Fig. 1

Cortisol up-regulates AChE-R expression in human CD34⁺ cells. (A) The upstream human ACHE sequence includes clusters of hematopoietic and stress-related motifs. Depicted is the reverse sequence of the cosmid insert (accession no. AF002993) of the human ACHE promoter. The arrow represents the position of a transcription start site. Two potentially relevant regions are shown, one beginning at nucleotide 5267 and one following the first exon (black box). Fully conserved consensus sequences are marked by triangles, and the scale in base pairs (bp) is shown below. Note the presence of a GRE half site and two functionally interdependent sites for binding of hepatic nuclear factor (HNF₃) [based on (40)]. (B) Enrichment of UCB CD34¹ cells. CD34¹ cells were enriched from human UCB cells using bead-attached antibodies to the CD34 protein. Shown is a representative flow cytometry of the recovered cells, demonstrating that 89% express the CD34 antigen. Inset: Example photograph of enriched CD34¹ cells stained by May-Grunwald-Giemsa. Note the large nuclei surrounded by thin rims of cytoplasm, characteristic of stem cells. (C, D) Cortisol stimulates expression of AChE mRNA splicing variants. Shown are CD34¹ cells treated with the noted concentrations of cortisol equivalent to physiologically normal (0.1 µM), mild stress (0.6 µM), and acute stress conditions (1.2 µM). (C) Presented are cytochemically stained cells (top) and pseudocolor representations of 3-dimensional projections created from confocal scanned images of CD34¹ cells following in situ hybridization with the noted 5 9-biotinylated 29O-methyl cRNA probes selective for the synaptic AChE-S and readthrough AChE-R mRNA variants. Note increasing red cytoplasmic labeling under high cortisol levels. Each photograph represents one of 10–20 analyzed cells with deviations in labeling of less than 6%. (D) Shown are the results of semiquantitative image analysis of in situ hybridization for each AChE mRNA transcript under stress-relevant concentrations. Note that only AChE-R mRNA accumulated under moderate cortisol concentrations. Asterisks denote statistical significance (p, 0.05, ANOVA).