Use of I region-restricted, antigen-specific T cell hybridomas to produce idiotypically specific anti-receptor antibodies

Murine T cell hybridomas bearing receptors for antigen plus I region gene products were used as immunogens in mice in an effort to raise anti-receptor antisera. The antisera were assayed for anti-receptor activity by the ability to inhibit interleukin 2 production by the T cell hybridomas stimulated by antigen and I region expressing antigen-presenting cells. The T cell hybridomas used in these experiments were made by fusing antigen-specific, I region-restricted BALB/c T cell blasts to the AKR thymoma, BW5147. Three groups of mice were immunized with the T cell hybridomas: (BALB/c X AKR)F1 animals, syngeneic to the hybridoma; (BALB.B X aKR)F1 animals, differing from the hybridomas at H2; and (C.B20 X AKR)F1 animals, differing from the hybridomas at Igh. Mice were immunized multiple times and sera from individual animals were assayed for anti-receptor antibodies. In all groups, some mice produced anti-receptor antibodies by the criterion that they were inhibitory in the assay mentioned above. The frequency of mice producing these inhibitory antibodies varied considerably between groups, with the (BALB.B X AKR)F1 animals producing these antibodies most frequently, and the (BALB/c X AKR)F1 animals producing them least often. All inhibitory antisera were idiotypically specific; they inhibited the response of the immunizing T cell hybridomas, but not the responses of closely related hybridomas with different specificities. Moreover, when they could be absorbed, the inhibitory antibodies could only be absorbed by the immunizing hybridoma. It is hoped that these antisera, and B cell hybridomas prepared from the immunized animals, will be useful in the elucidation of the structure of the receptors for antigen plus I region products on T cells.     

The use of negative ion thermospray liquid chromatography/tandem mass spectrometry for the determination of bile acids and their glycine conjugates.

A study was carried out on the negative ion thermospray liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry of a group of bile acids and their glycine conjugates. The liquid chromatographic/tandem mass spectrometric experiments were performed using low-energy collisions on a triple-quadrupole mass spectrometer. It was found that relatively high collision energies and collision gas pressures were required to produce fragmentation and that some unusual fragments were produced.     

High voltage electron microscopical analysis of chromosomal number in the slime mold Echinostelium minutum de bary

Utilizing serial thick sectioning techniques in combination with high voltage electron microscopy and stereopair analysis a count of 124 chromosomes was determined for a metaphasic plasmodial nucleus of the slime mold E. minutum.     

Comparison of a T cell clone and of T cells from a TCR transgenic mouse: TCR transgenic T cells specific for self-antigen are atypical

It has been widely assumed that T cells from TCR-transgenic (Tg) mice better represent the behavior of T cells from normal mice than do in vitro cultures of T cell clones. We have found that autoreactive T cells arising in the presumably more physiological environment of the BDC-2.5 TCR-Tg mouse, despite being apparently "naive" in surface phenotype, are highly activated functionally and do not resemble CD4(+) T cells from a spontaneously diabetic nonobese diabetic (NOD) mouse or the NOD-derived, diabetogenic CD4(+) T cell clone of origin, BDC-2.5. Our results suggest that autoreactive T cells cloned from the spontaneously diabetic NOD mouse more closely resemble effector T cells arising during the natural disease process.     

Protein interactions at the tight junction. Actin has multiple binding partners, and ZO-1 forms independent complexes with ZO-2 and ZO-3

Defining how the molecular constituents of the tight junction interact is a prerequisite to understanding tight junction physiology. We utilized in vitro binding assays with purified recombinant proteins and immunoprecipitation analyses to define interactions between ZO-1, ZO-2, ZO-3, occludin, and the actin cytoskeleton. Actin cosedimentation studies showed that ZO-2, ZO-3, and occludin all interact directly with F-actin in vitro, indicating that actin is engaged in multiple interactions at the tight junction. Low speed sedimentation analyses demonstrated that neither ZO-2, ZO-3, nor occludin act as F-actin cross-linking proteins, and further evidence indicates that these proteins do not bind to actin filament ends. The binding interactions of ZO-2, ZO-3, and occludin were corroborated in vivo by immunofluorescence colocalization experiments which showed that all three proteins colocalized with actin aggregates at cell borders in cytochalasin D-treated Madin-Darby canine kidney cells. Exploration of other tight junction protein interactions demonstrated that ZO-2 binds directly to both ZO-1 and occludin. Contrary to previous beliefs, our immunoprecipitation results indicate that ZO-1, ZO-2, and ZO-3 exist in situ primarily as independent ZO-1.ZO-2 and ZO-1.ZO-3 complexes rather than a trimeric ZO-1.ZO-2.ZO-3 grouping. These studies elucidate direct binding interactions among tight junction-associated proteins, giving insight into their organization as a multimolecular structure.     

