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S K Pierce

Publications and source records attributed to S K Pierce.

At least 55 records · Page 3Linked to original sources
PubMed1995

Generation and characterization of monoclonal antibodies specific for members of the mammalian 70-kDa heat shock protein family.

The 70-kDa heat shock proteins (hsp70) are a highly conserved, abundant, and ubiquitous family of proteins expressed by all organisms from bacteria to humans. It is well established that hsp70 family members function as molecular chaperones and aid in the intracellular folding of newly synthesized or denatured proteins. Current evidence suggests an emerging role for hsp70 family members in immune responses and in clinically important responses to stress and tissue damage. Here we report the generation and characterization of several MAbs to hsp70 family members. Immune responses to this highly conserved family were induced in mice by immunization with synthetic peptides that contain regions of the mouse mitochondrial hsp70 coupled to a potent helper T cell epitope derived from tetanus toxoid. The resulting MAbs include ones specific for the human and mouse mitochondrial hsp70 and others that show cross-reactivity among the family members and recognize the mitochondrial hsp70, the endoplasmic reticulum resident hsp70, Bip/grp78, the constitutively expressed cytosolic hsp70, hsc70, and the heat-induced member, hsp70. Significantly, these MAbs are effective in Western blotting, in immunoprecipitation, and in immunofluorescence, and thus should find applications in the purification and detection of members of this important family.

Amino Acid Sequence↗
PubMed1995

The intracellular assembly of antigenic-peptide-class II complexes.

The immune system employs remarkable strategies to ensure that foreign antigens, from the most complex pathogens to the simplest proteins, are displayed on the surfaces of cells which are targets of T lymphocyte recognition. At the heart of these strategies is the molecular transformation of a soluble protein antigen to a complex of a small peptide containing the antigenic determinant bound to a cell surface Major Histocompatibility Complex class I or class II protein. This process is termed antigen presentation. Progress in a variety of laboratories over the last several years has yielded a wealth of information about the molecular mechanisms underlying antigen presentation, providing potential new approaches to vaccine design. Here we describe recent studies in our laboratory aimed at elucidating the intracellular site in B lymphocytes in which antigenic peptide-class II complexes are assembled for recognition by helper T cells and the regulation of this assembly process. Our results suggest that processed antigen-class II complexes are assembled in a unique compartment in the endocytic route which contains all the necessary cellular and molecular machinery for assembly and that B cells regulate the assembly process in response to external and internal signals.

Antigen-Presenting Cells↗
PubMed1994

Molecular chaperones in the processing and presentation of antigen to helper T cells.

Helper T lymphocytes recognize peptide fragments of antigen bound to Major Histocompatibility Complex (MHC) class II molecules on the surfaces of antigen presenting cells (APC). Antigen processing involves internalization of the antigen into an acidic compartment where the antigen is degraded and the resulting peptide fragments of the antigen are bound to MHC class II molecules and the complexes subsequently displayed at the APC surface. Thus, antigen processing represents a complex, intracellular assembly process which may, like many intracellular protein folding and assembly processes, require the function of molecular chaperones. This contribution focuses on the evidence which suggests that members of the heat shock protein family of molecular chaperones play a role in this pathway.

Animals↗
PubMed1994

Heat shock enhances antigen processing and accelerates the formation of compact class II alpha beta dimers.

The heat shock response is a universal and highly conserved cellular response to stress. Here we describe the effect of heat shock induced by elevated temperatures on the processing and presentation of an exogenous Ag, cytochrome c, to an Ag-specific class II-restricted T cell hybrid. Heat shock markedly enhances processing of Ag entering the B cell either through fluid phase pinocytosis or through receptor-mediated endocytosis. B cells undergoing a stress response require less time to process and present Ag and achieve higher levels of T cell activation as compared with control cells. Augmented processing and presentation requires that the Ag be present during the stress response. Heat shock has no effect on the presenting ability of B cells that had already processed Ag; and heat shock in the absence of Ag has little effect on subsequent processing or presentation of Ag. Heat shock has no measurable effect on the cell surface expression of class II as measured by flow cytometry but markedly accelerates the formation of compact alpha beta dimers in B cells. The class II purified from heat shocked cells is more active in Ag presentation assays in vitro as compared with class II purified from cells grown at 37 degrees C, indicating that class II formed during a stress response is not identical to that formed under normal conditions. The effect of heat shock on B cell Ag processing reported here is likely to be relevant to processing in vivo, which may often proceed under conditions that induce the heat shock response, such as during viral or bacterial infections, inflammation, and fever.

