and transurethral drug delivery, and penile prostheses. Promising oral treatments are currently being investigated. Both doctors and the public need to be better informed about erectile dysfunction and its treatment.
Identification of the occluded artery in patients with myocardial ischemia induced by prolonged percutaneous transluminal coronary angioplasty using traditional vs transformed ECG-based indexes.
We have studied the spatial properties of ischemic changes as induced by prolonged angioplasty and how the changes are related to different ECG indexes. Indexes based on measurements at specific points in time (ST level at J + 60 ms point, maximal T wave amplitude and position, QT interval, and QRS duration) and global indexes (based on the Karhunen-Loève transform and applied to the QRS complex, ST-T complex, ST segment, and T wave), considering both repolarization and depolarization information, were analyzed. The changes during the occlusion period of the different indexes were used as variables in a multivariate discriminant analysis to determine which indexes showed the best discrimination of the three major occlusion sites (corresponding to LAD, RCA, and LCX coronary arteries). Occlusions in LCX artery were the most difficult to classify. With three local indexes (ST60 level measured in lead V3, T wave amplitude in I, and ST60 in III) it was possible to correctly classify 76% of patients by the occlusion site, and with three KLT-derived indexes (first-order KLT index for ST-T complex in I and for QRS in leads V3 and I) 83% of correct classification was obtained. Using six indexes for local and KLT-derived indexes the correct classification was increased to 85 and 90% of patients, respectively. The use of different ECG indexes (from different intervals) on quasiorthogonal leads permitted the identification of the occluded artery in patients undergoing PTCA and may be extended to more general use.
Admission standard electrocardiogram for early risk stratification in patients with unstable coronary artery disease not eligible for acute revascularization therapy: a TRIM substudy. ThRombin Inhibition in Myocardial Infarction.
OBJECTIVES: The purpose of this study was to determine the prognostic capacity of a single electrocardiogram (ECG) obtained early after admission to the hospital in patients suspected of non-Q-wave myocardial infarction and unstable angina pectoris. METHODS: Six hundred twenty-nine patients from the TRIM study were included. The patients were divided into subgroups on the basis of ST-segment changes in the inclusion ECG. Death, acute myocardial infarction, or refractory angina (despite treatment) were registered during a follow-up period of 30 days. RESULTS: Patients with ST depression had a significantly higher event rate compared with patients with other ECG changes or with normal ECG results. The difference in event rates between patients with ST depression and patients without ST depression regarding the composite of death and acute myocardial infarction was highly significant (P =.0008). A significant association between the magnitude of the ST-segment depression (in millimeters) and the risk of cardiac events was also demonstrated. Multivariate analysis proved ST depression early after admission to be an independent predictor of high risk. CONCLUSION: In patients with unstable coronary artery disease, ST-segment depression at admission is a strong predictor of early (30 days) cardiac events and the extent of ST depression carries important prognostic information as well.