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R L Stanfield

Publications and source records attributed to R L Stanfield.

47 records · Page 3Linked to original sources
PubMed1992

Crystallization, sequence, and preliminary crystallographic data for an antipeptide Fab 50.1 and peptide complexes with the principal neutralizing determinant of HIV-1 gp120.

X-ray quality crystals of an Fab fragment from an antipeptide monoclonal antibody (R/V3-50.1) that recognizes the principal neutralizing determinant (PND) of the gp120 glycoprotein of human immunodeficiency virus type 1 (HIV-1) (MN isolate) were grown as uncomplexed and peptide complexed forms. Crystals of the free Fab grew from high salt in orthorhombic space groups P2(1)2(1)2(1) and I222 and from polyethylene glycol in space groups P1 and P2(1). Seeds from either the P1 and P2(1) native (uncomplexed) Fab crystals induced nucleation of crystals of the Fab complexed to a 16-residue synthetic peptide corresponding to the PND when streak seeded into preequilibrated solutions of this complex. Data were collected from these complex crystals and from each of the four native Fab forms to at least 2.8 A resolution. The genes for the variable domain of the Fab were cloned and sequenced and the primary amino acid sequence was deduced from this information. Knowledge of the three-dimensional structure of this Fab-peptide complex will be important in the understanding of the PND of HIV-1 and its recognition by neutralizing monoclonal antibodies.

Amino Acid Sequence↗
PubMed1992

Effect of the ACAT inhibitor CI-976 on plasma cholesterol concentrations and distribution in hamsters fed zero- and low-cholesterol diets.

The overall objective of the present study was to determine if the ACAT inhibitor CI-976 can lower plasma cholesterol in hamsters fed zero or low, "human-like" levels of cholesterol. With a purified diet containing zero dietary cholesterol, CI-976 significantly lowered VLDL cholesterol (VLDL-C), but not total plasma cholesterol (TPC). When 0.06% cholesterol was added to this diet, reductions in both VLDL and LDL cholesterol (LDL-C) lowered TPC. Efficacy was still greater with 0.2% dietary cholesterol, but not potency. Mixing CI-976 into the purified diet resulted in greater decreases in VLDL-C compared to gavage administration, but LDL-C reductions with 0.2% cholesterol were optimal with gavage. With nonpurified, chow-based diets efficacy was markedly greater with diet-admix administration, regardless of the amount of dietary cholesterol. CI-976 inhibited cholesterol absorption with chow-based diets more potently compared to nonabsorbable agents (e.g., beta-sitosterol, tigogenin cellobioside), and the lowering of LDL-C was greatest when inhibition of cholesterol absorption was maximal. We conclude that the ACAT inhibitor CI-976 is efficacious in hamster models which utilize human-like levels of dietary cholesterol. Moreover, the data suggest that the pharmacologic responses to lipophilic ACAT inhibitors in the hamster, or even other lipid-regulating drugs, are likely to depend not only on the type of basal diet but also on the mode of drug administration.

Anilides↗
PubMed1991

Structural aspects of antibodies and antibody-antigen complexes.

The structures of several Fab fragments and Fab-antigen complexes have now been solved at high resolution. These structures of antibodies in complex with proteins, peptides and various other haptens have enabled us to gain insights into the structural basis of immune recognition. Early structures of Fab fragments with and without bound haptens showed the antibody combining sites to be pockets or grooves. More recent Fab-protein complex structures have shown the antibody-antigen interactions to be more extensive with flatter, more undulating binding surfaces. We have solved the structures of three Fab fragments in their native form and as complexes with their respective antigens. Two of these are anti-peptide Fab fragments, the other an anti-progesterone Fab. Comparison of the free and bound structures indicates small but significant changes in the antibody on ligand binding. An analysis of the Fab complexes solved so far indicates that the antibodies can have very differently shaped binding sites, depending on the antigen.

Antibodies↗
PubMed1990

Crystal structures of an antibody to a peptide and its complex with peptide antigen at 2.8 A.

