Spontaneous Aggregation and Cytotoxicity of the β-Amyloid Aβ1–40: A Kinetic Model

The time dependency of the spontaneous aggregation of the fibrillogenic β-Amyloid peptide, Aβ^1–40, was measured by turbidity, circular dichroism, HPLC, and fluorescence polarization. The results by all methods were comparable and they were most consistent with a kinetic model where the peptide first slowly forms an activated monomeric derivative (AM), which is the only species able to initiate, by tetramerization, the formation of linear aggregates. The anti-Aβ antibody 6E10, raised against residues 1–17, at concentrations of 200–300 nM delayed significantly the aggregation of 50 μM amyloid peptide. The anti–Aβ antibody 4G8, raised against residues 17–24, was much less active in that respect, while the antibody A162, raised against the C-terminal residues 39–43 of the full-length Aβ was totally inactive at those concentrations. Concomitant with the aggregation experiments, we also measured the time dependency of the Aβ^1–40–induced toxicity toward SH-EP1 cells and hippocampal neurons, evaluated by SYTOX Green fluorescence, lactate dehydrogenase release, and activation of caspases. The extent of cell damage measured by all methods reached a maximum at the same time and this maximum coincided with that of the concentration of AM. According to the kinetic scheme, the latter is the only transient peptide species whose concentration passes through a maximum. Thus, it appears that the toxic species of Aβ^1–40 is most likely the same transient activated monomer that is responsible for the nucleation of fibril formation. These conclusions should provide a structural basis for understanding the toxicity of Aβ^1–40 in vitro and possibly in vivo.     

The binding of pentaammineruthenium (III) to RNase B and RNase A+d(pA)4 in the crystalline state

The binding of pentaammineruthenium (III) to ribonuclease A and B both free and complexed with d(pA)₄ has been examined in the crystalline state through the application of X-ray diffraction and difference Fourier techniques. In crystals of native RNase B, the reagent was observed to have many binding sites, some entirely electrostatic in nature and others consistent with coordination to histidine residues. The primary histidine in the latter case was 105 with 119 also partially substituted. In crystals of RNase A+d(pA)₄ complex only a single, extremely strong site of substitution was observed, and this was 2.4 Å from the native position of the imidazole ring of histidine 105. Thus, the results of these X-ray diffraction studies appear to be quite consistent with the findings of earlier NMR studies and with the results obtained in crystals of the gene 5 DNA binding protein.     

Recovery of a bacterial sub-population from sewage using immunofluorescent flow cytometry and cell sorting

Abstract The effectiveness of immunofluorescence flow cytometry and cell sorting to detect, quantify and separate indigenous bacterial populations present in low concentrations in sewage outflow was investigated. Preparatory experiments for targeted recovery revealed indigenous, immunoglobulin-G-binding particles present at low levels in sewage outflow samples taken from Coniston Water. Fluorescence-activated cell sorting of this population was employed to enrich for these particles, which were confirmed as bacterial cells. This cell population comprised approximately 23% of the total plate count on MacConkey agar before cell sorting, rising to approximately 95% after sorting. These results corresponded to cell densities of less than 5% of the total plate count on R2A agar. Taxonomic tests suggested the bacterium to be Ochrobactrum anthropi .     

X-ray absorption spectroscopy of selenium-containing amino acids

Received: 2 April 1999 / Accepted: 17 September 1999     

Presence of prostaglandins E2 and A2 in canine gastric secretions

The presence of prostaglandins (PGs) was determined in gastric juice obtained from 3 conscious dogs, provided with a chronic gastric fistula. Outputs of acid (mequiv min^?1) and PGs (pg min^?1) were measured in gastric secretions stimulated by pentagastrin (100 or 200 ng kg^?1min^?1). Prostaglandin activity was estimated, after extraction and thin layer chromatography, by radioimmuno-assay of the PGB formed by treatment with alkali. Tritiated PGs were added to gastric juice for the purpose of correcting for PGs recovery. Using this method, the minimum mass of PGB which could be satisfactorily distinguished from zero was 25 pg. Prostaglandins A₂ and E₂ were present in pentagastrin-activated gastric secretions and averaged (mean ± SE, n = 8) 200.7 ± 18.1 and 260.1 ± 18.0 pg min^?1 respectively. The identity of PGA₂ and PGE₂ was confirmed by gas liquid chromatography combined with mass spectrometry. The amount of PGE₂ converted to PGA₂ during extraction, separation and conversion procedures was estimated from the amount of [³H] PGA₂ found when only [³H] PGE₂ had been added to a sample of gastric juice and averaged 14.5% ± 2.0. Our preliminary results support the possibility that PGE₂ and PGA₂ may be of physiological importance in the regulation of canine gastric secretions.