Psychosine-induced apoptosis and cytokine activation in immune peripheral cells of Krabbe patients

Globoid cell leukodystrophy or Krabbe disease (KD), is a hereditary disorder caused by galactosylceramidase deficiency. Progressive accumulation of psychosine is considered to be the critical pathogenetic mechanism of cell death in the Krabbe brain. Psychosine mechanism of action has not been fully elucidated. It seems to induce apoptosis in oligodendrocytes through a mitochondrial pathway and to up-regulate inflammatory cytokines production resulting in oligodendrocyte loss. Our aim was to evaluate the role of psychosine in apoptotic cell death and inflammatory response in a group of patients affected by KD using peripheral blood lymphocytes (PBLs) and peripheral blood mononuclear cells (PBMCs) as a cellular model. PBLs from KP and healthy controls were exposed to 20 microM psychosine and analysed by flow cytometry, agarose gel electrophoresis and fluorescence microscopy. Our results showed that psychosine induces apoptosis in PBLs through a mitochondrial pathway, but the apoptotic response was quite low especially KP. The role of psychosine in the up-regulation of cytokines (TNFalpha, IL8 and MCP1) has been evaluated by ELISA in PBMCs from KP and controls after stimulation with LPS and phytohemagglutinin. Both in basal condition and after LPS stimulation, cells from KP showed a significant increase in TNF-alpha production, reduced MCP1 levels and no modification in IL8. These results indicate that lymphomonocytes from KP had a basal proinflammatory pattern that was amplified by psychosine. In conclusion, the reduced apoptotic response and the atypical cytokine production observed in our experiments, suggest an involvement of inflammatory pattern in immune peripheral cells of KP.     

Structural characterization of the ceruloplasmin: lactoferrin complex in solution

Ceruloplasmin is a copper protein found in vertebrate plasma, which belongs to the family of multicopper oxidases. Like transferrin of the blood plasma, lactoferrin, the iron-containing protein of human milk, saliva, tears, seminal plasma and of neutrophilic leukocytes tightly binds two ferric ions. Human lactoferrin and ceruloplasmin have been previously shown to interact both in vivo and in vitro forming a complex. Here we describe a study of the conformation of the human lactoferrin/ceruloplasmin complex in solution using small angle X-ray scattering. Our ab initio structural analysis shows that the complex has a 1:1 stoichiometry and suggests that complex formation occurs without major conformational rearrangements of either protein. Rigid-body modeling of the mutual arrangement of proteins in the complex essentially yields two families of solutions. Final discrimination is possible when integrating in the modeling process extra information translating into structural constraints on the interaction between the two partners.     

One drug for two targets: Biological evaluation of antiretroviral agents endowed with antiproliferative activity

AIDS-related cancer diseases are malignancies with low incidence on healthy people that affect mostly subjects already immunocompromised. The connection between HIV/AIDS and these cancers has not been established yet, but a weakened immune system is certainly the main cause. We envisaged the possibility to screen a small library of compounds synthesized in our laboratory against opportunistic tumors mainly due to HIV infection like Burkitt’s Lymphoma. From cellular assays and gene expression analysis we identified two promising compounds. These derivatives have the dual action required inhibiting HIV replication in human TZM-bl cells infected with HIV-1 NL4.3 and showing cytotoxic activity on human colon HT-29 and breast adenocarcinoma MCF-7 cells. In addition, preclinical in vitro adsorption, distribution, metabolism, and excretion studies highlighted a satisfactory pharmacokinetic profile.     

Changes in mean erythrocyte volume and 2,3-diphosphoglycerate in two groups of diabetic subjects

Mean corpuscular volume and Red Blood Cell 2-3, diphosphoglycerate in insulin dependent diabetics and in non insulin dependent diabetics were evaluated. Only in insulin dependent diabetics an increased mean corpuscular volume was found while in non insulin dependent diabetics red blood cell 2-3, DPG level appears to be reduced. These findings were not correlated with the metabolic parameters neither they seemed to be dependent upon sex, age or vascular disease. The Authors suggest that the increase of mean corpuscular volume might be indicative of a tendency to macromegaloblastosis in insulin dependent diabetics; with regard to RBC 2-3, DPG level it seems to be extremely variable in dependence on oxygen request at the tissue level.     

