Theres Jägerbrink

Differential protein expression in pancreatic islets after treatment with an imidazoline compound

Abstract.The effects of an imidazoline compound (BL11282) on protein expression in rat pancreatic islets were investigated with a proteomic approach. The compound increases insulin release selectively at high glucose concentrations and is therefore of interest in type 2 diabetes. Whole cell extracts from isolated drug-treated and native pancreatic rat islets were compared after separation by 2-D gel electrophoresis. Differentially expressed proteins were identified by mass spectrometry; 15 proteins were selectively up-regulated and 7 selectively down-regulated in drug-treated islets. Of special interest among the differentially expressed proteins are those involved in protein folding (Hsp60, protein disulfide isomerase, calreticulin), Ca2+ binding (calgizzarin, calcyclin and annexin I) and metabolism or signalling (pyruvate kinase, alpha enolase and protein kinase C inhibitor 1).

Proteome Analysis of Vernix Caseosa

Vernix caseosa (vernix) is a white creamy substance covering the skin of the fetus during the last trimester of pregnancy. The function of vernix has long been debated but no consensus has been reached. We here report a proteome analysis of vernix using two-dimensional gel electrophoresis, matrix-assisted laser desorption/ionization mass spectrometry and liquid chromatography coupled to tandem mass spectrometry. We have identified 41 proteins, of which 25 are novel to vernix. Notably, 39% of the identified vernix proteins are components of innate immunity, and 29% have direct antimicrobial properties. These results form a substantial contribution to the knowledge of vernix composition and demonstrate that antimicrobial protection of the fetus and the newborn child is a major and important function of vernix.

Proteome patterns in uremic plasma.

In patients with chronic kidney disease (CKD), peptides and proteins circulate at altered concentrations versus in healthy individuals. We have characterized proteome samples from 7 pooled CKD stage 5 patients not yet on dialysis and with no known co-morbidities. We also analyzed pooled plasma samples from 7 healthy age- and sex-matched controls. After immunodepletion of the 6 most abundant plasma proteins, HPLC and SDS-PAGE patterns differed between the healthy and disease groups. The differing proteins were identified by peptide mass fingerprinting using MALDI mass spectrometry and verified with electrospray tandem mass spectrometry sequence analysis. Multiple differences in at least 19 HPLC fractions were observed, from which we identified 29 proteins, 25 in greater yield and 4 in lower yield than in the healthy controls, adding at least 6 protein components to those that were previously known to be altered in CKD.

Proinsulin C-peptide interaction with protein tyrosine phosphatase 1B demonstrated with a labeling reaction.

Based on nickel-catalyzed cross-labeling where binding partners become biotinylated, we have studied molecular interactions with an N-terminally fused GGH-tag proinsulin C-peptide. Since C-peptide has been reported to influence phosphatase activity in intact cells, we employed this method to study possible binding of the peptide to protein tyrosine phosphatase 1B (PTP-1B). C-peptide was found to interact with PTP-1B (and for control, also with antibodies to C-peptide), as did also the N- and C-terminal fragments of C-peptide which have sequence similarities with PTP-1B binding proteins. The labeling data combined with enzyme activity analysis indicate a functional interaction between acidic regions of C-peptide and specific sites of PTP-1B. Results highlight the importance of possible phosphatase/C-peptide roles in diabetes, and the usefulness of the cross-labeling reaction also for acidic peptides like C-peptide.

Proteins in the insulin-secreting cell line MIN6 bind the imidazoline compound BL11282

The imidazoline BL11282 stimulates insulin release and alters islet proteomes. Subcellular fractions of MIN6 cells showed that the membrane fraction exhibited binding to BL11282 on a Biacore chip and to BL11282‐labelled magnetic beads. Bound material extracted from the beads showed a ∼50 kDa differential band upon SDS–PAGE and a weaker 100 kDa band. The former was sensitive to competitive removal by preincubation of the fraction with BL11282, then highlighting the ∼100 kDa band. Masspectrometric analysis revealed the ∼50 kDa band to be EF1A and the ∼100 kDa band to be glucose regulated P94, both of interest in insulin synthesis and secretion.

Differential hemoglobin A sequestration between hemodialysis modalities

Abstract This report evaluates plasma protein patterns, dialysates and protein analysis of used dialysis membranes from the same patient under hemodialysis in three separate modalities, using high-flux membranes in concentration-driven transport (HD), convection-driven hemofiltration (HF) and combined hemodialfiltration (HDF). The plasma protein changes induced by each of the three dialysis modalities showed small differences in proteins identified towards our previous plasma analyses of chronic kidney disease (CKD) patients. The used dialysate peptide concentrations likewise exhibited small differences among the modalities and varied in the same relative order as the plasma changes, with protein losses in the order HD>HDF>HF. The membrane protein deposits allowed quantification of the relative Hb removal ratios as ~1.7 for HD and ~1.2 for HDF vs. ~1.0 for HF. Hence, plasma protein alterations, dialysate peptide contents and membrane Hb deposits all identify HD as the modality with the most extensive filtration results and exemplifies the accessibility of protein analysis of used membrane filters for evaluation of dialysis efficiencies.This report evaluates plasma protein patterns, dialysates and protein analysis of used dialysis membranes from the same patient under hemodialysis in three separate modalities, using high-flux membranes in concentration-driven transport (HD), convection-driven hemofiltration (HF) and combined hemodialfiltration (HDF). The plasma protein changes induced by each of the three dialysis modalities showed small differences in proteins identified towards our previous plasma analyses of chronic kidney disease (CKD) patients. The used dialysate peptide concentrations likewise exhibited small differences among the modalities and varied in the same relative order as the plasma changes, with protein losses in the order HD>HDF>HF. The membrane protein deposits allowed quantification of the relative Hb removal ratios as ~1.7 for HD and ~1.2 for HDF vs. ~1.0 for HF. Hence, plasma protein alterations, dialysate peptide contents and membrane Hb deposits all identify HD as the modality with the most extensive filtration results and exemplifies the accessibility of protein analysis of used membrane filters for evaluation of dialysis efficiencies.

Contents Vol. 26, 2008

163 13th International Conference on Continuous Renal Replacement Therapies (CRRT) February 27–March 1, 2008, San Diego, Calif. Editor: Mehta, R.L. (San Diego, Calif.) (Available online only)