Patient acceptance of transvaginal sonographic endometrial thickness assessment compared with hysteroscopy and biopsy for exclusion of endometrial cancer in cases of postmenopausal bleeding.

INTRODUCTION: Available examinations for women with postmenopausal bleeding include transvaginal sonography to measure endometrial thickness (TVS-ET), and invasive endometrial assessment using hysteroscopy/endometrial biopsy. However, selection of the examination method seldom involves consideration of patient preferences. The aim of this study was to examine patient preferences for the method used to investigate postmenopausal bleeding. METHODS: Women were asked to complete an interviewer-administered structured survey before they underwent clinical investigations at a university gynaecology unit from June 2016 to June 2017. Using the standard gamble approach, women were asked to choose between invasive assessment by hysteroscopy/endometrial biopsy (gold standard) or TVS-ET with a risk of missing endometrial cancer. The risk of missing endometrial cancer during TVS-ET was varied until each woman was indifferent to either option. RESULTS: The median detection rate for endometrial cancer required using TVS-ET was 95% (interquartile range=80%-99.9%). In total, 200 women completed the survey, and 77 (38.5%) women required TVS-ET to have a 99.9% detection rate for endometrial cancer. Prior hysteroscopy experience was the only factor that influenced the women's decisions: a significantly higher detection rate was required by this patient group than by patients without previous hysteroscopy experience (P=0.047). CONCLUSION: A substantial proportion of women would accept TVS-ET alone for the investigation of postmenopausal bleeding. In the era of patientcentred care, clinicians should incorporate patient preferences and enable women to make informed choices concerning the management of postmenopausal bleeding.

Substrate independent ATPase activity may complicate high throughput screening.

Inorganic phosphate release, [Pi], is often measured in an enzymatic reaction in a high throughput setting. Based on the published mechanism, we designed a protocol for our screening for inhibitors of SAICAR synthetase (PurC), and we found a gradual increase in [Pi] in positive control samples over the course of the day. Further investigation indicated that hydrolysis of ATP catalyzed by PurC, rather than substrate-related phosphate release, was responsible for a partial contribution to the signals in the control samples. Thus substrate-independent ATPase activity may complicate high throughput screening.

Structure of N5-carboxyaminoimidazole ribonucleotide synthase (PurK) from Bacillus anthracis.

The apo structure of N5-carboxyaminoimidazole ribonucleotide synthase (PurK) from Bacillus anthracis (baPurK) with Mg2+ in the active site is reported at 1.96 Šresolution. PurK is an enzyme in the purine-biosynthetic pathway, unique to prokaryotes, that converts 5-aminoimidazole ribonucleotide to N5-carboxyaminoimidazole ribonucleotide and has been suggested as a potential antimicrobial drug target. Two interesting features of baPurK are a flexible B-loop (residues 149/150-157) that is in close contact with the active site and the binding of Mg2+ to the active site without additional ligands.

Structural and dynamic study of the tetramerization region of non-erythroid alpha-spectrin: a frayed helix revealed by site-directed spin labeling electron paramagnetic resonance.

The N-terminal region of alpha-spectrin is responsible for its association with beta-spectrin in a heterodimer, forming functional tetramers. Non-erythroid alpha-spectrin (alphaII-spectrin) has a significantly higher association affinity for beta-spectrin than the homologous erythroid alpha-spectrin (alphaI-spectrin). We have previously determined the solution structure of the N-terminal region of alphaI-spectrin by NMR methods, but currently no structural information is available for alphaII-spectrin. We have used cysteine scanning, spin labeling electron paramagnetic resonance (EPR), and isothermal titration calorimetry (ITC) methods to study the tetramerization region of alphaII-spectrin. EPR data clearly show that, in alphaII-spectrin, the first nine N-terminal residues were unstructured, followed by an irregular helix (helix C'), frayed at the N-terminal end, but rigid at the C-terminal end, which merges into the putative triple-helical structural domain. The region corresponding to the important unstructured junction region linking helix C' to the first structural domain in alphaI-spectrin was clearly structured. On the basis of the published model for aligning helices A', B', and C', important interactions among residues in helix C' of alphaI- and alphaII-spectrin and helices A' and B' of betaI- and betaII-spectrin are identified, suggesting similar coiled coil helical bundling for spectrin I and II in forming tetramers. The differences in affinity are likely due to the differences in the conformation of the junction regions. Equilibrium dissociation constants of spin-labeled alphaII and betaI complexes from ITC measurements indicate that residues 15, 19, 37, and 40 are functionally important residues in alphaII-spectrin. Interestingly, all four corresponding homologous residues in alphaI-spectrin (residues 24, 28, 46, and 49) have been reported to be clinically significant residues involved in hematological diseases.

