Identification and enrichment of colony-forming cells from the adult murine pituitary

Stem and progenitor cells have been identified in many adult tissues including bone marrow, the central nervous system, and skin. While there is direct evidence to indicate the activity of a progenitor cell population in the pituitary gland, this putative subpopulation has not yet been identified. Herein we describe the isolation and characterization of a novel clonogenic cell type in the adult murine pituitary, which we have termed Pituitary Colony-Forming Cells (PCFCs). PCFCs constitute 0.2% of pituitary cells, and generate heterogeneous colonies from single cells. PCFCs exhibit variable proliferative potential, and may exceed 11 population doublings in 14 days. Enrichment of PCFCs to 61.5-fold with 100% recovery can be obtained through the active uptake of the fluorescent dipeptide, beta-Ala-Lys-Nepsilon-AMCA. PCFCs are mostly contained within the large, agranular subpopulation of AMCA+ cells, and constitute 28% of this fraction, corresponding to 140.5-fold enrichment. Interestingly, the AMCA+ population contains rare cells that are GH+ or PRL+. GH+ cells were also identified in PCFC single cell colonies, suggesting that PCFCs have the potential to differentiate into GH+ cells. Together, these data show that the pituitary contains a rare clonogenic population which may correspond to the somatotrope/lactotrope progenitors suggested by previous experiments.     

Analytical precision,biological variation,and mathematical normalization in high data density metabolomics

Metabolic serotypes sensitive to caloric intake may enable sera metabolomic profiles to validate epidemiological parameters and predict disease risk in humans. This long-range goal is complicated by the lack of known state markers and the requirement for simultaneous monitoring of multiple small changes. Therefore, analytical precision for appropriate high data density studies using HPLC separations coupled with coulometric array detectors was evaluated over a two month period in pooled rat sera samples (previously collected and stored at –80 °C), and in authentic biochemical standards. In sera, mean coefficients of variation (CV) of retention time and ratio accuracy within the established metabolic serotype varied within ±1% and ±3%, respectively. In sets of purified standards, the same parameters fluctuated, correspondently, in ranges of ±0.1% and ±1%. Median CV of the metabolite concentrations were ~13% in standards and ~11–19% in sera, and varied non-monotonically with the analytical system status and experimental design. These parameters were shown to be sufficiently controlled so as not to dominate intra-group biological variability in serum metabolomics studies. Continuation of experimental runs across an analytical breakpoint (column replacement) was associated with disproportionate changes in metabolite concentrations, independent of maintained analytical precision. These changes were sufficient to shift overall profile localization in megavariate projection analyses. We developed a mathematical approach to normalize this break and use partial least squares projection to latent structure discriminant analysis to confirm validity of this normalization approach. This generally applicable mathematical correction helps enable longer term high data density studies by removing a critical source of systemic variation.     

Investigation of a potential mechanism for the inhibition of SmTGR by Auranofin and its implications for Plasmodium falciparum inhibition

Schistosoma mansoni and Plasmodium falciparum are pathogen parasites that spend part of their lives in the blood stream of the human host and are therefore heavily exposed to fluxes of toxic reactive oxygen species (ROS). SmTGR, an essential enzyme of the S. mansoni ROS detoxification machinery, is known to be inhibited by Auranofin although the inhibition mechanism has not been completely clarified. Auranofin also kills P. falciparum, even if its molecular targets are unknown. Here, we used computational and docking techniques to investigate the molecular mechanism of interaction between SmTGR and Auranofin. Furthermore, we took advantage of the homology relationship and of docking studies to assess if PfTR, the SmTGR malaria parasite homologue, can be a putative target for Auranofin. Our findings support a recently hypothesized molecular mechanism of inhibition for SmTGR and suggest that PfTR is indeed a possible and attractive drug target in P. falciparum.     

Anaerobic and aerobic relative contribution to total energy release during supramaximal effort in patients with left ventricular dysfunction.

