In order to study whether the endothelial cells (ECs) with lipid peroxidation induced by diamide can express and secrete macrophage inflammatory protein 1 alpha (MIP-1 alpha), the expression of MIP-1 alpha protein in the cells was detected by cell enzyme-linked immunosorbent assay (ELISA) and that of MIP-1 alpha mRNA was determined by cell in situ hybridization and nuclease S1 protection assay after the ECs were exposed to different concentrations of diamide for 4 h. The chemotactic activity of MIP-1 alpha was tested by micropore filter method using modified Boyden chambers. Cell ELISA showed that the expression of MIP-1 alpha protein in endothelial cells exposed to 1 mumol/L, 5 mumol/L and 10 mumol/L diamide was 1.9-fold, 2.3-fold and 1.7-fold respectively as much as that in the control cells, which was statistically significant by analysis of variance. In situ hybridization revealed that the mRNA expression of ECs treated with 1 mumol/L, 5 mumol/L and 10 mumol/L diamide was 1.3-fold, 3.0-fold and 1.7-fold as much as that in the control group, which had statistical significance (F = 188.93, P < 0.01). The mRNA expression in 5 mumol/L dimide treated ECs, measured by nuclease S1 protection assay, was 3.4-fold as much as that in the control group (t = 8.70, P < 0.05). Chemotactic response(99.50 +/- 4.31 microns) to the culture medium conditioned by 5 mumol/L diamide treated ECs, which was stronger than that(66.47 +/- 3.25 microns) conditioned by the ECs (F = 404.31, P < 0.05), was significantly decreased (F = 192.25, P < 0.05) after adding MIP-1 alpha antibody. It suggests that diamide, a lipid peroxidation inducer, could stimulate ECs to produce high level of MIP-1 alpha, and might play an important role in atherogenesis by promoting the migration of peripheral blood monocytes into arterial intima.
Cells, Cultured ; Chemokine CCL3 ; Chemokine CCL4 ; Chemotaxis, Leukocyte ; physiology ; Diamide ; pharmacology ; Endothelium, Vascular ; cytology ; metabolism ; Humans ; Lipid Peroxidation ; Macrophage Inflammatory Proteins ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Sulfhydryl Reagents ; pharmacology ; Umbilical Veins ; cytology

OBJECTIVES: This study was conducted in order to understand the cellular events associated with silica-induced pathogenesis of the rat lung. METHODS: Silicosis was induced by an intratracheal instillation of 50 mg of silica (SiO2, 0.15 - 10 micrometer) suspended in 500 microliter of a sterile saline solution in Sprague-Dawley rats weighing 200g. Silicotic nodules were excised from the rat lungs 4 weeks after silica instillation, then boiled for 4 days at 110 degrees in solution containing 2% SDS, 10 M urea and 40 mM DTT. The insoluble cellular encapsulates were electrophoresed on 4-12 % gradient SDS-PAGE, and the amino acid composition was analyzed. Affinity chromatographies of the homogenate supernatants of the control lung, silicotic nodule, and normal rat plasma were performed using rabbit IgG, anti-rat, cross-linked protein from the silicotic nodule. The amounts of N epsilon-(gamma-glutamyl) lysine cross-linked in the control lungs and silicotic nodules were determined using HPLC analysis. RESULTS: The remaining cross-linked protein was insoluble in the 10 M urea and 40 mM sulfhydryl reagents even under prolonged boiling conditions. The encapsulate revealed the retention of silica particles within the protein whose amino acid composition showed a high percentage of alanine, leucine and glycine. A 46 KDa protein was identified as a cross-linked protein in the silicotic nodule by affinity chromatography. The level of N epsilon-(gamma-glutamyl) lysine dipeptide in the nodule digest was prominently increased compared with that in the control lung. CONCLUSIONS: Transglutaminase (TGase)-catalyzed cross-linking appears to be involved in the silicotic nodule formation, and the 46 KDa protein may be cross-linked to itself and other extracellular matrix proteins during fibrosis and the formation of eventually insoluble nodule.
Alanine ; Animals ; Chromatography ; Chromatography, Affinity ; Chromatography, High Pressure Liquid ; Electrophoresis, Polyacrylamide Gel ; Extracellular Matrix Proteins ; Fibrosis ; Glycine ; Immunoglobulin G ; Leucine ; Lung* ; Lysine ; Plasma ; Rats* ; Rats, Sprague-Dawley ; Silicon Dioxide ; Silicosis ; Sodium Chloride ; Sulfhydryl Reagents ; Urea

