Kinematics and modeling of leech crawling: evidence for an oscillatory behavior produced by propagating waves of excitation.

Many well characterized central pattern generators (CPGs) underlie behaviors (e.g., swimming, flight, heartbeat) that require regular rhythmicity and strict phase relationships. Here, we examine the organization of a CPG for leech crawling, a behavior whose success depends more on its flexibility than on its precise coordination. We examined the organization of this CPG by first characterizing the kinematics of crawling steps in normal and surgically manipulated animals, then by exploring its features in a simple neuronal model. The behavioral observations revealed the following. (1) Intersegmental coordination varied considerably with step duration, whereas the rates of elongation and contraction within individual segments were relatively constant. (2) Steps were generated in the absence of both head and tail brains, implying that midbody ganglia contain a CPG for step production. (3) Removal of sensory feedback did not affect step coordination or timing. (4) Imposed stretch greatly lengthened transitions between elongation and contraction, indicating that sensory pathways feed back onto the CPG. A simple model reproduced essential features of the observed kinematics. This model consisted of an oscillator that initiates propagating segmental waves of activity in excitatory neuronal chains, along with a parallel descending projection; together, these pathways could produce the observed intersegmental lags, coordination between phases, and step duration. We suggest that the proposed model is well suited to be modified on a step-by-step basis and that crawling may differ substantially from other described CPGs, such as that for swimming in segmented animals, where individual segments produce oscillations that are strongly phase-locked to one another.

pFab-CMV, a single vector system for the rapid conversion of recombinant Fabs into whole IgG1 antibodies.

We have constructed a single vector system for the rapid conversion of recombinant Fabs into whole IgG1 antibodies and their expression in eukaryotic cells. This vector, named pFab-CMV, utilizes the same unique cloning sites present on the pComb3 phagemid thus allowing for the direct subcloning of light chains and heavy chain Fd regions. pFab-CMV also allows for the expression of recombinant Fabs in eukaryotic cells by removal of a cassette containing part of the hinge, CH2 and CH3 sequences. Stable cell lines are rapidly obtained with pFab-CMV by NEO selection without the need for co-transfection of heavy and light chain expressing vectors.

Characterization of a type-common human recombinant monoclonal antibody to herpes simplex virus with high therapeutic potential.

We report the characterization of a type-common human recombinant monoclonal antibody previously isolated by antigen selection from a phage-displayed combinatorial antibody library established from a herpes simplex virus (HSV)-seropositive individual. Competition with well-characterized murine monoclonal antibodies and immunodetection of gD truncations revealed that this antibody recognizes the group Ib antigenic site of glycoprotein D, a highly conserved and protective type-common determinant. To our knowledge, this is the first human group Ib monoclonal antibody ever described. The antibody also displayed first-order neutralization kinetics and a high neutralization rate constant, was capable of completely inhibiting syncytium formation by a fusogenic strain of HSV type 1, and efficiently neutralized low-passage clinical isolates of both HSV serotypes. Taken together with our earlier observations of the in vivo antiviral activities of this human recombinant antibody in animal models of HSV infection, the present results support the high therapeutic potential of this antibody.

Mapping the prion protein using recombinant antibodies.

The fundamental event in prion disease is thought to be the posttranslational conversion of the cellular prion protein (PrPC) into a pathogenic isoform (PrPSc). The occurrence of PrPC on the cell surface and PrPSc in amyloid plaques in situ or in aggregates following purification complicates the study of the molecular events that underlie the disease process. Monoclonal antibodies are highly sensitive probes of protein conformation which can be used under these conditions. Here, we report the rescue of a diverse panel of 19 PrP-specific recombinant monoclonal antibodies from phage display libraries prepared from PrP deficient (Prnp0/0) mice immunized with infectious prions either in the form of rods or PrP 27-30 dispersed into liposomes. The antibodies recognize a number of distinct linear and discontinuous epitopes that are presented to a varying degree on different PrP preparations. The epitope reactivity of the recombinant PrP(90-231) molecule was almost indistinguishable from that of PrPC on the cell surface, validating the importance of detailed structural studies on the recombinant molecule. Only one epitope region at the C terminus of PrP was well presented on both PrPC and PrPSc, while epitopes associated with most of the antibodies in the panel were present on PrPC but absent from PrPSc.

A conformational transition at the N terminus of the prion protein features in formation of the scrapie isoform.

The scrapie prion protein (PrPSc) is formed from the cellular isoform (PrPC) by a post-translational process that involves a profound conformational change. Linear epitopes for recombinant antibody Fab fragments (Fabs) on PrPC and on the protease-resistant core of PrPSc, designated PrP 27-30, were identified using ELISA and immunoprecipitation. An epitope region at the C terminus was accessible in both PrPC and PrP 27-30; in contrast, epitopes towards the N-terminal region (residues 90 to 120) were accessible in PrPC but largely cryptic in PrP 27-30. Denaturation of PrP 27-30 exposed the epitopes of the N-terminal domain. We argue from our findings that the major conformational change underlying PrPSc formation occurs within the N-terminal segment of PrP 27-30.