Blocking the NGF-TrkA Interaction Rescues the Developmental Loss of LTP in the Rat Visual Cortex Role of the Cholinergic System

AbstractAlthough nerve growth factor (NGF) is a crucial factor in the activity-dependent development and plasticity of visual cortex, its role in synaptic efficacy changes is largely undefined. We demonstrate that the maintenance phase of long-term potentiation (LTP) is blocked by local application of exogenous NGF in rat visual cortex at an early stage of postnatal development. Long-term depression (LTD) and bidirectional plasticity are unaffected. At later postnatal ages, blockade of either endogenous NGF by immunoadhesin (TrkA-IgG) or TrkA receptors by monoclonal antibody rescues LTP. Muscarinic receptor activation/inhibition suggests that LTP dependence on NGF is mediated by the cholinergic system. These results indicate that NGF regulates synaptic strength in well-characterized cortical circuitries

Antibody binding loop insertions as diversity elements

In the use of non-antibody proteins as affinity reagents, diversity has generally been derived from oligonucleotide-encoded random amino acids. Although specific binders of high-affinity have been selected from such libraries, random oligonucleotides often encode stop codons and amino acid combinations that affect protein folding. Recently it has been shown that specific antibody binding loops grafted into heterologous proteins can confer the specific antibody binding activity to the created chimeric protein. In this paper, we examine the use of such antibody binding loops as diversity elements. We first show that we are able to graft a lysozyme-binding antibody loop into green fluorescent protein (GFP), creating a fluorescent protein with lysozyme-binding activity. Subsequently we have developed a PCR method to harvest random binding loops from antibodies and insert them at predefined sites in any protein, using GFP as an example. The majority of such GFP chimeras remain fluorescent, indicating that binding loops do not disrupt folding. This method can be adapted to the creation of other nucleic acid libraries where diversity is flanked by regions of relative sequence conservation, and its availability sets the stage for the use of antibody loop libraries as diversity elements for selection experiments

A comprehensive analysis of filamentous phage display vectors for cytoplasmic proteins: an analysis with different fluorescent proteins

Filamentous phage display has been extensively used to select proteins with binding properties of specific interest. Although many different display platforms using filamentous phage have been described, no comprehensive comparison of their abilities to display similar proteins has been conducted. This is particularly important for the display of cytoplasmic proteins, which are often poorly displayed with standard filamentous phage vectors. In this article, we have analyzed the ability of filamentous phage to display a stable form of green fluorescent protein and modified variants in nine different display vectors, a number of which have been previously proposed as being suitable for cytoplasmic protein display. Correct folding and display were assessed by phagemid particle fluorescence, and with anti-GFP antibodies. The poor correlation between phagemid particle fluorescence and recognition of GFP by antibodies, indicates that proteins may fold correctly without being accessible for display. The best vector used a twin arginine transporter leader to transport the displayed protein to the periplasm, and a coil-coil arrangement to link the displayed protein to g3p. This vector was able to display less robust forms of GFP, including ones with inserted epitopes, as well as fluorescent proteins of the Azami green series. It was also functional in mock selection experiments

Modulation of Cortical Synaptic Plasticity by the Cholinergic System and Nerve-Growth Factor

Long lasting modifications in the efficacy of synaptic transmission among neurons are thought to be the basic changes that account for complex processes such as learning and memory. Thus, the comprehension of the mechanisms and factors controlling synaptic plasticity is fundamental to understand higher cognitive functions. The aim of the present work was to investigate possible factors modulating synaptic plasticity in the cortex. One likely candidate is the cholinergic system, arising in the basal forebrain (BFB) and projecting to the cortex, whose deficits are known to impair cognitive functions. It has been found that either disruption of cholinergic neurons or blockade of cholinergic transmission impair Long-Term Potentiation (L TP) in rat visual cortex slices. Conversely, activation of the cholinergic receptors has a facilitatory effect in synaptic strengthening. In addition, by using a transgenic mouse expressing an anti-NGF monoclonal antibody, it has been investigated the long-term effects of Nerve-Growth Factor (NGF) deprivation in cholinergic system functionality, for which NGF is known to exert atrophic action. In these mice, it has been observed an impairment of L TP that can be rescued by application of cholino,mimetic drugs. Beside its action as trophic factor, NGF is known to exert a crucial role in the activity-dependent development and plasticity of visual cortex. This observation prompted me to investigate its possible effect as modulator of synaptic plasticity. It has been found that blocking the NGF-TrkA interaction rescues the developmental loss of L TP in the rat visual cortex. In contrast, an increase in the levels of NGF reduces the capability of synapses to be potentiated. Long-Term Depression and bidirectional plasticity are unaffected. These results indicate that both the cholinergic system and NGF are effective regulators of synaptic strength in the visual cortex. Moreover, the evidence that cholinergic antagonist avoids L TP rescue mediated by blockade of NGF-TrkA interaction and that BFB lesion masks the action of NGF on L TP expression suggest that NGF modulates L TP by means of the BFB cholinergic system

Controlled Manipulation of Bacteriophages Using Single-Virus Force Spectroscopy.

A method is described for the site-directed manipulation of single filamentous bacteriophages, by using phage display technology and atomic force microscopy. f1 filamentous bacteriophages were genetically engineered to display His-tags on their pIX tail. Following adsorption on nitrilotriacetate-terminated surfaces, force spectroscopy with tips bearing monoclonal anti-pIII antibodies was used to pull on individual phages via their pIII head. Analysis of the force-extension profiles revealed that upon pulling, the phages are progressively straightened into an extended orientation until rupture of the anti-pIII/pIII complex. The single-virus manipulation technique presented here provides new opportunities for understanding the forces driving cell-virus and material-virus interactions, and for characterizing the binding properties of polypeptide sequences or proteins selected by the phage display technology

Monoclonal antibodies directed against human FcRn and their applications.

The MHC class I-like Fc receptor (FcRn) is an intracellular trafficking Fc receptor that is uniquely responsible for the extended serum half-life of antibodies of the IgG subclass and their ability to transport across cellular barriers. By performing these functions, FcRn affects numerous facets of antibody biology and pathobiology. Its critical role in controlling IgG pharmacokinetics has been leveraged for the design of therapeutic antibodies and related biologics. FcRn also traffics serum albumin and is responsible for the enhanced pharmacokinetic properties of albumin-conjugated therapeutics. The understanding of FcRn and its therapeutic applications has been limited by a paucity of reliable serological reagents against human FcRn. Here, we describe the properties of a new panel of highly specific monoclonal antibodies (mAbs) directed against human FcRn with diverse epitope specificities. We show that this antibody panel can be used to study the tissue expression pattern of human FcRn, to selectively block IgG and serum albumin binding to human FcRn in vitro and to inhibit FcRn function in vivo. This mAb panel provides a powerful resource for probing the biology of human FcRn and for the evaluation of therapeutic FcRn blockade strategies