Leptin ameliorates burn-induced multiple organ damage and modulates postburn immune response in rats

The present study was designed to determine whether exogenous leptin reduces remote organ injury in the rats with thermal burn trauma. Leptin (10 microg/kg) or saline was administered intraperitoneally after burn injury, and the rats were decapitated at either 6 or 24 h. Plasma samples of 24-h burn group were assayed for the determination of monocyte and neutrophil apoptosis. Thermal injury increased tissue-associated myeloperoxidase (MPO) activity and microscopic damage scores in the lung, liver, stomach, colon and kidney of both 6- and 24-h burn groups. In the 6-h burn group, leptin reduced microscopic damage score in the liver and kidney only, while damage scores in the 24-h burn group were reduced in all the tissues except the lung. Also, in both burn groups, leptin reduced elevated MPO activity in all tissues except the lung. The percentage of mononuclear cells was significantly reduced at the 24 h of burn injury, while the granulocyte percentage was increased. Leptin treatment, however, had no significant effect on burn-induced reversal of white blood cell ratios. On the other hand, burn-induced increase in the death of mononuclear cells and granulocytes was significantly reduced in leptin-treated rats. The results of the present study suggest that leptin may provide a therapeutic benefit in diminishing burn-induced inflammation and associated multiple organ failure.     

Monitoring proton dissociation and solution conformation of chiral ytterbium complexes with near-IR CD

The ytterbium complex [Yb((S)-THP)](3+) ((S)-THP = (1S,4S,7S,10S-tetrakis(2-hydroxypropyl)-1,4,7,10-tetraazacyclododecane) is investigated in solution through NMR, near-IR absorption, and CD spectroscopy. Quantitative analysis of the paramagnetic pseudocontact NMR shift shows Lambda helicity of the ligand cage around the metal. The NIR CD spectrum recorded at acidic pH is found to be very similar to that of [Yb((R)-DOTMA)](-) ((R)-DOTMA = (1R,4R,7R,10R)-alpha,alpha',alpha',alpha'-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), which in solution assumes a twisted square antiprism (TSA) conformation. The similarity of the NIR CD spectra is discussed, and it is the first proof of the Lambda(lambda,lambda,lambda,lambda) conformation of [Yb((S)-THP)](3+). NIR CD spectra recorded in the pH range of 2-9 allow one to easily follow proton dissociation and to calculate the pK of this equilibrium in water (pK(A) = 6.4 +/- 0.1). This value agrees well with that determined for [Lu((S)-THP)](3+) using potentiometric methods. This demonstrates once again that NIR CD spectroscopy is a powerful technique for investigating the solution structure and dynamics of these complexes.     

Mesenchymal stromal cell supported umbilical cord blood ex vivo expansion enhances regulatory T cells and reduces graft versus host disease

Background aimsDouble cord blood transplantation (DCBT) may shorten neutrophil and platelet recovery times compared with standard umbilical cord blood transplantation. However, DCBT may be associated with a higher incidence of graft versus host disease (GVHD). In this study, we explored the effect of ex vivo expansion of a single cord blood unit (CBU) in a DCBT setting on GVHD and engraftment.MethodsPost-thaw cryopreserved CBUs from cord blood banks, hereinafter termed “banked” CBUs, were co-cultured with confluent bone marrow mesenchymal stromal cells (MSCs) supplemented with a cytokine cocktail comprising 100 ng/mL stem cell factor, 50 ng/mL flt3-ligand, 100 ng/mL thrombopoietin and 20 ng/mL insulin-like growth factor binding protein 2 for 12 days.ResultsWhen DCBT of one unexpanded and one expanded CBU was performed in non-obese diabetic/severe combined immunodeficient-IL2Rgamma^null (NOD/SCID-IL2γ^?/?, NSG) mice, the expanded CBU significantly boosted in vivo hematopoiesis of the unexpanded CBU. The median survival of NSG mice was significantly improved from 63.4% (range, 60.0–66.7%) for mice receiving only unexpanded units to 86.5% (range, 80.0–92.9%) for mice receiving an expanded unit (P < 0.001). The difference in survival appeared to be due to a lower incidence of GVHD in the mice receiving expanded cells. This effect on GVHD was mediated by a significant increase in regulatory T cells seen in the presence of MSC co-culture.ConclusionsMSC-supported ex vivo expansion of “banked” CBU boosted unexpanded CBU hematopoiesis in vivo, increased regulatory T cell content and decreased the incidence of GVHD.     

