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S K Pierce

Publications and source records attributed to S K Pierce.

At least 73 records · Page 4Linked to original sources
PubMed1992

Virus infection blocks the processing and presentation of exogenous antigen with the major histocompatibility complex class II molecules.

Helper T cell recognition of antigen requires that antigen be processed and presented by class II expressing antigen-presenting cells (APC). Many antigens presented by the immune system are part of infectious organisms, for example, bacteria and viruses, which themselves may affect APC function. Here we show that infection of B cell lines as APC with viruses of two different families, namely, influenza A or vaccinia, completely block processing and presentation of an exogenous globular protein antigen pigeon cytochrome c. The block appears to be primarily within the processing pathway, as virus infection has little effect on the presentation of an antigenic peptide of pigeon cytochrome c which does not require processing. It is likely that several steps in the processing pathway are affected. Only live infectious virus, not UV-inactivated virus blocks APC function, indicating that there is no competition of viral particles with cytochrome c for the class II processing machinery. As compared to uninfected cells, virus-infected cells internalize less antigen bound to surface Ig but degrade a similar portion of that which enters the cell. Virus infection results in reduced protein synthesis in APC which may also be a factor in decreasing APC function. Significantly, we show that the processing of a high affinity evolutionary variant of cytochrome c from Drosophila melanogaster is reduced less by virus infection as compared to c. Such knowledge may guide the selection of antigenic epitopes in vaccine design.

Animals↗
PubMed1992

Antigen presentation for T cell interleukin-2 secretion is a late acquisition of neonatal B cells.

The ability of B lymphocytes to process and present antigen to helper T cells is essential to initiate T cell-B cell interactions in humoral immune responses. Here we describe the developmental acquisition of the antigen-presenting function of B cells as measured by the ability of B cells to stimulate a T cell hybrid to interleukin (IL)-2 secretion. Neonatal splenic B cells are not adult-like in their ability to process and present the model protein antigen pigeon cytochrome (Pc), which enters the B cell through fluid-phase pinocytosis, until 21 to 28 days of life. The ability of neonatal B cells to process and present antigen which enters the cell bound to surface Ig is not adult-like until 28 days of age. When neonatal B cells acquire antigen-presenting cell (APC) function, surface IgM facilitates antigen processing. The delayed acquisition of APC function cannot be accounted for solely by a deficiency in major histocompatibility complex MHC class II, ICAM-1, or LFA-1 as neonatal B cells express adult levels of these molecules by 7-14 days after birth. Moreover, the ability of neonatal B cells to present a peptide fragment of Pc which does not require processing is adult like by day 14. Furthermore, neonatal B cells are capable of binding, internalizing and degrading radiolabeled antigen, suggesting a more subtle level of regulation. In contrast to neonatal B cells, immature B cells in the adult bone marrow and adult B cells undergoing antigen-driven differentiation to memory B cells, as defined by the loss of the J11D marker, are competent to process and present antigen resulting in T cell IL-2 secretion. Thus, developing B cell subpopulations in the adult and in the neonate can be distinguished. Only neonatal B cells are deficient in their ability to stimulate T cells to IL-2 production.

Animals↗
PubMed1992

Ionomycin produces an improved volume recovery by an increased efflux of taurine from hypoosmotically stressed molluscan red blood cells.

Nucleated erythrocytes of the blood clam, Noetia ponderosa, recover cell volume after a hypoosmotic stress by an efflux of K+, Cl- and taurine. When the cells are exposed to ionomycin followed by hypoosmotic stress, swelling is less and volume recovery is both faster and more complete than in control cells without the ionophore. The improved volume recovery is caused by a large increase in the efflux of taurine. The taurine efflux is altered by changing Ca2+ concentrations in the presence of the ionophore. Potassium regulation by the osmotically stressed erythrocytes is also increased in the presence of ionomycin, but only by a small amount, perhaps accounting for the initial decrease in swelling. Variation of Ca2+ in the presence of ionomycin without osmotic stress produces no change in the regulation of either osmolyte. These results indicate that both the osmotic stress and an increase in [Ca2+]i are required for the permeability change that produces taurine efflux.

Animals↗
PubMed1992

A case for chaperones in antigen processing.

The assembly of peptide-MHC-class-II molecule complexes by antigen-presenting cells is far more efficient than would be predicted from studies of peptide binding to purified MHC class II molecules in vitro. One possible explanation for this discrepancy is that proteins in the antigen-presenting cell facilitate the assembly process. Here, Diane DeNagel and Susan Pierce present the case for involvement of members of the chaperone/heat shock protein 70 family in the intracellular assembly of processed-antigen-MHC-class-II-molecule complexes.

