Lateral diffusion of GFP-tagged H2Ld molecules in the ER membrane reports on their interaction with the TAP complex during synthesis and peptide loading. Peptide-loaded H2Ld molecules diffuse rapidly, near the theoretical limit for proteins in a bilayer. However, these molecules are retained in the ER for some time after assembly. H2Ld molecules, associated with the TAP complex, diffuse slowly, as does GFP-tagged TAP1. This implies that the association of H2Ld molecules with the TAP complex is stable for at least several minutes. It also suggests that the TAP complex is very large, perhaps containing hundreds of proteins.

Learning by observation involves automatic creation of categories that summarize experience. In this paper we present UNIMEM, an artificial intelligence system that learns by observation. UNIMEM is a robust program that can be run on many domains with real-world problem characteristics such as uncertainty, incompleteness, and large numbers of examples. We give an overview of the program that illustrates several key elements, including the automatic creation of non-disjoint concept hierarchies that are evaluated over time. We then describe several experiments that we have carried out with UNIMEM, including tests on different domains (universities, Congressional voting records, and terrorist events) and an examination of the effect of varying UNIMEM's parameters on the resulting concept hierarchies. Finally we discuss flmlre directions for our work with the program.

SummaryThe interaction of the T cell receptor (TCR) with its cognate peptide-major histocompatibility complex (MHC) on the surface of antigen presenting cells (APCs) is a primary event during T cell activation. Here we used a dimeric IE k -MCC molecule to study its capacity to activate antigen-specific T cells and to directly analyze the role of CD4 in physically stabilizing the TCR-MHC interaction. Dimeric IE k -MCC stably binds to specific T cells. In addition, immobilized dimeric IE k -MCC can induce TCR downregulation and activate antigen-specific T cells more efficiently than anti-CD3. The potency of the dimeric IE k -MCC is significantly enhanced in the presence of CD4. However, CD4 does not play any significant role in stabilizing peptide-MHC-TCR interactions as it fails to enhance binding of IE k -MCC to specific T cells or influence peptide-MHC-TCR dissociation rate or TCR downregulation. Moreover, these results indicate that dimerization of peptide-MHC class II using an IgG molecular scaffold significantly increases its binding avidity leading to an enhancement of its stimulatory capacity while maintaining the physiological properties of cognate peptide-MHC complex. These peptide-MHC-IgG chimeras may, therefore, provide a novel approach to modulate antigen-specific T cell responses both in vitro and in vivo.

The generation of extended plots for melodramatic fiction is an interesting task for Artificial Intelligence research, one that requires the application of generalization techniques to carry out fully. UNIVERSE is a story-telling program that uses plan-like units, "plot fragments", to generate plot outlines. By using a rich library of plot fragments and a well-developed set of characters, UNIVERSE can create a wide range of plot outlines. In this paper, we illustrate how UNIVERSE's plot fragment library is used to create plot outlines and how it might be automatically extended using explanation-based • generalization methods. Our methods are based on analysis of a television melodrama, including comparisons of similar stories.

Extended story generation, i.e., the creation of continuing serials, presents difficult and interesting problems for Artificial Intelligence. We present here the first phase or the development of a program, UNIVERSE, that will ultimately tell extended stories. In particular, alter describing our overall model of story telling, we present a method Cor creating universes of characters appropriate for extended story generation. This method concentrates on the need to keep story-telling universes consistent and coherent. We also describe the information that must be maintained ror characters and interpersonal relationships, and the use of stereotypical information about people to help motivate trait values. The use of historical events for motivation is also described. Finally, we present an example of a character generated by UNIVERSE.

The F1 moiety of rat liver ATP synthase has a molecular mass of 370,000, exhibits the unique substructure alpha 3 beta 3 gamma delta epsilon, and fully restores ATP synthesis to F1-depleted membranes. Here we provide new information about rat liver F1 as it relates to the relationship of its unique substructure to its nucleotide binding properties, enzymatic states, and crystalline form. Seven types of experiments were performed in a comprehensive study. First, the capacity of F1 to bind [3H]ADP, the substrate for ATP synthesis and [32P]AMP-PNP (5'-adenylyl-beta,gamma-imidodiphosphate), a nonhydrolyzable ATP analog, was quantified. Second, double-label experiments were performed to establish whether ADP and AMP-PNP bind to the same or different sites. Third, total nucleotide binding was assessed by the luciferin-luciferase assay. Fourth, F1 was subfractionated into an alpha gamma and a beta delta epsilon fraction, both of which were subjected to nucleotide binding assays. Fifth, the nucleotide binding capacity of F1 was quantified after undergoing ATP hydrolysis. Sixth, the intensity of the fluorescence probe pyrene maleimide bound at alpha subunits was monitored before and after F1 experienced ATP hydrolysis. Finally, the catalytic activity and nucleotide content of F1 obtained from crystals being used in x-ray crystallographic studies was determined. The picture of rat liver F1 that emerges is one of an enzyme molecule that 1) loads nucleotide readily at five sites; 2) requires for catalysis both the alpha gamma and the beta delta epsilon fractions; 3) directs the reversible binding of ATP and ADP to different regions of the enzyme's substructure; 4) induces inhibition of ATP hydrolysis only after ADP fills at least five sites; and 5) exists in several distinct forms, one an active, symmetrical form, obtained in the presence of ATP and high P(i) and on which an x-ray map at 3.6 A has been reported (Bianchet, M., Ysern, X., Hullihen, J., Pedersen, P. L., and Amzel, L. M. (1991) J. Biol. Chem. 266, 21197-21201). These results are discussed within the context of a multistate model for rat liver F1 and also discussed relative to those reported for bovine heart F1, which has been crystallized with inhibitors in an asymmetrical form and has a propensity for binding nucleotides more tightly.

Background and aimsHepatocellular carcinoma (HCC) is a difficult to treat tumor with a poor prognosis. Aspartate β-hydroxylase (ASPH) is a highly conserved enzyme overexpressed on the cell surface of both murine and human HCC cells.MethodsWe evaluated therapeutic effects of nanoparticle lambda (λ) phage vaccine constructs against ASPH expressing murine liver tumors. Mice were immunized before and after subcutaneous implantation of a syngeneic BNL HCC cell line. Antitumor actively was assessed by generation of antigen specific cellular immune responses and the identification of tumor infiltrating lymphocytes.ResultsProphylactic and therapeutic immunization significantly delayed HCC growth and progression. ASPH-antigen specific CD4+ and CD8+ lymphocytes were identified in the spleen of tumor bearing mice and cytotoxicity was directed against ASPH expressing BNL HCC cells. Furthermore, vaccination generated antigen specific Th1 and Th2 cytokine secretion by immune cells. There was widespread necrosis with infiltration of CD3+ and CD8+ T cells in HCC tumors of λ phage vaccinated mice compared to controls. Moreover, further confirmation of anti-tumor effects on ASPH expressing tumor cell growth were obtained in another murine syngeneic vaccine model with pulmonary metastases.ConclusionsThese observations suggest that ASPH may serve as a highly antigenic target for immunotherapy.

hypoglycemia͞hypoxia ͉ neuroprotection ͉ PAN-811͞Triapine ͉ oxidative ͉ calcium