Dynamic membrane studies in individuals at risk for Huntington's disease

Lymphocyte plasma membrane dynamics were studied by energy-transfer polarization in twenty-three neurologically normal individuals at-risk for Huntington's disease (HD). The results were compared to 10 normal controls and 10 known HD patients. The normal and HD subjects segregated into two distinct groups. The at-risk group had findings distributed along a continuum with values similar to known HD patients or to normal controls. These findings suggest that further studies of membrane dynamics will contribute to understanding the molecular defect in HD and to the development of a potential molecular marker.     

Two simple methods for quantifying low-affinity dye-substrate binding

Binding with low-affinity ligands, such as histological dyes, can be difficult to quantitate owing to the dissociation of bound ligand with washing or the retention of nonspecifically bound ligand because of incomplete washing. The present report describes two simple, rapid methods of discriminating bound from free ligand without the need for washing steps. One method is based on the spectral changes induced in a dye ligand, Congo red, on binding to the "receptor" insulin fibrils. This method discriminates spectrophotometrically between bound and free ligand without requiring any physical separation of the two forms. No radioactive ligands are necessary, and, by using disposable cuvettes, the entire binding assay can be done in a single container without the need for transfers. The second method employs a non-traditional filtration approach that avoids the need for a washing step by measuring the decrease in concentration of the dye ligand in the filtrate rather than by applying the usual approach of measuring the absolute amount of ligand bound to the precipitated "receptor." Both methods show saturation of binding sites and give similar values for the KD and Bmax.     

Aggregation of β-Amyloid Peptide Is Promoted by Membrane Phospholipid Metabolites Elevated in Alzheimer's Disease Brain

Abstract: Increased amounts of β-amyloid (Aβ) peptide deposits are found in Alzheimer's disease brain. These amyloid deposits have been implicated in the pathophysiology of this common dementing illness. Aβ peptides have been shown to be toxic to neurons in cell culture, and this toxicity is critically dependent on the aggregation of the peptide into cross-β-pleated sheet fibrils. Also, in vivo and postmortem NMR studies have shown changes in certain brain membrane phospholipid metabolites in normal aging and more extensive alterations in patients with Alzheimer's disease. The finding that membrane phospholipids affect the aggregation of Aβ suggests that the abnormalities in membrane metabolism found in Alzheimer's disease could affect the deposition of Aβ in vivo. Therefore, we examined the effect of membrane phospholipid metabolites that are altered in Alzheimer's disease brain on the aggregation of Aβ(1–40) using a light scattering method. Certain metabolites (glycerophosphocholine, glycerophosphoethanolamine, and α-glycerophosphate) augment the aggregation of Aβ. Other membrane phospholipid metabolites (phosphocholine, phosphoethanolamine, and inositol-1-phosphate) have no effect. We conclude that increased membrane phospholipid metabolite concentrations may play a role in the deposition of Aβ seen in normal aging and the even greater deposition of Aβ observed in Alzheimer's disease.     

Fluorescence probes specific for the plasma membrane of intact human erythrocytes and lymphocytes

Human erythrocytes and lymphocytes were isolated from venous blood and subjected to one of two protocols. In one protocol the suspended cells were labeled with fluorophore (fluorescamine or 12(9)AS). This procedure was followed sequentially by cellular lysis, cellular fractionation, and fluorescence and absorption readings. In the other protocol the suspended cells were lysed, and then the cellular homogenate labeled with fluorophore followed by cellular fractionation and spectroscopy readings. The lymphocytes were fractionated into plasma membrane, cytosol, and nuclear-mitochondrial fractions and the erythrocytes into plasma membrane and cytosol fractions. The results demonstrate that under the given labeling conditions, both fluorescamine and 12(9)AS are highly localized to the plasma membrane of intact human erythrocytes and lymphocytes. Furthermore, by P-31 NMR analysis, fluorophore labeling did not alter cellular high energy phosphate metabolism or cellular permeability to Mn²⁺. Therefore, these fluorophores are potentially powerful probes of human erythrocyte and lymphocyte plasma membrane dynamics in inherited and acquired disease states.     

