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Biomedical subjects

G A Heavner

Publications and source records attributed to G A Heavner.

At least 19 recordsLinked to original sources
PubMed2000

Antibodies against the first Ig-like domain of human platelet endothelial cell adhesion molecule-1 (PECAM-1) that inhibit PECAM-1-dependent homophilic adhesion block in vivo neutrophil recruitment.

Platelet endothelial cell adhesion molecule (PECAM-1), a member of the Ig superfamily, is found on endothelial cells and neutrophils and has been shown to be involved in the migration of leukocytes across the endothelium. Adhesion is mediated, at least in part, through binding interactions involving its first N-terminal Ig-like domain, but it is still unclear which sequences in this domain are required for in vivo function. Therefore, to identify functionally important regions of the first Ig-like domain of PECAM-1 that are required for the participation of PECAM-1 in in vivo neutrophil recruitment, a panel of mAbs against this region of PECAM-1 was generated and characterized in in vitro adhesion assays and in an in vivo model of cutaneous inflammation. It was observed that mAbs that disrupted PECAM-1-dependent homophilic adhesion in an L cell aggregation assay also blocked TNF-alpha-induced intradermal accumulation of neutrophils in a transmigration model using human skin transplanted onto SCID mice. Localization of the epitopes of these Abs indicated that these function-blocking Abs mapped to specific regions on either face of domain 1. This suggests that these regions of the first Ig-like domain may contain or be close to binding sites involved in PECAM-1-dependent homophilic adhesion, and thus may represent potential targets for the development of antiinflammatory reagents.

Amino Acid Sequence↗
PubMed1999

Identification of TNF-alpha binding peptides from a D-amino acid hexapeptide library that specifically inhibit TNF-alpha binding to recombinant p55 receptor.

Tumour necrosis factor alpha (TNF-alpha) is a pro-inflammatory cytokine with pleiotropic activity that binds to two transmembrane receptors. Its role in mediating the inflammatory response to injury or infection has been well documented and it has been shown to be a causative factor in rheumatoid arthritis, inflammatory bowel disease and septic shock. Using synthetic peptide libraries composed exclusively of D-amino acids, two distinct hexapeptide families that block the binding of TNF-alpha to its receptors were identified. In the deconvolution of the library, activity increased from submillimolar to the low micromolar range with the most active compound having an IC50 of 0.33 microM. With the aid of biotinylated constructs of these hexapeptides it was possible to demonstrate that their antagonistic effect is due to specific binding to TNF-alpha and not to its receptor.

Amino Acids↗
PubMed1996

Structure-function studies on synthetic peptides derived from the 109-118 lectin domain of selectins.

Previously, we identified several peptides corresponding to amino acid sequences within the lectin domains of selectins that inhibit neutrophil (PMN) adhesion to P-selectin. Here we focused on one of the active regions, 109-118, which contains residues that have been identified as critical for E-selectin binding to the sialyl Lexis X (sLex) counter receptor. Analogues were synthesized and examined for their inhibitory effect on PMN binding to P-selectin and E-selectin immunoglobulin fusion proteins (P-IgG, E-IgG) and also on P-IgG and E-IgG binding to sLex coated surfaces. Peptide sequences which inhibited PMN binding to the fusion proteins were not necessarily those that inhibited fusion protein binding to sLex. In addition, various amino acid substitutions could be tolerated at the 111 and 113 positions without altering inhibitory activity. Modeling suggests that structural conformations of peptide analogues could explain the differences in biological activity of peptide analogues compared to mutants of the native protein.

Amino Acid Sequence↗
PubMed1996

Determination of the core sequence of an antagonist of selectin-dependent leukocyte adhesion and correlation of its structure with molecular modeling studies.

The sequence 36-50 from the lectin domain of human P-selectin has been previously identified as a weak inhibitor of selectin-dependent leukocyte adhesion. A series of C- and N-terminally truncated peptides was synthesized to determine the limits of the active core region within the parent sequence. Deletions from both the N- and C-termini gave significant increases in inhibitory activity and identified 41-50 or 36-49 as minimum active sequences, but surprisingly not the common 41-49 peptide. All peptides tested showed parallel inhibition of both P- and E-selectin-dependent adhesion. A molecular model of the lectin domain was constructed using homology modeling. Examination of this model suggests one hypothesis to explain the increase in activity on deletion of Asp36.

Amino Acid Sequence↗
PubMed1996

Peptides derived from the lectin domain of selectin adhesion molecules inhibit leukocyte rolling in vivo.

OBJECTIVE: The selectins are a family of adhesion molecules that mediate leukocyte rolling, a prerequisite for their later firm adhesion and migration to sites of inflammation. The N-terminal lectin domain of selectins is important for Ca(2+)-dependent binding to oligosaccharide ligands. We set out to study the effect of peptides corresponding to residues 11-20, 23-30, 36-50, 54-63, 70-79 and 109-118 (counting from the N-terminus of the mature proteins) of the lectin domain of human L-, P- and E-selectins on leukocyte rolling in vivo. METHODS: Peptides were applied by local intravascular microinfusion via a glass micropipette into rat mesenteric venules. Visibly rolling cells were counted off-line and compared with rolling cells counted during control periods. RESULTS: Peptides corresponding to residues 70-79 of P-selectin and 11-20 of L-selectin reduced leukocyte rolling flux in rat mesenteric venules to less than 30% of that measured during control infusion. Peptides corresponding to residues 109-118 of P-selectin, 54-63 of L-selectin and 23-30 of E-selectin also reduced leukocyte rolling flux, although to a lesser degree. CONCLUSIONS: We have shown that small peptides based on the lectin domain of all three selectins can be effective inhibitors of leukocyte rolling in vivo.

