Cumulative mutagenesis of the basic residues in the 201-218 region of insulin-like growth factor (IGF)-binding protein-5 results in progressive loss of both IGF-I binding and inhibition of IGF-I biological action

We have reported previously that mutation of two conserved nonbasic amino acids (G203 and Q209) within the highly basic 201–218 region in the C-terminal domain of IGF-binding protein-5 (IGFBP-5) decreases binding to IGFs. This study reveals that cumulative mutagenesis of the 10 basic residues in this region, to create the C-Term series of mutants, ultimately results in a 15-fold decrease in the affinity for IGF-I and a major loss in heparin binding. We examined the ability of mutants to inhibit IGF-mediated survival of MCF-7 cells and were able to demonstrate that this depended not only upon the affinity for IGF-I, but also the kinetics of this interaction, because IGFBP-5 mutants with similar affinity constants (KD) values, but with different association (Ka) and dissociation (Kd) rate values, had markedly different inhibitory properties. In contrast, the affinity for IGF-I provided no predictive value in terms of the ability of these mutants to enhance IGF action when bound to the substratum. Instead, these C-Term mutants appeared to enhance the actions of IGF-I by a combination of increased dissociation of IGF-IGFBP complexes from the substratum, together with dissociation of IGF-I from IGFBP-5 bound to the substratum. These effects of the IGFBPs were dependent upon binding to IGF-I, because a non-IGF binding mutant (N-Term) was unable to inhibit or enhance the actions of IGF-I. These results emphasize the importance of the kinetics of association/dissociation in determining the enhancing or inhibiting effects of IGFBP-5 and demonstrate the ability to generate an IGFBP-5 mutant with exclusively IGF-enhancing activity

Plasma levels of GH and PRL and concentrations in the fluids of bovine ovarian cysts and follicles

Prolactin and GH have been detected within the ovary, and it has become increasingly evident that they have a role as intrafollicular regulatory factors. The aim of the present work was to gain an insight into the elements influencing intraovarian GH and PRL in bovine species and to see whether cystic degeneration was accompanied by abnormal bovine GH (bGH) and PRL (bPRL) plasma patterns. We followed the relationships between plasma and ovarian fluid bGH and bPRL concentrations over an entire year in Friesian cows whose ovaries showed distinct types of structures. To assess the presence of bGH and bPRL within ovarian cells, we assayed selected ovarian structures by immunohistochemistry. The results demonstrated that: 1) plasma and ovarian fluid hormonal concentrations were independent, and their ratio was independent of the ovarian structure classes, subclasses and period of the year; 2) in the majority of the cows the concentration of bGH in ovarian fluid was no more than 80% of the level in plasma, whereas in about half the animals bPRL concentrations were higher in the ovary than in peripheral plasma; 3) mean bPRL concentrations in ovarian fluids were significantly higher in summer than in winter; 4) immunoreactive bGH and bPRL were present within granulosa and luteal cells. Thus, it is suggested that in the cow bGH and bPRL levels in the ovary might be regulated in some way independently of the pituitary

Anthropometric or ultrasonic measurements in assessment of visceral fat. A comparative study

The main aim of the study (study 1) was to compare the accuracy of anthropometric and ultrasonic measurements in assessing the amount of visceral adipose tissue. An additional aim (study 2) was to verify ultrasound technique precision. Study 1: using computed tomography (CT) L4-L5 adipose tissue area as a gold standard we compared the accuracy of waist/hip circumference ratio, sagittal diameter and ultrasonic measurements of intra-abdominal depth in assessing the amount of visceral adipose tissue. Study 2: ultrasonic measurements of the intra-abdominal muscle-vertebra distance were made in triplicate by three different operators. In study 1, 24 volunteers were used; body mass index (BMI): 19-43. In study 2, 22 volunteers were used; BMI 20-42. In study 1, ultrasonic measurements of the abdominal depth correlated best with CT visceral adipose tissue area (r = 0.89-0.91). In study 2, inter-operator and intra-operator mean variation coefficients were about 7% and 5% respectively. We concluded that using a well standardized technique and properly trained operators, ultrasonic measurement of intra-abdominal depth is a valid method in assessing the amount of visceral adipose tissue

Specific amino acid substitutions determine the differential contribution of the N- and C-terminal domains of insulin-like growth factor (IGF)-binding protein-5 in binding IGF-I

We have previously reported that two highly conserved amino acids in the C-terminal domain of rat insulin-like growth factor-binding protein (IGFBP)-5, Gly203 and Gln209, are involved in binding to insulin-like growth factor (IGF)-1. Here we report that mutagenesis of both amino acids simultaneously (C-Term mutant) results in a cumulative effect and an even greater reduction in IGF-I binding: 30-fold measured by solution phase IGF binding assay and 10-fold by biosensor analysis. We compared these reductions in ligand binding to the effects of specific mutations of five amino acids in the N-terminal domain (N-Term mutant), which had previously been shown by others to cause a very large reduction in IGF-I binding (1). Our results confirm this as the major IGF-binding site. To prove that the mutations in either N- or C-Term were specific for IGF-I binding, we carried out CD spectroscopy and showed that these alterations did not lead to gross conformational changes in protein structure for either mutant. Combining these mutations in both domains (N+C-Term mutant) has a cumulative effect and leads to a 126-fold reduction in IGF-I binding as measured by biosensor. Furthermore, the equivalent mutations in the C terminus of rat IGFBP-2 (C-Term 2) also results in a significant reduction in IGF-I binding, suggesting that the highly conserved Gly and Gln residues have a conserved IGF-I binding function in all six IGFBPs. Finally, although these residues lie within a major heparin-binding site in IGFBP-5 and -3, we also show that the mutations in C-Term have no effect on heparin binding