Basic fibroblast growth factor (FGF-2) is one of a select group of proteins that can exit cells through an alternate, endoplasmic reticulum/Golgi apparatus independent exocytic pathway. This alternate pathway has been termed protein export. In an attempt to better understand this process, we have identified a family of related compounds, "cardenolides," that inhibit FGF-2 export. The cardenolides inhibit FGF-2 export in a time and concentration dependent fashion. Inhibition of FGF-2 export is specific in that the cardenolides have no effect on conventional protein secretion as measured by their inability to block release of the secreted protein human chorionic gonadotropin-␣.Because cardenolides are known to inhibit ion transport activity mediated by Na Taken together, these data: 1) identify a novel activity for cardenolides; 2) suggest a previously unknown role for the ␣-subunit of Na ؉ , K ؉

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Monoclonal antibody 2F10 is an "internalimage" anti-idiotype (anti-id) antibody capable of micking the group-specific "a" determinant of human hepatitis B surface antigen (HBsAg). By mRNA sequencing and computer-assisted molecular modeling of monoclonal antibody 2F10, we identified a 15-amino acid region of the heavy-chain hypervariable region that has partial residue homology with sequences of the "a" determinant epitopes of HsAg. We have established that a linear 15-mer peptide from a contiguous region on the anti-id antibody can (i) generate anti-HBsAg-specific antibodies when injected into mice, (ii) prime murine lymph node cells for in vitro HBsAg-speciflc T-ceff prolerative responses, and (ii) stimulate in vitro human CD4+ T cells that were primed in vivo to HBsAg by natural infection with hepatitis B virus or v itio with a commercially available lBsAg vaccine. Siflcant y, this peptide could also stimulate CD4+ T cells of human hepatitis B virus carriers. We conclude that a 15-mer peptide derived frot the anti-id sequence can duplicate the B-and T-cell stimulatory activity of the intact anti-id antibody and the antigen that is mimicked, HBsAg.Infection with human hepatitis B virus (HBV) results in a gamut of clinical symptoms ranging from minor flu-like symptoms to death. The wide spectrum of responses is believed to be accounted for by the host's immune response to the virus because HBV is not directly cytopathic for hepatocytes (1). The specific serologic marker of HBV infection is the envelope protein, hepatitis B surface antigen (HBsAg), which contains three related proteins designated S, M (S plus pre-52), and L (M plus pre-Sl). All ofthese proteins share the 226-amino acid sequence of the S protein (in this paper, HBsAg will refer to the S protein). HBsAg possesses a common group-specific a determinant and two sets of mutually exclusive subtype-specific determinants d/y and w/r. Because antibodies directed toward the a determinant confer protection against HBV infection, regardless of subtype (2), any vaccine developed to prevent HBV infection should elicit immunity to the a determinant.The immune network theory proposed by Jerne (3) predicts the appearance of several types of anti-idiotype (anti-id) antibodies during the immune response to a given antigen. The subset of "internal-image" anti-id antibodies (termed Ab2f3) has been proposed to be anti-paratopic and to mimic the molecular features of the original antigen (4, 5). This working hypothesis is based on the concept that certain homologous or analogous molecular motifs of the anti-id sequence can mimic specific immunogenic epitopes of the infectious organism, thereby inducing a protective immune response (4, 5). Such anti-id antibodies have been used in various experimental systems as surrogate vaccines against specific bacterial, viral, and parasitic organisms (for review, see refs. 6 and 7).We produced six monoclonal anti-id antibodies against a monoclonal antibody (mAb), designated H3F5 (id) (8), which recognizes the protective a de...

Immunoglobulin (Ig) amino acid sequences are highly conserved and often have sequence homology ranging from 70 to 95%. Antigen binding fragments (Fab), variable region fragments (Fv), and single chain Fv (scFv) of more than 50 myeloma proteins and monoclonal antibodies (mAb) have been crystallized and display a high degree of structural similarity. Based on this observation, several homology modeling approaches have been developed for the prediction of Fab and Fv structures prior to their experimental determination. We have extracted features from existing Ig sequences, 44 known Fab and Fv structures to create an automated AntiBody structure GENeration (ABGEN) algorithm for obtaining structural models of antibody fragments. ABGEN utilizes a homology based scaffolding technique, and includes the use of invariant and strictly conserved residues, structural motifs of known Fab, canonical features of hypervariable loops, torsional constraints for residue replacements and key inter-residue interactions. The validity of the ABGEN algorithm has been tested using a five-fold cross validation with the existing Fab structures. Molecular mechanics and dynamics methods have been implemented with ABGEN models to accurately predict two Fab structures of anti-sweetener antibodies prior to crystallographic determinations.

The importance of basic fibroblast growth factor (bFGF) in several pathophysiological processes has stimulated interest in the design of receptor antagonists to mitigate such effects. Of key importance in this connection is the characterization of the functional binding epitopes of the growth factor for its receptor. Based on peptide mapping and molecular dynamics calculations of the three-dimensional structure of basic fibroblast growth factor, we employed site-directed mutagenesis to investigate the effect of altering residues at positions 107, 109 -114, and 96 on bFGF on receptor binding affinity. All muteins were cloned and expressed in Escherichia coli, purified to homogeneity employing heparinSepharose columns, and evaluated for receptor binding affinity. We found that replacement of residues at positions 107 and 109 -114 by alanine or phenylalanine had little effect on receptor binding affinities compared with wild type bFGF, in agreement with previous evidence that bFGF residues 109 -114 comprise a low affinity binding site. By contrast, substitution of Glu-96 with alanine yielded a molecule having about 0.1% of the affinity of the wild type bFGF. The affinity of the corresponding lysine and glutamine muteins was 0.3 and 10%, respectively, emphasizing the importance of a negative charge at this position. Our findings are consistent with the view that residues 106 -115 on bFGF represent a low affinity binding site on bFGF. In addition, we identify Glu-96 as a crucial residue for binding to fibroblast growth factor receptor-1.