Amino Acid Sequence↗
PubMed1992

The ionic basis of the hypo-osmotic depolarization in neurons from the opisthobranch mollusc Elysia chlorotica.

The resting potential of identified cells (Parker cells) in the abdominal ganglion of Elysia chlorotica (Gould) depolarizes by about 30 mV in response to a 50% reduction in osmolality and returns to the original potential in 20 min. Cell volume recovery requires approximately 2 h. Thus, recovery of the resting potential is not dependent on recovery of cell volume. The hypo-osmotic depolarization persists following inhibition of the electrogenic Na+/K(+)-ATPase with ouabain, and the levels of extracellular K+ and Cl- have little effect on the magnitude of the depolarization, while decreasing extracellular Na+ concentration produces a depolarization of only 10 mV. This suggests that the hypo-osmotic depolarization in Parker cells results mostly from increased relative permeability to Na+. Following transfer from 920 to 460 mosmol kg-1, Na+, Cl- and proline betaine leave the cells while intracellular K+ is conserved. Loss of intracellular Na+ and conservation of intracellular K+ are dependent on active transport by the Na+/K(+)-ATPase. Na+ and proline betaine leave the cells with a time course that is much longer than that of the hypo-osmotic depolarization. Unlike the other solutes, most of the reduction in intracellular Cl- concentration occurs coincidentally with the hypo-osmotic depolarization. However, unlike the hypo-osmotic depolarization, bulk loss of Cl- does not require the reduction in osmolality, only the reduction in extracellular ion concentrations. There is no apparent relationship between membrane depolarization and the regulation of intracellular osmolytes in Elysia neurons following hypo-osmotic stress.

Animals↗
PubMed1991

Characterization of naturally processed antigen bound to major histocompatibility complex class II molecules.

Helper T lymphocytes recognize peptide fragments of antigen bound to major histocompatibility complex (MHC) class II molecules presented on the surface of antigen-presenting cells (APCs). Previous studies showed that the MHC class II, I-Ek molecules purified from APCs that had processed Drosophila melanogaster cytochrome c (DMc) contained functional, processed antigen-I-Ek complexes. This was demonstrated by the ability of purified I-Ek, incorporated into liposomes, to stimulate DMc-specific T cells in the absence of any additional antigen. Here we describe the isolation and characterization of the processed antigen bound to I-Ek. This was accomplished using DMc radiolabeled across its entire length by reductive methylation of its lysine residues, allowing an analysis of the totality of processed antigen bound to MHC class II molecules. After processing, only about 0.2% of the APC I-Ek molecules contained processed DMc (approximately 800 per cell), yet these were sufficient to stimulate specific T cells. The DMc peptides isolated from the I-Ek molecules showed only two predominant radioactive peaks as analyzed by reverse-phase chromatography. Less processed antigen was bound to purified I-Ak molecules, and these peptides were distinct from those bound to I-Ek. The association of processed DMc with the I-Ek and I-Ak molecules appears highly specific in that no radiolabeled peptides were isolated from purified MHC class I molecules, Kk and Dk, or from the B-cell differentiation antigen B220. The majority of processed antigen-I-Ek complexes migrated more slowly than the majority of the I-Ek protein as analyzed by SDS/PAGE under nonreducing conditions without heating of the sample. This form of I-Ek may be analogous to the earlier described "floppy" form of MHC class II molecules [Dormair, K., Rothenhausler, B. & McConnell, H. M. (1990) Cold Spring Harbor Symp. Quant. Biol. 54, 409-416]. Since newly processed antigen binds nearly exclusively to this slow-migrating form, it may be of functional significance.

Amino Acid Sequence↗
PubMed1991

Cellular and subcellular distribution of PBP72/74, a peptide-binding protein that plays a role in antigen processing.

