Identification of UDP-linked murein precursors as contaminants in recombinant proteins of low molecular weight.

The A(280)/A(260) ratio of a purified protein is frequently used as an indication of the purity of the preparation with respect to nucleic acids. We show here that for low-molecular-weight recombinant proteins purified from Escherichia coli, a low A(280)/A(260) ratio can also result from contamination with UDP-linked murein precursors derived from bacterial cell wall metabolism. Although these precursors are small molecules of molecular weight 1000-1200, they comigrate in gel filtration with recombinant human FKBP (MW 11,820). This gel filtration behavior, which is distinct from that of unmodified mononucleotides, does not reflect binding interactions with FKBP, but is an intrinsic property of these precursors. Therefore, these molecules would be expected to copurify with other low-molecular-weight proteins, especially in the abbreviated purification protocols made possible by freeze-thaw release of recombinant proteins from E. coli (Johnson, B. H., and Hecht, M. H. (1994) BioTechnology 12, 1357-1360). Several alternative strategies are discussed for integrating these findings into the design of improved purification procedures for low-molecular-weight recombinant proteins.     

Lesions of the Basal Forebrain Cholinergic System in Mice Disrupt Idiothetic Navigation

Loss of integrity of the basal forebrain cholinergic neurons is a consistent feature of Alzheimer’s disease, and measurement of basal forebrain degeneration by magnetic resonance imaging is emerging as a sensitive diagnostic marker for prodromal disease. It is also known that Alzheimer’s disease patients perform poorly on both real space and computerized cued (allothetic) or uncued (idiothetic) recall navigation tasks. Although the hippocampus is required for allothetic navigation, lesions of this region only mildly affect idiothetic navigation. Here we tested the hypothesis that the cholinergic medial septo-hippocampal circuit is important for idiothetic navigation. Basal forebrain cholinergic neurons were selectively lesioned in mice using the toxin saporin conjugated to a basal forebrain cholinergic neuronal marker, the p75 neurotrophin receptor. Control animals were able to learn and remember spatial information when tested on a modified version of the passive place avoidance test where all extramaze cues were removed, and animals had to rely on idiothetic signals. However, the exploratory behaviour of mice with cholinergic basal forebrain lesions was highly disorganized during this test. By contrast, the lesioned animals performed no differently from controls in tasks involving contextual fear conditioning and spatial working memory (Y maze), and displayed no deficits in potentially confounding behaviours such as motor performance, anxiety, or disturbed sleep/wake cycles. These data suggest that the basal forebrain cholinergic system plays a specific role in idiothetic navigation, a modality that is impaired early in Alzheimer’s disease.     

Polymorphism due to variable number of repeats in the human involucrin gene.

The coding region of the involucrin gene in higher primates contains a segment consisting of numerous tandem repeats of a 10-codon sequence. The process of repeat addition began in a common ancestor of all higher primates and subsequent repeats were added vectorially. As a result, the principal site of repeat addition has moved in the 3' to 5' direction and the most recently generated repeats (the late region) are close to the 5' end of the segment of repeats. In the human, most of the late region is made up of two different blocks, each consisting of nearly identical repeats. We describe here five polymorphic forms resulting from the addition of differing numbers of repeats to each block. As the variety and nature of the polymorphic alleles are different in different human populations, we postulate that the process of repeat addition is genetically determined.     

Fickean and Non-Fickean Water Desorption During Vacuum Drying of <Emphasis Type="Italic">L. bulgaricus</Emphasis>

The recovery of Lactobacillus bulgaricus was studied in correlation to the kinetics of cell drying. When bacteria were dehydrated at 30 °C, either in the presence or the absence of sucrose, the drying kinetics corresponds to a Fickean diffusion in correspondence with a short lag time. In contrast, when the bacteria were dehydrated at 70 °C in the absence of sugar, the kinetics corresponds to an anomalous diffusion, and the lag time is four to five times higher than that at 30 °C. However, when drying at 70 °C was carried out in the presence of sucrose, drying kinetics turned into a Fickean process parallel to a substantial decrease in the lag time. The pattern of water desorption was correlated with the critical water activity. When the drying kinetics corresponds to a Fickean diffusion, the lag time started to increase at 0.7 water activity, but when the cells were dried at 70 °C, the damage started at 0.5 water activity. This observation indicates that the drying rate affects the pattern of water desorption, and it can change the value of critical water activity. These results put into relevance that the cell recovery is due to the drying history and that the recovery increase produced by sucrose can be related to the maintenance of kinetic barriers for water desorption.