No abstract

In this study, we describe the identification and in vitro functional activity of a novel multiple domain complement regulatory protein discovered based on its homology to short consensus repeat (SCR)-containing proteins of the regulators of complement activation (RCA) gene family. The rat cDNA encodes a predicted 388-kDa protein consisting of 14 N-terminal CUB domains that are separated from each other by a SCR followed by 15 tandem SCR domains, a transmembrane domain, and a short cytoplasmic tail. This protein is the homolog of the human protein of unknown function called the CUB and sushi multiple domains 1 (CSMD1) protein. A cloning strategy that incorporates the two C-terminal CUB-SCR domains and 12 of the tandem SCR repeats was used to produce a soluble rat CSMD1 protein. This protein blocked classical complement pathway activation in a comparable fashion with rat Crry but did not block alternative pathway activation. Analysis of CSMD1 mRNA expression by in situ hybridization and immunolabeling of neurons indicates that the primary sites of synthesis are the developing CNS and epithelial tissues. Of particular significance is the enrichment of CSMD1 in the nerve growth cone, the amoeboid-leading edge of the growing neuron. These results suggest that CSMD1 may be an important regulator of complement activation and inflammation in the developing CNS, and that it may also play a role in the context of growth cone function.

T cell activation and tolerance are regulated by costimulatory molecules. Although PD-1 serves as a crucial negative regulator of T cells, the function of its ligands, PDL1 and PDL2, is still controversial. In this study, we created a PDL2-deficient mouse to characterize its function in T cell activation and tolerance. Antigenpresenting cells from PDL2؊͞؊ mice were found to be more potent in activation of T cells in vitro over the wild-type controls, which depended on PD-1. Upon immunization with chicken ovalbumin, PDL2؊͞؊ mice exhibited increased activation of CD4 ؉ and CD8 ؉ T cells in vivo when compared with WT animals. In addition, T cell tolerance to an oral antigen was abrogated by the lack of PDL2. Our results thus demonstrate that PDL2 negatively regulates T cells in immune responses and plays an essential role in immune tolerance.costimulation ͉ cytokines ͉ PD-1 D uring infection, pathogen-specific T cells are activated, undergo robust clonal expansion, and subsequently differentiate into effector cells. In contrast, peripheral tolerance mechanisms have been found to prevent autoreactive T cell function (1, 2). Oral tolerance is a form of peripheral tolerance, in which antigen-specific T cell tolerance is induced against oral antigens (3). Although oral tolerance has been tested for protection against autoimmune and allergic diseases, the cellular and molecular mechanisms underlying oral tolerance induction have remained unclear.T cell activation and tolerance are critically regulated by costimulatory molecules, especially those in the B7 and CD28 superfamilies (4). PD-1, a novel member of the CD28 family, is expressed on activated T cells and B cells (5). PD-1 has been shown to be a negative regulator of T cell activation and is crucial for maintaining immune tolerance. PD-1 deficiency in mouse results in spontaneous autoimmune diseases (6, 7). Moreover, PD-1 deficiency (8) or blockade (9) accelerated autoimmune diabetes on NOD background. Blocking PD-1 also enhanced experimental autoimmune encephalomyelitis (EAE) disease (10).Two ligands, B7-H1͞PDL1 and PDL2͞B7DC, have been found to bind to . The function of PDL1 and PDL2 in T cell activation is still in debate. Contradictory results have suggested PDL2 serves as a negative and a positive regulator of T cell function. Latchman et al. (13) have shown that recombinant PDL2 protein inhibited the activation and cytokine production of CD4 ϩ T cells via cell-cycle arrest, whereas Tseng et al. (14) published that B7DC-Ig costimulated the proliferation of naïve T cells at suboptimal anti-CD3 concentrations, and that it increased IFN-␥ secretion. Others studied PDL2 function by using antibodies that block PDL2 binding to PD-1. Salama et al. (10) reported exacerbation of EAE disease when PDL2, but not PDL1, was blocked. In a model of airway hypersensitivity, Matsumoto et al. (15) found that anti-PDL2 antibody administered at the time of challenge increased eosinophilia. These data suggest that PDL2, through engaging PD-1, negatively regulates T cell priming...