A 72/74-kDa peptide binding protein (PBP72/74) was previously described which plays a role in the processing and/or presentation of Ag, possibly by facilitating the association of processed Ag with the MHC class II molecules. PBP72/74 was recently shown to be related to the 70-kDa family of heat shock proteins (hsp70), whose members show the general characteristic of binding to denatured or inappropriately folded proteins. Here we describe the cellular and subcellular distribution of PBP72/74. By flow cytometry with PBP72/74-specific rabbit antisera, PBP72/74 is detected on the surfaces of mouse Ig+ B cells and MAC-1+ macrophages. PBP72/74 74 was not detected on the surfaces of Thy-1+ T cells or NK1.1+ NK cells. The cell surface expression of PBP72/74 does not require MHC class II expression. Indeed, the Ia- variant B cell lymphoma cell line, M12.C3, expresses PBP72/74 at levels equivalent to that of the Ia+ parent cell line, M12.4.1, from which it was derived. Furthermore, the fibroblast L cell line, DAP.3, shows no cell surface expression of PBP72/74, nor do DAP.3 lines transfected with and expressing genes encoding the alpha- and beta-chain of the I-Ad and I-Ed molecules. Moreover, treatment of B cells with either IL-4 or LPS, which increases Ia expression severalfold, does not affect PBP72/74 expression. Thus, PBP72/74 cell surface expression appears to be a property of B cells and macrophages, independent of Ia expression. In addition, the B cell surface expression of PBP72/74 is not altered by stress in the form of heat shock. Thus, PBP72/74 appears to be a constitutive noninducible member of the hsp70 family. By immunoelectron microscopy, PBP72/74 is detected in approximately 36% of early endocytic vesicles into which surface Ig is internalized after binding to anti-Ig antibodies. This compartment was previously shown to contain class II en route to the cell surface associated with invariant chain and the proteases cathepsin B and D and is suggested to be a subcellular site of antigen processing. PBP72/74 is also found associated with the plasma membrane, endoplasmic reticulum, and membranes proximal to the Golgi stacks. The cellular and subcellular distribution of PBP72/74 is consistent with its playing a role in the processing of presentation of Ag with the MHC class II molecules.

Animals↗
PubMed1991

Specific protein phosphorylation occurs in molluscan red blood cell ghosts in response to hypoosmotic stress.

The regulation of cellular volume upon exposure to hypoosmotic stress is accomplished by specific plasma membrane permeability changes that allow the efflux of certain intracellular solutes (osmolytes). The mechanism of this membrane permeability regulation is not understood; however, previous data implicate Ca2+ as an important component in the response. The regulation of protein phosphorylation is a pervasive aspect of cellular physiology that is often Ca2+ dependent. Therefore, we tested for osmotically induced protein phosphorylation as a possible mechanism by which Ca2+ may mediate osmotically dependent osmolyte efflux. We have found a rapid increase in 32Pi incorporation into two proteins in clam blood cell ghosts after exposure of the intact cells to a hypoosmotic medium. The osmotic component of the stress, not the ionic dilution, was the stimulus for the phosphorylations. The osmotically induced phosphorylation of both proteins was significantly inhibited when Ca2+ was omitted from the medium, or by the calmodulin antagonist, chlorpromazine. These results correlate temporally with cell volume recovery and osmolyte (specifically free amino acid) efflux. The two proteins that become phosphorylated in response to hypoosmotic stress may be involved in the regulation of plasma membrane permeability to organic solutes, and thus, contribute to hypoosmotic cell volume regulation.

Analysis of Variance↗
PubMed1991

Heat shock proteins implicated in antigen processing and presentation.

The recognition of antigen by helper T lymphocytes requires that the antigen be processed and presented by a cell expressing the Major Histocompatibility Complex (MHC) class II molecules. Antigen is taken into an acidic intracellular compartment where it is proteolytically degraded, releasing peptide fragments which become displayed on the cell surface in association with the MHC class II molecules. At present, little is known of the discrete steps in this process or the molecular mechanisms underlying the assembly of the antigenic peptide-MHC class II complex. Although antigenic peptides have been shown to bind directly to the MHC class II molecules, the unusual characteristics of this binding, namely extraordinarily slow association and dissociation rates, make it likely that the binding of peptide to MHC is facilitated within the cell by unknown mechanisms. The functions recently ascribed to several members of the heat shock proteins, in particular their binding to newly synthesized, denatured or inappropriately folded proteins, make them attractive candidates to play a role in antigen processing. In searching for proteins which might facilitate the binding of peptides to the MHC class II molecules, we isolated a peptide binding protein of 72/74 kDa Mr (PBP72/74). Antibodies raised to PBP72/74 block antigen processing and/or presentation, indicating a role for PBP72/74 in this process. Recent studies show that PBP72/74 is serologically related to the heat shock protein (hsp) family and that PBP72/74 shares a second characteristic of this family, namely ATP binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Purification and characterization of recombinant plasminogen activator inhibitor-1 from Escherichia coli.