Animals↗
PubMed1994

Evidence for dimers of MHC class II molecules in B lymphocytes and their role in low affinity T cell responses.

The crystallographic structure of the MHC class II molecule showed that the alpha beta heterodimer can itself dimerize to form a four chain (alpha beta)2 complex of 120 kDa. Here we provide evidence for the existence of a 120 kDa (alpha beta)2 complex of the class II I-Ek molecules in mouse B cells. Both a 60 kDa and a 120 kDa form of I-Ek are detected by Western blotting and by immunoprecipitation under conditions in which class II alpha beta heterodimers are stable. The 120 kDa I-Ek complex does not contain Ii and, upon warming, dissociates into free alpha and beta chains. The 120 kDa I-Ek complex is expressed at the cell surface, is active in antigen presentation, and appears to play a significant role in T cell responses to low affinity but not to high affinity antigens, possibly by facilitating cross-linking of the T cell receptors.

Animals↗
PubMed1994

Separation of subcellular compartments containing distinct functional forms of MHC class II.

Antigen processing in B lymphocytes entails initial binding of antigen to the surface Ig and internalization of the antigen into acidic compartments where the antigen is degraded, releasing peptides for binding to major histocompatibility complex class II molecules. Using subcellular fractionation techniques we show that functional, processed antigen-class II complexes capable of activating antigen-specific T cells in vitro are first formed in dense vesicles cosedimenting with lysosomes which are distinct from early endosomes and the bulk of late endosomes. With time, processed antigen-class II complexes appear in vesicles sedimenting with early endosomes and finally cofractionate with plasma membrane. A separate compartment is identified which contains major histocompatibility complex class II receptive to peptide binding but which does not have access to processed antigen in the B cell. These class II molecules are in the so-called "floppy" form in contrast to the class II molecules in the very dense vesicles which are in the "compact" form. These results demonstrate a correlation between the floppy and compact forms of class II molecules and their association with processed antigen and show that floppy and compact forms of class II reside in distinct and physically separable subcellular compartments.

Amino Acid Sequence↗
PubMed1994

PBP74, a new member of the mammalian 70-kDa heat shock protein family, is a mitochondrial protein.

The cloning of a cDNA encoding a new member of the highly conserved mammalian 70-kDa heat shock protein (hsp 70) family termed PBP74 was recently reported. Critical to an understanding of the function of this new hsp 70 is delineating its subcellular localization. Here we use a variety of immunological and biochemical approaches both in vitro and in vivo to demonstrate that PBP74 is imported into and resides in mitochondria. By confocal immunofluorescence microscopy PBP74 is detected in mitochondria, colocalizing with the mitochondrial 60-kDa heat shock protein. To address the inherent problem of serological cross-reactivity among the hsp70 family members, an influenza virus hemagglutinin epitope tag was introduced into the PBP74 cDNA. The epitope-tagged PBP74 protein transiently expressed in L cells localized to mitochondria. Moreover, deletion of the N-terminal 46-amino acid presequence results in a cytosolic localization of the epitope-tagged protein. Cell fractionation studies demonstrated PBP74 in purified mitochondria in a protease-protected location. After coupled transcription-translation the precursor of PBP74 is imported into isolated yeast mitochondria, where it becomes processed to the mature protein. According to a subfractionation of the mitochondria, the imported protein was found to be localized in the matrix space. Import in vitro is time- and temperature-dependent, requires matrix ATP, and is abolished upon depletion of the membrane potential across the mitochondrial inner membrane. Similarly, in mammalian cells PBP74 is synthesized as a pre-protein that requires membrane potential-dependent import into mitochondria for its maturation. Taken together, our data demonstrate that PBP74 is a mammalian mitochondrial hsp70.

Amino Acid Sequence↗
PubMed1994

Antiplatelet and antithrombotic efficacy of DMP 728, a novel platelet GPIIb/IIIa receptor antagonist.

