Neuroendocrine peptide hormone regulation of immunity.
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Amino Acid Sequence↗
Biomedical subjects
H M Johnson
Publications and source records attributed to H M Johnson.
Antiproliferative activity of a pregnancy recognition hormone, ovine trophoblast protein-1.
Ovine trophoblast protein-1 (oTP-1) is the alpha-interferon (IFN alpha) variant, secreted by conceptuses and referred to as type I trophoblast interferon, that is responsible for maternal recognition of pregnancy in sheep. We have previously shown that oTP-1 is as potent an antiviral agent as any known IFN. IFNs also possess anticellular activity and are, in fact, used in cancer therapy and have been found to be effective in the treatment of cancer such as myelogenous and hairy cell leukemias. A significant problem with the currently used IFNs is the undesirable side effect of toxicity at high concentrations. In this study, we examined the anticellular activity and toxicity of oTP-1. It inhibited proliferation but did not exhibit toxicity at high concentrations, unlike known IFN alpha S. In an anticellular assay using colony formation of both the human amnionic line, WISH, and the bovine epithelial line, MDBK, oTP-1 inhibited both colony size and number. oTP-1 was as effective as human and bovine IFN alpha s on human and bovine cells, respectively; thus, it displays potent cross-species activity. Its activity was dose dependent, and inhibition of proliferation could be observed at concentrations as low as 1 unit/ml. Concentrations as high as 50,000 units/ml stopped proliferation, while viability was not impaired. Cell cycle analysis revealed an increased proportion of cells in S phase and a corresponding decreased proportion of cells in G2/M after 48 h of oTP-1 treatment. Therefore, oTP-1 appears to inhibit progress of cells through S phase. oTP-1 antiproliferative effects can be observed as early as 12 h after after the initiation of culture and are maintained through 6 days. Thus, oTP-1 exhibits potent anticellular activity without toxicity across species and may have therapeutic potential as an antitumor agent without the toxic effects generally associated with IFNs.
Animals↗
Both alpha-helices along the major histocompatibility complex binding cleft are required for staphylococcal enterotoxin A function.
The superantigen staphylococcal enterotoxin A (SEA) requires interaction with class II major histocompatibility complex (MHC) molecules to activate T cells. We have previously used the synthetic peptide approach to establish one side of the hypothetical class II foreign-antigen binding cleft, alpha-helical region 65-85 of the beta chain, as a binding site involved in accessory cell presentation of SEA to T cells. To further characterize the structural basis for MHC-SEA interaction we have examined the role of the alpha-helical regions of the class II alpha and beta chains in SEA function. Using the synthetic peptide approach, we have found that both alpha-helical regions are required for SEA-induced proliferation. Their corresponding peptides directly bound SEA. Although the beta-chain peptides were able to inhibit SEA binding to human and mouse cells, the alpha-chain peptides were not. The data suggest that the alpha-helices along both sides of the hypothetical class II MHC molecule binding cleft are required for SEA-induced function, whereas the beta-chain alpha-helix is sufficient for SEA binding. A model of superantigen presentation is proposed wherein the MHC beta chain, possibly region 70-80, interacts with SEA region 1-45, whereas another region of SEA binds region 51-80 of the alpha chain.
Binding Sites↗
Characterization of a synthetic peptide corresponding to a receptor binding domain of mouse interferon gamma.
A receptor binding region of mouse interferon gamma (IFN gamma) has previously been localized to the N-terminal 39 amino acids of the molecule by use of synthetic peptides and monoclonal antibodies. In this report, a detailed analysis of the synthetic peptide corresponding to this region, IFN gamma (1-39), is presented. Circular dichroism (CD) spectroscopy indicated that the peptide has stable secondary structure under aqueous conditions and adopts a combination of alpha-helical and random structure. A peptide lacking two N-terminal amino acids, IFN gamma (3-39), had similar secondary structure and equivalent ability to compete for receptor binding, while peptides lacking four or more N-terminal residues had reduced alpha-helical structure and did not inhibit 125I-IFN gamma binding. Substitution of proline, a helix-destabilizing amino acid, for leucine (residue 8) of a predicted amphipathic alpha-helix (residues 3-12), IFN gamma (1-39) [Pro]8, resulted in a substantial reduction in the helical content of the peptide, supporting the presence of helical structure in this region. However, destabilization of the helix did not reduce the competitive ability of the peptide. A peptide lacking eight C-terminal residues, IFN gamma (1-31), did not block 125I-IFN gamma binding and had no detectable alpha-helical structure, suggesting a requirement of the predicted second alpha-helix (residues 20-34) for receptor interaction and helix stabilization. Substitution of phenylalanine for tyrosine at position 14, IFN gamma (1-39) [Phe]14, a central location of a predicted omega-loop structure, did not affect the secondary structure associated with the region yet resulted in a 30-fold increase in receptor competition.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Structural basis for differential binding of staphylococcal enterotoxin A and toxic shock syndrome toxin 1 to class II major histocompatibility molecules.
