Molecular pathways of early CD105-positive erythroid cells as compared with CD34-positive common precursor cells by flow cytometric cell-sorting and gene expression profiling.

Special attention has recently been drawn to the molecular network of different genes that are responsible for the development of erythroid cells. The aim of the present study was to establish in detail the immunophenotype of early erythroid cells and to compare the gene expression profile of freshly isolated early erythroid precursors with that of the CD34-positive (CD34(+)) compartment. Multiparameter flow cytometric analyses of human bone marrow mononuclear cell fractions (n=20) defined three distinct early erythroid stages. The gene expression profile of sorted early erythroid cells was analyzed by Affymetrix array technology. For 4524 genes, a differential regulation was found in CD105-positive erythroid cells as compared with the CD34(+) progenitor compartment (2362 upregulated genes). A highly significant difference was observed in the expression level of genes involved in transcription, heme synthesis, iron and mitochondrial metabolism and transforming growth factor-ß signaling. A comparison with recently published data showed over 1000 genes that as yet have not been reported to be upregulated in the early erythroid lineage. The gene expression level within distinct pathways could be illustrated directly by applying the Ingenuity software program. The results of gene expression analyses can be seen at the Gene Expression Omnibus repository.

On-line analysis of nitrogen stable isotopes in NO from ambient air samples.

A method was developed for the on-line analysis of nitrogen stable isotopes at the natural abundance level in NO in order to study the NO contribution to the nitrogen cycle in ecosystems and in the atmosphere. The method enables a quick and accurate determination of 15N/14N ratios for NO and consists of the following steps: (a) accumulation of NO from air samples on a molecular sieve of 5 A, (b) desorption of NO from the molecular sieve during 15 min of heating at 350 degrees C (an offset of Deltadelta 4.6% must be corrected for), (c) trapping and cryofocusation of the desorbed NO on a PoraPlot Q matrix at -196 degrees C during heating, (d) release of the trapped NO from the PoraPlot Q matrix followed by chromatographic separation, reduction to N2, and isotopic composition analysis. A minimum sample size of 125 nmol of NO is recommended. A correction function for the calculation of the delta15N-NO values was introduced for sample sizes from 125 to 220 nmol of NO. Measurements of NO in automobile exhaust have proven the applicability of the developed method.

On-Line Analysis of Stable Isotopes of Nitrogen in NH(3), NO, and NO(2) at Natural Abundance Levels.

Methods were developed for the on-line analysis of stable isotopes of nitrogen (at natural abundance levels) in NH(3), NO and NO(2) in order to study the contribution of these trace gases to nitrogen cycling in ecosystems. Standard methods for the combustion of organic substances to N(2) (for the determination of the (15)N/(14)N ratios by mass spectrometry) failed to satisfactorily oxidize or reduce the respective trace gases. The following oxidation or reduction techniques ensured quantitative conversion of the trace gases to N(2) and analytical precision close to the internal precision of the instrument (precision ±0.15‰): (1) oxidation of NH(3) to N(2) at 1150 °C on a NiO surface (which needs a reoxidation before each NH(3) analysis; measuring range 11.7-58.8 nmol of NH(3), precision ±0.30‰); (2) reduction of NO to N(2) at 1150 °C on a Ni surface (measuring range 33.3-133.3 nmol of NO, precision ±0.28‰); (3) reduction of NO(2) to form NO at 420 °C on a Mo surface followed by further treatment as for NO (measuring range 26.0-129.9 nmol of NO(2), precision ±0.90‰). This last technique was developed due to the poor chromatographic properties of NO(2).

Insulin-like growth factor induces phosphorylation of immunoreactive insulin receptor substrate and its association with phosphatidylinositol-3 kinase in human thymocytes.

Insulin receptor substrate 1 (IRS-1) is the principle cellular substrate for insulin and insulin-like growth factor I (IGF-I) receptor signaling. After phosphorylation of tyrosine residues within the YMXM or YXXM motifs, IRS-1 associates with phosphatidylinositol-3 kinase (PI3K). This signaling pathway and the presence of an IRS-1-like molecule have been demonstrated in IRS-1-transfected and in nontransfected hematopoietic cell lines, respectively. IGF-I has been implicated in lymphocyte development and function, and recently, we showed that functional type-I IGF receptors are present on human thymocytes and peripheral T cells. In this study, we addressed IGF-I signal transduction in nontransformed, freshly isolated, human thymocytes, as well as in blood T cells. Using Western blot analysis, we found that IGF-I induced phosphorylation of a 160-180-kD protein (pp170) in human thymocytes and that phosphorylated pp170 becomes associated with PI3K and is recognized by anti-IRS-1. In blood T cells, this immunoreactive IRS-1 (irIRS-1) is less abundantly expressed than in thymocytes, as assessed with immunoblotting. As a consequence, phosphorylated pp170 was not or hardly detectable after stimulation with IGF-I, and irIRS-1 was not detected in PI3K immunoprecipitates from lysates of IGF-I-stimulated T cells. However, IGF-I induced the tyrosine phosphorylation of other cellular proteins, indicating that differential expression of irIRS-1 contributes to a distinct signaling pathway in T cells.