Active oxygen forms in photoreaction between DNA and furanochromones khellin and visnagin

The two furanochromones khellin and visnagin react with DNA under irradiation by 365 nm light, forming photoadducts. Recently, the use of khellin as therapeutic agent for skin diseases has been proposed. It is well known that during the formation of photoadducts toxic active oxygen forms are produced. We studied therefore the behaviour of the two furanochromones as producers of 1O-2 and O-2. Our results indicate that visnagin is a strong generator of both superoxide radicals and singlet oxygen, while khellin does not exhibit strong production of OO-2, which is promptly quenched by superoxide dismutase.     

Synthesis,Structural Characterization,and Chiroptical Studies of Bidentate Salen‐Type Lanthanide (III) Complexes

The salen‐type ligand prepared with (R,R) diphenylethan‐1,2‐diamine and salicylaldehyde provides stable and inert complexes KLnL₂ upon simple reaction with lanthanide halides or pseudohalides LnX₃ (Ln = Tb³⁺‐Lu³⁺; X = Cl^? or TfO^?) of its potassium salt. All the complexes were completely characterized through nuclear magnetic resonance (NMR), electronic circular dichroism (ECD) in the UV and some (Er³⁺, Tm³⁺, Yb³⁺) also with Near‐IR ECD (NIR‐ECD) and luminescence (Tb³⁺, Tm³⁺). Careful analysis of the NMR shifts demonstrated that the complexes are isostructural in solution and afforded an accurate geometry. This was further confirmed by means of Density Functional Theory (DFT) optimization of the Lu³⁺ complex, and by comparing the ligand‐centered experimental and time‐dependent TD‐DFT computed UV‐ECD spectra. As final validation, we used the NIR‐ECD spectrum of the Yb³⁺ derivative calculated by means of Richardson's equations. The excellent match between calculated and experimental ECD spectra confirm the quality of the NMR structure.  Chirality 27:857–863, 2015. © 2015 Wiley Periodicals, Inc.     

M2 receptor activation inhibits cell cycle progression and survival in human glioblastoma cells

Muscarinic receptors, expressed in several primary and metastatic tumours, appear to be implicated in their growth and propagation. In this work we have demonstrated that M2 muscarinic receptors are expressed in glioblastoma human specimens and in glioblastoma cell lines. Moreover, we have characterized the effects of the M2 agonist arecaidine on cell growth and survival both in two different glioblastoma cell lines (U251MG and U87MG) and in primary cultures obtained from different human biopsies. Cell growth analysis has demonstrated that the M2 agonist arecaidine strongly decreased cell proliferation in both glioma cell lines and primary cultures. This effect was dose and time dependent. FACS analysis has confirmed cell cycle arrest at G1/S and at G2/M phase in U87 cells and U251 respectively. Cell viability analysis has also shown that arecaidine induced severe apoptosis, especially in U251 cells. Chemosensitivity assays have, moreover, shown arecaidine and temozolomide similar effects on glioma cell lines, although IC50 value for arecaidine was significantly lower than temozolomide. In conclusion, we report for the first time that M2 receptor activation has a relevant role in the inhibition of glioma cell growth and survival, suggesting that M2 may be a new interesting therapeutic target to investigate for glioblastoma therapy.     

