Biomedical subjects

H Slayter

Publications and source records attributed to H Slayter.

10 recordsLinked to original sources

PubMed1996

Structures of class A macrophage scavenger receptors. Electron microscopic study of flexible, multidomain, fibrous proteins and determination of the disulfide bond pattern of the scavenger receptor cysteine-rich domain.

Structures of secreted forms of the human type I and II class A macrophage scavenger receptors were studied using biochemical and biophysical methods. Proteolytic analysis was used to determine the intramolecular disulfide bonds in the type I-specific scavenger receptor cysteine-rich (SRCR) domain: Cys2-Cys7, Cys3-Cys8, and Cys5-Cys6. This pattern is likely to be shared by the highly homologous domains in the many other members of the SRCR domain superfamily. Electron microscopy using rotary shadowing and negative staining showed that the type I and II receptors are extended molecules whose contour lengths are approximately 440 A. They comprised two adjacent fibrous segments, an alpha-helical coiled-coil ( approximately 230 A, including a contribution from the N-terminal spacer domain) and a collagenous triple helix ( approximately 210 A). The type I molecules also contained a C-terminal globular structure ( approximately 58 x 76 A) composed of three SRCR domains. The fibrous domains were joined by an extremely flexible hinge. The angle between these domains varied from 0 to 180 degrees and depended on the conditions of sample preparation. Unexpectedly, at physiologic pH, the prevalent angle seen using rotary shadowing was 0 degrees , resulting in a structure that is significantly more compact than previously suggested. The apparent juxtaposition of the fibrous domains at neutral pH provides a framework for future structure-function studies of these unusual multiligand receptors.

Amino Acid Sequence↗

PubMed1988

Receptor-like nature of class I HLA: endocytosis via coated pits.

The present investigations show that class I HLA are internalized by macrophage/monocyte type cells. Anti-class I antibody-binding assays show that about 30% of class I Ag present on cell surface are endocytosed within 1 h. Electronmicroscopic investigations reveal that, like other well established receptor molecules, internalization of HLA is mediated by coated pits and coated vesicles. The endocytosed Ag are transferred from endosomes to trans-Golgi reticulum and trans-Golgi cisternae, suggesting recycling of these Ag back to the cell surface. In the presence of phorbol ester tetradecanoyl phorbol acetate, there is a modest increase in the rate of internalization. These results are consistent with the hypothesis that class I Ag on monocytes/macrophages behave like receptor molecules. Malignant transformation of monocytic cells apparently causes the loss of this property of class I Ag.

Antigens, Surface↗

PubMed1988

Size and structure of antigen-antibody complexes. Electron microscopy and light scattering studies.

Size parameters of model antigen-antibody (Ag-Ab) complexes formed by the interaction of bovine serum albumin (BSA) and pairs of monoclonal anti-BSA antibodies (mAb) were evaluated by quasielastic light scattering, classical light scattering, and electron microscopy (EM). Mean values for the hydrodynamic radius, radius of gyration, and molecular weight were determined by light scattering. Detailed information regarding the molecular weight distribution and the presence of cycles or open chains was obtained with EM. Average molecular weights were calculated from the EM data, and the Porod-Kratky wormlike chain theory was used to model the conformational behavior of the Ag-mAb complexes. Ag-mAb complexes prepared from three different mAb pairs displayed significantly different properties as assessed by each of the techniques employed. Observations and size parameter calculations from EM photomicrographs were consistent with the results from light scattering. The differences observed between the mab pairs would not have been predicted by idealized thermodynamic models. These results suggest that the geometric constraints imposed by the individual epitope environment and/or the relative epitope location are important in determining the average size of complexes and the ratio of linear to cyclic complexes.

Antibodies, Monoclonal↗

PubMed1987

Intracellular pathways of ricin A chain cytotoxins.

Ricin A chain, a potent ribosomal poison, was disulfide linked either to the iron transport protein, transferrin, or to anti-transferrin receptor antibodies to produce highly specific derivative toxins, Tf-A and TfR-A, respectively. The ability of these agents to gain access to and damage ribosomes within the cell was accelerated in the presence of carboxylic ionophores. Their effectiveness for killing clonogenic target cells was correspondingly enhanced by 5 logs after a brief treatment with Tf-A plus ionophore. Intracellular trafficking of Tf-A and TfR-A was monitored by a variety of methods to better understand their mechanism of action. Data obtained with 125I-labeled A chain and 59Fe3+-labeled toxin probes indicated that the natural iron delivery pathway was initially followed. This was characterized by specific attachment to surface receptors, internalization, entry into low-density acidic vesicles, uncoupling of iron, an absence of lysosomal degradation, and sustained cycling. Ultrastructural studies using a colloidal gold-labeled anti-A chain probe confirmed the presence of these toxins within the structural elements associated with endocytosis. Toxic Tf-A molecules, however, diverged from this pathway (t1/2 = 88 min) to eventually kill cells as witnessed by a gradual loss in the ability to rescue cells using excess transferrin. Potentiating agents, such as carboxylic ionophores or B chain, seem to act by speeding the divergence of Tf-A and TfR-A from the normal endocytotoxic cycle.

Animals↗

PubMed1985

Native conformation of human von Willebrand protein. Analysis by electron microscopy and quasi-elastic light scattering.

von Willebrand factor (vWF) was analyzed by electron microscopic and quasi-elastic light scattering techniques in order to evaluate the size and shape of this heterogeneous polymeric plasma glycoprotein. Electron micrographs demonstrated that native vWF molecules are flexible, linear polymers, ranging in contour length from 100 to 1300 nm. In their typical configuration, the polymers were coiled upon themselves with maximal diameters ranging from 60 to 200 nm. Individual repeating protomeric subunits were discernible in occasionally noted, uncoiled polymers and measured 100 nm X 1.5-2.0 nm. Quasi-elastic light scattering analysis confirmed that measurements of the size and shape of purified vWF molecules in solution were similar to those obtained with electron microscopic techniques. In addition, the mean Stokes radius and mean radius of gyration assessed by quasielastic light scattering were directly related over a wide range of values, as were the diameter and contour length measured from electron micrographs, suggesting that the overall shape of polymers does not change with increasing size. This study supports the concept that native vWF molecules are flexible, linear polymers. In addition, this study clearly shows that the polymer configuration assessed from electron micrographs is a valid representation of the configuration of the polymer in solution. The data presented also provide the first evidence for a well-defined, repeating protomeric subunit.

Blood Coagulation Factors↗

PubMed1982