The related staphylococcal toxins staphylococcal enterotoxin A (SEA) and toxic shock syndrome toxin 1 (TSST-1) are microbial superantigens. They require interaction with class II major histocompatibility complex (MHC) molecules to activate T cells. We have previously identified a binding site on SEA, the N-terminal 45 amino acids, as well as its corresponding receptor on the MHC antigen, residues 65-85 of the beta chain. To further characterize the structural basis for SEA binding to class II MHC molecules we have examined its relationship to TSST-1 binding. Both toxins bound similarly to murine A20 cells, but blockage of binding was observed only with the homologous toxin, which suggests that the binding sites for the two toxins on A20 cells are distinct. In contrast, specific binding of SEA was greater than that of TSST-1 on human Raji cells. Further, SEA was a better inhibitor of TSST-1 binding than was TSST-1 itself at low concentrations, but TSST-1 only minimally inhibited SEA binding. The data suggest that TSST-1 interacts with Raji cells at an SEA binding site, but with a lower affinity. The peptides SEA-(1-45) and I-A beta b-(65-85) were capable of blocking SEA binding on both A20 and Raji cells, but blockage was more effective on A20 cells. Neither peptide was capable of blocking TSST-1 binding on either cell line. The data are compatible with a model in which SEA has a binding site on A20 cells involving SEA-(1-45) and I-A beta b-(65-85) which is distinct from that which binds TSST-1, while at least two binding sites are present on Raji cells. One site involves predominantly the residue 1-45 region on SEA and the 65-85 region of the MHC beta chain, while the other site involves both a different region on the SEA molecule and a different site on the class II MHC molecule to which it binds. This latter site also binds TSST-1.
Animals↗
Cloning and expression in Saccharomyces cerevisiae of a synthetic gene for the type-I trophoblast interferon ovine trophoblast protein-1: purification and antiviral activity.
Ovine trophoblast protein-1 (oTP-1) is a unique, Type I, trophoblast interferon (IFN) that possesses potent antiviral activity and is thought to be primarily responsible for maternal recognition of pregnancy in sheep. To provide sufficient amounts of protein for detailed studies, a synthetic gene for oTP-1 was designed and assembled in Escherichia coli, subcloned into a yeast expression plasmid, and used to overproduce recombinant oTP-1 in Saccharomyces cerevisiae. Recombinant oTP-1 was purified from soluble yeast extract using sequential ion-exchange and molecular sieve chromatography. Recombinant oTP-1 purified in this fashion exhibited potent antiviral activity (0.6 x 10(8) U/mg) similar to native oTP-1. This expression system will enable production of large quantities of soluble, biologically active, and correctly processed recombinant oTP-1. Furthermore, the synthetic gene construct facilitates introduction of mutations for ongoing structure/function studies of this unique, Type I, trophoblast IFN.
Amino Acid Sequence↗
Staphylococcal enterotoxin microbial superantigens.
Staphylococcal enterotoxins are a family of structurally related proteins that are produced by Staphylococcus aureus. In addition to their role in the pathogenicity of food poisoning, these microbial superantigens have profound effects on the immune system, which makes them useful tools for understanding its mechanism of action. These molecules (24-30 kDa) are highly hydrophilic and exhibit low alpha helix and high beta pleated sheet content, suggesting a flexible, accessible structure. Staphylococcal enterotoxins are among the most potent activators of T lymphocytes known. The receptors for staphylococcal enterotoxins on antigen-presenting cells are major histocompatibility complex (MHC) class II molecules. Further, the alpha-helical regions of the class II molecule are essential for function and appear to interact directly with the NH2-terminal region of staphylococcal enterotoxins such as SEA. Recent studies have shown that a complex of staphylococcal enterotoxin and MHC class II molecules is required for binding to the V beta region of the T cell antigen receptor. Staphylococcal enterotoxin mitogenic activity is dependent on induction of interleukin 2, which may be intimately involved in the mechanism of toxicity. The mouse minor lymphocyte stimulating (M1s) "endogenous" self-superantigen has been shown to be a retroviral gene product, so this too is apparently a microbial superantigen. An understanding of the mechanisms of action of these microbial superantigens has implications for normal and pathological immune functions.
