Differentiation-associated changes of cation-transport activities in myeloid leukemic cell lines

AbstractInduction of differentiation in HL-60 and U-937 leukemic cell lines, resulted in 1.5–10-fold increase in 45Ca2+ uptake. The increased 45Ca2+ uptake in the differentiating cells was inhibited by verapamil, cromolyn and amiloride. Elevation in Ca2+ uptake in differentiating cells was also demonstrated using the fluorescent probe, fura-2 acetoxymethyl ester. The increased 45Ca2+ uptake was accompanied by a decrease in ouabain-insensitive and -sensitive 86Rb+ uptake. Furthermore, correlation between the changes in these activities was observed. Modulation of extracellular pH affected differentiation: higher pH increased the extent of differentiation

Carbon Monoxide Induced Erythroid Differentiation of K562 Cells Mimics the Central Macrophage Milieu in Erythroblastic Islands

Growing evidence supports the role of erythroblastic islands (EI) as microenvironmental niches within bone marrow (BM), where cell-cell attachments are suggested as crucial for erythroid maturation. The inducible form of the enzyme heme oxygenase, HO-1, which conducts heme degradation, is absent in erythroblasts where hemoglobin (Hb) is synthesized. Yet, the central macrophage, which retains high HO-1 activity, might be suitable to take over degradation of extra, harmful, Hb heme. Of these enzymatic products, only the hydrophobic gas molecule - CO can transfer from the macrophage to surrounding erythroblasts directly via their tightly attached membranes in the terminal differentiation stage

Mechanochemistry of single red blood cells monitored using Raman tweezers

Two microparticles were biochemically attached to a red blood cell at diametrically opposite parts and held by optical traps allowing to impose deformations. The cell deformation was monitored from the microscopy images. Raman spectra of the cell under tunable deformations were studied. Vibrational spectra analysis at different stretching states was supported with two statistical methods. Principal Component Analysis distinguishes the most prominent changes in spectra while 2D correlation technique monitors the evolution of Raman bands during stretching. The measurements show significant changes in the cell chemical structure with stretching however the changes saturate above 20% of cell deformation. Mechanical deformation of the cell mainly affects the bands corresponding to hemoglobin but contributions from spectrin and membrane proteins can not be excluded. The saturation of bands at higher deformations suggests some structural relaxation that RBC has to undergo to bear extra load. The results confirm widely accepted belief that spectrin released from membrane proteins allows for significant shape changes of the cells. We therefore tentatively suggest that interaction between membrane and cytoskeleton during deformation can be efficiently probed by confocal Raman spectroscopy, in particular via the peak around 1035 cm−1

External antigen uptake by Langerhans cells with reorganization of epidermal tight junction barriers

Outermost barriers are critical for terrestrial animals to avoid desiccation and to protect their bodies from foreign insults. Mammalian skin consists of two sets of barriers: stratum corneum (SC) and tight junctions (TJs). How acquisition of external antigens (Ags) by epidermal Langerhans cells (LCs) occur despite these barriers has remained unknown. We show that activation-induced LCs elongate their dendrites to penetrate keratinocyte (KC) TJs and survey the extra-TJ environment located outside of the TJ barrier, just beneath the SC. Penetrated dendrites uptake Ags from the tip where Ags colocalize with langerin/Birbeck granules. TJs at KC–KC contacts allow penetration of LC dendrites by dynamically forming new claudin-dependent bicellular- and tricellulin-dependent tricellular TJs at LC–KC contacts, thereby maintaining TJ integrity during Ag uptake. Thus, covertly under keratinized SC barriers, LCs and KCs demonstrate remarkable cooperation that enables LCs to gain access to external Ags that have violated the SC barrier while concomitantly retaining TJ barriers to protect intra-TJ environment

Carbon Monoxide Promotes Respiratory Hemoproteins Iron Reduction Using Peroxides as Electron Donors

