Characterization of extracellular vesicle miRNA identified in peripheral blood of chronic pancreatitis patients

Molecular and Cellular Biochemistry - Plasma-derived extracellular vesicles (EV) can serve as markers of cell damage/disease but can also have therapeutic utility depending on the nature of their...     

Effect of Circulating Forms of Soy Isoflavones on the Oxidation of Low Density Lipoprotein

Soy isoflavones are thought to have a cardioprotective effect that is partly mediated by an inhibitory influence on the oxidation of low density lipoprotein (LDL). However, the aglycone forms investigated in many previous studies do not circulate in appreciable quantities because they are metabolised in the gut and liver. We investigated effects of various isoflavone metabolites, including for the first time the sulphated conjugates formed in the liver and the mucosa of the small intestine, on copper-induced LDL oxidation. The parent aglycones inhibited oxidation, although only 5% as well as quercetin. Metabolism increased or decreased their effectiveness. Equol inhibited 2.65-fold better than its parent compound daidzein and 8-hydroxydaidzein, not previously assessed, was 12.5-fold better than daidzein. However, monosulphated conjugates of genistein, daidzein and equol were much less effective and disulphates completely ineffective. Since almost all isoflavones circulate as conjugates, these data suggest that despite the increased potency produced by some metabolic changes, isoflavones may not be effective antioxidants in vivo unless they are deconjugated again.     

Evaluation of activity of selected antioxidants on proteins in solution and in emulsions

Protection against protein oxidation by lipophilic and hydrophilic antioxidants in model systems using bovine serum albumin (BSA) in solution alone, or in an emulsion with linolenic acid methyl ester (LnMe) was found to be strongly dependent on the oxidation initiator. Tocopherol, Trolox, or the carotenoids astaxanthin and canthaxanthin were incubated with BSA or BSA/LnMe and oxidation was initiated either with the water-soluble azo-initiator 2,2′ azo-bis-(2-amidinopropane) hydrochloride (AAPH), or FeCl₃ and ascorbate, or the Fenton system using FeCl₂/EDTA/H₂O₂, or with the singlet oxygen generating species anthracene-9,10-dipropionic acid disodium 1,4 endoperoxide (NDPO₂).

The results show that all the antioxidants tested were inefficient in the system with FeCl₃/ascorbate. However, with the other initiating agents, the hydrophilic antioxidant, Trolox, was the most effective in preventing both protein and lipid oxidation. In contrast the lipophilic antioxidants were ineffective in preventing oxidation of BSA in aqueous solution, but did show some moderate antioxidative activity on protein and lipid in the BSA/LnMe system. Using the singlet oxygen generating system it was also demonstrated that Trolox always provided better protection of the protein than tocopherol and the carotenoids in both the BSA and the BSA/LnMe systems. In conclusion, prevention of protein oxidation using a water-soluble antioxidant has a protective effect on the lipid fraction and this approach deserves further attention in complex biological systems.     

Impact of bone marrow-derived mesenchymal stromal cells on experimental xenogeneic graft-versus-host disease

Background aimsGraft-versus-host disease (GVHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation caused by donor T cells reacting against host tissues. Previous studies have suggested that mesenchymal stromal cells (MSCs) could exert potent immunosuppressive effects.MethodsThe ability of human bone marrow derived MSCs to prevent xenogeneic GVHD in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice and in NOD/SCID/interleukin-2Rγ(null) (NSG) mice transplanted with human peripheral blood mononuclear cells (PBMCs) was assessed.ResultsInjection of 200 × 10⁶ human PBMCs intraperitoneally (IP) into sub-lethally (3.0 Gy) irradiated NOD/SCID mice also given anti-asialo GM₁ antibodies IP 1 day prior and 8 days after transplantation induced lethal xenogeneic GVHD in all tested mice. Co-injection of 2 × 10⁶ MSCs IP on day 0 did not prevent lethal xenogeneic GVHD induced by injection of human PBMCs. Similarly, injection of 30 × 10⁶ human PBMCs IP into sub-lethally (2.5 Gy) irradiated NSG mice induced a lethal xenogeneic GVHD in all tested mice. Injection of 3 × 10⁶ MSCs IP on days 0, 7, 14 and 21 did not prevent lethal xenogeneic GVHD induced by injection of human PBMCs.ConclusionsInjection of MSCs did not prevent xenogeneic GVHD in these two humanized mice models.     

Kinetics of IL-7 and IL-15 Levels after Allogeneic Peripheral Blood Stem Cell Transplantation following Nonmyeloablative Conditioning

Background

We analysed kinetics of IL-7 and IL-15 levels in 70 patients given peripheral blood stem cells after nonmyeloablative conditioning.

Methods

EDTA-anticoagulated plasma and serum samples were obtained before conditioning and about once per week after transplantation until day 100. Samples were aliquoted and stored at −80°C within 3 hours after collection until measurement of cytokines. IL-7 and IL-15 levels were measured by ELISAs.