A recombinant form of plasminogen activator inhibitor-1 (rPAI-1) has been purified from lysates of pCE1200, a bacterial expression vector containing the full length PAI-1 gene, by utilizing sequential anion exchange and cation exchange chromatography on Q-Sepharose and S-Sepharose columns. Approximately 140 mg of rPAI-1, estimated at 98% purity on the basis of analytical high performance liquid chromatography, could be obtained from 200 g wet weight of cells. The purified protein exhibited a single Coomassie Blue-stainable band at the region of Mr = 42,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an NH2-terminal amino acid sequence consistent with the expected translation product of the pCE1200 PAI-1 insert. The rPAI-1 rapidly inhibited single- and two-chain tissue plasminogen activators, as well as urokinase, with apparent second order rate constants in the range of 2-5 x 10(7) M-1 s-1. A specific activity measurement of 250,000 units/mg was calculated for the rPAI-1 based on its ability to inhibit the enzymatic activity of a single-chain tissue plasminogen activator. Stability studies showed that the activity of the rPAI-1 was very stable when stored at temperatures of 25 degrees C or lower, but decayed within hours when stored at 37 degrees C. Sodium dodecyl sulfate treatment, which partially activates the latent form of natural PAI-1, inactivated rPAI-1. These results show that the purified rPAI-1 produced from pCE1200 displays many of the properties associated with the biologically active form of natural PAI-1.

Amino Acid Sequence↗
PubMed1990

Characteristics of peptides which compete for presented antigen-binding sites on antigen-presenting cells.

The T cell recognition of globular protein antigens requires the cell surface presentation of the protein by Ia-expressing antigen-presenting cells (APC). The mechanisms by which APC function remain to be elucidated. To gain a better understanding of association of antigen with APC surfaces, a large panel of peptides of diverse physicochemical properties was assayed for the ability to compete with presented antigen for binding sites on the APC surface. Competition was measured by the ability of a peptide to block the I-Ek-restricted T cell response to pigeon cytochrome c (Pc) as presented by APC. The panel assayed included overlapping peptides representing the entire length of sperm whale myoglobin and the alpha and beta chains of human adult hemoglobin as well as synthetic conformational peptides of lactate dehydrogenase C4 exhibiting stable secondary, alpha-helical structures. The results presented here show that several peptides of this group compete with the presented form of Pc for binding sites on the APC. However, there is no single biochemical property or amino acid sequence algorithm which predicts the blocking ability. The peptides which compete with presented Pc are not predicted to assume the amphipathic alpha-helical conformation hypothesized by De Lisi and Berzofsky (Proc. Natl. Acad. Sci. USA 1986. 82: 7048) for T cell antigenic peptides. However, peptides designed and synthesized to adopt a stable alpha-helical secondary structure show more potent blocking activity than the corresponding linear peptides, suggesting that the secondary structure may indeed be a contributing factor in the ability of presented antigenic peptides to be bound by the APC. The results with the myoglobin and hemoglobin peptides show no connection between any particular secondary structure of the peptide in the native proteins and the ability of the peptides to block presentation. Further, there is no correlation between the major histocompatibility complex restriction of the competing peptides and their ability to block the I-Ek-restricted Pc-specific T cell response. This suggests that antigen presented by the APC may be bound to APC structures other than Ia prior to association with Ia. Such additional binding sites for presented antigen may be necessary to facilitate association with Ia.

Amino Acid Sequence↗
PubMed1990

Isolation of a functional antigen-Ia complex.

The helper T-cell recognition of globular protein antigens requires that the antigen be processed and presented by an I-region associated (Ia)-expressing antigen-presenting cell (APC). Processing involves the uptake of antigen into an intracellular, proteolytic, acidic compartment; release of peptide fragments containing the T-cell antigenic determinant; association of these peptides with Ia; and presentation of these complexes on the cell surface for recognition by the specific T cells. The molecular mechanisms by which processed antigenic peptides associate with Ia within the APC are poorly understood. To date, functional antigen-Ia complexes have not been isolated from cells that have processed native antigens, although the resolution of the structure of a major histocompatibility complex (MHC) class I protein indicates that peptide is bound in a groove between two alpha-helical regions of the molecule and synthetic peptides have been demonstrated to bind purified MHC both in detergent solution and incorporated into planar membranes, where the MHC-peptide complexes function to activate specific T cells. Here we demonstrate that Ia purified from APCs that have processed the native globular protein antigen cytochrome c, when incorporated into lipid membranes, stimulates cytochrome c-specific T cells in the absence of exogenous antigenic peptide. The T-cell response to Ia purified from cytochrome c-pulsed APCs shows the same MHC restriction and antigen fine specificity as the response to antigen-pulsed APCs. Indeed, T-cell recognition of pigeon cytochrome c (Pc) shows a well documented high-affinity heteroclitic cross-reaction to insect cytochromes c-namely, those of Drosophila melanogaster (DMc) and tobacco hornworm moth (THMc). The same heteroclitic response is observed when purified Ia from unpulsed cells, incorporated into lipid membranes, is used to present antigenic peptides of Pc and of THMc. Significantly, Ia purified from APCs that have processed DMc is approximately 50-fold more active in stimulating specific T cells compared to Ia purified from APCs that have processed Pc. The peptide-Ia complex isolated here may provide the necessary material for analysis of the physiochemical properties of the processed form of the antigen that is produced by the APC and associates with Ia.