Effect of stent coating alone on in vitro vascular smooth muscle cell proliferation and apoptosis

Synthetic polymers, like methacrylate (MA) compounds, have been clinically introduced as inert coatings to locally deliver drugs that inhibit restenosis after stent. The aim of the present study was to evaluate the effects of MA coating alone on vascular smooth muscle cell (VSMC) growth in vitro. Stainless steel stents were coated with MA at the following doses: 0.3, 1.5, and 3 ml. Uncoated/bare metal stents were used as controls. VSMCs were cultured in dishes, and a MA-coated stent or an uncoated bare metal stent was gently added to each well. VSMC proliferation was assessed by bromodeoxyuridine (BrdU) incorporation. Apoptosis was analyzed by three distinct approaches: 1) annexin V/propidium iodide fluorescence detection; 2) DNA laddering; and 3) caspase-3 activation and PARP cleavage. MA-coated stents induced a significant decrease of BrdU incorporation compared with uncoated stents at both the low and high concentrations. In VSMCs incubated with MA-coated stents, annexin V/propidium iodide fluorescence detection showed a significant increase in apoptotic cells, which was corroborated by the typical DNA laddering. Apoptosis of VSMCs after incubation with MA-coated stents was characterized by caspase-3 activation and PARP cleavage. The MA-coated stent induced VSMC growth arrest by inducing apoptosis in a dose-dependent manner. Thus MA is not an inert platform for eluting drugs because it is biologically active per se. This effect should be taken in account when evaluating an association of this coating with antiproliferative agents for in-stent restenosis prevention.     

Structural stability of soybean lipoxygenase-1 in solution as probed by small angle X-ray scattering

Soybean lipoxygenase-1 (LOX-1) is used widely as a model for studying the structural and functional properties of the homologous family of lipoxygenases. The crystallographic structure revealed that LOX-1 is organized in a beta-sheet N-terminal domain and a larger, mostly helical, C-terminal domain. Here, we describe the overall structural characterization of native unliganded LOX-1 in solution, using small angle X-ray scattering (SAXS). We show that the scattering pattern of the unliganded enzyme in solution does not display any significant difference compared with that calculated from the crystal structure, and that models of the overall shape of the protein calculated ab initio from the SAXS pattern provide a close envelope to the crystal structure. These data, demonstrating that LOX-1 has a compact structure also in solution, rule out any major motional flexibility of the LOX-1 molecule in aqueous solutions. In addition we show that eicosatetraynoic acid, an irreversible inhibitor of lipoxygenase used to mimic the effect of substrate binding, does not alter the overall conformation of LOX-1 nor its ability to bind to membranes. In contrast, the addition of glycerol (to 5%, v/v) causes an increase in the binding of the enzyme to membranes without altering its catalytic efficiency towards linoleic acid nor its SAXS pattern, suggesting that the global conformation of the enzyme is unaffected. Therefore, the compact structure determined in the crystal appears to be essentially preserved in these various solution conditions. During the preparation of this article, a paper by M. Hammel and co-workers showed instead a sharp difference between crystal and solution conformations of rabbit 15-LOX-1. The possible cause of this difference might be the presence of oligomers in the rabbit lipoxygenase preparations.     