The three-dimensional structures of an antibody to a peptide and its complex with the peptide antigen have been determined at 2.8 A resolution. The antigen is a synthetic 19-amino acid peptide homolog of the C helix of myohemerythrin (Mhr). The unliganded Fab' crystals are orthorhombic with two molecules per asymmetric unit, whereas the complex crystals are hexagonal with one molecule per asymmetric unit. The Fab' and the Fab'-peptide complex structures have been solved independently by molecular replacement methods and have crystallographic R factors of 0.197 and 0.215, respectively, with no water molecules included. The amino-terminal portion of the peptide sequence (NH2-Glu-Val-Val-Pro-His-Lys-Lys) is clearly interpretable in the electron density map of the Fab'-peptide complex and adopts a well-defined type II beta-turn in the concave antigen binding pocket. This same peptide amino acid sequence in native Mhr is alpha-helical. The peptide conformation when bound to the Fab' is inconsistent with binding of the Fab' to native Mhr, and suggests that binding of the Fab' to conformationally altered forms of the native Mhr or to apo-Mhr. Immunological mapping previously identified this sequence as the peptide epitope, and its fine specificity correlates well with the structural analysis. The binding pocket includes a large percentage of hydrophobic residues. The buried surfaces of the peptide and the antibody are complementary in shape and cover 460 A2 and 540 A2, respectively. These two structures now enable a comparison of a specific monoclonal Fab' both in its free and antigen complexed state. While no major changes in the antibody were observed when peptide was bound, there were some small but significant side chain and main chain rearrangements.

Amino Acid Sequence↗
PubMed1989

Preliminary crystallographic data and primary sequence for anti-peptide Fab' B13I2 and its complex with the C-helix peptide from myohemerythrin.

Crystals of the Fab' fragment from the monoclonal anti-peptide antibody B1312 and of the Fab'-peptide antigen complex have been characterized. The monoclonal antibodies were raised against a synthetic homologue of the C-helix of myohemerythrin (residues 69-87 in myohemerythrin). The Fab'-peptide complex crystallizes in space group P6322 with unit cell dimensions a = b = 142.5 A, c = 101.5 A, alpha = beta = 90 degrees, gamma = 120 degrees, and Z = 1. The native Fab' crystallizes in space group P212121 with unit cell dimensions a = 98.0 A, b = 151.7 A, c = 80.8 A, alpha = beta = gamma = 90 degrees, and Z = 2. Both crystal forms diffract to beyond 2.6 A resolution. We also report the cDNA and predicted amino acid sequences for the variable regions of both the light and heavy chains of this anti-peptide antibody.

Amino Acid Sequence↗
PubMed1988

Characterization of two crystal forms of human defensin neutrophil cationic peptide 1, a naturally occurring antimicrobial peptide of leukocytes.

Two orthorhombic crystal forms (P2(1)2(1)2, a = 30.5 A, b = 44.5 A, c = 40.5 A; I2(1)2(1)2(1) (or I222), a = 30.1 A, b = 66.5 A, c = 35.5 A) of human neutrophil cationic peptide 1 have been characterized. The P2(1)2(1)2 form contains two peptides (Mr = 3425) per asymmetric unit; the I2(1)2(1)2(1) form contains one peptide per asymmetric unit. Both crystal forms diffract to beyond 1.8 A resolution.

Blood Bactericidal Activity↗
PubMed1988

Novel subunit structure observed for noncooperative hemoglobin from Urechis caupo.

Tetrameric hemoglobin from the "fat innkeeper" worm Urechis caupo possesses a novel subunit arrangement having an "inside out" quaternary structure in that the G/H helices are located on the outer surface of the tetramer. A 5-A resolution crystal structure reveals that although the individual subunits are beta-like, having a distinct D helix and the general myoglobin fold, the subunit contacts are very different from those previously observed for hemoglobins. Furthermore, the hemoglobin from U. caupo is also quite different from the unusual hemoglobin tetramer from clam which also has its G/H helices on the outer surface but with the hemes in close proximity through E-F helical contacts (Royer, W. E., Jr., Love, W. E., and Fenderson, F. F. (1985) Nature 316, 277-280).

Animals↗
PubMedDate not supplied

Immune recognition of viral antigens.

The response exhibited by the immune system to viral and other foreign antigens consists of antibody-mediated and T cell-mediated immunity. Structural and molecular biological studies have shown that the antibody response is tailored to provide exquisite specificity by generating binding pockets that are complementary in shape as well as in charge to the antigen. On the other hand, the cellular response uses T-cell receptors (TCRs) and the major histocompatibility complex (MHC) antigens. Structural information on the TCRs is not yet available, but the crystal structures of several MHC class I molecules have shown how one MHC molecule can bind many different peptide sequences that share only the common anchor residue positions that determine allele specificity. MHC class I interactions with the peptide backbone at the N and C termini explain the high specificity of the binding groove for peptide ligands and suggest a universal mode of recognition for peptides to MHC class I molecules. Peptide-MHC class II interactions are less well understood, although recent structural work has shown important differences in the binding clefts of MHC class I and II that lead to longer peptides being bound to class II molecules. Detailed analysis at the molecular level has indicated that conformational changes in both antibodies and MHC molecules occur upon antigen binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