Alzheimer''s β-Amyloid Protein Is Covalently Modified when Dissolved in Formic Acid

beta-Amyloid protein is a major protein component of neuritic plaques in the brain of Alzheimer's disease patients. A major advance in understanding the molecular biology of Alzheimer's disease came with the purification and sequencing of this protein. Because beta-amyloid protein is very insoluble, extreme conditions such as 88% formic acid were commonly used to dissolve its fibrils. We now report that 88% formic acid covalently modifies beta-amyloid protein fragments, probably by the formation of a formate ester to a serine in the protein. The t1/2 of the formylation is approximately 3.5 h, and the t1/2 for hydrolysis of the formylated peptide is much longer, being 9.9 h in water and 66 h in HPLC eluant. This suggests that if formic acid is used in the purification of beta-amyloid protein or peptide fragments of this protein, it is likely that some formylated peptide will be present in subsequent studies. Although unrecognized modification of a protein is inherently undesirable, it is uncertain what effects this formylation will have on ensuing studies. Certainly, investigations into the immunologic, physical, and physiologic properties of beta-amyloid protein could be influenced.     

Brain membrane phospholipid alterations in Alzheimer's disease

Studies have demonstrated alterations in brain membrane phospholipid metabolite levels in Alzheimer's disease (AD). The changes in phospholipid metabolite levels correlate with neuropathological hallmarks of the disease and measures of cognitive decline. This ³¹P nuclear magnetic resonance (NMR) study of Folch extracts of autopsy material reveals significant reductions in AD brain levels of phosphatidylethanolamine (PtdEtn) and phosphatidylinositol (PtdIns), and elevations in sphingomyelin (SPH) and the plasmalogen derivative of PtdEtn. In the superior temporal gyrus, there were additional reductions in the levels of diphosphatidylglycerol (DPG) and phosphatidic acid (PtdA). The findings are present in 3/3 as well as 3/4 and 4/4 apolipoprotein E (apoE) genotypes. The AD findings do not appear to reflect non-specific neurodegeneration or the presence of gliosis. The present findings could possibly contribute to an abnormal membrane repair in AD brains which ultimately results in synaptic loss and the aggregation of A peptide.     

Transfer Buffer Containing Methanol Can Be Reused Multiple Times in Protein Electrotransfer

We investigated the feasibility of repeated use of transfer buffer containing methanol in electrotransfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to polyvinylidene difluoride (PVDF) membrane using a prestained protein marker of broad molecular sizes. Transfer of the antitumor protein p53 in HEK293T cell extracts, using fresh and used transfer buffer, followed by detection with anti-p53 antibody was also performed to test detectability in immunoblot. Results from these experiments indicate that the transfer buffer can be reused at least five times and maintain a similar extent of protein transfer to PVDF membrane. Repeated use of the transfer buffer containing methanol will significantly reduce the volume of hazardous waste generated and its disposal cost as well as its adverse effect on environment.     

Clinically Relevant Concentration Determination of Inhaled Anesthetics (Halothane,Isoflurane, Sevoflurane,and Desflurane) by <Superscript>19</Superscript>F NMR

Biophysical studies of protein–anesthetic interactions using nuclear magnetic resonance (NMR) spectroscopy are often conducted by the addition of micro amounts of neat inhaled anesthetic which yields much higher than clinically relevant (0.2–0.5 mM) anesthetic concentrations. We report a ¹⁹F NMR technique to measure clinically relevant inhaled anesthetic concentrations from saturated aqueous solutions of these anesthetics (halothane, isoflurane, sevoflurane, and desflurane). We use a setup with a 3-mm NMR tube (containing trifluoroacetic acid as standard), coaxially inserted in a 5-mm NMR tube containing anesthetic solution under investigation. All experiments are conducted in a 5-mm NMR probe. We also have provided standard curves for four inhaled anesthetics using NMR technique. The standard curve for each of these anesthetics is helpful in determining the prerequisite amount of aqueous anesthetic solution required to prepare clinically relevant concentrations for protein–anesthetic interaction studies. Parts of the results to be presented at Society for Neuroscience meeting, 2008.     

Al-ATP as an intracellular carrier of Al(III) ion.

1. Using 27Al and 31P NMR spectroscopy in conjunction with an Al lactate aqueous reagent at pH 7.2, Al complexes of ATP and of phospholipids were characterized in synthetic-aqueous and organic-phospholipid chemical systems and in the intact human red blood cell. 2. The observed 31P NMR chemical shifts of the Al-ATP complex in aqueous laboratory preparations or the intact human red blood cell were, respectively, alpha phosphate, -11.53 delta; beta phosphate, -22.65 delta; and gamma phosphate, -10.95 delta. 3. The observed complexed 27Al chemical shift was -2.22 delta. 4. The relative affinities for Al of the phospholipids determined from 31P NMR spectroscopic titrations were PA much greater than Cl much greater than PS greater than PG approximately equal to PI greater than PE plus approximately equal to PE much greater than SPH greater than PC.