Retroviruses contain three genes for replication: gag, pol, and env, which encode polyprotein precursors for the internal capsid proteins, reverse transcriptase, and envelope glycoprotein, respectively (1). Murine leukemia virus (MuLV) differs from most other retroviruses in that it encodes two different pathways for gag gene expression (2). These two pathways begin with two independent translation products, gPr80gag and Pr65ag. Pr65gag is processed by proteolytic cleavage to yield the internal capsid proteins of the virus particle and is analogous to the gag polyprotein precursors of other retroviruses (3-5). gPr8Ogg contains the amino acid sequences of Pr65gag as well as 4-6 kilodaltons (kDa) of amino-terminal protein (6, 7). The additional amino-terminal peptides result in glycosylation of gPr8O'ag during translation (8, 9). gPr8 gag is processed by the further addition of carbohydrate and exported to the cell surface where it appears as a glycoprotein of 95 kDa (8-11). It may be also be released into the extracellular medium as cleavage products of 55 and 40 kDa (8,12). Glycosylated gag products are not incorporated into MuLV virions, but they associate with the extracellular matrix (13).While the function of Pr65gag is known, the role of gPr809ae in MuLV infection is not known. Glycosylated gag might provide some function required for viral replication, or it might play an accessory role. We previously isolated mutants of Moloney MuLV (M-MuLV)-infected mouse fibroblasts that did not express gag proteins at the cell surface, and they were deficient for virus production. However, these mutants were cellular, not viral, in nature and they produced normal amounts of gPr8g'ag intracellularly (14). To obtain a more definitive answer, we constructed two mutants of M-MuLV at the recombinant DNA level and recovered virus by transfection. The constructions. and characterizations of the mutant viruses are described here.MATERIALS AND METHODS Cells and Viruses. All cells were grown in Dulbecco-modified Eagle's medium/10% calf serum; Mouse NIH-3T3 cells were described previously (15), as were M-MuLV-infected NIH-3T3 cells (clone A9) (16).The UV-XC assay for MuLV was carried out as described (17). Assay of viral reverse transcriptase by the addition of exogenous poly(rA):oligo(dT) template-primer (16) and banding of virus in sucrose density gradients (18) was carried out as described.Recombinant DNA Cloning. A phage recombinant DNA clones (A-MLV clones 48, 61, and 63) carrying integrated copies of M-MuLV provirus were described previously (19). pMSV-1 is a plasmid clone of unintegrated Moloney murine sarcoma virus (M-MSV) DNA permuted about the unique HindIII site (20). pSLT is a subclone of pMSV-1 deleted from the Sal I site in M-MSV DNA to the Sal I site in the pBR322.vector (see Fig. 2) and was kindly provided by Inder Verma. P90(Abl), a plasmid clone of unintegrated Abelson MuLV (Ab-MuLV) (P90 strain) DNA, was kindly provided by Owen Witte. Plasmid vector pBR328 has been described by Soberon et al. (21).Restriction ...

Transposon TnlO was used to mutagenize the fadR gene in Escherichia coli. Mutants bearing fadR::TnlO insertion mutations were found to (i) utilize the noninducing fatty acid decanoate as sole carbon source, (ii) f8-oxidize fatty acids at constitutive rates, and (iii) contain constitutive levels of the five key ,B-oxidative enzymes. These characteristics were identical to those observed in spontaneous fadR mutants. The constitutive phenotype presented by the fadR: :TnlO mutants was shown to be genetically linked to the associated transposon-encoded drug resistance. These results suggest that the fadR gene product exerts negative control over the fatty acid degradative regulon. The fadR gene of E. coli has been mapped through the use of transposon-mediated fadR insertion mutations. The fadR locus is at 25.5 min on the revised map and cotransduces with purB, hemA, and trp. Three-factor conjugational and transductional crosses indicate that the order of loci in this region of the chromosome is purB-fadR-hemA-trp. Spontaneous fadR mutants were found to map at the same location. Strains that exhibit alterations in the control of the fad regulon in response to changes in temperature were also isolated and characterized. These fadR(Ts) mutants were constitutive for the fad enzymes at elevated temperatures and inducible for these activities at low temperatures. The fadR(Ts) mutations also map at the fadR locus. These results strongly suggest that the fadR gene product is a repressor protein. Wild-type Escherichia coli K-12 is able to grow on long-chain (12 or more carbons) but not on short-(C4 to C5) or medium-(C6 to Cll) chain fatty acids as a sole carbon source (21, 22, 26). Growth in media containing long-chain fatty acids induces the coordinate synthesis of at least five key enzymes involved in the f8-oxidation of fatty acids (21, 22, 26). Mutants unable to grow on fatty acids of any chain length have been obtained and shown to harbor lesions in structural genes for the fi-oxidative enzymes (21, 22) as well as for genes involved in fatty acid activation (11, 15) and transport (11, 19, 20). These fatty acid degradation (fad) lesions map at no fewer than four separate locations on the E. coli chromosome (15, 19, 21). Strains harboring a lesion in one of the structural genes, fadD, lack fatty acyl-coenzyme A synthetase activity and cannot be induced for the other fi-oxidative enzymes (15). These results led Klein et al. (15) to propose that long-chain acyl-coenzyme As serve to induce the fatty acid degradative (fad) system in E. coli. Medium-chain fatty acids can serve as substrates for the ,8-oxidative enzymes, but cannot