Animals↗
PubMed1989

A peptide binding protein having a role in antigen presentation is a member of the HSP70 heat shock family.

The T cell recognition of globular protein antigens requires the processing and presentation of the antigen by Ia-expressing APCs. Processing is believed to involve the uptake of antigen into an acidic compartment where proteolysis occurs. The resulting peptides containing the T cell antigenic determinant are associated with Ia and presented at the cell surface to the specific T cells. The mechanisms by which antigenic peptides become associated with Ia is not known. We previously described a peptide binding protein of 72/74 x 10(3) Mr (PBP72/74) that plays a role in antigen presentation as shown by the ability of an antiserum raised in rabbits to affinity-purified PBP72/74 to block presentation of cytochrome c to a cytochrome c-specific T cell hybrid. Here we show that PBP72/74 is recognized by mAbs specific for members of the HSP70 family of proteins. In Western blots PBP72/74 is bound by mAb 7.10, specific for an evolutionarily conserved epitope of HSP proteins and by mAb N27, specific for both the constitutively expressed and inducible 72/73 x 10(3) Mr HSP70 proteins. In addition, PBP72/74 shares a second common feature of the HSP proteins, that of binding to ATP. Indeed, ATP causes the release of PBP72/74 from binding to a peptide fragment of cytochrome c (Pc 81-104) and PBP72/74 can be eluted from ATP columns by Pc 81-104. Finally, a portion of PBP72/74 is shown to be present on B cell surfaces by immunofluorescence staining. Thus, it appears that characteristics of the heat shock proteins are shared by a protein playing a role in antigen presentation, suggesting some commonality in function.

Adenosine Triphosphate↗
PubMed1989

Free amino acids and cell volume regulation in the euryhaline ciliate Paramecium calkinsi.

Paramecium calkinsi was isolated from a tidal marsh in which the salinity fluctuated widely on a daily basis. In the laboratory, this ciliate survived for days in sea water ranging in osmotic strength from 10 to 2,000 mOsm and divided in nutritive media of 1,000 mOsm or less. When transferred from 750 to 250 mOsm, cells swelled but regained 78% of the original volume within 60 min and the original volume within 1 day. Cells acclimated to 250 mOsm and transferred to 750 mOsm shrank, regained 40% of the original volume in 60 min, and regained little more volume during the next 24 hr. Free amino acids (FAA), principally proline and alanine, are osmolytes in P. calkinsi. In cells that have been acclimated for more than 1 month, Pro is undetectable at 10 mOsm but at 250 mOsm is present in substantial amounts and is still higher at 750 mOsm. Ala is found in cells at all three salinities and increases dramatically with increasing salinity. A complex pattern of amino acid changes occurs during the 4 hr following a transfer from 250 to 750 mOsm, resulting in a marked increase in Ala but no change in Pro. Thus the metabolic changes that lead to the increased FAA levels of acclimated cells are apparently long-term and complex. After transfer of cells from 750 to 250 mOsm there is a rapid and selective loss of Pro and Ala from the cells to the medium.

Amino Acids↗
PubMed1989

Evidence of calmodulin involvement in cell volume recovery following hypo-osmotic stress.

An influx of Ca2+ into red blood cells of the bivalve mollusc Noetia ponderosa occurs immediately following a hypo-osmotic stress. The volume recovery response to the stress is dependent upon [Ca2+]o and is inhibited by phenothiazines. The action of these drugs is on the amino acid regulation portion of the recovery rather than on the ionic portion. Since the phenothiazines are non-specific in action, we have conducted several experiments to decide the site of phenothiazine action on the volume recovery response. The sulfoxide derivatives of both chlorpromazine and trifluoperazine have no effect on volume regulation at the same dose where the parent compound inhibits. At 50-100 times the concentration of the parent compound, the derivatives block both volume regulation and taurine efflux. The phorbol ester, TPA, an activator of protein kinase C, alters the volume recovery, but does so by affecting K+ rather than amino acid regulation. The only phenothiazine target that we can not rule out is calmodulin, which we also demonstrate to be present in the clam red cells. Thus, the data presented suggest that calmodulin is involved in the amino acid regulatory portions of the volume recovery in response to hypo-osmotic swelling.

Animals↗