Activated c-Kit receptor in the heart promotes cardiac repair and regeneration after injury

The role of endogenous c-Kit receptor activation on cardiac cell homeostasis and repair remains largely unexplored. Transgenic mice carrying an activating point mutation (TgD814Y) in the kinase domain of the c-Kit gene were generated. c-Kit^TgD814Y receptor was expressed in the heart during embryonic development and postnatal life, in a similar timing and expression pattern to that of the endogenous gene, but not in the hematopoietic compartment allowing the study of a cardiac-specific phenotype. c-Kit^TgD814Y mutation produced a constitutive active c-Kit receptor in cardiac tissue and cells from transgenic mice as demonstrated by the increased phosphorylation of ERK1/2 and AKT, which are the main downstream molecular effectors of c-Kit receptor signaling. In adult transgenic hearts, cardiac morphology, size and total c-Kit⁺ cardiac cell number was not different compared with wt mice. However, when c-Kit^TgD814Y mice were subjected to transmural necrotic heart damage by cryoinjury (CI), all transgenic survived, compared with half of wt mice. In the sub-acute phase after CI, transgenic and wt mice showed similar heart damage. However, 9 days after CI, transgenic mice exhibited an increased number of c-Kit⁺CD31⁺ endothelial progenitor cells surrounding the necrotic area. At later follow-up, a consistent reduction of fibrotic area, increased capillary density and increased cardiomyocyte replenishment rate (as established by BrdU incorporation) were observed in transgenic compared with wt mice. Consistently, CD45⁻c-Kit⁺ cardiac stem cells isolated from transgenic c-Kit^TgD814Y mice showed an enhanced endothelial and cardiomyocyte differentiation potential compared with cells isolated from the wt. Constitutive activation of c-Kit receptor in mice is associated with an increased cardiac myogenic and vasculogenic reparative potential after injury, with a significant improvement of survival.c-Kit is a tyrosine kinase receptor essential for proliferation, survival and migration of several stem cell types such as melanocyte precursors, hematopoietic and germ stem cells.^{1, 2, 3, 4} More recently, c-Kit receptor was reported to be expressed in cardiac and neuronal stem cells.^{5, 6} Mice lacking c-Kit gene present germ cell and melanocyte defects and die in the first days of postnatal life because of impaired hematopoiesis.^{7, 8} The binding of c-Kit ligand (KL) induces receptor homodimerization and autophosphorylation of the intracellular tyrosine kinase domains leading to the modulation of different signaling pathways such as AKT and MAPKs.^{9, 10, 11}In the past 15 years, several studies have shown that c-Kit⁺ cardiac stem cells (CSCs) have beneficial effects in cardiac repair and regeneration.¹² Genetically mutant mice deficient in c-Kit signaling (W/Wv) show a worsened cardiac remodeling after myocardial infarction, conversely transgenic mice overexpressing KL in the heart exhibit an improved myocardial repair compared with their wt littermates after myocardial infarction.^{13, 14, 15} Increased expression of KL occurring in ischemic heart favors migration of c-Kit⁺ bone marrow-derived and CSCs via activation of p38 MAPK.^{16, 17, 18} Furthermore, c-Kit⁺ CSCs have been recently used in the treatment of patients with ischemic cardiomyopathy and heart failure with significant improvement in systolic function and a reduction in infarct size.^{19, 20, 21}Although c-Kit is widely used as a ''cell surface marker'' to identify stem cells from the embryonic as well as adult heart,^{22, 23} little information^{14, 15} is available on the function that c-Kit signaling has in the activation of CSCs in cardiac repair.To investigate the role of c-Kit in heart repair, transgenic mice were generated carrying a mutated version of c-Kit gene. The substitution of tyrosine for aspartic acid 814 in the phosphotransferase domain leads to constitutive activation of the receptor. Decreased fibrotic area in cryoinjured hearts, reduced inflammatory myeloid cells in the blood, increased number of c-Kit⁺CD31⁺ endothelial cells and isolectin B4 (IB-4)-labeled capillaries as well as BrdU-positive newly formed cardiomyocytes in damaged cardiac area of transgenic mice were observed. MAPK and AKT activation was significantly enhanced in the hearts and CSCs of transgenic mice, whereby the two kinases modulate the activation and endothelial/myogenic differentiation of CSCs. Overall, these data indicate that the activated c-Kit receptor exerts a beneficial protective/regenerative role for myocardial tissue after injury improving cardiac remodeling and repair while fostering differentiation of cardiac progenitor cells likely due to MAPK and AKT signaling activation.