Thomas G. Beito

Characterization of PKD Protein-Positive Exosome-Like Vesicles

Proteins associated with autosomal dominant and autosomal recessive polycystic kidney disease (polycystin-1, polycystin-2, and fibrocystin) localize to various subcellular compartments, but their functional site is thought to be on primary cilia. PC1+ vesicles surround cilia in Pkhd1(del2/del2) mice, which led us to analyze these structures in detail. We subfractionated urinary exosome-like vesicles (ELVs) and isolated a subpopulation abundant in polycystin-1, fibrocystin (in their cleaved forms), and polycystin-2. This removed Tamm-Horsfall protein, the major contaminant, and subfractionated ELVs into at least three different populations, demarcated by the presence of aquaporin-2, polycystin-1, and podocin. Proteomic analysis of PKD ELVs identified 552 proteins (232 not yet in urinary proteomic databases), many of which have been implicated in signaling, including the molecule Smoothened. We also detected two other protein products of genes involved in cystic disease: Cystin, the product of the mouse cpk locus, and ADP-ribosylation factor-like 6, the product of the human Bardet-Biedl syndrome gene (BBS3). Our proteomic analysis confirmed that cleavage of polycystin-1 and fibrocystin occurs in vivo, in manners consistent with cleavage at the GPS site in polycystin-1 and the proprotein convertase site in fibrocystin. In vitro, these PKD ELVs preferentially interacted with primary cilia of kidney and biliary epithelial cells in a rapid and highly specific manner. These data suggest that PKD proteins are shed in membrane particles in the urine, and these particles interact with primary cilia.

Characterization of monoclonal antibodies against the glaucoma-associated protein myocilin

Although the glaucoma-associated protein myocilin has been the focus of intensive research, its biological function is still unknown. One of the limiting factors has been the lack of well-characterized antibodies, particularly monoclonal antibodies. We describe the development of six monoclonal antibodies specific to myocilin and characterize their suitability in Western blot and immunohistochemical applications. Three of the six monoclonal antibodies recognize the N-terminus of myocilin (amino acids 33-214), two antibodies recognize the middle third of the protein (amino acids 215-368), and one antibody recognizes the C-terminus (amino acids 369-504). Isotyping revealed that all antibodies are of the IgG1 kappa class except one, which is IgG2b kappa. Purified myocilin monoclonal antibodies were able to recognize myocilin in human aqueous humor separated on denatured/reduced and native gels, and human trabecular meshwork lysate by Western blot. Myocilin was also detected by immunohistochemistry in trabecular meshwork, ciliary body, iris, cornea, sclera, choroid, and retinal pigment epithelial cells.

Determination of tolerance to self Eα peptides by clonal elimination of H-2E-reactive T cells and antigen presentation by H-2A molecules

A series of three synthetic peptides spanning H-2Eka chain residues (90–110), (110–130), and (130–150) were synthesized and purified. Mice representative of H-2E− (B6, B10, B10.M, B10.Q, B10.S) and H-2E+ (B10.D2, B10.K, B10.RIII) were immunized with individual peptides and lymph node cells challenged in vitro. Both B6 and B10 mice respond to in vitro challenge to peptides (90–110) (cpm 20,000), (110–130) (cpm 40,000), and (130–150) (cpm 60,000). In contrast all H-2E+ haplotypes were unresponsive to all three peptides (cpms <10,000). Furthermore, B10 mice could be rendered hyporesponsive to Eka peptide challenge following expression of an Eka transgene or mating to an H-2E+ strain. The H-2Ad,k,f,q,s alleles were associated with reduced peptide recognition. Furthermore, alteration of the H-2Aβ chain in bm12 mutant mice resulted in impaired responses to all three peptides. Immunization with synthetic peptides comprising major histocompatibility molecules may yield insights into mechanisms of self-tolerance.

Immunodetection of human topoisomerase I-DNA covalent complexes

A number of established and investigational anticancer drugs slow the religation step of DNA topoisomerase I (topo I). These agents induce cytotoxicity by stabilizing topo I-DNA covalent complexes, which in turn interact with advancing replication forks or transcription complexes to generate lethal lesions. Despite the importance of topo I-DNA covalent complexes, it has been difficult to detect these lesions within intact cells and tumors. Here, we report development of a monoclonal antibody that specifically recognizes covalent topo I-DNA complexes, but not free topo I or DNA, by immunoblotting, immunofluorescence or flow cytometry. Utilizing this antibody, we demonstrate readily detectable topo I-DNA covalent complexes after treatment with camptothecins, indenoisoquinolines and cisplatin but not nucleoside analogues. Topotecan-induced topo I-DNA complexes peak at 15–30 min after drug addition and then decrease, whereas indotecan-induced complexes persist for at least 4 h. Interestingly, simultaneous staining for covalent topo I-DNA complexes, phospho-H2AX and Rad51 suggests that topotecan-induced DNA double-strand breaks occur at sites distinct from stabilized topo I-DNA covalent complexes. These studies not only provide new insight into the action of topo I-directed agents, but also illustrate a strategy that can be applied to study additional topoisomerases and their inhibitors in vitro and in vivo.

