Co-incorporation of A beta 40 and A beta 42 to form mixed pre-fibrillar aggregates.

Senile plaques, the invariable hallmark and likely proximal cause of Alzheimer's disease (AD), are structured depositions of the 40- and 42-residue forms of the A beta peptide. Conversely, diffuse plaques, which are not associated with neurodegeneration, consist mainly of unstructured A beta 42. We have investigated the interaction between A beta 40 and A beta 42 through an assay, which involves labeling both variants with an environment-sensitive fluorophore. We have monitored association of A beta without fibrillar seeds, which allows investigation of molecular species preceding fibrils. Immediately upon mixture, A beta 40 and A beta 42 associate into mixed aggregates, in which the peptides are unstructured and relatively accessible to water. When left to incubate for an extended period, larger, more tightly packed aggregates, which show secondary structure, replace the small, unstructured aggregates formed earlier. Our results show that in vitro the two A beta variants coassemble early in the fibrillogenesis pathway. The ease of formation for mixed and homogeneous aggregates is similar. A change in the local A beta variant ratio can therefore have a significant impact on A beta aggregation; indeed such a change has been reported in some types of familial AD.     

Developmental changes in BDNF protein levels in the hamster retina and superior colliculus.

Quantitative studies of ontogenetic changes in the levels of brain-derived neurotrophic factor (BDNF) mRNA and its effector, BDNF protein, are not available for the retinal projection system. We used an electrochemiluminescence immunoassay to measure developmental changes in the tissue concentration of BDNF within the hamster retina and superior colliculus (SC). In the SC, we first detected BDNF (about 9 pg/mg tissue) on embryonic day 14 (E14). BDNF protein concentration in the SC rises about fourfold between (E14) and postnatal day 4 (P4), remains at a plateau through P15, then declines by about one-third to attain its adult level by P18. By contrast, BDNF protein concentration in the retina remains low (about 1 pg/mg tissue) through P12, then increases 4.5-fold to attain its adult level on P18. The developmental changes in retinal and collicular BDNF protein concentrations are temporally correlated with multiple events in the structural and functional maturation of the hamster retinal projection system. Our data suggest roles for BDNF in the cellular mechanisms underlying some of these events and are crucial to the design of experiments to examine those roles.     

3-Deoxy-D-arabino-heptulosonic acid 7-phosphate: chemical synthesis and isolation from Escherichia coli auxotrophs

A new chemical synthesis of 3-deoxy-D-arabino-heptulosonic acid 7-phosphate is described and contrasted to isolation of the same molecule from the growth medium of several different auxotrophic strains of Escherichia coli. The higher yielding chemical synthesis begins with 2-deoxyglucose while the less time-intensive biological approach proceeds directly from glucose. Growth and accumulation aspects of whole cell biological synthesis are discussed along with various aspects of the biological purification protocol. Both approaches can be utilized to produce substantial quantities of methyl (methyl 3-deoxy-D-arabino-heptulopyranosid)onate, a key intermediate for semisynthetic 3-deoxy-D-arabino-heptulosonic acid 7-phosphate and a number of its derivatives.     

Conservation of Glycerolated Cultures of Ochromonas danica and O. malhamensis at −10 C

SYNOPSIS. Ochromonas danica in a complex natural growth medium dies at 6–10 C in 4 days; O. malhamensis in ∼2 days. O. danica grown in the medium supplemented with 4.0% glycerol survived at −10±2 C for 35 days, and with 8% glycerol 29 days. O. malhamensis lasted only to 5 days in these media supplemented with 4% glycerol. Ethylene glycol and dimethylsulfoxide were too toxic to be effective. Difficulties in freeze-preservation of certain other phagocytic cells, notably blood granulocytes having comparatively simple flexuous outer membranes, add interest to use of O. danica and O. malhamensis as test organisms for preservation methods, especially in the convenient, inexpensive -10 to -20 C range. Biphasic media with an overlay of distilled water serve for conservation at room temperature. Problems of mutational erosion of these photosynthetic phagotrophs are discussed.