Aggregation and Metal-Binding Properties of Mutant Forms of the Amyloid Aβ Peptide of Alzheimer's Disease

Abstract: The fibrillogenic properties of Alzheimer's Aβ peptides corresponding to residues 1–40 of the normal human sequence and to two mutant forms containing the replacement Ala²¹ to Gly or Glu²² to Gln were compared. At pH 7.4 and 37°C the Gln²² peptide was found to aggregate and precipitate from solution faster than the normal Aβ, whereas the Gly²¹ peptide aggregated much more slowly. Electron microscopy showed that the aggregates all had fibrillar structures. Circular dichroism spectra of these peptides revealed that aggregation of the normal and Gln²² sequences was associated with spectral changes consistent with a transformation from random coil to β sheet, whereas the spectrum of the Gly²¹ peptide remained almost unchanged during a period in which little or no aggregation occurred. When immobilised by spotting onto nitrocellulose membranes the peptides bound similar amounts of the radioisotope ⁶⁵Zn²⁺. Of several competing metal ions, tested at 20× the concentration of Zn²⁺, Cu²⁺ displaced >95% of the radioactivity from all three peptides and Ni²⁺ produced >50% displacement in each case. Some other metal ions tested caused lesser displacement, but Fe²⁺ and Al³⁺ were without effect. In a saturation binding assay, a value of 3.2 µM was obtained for the binding of Zn²⁺ to Aβ but our data provided no evidence for a reported higher affinity site (107 nM). The results suggest that the neuropathology associated with the Gly²¹ mutation is not due to enhanced fibrillogenic or different metal-binding properties of the peptide and that the binding of zinc to amyloid peptides is not a specific phenomenon.     

Characterization of the sources of protein-ligand affinity: 1-sulfonato-8-(1'')anilinonaphthalene binding to intestinal fatty acid binding protein.

1-Sulfonato-8-(1')anilinonaphthalene (1,8-ANS) was employed as a fluorescent probe of the fatty acid binding site of recombinant rat intestinal fatty acid binding protein (1-FABP). The enhancement of fluorescence upon binding allowed direct determination of binding affinity by fluorescence titration experiments, and measurement of the effects on that affinity of temperature, pH, and ionic strength. Solvent isotope effects were also determined. These data were compared to results from isothermal titration calorimetry. We obtained values for the enthalpy and entropy of this interaction at a variety of temperatures, and hence determined the change in heat capacity of the system consequent upon binding. The ANS-1-FABP is enthalpically driven; above approximately 14 degrees C it is entropically opposed, but below this temperature the entropy makes a positive contribution to the binding. The changes we observe in both enthalpy and entropy of binding with temperature can be derived from the change in heat capacity upon binding by integration, which demonstrates the internal consistency of our results. Bound ANS is displaced by fatty acids and can itself displace fatty acids bound to I-FABP. The binding site for ANS appears to be inside the solvent-containing cavity observed in the x-ray crystal structure, the same cavity occupied by fatty acid. From the fluorescence spectrum and from an inversion of the Debye-Hueckel formula for the activity coefficients as a function of added salt, we inferred that this cavity is fairly polar in character, which is in keeping with inferences drawn from the x-ray structure. The binding affinity of ANS is considered to be a consequence of both electrostatic and conditional hydrophobic effects. We speculate that the observed change in heat capacity is produced mainly by the displacement of strongly hydrogen-bonded waters from the protein cavity.     

Formation of the sea urchin male pronucleus in vitro: Membrane-independent chromatin decondensation and nuclear envelope-dependent nuclear swelling

We demonstrate that complete sea urchin male pronuclear development in vitro is a two-step process involving membrane-independent chromatin decondensation and nuclear envelope-dependent pronuclear swelling. In the absence of cytoplasmic membrane vesicles (MVs), permeabilized sperm chromatin decondenses into a spherical nucleus of ≈4 μm in diameter. Pronuclear swelling to ≈7 μm requires an intact nuclear envelope, and the degree of swelling is limited by the amount of MVs assembled on the chromatin. Furthermore, after a nuclear envelope is formed, swelling can occur in the absence of additional cytoplasmic MVs. Nuclear swelling also requires ATP hydrolysis, Ca²⁺ and cytosolic factors, some of which are sensitive to heat and to the sulfhy-dryl alkylating agent, N-ethylmaleimide. The requirement for a nuclear envelope and the rate of pronuclear swelling are consistent with previous in vivo observations. © 1995 wiley-Liss, Inc.     

Using real-world data to predict health outcomes—The prediction design: Application and sample size planning

The gold standard for investigating the efficacy of a new therapy is a (pragmatic) randomized controlled trial (RCT). This approach is costly, time-consuming, and not always practicable. At the same time, huge quantities of available patient-level control condition data in analyzable format of (former) RCTs or real-world data (RWD) are neglected. Therefore, alternative study designs are desirable. The design presented here consists of setting up a prediction model for determining treatment effects under the control condition for future patients. When a new treatment is intended to be tested against a control treatment, a single-arm trial for the new therapy is conducted. The treatment effect is then evaluated by comparing the outcomes of the single-arm trial against the predicted outcomes under the control condition. While there are obvious advantages of this design compared to classical RCTs (increased efficiency, lower cost, alleviating participants’ fear of being on control treatment), there are several sources of bias. Our aim is to investigate whether and how such a design—the prediction design—may be used to provide information on treatment effects by leveraging external data sources. For this purpose, we investigated under what assumptions linear prediction models could be used to predict the counterfactual of patients precisely enough to construct a test and an appropriate sample size formula for evaluating the average treatment effect in the population of a new study. A user-friendly R Shiny application (available at: https://web.imbi.uni-heidelberg.de/PredictionDesignR/ ) facilitates the application of the proposed methods, while a real-world application example illustrates them.     

