Biomedical subjects

S Perampalam

Publications and source records attributed to S Perampalam.

3 recordsLinked to original sources

PubMed2001

Probing the surface of eukaryotic cells using combinatorial toxin libraries.

The success of proteomics hinges in part on the development of approaches able to map receptors on the surface of cells. One strategy to probe a cell surface for the presence of internalized markers is to make use of Shiga-like toxin 1 (SLT-1), a ribosome-inactivating protein that kills eukaryotic cells [1, 2]. SLT-1 binds to the glycolipid globotriaosylceramide [3, 4], which acts as a shuttle, allowing the toxin to be imported and routed near ribosomes. We investigated the use of SLT-1 as a structural template to create combinatorial libraries of toxin variants with altered receptor specificity. Since all SLT-1 variants retain their toxic function, this property served as a search engine enabling us to identify mutants from these libraries able to kill target cells expressing internalizable receptors. Random mutations were introduced in two discontinuous loop regions of the SLT-1 receptor binding subunit. Minimal searches from screening 600 bacterial colonies randomly picked from an SLT-1 library identified toxin mutants able to kill cell lines resistant to the wild-type toxin. One such mutant toxin was shown to bind to a new receptor on these cell lines by flow cytometry. Toxin libraries provide a strategy to delineate the spectrum of receptors on eukaryotic cells.

Amino Acid Sequence↗

PubMed1999

Shiga-like toxin-1 receptor on human breast cancer, lymphoma, and myeloma and absence from CD34(+) hematopoietic stem cells: implications for ex vivo tumor purging and autologous stem cell transplantation.

The ribosome-inactivating protein, Shiga-like toxin-1 (SLT-1), targets cells that express the glycolipid globotriaosylceramide (CD77) on their surface. CD77 and/or SLT-1 binding was detected by flow cytometry and immunocytochemistry on lymphoma and breast cancer cells recovered from biopsies of primary human cancers as well as on B cells or plasma cells present in blood/bone marrow samples of multiple myeloma patients. Breast cancer cell lines also expressed receptors for the toxin and were sensitive to SLT-1. Treatment of primary B lymphoma, B-cell chronic lymphocytic leukemia, and myeloma B or plasma cells with SLT-1-depleted malignant B cells by 3- to 28-fold, as measured by flow cytometry. Depletion of myeloma plasma cells was confirmed using a cellular limiting dilution assay followed by reverse transcriptase-polymerase chain reaction analysis of clonotypic IgH transcripts, which showed a greater than 3 log reduction in clonotypic myeloma cells after SLT-1 treatment. Receptors for the toxin were not detected on human CD34(+) hematopoietic progenitor cells (HPC). HPC were pretreated with a concentration of SLT-1 known to purge primary malignant B cells and cultured for 6 days. The number of HPC was comparable in toxin-treated and untreated cultures. HPC were functionally intact as well. Colony-forming units (CFU) were present at an identical frequency in untreated and SLT-1 pretreated cultures, confirming that CFU escape SLT-1 toxicity. The results suggest the ex vivo use of SLT-1 in purging SLT-1 receptor-expressing malignant cells from autologous stem cell grafts of breast cancer, lymphoma, and myeloma patients.

Antibodies, Monoclonal↗

PubMed1999

Identification of a unique glomerular factor X activator in murine lupus nephritis.

The role of glomerular procoagulant activity (PCA) was studied in mice (MRL/lpr, NZBxWF,, and BXSB) that are known to develop lupus nephritis. In young mice (6 to 8 wk) without renal disease, there was no increase in spontaneous glomerular PCA. In contrast, older (5 to 8 mo) autoimmune mice had significant augmentation in glomerular PCA, coinciding with the histologic appearance of severe glomerulonephritis and renal fibrin deposition. The PCA was characterized as a serine protease that directly activated factor X. This factor X activator is not tissue factor because (1) expression of PCA was not dependent on factor VII; (2) a monoclonal antibody against the factor X activator inhibited glomerular PCA, but not tissue factor; (3) the molecular weight (66 kD) of the activator was different from that of tissue factor; and (4) concanavalin A inhibited tissue factor but not glomerular PCA. Immunohistochemical studies localized the factor X activator to the glomerular mesangium and capillary wall of 4- to 6-mo-old diseased MRL/lpr mice. Immunogold-labeled antibody bound to the dense deposits, macrophages, and endothelial cells of diseased glomeruli. These studies define the role of a unique glomerular factor X activator in murine lupus nephritis.

Animals↗