BACKGROUND: Currently used antiplatelet drugs, including aspirin, ticlopidine, and others, are effective against certain but not all of the many endogenous platelet activators. Because of their limited efficacy, a significant number of serious thromboembolic complications still occur, highlighting the need for a more effective therapy. Thus, we have identified a systemically active peptide analogue (DMP 728) of the arginine-glycine-aspartic acid (RGD) recognition sequence that mediates the binding of ligands such as fibrinogen to the platelet glycoprotein (GP) IIb/IIIa receptors. The goals of the present study were to determine the antiplatelet and antithrombotic efficacies of DMP 728 in various arterial thrombosis models. METHODS AND RESULTS: DMP 728 demonstrated antiplatelet efficacy in vitro in inhibiting ADP-induced human platelet aggregation (IC50, 46 +/- 2 nmol/L) and fibrinogen binding to human platelets (IC50, 2.3 +/- 0.8 nmol/L) or purified human GPIIb/IIIa receptors (IC50, 0.6 +/- 0.1 nmol/L). DMP 728 demonstrated high affinity and specificity for human platelet GPIIb/IIIa over other adhesion molecules. In anesthetized mongrel dogs, DMP 728 at 0.001 to 1.0 mg/kg IV produced dose-dependent antiplatelet effects in inhibiting ex vivo platelet aggregation induced by ADP and in prolonging template bleeding time. DMP 728 effects on bleeding time prolongation were more rapidly reversible than those on platelet aggregation inhibition. A maximal antiplatelet effect for DMP 728 was demonstrated at 0.01 mg/kg IV bolus. The antithrombotic efficacy of DMP 728 was examined in vitro and in vivo after IV administration at different doses in various models of arterial thrombosis. In the coronary artery Folts model in dogs, DMP 728 demonstrated maximal antithrombotic efficacy at 0.01 mg/kg IV bolus with an ED50 of 0.005 mg/kg IV bolus in inhibiting cyclic flow reductions. Additionally, DMP 728 demonstrated 100% prevention of primary thrombosis and rethrombosis (P < .01) after treatment with different thrombolytics, including tissue plasminogen activator and streptokinase, in an electrolytically induced femoral artery thrombosis model in dogs. CONCLUSIONS: Acute intravenous DMP 728 administration (0.001 to 1.0 mg/kg) has dose-dependent antiplatelet and antithrombotic effects in different arterial thrombosis models. These data suggest that DMP 728, a low-molecular-weight GPIIb/IIIa receptor antagonist, may have therapeutic potential as an effective antithrombotic agent in coronary and peripheral artery thromboembolic disorders.

Angina, Unstable↗
PubMed1994

Extraction of DNA from mucilaginous tissues of a sea slug (Elysia chlorotica).

Efforts to study the cellular and molecular biology of the symbiotic association between opisthobranch molluscs and algal chloroplasts have been hampered by the copious amounts of mucus produced by the animals. We report for the first time a procedure for isolating total DNA free of contaminating mucilaginous compounds from the mollusc Elysia chlorotica Gould that harbors photosynthetically active chloroplasts from the siphonaceous alga, Vaucheria litorea C. Agardh. This method involves an initial extraction of fresh or freeze-dried Elysia tissue in absolute ethanol and differential processing of the resultant two-phase pellet. Final purification by CsCl-gradient centrifugation produces high molecular weight DNA suitable for molecular analysis.

Animals↗
PubMed1993

Antigen entry into early endosomes is insufficient for MHC class II processing.

Helper T-cell recognition of Ag requires that the Ag be processed and presented by class II-expressing Ag-presenting cells. Processing involves the introduction of Ag into acidic compartments where proteolysis occurs producing peptides that bind to the class II molecules. Although Ag can enter the processing pathway through fluid phase pinocytosis, Ag processing can be made over 1000-fold more efficient by binding the Ag to a variety of Ag-presenting cell surface structures. The increased efficiency in processing is presumably the result of the ability of such structures to deliver the bound Ag to compartments involved in processing. Here we report that Ag bound to the transferrin receptor (TfR), which cycles predominantly through early endosomal compartments, does not enter the processing pathway. We found that cytochrome c(c)covalently coupled to monovalent iron-saturated transferrin (Tf), (c-Tf), is not processed or presented significantly better than unconjugated c, indicating that the majority of cycling TfR does not enter compartments where processing proceeds. The conjugation of Tf to c does not affect its binding to the TfR, as the binding is both saturable and compatible with unmodified Tf. Moreover, c-Tf and unmodified Tf cycle equivalently with a t1/2 of internalization of 3 to 5 min and are released outside the cell with little detectable degradation. Significantly, we found that c-Tf is efficiently processed and presented when the TfR is cross-linked, altering its normal cycling. Indeed, c covalently coupled to polymerized Tf is presented at 1/100th the concentration of c alone. Cross-linking of c-Tf bound to the TfR using c-specific antibodies also results in efficient processing and presentation. Thus, the endosomal compartments through which Tf normally cycles are not sites of processing, whereas compartments into which cross-linked Tf is diverted allow efficient processing and presentation of Ag.

