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Human serum albumin (HSA), the most prominent protein in plasma, is best known for its extraordinary ligand binding capacity. The three homologous domains of HSA (labeled I, II, and III), each in turn composed of two subdomains (named A and B), give rise to the three-dimensional structure of HSA. This flexible structural organization allows the protein structure to adapt to a variety of ligands. As conformational adaptability of HSA extends well beyond the immediate vicinity of the binding site(s), cooperativity and allosteric modulation arise among binding sites; this makes HSA similar to a multimeric protein. Although kinetic and thermodynamic parameters for ligand binding to HSA calculated by quantitative structure-activity relationship models are in excellent agreement with those obtained in vitro, cooperative and allosteric equilibria between different binding sites and competition between drugs or between drugs and endogenous ligands make difficult the interpretation of HSA binding properties in vivo. Binding of exogenous and endogenous ligands to HSA appears to be relevant in drug therapy and management. Here, the allosteric modulation of drug binding to HSA is briefly reviewed.

Adhesion of monocytes to the extracellular matrix is mediated by a direct high affinity interaction between cell-surface urokinase-type plasminogen activator (uPA) receptor (uPAR) and the extracellular matrix protein vitronectin. We demonstrate a tight connection between uPA-regulated uPAR oligomerization and high affinity binding to immobilized vitronectin. We find that binding of soluble uPAR (suPAR) to immobilized vitronectin is strictly ligand-dependent with a linear relationship between the observed binding and the concentration of ligand added. Nevertheless, a comparison of experimentally obtained binding curves to those generated using a simple equilibrium model suggests that the high affinity vitronectin-binding pro-uPA⅐suPAR complex contains two molecules of suPAR. In co-immunoprecipitation experiments, using different epitopetagged suPAR molecules, suPAR/suPAR co-immunoprecipitation displayed a similar uPA dose dependence as that observed for vitronectin binding, demonstrating that the high affinity vitronectin-binding complex indeed contains oligomeric suPAR. Structurally, the kringle domain of uPA was found to be critical for the formation of the vitronectin-binding competent complex because the amino-terminal fragment, but not the growth factor-like domain, behaved as a full-length uPA. Our data represent the first demonstration of functional, ligand-induced uPAR oligomerization having extensive implications for glycosylphosphatidylinositolanchored receptors in general, and for the biology of the uPA/uPAR system in particular.Cell migration and invasion are important processes in many patho/physiological conditions such as tumor invasion, angiogenesis, and inflammation. Plasminogen activators, their inhibitors, and their cell-surface receptor(s) play central roles in these processes by regulating extracellular proteolysis, cell adhesion, and signal transduction. In tissues, extracellular proteolysis is controlled by the production of plasmin that is generated by plasminogen activators, mainly urokinase (uPA) 1 (1), which binds to a specific membrane receptor, uPAR.Fully processed human uPAR is a 45-55-kDa glycoprotein linked to the outer membrane leaflet by a glycosylphosphatidylinositol lipid anchor (2). The protein is composed of three homologous domains with a disulfide bonding pattern characteristic of the uPAR/Ly-6 superfamily (3).Besides providing the cells with the means to perform directed extracellular matrix degradation, binding of uPA to uPAR has profound effects on cell adhesion, migration, and proliferation (4 -6). Although binding to uPAR is always required, these latter processes are often independent of the proteolytic activity of uPA, strongly suggesting that other protein interactions are involved.Indeed, several data indicate that a conformational change in uPAR is capable of profoundly modifying its biological properties. First, it has been shown that uPA binding to uPAR causes the appearance of novel binding sites for vitronectin (Vn) (7-10), thrombospondin (8), uPAR-associated ...

A method based upon an extension of Campbell's theorem is used to measure the amplitude, waveform, and frequency of occurrence of miniature endplate potentials (mepps) at rapidly secreting neuromuscular junctions of frog cutaneous pectoris muscles. Measurements of the variance, skew, and power spectrum of the fluctuations in membrane potential are used to deduce the mepp parameters. These estimates of mepp amplitude and frequency are insensitive to slow drifts in membrane potential that preclude the conventional application of Campbell's theorem, which uses the mean and variance. The new method becomes unreliable at high mepp frequencies because the distribution of the values of membrane potential approaches a Gaussian thereby reducing the accuracy of skew measurements. Frequencies approaching 10(4) s-1 can be measured, however, if the data are high-pass filtered. The method has been tested with computer simulated data and applied to junctions exposed to La3+; the effects of Ca2+ on the La3+-induced secretion have been explored. Some muscles were fixed after treatment with La3+, and changes in nerve terminal ultrastructure were assessed by morphometric analysis of electron micrographs. Horseradish peroxidase was used to obtain information about vesicle recycling.

of glutamate-glutamine cycle between astrocytes and neurons inhibits epileptiform activity in hippocampus. 88: 2302-2310, 2002; 10.1152/jn.00665.2001. Recurrent epileptiform activity occurs spontaneously in cultured CNS neurons and in brain slices in which GABA inhibition has been blocked. We demonstrate here that pharmacological treatments resulting in either the block of glutamine production by astrocytes or the inhibition of glutamine uptake by neurons suppress or markedly decrease the frequency of spontaneous epileptiform discharges both in primary hippocampal cultures and in disinhibited hippocampal slices. These data point to an important role for the neuron-astrocyte metabolic interaction in sustaining episodes of intense rhythmic activity in the CNS, and thereby reveal a new potential target for antiepileptic treatments. J Neurophysiol

Abstract. Recycling of synaptophysin (p38), a synaptic vesicle integral membrane protein, was studied by the use of antisera raised against the protein purified from frog brain. When frog cutaneous pectoris muscles were fixed at rest, a bright, specific immunofluorescent signal was observed in nerve-terminal regions only if their plasma membranes had been previously permeabilized. When muscles were fixed after they had been treated for 1 h with a low dose of ct-latrotoxin in Ca2÷-free medium, an equally intense fluorescence could be observed without previous permeabilization. Under this condition, ~t-latrotoxin depletes nerve terminals of their quantal store of acetylcholine and of synaptic vesicles. These results indicate that fusion of synaptic vesicles leads to the exposure of intravesicular antigenic determinants of synaptophysin on the outer surface of the axolemma, and provide direct support for the vesicle hypothesis of neurotransmitter release. After 1 h treatment with the same dose of a-latrotoxin in the presence of 1.8 mM extracellular Ca 2+, immunofluorescent images were obtained only after permeabilization with detergents. Under this condition, the vesicle population was maintained by an active process of recycling and more than two times the initial store of quanta were secreted. Thus, despite the active turnover of synaptic vesicles and of quanta of neurotransmitter, no extensive intermixing occurs between components of the vesicle and presynaptic plasma membrane.

We provide evidence that maternal immune activation hits a key neurodevelopmental process, the excitatory-to-inhibitory gamma-aminobutyric acid switch; defects in this switch have been unequivocally linked to diseases such as autism spectrum disorder or epilepsy. These data open the avenue for a safe pharmacological treatment that may prevent the neurodevelopmental defects caused by prenatal immune activation in a specific pregnancy time window.

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