Biochemical analysis of the receptor for ubiquitin-like polypeptide.
(17/1806)
Monoclonal nonspecific suppressor factor (MNSF), a lymphokine produced by murine T cell hybridoma, possesses pleiotrophic antigen-nonspecific suppressive functions. A cDNA clone encoding MNSF-beta, an isoform of the MNSF, has been isolated and characterized. MNSF-beta cDNA encodes a fusion protein consisting of a ubiquitin-like segment (Ubi-L) and ribosomal protein S30. Ubi-L appears to be cleaved from the ribosomal protein and released extracellularly in association with T cell receptor-like polypeptide. In the current study we have characterized the biochemical nature of the Ubi-L receptor on D.10 G4.1, a murine T helper clone type 2. Biotinylated Ubi-L bound preferentially to concanavalin A-stimulated but not to unstimulated D.10 cells. Detergent-extracted membrane proteins were applied to an immobilized Ubi-L column. SDS-polyacrylamide gel electrophoresis of eluted fraction revealed a band of Mr = 82,000. Biotinylated Ubi-L specifically recognized this band, confirming that the 82-kDa protein is the Ubi-L receptor. A complex of Mr = 90,000 was visualized by immunoprecipitation of 125I-Ubi-L cross-linked to the purified receptor followed by SDS-polyacrylamide gel electrophoresis and autoradiography. In addition, a 105-kDa protein was coimmunoprecipitated by anti-Ubi-L receptor (82-kDa polypeptide) antibody, indicative of the association of this protein with the Ubi-L receptor complex. Amino acid sequence analysis of the 82-kDa polypeptide revealed that the Ubi-L receptor may be a member of a cytokine receptor family. (+info)
Altered cell-surface targeting of stem cell factor causes loss of melanocyte precursors in Steel17H mutant mice.
(18/1806)
The normal products of the murine Steel (Sl) and Dominant white spotting (W) genes are essential for the development of melanocyte precursors, germ cells, and hematopoietic cells. The Sl locus encodes stem cell factor (SCF), which is the ligand of c-kit, a receptor tyrosine kinase encoded by the W locus. One allele of the Sl mutation, Sl17H, exhibits minor hematopoietic defects, sterility only in males, and a complete absence of coat pigmentation. The Sl17H gene encodes SCF protein which exhibits an altered cytoplasmic domain due to a splicing defect. In this paper we analyzed the mechanism by which the pigmentation phenotype in Sl17H mutant mice occurs. We show that in embryos homozygous for Sl17H the number of melanocyte precursors is severely reduced on the lateral neural crest migration pathway by e11.5 and can no longer be detected by e13.5 when they would enter the epidermis in wildtype embryos. The reduced number of dispersing melanocyte precursors correlates with a reduction of SCF immunoreactivity in mutant embryos in all tissues examined. Regardless of the reduced amount, functional SCF is present at the cell surface of fibroblasts transfected with Sl17H mutant SCF cDNA. Since SCF immunoreactivity normally accumulates in basolateral compartments of SCF-expressing embryonic epithelial tissues, we analyzed the localization of wildtype and Sl17H mutant SCF protein in transfected epithelial (MDCK) cells in vitro. As expected, wildtype forms of SCF localize to and are secreted from the basolateral compartment. In contrast, mutant forms of SCF, which either lack a membrane anchor or exhibit the Sl17H altered cytoplasmic tail, localize to and are secreted from the apical compartment of the cultured epithelium. We suggest, therefore, that the loss of melanocyte precursors prior to epidermal invasion, and the loss of germ cells from mature testis, can be explained by the inability of Sl17H mutant SCF to be targeted to the basolateral compartment of polarized epithelial keratinocytes and Sertoli cells, respectively. (+info)
Improved methods for immunoassay of mycothiol.
