Interactions between an amphipathic di-histidine peptide and a metal affinity chromatographic resin derived from a bis(tacn)butane chelating ligand.

The interactive behavior of an amphipathic peptide with the Cu2+ , Ni2+ , and Zn2+ complexes of 1,4-bis(triazacyclonon-1-yl)butane), bis(tacn)but , immobilized onto Sepharose CL-4B, has been investigated. The effects of incubation time, as well as the incubation buffer pH and ionic strength, have been examined. The binding data have been interrogated using Langmuir, Langmuir-Freundlich, bi-Langmuir, and Temkin isothermal models and Scatchard plots. These results confirm that this amphipathic peptide binds with relatively high capacities to the immobilized Cu2+ - and Ni2+ -1,4-bis(triazacyclonon-1-yl)butane)-Sepharose CL-4B sorbents via at least two discrete sites. However, the corresponding immobilized Zn2+ -sorbent had low binding capacity. Moreover, the magnitude of the binding capacities of these sorbents was dependent on the pH and ionic strength of the incubation buffer. These results are relevant to the isolation of E. coli expressed recombinant proteins that incorporate this and related amphipathic peptide tags, containing two or more histidine residues, located at the N- or C-terminus of the recombinant protein, and the co-purification of low abundance host cell proteins of diverse structure, by immobilized metal ion affinity chromatographic methods.

Electron microscopic 3D-reconstruction of dendritic spines in cultured hippocampal neurons undergoing synaptic plasticity.

Dendritic spines are assumed to constitute the locus of neuronal plasticity, and considerable effort has been focused on attempts to demonstrate that new memories are associated with the formation of new spines. However, few studies that have documented the appearance of spines after exposure to plasticity-producing paradigms could demonstrate that a new spine is touched by a bona fida presynaptic terminal. Thus, the functional significance of plastic dendritic spine changes is not clearly understood. We have used quantitative time lapse confocal imaging of cultured hippocampal neurons before and after their exposure to a conditioning medium which activates synaptic NMDA receptors. Following the experiment the cultures were prepared for 3D electron microscopic reconstruction of visually identified dendritic spines. We found that a majority of new, 1- to 2-h-old spines was touched by presynaptic terminals. Furthermore, when spines disappeared, the parent dendrites were sometime touched by a presynaptic bouton at the site where the previously identified spine had been located. We conclude that new spines are most likely to be functional and that pruned spines can be transformed into shaft synapses and thus maintain their functionality within the neuronal network.

Is fragile X mental retardation protein involved in activity-induced plasticity of dendritic spines?

Dendritic morphology of 2-week-old cultured neurons, taken from postnatal day 1 fragile X mental retardation gene1 knock out (FMR1-/-) mice hippocampus, were compared with cells taken from wild type mice. Under control conditions the FMR1-/- neurons displayed significantly lower spine densities compared to wild type neurons. Pharmacological stimulation of electrical activity, induced by bicuculline, caused a reduction in dendritic spine density in both the FMR1-/- and the wild type cells. In both groups, bicuculline induced a significant shrinkage of spines that were occupied by one or more synaptophysin-immunoreactive presynaptic terminals. The concentration of FMR1 in the wild type cultures was not affected by bicuculline treatment. These experiments indicate that FMR1 is not likely to be an essential factor in activity-modulated morphological plasticity of dendritic spines in cultured hippocampal neurons.