Activity differentially regulates the surface expression of synaptic AMPA and NMDA glutamate receptors

Distinct subtypes of glutamate receptors often are colocalized at individual excitatory synapses in the mammalian brain yet appear to subserve distinct functions. To address whether neuronal activity may differentially regulate the surface expression at synapses of two specific subtypes of ionotropic glutamate receptors we epitope-tagged an AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) receptor subunit (GluR1) and an NMDA (N-methyl-d-aspartate) receptor subunit (NR1) on their extracellular termini and expressed these proteins in cultured hippocampal neurons using recombinant adenoviruses. Both receptor subtypes were appropriately targeted to the synaptic plasma membrane as defined by colocalization with the synaptic vesicle protein synaptophysin. Increasing activity in the network of cultured cells by prolonged blockade of inhibitory synapses with the γ-aminobutyric acid type A receptor antagonist picrotoxin caused an activity-dependent and NMDA receptor-dependent decrease in surface expression of GluR1, but not NR1, at synapses. Consistent with this observation identical treatment of noninfected cultures decreased the contribution of endogenous AMPA receptors to synaptic currents relative to endogenous NMDA receptors. These results indicate that neuronal activity can differentially regulate the surface expression of AMPA and NMDA receptors at individual synapses.

Independent mobility of catalytic and regulatory domains of myosin heads

The recent determination of the myosin head atomic structure has led to a new model of muscle contraction, according to which mechanical torque is generated in the catalytic domain and amplified by the lever arm made of the regulatory domain [Fisher, A. J., Smith, C. A., Thoden, J., Smith, R., Sutoh, K., Holden, H. M. & Rayment, I. (1995) Biochemistry 34, 8960–8972]. A crucial aspect of this model is the ability of the regulatory domain to move independently of the catalytic domain. Saturation transfer–EPR measurements of mobility of these two domains in myosin filaments give strong support for this notion. The catalytic domain of the myosin head was labeled at Cys-707 with indane dione spin label; the regulatory domain was labeled at the single cysteine residue of the essential light chain and exchanged into myosin. The mobility of the regulatory domain in myosin filaments was characterized by an effective rotational correlation time (τR) between 24 and 48 μs. In contrast, the mobility of the catalytic domain was found to be τR = 5–9 μs. This difference in mobility between the two domains existed only in the filament form of myosin. In the monomeric form, or when bound to actin, the mobility of the two domains in myosin was indistinguishable, with τR = 1–4 μs and >1,000 μs, respectively. Therefore, the observed difference in filaments cannot be ascribed to differences in local conformations of the spin-labeled sites. The most straightforward interpretation suggests a flexible hinge between the two domains, which would have to stiffen before force could be generated.

Ras Family GTPases Control Growth of Astrocyte Processes

Astrocytes in neuron-free cultures typically lack processes, although they are highly process-bearing in vivo. We show that basic fibroblast growth factor (bFGF) induces cultured astrocytes to grow processes and that Ras family GTPases mediate these morphological changes. Activated alleles of rac1 and rhoA blocked and reversed bFGF effects when introduced into astrocytes in dissociated culture and in brain slices using recombinant adenoviruses. By contrast, dominant negative (DN) alleles of both GTPases mimicked bFGF effects. A DN allele of Ha-ras blocked bFGF effects but not those of Rac1-DN or RhoA-DN. Our results show that bFGF acting through c-Ha-Ras inhibits endogenous Rac1 and RhoA GTPases thereby triggering astrocyte process growth, and they provide evidence for the regulation of this cascade in vivo by a yet undetermined neuron-derived factor.

Immunization of metastatic breast cancer patients with a fully synthetic globo H conjugate: A phase I trial

The carbohydrate antigen globo H commonly found on breast cancer cells is a potential target for vaccine therapy. The objectives of this trial were to determine the toxicity and immunogenicity of three synthetic globo H-keyhole limpet hemocyanin conjugates plus the immunologic adjuvant QS-21. Twenty-seven metastatic breast cancer patients received five vaccinations each. The vaccine was well tolerated, and no definite differences were observed among the three formulations. Serologic analyses demonstrated the generation of IgM antibody titers in most patients, with minimal IgG antibody stimulation. There was significant binding of IgM antibodies to MCF-7 tumor cells in 16 patients, whereas IgG antibody reactivity was observed in a few patients. There was evidence of complement-dependent cytotoxicity in several patients. Affinity column purification supported the specificity of IgM antibodies for globo H. On the basis of these data, globo H will constitute one component of a polyvalent vaccine for evaluation in high-risk breast cancer patients.