(19/1806)
Improved enzyme-linked immunosorbent assay (ELISA) methods have been developed for the determination of femtomole amounts of mycothiol (MSH), the main low-molecular-weight thiol in mycobacteria. The immunoassays utilize an affinity-purified rabbit polyclonal antibody that is highly specific for the pseudodisaccharide moiety of MSH. MSH was first biotinylated by the thiol-specific reagent 3-(N-maleimidopropionyl)biocytin. The MSH-biotin adduct was then captured with immobilized avidin and detected with anti-MSH antibody (biotin-capture ELISA) or was captured with immobilized anti-MSH antibody and detected with alkaline phosphatase-labelled avidin (MSH-capture ELISA). The MSH-capture ELISA was the most sensitive method, measuring as little as 0.3 fmol of MSH. Methods for biotinylating MSH directly from Mycobacterium spp. are described. The MSH-capture ELISA was tested for the detection of M. avium seeded in human urine or cerebrospinal fluid samples and for screening mutant M. smegmatis strains to detect MSH production. (+info)
Bovine polymorphonuclear neutrophil-mediated phagocytosis and an immunoglobulin G2 protease produced by Porphyromonas levii.
(20/1806)
Acute interdigital phlegmon (AIP) is a commonly occurring anaerobic bacterial infection in cattle. This study examined in vitro the interaction of bovine polymorphonuclear granulocytic neutrophils (PMN) from blood with bacterial species involved in AIP. Polymorphonuclear neutrophils were purified from whole bovine blood, exposed to one of the three putative etiologic agents of AIP and comparatively assessed for phagocytosis using light microscopy. Fusobacterium necrophorum and Prevotella intermedia were effectively phagocytosed by PMN, but Porphyromonas levii was phagocytosed significantly less effectively by PMN. The effect of high titre anti-P. levii bovine serum on antibody-mediated phagocytosis by PMN was also evaluated. High titre serum increased the efficiency of phagocytosis of P. levii by bovine PMN. This was independent of heat labile complement factors. Antibodies specific for P. levii were assessed for protease activity capable of cleaving bovine immunoglobulins (IgG, IgG1, IgG2, and IgM). Partially purified supernatant from broth cultures of P. levii were incubated with biotinylated immunoglobulins (Igs). Samples were taken from times 0 to 72 h and examined using SDS-PAGE followed by Western blot analysis. Streptavidin-alkaline phosphatase and NBT-BCIP were used to visualize the Igs for heavy and light chains as well as lower molecular weight fragments of these glycoproteins. Porphyromonas levii produced an immunoglobulin protease which readily cleaved bovine IgG into fragments, but did not act against IgM. Specifically, the enzyme may be a significant virulence factor as it may act to neutralize the antibodies demonstrated necessary for effective PMN-mediated phagocytosis. (+info)
Congenital insulin resistance associated with a conformational alteration in a conserved beta-sheet in the insulin receptor L1 domain.
(21/1806)
The hormone binding site of members of the insulin receptor family is contained within a highly conserved extracellular region of the receptor. Recent crystallization of the N-terminal region of the binding site revealed two large domains (L1, L2), each organized as a single-stranded right-handed beta-helix, connected by a rod-shaped cysteine-rich domain. Here, we analyze two new naturally occurring mutations in a single beta-sheet within L1, D59G and L62P, that we previously identified in a young woman with classic congenital insulin resistance (type A). Substitution of D59G, a beta-sheet connecting loop residue, caused decreased hormone binding but did not disrupt overall folding, assembly, or movement to the cell surface. In contrast, replacement of the adjacent residue L62P, which is located within the beta-sheet, and positioned in a hormone binding surface, completely disrupted intracellular folding, oligomerization, and trafficking and resulted in aberrant proteolytic degradation. Immunohistochemistry in combination with biosynthetic studies showed that misfolded receptors were retained in an incorrect cellular location and that they colocalized with the resident endoplasmic reticulum chaperone calnexin. This study, together with other mutagenesis data, shows that formation of beta-sheet elements within the L1 beta-helix are critical for the folding of the entire extracellular domain of the receptor and that the hormone contact site is composed in part by residues in this domain. (+info)
Chemical cleavage of the overexpressed mitochondrial F1beta precursor with CNBr: a new strategy to construct an import-competent preprotein.