Animals↗
Type I conceptus interferons: maternal recognition of pregnancy signals and potential therapeutic agents.
In summary, Type I conceptus interferons of sheep, cows, and goats (perhaps all ruminants), which signal maternal recognition of pregnancy, may be useful for enhancing fertility in animal agriculture and may have a role in human and veterinary medicine as well. The Type I conceptus interferons share antiviral, anticellular, and immunosuppressive properties with Type I leukocyte interferons, but lack their cytotoxic effects, which may make them especially useful as therapeutic agents in human and veterinary medicine. The unique N-terminal structure of oTP-1 may account for its distinctive biological properties, including its antiluteolytic activity.
Animals↗
A qualitative comparison of paper flowsheets vs a computer-based clinical information system.
Demonstrating the value of computerized clinical information systems (CIS) relative to its cost has been difficult. Decreased mortality in the intensive care unit or a reduction in nursing staff have not been apparent, but CIS does lead to a significant improvement in documentation over handwritten flowsheets, both in volume and accuracy. This may have a medicolegal and quality assurance impact, as well as enhancing patient care.
Burn Units↗
Staphylococcal enterotoxin superantigens.
Staphylococcal enterotoxins (SE) are a family of structurally related proteins that are produced by Staphylococcus aureus. They play a role in the pathogenesis of food poisoning and are the most potent activators of T lymphocytes known. The receptors for SE on antigen-presenting cells are major histocompatibility complex class II molecules. Recent studies have shown that a complex of SE and major histocompatibility complex class II molecules is required for binding to the variable region of the T cell antigen receptor beta-chain. SE mitogenic activity is dependent on induction of interleukin 2, which may be intimately involved in the mechanism of SE toxicity. The minor lymphocyte-stimulating "endogenous" self-superantigen has recently been shown to be a retroviral gene product, so that this too is apparently a microbial superantigen. An understanding of the mechanism of action of these microbial superantigens has implications for normal and pathological immune functions.
Animals↗
Clinical evaluation of a computer-based patient monitoring and data management system.
Clinical information systems can extend the scope of computerization beyond laboratory data collection and storage. An automated and computerized intensive care unit flowsheet and patient chart can reduce nonnursing work and improve the quality, quantity, and recall of clinical information.
Burn Units↗
Topology of receptor binding domains of mouse IFN-gamma.
IFN-gamma is an essential immunoregulatory lymphokine for a variety of immunologic functions including upregulation of MHC Ag. The elucidation of the structure, particularly the receptor binding domains, should further enhance our understanding of its mechanism of action, and provide a rational basis for modulation of its activity by alteration of its structure. A predicted model of murine IFN-gamma structure has been constructed based on data derived from our synthetic peptide studies, circular dichroism spectra, and predictive algorithms for secondary structure, surface accessibility, and tertiary structure, as well as predicted structural similarities to IL-2. Direct synthetic peptide competition studies using five overlapping peptides that encompassed the entire IFN-gamma sequence of 133 amino acids showed that only the N-terminus of IFN-gamma, IFN-gamma(1-39), blocked binding to receptor, suggesting that the N-terminus is directly involved in receptor binding. Rabbit antibodies to the N-terminal (IFN-gamma(1-39)) and C-terminal (IFN-gamma(95-133)) peptides neutralized IFN-gamma activity, whereas antibodies to the three peptides that spanned the internal region, sequence 36-107, were without effect. Thus, the antibody data support the involvement of the N-terminus as a receptor binding domain and also suggest that the C-terminus of the molecule is also a binding domain. Predictive algorithms assign six alpha-helices and five turns to the molecule and circular dichroism spectra of both intact human and murine IFN-gamma and synthetic peptides of murine IFN-gamma showed that the molecule is mainly alpha-helical in structure. Drawing mainly on the four alpha-helix bundle model, a common motif in globular proteins such as IFN-gamma and IL-2 (whose crystalline structure is known), we constructed a simple model of the tertiary structure of IFN-gamma that fits well with our synthetic peptide and circular dichroism data. The model consists of the four-alpha-helical bundle along with N- and C-terminal helices that are predicted to be closely associated and together form the receptor binding domains. The model presented should contribute to further understanding the molecular basis of IFN-gamma action and allow us to begin modulating the function of IFN-gamma through the design of agonists and antagonists.
