Bone marrow derived cells in the tumour microenvironment contain cells with primitive haematopoietic phenotype

Infiltration of bone marrow derived cells is part of the angiogenic switch required for uncontrolled tumour growth. However, the nature of the tumour-infiltrating cells from bone marrow has not been fully elucidated. To investigate the phenotype of bone marrow derived cells within a tumour, we employed the Lewis lung carcinoma (LLC) murine tumour model. We followed bone marrow derivation of tumour-infiltrating cells through transplantation of CD45.2 bone marrow cells into pre-irradiated CD45.1 mice. We found robust CD45.2 donor type chimerism in bone marrow and blood of CD45.1 recipient tumour-bearing mice. Flow cytometric analysis of LLC tumours showed, in addition to previously described pro-angiogenic CD45⁺VEGFR2⁺‘endothelial progenitor cells’ (EPC), or CD45⁺Tie2⁺‘Tie2-expressing monocytes’ (TEM), incorporation of donor type lineage marker negative (Lin⁻) and Lin⁻Sca1⁺ undifferentiated haematopoietic cell types. Immunohistochemical analysis confirmed the extravasal location of the primitive haematopoietic cells. Flow-cytometric sorting of bone marrow cells and subsequent analysis in haematopoietic colony-forming assays revealed that cells with a Lin⁻Sca1⁺ phenotype, which were initially negative for VEGFR2 and Tie2, gave rise to VEGFR2⁺ and/or Tie2⁺ cells. Moreover, Lin⁻ bone marrow cells pre-labelled with the membrane dye PKH26 (a red fluorochrome) and transplanted i.v. into tumour-bearing mice were found to extravasate and incorporate into LLC tumours within 24 hrs. Thus, primitive haematopoietic precursors which are thought to be precursors of EPC and TEMs, constitute a part of the tumour microenvironment. This makes them an attractive target cell population for tumour-directed cellular therapies.     

Probing the Conformation of a Prion Protein Fibril with Hydrogen Exchange

A fragment of the prion protein, PrP(89–143, P101L), bearing a mutation implicated in familial prion disease, forms fibrils that have been shown to induce prion disease when injected intracerebrally into transgenic mice expressing full-length PrP containing the P101L mutation. In this study, we utilize amide hydrogen exchange measurements to probe the organization of the peptide in its fibrillar form. We determined the extent of hydrogen exchange first by tandem proteolysis, liquid chromatography, and mass spectrometry (HXMS) and then by exchange-quenched NMR. Although single amide resolution is afforded by NMR measurements, HXMS is well suited to the study of natural prions because it does not require labeling with NMR active isotopes. Thus, natural prions obtained from infected animals can be compared with model systems such as PrP(89–143, P101L) studied here. In our study, we find two segments of sequence that display a high level of protection from exchange, residues 102–109 and 117–136. In addition, there is a region that displays exchange behavior consistent with the presence of a conformationally heterogeneous turn. We discuss our data with respect to several structural models proposed for infectious PrP aggregates and highlight HXMS as one of the few techniques well suited to studying natural prions.     

NMR investigation of the dynamics of tryptophan side-chains in hemoglobins

NMR relaxation measurements of 15N spin-lattice relaxation rate (R(1)), spin-spin relaxation rate (R(2)), and heteronuclear nuclear Overhauser effect (NOE) have been carried out at 11.7T and 14.1T as a function of temperature for the side-chains of the tryptophan residues of 15N-labeled and/or (2H,15N)-labeled recombinant human normal adult hemoglobin (Hb A) and three recombinant mutant hemoglobins, rHb Kempsey (betaD99N), rHb (alphaY42D/betaD99N), and rHb (alphaV96W), in the carbonmonoxy and the deoxy forms as well as in the presence and in the absence of an allosteric effector, inositol hexaphosphate (IHP). There are three Trp residues (alpha14, beta15, and beta37) in Hb A for each alphabeta dimer. These Trp residues are located in important regions of the Hb molecule, i.e. alpha14Trp and beta15Trp are located in the alpha(1)beta(1) subunit interface and beta37Trp is located in the alpha(1)beta(2) subunit interface. The relaxation experiments show that amino acid substitutions in the alpha(1)beta(2) subunit interface can alter the dynamics of beta37Trp. The transverse relaxation rate (R(2)) for beta37Trp can serve as a marker for the dynamics of the alpha(1)beta(2) subunit interface. The relaxation parameters of deoxy-rHb Kemspey (betaD99N), which is a naturally occurring abnormal human hemoglobin with high oxygen affinity and very low cooperativity, are quite different from those of deoxy-Hb A, even in the presence of IHP. The relaxation parameters for rHb (alphaY42D/betaD99N), which is a compensatory mutant of rHb Kempsey, are more similar to those of Hb A. In addition, TROSY-CPMG experiments have been used to investigate conformational exchange in the Trp residues of Hb A and the three mutant rHbs. Experimental results indicate that the side-chain of beta37Trp is involved in a relatively slow conformational exchange on the micro- to millisecond time-scale under certain experimental conditions. The present results provide new dynamic insights into the structure-function relationship in hemoglobin.     