Conformational changes at the tetramerization site of erythroid alpha-spectrin upon binding beta-spectrin: a spin label EPR study.

We used cysteine scanning, isothermal titration calorimetry (ITC) and spin label EPR methods to study the two regions that flank the partial domain Helix C' of the N-terminal end of alpha-spectrin (residues 14-20 and residues 44-54) in the absence and presence of a model protein of the beta-spectrin C-terminal end. In the absence of beta-spectrin, residues 14-20 and 46-52 were known to be unstructured. The EPR spectral values of the inverse line width (Delta H (-1)) and of the width between the low field peak and the central peak ( aZ) of residues in part of the first unstructured region (residues 17-20) and of most residues in the second unstructured junction region (residues 46-52) changed dramatically upon association with beta-spectrin, suggesting that the two regions undergo a conformational change, becoming more rigid and likely becoming helical. ITC results showed that three of the seven residues in the junction region (residues 46-52) were very important in its association with beta-spectrin, in the following order: L49 > G46 > K48. In general, our results suggest that any mutations that affect the propensity of helical formation in the region spanning residues 17-52 in alpha-spectrin, or that affect hydrophobic clustering and/or salt-bridge stabilization of the bundled helices, would affect spectrin tetramer formation, and may lead to blood disorders.

Potential artifacts in using a glutathione S-transferase fusion protein system and spin labeling electron paramagnetic resonance methods to study protein-protein interactions.

Site-directed spin labeling electron paramagnetic resonance methods have been an important tool in studying protein-protein interactions. Labels are often attached to a cysteine residue, and spectra are acquired with and without binding partner(s) to provide information on the binding. This requires a knowledge of the label location which is simplified if the label remains faithfully attached to the designated residue in the complex. We report a system where this is not the case because the label was extracted by dialysis-resistant glutathione molecules. Once this artifact is identified, spectral subtraction provides a solution for meaningful data interpretation.

Sickle hemoglobin is more fusogenic than normal hemoglobin at physiological pH and ionic strength conditions.

We used electron microscopy, quasi-elastic light scattering and static light scattering to show that human hemoglobin (Hb) interacts with bovine brain phosphatidylserine lipid vesicles and promotes vesicle fusion in an isotonic buffer at pH 7.4. The fusogenic properties of Hb were observed in both small unilamellar vesicles (SUVs) and large unilamellar vesicles (LUVs). A simple turbidity measurement method was used to follow increases in vesicle size (scattering diameter) as a function of time. For the first 3 h, upon incubation with oxygenated Hb, the scattering diameters of vesicles increased at a rate of 7.8 nm/h for LUVs. Continuous incubation with Hb led to complicated vesicle fusion, probably due to the oxidation products of Hb and lipid molecules. In the absence of both Hb and lipid oxidation, using Hb liganded with carbon monoxide, we obtained, for the entire 20 h incubation period, a fusion rate of 2.9 nm/h for LUVs. We also studied interactions between sickle Hb and vesicles under the same conditions and found that the vesicle fusion rates for sickle Hb were about 2 times faster than those for normal Hb. These results showed that sickle Hb exhibited more extensive interactions with lipid bilayer than normal Hb at physiological pH and ionic strength conditions, and provide insights toward understanding the molecular mechanisms in sickle cell abnormalities.

Reduced water exchange in sickle cell anemia red cells: a membrane abnormality.

We have measured the diffusional water permeability of sickle cell anemia red blood cells under isotonic conditions using pulsed nuclear magnetic resonance (NMR) techniques. We have found that the equilibrium diffusional permeability for sickle cells is about 1.61.10(-3) cm/s, or about 60% of the value measured for normal cells. This abnormality is not related to the heterogeneity generally found in cell populations in sickle red cells with different mean corpuscular hemoglobin concentrations. We speculate that the abnormality of water exchange under isotonic conditions in sickle cells reflects an alteration of membrane proteins responsible for water exchange, possibly caused by oxidation of Band 3 proteins.

A method to evaluate the antioxidant system for radicals in erythrocyte membranes.

The erythrocyte defense system against cellular oxidants is complex and efficient. Free radicals generated in cell membranes, however, are relatively sequestered from the cell's antioxidant mechanisms. When an oxidant challenge exceeds the capacity of the erythrocyte's antioxidant system, membrane damage may occur, causing red cell destruction and hemolytic anemia. In this study, we present a method for monitoring radical reduction in erythrocyte membranes, using fatty acid spin labels with nitroxide radicals on the hydrocarbon chains. About 50 microL of packed (about 5-6 x 10(8)), carbon monoxide (CO)-gassed red blood cells are used. The electron paramagnetic resonance signals of the 5-doxylstearic acid spin labels in the intact cells are obtained as a function of time, at 37 degrees C over a period of 2 h. The pseudo first-order rate constant for reduction of the spin label in normal adult intact cells under our experimental conditions is 4.3 +/- 1.8 x 10(-3)/min. The reproducibility and variability of the measurements are discussed. Since the measurements we describe reflect the extent of radical reductions occurring in cell membranes, we suggest that this method can be used to measure the ability to defend oxidants in membranes of erythrocytes with defective antioxidant systems. This method is particularly useful for measuring the modification of the antioxidant system toward radicals in membranes by drugs, chemicals, or environmental toxins.