Amino Acid Sequence↗
PubMed1993

Peptides from multiple regions of the lectin domain of P-selectin inhibiting neutrophil adhesion.

The selectins are a family of three structurally related glycoproteins that are integral components of leukocyte adhesion to the vascular endothelium. Their involvement in the recruitment and extravasation of neutrophils is critical in mounting an inflammatory reaction. The carbohydrate nature of the selectin ligands suggests that the binding regions of the selectins are contained within the lectin-like domains of the selectins. The synthesis and evaluation for inhibition of selectin binding of overlapping peptides of the lectin and adjacent EGF-like domains of P-selectin have been used to identify small peptides that completely inhibit P-selectin-dependent neutrophil adhesion. These peptides span a region of more than 100 amino acids and may define the carbohydrate recognition domain of P-selectin.

Amino Acid Sequence↗
PubMed1992

Lectin domain peptides from selectins interact with both cell surface ligands and Ca2+ ions.

Selectins are receptors that mediate leukocyte adhesion to platelets or endothelial cells through Ca(2+)-dependent interactions with cell surface oligosaccharides. We found that peptides corresponding to residues 23-30, 54-63, and 70-79 of the N-terminal lectin domain of P-selectin inhibited leukocyte adhesion to P-selectin. Peptides corresponding to the homologous 23-30 and 54-63 regions of E-selectin and L-selectin also prevented cell binding to P-selectin. Immobilized albumin conjugates of the three P-selectin peptides supported adhesion of myeloid cells and certain other cells expressing fucosylated oligosaccharides. Ca2+ was required for optimal cell adhesion to the conjugates containing the 23-30 and 54-63 sequences. Furthermore, Ca2+ interacted with the 23-30 and 54-63 peptides of all three selectins, as detected by changes in intrinsic fluorescence emission intensity. These data suggest that residues contained within the 23-30 and 54-63 regions of the selectins represent contact sites for carbohydrate structures on target cells. Furthermore, binding of Ca2+ to these sequences may directly enhance their ability to interact with cell surface ligands.

Amino Acid Sequence↗
PubMed1991

Biologically active conformations of thymopentin. Studies with conformationally restricted analogs.

Four cyclic analogs of thymopentin were synthesized and evaluated for biological activity on the human T cell line CEM. Three of these conformationally restricted analogs were biologically active. The one analog which most closely mimicked the conformation predicted from NMR and theoretical energy minimization calculations proved to be inactive. These studies establish that the biologically active conformations of thymopentin differ from the most probable conformation predicted from solution NMR and theoretical energy minimization studies.

Amino Acid Sequence↗
PubMed1990

Peptide analogs of thymopentin distinguish distinct thymopoietin receptor specificities on two human T cell lines.

Thymopoietin, a polypeptide hormone of the thymus, and the synthetic pentapeptide thymopentin, corresponding to thymopoietin32-36, both induced elevations of intracellular cyclic GMP in two human T cell lines, CEM and MOLT-4. In contrast, the closely related polypeptide thysplenin, which differs from thymopoietin at position 34, induced intracellular cyclic GMP elevation in MOLT-4 but not in CEM. We synthesized a series of penta- and tetrapeptide analogs of amino acids 32-36 of human thymopoietin and thysplenin, and now show that distinct patterns of activity can be obtained in these small peptides, with selectivity for cyclic GMP elevation in MOLT-4 alone or CEM alone. This suggests that the thymopoietin receptors (TPR) on these two human T cell lines are distinguishable by their differing ligand specificities, and we have termed them alpha TPR and beta TPR for CEM and MOLT-4 receptors, respectively.

Cell Line↗
PubMed1990

Isolation, identification, and characterization of a palladium complex in the catalytic deprotection of a protected peptide.

The final catalytic deprotection in the large scale synthesis of thymopentin (Arg-Lys-Asp-Val-Tyr) produced an impurity which had not previously been observed. Isolation by preparative HPLC and spectroscopic characterization led to a postulated structure of the impurity as the 1:1 thymopentin-palladium complex. A complex corresponding to the proposed structure was independently synthesized and shown to have identical chromatographic and spectroscopic properties with the isolated material. Proton and carbon (13) NMR were used to determine the coordination sites of the peptide with palladium. The susceptibility of the complex to hydrogenation indicated a possible source for its formation.

Catalysis↗
PubMed1985

Detection and quantitation of low levels of protected and unprotected (R)-amino acids in the synthesis of thymopentin, an immunoregulatory peptide.