The physiological role of the respiratory hemoproteins (RH), hemoglobin and myoglobin, is to deliver O2 via its binding to their ferrous (FeII) heme-iron. Under variety of pathological conditions RH proteins leak to blood plasma and oxidized to ferric (FeIII, met) forms becoming the source of oxidative vascular damage. However, recent studies have indicated that both metRH and peroxides induce Heme Oxygenase (HO) enzyme producing carbon monoxide (CO). The gas has an extremely high affinity for the ferrous heme-iron and is known to reduce ferric hemoproteins in the presence of suitable electron donors. We hypothesized that under in vivo plasma conditions, peroxides at low concentration can assist the reduction of metRH in presence of CO. The effect of CO on interaction of metRH with hydrophilic or hydrophobic peroxides was analyzed by following Soret and visible light absorption changes in reaction mixtures. It was found that under anaerobic conditions and low concentrations of RH and peroxides mimicking plasma conditions, peroxides served as electron donors and RH were reduced to their ferrous carboxy forms. The reaction rates were dependent on CO as well as peroxide concentrations. These results demonstrate that oxidative activity of acellular ferric RH and peroxides may be amended by CO turning on the reducing potential of peroxides and facilitating the formation of redox-inactive carboxyRH. Our data suggest the possible role of HO/CO in protection of vascular system from oxidative damage

COMPARISON OF MODIFICATION SITES FORMED ON HUMAN SERUM ALBUMIN AT VARIOUS STAGES OF GLYCATION

Background—Many of the complications encountered during diabetes can be linked to the nonenzymatic glycation of proteins, including human serum albumin (HSA). However, there is little information regarding how the glycation pattern of HSA changes as the total extent of glycation is varied. The goal of this study was to identify and conduct a semi-quantitative comparison of the glycation products on HSA that are produced in the presence of various levels of glycation. Methods—Three glycated HSA samples were prepared in vitro by incubating physiological concentrations of HSA with 15 mmol/l glucose for 2 or 5 weeks, or with 30 mmol/l glucose for 4 weeks. These samples were then digested and examined by matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF MS) to identify the glycation products that were formed. Results—It was found that the glycation pattern of HSA changed with its overall extent of total glycation. Many modifications including previously-reported primary glycation sites (e.g., K199, K281, and the N-terminus) were consistently found in the tested samples. Lysines 199 and 281, as well as arginine 428, contained the most consistently identified and abundant glycation products. Lysines 93, 276, 286, 414, 439, and 524/525, as well as the N-terminus and arginines 98, 197, and 521, were also found to be modified at various degrees of HSA glycation. Conclusions—The glycation pattern of HSA was found to vary with different levels of total glycation and included modifications at the 2 major drug binding sites on this protein. This result suggests that different modified forms of HSA, both in terms of the total extent of glycation and glycation pattern, may be found at various stages of diabetes. The clinical implication of these results is that the binding of HSA to some drug may be altered at various stages of diabetes as the extent of glycation and types of modifications in this protein are varied

Expression of basic fibroblast growth factor is associated with poor outcome in non-Hodgkin's lymphoma

It is now clear that angiogenesis and angiogenesis factors are important in the pathogenesis of haematological malignancies. High pretreatment levels of serum basic fibroblast growth factor have been shown to be associated with poor prognosis in patients with non-Hodgkin's lymphoma. The aim of this study was to evaluate whether non-Hodgkin's lymphoma cells express basic fibroblast growth factor and/or its receptor (fibroblast growth factor receptor-1) and whether basic fibroblast growth factor expression correlates with basic fibroblast growth factor serum levels, intratumoral microvessel density, and patient outcome. We measured basic fibroblast growth factor by enzyme-linked immunosorbent assay in sera taken from 58 patients with non-Hodgkin's lymphoma before treatment and in 19 of them also after treatment. Pathological specimens at diagnosis were evaluated by immunohistochemistry staining using polyoclonal antibody against factor-VIII-related antigen, basic fibroblast growth factor and fibroblast growth factor receptor-1 to determine the expression of the microvessel count and basic fibroblast growth factor and fibroblast growth factor receptor-1. The lymphoma specimens demonstrated positive staining for basic fibroblast growth factor (in 23%) and fibroblast growth factor receptor-1 (in 58.5%). The patients who expressed basic fibroblast growth factor had a significantly worse progression-free and overall survival than those who did not (P=0.003 and P=0.03 respectively), while patients expressing fibroblast growth factor receptor-1 were less likely to achieve complete remission than those lacking the receptor (33% vs 65% , P=0.047). There was no correlation of basic fibroblast growth factor staining with either serum basic fibroblast growth factor levels or microvessel count. Basic fibroblast growth factor serum levels did not change significantly after treatment These results suggest that non-Hodgkin's lymphoma specimens express basic fibroblast growth factor and its receptor (fibroblast growth factor receptor-1) and this expression is associated with poor patient outcome