Energetic metabolism during effort is impaired in patients with left ventricular dysfunction (Dysf), but data have been lacking up to now on the relative anaerobic vs. aerobic contribution to total energy release during supramaximal effort. Recently, the maximal accumulated oxygen deficit (MAOD) has been shown to be measurable in Dysf patients, making it possible to evaluate the anaerobic/aerobic interaction under conditions of maximal stress of both anaerobic and aerobic metabolic pathways in this population. Nineteen Dysf patients and 17 normal patients (N) underwent one ramp cardiopulmonary, three moderate-intensity constant-power, and three supramaximal constant-power (1- to 2-min, 2- to 3-min, and 3- to 4-min duration) exercise tests. MAOD was the difference between accumulated O(2) demand (accO(2)dem; estimated from the moderate-intensity O(2) uptake/watt relationship) and uptake during supramaximal tests. Percent anaerobic (%Anaer) and aerobic (%Aer) energetic release were [(MAOD/accO(2)dem).100] and 100 - %Anaer, respectively. MAOD did not vary between 1-2, 2-3, and 3-4 min supramaximal tests, whereas accO(2)dem increased significantly with and was linearly related to test duration in both Dysf and N. Consequently, %Anaer and %Aer decreased and increased, respectively, with increasing test duration but did not differ between Dysf and N in 1-2 min, 2-3 min, and 3-4 min tests. Our study demonstrates a similar relative anaerobic vs. aerobic contribution to total energy release during supramaximal effort in Dysf and N. This finding indicates that energetic metabolism during supramaximal exercise is exercise tolerance independent and that relative anaerobic vs. aerobic contribution in this effort domain remains the same within the physiology- or pathology-induced limits to individual peak exercise performance.     

D-glucose induces microtubular changes in C1300 neuroblastoma cell line through the incorporation of 3-nitro-L-tyrosine into tubulin

The microtubular network of neurons is involved in several functions such as formation and tropism of cellular processes, cell division and intracellular transport. A lot of evidences testify that the microtubular network of neurons can be impaired by oxidative stress. A condition of oxidative stress is often possible when D-glucose overloads its metabolic pathway, resulting in an increase in reactive oxygen species and subsequent neurological disorders. The aim of this work was to check in undifferentiated mouse neuroblastoma cells (C1300) the possible oxidative effects of D-glucose on microtubules. Using a concentration of 110mM D-glucose, cell morphology, growth rate, viability and catalase activity were seriously altered. Noteworthy, an increase in 3-nitro-L-tyrosine and a downregulation of tubulins was found in D-glucose-exposed cells, whereas another cytoskeletal proteins, namely actin, did not show any changes. In conclusion, microtubular network can be impaired by D-glucose through specific nitrosative effects, suggesting a possible mechanism at the basis of hyperglycemia-induced neuronal damage.     

Circulating hematopoietic progenitor cells in runners.

Because endurance exercise causes release of mediators and growth factors active on the bone marrow, we asked whether it might affect circulating hematopoietic progenitor cells (HPCs) in amateur runners [n = 16, age: 41.8 +/- 13.5 (SD) yr, training: 93.8 +/- 31.8 km/wk] compared with sedentary controls (n = 9, age: 39.4 +/- 10.2 yr). HPCs, plasma cortisol, interleukin (IL)-6, granulocyte colony-stimulating factor (G-CSF), and the growth factor fms-like tyrosine kinase-3 (flt3)-ligand were measured at rest and after a marathon (M; n = 8) or half-marathon (HM; n = 8). Circulating HPC counts (i.e., CD34(+) cells and their subpopulations) were three- to fourfold higher in runners than in controls at baseline. They were unaffected by HM or M acutely but decreased the morning postrace. Baseline cortisol, flt3-ligand, IL-6, and G-CSF levels were similar in runners and controls. IL-6 and G-CSF increased to higher levels after M compared with HM, whereas cortisol and flt3-ligand increased similarly postrace. Our data suggest that increased HPCs reflect an adaptation response to recurrent, exercise-associated release of neutrophils and stress and inflammatory mediators, indicating modulation of bone marrow activity by habitual running.