Two chiral chromatographic procedures (HPLC and capillary GC) were developed to monitor the extent of epimerization of protected and unprotected amino acids used in a synthesis of thymopentin, an immunoregulatory peptide currently in clinical trials. The capillary GC method allowed the detection of the (R)-enantiomer in the presence of the (S)-enantiomer at levels of greater than or equal to 0.2%, while the HPLC method allowed similar detection at levels of greater than or equal to 0.1%.

Amino Acids↗
PubMed1985

Thymopentin structure and relation to thymic factors.

High-performance liquid chromatography (HPLC) was used to evaluate the purity and batch-to-batch consistency of four commercially available thymic extracts and thymopentin, the synthetic pentapeptide corresponding to the biologically active region of the thymic hormone thymopoietin. The thymus extracts were all extremely heterogeneous, differing one from the other. Additionally, they showed high batch-to-batch variation. In contrast, thymopentin was homogeneous and this homogeneity was consistent in different batches.

Animals↗
PubMed1985

Structural requirements for the biological activity of thymopentin analogs.

Thymopentin, the synthetic pentapeptide Arg-Lys-Asp-Val-Tyr, corresponds to residues 32-36 of the thymic hormone thymopoietin. Thymopentin, like thymopoietin, induces intracellular cGMP elevations in the human T-cell line, CEM. Thymopentin also displaces radiolabeled thymopoietin from a receptor glycoprotein prepared from CEM cells, provided that a nonapeptide corresponding to thymopoietin is added to block thymopoietin binding to an additional binding site. Twenty nine analogs with single position substitutions were synthesized by solid-phase or classical solution synthesis, and are evaluated in these assays. All analogs that were active gave positive effects in both assays. A number of substitutions were tolerated at positions 2, 4, and 5, but there was an absolute requirement for L- or D-Arg at position 1 and L- or D-Asp at position 3 to maintain biological activity.

Amino Acid Sequence↗
PubMed1984

Cooperativity of thymopoietin 32-36 (the active site) and thymopoietin 38-45 in receptor binding.

Thymopoietin is a 49 amino acid polypeptide hormone of the thymus whose biological activity is reproduced by the synthetic pentapeptide thymopentin, corresponding to amino acids 32-36. Thymopentin requires the addition of an octapeptide corresponding to thymopoietin 38-45 for full competition with native thymopoietin in a radioreceptor assay with receptor derived from the human T-cell line CEM. Thus thymopoietin appears to bind to its receptor on T-cells by two regions (32-36 and 38-45). Thymopentin alone is biologically active and induces elevations of intracellular cyclic GMP. Whilst occupancy of the adjacent site by thymopoietin 37-45 does not of itself cause an elevation of intracellular cyclic GMP this peptide is not biologically silent as it does enhance the potency of thymopentin.

Amino Acid Sequence↗
PubMed1981

Proton NMR investigation of Ln3+ complexes of thymopoietin 32-36.

The pentapeptide Arg-Lys-Asp-Val-Tyr (TP5) is a biologically active fragment of thymopoietin, the thymic hormone that induces selective T-cell differentiation. The formation of lanthanide(III) complexes of TP5 is demonstrated through the observation of Tb3+ fluorescence enhancement. The equilibria, stoichiometry and solution conformation of the La3+, Pr3+ and Yb3+ complexes of TP5 have been investigated using NMR spectroscopy. In addition, the dissociation constants of two methyl ester analogs of TP5 have been studied. Evidence is presented supporting an interaction between the arginine guanidino N epsilon H and the aspartate carboxylate of TP5. Binding of Ln3+ appears to be accompanied by a disruption (or weakening) of this interaction and a concomitant increase in the 180 degrees rotamer population for the aspartate carboxylate group. The observed trends in the magnitudes of the dissociation constants and the rotamer populations appear to suggest that, although a significant amount of monodentate complexes may also exist, the metal ion binds predominantly to both carboxylates in a bidentate fashion.

Amino Acids↗
PubMed1981

Proton nuclear magnetic resonance study of an active pentapeptide fragment of ubiquitin.

The aqueous solution conformation of Tyr-Asn-Ile-Gln-Lys (UB5) corresponding to positions 59-63 of the polypeptide, ubiquitin, has been investigated by proton NMR. Like the parent protein, UB5 induces nonspecifically both T and B lymphocyte differentiation. The various NH and CH resonances of this pentapeptide have been assigned, and its solution conformation has been probed through a study of chemical shift variations with pH, temperature dependence of amide hydrogen chemical shifts, vicinal NH--C alpha H and C alpha H--C beta H2 coupling constant data, and amide hydrogen-exchange rates. The latter were measured in H2O by using a combination of transfer of solvent saturation and saturation recovery NMR experiments. The data are compatible with the assumption of a highly motile dynamic equilibrium among different conformations for this peptide. The various secondary amide hydrogens remain essentially exposed to the solvent. The temperature-dependence study of the amide hydrogen chemical shifts also did not reveal any strong internal hydrogen bonds. A rotamer population analysis of tyrosine and asparagine side chains suggests that two of the rotomers are predominantly populated for each of these residues. From these results, a picture emerges of the dynamic conformation of UB5 in aqueous solution.

Chromosomal Proteins, Non-Histone↗