Differential removal of insulin-like growth factor binding proteins in rat serum by solvent extraction procedures

Solvent extraction of serum and other biological fluids at an acidic pH is a convenient method to remove the insulin-like growth factor binding proteins (IGFBPs); however, an incomplete removal of IGFBPs can occur and this can potentially interfere with the radioimmunoassay of insulin-like growth factors (IGFs). This study compared the removal of IGFBPs from normal adult rat serum and 5-day old neonatal rat serum by acid-gel filtration, and three solvent extraction methods, i.e., acid-ethanol (AE), acid-cryo-ethanol (ACE) and formic acid-acetone (FAA) treatments by western ligand blotting and slot-blotting analysis. In adult rat serum all three extraction methods removed nearly 75% of total IGFBPs present. For the neonatal serum, AE and FAA were very inefficient in eliminating the IGFBPs, while ACE was somewhat better, as it removed nearly 30% of IGFBPs. Ligand blots of extracted samples showed that IGFBPs of lower size range, 24 to 32 kDa (IGFBP-4, IGFBPs-1 and-2), were resistant to solvent extraction. Acid-gel filtration, in contrast, eliminated >95% of IGF-binding components in both sera. Determination of IGF-I concentrations in samples after gel filtration and extraction methods revealed lower IGF-I values in neonatal serum in acid extracted samples. These data caution against using solvent extractions for IGFBP removal in fetal/neonatal serum.     

Glucose does not influence the insulin-like growth factor (IGF) binding to carrier proteins (IGFBPs): Analysis of rat and human serum by western ligand blotting

The insulin-like growth factors (IGFs) circulate bound to specific proteins (termed IGFBP-1 through IGFBP-6) that modulate IGF bioactivity in tissues. The aim of this study was to analyse the effects of glucose on IGF binding to IGFBPs in rat and human serum by means of western ligand blotting. Serum samples were incubated with increasing concentrations of glucose (0 to 50 mmol/l), and EDTA (25 mmol/l) was added to inhibit protease activity. To analyse the effect of glucose on protection of IGFBPs from protease activity, serum from pregnant women (reported to be very rich in proteolytic activity against IGFBPs) was added to rat serum previously incubated with glucose. Glucose did not affect the¹²⁵I-IGF binding to rat and human serum IGFBPs. The intensity of IGFBP-3 bands decreased considerably during the incubation. This appeared to be due to endogenous protease activity, since the decrease was blocked by addition of EDTA. The incubationi of rat serum with pregnant human serum produced a marked attenuation of IGFBP-3 and disappearance of IGFBP-4 bands. In conclusion, our study shows that glucose does not influence the IGF binding to IGFBP-3 either in rat or in human serum, confirms the presence of endogenous proteolytic activity in normal non-pregnant serum, and demonstrates that glucose has no protective action against protease activity.     

A 30-kDa fragment of insulin-like growth factor (IGF) binding protein-3 in human pregnancy serum with strongly reduced IGF-I binding

Proteolytic cleavage of insulin-like growth factor (IGF) binding protein (IGFBP)-3 during pregnancy is likely to have both IGF-dependent and -independent effects on maternal, placental and fetal growth and metabolism. A 30-kDa proteolytic IGFBP-3 fragment was isolated from third trimester pregnancy human serum and identified by N- and C-terminal amino acid sequence analysis and mass spectrometry to correspond to residues 1–212 of the parent protein. This fragment is the dominating IGFBP-3 immunoreactive species in pregnancy serum. The 30-kDa fragment was also detected in serum of non-pregnant women where it coexists with intact IGFBP-3. Using biosensor technology, (1–212)IGFBP-3 was found to have 11-fold lower affinity for IGF-I compared to intact IGFBP-3, while a 4-fold decrease in affinity was found for IGF-II. Tests with des(1–3)IGF-I suggest fast binding of IGF-I to the N-terminal region of IGFBP-3 and similar affinity to a slow binding site in the C-terminal region. Received 24 April 2007; received after revision 11 June 2007; accepted 13 June 2007     

SNX3-dependent regulation of epidermal growth factor receptor (EGFR) trafficking and degradation by aspirin in epidermoid carcinoma (A-431) cells

Since being introduced globally as aspirin in 1899, acetylsalicylic acid has been widely used as an analgesic, anti-inflammation, anti-pyretic, and anti-thrombotic drug for years. Aspirin had been reported to down-regulate surface expression of CD40, CD80, CD86, and MHCII in myeloid dendritic cells (DC), which played essential roles in regulating the immune system. We hypothesized that the down-regulation of these surface membrane proteins is partly due to the ability of aspirin in regulating trafficking/sorting of endocytosed surface membrane proteins. By using an established epidermoid carcinoma cell line (A-431), which overexpresses the epidermal growth factor receptor (EGFR) and transferrin receptor (TfnR), we show that aspirin (1) reduces cell surface expression of EGFR and (2) accumulates endocytosed-EGFR and -TfnR in the early/sorting endosome (ESE). Further elucidation of the mechanism suggests that aspirin enhances recruitment of SNX3 and SNX5 to membranes and consistently, both SNX3 and SNX5 play essential roles in the aspirin-mediated accumulation of endocytosed-TfnR at the ESE. This study sheds light on how aspirin may down-regulate surface expression of EGFR by inhibiting/delaying the exit of endocytosed-EGFR from the ESE and recycling of endocytosed-EGFR back to the cell surface.     

Production of passive cutaneous anaphylaxis (PCA) and reversed PCA by rat IgE antibody in the mouse

Summary Although IgE antibody is generally characterized as a homocytotropic antibody, it has been well known for some time that mouse IgE antibody causes potent sensitization of rat skin for PCA. The present study clearly shows, the reciprocal cross-sensitization of mouse skin with rat IgE molecules. PCA and RPCA were produced by rat IgE antibody in an inbred mouse strain, DS/Shi, though not in C3H/HeShi, C57BL/6JShi and BALB/cCrj strains. Sensitization of DS/Shi mouse skin for PCA with rat IgE antibody was comparable in sensitivity with that of rat skin, but lasted only for a short term in comparison with the long persistence in rat skin.