Biomedical subjects

P M Theiss

Publications and source records attributed to P M Theiss.

5 recordsLinked to original sources

PubMed1998

Structural basis of agonist-induced desensitization and sequestration of the P2Y2 nucleotide receptor. Consequences of truncation of the C terminus.

Molecular determinants of P2Y2 receptor desensitization and sequestration have been investigated. Wild-type P2Y2 receptors and a series of five C-terminal truncation mutants of the receptor were epitope-tagged and stably expressed in 1321N1 cells. These constructs were used to assess the importance of the intracellular C terminus on 1) UTP-stimulated increases in intracellular calcium concentration, 2) homologous desensitization of the receptor, and 3) agonist-induced decreases in cell-surface density (receptor sequestration) of epitope-tagged receptors using fluorescence-activated cell sorting. The potency and efficacy of UTP were similar for the wild-type and all mutant P2Y2 receptors. Truncation of 18 or more amino acids from the C terminus increased by approximately 30-fold the concentration of UTP necessary to desensitize the receptor. Both the rate and magnitude of UTP-induced receptor sequestration were decreased with progressively larger truncations of the C terminus. Furthermore, the recovery from sequestration was slower for the most extensively truncated receptor. Complete desensitization was obtained with >50% of the original receptor complement remaining on the cell surface. Protein kinase C activation, which desensitizes the P2Y2 receptor, had no effect on sequestration, consistent with the ideas that desensitization and sequestration are discrete events and that agonist occupancy is required for receptor sequestration.

Amino Acid Sequence↗

PubMed1993

Isolation and characterization of gamma delta T lymphocyte cell lines from Sinclair swine peripheral blood.

Sinclair miniature swine represent a breed of miniature swine which display a significant incidence of inheritable melanoma which undergo a developmentally regulated spontaneous regression. In an attempt to characterize the host cellular immune response to the melanoma, lymphocyte cell lines have been generated from peripheral blood and designated as peripheral blood lymphocyte cell lines (PBLCLs). The cell lines were expanded in vitro without the addition of exogenous mediators, cloned by limiting dilution, and characterized by flow microfluorimetry, Western, and Northern blot analysis. The cell lines were shown to be CD2-, CD4-, CD8-, and slg-, a phenotype consistent with a null cell population described in swine. The null cell population in swine has been reported to consist of a subpopulation of cells which express the gamma delta T cell receptor (TCR) heterodimer, swine gamma delta T lymphocytes. The PBLCLs were further analyzed by flow microfluorimetry and observed to express the IL-2R, swine MHC Class II antigens, and the endothelial lymphocyte adhesion marker (CD44), which can function as a homing receptor for the skin. In addition, the PBLCLs were observed to express the antigen which is recognized by mAb 86D, an antibody that has been reported to recognize an external epitope on a subset of gamma delta TCR bearing swine T lymphocytes. Western blot analysis of Triton X-114 phase fractions of a PBLCL revealed a protein recognized by the W6 antibody, an antibody which recognizes a conserved region of the C delta chain. Furthermore, Southern and Northern blot analysis indicated that the PBLCL have rearranged the TCR gamma chain gene and express mRNA from the TCR gamma and delta chain genes prior to and following treatment with ionomycin or Concanavalin A. Therefore, the data indicates that the PBLCLs represent swine gamma delta T lymphocyte cell lines which should enable us to enhance our understanding of the role of gamma delta T lymphocytes in the porcine immune system.

Animals↗

PubMed1993

Differential protein expression and surface presentation generate high-frequency antigenic variation in Mycoplasma fermentans.

Mycoplasma fermentans, a wall-less prokaryote, is currently under investigation as a potential human pathogen. Recently, several surface lipoproteins have been shown to vary in expression between M. fermentans strains. Using specific antibodies to these lipoproteins, we investigated the extent and nature of antigenic variation within this species. Immunoscreening of type strain PG18 agar-grown colonies revealed marked heterogeneity in expression of distinct surface lipoproteins. Subsequent isolation and propagation of clonal isolates established isogenic lineages which displayed high-frequency (10(-2) to 10(-5) per generation) antigenic phase variation. [35S]cysteine-labeled protein profiles and Western immunoblots of phase-variant clones showed that several distinct integral membrane proteins undergo noncoordinate variation in expression. In addition to differential expression of epitope-bearing lipoproteins, differential accessibility of epitopes to antibodies was also documented as a mechanism generating surface phenotypic variation. Examination of one strain-variant antigen showed high-frequency phase variation to underlie previously observed antigenic differences between strains of this species. Thus, M. fermentans has a complex system capable of creating rapid changes in surface mosaics. This may profoundly affect mycoplasma-host interactions and may limit the methods by which populations of M. fermentans may be studied in vivo.

Antibodies, Monoclonal↗

PubMed1993

Antigenic variation in Mycoplasma hyorhinis: increased repertoire of variable lipoproteins expanding surface diversity and structural complexity.

VlpE is characterized as a new member in a family of variable surface lipoproteins (Vlps) of Mycoplasma hyorhinis. VlpE shows phenotypic variation in expression and size within isogenic lineages of some strains but is absent from lineages of other strains that express only three previously known Vlps. Expression of four Vlps in some cells further indicates the presence and usage of an expanded reservoir of Vlp coding sequences, which greatly increases the capacity for surface diversification.

Antibodies, Monoclonal↗

PubMed1993

A family of strain-variant surface lipoproteins of Mycoplasma fermentans.

The wall-less procaryote Mycoplasma fermentans is currently being examined as an agent potentially associated with human disease, including infectious processes affecting immunocompromised individuals. To delineate and understand the interactions of M. fermentans with its host, specific membrane surface components were characterized as markers for detecting the organism and for assessing heterogeneity in antigenic surface architecture within this mycoplasma species. Detergent phase fractionation of metabolically labeled organisms of type strain PG18 identified a family of prominent integral membrane proteins; several of these labeled with 35S-cysteine and 3H-palmitate, which are characteristics of procaryotic lipoproteins. Specific monoclonal and polyclonal antibodies raised to strain PG18 components further distinguished seven of these membrane proteins, which were localized on the organism's surface by monitoring their selective susceptibility during trypsin treatment of intact cells. With these antibodies, Western immunoblot profiles of surface membrane antigens expressed on strain PG18 were compared with those expressed on the recently identified Incognitus strain of M. fermentans, as well as with several other human and animal mycoplasma species. While the antibodies were specific for M. fermentans, marked differences were observed between the strains in the size of one surface lipoprotein and in the apparent levels of several antigens expressed in the cultured populations analyzed. Some monoclonal antibodies to strain PG18 and a previously described monoclonal antibody to strain Incognitus showed apparent selectivity for the strain used for immunization. Monoclonal antibodies developed here recognize stable epitopes defining a family of surface lipoproteins and provide critical tools to determine the basis of surface variation in this mycoplasma species and to assess the location and antigenic phenotypes of organisms in the human host.

Animals↗