Animals↗
PubMed1993

Peptides of 23 residues or greater are required to stimulate a high affinity class II-restricted T cell response.

Helper T cells recognize fragments of antigen bound to the class II molecules on the surface of antigen-presenting cells. Naturally processed antigenic fragments have been isolated from the class II molecules and shown to be heterogeneous in length, ranging from 13 to 25 residues, and to vary at both the N and C termini. A 15-residue peptide in an extended conformation is predicted to fit in an open peptide-binding cleft of the class II molecules. Thus, the longer peptides observed bound to class II presumably have regions which reside outside the cleft. It is not known if the additional length contributes significantly to T cell activation. We have carried out a systematic analysis of the antigenicity of peptides of increasing length beyond the minimally defined T cell antigenic peptide. Here we show that the full functional activities of peptides representing the major antigenic determinant of the protein antigen, cytochrome c, minimally require that the peptides be 23 amino acids long. The long peptides do not require processing and are presented by purified class II molecules incorporated into synthetic membranes, indicating that such peptides associate directly with class II and require no additional cellular machinery for presentation. We also show that a hybrid peptide, 51 residues in length, containing a 29-residue cytochrome c peptide and a "promiscuous" peptide of tetanus toxoid, is more antigenic than the 23-residue peptide alone and significantly, does not require processing. Thus, the additional peptide length, although not predicted to bind in the peptide-binding groove of the MHC class II molecule, has a significant impact on the ability of the peptides to stimulate T cell responses maximally.

Amino Acid Sequence↗
PubMed1993

Cloning of the gene encoding peptide-binding protein 74 shows that it is a new member of the heat shock protein 70 family.

We have previously described peptide-binding proteins of 72 and 74 kDa (PBP72/74), which have been implicated as playing a role in antigen processing and are serologically related to the 70-kDa heat shock protein (hsp70) family. Here we report the cloning and sequencing of the cDNA encoding PBP74 in mice and in humans, accomplished by using amino acid sequence information obtained from the purified protein. We show that PBP74 is highly homologous to members of the hsp70 family but, significantly, is not identical to any known member of this family. Inspection of the cDNA nucleotide sequence indicates that it encodes a 46-residue N-terminal peptide which is not present in the mature protein. Transcription and translation in vitro of the PBP74 cDNA verified that it encodes a form of PBP74 which is larger than the mature protein. The presequence does not conform to known motifs for organelle-targeting sequences, and at present, its function is not known. By confocal microscopy, PBP74 was localized to cytoplasmic vesicles but not to the nucleus, mitochondria, or plasma membrane by using antibodies specific for the N-terminal 16 residues of PBP74. By RNA filter hybridization analysis, PBP74 mRNAs are detected in all cell types tested. Exposure of cells to heat shock does not result in an increase in the mRNA levels of PBP74, unlike the dramatic increase observed for the stress-inducible hsp70 mRNA. Thus, PBP74 appears to be a constitutive, new member of the hsp70 family.

Amino Acid Sequence↗
PubMed1993

Heat shock proteins in immune responses.

The assembly of functional MHC-I and MHC-II complexes is rapid, specific, and efficient. Recent advances in the area of antigen processing and presentation suggest that members of the heat shock protein (hsp) family facilitate the molecular events in the assembly process. Furthermore, hsps themselves have been shown to be the dominant antigens of a variety of pathogens as well as serving as targets of the immune system in healthy individuals. Hsps may play a fundamental role in immune responses, serving as an early warning to the host's immune system during the onset of infection.

Animals↗
PubMed1992

Monoclonal antibodies to the nonpeptide angiotensin II receptor antagonist, losartan.