Effects of Menadione, Hydrogen Peroxide, and Quercetin on Apoptosis and Delayed Luminescence of Human Leukemia Jurkat T-Cells

Menadione (MD) is an effective cytotoxic drug able to produce intracellularly large amounts of superoxide anion. Quercetin (QC), a widely distributed bioflavonoid, can exert both antioxidant and pro-oxidant effects and is known to specifically inhibit cell proliferation and induce apoptosis in different cancer cell types. We have investigated the relation between delayed luminescence (DL) induced by UV-laser excitation and the effects of MD, hydrogen peroxide, and QC on apoptosis and cell cycle in human leukemia Jurkat T-cells. Treatments with 500 μM H₂O₂ and 250 μM MD for 20 min produced 66.0 ± 4.9 and 46.4 ± 8.6% apoptotic cell fractions, respectively. Long-term (24 h) pre-exposure to 5 μM, but not 0.5 μM QC enhanced apoptosis induced by MD, whereas short-term (1 h) pre-incubation with 10 μM QC offered 50% protection against H₂O₂-induced apoptosis, but potentiated apoptosis induced by MD. Since physiological levels of QC in the blood are normally less than 10 μM, these data can provide relevant information regarding the benefits of flavonoid-combined treatments of leukemia. All the three drugs exerted significant effects on DL. Our data are consistent with (1) the involvement of Complex I of the mitochondrial respiratory chain as an important source of delayed light emission on the 10 μs–10 ms scale, (2) the ability of superoxide anions to quench DL on the 100 μs–10 ms scale, probably via inhibition of reverse electron transfer at the Fe/S centers in Complex I, and (3) the relative insensitivity of DL to intracellular OH^? and H₂O₂ levels.     

Site mutations disrupt inter-helical H-bonds (α14W–α67T and β15W–β72S) involved in kinetic steps in the hemoglobin R→T transition without altering the free energies of oxygenation

Three recombinant mutant hemoglobins (rHbs) of human normal adult hemoglobin (Hb A), rHb (αT67V), rHb (βS72A), and rHb (αT67V, βS72A), have been constructed to test the role of the tertiary intra-subunit H-bonds between α67T and α14W and between β72S and β15W in the cooperative oxygenation of Hb A. Oxygen-binding studies in 0.1 M sodium phosphate buffer at 29 °C show that rHb (αT67V), rHb (βS72A), and rHb (αT67V, βS72A) exhibit oxygen-binding properties similar to those of Hb A. The binding of oxygen to these rHbs is highly cooperative, with a Hill coefficient of approximately 2.8, compared to approximately 3.1 for Hb A. Proton nuclear magnetic resonance (NMR) studies show that rHb (αT67V), rHb (βS72A), rHb (αT67V, βS72A), and Hb A have similar quaternary structures in the α₁β₂ subunit interfaces. In particular, the inter-subunit H-bonds between α42Tyr and β99Asp and between β37Trp and α94Asp are maintained in the mutants in the deoxy form. There are slight perturbations in the distal heme pocket region of the α- and β-chains in the mutants. A comparison of the exchangeable ¹H resonances of Hb A with those of these three rHbs suggests that α67T and β72S are H-bonded to α14W and β15W, respectively, in the CO and deoxy forms of Hb A. The absence of significant free energy changes for the oxygenation process of these three rHbs compared to those of Hb A, even though the inter-helical H-bonds are abolished, indicates that these two sets of H-bonds are of comparable strength in the ligated and unligated forms of Hb A. Thus, the mutations at αT67V and βS72A do not affect the overall energetics of the oxygenation process. The preserved cooperativity in the binding of oxygen to these three mutants also implies that there are multiple interactions involved in the oxygenation process of Hb A.     

