Identification of a Potent Endothelium-Derived Angiogenic Factor

The secretion of angiogenic factors by vascular endothelial cells is one of the key mechanisms of angiogenesis. Here we report on the isolation of a new potent angiogenic factor, diuridine tetraphosphate (Up₄U) from the secretome of human endothelial cells. The angiogenic effect of the endothelial secretome was partially reduced after incubation with alkaline phosphatase and abolished in the presence of suramin. In one fraction, purified to homogeneity by reversed phase and affinity chromatography, Up₄U was identified by MALDI-LIFT-fragment-mass-spectrometry, enzymatic cleavage analysis and retention-time comparison. Beside a strong angiogenic effect on the yolk sac membrane and the developing rat embryo itself, Up₄U increased the proliferation rate of endothelial cells and, in the presence of PDGF, of vascular smooth muscle cells. Up₄U stimulated the migration rate of endothelial cells via P2Y2-receptors, increased the ability of endothelial cells to form capillary-like tubes and acts as a potent inducer of sprouting angiogenesis originating from gel-embedded EC spheroids. Endothelial cells released Up₄U after stimulation with shear stress. Mean total plasma Up₄U concentrations of healthy subjects (N = 6) were sufficient to induce angiogenic and proliferative effects (1.34±0.26 nmol L^-1). In conclusion, Up₄U is a novel strong human endothelium-derived angiogenic factor.     

Diversity of Plant Methionine Sulfoxide Reductases B and Evolution of a Form Specific for Free Methionine Sulfoxide

Methionine can be reversibly oxidized to methionine sulfoxide (MetO) under physiological conditions. Organisms evolved two distinct methionine sulfoxide reductase families (MSRA & MSRB) to repair oxidized methionine residues. We found that 5 MSRB genes exist in the soybean genome, including GmMSRB1 and two segmentally duplicated gene pairs (GmMSRB2 and GmMSRB5, GmMSRB3 and GmMSRB4). GmMSRB2 and GmMSRB4 proteins showed MSRB activity toward protein-based MetO with either DTT or thioredoxin (TRX) as reductants, whereas GmMSRB1 was active only with DTT. GmMSRB2 had a typical MSRB mechanism with Cys121 and Cys 68 as catalytic and resolving residues, respectively. Surprisingly, this enzyme also possessed the MSRB activity toward free Met-R-O with kinetic parameters similar to those reported for fRMSR from Escherichia coli, an enzyme specific for free Met-R-O. Overexpression of GmMSRB2 or GmMSRB4 in the yeast cytosol supported the growth of the triple MSRA/MSRB/fRMSR (Δ3MSRs) mutant on MetO and protected cells against H₂O₂-induced stress. Taken together, our data reveal an unexpected diversity of MSRBs in plants and indicate that, in contrast to mammals that cannot reduce free Met-R-O and microorganisms that use fRMSR for this purpose, plants evolved MSRBs for the reduction of both free and protein-based MetO.     

LPS Unmasking of Shigella flexneri Reveals Preferential Localisation of Tagged Outer Membrane Protease IcsP to Septa and New Poles

The Shigella flexneri outer membrane (OM) protease IcsP (SopA) is a member of the enterobacterial Omptin family of proteases which cleaves the polarly localised OM protein IcsA that is essential for Shigella virulence. Unlike IcsA however, the specific localisation of IcsP on the cell surface is unknown. To determine the distribution of IcsP, a haemagglutinin (HA) epitope was inserted into the non-essential IcsP OM loop 5 using Splicing by Overlap Extension (SOE) PCR, and IcsP^HA was characterised. Quantum Dot (QD) immunofluorescence (IF) surface labelling of IcsP^HA was then undertaken. Quantitative fluorescence analysis of S. flexneri 2a 2457T treated with and without tunicaymcin to deplete lipopolysaccharide (LPS) O antigen (Oag) showed that IcsP^HA was asymmetrically distributed on the surface of septating and non-septating cells, and that this distribution was masked by LPS Oag in untreated cells. Double QD IF labelling of IcsP^HA and IcsA showed that IcsP^HA preferentially localised to the new pole of non-septating cells and to the septum of septating cells. The localisation of IcsP^HA in a rough LPS S. flexneri 2457T strain (with no Oag) was also investigated and a similar distribution of IcsP^HA was observed. Complementation of the rough LPS strain with rmlD resulted in restored LPS Oag chain expression and loss of IcsP^HA detection, providing further support for LPS Oag masking of surface proteins. Our data presents for the first time the distribution for the Omptin OM protease IcsP, relative to IcsA, and the effect of LPS Oag masking on its detection.     

Effect of Urea and Thiourea on Migration Activity of Climbing Perch Anabas testudineus

Journal of Ichthyology - The influence of urea and thiourea on the dynamics of the migration activity of climbing perch Anabas testudineus was studied. The exposure of climbing perch to 0.05%...     

Harvesting and seeding of stochastic populations: analysis and numerical approximation

Journal of Mathematical Biology - We study an ecosystem of interacting species that are influenced by random environmental fluctuations. At any point in time, we can either harvest or seed...     