Two murine monoclonal antibodies were produced to losartan (DuP 753), a nonpeptide angiotensin II receptor antagonist. Using a solid phase competitive enzyme-linked immunosorbent assay (ELISA), each antibody was examined for its ability to bind to a set of losartan analogs that differ structurally in varying degrees. Both antibodies distinguished fine structural changes in the analogs, particularly at the R5 position of the imidazole ring. No cross-reactivity towards either antibody was observed with the natural ligand angiotensin II, the peptide antagonist saralasin, or the AT2 selective nonpeptide antagonist PD123177.

Angiotensin II↗
PubMed1992

Design and immunological properties of topographic immunogenic determinants of a protein antigen (LDH-C4) as vaccines.

Antibodies elicited by immunization with short peptides containing antigenic determinants have been shown, in general, to bind with greatly reduced affinity to the corresponding region in the native proteins. Thus, contiguous linear peptides have not proven to be effective immunogens in generating high affinity neutralizing or protective antibodies and consequently appear to be poor prospects for vaccines. The molecular basis for such reduced reactivity is clear from the crystal structure determination of antibody Fabs bound to protein antigens, which showed the complementarity between interfaces to be lock-and-key-like and extending over a large area (750 A2) involving discontinuous segments of the polypeptide chain. Thus, small perturbations in the secondary and tertiary structure of the antigen have profound effects on the fit of the antigen and its corresponding antibody. Because short peptides are unlikely to assume any particular conformation in solution, the fit is likely to be poor. New strategies are therefore required to produce conformationally stable peptides that mimic the critical structural features of the protein antigenic site. Here we show that a putative topographic determinant of the testis-specific isozyme of lactate dehydrogenase C4 (LDH-C4), designed and synthesized to adopt a well defined alpha-helical secondary and tertiary structure (four-helix bundle motif) in aqueous solutions, is highly immunogenic in both rabbits and mice, inducing IgG antibodies that bind to native LDH-C4. This engineered conformational 40-residue peptide is considerably more effective in inducing antibodies, as compared with the corresponding linear peptide. The antibody response is obtained without coupling the peptide to a carrier protein, suggesting that the peptide contains a T-cell antigenic determinant. The strategy described here to produce a conformationally stable peptide that mimics the native structure may have general applications in vaccine design.

Amino Acid Sequence↗
PubMed1992

Biochemical evidence for the rapid assembly and disassembly of processed antigen-major histocompatibility complex class II complexes in acidic vesicles of B cells.

Helper T cell recognition of antigen requires that it be processed within antigen-presenting cells (APC) to peptide fragments that subsequently bind to major histocompatibility complex (MHC) class II molecules and are displayed on the APC surface. Heretofore, processed antigen-MHC class II complexes have been detected by functional assays, measuring the activation of specific T cells. We now report direct, biochemical evidence for the assembly of processed antigen-MHC class II complexes within splenic B cells as APC. The I-Ek MHC class II molecules were immunoprecipitated from B cells that had processed the model protein antigen cytochrome c radiolabeled across its entire length by reductive methylation of lysine residues and covalently coupled to Ig-specific antibodies, allowing internalization after binding to surface Ig. Our previous studies showed that I-Ek immunoaffinity purified from B cells that had processed cytochrome c contains functional processed antigen--MHC class II complexes and that approximately 0.2% of the I-Ek molecules are specifically associated with one of two predominant processed antigenic fragments. Here we show that these complexes are rapidly assembled, within 30-60 min after antigen binding to surface Ig on splenic B cells. Maximal numbers of complexes are assembled by 2 h in a process that is sensitive to acidic vesicle inhibitors but not to inhibitors of protein synthesis. The processed antigen-I-Ek complexes have a relatively short half-life of 2-4 h and are disassembled or degraded within 8 h after antigen is first internalized. The disassembly or degradation of the processed antigen-I-Ek complexes requires acidic vesicle function, and in the presence of an acidic vesicle inhibitor the complexes are long lived. Thus, using a biochemical assay to monitor processed antigen-I-Ek complexes, we find that, in B cells, processed antigen is relatively rapidly associated in acidic vesicles with preexisting MHC class II molecules, and the complexes are disassembled 4-6 h later in processes that also require acid vesicle function.

Acids↗