Biomedical subjects

David Glesne

Publications and source records attributed to David Glesne.

5 recordsLinked to original sources

PubMed2006

Regulatory properties and cellular redistribution of zinc during macrophage differentiation of human leukemia cells.

Many proteins require the binding of trace metals such as Ca, Fe, Cu, or Zn, which may modulate their structure, function, or activity. To determine if there were any overall changes in metalloprotein distribution or metal concentration during the process of macrophage differentiation we induced human myeloid HL-60 leukemia cells with phorbol 12-myristate 13-acetate (PMA) and quantitatively mapped their metal content using hard X-ray fluorescence micro-analysis. We found a transient increase in the zinc content of HL-60 cell nuclei during the early stages of differentiation induction. This finding was confirmed by spectrofluorometry in HL-60 cells and extended to U-937 leukemia cells. A role for protein kinase C-beta (PKC-beta) in this process was established by examining zinc content in an HL-60 variant, HL-525, which is PKC-beta deficient, and in HL-525 cells in which PKC-beta was restored by stable overexpression. Chemical chelation of both Cu and Zn served to inhibit macrophage differentiation in HL-60 cells, indicating a requirement for these metals during this process. Finally, we demonstrate that growth of HL-60 cells in a low-zinc environment removes their susceptibility to PMA-induced differentiation, and that this capacity can be partially restored by the addition of exogenous zinc.

Active Transport, Cell Nucleus↗

PubMed2005

Analysis of protein interaction and function with a 3-dimensional MALDI-MS protein array.

Protein profiling and characterization of protein interactions in biological samples ultimately require indicator-free methods of signal detection, which likewise offer an opportunity to distinguish specific interactions from nonspecific protein binding. Here we describe a new 3-dimensional protein microchip for detecting biomolecular interactions with matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS); the microchip comprises a high-density array of methacrylate polymer elements containing immobilized proteins as capture molecules and directly interfaces with a commercially available mass spectrometer. We demonstrated the performance of the chip in three types of experiments by detecting antibody-antigen interactions, enzymatic activity, and enzyme-inhibitor interactions. MALDI-MS biochip-based tumor necrosisfactor alpha (TNF-alpha) immunoassays demonstrated the feasibility of detecting antigens in complex biological samples by identifying molecular masses of bound proteins even at high nonspecific protein binding. By detecting model interactions of trypsin with trypsin inhibitors, we showed that the protein binding capacity of methacrylate polymer elements and the sensitivity of MALDI-MS detection of proteins bound to these elements surpassed that of other 2- and 3-dimensional substrates tested Immobilized trypsin retained functional (enzymatic) activity within the protein microchip and the specificity of macromolecular interactions even in complex biological samples. We believe that the underlying technology should therefore be extensible to whole-proteome protein expression profiling and interaction mapping.

Equipment Design↗

PubMed2004

Spermine acts as a negative regulator of macrophage differentiation in human myeloid leukemia cells.

The role of putrescine, spermidine and spermine in phorbol 12-myristate-13-acetate (PMA)-induced macrophage differentiation was examined in human HL-60 and U-937 myeloid leukemia cells. Unlike other polyamines, spermine affected this differentiation by acting as a negative regulator. This negative regulation was established by showing that the PMA-induced macrophage phenotype, but not PMA-associated replication arrest, was abrogated (a) by replenishing the PMA-evoked decrease in cellular spermine levels with this polyamine from an exogenous source and (b) by blocking PMA-induced expression of the polyamine catabolic enzyme N(1)-spermidine/spermine acetyltransferase (SSAT) with antisense oligonucleotides in the presence of low substrate level. The PMA-evoked reduction in cellular spermine appears to result from an increase in the activity of SSAT and a decrease in the activity of ornithine decarboxylase, the polyamine biosynthetic enzyme. To a degree, these changes are due to corresponding changes in the expression of the genes that code for these enzymes. When cell differentiation is initiated, SSAT expression is increased after PMA-evoked activation of protein kinase C-beta. The present studies raise the possibility that agents able to reduce spermine levels in patients' myeloid leukemia cells may enhance the activity of differentiation therapy drugs for this type of leukemia.

Acetyltransferases↗

PubMed2004

The crystal structure of Escherichia coli MoaB suggests a probable role in molybdenum cofactor synthesis.

The crystal structure of Escherichia coli MoaB was determined by multiwavelength anomalous diffraction phasing and refined at 1.6-A resolution. The molecule displayed a modified Rossman fold. MoaB is assembled into a hexamer composed of two trimers. The monomers have high structural similarity with two proteins, MogA and MoeA, from the molybdenum cofactor synthesis pathway in E. coli, as well as with domains of mammalian gephyrin and plant Cnx1, which are also involved in molybdopterin synthesis. Structural comparison between these proteins and the amino acid conservation patterns revealed a putative active site in MoaB. The structural analysis of this site allowed to advance several hypothesis that can be tested in further studies.

Arabidopsis Proteins↗

PubMed2003

A human peripheral blood monocyte-derived subset acts as pluripotent stem cells.

We have identified, cultured, characterized, and propagated adult pluripotent stem cells (PSC) from a subset of human peripheral blood monocytes. These cells, which in appearance resemble fibroblasts, expand in the presence of macrophage colony-stimulating factor and display monocytic and hematopoietic stem cell markers including CD14, CD34, and CD45. We have induced these cells to differentiate into mature macrophages by lipopolysaccharide, T lymphocytes by IL-2, epithelial cells by epidermal growth factor, endothelial cells by vascular endothelial cell growth factor, neuronal cells by nerve growth factor, and liver cells by hepatocyte growth factor. The pluripotent nature of individual PSC was further confirmed by a clonal analysis. The ability to store, expand, and differentiate these PSC from autologous peripheral blood should make them valuable candidates for transplantation therapy.

Antigens, CD34↗