Results

Median IL-7 plasma levels remained below 6 pg/L throughout the first 100 days, although IL-7 plasma levels were significantly higher on days 7 (5.1 pg/mL, P = 0.002), 14 (5.2 pg/mL, P<0.001), and 28 (5.1 pg/mL, P = 0.03) (but not thereafter) than before transplantation (median value of 3.8 pg/mL). Median IL-15 serum levels were significantly higher on days 7 (12.5 pg/mL, P<0.001), 14 (10.5 pg/mL, P<0.001), and 28 (6.2 pg/mL, P<0.001) than before transplantation (median value of 2.4 pg/mL). Importantly, IL-7 and IL-15 levels on days 7 or 14 after transplantation did not predict grade II–IV acute GVHD.

Conclusions

These data suggest that IL-7 and IL-15 levels remain relatively low after nonmyeloablative transplantation, and that IL-7 and IL-15 levels early after nonmyeloablative transplantation do not predict for acute GVHD.     

Two-Dimensional 1H NMR Study of a Tetradecapeptide with the Consensus Sequence Arg5-Asp-Val-Arg-Gly9: Structural Effects of the Outside Substitution Ser12 by Ala12

Abstract

Conformation of a tetradecapeptide with a RXVRG consensus sequence, Arg^s-Asp-Val-Arg-Gly⁹, found in several precursors of antibacterian peptides, was investigated in dimethylsulfoxide solution by proton NMR spectroscopy. Complete resonance assignments and conformational parameters were obtained through correlated (COSY) and nuclear Overhauser (NOESY) techniques. The ³J(αH, βH) coupling constants and the intramolecular NOE, NH…βH, were used to analyse the conformers around the Cα-Cβ bond and, in four cases, to obtain stereospecific assignments.

Use of restraints derived from NOE connectivities and ³J(NH, αH) coupling constants allows the determination of a range of φ and ψ dihedral angles for all the residues in the sequence. The present NMR results provide favourable evidence for the formation of two bends in the consensus sequence of the tetradecapeptide. The first one has most of the features of a Glu⁴- Val⁷ β–turn (low temperature coefficient of the Val⁷NH chemical shift, Arg⁵αH…Val⁷NH and Asp⁶NH.-.Val⁷NH NOE correlations). The second one exhibits only the Asp⁶αH…Arg⁷NH and Val⁷NH…Arg⁸NH NOE interactions. These consensus sequence organizations proposed were confirmed by molecular modeling based on low potential energy structure on the [4–9] fragment with high agreement of NOE data.

Overall, the substitution of Ser¹² by Ala¹² shifts the conformation of the hydrophobic moiety [10–14] towards a quite random coil structure in this fragment and strongly destabilizes the folded structures of the consensus domain where only one NH (Val⁷) is solvent-shielded opposed to three (Asp⁶ to Arg⁸) in the [Ser¹²] tetradecapeptide. These conformational changes could be related to the processing enzyme activities on these model oligopeptides.     

Tethered Spectroscopic Probes Estimate Dynamic Distances with Subnanometer Resolution in Voltage-Dependent Potassium Channels

Measurements of inter- and intramolecular distances are important for monitoring structural changes and understanding protein interaction networks. Fluorescence resonance energy transfer and functionalized chemical spacers are the two predominantly used strategies to map short-range distances in living cells. Here, we describe the development of a hybrid approach that combines the key advantages of spectroscopic and chemical methods to estimate dynamic distance information from labeled proteins. Bifunctional spectroscopic probes were designed to make use of adaptable-anchor and length-varied spacers to estimate molecular distances by exploiting short-range collisional electron transfer. The spacers were calibrated using labeled polyproline peptides of defined lengths and validated by molecular simulations. This approach was extended to estimate distance restraints that enable us to evaluate the resting-state model of the Shaker potassium channel.     

The Middle Jurassic stephanoceratid ammonite Kumatostephanus from Dorset and Somerset,UK, and its evolution

A new exposure at Oborne, Dorset has yielded a rich fossil assemblage of the Lower Bajocian ammonite Kumatostephanus from the Laeviuscula and Humphriesianum zones. The principal bed containing this ammonite, the Green Grained White Marl is locally diachronous; it is an important local marker horizon and it is herein proposed as a formal stratigraphical unit. The specimens obtained allow a better understanding of the evolution of the genus Kumatostephanus and its relationships to the other stephanoceratids, notably Stemmatoceras.     

Novel imino sugar α-glucosidase inhibitors as antiviral compounds

Deoxynojirimycin (DNJ) based imino sugars display antiviral activity in the tissue culture surrogate model of Hepatitis C (HCV), bovine viral diarrhoea virus (BVDV), mediated by inhibition of ER α-glucosidases. Here, the antiviral activities of neoglycoconjugates derived from deoxynojirimycin, and a novel compound derived from deoxygalactonojirimycin, by click chemistry with functionalised adamantanes are presented. Their antiviral potency, in terms of both viral infectivity and virion secretion, with respect to their effect on α-glucosidase inhibition, are reported. The distinct correlation between the ability of long alkyl chain derivatives to inhibit ER α-glucosidases and their anti-viral effect is demonstrated. Increasing alkyl linker length between DNJ and triazole groups increases α-glucosidase inhibition and reduces the production of viral progeny RNA and the maturation of the envelope polypeptide. Disruption to viral glycoprotein processing, with increased glucosylation on BVDV E2 species, is representative of α-glucosidase inhibition, whilst derivatives with longer alkyl linkers also show a further decrease in infectivity of secreted virions, an effect proposed to be distinct from α-glucosidase inhibition.