The roles of ascorbic acid and other antioxidants in the erythrocyte in reducing membrane nitroxide radicals.

Fatty acid nitroxide radicals in CO-gassed erythrocytes are reduced by intracellular components with a half-life of about 160 min. In this study, using reduction rate constants of fatty acid spin labels to determine the reduction quantitatively, we found that catalase, glutathione, glutathione peroxidase, superoxide dismutase, and vitamin E--as well as hemoglobin, individually or in concerted manner, contributed little in reducing membrane nitroxides. Ascorbic acid appeared to be the predominant component in the erythrocyte to reduce membrane nitroxides. However, ascorbic acid solutions at 0.1 mM or less, concentrations similar to those found in the erythrocyte, produced no observable reduction in spin labeled membranes during the 2 h monitoring period. Ascorbic acid solutions at about 1 mM were needed to exhibit rate constants similar to those observed in labeled erythrocyte samples. It was also found that beta-nicotinamide adenine dinucleotide, beta-nicotinamide adenine dinucleotide phosphate, and heat-sensitive components in the erythrocyte enhanced the ability of ascorbic acid to scavenge nitroxide radicals in the erythrocyte membrane near the membrane surface.

NMR analysis of secondary structure and dynamics of a recombinant peptide from the N-terminal region of human erythroid alpha-spectrin.

We have studied the nuclear magnetic resonance solution secondary structure of the N-terminal region in human erythroid alpha-spectrin using a recombinant model peptide of alpha-spectrin consisting of residues 1-156. Pulsed field gradient diffusion coefficient measurements show that the model peptide exists as a monomer under the solution conditions used. The first 20 residues are in a random coil conformation, followed by a helix of 25 residues and then a random coil segment before the next helix. The random coil nature of this linker was confirmed by the presence of fast internal motion from (15)N relaxation measurements. The second, third and fourth helices are thought to form the triple helical bundle structural domain, consistent with previous studies. Our study shows that the N-terminal region of alpha-spectrin prior to the first structural domain forms a well behaved helix without its beta-spectrin partner.

Spin label EPR structural studies of the N-terminus of alpha-spectrin.

Spectrin, a vital component in human erythrocyte, is composed of alpha- and beta-subunits, which associate to form (alphabeta)2 tetramers. The tetramerization site is believed to involve the alpha-spectrin N-terminus and the beta-spectrin C-terminus. Abnormal interactions in this region may lead to blood disorders. It has been proposed that both termini consist of partial structural domains and that tetramerization involves the association of these partial domains. We have studied the N-terminal region of a model peptide for alpha-spectrin by making a series of double spin-labeled peptides and studying their dipolar interaction by electron paramagnetic resonance methods. Our results indicate that residues 21-42 of the N-terminus region exhibit an alpha-helical conformation, even in the absence of B-spectrin.

Selective detection of rapid motions in spectrin by NMR.

Human erythrocyte spectrin molecules exhibit relatively sharp (30-50 Hz) proton NMR signals in the aliphatic region. A standard solvent presaturation pulse sequence that also partially suppresses the broad envelope from protons with rigid structures in spectrin and selectively enhances the sharp resonances has been used to characterize the behavior of these resonances. The overall resonance pattern strongly resembles that of the denatured spectrin. The observed spectra are also quite similar to the line-broadened spectrum from a mixture of amino acids that corresponds to the composition of the spectrin molecule. These data indicate the existence of regions exhibiting rapid internal motions within the intact spectrin molecule, and suggest that the amino acid composition of the residues giving rise to the sharp resonances is quite similar to that of the full spectrin molecule.

Hemoglobin-membrane interaction at physiological ionic strength and temperature.

The spin-label electron paramagnetic resonance (EPR) technique has been used to study the interaction between human hemoglobin and erythrocyte membranes as a function of temperature and ionic strength. We show, for the first time, experimental evidence for the existence of the interaction at physiological pH, ionic strength and temperature. In addition to the pH dependence that we have previously reported, the interactions are also temperature and ionic strength dependent. Using a simple two-state equilibrium model to analyze the EPR data, we obtain an equilibrium dissociation constant of about 8.1 +/- 5.6 X 10(-5) M for hemoglobin-membrane systems in 5 mM phosphate with 150 mM NaCl at pH 7.4 and 37 degrees C.