Variable Spontaneous Mutation and Loss of Heterozygosity among Heterozygous Genomes in Yeast

Mutation and recombination are the primary sources of genetic variation. To better understand the evolution of genetic variation, it is crucial to comprehensively investigate the processes involving mutation accumulation and recombination. In this study, we performed mutation accumulation experiments on four heterozygous diploid yeast species in the Saccharomycodaceae family to determine spontaneous mutation rates, mutation spectra, and losses of heterozygosity (LOH). We observed substantial variation in mutation rates and mutation spectra. We also observed high LOH rates (1.65–11.07×10⁻⁶ events per heterozygous site per cell division). Biases in spontaneous mutation and LOH together with selection ultimately shape the variable genome-wide nucleotide landscape in yeast species.     

Lenalidomide overcomes suppression of human natural killer cell anti-tumor functions by neuroblastoma microenvironment-associated IL-6 and TGFβ1

Background

Treatment for children with high-risk neuroblastoma with anti-disialoganglioside mAb ch14.18, IL-2, and GM-CSF plus 13-cis-retinoic acid after myeloablative chemotherapy improves survival, but 40 % of patients still relapse during or after this therapy. The microenvironment of high-risk neuroblastoma tumors includes macrophages, IL-6, and TGFβ1. We hypothesized that this microenvironment suppresses anti-tumor functions of natural killer (NK) cells and that lenalidomide, an immune-modulating drug, could overcome suppression.

Methods

Purified NK cells were cultured with IL-2, neuroblastoma/monocyte-conditioned culture medium (CM), IL-6, TGFβ1, and lenalidomide in various combinations and then characterized using cytotoxicity (direct and antibody-dependent cell-mediated cytotoxicity), cytokine, flow cytometry, and Western blotting assays. Anti-tumor activity of NK cells with lenalidomide, ch14.18, or both was evaluated with a xenograft model of neuroblastoma.

Results

CM from neuroblastoma/monocyte co-cultures contains IL-6 and TGFβ1 that suppress IL-2 activation of NK cell cytotoxicity and IFNγ secretion. IL-6 and TGFβ1 activate the STAT3 and SMAD2/3 pathways in NK cells and suppress IL-2 induction of cytotoxicity, granzymes A and B release, perforin expression, and IFNγ secretion. Lenalidomide blocks IL-6 and TGFβ1 activation of these signaling pathways and inhibits their suppression of NK cells. Neuroblastoma cells in NOD/SCID mice exhibit activated STAT3 and SMAD2/3 pathways. Their growth is most effectively inhibited by co-injected peripheral blood mononuclear cells (PBMC) containing NK cells when mice are treated with both ch14.18 and lenalidomide.

Conclusion

Immunotherapy with anti-tumor cell antibodies may be improved by lenalidomide, which enhances activation of NK cells and inhibits their suppression by IL-6 and TGFβ1.     

Probing antioxidant activity of 2′-hydroxychalcones: Crystal and molecular structures, in vitro antiproliferative studies and in vivo effects on glucose regulation

In order to better understand the antioxidant behavior of a series of polyphenolic 2′-hydroxychalcones, we describe the results of several chemical and biological studies, in vitro and in vivo. Single crystal X-ray methods elucidated their molecular structures and important intermolecular interactions such as H-bonding and molecular stacking in the crystal structures that contribute to our knowledge in explaining antioxidant activity. The results of experiments using the 1,1-diphenyl-2-dipicrylhydrazyl (DPPH) UV–vis spectroscopic method indicate that a hydroxyl group in position 5′ induces the highest antioxidant activity. Consequently, 2,2′,5′-trihydroxychalcone was selected for further study in vitro towards ROS scavenging in L-6 myoblasts and THP-1 human monocytes, where it shows an excellent antioxidant activity in a concentration range lower than that reported by most studies of related molecules. In addition, this chalcone shows a very selective activity: it inhibits the proliferation of leukemic cells, but it does not affect the normal L-6 myoblasts and human fibroblasts. In studying 2,2′,5′-trihydroxychalcone's effect on weight gain and serum glucose and insulin levels in Zucker fatty (fa⁻/fa⁻) rats we found that supplementing the diet with a 10 mg/kg dose of this chalcone (3 times weekly) blunted the increase in glucose that co-occurs with weight gain over the 6-week treatment period. It is concluded that 2,2′,5′-trihydroxychalcone has the potential to serve as a protective agent for some debilitating diseases.     

Liposomal Delivery Systems for Encapsulation of Ferrous Sulfate: Preparation and Characterization

Liposomal delivery systems for water-soluble bioactives were prepared using the pro-liposome and the microfluidization technologies. Iron, an essential micronutrient as ferrous sulfate and ascorbic acid, as an antioxidant for iron were encapsulated in the liposomes. Liposomes prepared by the microfluidization technology using 6% (w/w) concentration of the lipid encapsulated with ferrous sulfate and ascorbic acid had particle size distributions around 150 to 200 nm, whereas liposomes from the pro-liposome technology resulted in particle sizes of about 5 μm. The encapsulation efficiency of ferrous sulfate was 58% for the liposomes prepared by the microfluidization using 6% (w/w) lipid and 7.5% of ferrous sulfate concentrations, and it was 11% for the liposomes from pro-liposome technology using 1.5% (w/v) lipid and 15% of ferrous-sulfate concentration. Both the liposomes exhibited similar levels of oxidative stability, demonstrating the feasibility of microfluidization-based liposomal delivery systems for large-scale food/nutraceutical applications.