In order to study whether the endothelial cells (ECs) with lipid peroxidation induced by diamide can express and secrete macrophage inflammatory protein 1 alpha (MIP-1 alpha), the expression of MIP-1 alpha protein in the cells was detected by cell enzyme-linked immunosorbent assay (ELISA) and that of MIP-1 alpha mRNA was determined by cell in situ hybridization and nuclease S1 protection assay after the ECs were exposed to different concentrations of diamide for 4 h. The chemotactic activity of MIP-1 alpha was tested by micropore filter method using modified Boyden chambers. Cell ELISA showed that the expression of MIP-1 alpha protein in endothelial cells exposed to 1 mumol/L, 5 mumol/L and 10 mumol/L diamide was 1.9-fold, 2.3-fold and 1.7-fold respectively as much as that in the control cells, which was statistically significant by analysis of variance. In situ hybridization revealed that the mRNA expression of ECs treated with 1 mumol/L, 5 mumol/L and 10 mumol/L diamide was 1.3-fold, 3.0-fold and 1.7-fold as much as that in the control group, which had statistical significance (F = 188.93, P < 0.01). The mRNA expression in 5 mumol/L dimide treated ECs, measured by nuclease S1 protection assay, was 3.4-fold as much as that in the control group (t = 8.70, P < 0.05). Chemotactic response(99.50 +/- 4.31 microns) to the culture medium conditioned by 5 mumol/L diamide treated ECs, which was stronger than that(66.47 +/- 3.25 microns) conditioned by the ECs (F = 404.31, P < 0.05), was significantly decreased (F = 192.25, P < 0.05) after adding MIP-1 alpha antibody. It suggests that diamide, a lipid peroxidation inducer, could stimulate ECs to produce high level of MIP-1 alpha, and might play an important role in atherogenesis by promoting the migration of peripheral blood monocytes into arterial intima.
Cells, Cultured ; Chemotaxis, Leukocyte/physiology ; Diamide/*pharmacology ; Endothelium, Vascular/cytology ; Endothelium, Vascular/*metabolism ; Lipid Peroxidation ; Macrophage Inflammatory Protein-1/*biosynthesis ; Macrophage Inflammatory Protein-1/genetics ; RNA, Messenger/biosynthesis ; RNA, Messenger/genetics ; Sulfhydryl Reagents/pharmacology ; Umbilical Veins/cytology

OBJECTIVETo investigate the effects of mitochondrial membrane permeability transition pore (MPT)-opened agent diamide and MPT-closed agent cyclosporin A on arsenic trioxide (As(2)O(3))-induced apoptosis in acute promyelocytic leukemia (APL) cell line NB4.

METHODSNB4 cells were treated with As(2)O(3) alone or in combination with diamide or cyclosporin A in different concentrations. Cell apoptosis was assessed by the morphological observation, Annexin-V assay, distribution of cellular DNA contents and genomic DNA electrophoresis. The mitochondrial transmembrane potentials (DeltaPsim) were detected by flow cytometry according to the intensity of rhodamine 123 uptake in cells.

RESULTSBoth diamide and cyclosporin A significantly enhanced As(2)O(3)-induced apoptosis in NB4 cells. The DeltaPsim collapse induced by As(2)O(3) was also enforced by combined treatment with diamide or cyclospo-rin A. 1 micromol/L As(2)O(3) alone treatment for 72 hours led to DeltaPsim disruption in 27.9% of cells, while combined treatment of As(2)O(3) and diamide or cyclosporin A increased DeltaPsim disruption cells to 59.7% and 42.2%, respectively.

CONCLUSIONSAs(2)O(3)-induced DeltaPsim disruption possibly involves with thiol oxidation or crosslink of important components especially ANT-related molecules.

Antineoplastic Agents ; pharmacology ; therapeutic use ; Apoptosis ; Arsenicals ; pharmacology ; therapeutic use ; Cyclosporine ; pharmacology ; Diamide ; pharmacology ; Drug Synergism ; Enzyme Inhibitors ; pharmacology ; Humans ; Leukemia, Promyelocytic, Acute ; drug therapy ; pathology ; Membrane Potentials ; drug effects ; physiology ; Mitochondria ; physiology ; Oxides ; pharmacology ; therapeutic use ; Sulfhydryl Reagents ; pharmacology ; Tumor Cells, Cultured

The purpose of this paper is to establish sulfhydryl site-directed PEGylation method for lysostaphin and to evaluate effects of mutagenesis and modification of amino acid residue within putative linker on enzyme activity. On the basis of structural analysis of lysostaphin, amino acid 133-154 of tentative linker between the N-terminal and C-terminal domain were chosen as the candidate residues for site-directed mutagenesis to cysteine. Subsequently, sulfhydryl site-directed PEGylation was performed by reacting PEG-maleimide reagent with the newly introduced cysteine residue of the mutant lysostaphin. The Cys-mutant and PEG-modified proteins were both purified, and their enzymatic activity were further PEGylated lysostaphins. The mono-PEGylated lysostaphins were separated from unmodified lysostaphins through highly efficient one step method with Ni(2+)-NTA column chromatography. However, both Cys-mutant and PEGylated lysostaphin only retained partial activities of the wild-type enzyme. It suggests that sulfhydryl site-directed PEGylation modification of the tentative linker between the N-terminal and C-terminal domain may affect the catalytic activity of lysostaphin.
Anti-Infective Agents, Local ; chemistry ; metabolism ; Base Sequence ; Catalysis ; Cysteine ; chemistry ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Lysostaphin ; biosynthesis ; chemistry ; metabolism ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; Mutant Proteins ; chemistry ; metabolism ; Polyethylene Glycols ; chemistry ; Recombinant Proteins ; biosynthesis ; genetics ; metabolism ; Staphylococcus ; metabolism ; Sulfhydryl Reagents ; pharmacology