(22/1806)
We have isolated a soluble import-competent 15 kDa N-terminal fragment of the overexpressed Nicotiana plumbaginifolia F1beta precursor of the ATP synthase (N15pF1beta). The isolation was achieved after chemical cleavage, with CNBr, of the insoluble precursor collected in inclusion bodies, followed by purification of the fragment using ion-exchange chromatography. The purity of the final product was estimated to be more than 99%. N15pF1beta contained a presequence of 54 amino acid residues (except for the N-terminal methionine residue) and 82 N-terminal residues of the mature protein. N15pF1beta was shown to be imported into isolated potato tuber mitochondria and to be processed by the isolated mitochondrial processing peptidase (MPP) integrated into the cytochrome bc1 complex of the respiratory chain. Addition of N15pF1beta at micromolar concentrations resulted in the inhibition of import of F1beta precursor and alternative oxidase precursor, synthesized in vitro, into isolated mitochondria as well as the processing of these precursors catalysed by the isolated MPP-bc1 complex. N15pF1beta conjugated via a biotin link to avidin blocked import sites even after the reisolation of mitochondria and inhibited the import of the mitochondrial precursors, indicating that it can be used as a substrate for the generation of a stable translocation intermediate. Our results present a novel procedure for the production of an N-terminal fragment of the F1beta precursor that contains all information necessary for mitochondrial targeting and processing and that can be used for structural and functional studies of the mitochondrial protein import system. This procedure has a general value because it can be used for the production of chemical quantities of any mitochondrial import substrate and presequence peptide. (+info)
Tumor pretargeting with avidin improves the therapeutic index of biotinylated tumor necrosis factor alpha in mouse models.
(23/1806)
The clinical use of tumor necrosis factor alpha (TNF) as an anticancer drug is limited to local or locoregional administration because of dose-limiting systemic toxicity. We investigated in animal models whether the therapeutic index of systemically administered human or murine TNF can be increased by tumor pretargeting strategies based on the biotin-avidin system. Pretargeting of s.c. mouse WEHI-164 fibrosarcoma and RMA lymphoma genetically engineered to express the Thy 1.1 antigen on the cell membrane was achieved by i.p. injection of a biotinylated anti-Thy 1.1 antibody and avidin. This pretreatment increased the antitumor activity of systemically administered biotin-TNF conjugates by at least 5-fold. In contrast, pretargeting did not increase the toxicity of biotin-TNF, as judged by animal survival and weight loss after treatment. Ex vivo analysis of tumor cells 24 h after treatment showed that biotin-TNF persisted for several hours on the surface of pretargeted tumors, but not when avidin was omitted. The potentiation of the antitumor effects was related primarily to indirect mechanisms, involving a host-mediated response. The results indicate that tumor pretargeting improves the antitumor activity of TNF. Tumor pretargeting with avidin, which is currently used to increase the uptake of radioactive-labeled biotin in patients, could represent a new strategy for improving the therapeutic index of TNF. (+info)
Architecture of the active DNA polymerase delta.proliferating cell nuclear antigen.template-primer complex.
(24/1806)
The relative positions of components of the DNA-dependent DNA polymerase delta (pol delta).proliferating cell nuclear antigen (PCNA).DNA complex were studied. We have shown that pol delta incorporates nucleotides close to a template biotin-streptavidin complex located 5' (downstream) to the replicating complex in the presence or absence of PCNA. PCNA-dependent synthesis catalyzed by pol delta was nearly totally (95%) inhibited by a biotin. streptavidin complex located at the 3'-end of a template with a 15-mer primer (upstream of the replicating complex), but was only partially inhibited with a 19-mer primer. With either primer, PCNA-independent synthesis was not affected by the biotin. streptavidin complex. Quantification of results with primers of varying length suggested that pol delta interacts with between 8 and 10 nucleotides of duplex DNA immediately proximal to the 3'-OH primer terminus. Using UV photocross-linking, we determined that the 125-kDa subunit of pol delta, but not the 50-kDa subunit, interacted with a photosensitive residue of a substrate oligonucleotide. Interaction apparently takes place through the C terminus of p125. Based on these results, we conclude that PCNA is located "behind" pol delta in the polymerization complex during DNA synthesis and that only the large subunit of pol delta (two-subunit form) interacts directly with DNA. A detailed model of the enzymatically active complex is proposed. (+info)