carbocyanines and Glomerulosclerosis--Focal-Segmental

Two new iodoacetamide-substituted cyanines, C3NIASO3 and C5NIASO3, were synthesized starting from hemicyanine and were utilized for labeling plasma proteins. Specificity, sensitivity and feasibility for SH residues was tested utilizing an equimolar mixture of standard proteins and with normal plasma. Oxidized plasma proteins following H(2)O(2 )exposure and plasma from patients with focal glomerulosclerosis were analyzed as models of altered protein oxido-redox status. Following optimization of the assay (dye/protein ratio, pH), C3NIASO3 and C5NIASO3 gave a sensitivity slightly better than N-hydroxysuccinimidyl dyes for plasma proteins and were successfully employed for differential display electrophoresis (DIGE). Twenty-nine proteins were detected in normal plasma after 2-DE while less proteins were detected in plasma of patients with glomerulosclerosis. Following massive 'in vitro' oxidation with H(2)O(2), C3NIASO3 and C5NIASO3 failed to detect any residual SH, implicating massive oxidation. In conclusion, this study describes the synthesis of two new iodoacetamide cyanines that can be utilized for the analysis of plasma proteins with 2-DE and DIGE. They are also indicated for the definition of the oxido-redox status of proteins and were successfully utilized to extend the analysis of oxidation damage in patients with glomerulosclerosis.

Topics: Blood Proteins; Carbocyanines; Cysteine; Electrophoresis, Polyacrylamide Gel; Fluorescent Dyes; Glomerulosclerosis, Focal Segmental; Humans; Iodoacetamide; Oxidation-Reduction; Proteomics; Reproducibility of Results; Sensitivity and Specificity; Sulfhydryl Compounds

Type A scavenger receptors (Scr) mediate the uptake of modified low-density lipoproteins by macrophages. The accumulation of lipids via this process is thought to lead to foam cell formation in atherosclerotic plaques. Human mesangial cells (HMCs) have not been previously shown to express Scr in normal culture. We therefore investigated whether there is an inducible form of Scr in a human mesangial cell line (HMCL).. Scr activity was analyzed by cellular uptake of fluorescently labeled acetylated low-density lipoprotein using a flow cytometer. Scr mRNA expression was examined using reverse transcription-polymerase chain reaction, followed by Southern blotting. To investigate the molecular mechanism of Scr expression, several reporter gene constructs were designed. The first contained a full Scr promoter, the second a part of the Scr promoter that has both AP-1 and ets transcription factor binding sites. Other constructs were identical to the second, except that they contained either AP-1 or ets motif mutations.. Phorbol 12-Myristate 13-acetate (PMA) and angiotensin II (Ang II) increased both the percentage of Scr-positive cells and the Scr mean fluorescence intensity. PMA and Ang II also increased Scr mRNA and promoter activity in a time- and dose-responsive manner. Protein kinase C and calmodulin transduction pathways were involved in Scr up-regulation induced by PMA and Ang II. Additionally, a serine/threonine kinase was involved in PMA stimulation. Functional analysis showed that both AP-1 and ets motifs were specific response elements to PMA stimulation in HMCLs.. This study suggests that HMCs may express an inducible Scr, by which cells can acquire lipids and convert to foam cells in developing glomerulosclerosis.

Topics: Angiotensin II; Arteriosclerosis; Carbocyanines; Carcinogens; Cell Adhesion Molecules; Cell Line; Cholesterol, LDL; DNA Probes; Dose-Response Relationship, Drug; Fluorescent Dyes; Foam Cells; Gene Expression Regulation; Genes, Reporter; Glomerular Mesangium; Glomerulosclerosis, Focal Segmental; Humans; Mutagenesis; Plasmids; Prolactin; Promoter Regions, Genetic; Receptors, Immunologic; Receptors, Scavenger; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Tetradecanoylphorbol Acetate; Transcription Factor AP-1; Vasoconstrictor Agents