Amino Acid Sequence↗
The I-A beta b region (65-85) is a binding site for the superantigen, staphylococcal enterotoxin A.
Ia antigen is a receptor for the superantigen staphylococcal enterotoxin A (SEA). Peptides I-A beta b(30-60), I-A beta b(50-70), I-A beta b(65-85), and I-A beta b(80-100) of the MHC class II antigen beta chain on mouse (H-2b) accessory cells were synthesized. Only I-A beta b(65-85) inhibited SEA binding to the mouse B-cell lymphoma line, A20 (H-2d) and the human Burkitt's lymphoma line, Raji (HLA-DR). The I-A beta b(65-85) sequence is a predicted alpha-helix along the hypothetical antigen binding cleft of the Ia molecule. I-A beta b(65-85) also directly and specifically bound both the intact SEA molecule and its Ia binding site, represented by the peptide SEA(1-45). The results suggest that I-A beta b region (65-85) is a necessary site for Ia molecular interaction with the superantigen SEA. Further, the data suggest that the same helical region of other Ia antigens binds SEA irrespective of haplotype and species.
Amino Acid Sequence↗
Arginine vasopressin-binding peptides derived from the bovine and rat genomes differ in their abilities to block arginine vasopressin modulation of murine immune function.
Previously we reported on a nonapeptide (binding peptide) derived by reading the complementary DNA strand of the bovine arginine vasopressin (AVP) gene in the 3'5' direction that specifically blocks the AVP helper signal for gamma-interferon (IFN gamma) production by helper cell-depleted mouse spleen cultures. Bovine 5'3' AVP-binding peptide, however, did not block AVP activity. We report here on the relative abilities of 5'3' and 3'5' AVP-binding peptides derived from the bovine and rat AVP genes to block the AVP helper signal for IFN gamma production. The sequences of the bovine and rat 5'3' AVP-binding peptides differ by two amino acids, whereas the 3'5' AVP-binding peptides derived from both genes are the same. In contrast to the lack of blocking activity of the bovine 5'3' AVP-binding peptide, the rat 5'3' AVP-binding peptide was almost as effective as the 3'5' AVP-binding peptide in blocking AVP function. No effect was seen with a 9-amino acid control peptide consisting of a 'scrambled' 3'5' AVP-binding peptide sequence. Further, polyclonal anti-rat 5'3' AVP-binding peptide antibodies blocked AVP activity, whereas polyclonal anti-bovine 5'3' AVP-binding peptide antibodies had no significant effect. Polyclonal antibodies generated against the 3'5' AVP-binding peptide also blocked AVP activity. These antibodies possibly blocked AVP function by interaction with the lymphocyte AVP receptor, since AVP specifically displaced binding of the antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Localization of an antiviral site on the pregnancy recognition hormone, ovine trophoblast protein 1.
Ovine trophoblast protein 1 (oTP-1) is the interferon alpha (IFN-alpha) variant with potent antiviral activity and low toxicity that is responsible for maternal recognition of pregnancy in sheep. To examine the structure/function basis for the potent antiviral activity of oTP-1, we have exploited the direct approach of synthetic peptide competition with oTP-1 for receptor, using N-terminal oTP-1-(1-37) and C-terminal oTP-1-(139-172) peptides. These peptides possess structures similar to those predicted for the intact molecule on the basis of circular dichroism. oTP-1-(1-37) at 1.5 mM specifically blocked oTP-1 antiviral activity without affecting the antiviral activity of natural ovine IFN-alpha, recombinant bovine IFN-alpha, and recombinant human IFN-alpha. At concentrations as low as 0.15 mM, oTP-1-(139-172) blocked the antiviral activity of oTP-1, as well as that of natural ovine IFN-alpha, recombinant bovine IFN-alpha, and recombinant human IFN-alpha, but not recombinant bovine interferon gamma. Further, binding of radiolabeled oTP-1 to endometrial membrane preparations could be effectively inhibited by polyclonal anti-C-terminal and anti-N-terminal antisera, with the anti-C-terminal antiserum being the more effective inhibitor. Consistent with peptide and antiserum functional data, oTP-1 and recombinant bovine IFN-alpha are predicted to possess similar C-terminal structure but different N-terminal structure by composite surface profile predictions. The findings suggest that the C-terminal regions of IFN-alpha s bind to a common site on the IFN-alpha receptor while the N-terminal region binds to a site unique for the particular IFN-alpha.