Gene therapy for lysosomal storage diseases: the lessons and promise of animal models

There are more than 40 different forms of inherited lysosomal storage diseases (LSDs) known to occur in humans and the aggregate incidence has been estimated to approach 1 in 7000 live births. Most LSDs are associated with high morbidity and mortality and represent a significant burden on patients, their families, and health care providers. Except for symptomatic therapies, many LSDs remain untreatable, and gene therapy is among the only viable treatment options potentially available. Therapies for some LSDs do exist, or are under evaluation, including heterologous bone marrow transplantation (BMT), enzyme replacement therapy (ERT), and substrate reduction therapy (SRT), but these treatment options are associated with significant concerns, including high morbidity and mortality (BMT), limited positive outcomes (BMT), incomplete response to therapy (BMT, ERT, and SRT), life-long therapy (ERT, SRT), and cost (BMT, ERT, SRT). Gene therapy represents a potential alternative therapy, albeit a therapy with its own attendant concerns. Animal models of LSDs play a critical role in evaluating the efficacy and safety of therapy for many of these conditions. Naturally occurring animal homologs of LSDs have been described in the mouse, rat, dog, cat, guinea pig, emu, quail, goat, cattle, sheep, and pig. In this review we discuss those animal models that have been used in gene therapy experiments and those with promise for future evaluations.     

Cutting edge: CD40-induced expression of recombination activating gene (RAG) 1 and RAG2: a mechanism for the generation of autoaggressive T cells in the periphery

It has been speculated that autoimmune diseases are caused by failure of central tolerance. However, this remains controversial. We have suggested that CD40 expression identifies autoaggressive T cells in the periphery of autoimmune prone mice. In this study, we report that CD40 was cloned from autoaggressive T cells and that engagement induces expression and nuclear translocation of the recombinases, recombination activating gene (RAG) 1 and RAG2 in the autoaggressive, but not in the nonautoaggressive, peripheral T cell population. Furthermore, we demonstrate that CD40 engagement induces altered TCR Valpha, but not Vbeta, expression in these cells. Therefore, CD40-regulated expression of RAG1 and RAG2 in peripheral T cells may constitute a novel pathway for the generation of autoaggressive T cells.     

Recognition of Dictyostelium discoideum lysosomal enzymes is conferred by the amino-terminal carbohydrate binding site of the insulin-like growth factor II/mannose 6-phosphate receptor

The insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-II/MPR) is a type I glycoprotein that mediates both the intracellular sorting of lysosomal enzymes bearing mannose 6-phosphate (Man-6-P) residues to the lysosome and the bioavailability of IGF-II. The extracytoplasmic region of the IGF-II/MPR contains 15 repeating domains; the two carbohydrate recognition domains (CRDs) have been localized to domains 1-3 and 7-9, and the high-affinity IGF-II binding site maps to domain 11. To characterize the carbohydrate binding properties of the IGF-II/MPR, regions of the receptor encompassing the individual CRDs were produced in a baculovirus expression system. Characterization of the recombinant proteins revealed that the pH optimum for carbohydrate binding is significantly more acidic for the carboxyl-terminal CRD than for the amino-terminal CRD (i.e., pH 6.4-6.5 vs 6.9). Equilibrium binding studies demonstrated that the two CRDs exhibit a similar affinity for Man-6-P. Furthermore, substitution of the conserved arginine residue in domain 3 (R435) or in domain 9 (R1334) with alanine resulted in a similar >1000-fold decrease in the affinity for the lysosomal enzyme, beta-glucuronidase. In contrast, the two CRDs differ dramatically in their ability to recognize the distinctive modifications (i.e., mannose 6-sulfate and Man-6-P methyl ester) found on Dictyostelium discoideum lysosomal enzymes: the amino-terminal CRD binds mannose 6-sulfate and Man-6-P methyl ester with a 14-55-fold higher affinity than the carboxyl-terminal CRD. Taken together, these results demonstrate that the IGF-II/MPR contains two functionally distinct CRDs.     

ZO-3, a Novel Member of the MAGUK Protein Family Found at the Tight Junction, Interacts with ZO-1 and Occludin

A 130-kD protein that coimmunoprecipitates with the tight junction protein ZO-1 was bulk purified from Madin-Darby canine kidney (MDCK) cells and subjected to partial endopeptidase digestion and amino acid sequencing. A resulting 19–amino acid sequence provided the basis for screening canine cDNA libraries. Five overlapping clones contained a single open reading frame of 2,694 bp coding for a protein of 898 amino acids with a predicted molecular mass of 98,414 daltons. Sequence analysis showed that this protein contains three PSD-95/SAP90, discs-large, ZO-1 (PDZ) domains, a src homology (SH3) domain, and a region similar to guanylate kinase, making it homologous to ZO-1, ZO-2, the discs large tumor suppressor gene product of Drosophila, and other members of the MAGUK family of proteins. Like ZO-1 and ZO-2, the novel protein contains a COOH-terminal acidic domain and a basic region between the first and second PDZ domains. Unlike ZO-1 and ZO-2, this protein displays a proline-rich region between PDZ2 and PDZ3 and apparently contains no alternatively spliced domain. MDCK cells stably transfected with an epitope-tagged construct expressed the exogenous polypeptide at an apparent molecular mass of ~130 kD. Moreover, this protein colocalized with ZO-1 at tight junctions by immunofluorescence and immunoelectron microscopy. In vitro affinity analyses demonstrated that recombinant 130-kD protein directly interacts with ZO-1 and the cytoplasmic domain of occludin, but not with ZO-2. We propose that this protein be named ZO-3.