No abstract

The measurement of serum insulin-like growth factors (IGFs) in serum is complicated by the presence of high affinity IGF-binding proteins. The accurate measurement of IGFs by radioligand binding assays requires that the interference from binding proteins be eliminated. Acid-gel chromatography, the standard method for removing binding proteins, is laborious and time consuming. Alternative methods for extracting serum IGFs include the use of HCl-ethanol treatment and reverse phase minicolumns. However, these methods are unsuitable for use with serum for some species, such as rat and sheep, due to incomplete removal of binding proteins. We developed a fast protein liquid chromatography size-exclusion chromatographic method for characterizing the presence of IGF-binding proteins in physiological fluids and used this method to systematically investigate different combinations of acids and organic solvents as potential extraction methods for IGFs. We developed and validated an improved extraction procedure that uses formic acid, Tween-20, and acetone. The new extraction method was used in conjunction with purified biosynthetic human IGF-II and a commercially available anti-IGF-II monoclonal antibody in the development of an improved RIA for IGF-II. The new RIA is sensitive (5.0 pg/tube), specific (IGF-I cross-reactivity, less than 1%), and reproducible [interassay precision (coefficient of variation), less than 9.2%). We measured the serum concentrations of IGF-II in adults and found a significant difference between normal subjects and individuals with insulin-dependent diabetes mellitus.

Pluripotential stem cells (PSCs) have been recently described in many tissues including skeletal muscle, brain, and bone marrow. However, the true nature of these cells is still unclear, and their precise definition has yet to be determined. We hypothesized that a common, rare population of PSCs with a broad tissue differentiation potential can be identified in multiple murine tissues and that these cells are capable of transdifferentiation into cells of different primordial germ layer origins in response to diverse microenvironmental cues. To examine this hypothesis, we isolated phenotypically defined cells from murine skeletal muscle and cultured these cells under different conditions tailored to promote differentiation into several cell types including myocytes. We report here that in conditions permissive for hematopoietic differentiation, muscle-derived CD45(-)Sca-1(+)c-kit(-) cells differentiated into cells expressing hematopoietic-specific mRNA; while in conditions promoting myogenic, neuronal, and adipocytic differentiation, cells morphologically typical of these cell types expressing tissue-specific markers were identified 9-14 days in culture. When CD45(-)Sca-1(+)c-kit(-) cells from muscle or bone marrow were transplanted intracerebellarly into Purkinje cell degenerative (pcd) mice, the behavior of these mice improved 28 days after transplantation relative to mice injected with vehicle alone, suggesting that these cells contributed to the appearance of functional neuronal cells that may have improved the ataxic condition characteristic of these mice. Phenotypic analysis of single cell suspensions prepared from brain, blood, and intestinal epithelium revealed the presence of CD45(-)Sca-1(+)c-kit(-) cells in varying degrees. These studies suggest that a phenotypically common, multipotent cell can be identified in different tissues and that this cell may represent a universal pluripotent stem cell residing at different levels in multiple murine tissues.

Hypoxia-ischemia induces apoptotic and necrotic cell death, which results partially from persistent alterations in cellular energy homeostasis. Insulin-like growth factor I (IGF-I) is an anabolic pleiotrophic factor required by developing neurons for their optimal proliferation, differentiation, and survival. To determine how cell death and changes in IGF-I gene expression relate to the extent of hypoxia-ischemia, we evaluated the time course of apoptosis in a neonatal hypoxia-ischemia model in relation to the cellular distribution of IGF-I and IGFBP5 mRNA. Severe hypoxia-ischemia results in an immediate decrease in neuronal IGF-I and IGFBP5 mRNA. The decrease in neuronal IGF-I mRNA was concurrent with an increase in the number of apoptotic cells. It is conceivable that the immediate decrease in IGF-I gene expression may contribute to the impending neuronal death and selective vulnerability of myelinogenesis during the perinatal period.