Mapping intersubunit interactions of the regulatory subunit (RIalpha) in the type I holoenzyme of protein kinase A by amide hydrogen/deuterium exchange mass spectrometry (DXMS)

Protein kinase A (PKA), a central locus for cAMP signaling in the cell, is composed of regulatory (R) and catalytic (C) subunits. The C-subunits are maintained in an inactive state by binding to the R-subunit dimer in a tetrameric holoenzyme complex (R(2)C(2)). PKA is activated by cAMP binding to the R-subunits which induces a conformational change leading to release of the active C-subunit. Enzymatic activity of the C-subunit is thus regulated by cAMP via the R-subunit, which toggles between cAMP and C-subunit bound states. The R-subunit is composed of a dimerization/docking (D/D) domain connected to two cAMP-binding domains (cAMP:A and cAMP:B). While crystal structures of the free C-subunit and cAMP-bound states of a deletion mutant of the R-subunit are known, there is no structure of the holoenzyme complex or of the cAMP-free state of the R-subunit. An important step in understanding the cAMP-dependent activation of PKA is to map the R-C interface and characterize the mutually exclusive interactions of the R-subunit with cAMP and C-subunit. Amide hydrogen/deuterium exchange mass spectrometry is a suitable method that has provided insights into the different states of the R-subunit in solution, thereby allowing mapping of the effects of cAMP and C-subunit on different regions of the R-subunit. Our study has localized interactions with the C-subunit to a small contiguous surface on the cAMP:A domain and the linker region. In addition, C-subunit binding causes increased amide hydrogen exchange within both cAMP-domains, suggesting that these regions become more flexible in the holoenzyme and are primed to bind cAMP. Furthermore, the difference in the protection patterns between RIalpha and the previously studied RIIbeta upon cAMP-binding suggests isoform-specific differences in cAMP-dependent regulation of PKA activity.     

Arachidonic acid accumulates in the stromal macrophages during thymus involution in diabetes

Diabetes is a debilitating disease with chronic evolution that affects many tissues and organs over its course. Thymus is an organ that is affected early after the onset of diabetes, gradually involuting until it loses most of its thymocyte populations. We show evidence of accumulating free fatty acids with generation of eicosanoids in the diabetic thymus and we present a possible mechanism for the involution of the organ during the disease. Young rats were injected with streptozotocin and their thymuses examined for cell death by flow cytometry and TUNEL reaction. Accumulation of lipids in the diabetic thymus was investigated by histology and electron microscopy. The identity and quantitation of accumulating lipids was done with gas chromatography-mass spectrometry and liquid chromatography. The expression and dynamics of the enzymes were monitored via immunohistochemistry. Diabetes causes thymus involution by elevating the thymocyte apoptosis. Exposure of thymocytes to elevated concentration of glucose causes apoptosis. After the onset of diabetes, there is a gradual accumulation of free fatty acids in the stromal macrophages including arachidonic acid, the substrate for eicosanoids. The eicosanoids do not cause thymocyte apoptosis but administration of a cyclooxygenase inhibitor reduces the staining for ED1, a macrophage marker whose intensity correlates with phagocytic activity. Diabetes causes thymus involution that is accompanied by accumulation of free fatty acids in the thymic macrophages. Excess glucose is able to induce thymocyte apoptosis but eicosanoids are involved in the chemoattraction of macrophage to remove the dead thymocytes.     

V-ATPase B1-subunit promoter drives expression of EGFP in intercalated cells of kidney, clear cells of epididymis and airway cells of lung in transgenic mice

Inward rectifier K⁺ channels (Kir) are a significant determinant of endothelial cell (EC) membrane potential, which plays an important role in endothelium-dependent vasodilatation. In the present study, several complementary strategies were applied to determine the Kir2 subunit composition of human aortic endothelial cells (HAECs). Expression levels of Kir2.1, Kir2.2, and Kir2.4 mRNA were similar, whereas Kir2.3 mRNA expression was significantly weaker. Western blot analysis showed clear Kir2.1 and Kir2.2 protein expression, but Kir2.3 protein was undetectable. Functional analysis of endothelial inward rectifier K⁺ current (I_K) demonstrated that 1) I_K current sensitivity to Ba²⁺ and pH were consistent with currents determined using Kir2.1 and Kir2.2 but not Kir2.3 and Kir2.4, and 2) unitary conductance distributions showed two prominent peaks corresponding to known unitary conductances of Kir2.1 and Kir2.2 channels with a ratio of 4:6. When HAECs were transfected with dominant-negative (dn)Kir2.x mutants, endogenous current was reduced 50% by dnKir2.1 and 85% by dnKir2.2, whereas no significant effect was observed with dnKir2.3 or dnKir2.4. These studies suggest that Kir2.2 and Kir2.1 are primary determinants of endogenous K⁺ conductance in HAECs under resting conditions and that Kir2.2 provides the dominant conductance in these cells. potassium channels; inward rectifier potassium channel