Saturation transfer EPR studies of membrane alteration in hereditary spherocytosis.

Our earlier spin label electron paramagnetic resonance (EPR) studies of hereditary spherocytosis (HS) erythrocyte revealed the existence of structural alteration(s) when the membrane is subjected to heat stress. We have now used saturation transfer EPR to show restricted motion in membrane proteins even without subjecting membrane to stress. The abnormal motional behavior is amplified when membranes are incubated at 47 degrees C and is readily detectable by conventional EPR. Gel electrophoresis and lipid assays show that proteins but not lipids are released upon heating. Thus, the more restricted motions in HS membranes may be due to a different membrane protein organization, ultimately resulting in the abnormal morphology of HS cells.

Secondary structure prediction for the spectrin 106-amino acid segment, and a proposed model for tertiary structure.

A collective secondary structure prediction for the human erythrocyte spectrin 106-residue repeat segment is developed, based on the sequences of nine segments that have been reported in the literature, utilizing a consensus of several secondary structure prediction methods for locating turn regions. The analysis predicts a five-fold structure, with three alpha-helices and two beta-strand regions, and differs from previous models on the lengths of the helices and the existence of beta-strand structure. We also demonstrate that this structural motif can be folded into tertiary structures that satisfy the experimental spectrin data and several general principles of protein organization.

Quantitative detection of rapid motions in spectrin by NMR.

Previous high resolution proton NMR data on human erythrocyte spectrin molecules has indicated the existence of regions exhibiting rapid internal motions within the intact molecules [L. W.-M. Fung, H.-Z. Lu, R. P. Hjelm, jr, M. E. Johnson, FEBS Lett., 197, 234 (1986)]. We have extended the studies by developing quantitative NMR methods to determine the fraction of spectrin protons exhibiting rapid internal motions, in both the isolated molecule and within the spectrin-actin network. Using both one-pulse and spin echo pulse sequences, we find that the fraction of the protons in rapid motion is about 15% of the total protons in the spectrin molecule at 37 degrees C in phosphate buffer with 150 mM NaCl at pH 7.4. Quantitative information on these rapid motions will be important in understanding the structural, mechanical and functional properties of spectrin molecules, as well as in understanding filamentous protein structures in general.

Molecular properties of cetiedil and its interactions with erythrocyte membranes.

Cetiedil, an antisickling agent and a vascular smooth muscle relaxant, is an amphiphilic molecule. The critical micelle concentration in 5 mM phosphate buffer with 150 mM NaCl is approximately 8.8 mM. The molecule, as the citrate salt, is highly acidic at millimolar concentrations. The UV absorption extinction coefficient at 233 nm, E233, is 2796 M-1 cm-1. The studies of free cetiedil concentrations in the presence of membrane ghosts show that large amounts of cetiedil associate with membrane samples. Spin label electron paramagnetic resonance experiments showed that the lipids and the proteins of erythrocyte membrane samples were both affected by the addition of cetiedil. However, the cetiedil effects on membrane components are reversible. The protein spin label results demonstrate the binding of cetiedil to the membrane with an apparent equilibrium dissociation constant of approximately 2 mM. The binding is saturable. The apparent half-saturation concentrations for the binding at physiological ionic strength and temperature are in the range of 1-3 mmoles of cetiedil per gram of membrane proteins. Our studies also indicate that binding is not affected by the removal of the spectrin and actin network from the membranes. Interaction of cetiedil with Band 3 molecules in the erythrocyte membrane is suggested. The regions near the lipid head groups in the membrane samples are also affected by cetiedil.

Titanium-porcelain system. Part II: Bond strength of fired porcelain on nitrided pure titanium.

Commercially pure titanium (CPT) substrate was subjected to porcelain firing and bond strengths under three-point bending mode (span length: 15 mm; crosshead speed: 0.5 mm/min) were evaluated. Experimental variables included surface treatments of CPT and porcelain firing schedules. Variables for the surface treatments were (1) sandblasting, (2) mono- and triple-layered nitridation, and (3) mono-layered chrome-doped nitridation. Variables for the porcelain firing schedule included (4) bonding agent application, (5) bonding agent plus gold bonding agent application, and (6) Procera porcelain application. All together eleven sample groups were prepared with different combination of aforementioned experimental variables. Statistically all of them exhibited no significant differences. Hence, we employed two further criteria; (I) the minimum bond strength should exceed the maximum porcelain strength per se, and (II) the CPT substrate should not be heated close to the beta-transus temperature. After applying these criteria, it was concluded that mono-layered nitridation and mono-layered application of chrome-doped nitridation on both (with and without) sandblasted and non-sandblasted surfaces were the most promising conditions for a successful Titanium-Porcelain System.