Albumin obscures sex differences in blood protein patterns of rats and humans.

Sex related differences in the blood protein patterns of male and female rats and humans have been studied by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. In rats, a prominent band of mol.wt. 74000--78000 is seen in females in far greater quantity than in males, castrated males or ovariectomized females. A secondary band of 100000 is seen under non-reducing conditions in female rats that is absent in males. In humans, bands of 92000 and 88000 mol.wt. appear to be variable in concentration in men although relatively constant in women. The above differences are observable only if serum albumin is removed from the samples before electrophoresis. The results suggest that each sex has its own characteristic blood protein pattern.     

Oxidant effect of acetylphenylhydrazine: a comparative study with erythrocytes of several animal species.

Red cells from several animal species were treated with acetylphenylhydrazine and a comparative study of the oxidation of hemoglobin (Hb) and glutathione (GSH) made. Wide interspecies differences were observed but the oxidation of GSH paralleled that of Hb. Added glucose protected both Hb and GSH from oxidation; GSH by itself exercises a protective effect on Hb. The characteristic rates of oxidation of GSH in the different species can be observed only in the presence of oxyhemoglobin but not carboxyhemoblobin or methemoglobin. The oxidation of Hb appears to be the primary event, the oxidation of GSH being a consequence thereof.     

Measurement of endogenous leucine enkephalin in canine caudate nuclei and hypothalami with high-performance liquid chromatography and field-desorption mass spectrometry

For the first time, endogenous amounts of Leu-enkephalin are measured in brain tissue with a technique preserving integrity of the entire molecular structure of the neuropeptide. Field-desorption mass spectrometry enables measurement of picomole amounts of endogenous, chemically underivatized Leu-enkephalin in canine caudate nuclei and hypothalami. The optimal sensitivity and resolution of high-performance liquid chromatography is coupled with maximal molecular specificity of field-desorption mass spectrometry to measure enkephalins in caudate nuclei and hypothalami from dog brains. This novel combination of two recent instrumental methodologies provides a firm molecular basis for calibrating the radioimmunoassay measurement of endogenous levels of biologically active brain neuropeptides.     

Analysis of methionine enkephalin in human pituitary by multi-dimensional reversed-phase high-performance liquid chromatography, radioreceptor assay, radioimmunoassay, fast atom bombardment mass spectrometry, and mass spectrometry—mass spectrometry

Methionine enkephalin (ME = YGGFM) was measured in five individual human post-mortem pituitaries using four different analytical methods, with the objective of comparing the molecular specificities of the methods. Radioreceptor assay (RRA) used a receptor-rich preparation from brain and [³H]etorphine as radioligand to determine ME-like receptoractivity (ME-LR). Radioimmunoassay (RIA) measured ME-like immunoreactivity (ME-LI). Pituitary samples analyzed by RRA and RIA were purified first with a high-performance liquid chromatography (HPLC) gradient on a polymer analytical column. Fast atom bombardment mass spectrometry (FAB-MS) in two different detection modes quantified ME using the protonated molecular ion MH⁺ of ME at 574 a.m.u. and B/E linked-field selected reaction monitoring (SRM) to monitor the specific unimolecular metastable transition that produced the unique amino acid sequence-determining tetrapeptide fragment ion YGGF⁺ from the MH⁺ precursor ion. Both FAB-MS methods used the deuterated internal standard YGG[²H₅-F]M. Samples analyzed with FAB-MS were purified first with multi-dimensional reversed-phase HPLC. The first dimension was an ODS gradient, and the second dimension was a polymer isocratic elution. The following ME amounts were measured (mean ± standard error of the mean): ME-LR, 7.0 ± 1.9 μg g⁻¹ tissue; ME-LI, 1.8 ± 0.7 μg g⁻¹ tissue; MH⁺, 2.7 ± 0.6 μg g⁻¹ tissue; SRM, 3.0 ± 0.8 μg g⁻¹ tissue. The FAB SRM method provided the highest level of molecular specificity amount these four analytical methods used to measure picomole amounts of endogenous ME in a human pituitary.     

Bromocriptine-induced implantation failure in mice: failure of the stud male to protect pregnancy.

The presence of the stud male failed to prevent implantation failure in newly inseminated females induced by administration of bromocriptine. This is in contrast with the ability of the stud male to prevent implantation failure in females induced by alien male exposure, and nutritional stress. Since bromocriptine is a potent inhibitor of hypophysial prolactin release by virtue of its stimulatory effect on hypothalamic dopaminergic activity, the results suggest that the stud male-originating luteotrophic stimulus is incapable of overriding the dopaminergic activity in bromocriptine-treated females.