Functional and total IGFBP3 for the assessment of disorders of the GH/IGF1 axis in children with chronic kidney disease, GH deficiency, or short stature after SGA status at birth.

OBJECTIVE: IGFBP3 immunoreactivity may appear elevated in patients with chronic kidney disease (CKD), in part due to accumulation of low molecular fragments. The importance of these IGFBP3 variants for binding and inactivation of IGF1 and their relevance for the impaired growth of uremic children are unclear. Nevertheless, IGFBP3, measured as total (t-)IGFBP3, is frequently used as a diagnostic parameter in pediatric CKD patients. A new assay for functional (f-)IGFBP3 exclusively detects IGFBP3 capable of IGF binding. The aim of the study was to evaluate the significance of f-IGFBP3 measurements for the assessment of uremic abnormalities of the GH/IGF1 axis. DESIGN: Prospective cross-sectional study. METHODS: t-IGFBP3, f-IGFBP3, and IGF1 were measured in pediatric CKD patients, including patients with CKD stage 3-4 not on dialysis (CKD, n=33), on dialysis treatment (DT, n=26), patients after renal transplantation (RTx, n=89), healthy children (n=29), children with GH deficiency (GHD, n=42), and small for gestational age (SGA) children (SGA, n=34). RESULTS: Mean t-IGFBP3 SDS was elevated in CKD, DT, and RTx children compared with controls and GHD patients (P≤0.0004). Highest values were reached in DT (P<0.0001 vs all groups). In contrast, mean f-IGFBP3 was similar in all groups (P=0.30). CONCLUSIONS: Pediatric CKD patients displayed elevated serum concentrations of t-IGFBP3 but not f-IGFBP3, supporting the hypothesis that IGFBP3 fragments not binding IGF1 accumulate during uremia. f-IGFBP3 is an indicator of IGFBP3 fragmentation and seems to reflect IGF1 binding in CKD better than t-IGFBP3. However, the role of f-IGFBP3 for the diagnosis of disturbances of the GH/IGF hormonal axis appears to be limited.

Validation of functional insulin-like growth factor binding protein-3 measurement by a ligand immunoassay.

BACKGROUND: Proteolysis of Insulin-like Growth Factor Binding Protein (IGFBP)--3 is a well known mechanism regulating IGF-I bioavailability and IGF-independent actions of IGFBP-3 fragments. Measurement of functional IGFBP-3 can be of use in diagnostics of growth failure or renal impairment. We herein characterize the properties of a commercially available immunoassay for the measurement of functional (IGF-I binding) IGFBP-3. METHOD: Fragmentation of IGFBP-3 is analyzed by gel filtration, SDS-PAGE, and western ligand and immunoblotting and compared with subsequent measurement of total and functional IGFBP-3 by ELISA/IFA. Furthermore, assay characteristics such as reproducibility, linearity, and sensitivity are surveyed. RESULTS: Functional IGFBP-3 was reproducibly measured (6.8/5.6% Inter-/Intra assay variance). A broad range of linearity (1:50-1:300) and a high sensitivity (0.18 microg/L) allowed reliable measurement of IGF-binding IGFBP-3. Analysis of IGFBP-3 fragments reveals that the assay described only detects intact IGFBP-3. Analysis of 189 serum samples from healthy blood donors showed that on average 84% and 69% of total IGFBP-3 was functional in men and women, respectively (p < 0.01). CONCLUSIONS: Functional IGFBP-3 can be measured reliably by the assay system used. Thus, this assay system is suited for the investigation of the diagnostic value of functional IGFBP-3 in human body fluids.

Amino acid preferences in the octapeptide subunit of the major histocompatibility complex class I heterotrimer H-2Ld.

Major histocompatibility complex class I (MHC-I) molecules are heterotrimers composed of polymorphic alpha-chains, monomorphic beta-chains, and peptides of eight or nine amino acids. The peptides are derived from various intracellularly occurring proteins and are very heterogeneous. They are essential for a stable conformation of the MHC-I protein at physiological temperature. This study presents results from stabilization experiments that were designed to determine the impact of the amino acids in every sequence position of octapeptides on the thermal stability of the mouse MHC-I molecule H2-Ld. OX7 octapeptide libraries with one defined and seven randomized positions were employed as they allow the effects of individual amino acids to be determined. The results confirm the importance of the motif amino acids proline and leucine for positions 2 and 8, respectively, of octapeptides. They are among the most efficient amino acids for these positions. However, with a few exceptions, all amino acids are permitted in all eight sequence positions. Hydrophobic amino acids are generally favored. Charged amino acids, especially aspartic acid and glutamic acid, are disfavored. Stabilization indices were defined as measures for the MHC stabilization power of the amino acids. These indices can serve to predict the efficiency of peptide binding to H-2Ld and can guide the design of T-cell epitopes.

Identification of the yeast ACC1 gene product (acetyl-CoA carboxylase) as the target of the polyketide fungicide soraphen A.

Soraphen A, a polyketide isolated from the myxobacterium Sorangium cellulosum, is a potent inhibitor of fungal growth. We have used a genetic approach to localize the target of this drug, employing Saccharomyces cerevisiae as a model organism. we have isolated soraphen A-resistant mutants and found that all of them map at the same genetic locus and exhibit a broad range of semidominant phenotypes. Data from genetic crosses of soraphen A-resistant clones with an acc1 mutant revealed that ACC1, coding for acetyl-CoA carboxylase (E.C. 6.4.1.2), is tightly linked to soraphen A resistance. Partially-purified enzyme extracts containing acetyl-CoA carboxylase were prepared and assayed for their soraphen A sensitivity. Our experiments showed that the catalytic activity of the wild-type enzyme is inhibited in vitro by soraphen A while the mutant enzyme remains catalytically active. Taken together these data strongly suggest that the ACC1 gene product is the primary target for soraphen A in vivo.