A Recessive Founder Mutation in Regulator of Telomere Elongation Helicase 1, RTEL1, Underlies Severe Immunodeficiency and Features of Hoyeraal Hreidarsson Syndrome

Dyskeratosis congenita (DC) is a heterogeneous inherited bone marrow failure and cancer predisposition syndrome in which germline mutations in telomere biology genes account for approximately one-half of known families. Hoyeraal Hreidarsson syndrome (HH) is a clinically severe variant of DC in which patients also have cerebellar hypoplasia and may present with severe immunodeficiency and enteropathy. We discovered a germline autosomal recessive mutation in RTEL1, a helicase with critical telomeric functions, in two unrelated families of Ashkenazi Jewish (AJ) ancestry. The affected individuals in these families are homozygous for the same mutation, R1264H, which affects three isoforms of RTEL1. Each parent was a heterozygous carrier of one mutant allele. Patient-derived cell lines revealed evidence of telomere dysfunction, including significantly decreased telomere length, telomere length heterogeneity, and the presence of extra-chromosomal circular telomeric DNA. In addition, RTEL1 mutant cells exhibited enhanced sensitivity to the interstrand cross-linking agent mitomycin C. The molecular data and the patterns of inheritance are consistent with a hypomorphic mutation in RTEL1 as the underlying basis of the clinical and cellular phenotypes. This study further implicates RTEL1 in the etiology of DC/HH and immunodeficiency, and identifies the first known homozygous autosomal recessive disease-associated mutation in RTEL1.     

Phosphorylation of Ser 402 impedes phosphatase activity of slingshot 1

By using mass spectrometry, we have identified Ser 402 as a new phosphorylation site within the catalytic domain of human slingshot 1 (SSH1). Phosphorylation at this site inhibits substrate binding and, thus, phosphatase activity in vitro, resulting in enrichment of phosphorylated cofilin in monolayer cell culture. We further demonstrate that protein kinase D (PKD) is upstream from Ser 402 phosphorylation. Accordingly, expression of active PKD in Drosophila phenotypically mimics the loss of SSH activity by inducing accumulation of phosphorylated cofilin and filamentous actin. We thus identify a universal mechanism by which PKD controls SSH1 phosphatase activity.     

Structure-Function Analysis of MurJ Reveals a Solvent-Exposed Cavity Containing Residues Essential for Peptidoglycan Biogenesis in Escherichia coli

Gram-negative bacteria such as Escherichia coli build a peptidoglycan (PG) cell wall in their periplasm using the precursor known as lipid II. Lipid II is a large amphipathic molecule composed of undecaprenyl diphosphate and a disaccharide-pentapeptide that PG-synthesizing enzymes use to build the PG sacculus. During PG biosynthesis, lipid II is synthesized at the cytoplasmic face of the inner membrane and then flipped across the membrane. This translocation of lipid II must be assisted by flippases thought to shield the disaccharide-pentapeptide as it crosses the hydrophobic core of the membrane. The inner membrane protein MurJ is essential for PG biogenesis and homologous to known and putative flippases of the MOP (multidrug/oligo-saccharidyl-lipid/polysaccharide) exporter superfamily, which includes flippases that translocate undecaprenyl diphosphate-linked oligosaccharides across the cytoplasmic membranes of bacteria. Consequently, MurJ has been proposed to function as the lipid II flippase in E. coli. Here, we present a three-dimensional structural model of MurJ generated by the I-TASSER server that suggests that MurJ contains a solvent-exposed cavity within the plane of the membrane. Using in vivo topological studies, we demonstrate that MurJ has 14 transmembrane domains and validate features of the MurJ structural model, including the presence of a solvent-exposed cavity within its transmembrane region. Furthermore, we present functional studies demonstrating that specific charged residues localized in the central cavity are essential for function. Together, our studies support the structural homology of MurJ to MOP exporter proteins, suggesting that MurJ might function as an essential transporter in PG biosynthesis.