Identification of a factor Xa-interactive site within residues 337-372 of the factor VIII heavy chain.

We recently demonstrated that the residues 337-372, comprising the acidic C-terminal region in A1 subunit, interact with factor Xa during the proteolytic inactivation of factor VIIIa (Nogami, K., Wakabayashi, H., and Fay, P.

Peptide ligands in antibacterial drug discovery: use as inhibitors in target validation and target-based screening.

There is an urgent need to develop novel classes of antibiotics to counter the inexorable rise of resistant bacterial pathogens. Modern antibacterial drug discovery is focused on the identification and validation of novel protein targets that may have a suitable therapeutic index. In combination with assays for function, the advent of microbial genomics has been invaluable in identifying novel antibacterial drug targets.

Interaction of the A1 subunit of factor VIIIa and the serine protease domain of factor X identified by zero-length cross-linking.

We have previously used a solid phase binding assay to localize a Factor X (FX) interactive site to the acidic C-terminus of the A1 subunit of FVIIIa (Lapan KA, Fay PJ. J Biol Chem 1997; 272: 2082-2088). The complex of FVIII-FX was made covalent following reaction with the zero-length cross-linking reagent 1-ethyl-3-(3dimethylaminopropyl-)carbodiimide hydrochloride (EDC).

Localization of a factor X interactive site in the A1 subunit of factor VIIIa.

The protein cofactor, factor (F) VIIIa, is required for the efficient conversion of the substrate FX to FXa by the serine protease FIXa. The interaction between human FVIII (and its constituent subunits) and FX was characterized using a solid phase binding assay performed in the absence of phospholipid and FIXa. Saturable binding of FX to heterodimeric FVIII, the FVIII heavy chain (contiguous A1-A2 domains), the FVIIIa-derived A1/A3-C1-C2 dimer, and the isolated A1 subunit was observed with estimated Kd values ranging from approximately 1 to 3 microM.

Expression of the C terminus of the amyloid precursor protein alters growth factor responsiveness in stably transfected PC12 cells.

The amyloid precursor protein (APP) is a molecule centrally involved in Alzheimer disease pathology, but whose normal function is still poorly understood. To investigate the consequences of increased intracellular production of various regions of APP on cellular physiology, we stably transfected PC12 cells with the C-terminal 100 amino acids of the human APP. In eight transfected clones that express the APP(C100) protein, exposure to nerve growth factor (NGF) did not promote differentiation.

Production and characterization of two new mouse monoclonal antibodies reactive with denatured mouse class II beta chains.

We report on the production and characterization of two murine peptide specific anti-major histocompatibility complex class II chain specific monoclonal antibodies. Two new mouse monoclonal antibodies reactive with two synthetic peptides corresponding to Abb amino acids (146-157) and Abs amino acids (146-157) were produced. KL295 is the mouse anti-Abb monoclonal antibody, which reacts with denatured beta chains of H-2b, H-2d, H-2p, and H-2q, but fail to react with spleen cell lysates from H-2f, H-2j, H-2k and H-2s mice.

LCMV-specific, class II-restricted cytotoxic T cells in beta 2-microglobulin-deficient mice.

Intracranial infection of normal mice with lymphocytic choriomeningitis virus (LCMV) causes meningitis and death mediated by CD8+ major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocytes (CTLs). beta 2-Microglobulin-deficient mice (beta 2M-/-) do not express functional MHC class I proteins and do not produce significant numbers of CD8+ T cells. When beta 2M-/- mice were infected with LCMV, many died from LCMV disease and produced a specific response to LCMV mediated by CD4+ CTLs that were class II-restricted.

Production and characterization of a peptide specific, anti-major histocompatibility complex class II, monoclonal antibody.

Monoclonal antibodies to major histocompatibility complex Class II proteins have been useful probes in understanding both the biochemistry and biology of these proteins. Almost all of the monoclonal antibodies previously described have been produced by immunization of mice with living cells. These antibodies react with native Class II proteins, but not usually with denatured material.

Cytotoxic T lymphocyte recognition of hybrid MHC class I molecules.

We have utilized a group of MHC class I genes produced by in vitro recombination between Dp and Dd to study recognition of MHC class I molecules by cytolytic T cells (CTLs). Both polyclonal allo-specific and H-2-restricted CTLs require that alpha 1 and alpha 2 of the target class I molecule be derived from the same haplotype for efficient killing. By using T-cell lines we showed that within the bulk population there must exist a fraction of T cells which can recognize epitopes in alpha 1 or alpha 2.

The alpha 1 and alpha 2 domains of H-2 class I molecules interact to form unique epitopes.

Mouse class I antigens are the major targets of cytolytic T lymphocytes in both major histocompatibility complex (MHC)-restricted and allogeneic responses. Considerable evidence has recently accumulated demonstrating that MHC class I molecules encoded by genes whose alpha 1 and alpha 2 coding exons were interchanged are not recognized by T lymphocytes specific for parental class I products. Along with the loss of T-cell reactivity, there is a loss of recognition by some, but not all monoclonal antibodies.