Amino Acid Sequence↗
Structure of an epitope in an immunodominant region of the interferon-gamma molecule that is involved in receptor interaction.
An amino-terminal immunodominant region of murine interferon-gamma (IFN-gamma) that may be involved in function is characterized using a neutralizing amino-terminal-specific monoclonal antibody (mAb) and synthetic peptides. Competition studies with peptide truncations determined that residues 3, 4, and 5 of IFN-gamma were required for binding to the mAb. These residues are predicted to participate in an amphipathic alpha-helix spanning residues 3-11 of IFN-gamma. Conservative peptide analogs that maintain the amphipathicity of the alpha-helix of IFN-gamma (1-20) retained the ability to block IFN-gamma binding to mAb. Peptide analogs with substitutions that disrupted the amphipathicity of the alpha-helix lost their ability to block binding. The tyrosine at position 14 was required as its removal in carboxy-terminal truncations caused the loss of blocking ability. We conclude that the IFN-gamma epitope for the neutralizing mAb involves residues 3-14, spanning 12 residues, and it appears that residues 3, 4, 5, and 14 are an important part of the epitope. Based on these characteristics and the observation that most continuous epitopes are reported to involve shorter regions of up to 8 residues, this epitope may be described as a linear discontinuous epitope that may require the amphipathic helix. The data presented here provide further insight to the structure of a site that is involved in receptor interaction.
Amino Acid Sequence↗
Site of nonrestrictive binding of SEA to class II MHC antigens.
We have used the synthetic peptide approach to show that the N-terminal 45-amino acids of staphylococcal enterotoxin A (SEA), SEA(1-45), constitute an important part of its binding site on class II major histocompatibility complex (MHC) molecules. SEA(1-45) and to a lesser extent SEA(1-27) were able to displace SEA from HLA-DR on Raji cells as assessed by flow cytometry and to compete with radiolabeled SEA for interaction with HLA-DR in a direct binding assay. Specific binding of SEA to Ia on murine A-20 cells could be inhibited by the same peptides [i.e. SEA(1-45) greater than SEA(1-27)] that blocked binding to HLA-DR. Therefore, different class II MHC molecules associate with the same functional site on SEA. Further, an ELISA system was used to demonstrate that SEA(1-45) is able to directly bind to a mouse synthetic I-A beta b peptide, I-A beta b (65-85), which contains a binding site of the class II MHC molecule involved in SEA presentation to T cells. Thus, we have localized a site on SEA that is involved in selective surface association with class II MHC antigens and identified the region on the class II MHC antigen to which that site binds.
Animals↗
Onset of secretion of proteins with antiviral activity by pig conceptuses.
In Exp. 1, antiviral activity was detected in Day-15 pregnant uterine flushings (6222 +/- 2167 units/ml) and in conceptus culture medium collected at 0, 1, 2, 4, 8, 16, 24, 32, 40, and 48 h (95, 375, 650, 1216, 1600, 2100, 2017, 2083, 3500 and 5000 units/ml, respectively; R2 = 0.81, P less than 0.01; y = 190.0 + 252.7x - 11.2x2 + 0.2x3. In Exp. 2, antiviral activity of Day-15 conceptus culture medium was reduced 99% after boiling for 20 min (P less than 0.01) and, after 18 h dialysis (6000-8000 Mr cut-off), 100% of the activity was in the retentate. In Exp. 3, antiviral activity was not detected in cultures of conceptuses from Days 10 and 11 and activity was maximal for Day 14 and Day 15 conceptuses (2100 and 2083 units/ml, respectively). Effects of day were best described by a quadratic regression equation (y = 17,652 - 3263x + 150x2; R2 = 0.55, P less than 0.01). In Exp. 4, changes in antiviral activity detected in uterine flushings from pregnant gilts on Days 8, 10, 11, 12, 14 and 15 (1.3, 0, 6.7, 63.3, 580 and 1663 units/ml, respectively) were described by the equation y = -20,743 + 6189x - 606x2 + 20x3 (R2 = 0.85, P less than 0.01). In Exp. 5, low antiviral activities (5-30 units/ml) were detected in all plasma samples collected from the uterine artery and uterine vein of pregnant and cyclic gilts, but values were not significantly influenced by pregnancy status, day or site of collection.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