Genetic control of the immune response to Trichinella spiralis: recognition of muscle larval antigens.

Summary Host antibody recognition of muscle larval (ML) antigens of Trichinella spiralis was examined. Monoclonal antibodies (MoAbs) to known host protective ML antigens have been produced in order to aid this examination. Eleven strains of mice with independent MHC haplotypes and seventeen T. spiralis infected human patients were all found to recognize the same three major antigens as the monoclonal antibodies; i.e., of mw 41, 46 and 55 kD. However all scrum samples tested also recognized further ML antigens and this recognition varied with the individual or strain. This variation in antigen recognition also applied to the MoAb. Mutual inhibition studies demonstrated that even where the MoAb apparently recognized the same antigens, specific epitope recognition was disparate. Hence some of the major antigens recognized by hosts of T. spiralis, regardless of whether vaccinated or infected, correspond with antigens which have considerable host protective properties. There also appear to be a number of epitopes upon these antigens and the biological implications of this are discussed.

Production and characterization of monoclonal antibodies to human sclerostin.

We developed and characterized monoclonal antibodies directed against the amino-terminal and carboxy-terminal regions of human and mouse sclerostin (scl). Amino-terminal and carboxy-terminal scl peptides with limited homology to scl domain-containing protein-1 were synthesized using f-moc chemistry. The peptides were conjugated to keyhole limpet hemocyanin and the conjugates were used for immunization of mice. Monoclonal antibodies were obtained and characterized using bacterially expressed and insect cell-expressed recombinant scl. The amino-terminal (IgG 2aK) and carboxy-terminal (IgG 2bK) antibodies bound bioactive sclerostin that was expressed in an insect-cell expression system with dissociation constants in the nanomolar range. The antibodies are potentially useful agents that can be used for modulating sclerostin bioactivity.

Synthesis of a peptide-universal nucleotide antigen: towards next-generation antibodies to detect topoisomerase I-DNA covalent complexes

The topoisomerase (topo) I-DNA covalent complex represents an attractive target for developing diagnostic antibodies to measure responsiveness to drugs. We report a new antigen, peptide , and four murine monoclonal antibodies raised against that exhibit excellent specificity for recognition of in comparison to structurally similar peptides by enzyme-linked immunosorbent assays. Although topo I-DNA complex detection was not achieved in cellular samples by these new antibodies, a new strategy for antigen design is reported.

Immunological Analysis of the Avian Progesterone Receptor

Although the avian progesterone receptor has been purified and investigated extensively, its structure and composition remain unclear. The nontransformed receptor in cytosol extracts exists as two major forms, I and II, which differ in size and in ionic properties (1,2). Both forms I and II are large 8S protein complexes which contain a common 90,000-dalton protein in addition to the hormone binding protein or receptor. This 90,000-dalton protein has been recently identified as heat shock protein 90 (hsp90) (3–5), one of the major heat shock proteins found in eukaryotic cells. Also, the same protein has been shown to form complexes with other steroid receptors from the chicken (6,7), and may thus be a common element of all nontransformed receptors.

The Use of Mouse Class II MHC Peptides to Produce Site-Specific Monoclonal Antibodies

We have attempted to produce site-specific monoclonal antibodies against defined regions on mouse class II (Ia) molecules. These studies were designed to produce monoclonal antibodies against defined sequences of Ia molecules which may have applications in the design of novel experimental , functional and therapeutic investigations into the biochemical role of Ia molecules in immunity and disease states (Rothbard and Taylor , 1988) . We describe here our initial results using peptides derived from the polymorphic regions 43–61 of the alpha chain subunit and polymorphic regions 57–74/78 of beta chain subunit of I-A molecules from three alleles .

The impact of Jack Stimpfling's recombinants on immunogenetics.

An angler dreams of a rising rainbow, chooses just the precise fly currently hatching in order to complete the dream of a mountain stream battle that would ensue. The fly must land and drift perfectly so that the trout will be fooled. A camper dreams of just the right location for a warm fire to rest his tent where he will be protected from the wind, yet be able to view the natural beauty surrounding him. A scientist hopes that by pursuing his love of truth and awe of the unknown, he will be contributing to the benefit of his fellow man. Once upon a time, just such a scientist began such a career on the island of Mount Desert. He not only fulfilled his scientific dreams, but also in later years would enjoy the tranquility of nature, open space and the big skies. This is a chronicle of one aspect of his scientific contribution to his fellow man.