Antiviral activity of benzimidazole derivatives. II. Antiviral activity of 2-phenylbenzimidazole derivatives

Seventy-six 2-phenylbenzimidazole derivatives were synthesized and evaluated in cell-based assays for cytotoxicity and antiviral activity against a panel of 10 RNA and DNA viruses. The most commonly affected viruses were, in decreasing order, CVB-2, BVDV, Sb-1, HSV-1, and YFV, while HIV-1 and VSV were not affected, and RSV, VV and Reo-1 were only susceptible to a few compounds. Thirty-nine compounds exhibited high activity (EC₅₀ = 0.1–10 μM) against at least one virus, and four of them were outstanding for their high and selective activity against VV (24, EC₅₀ = 0.1 μM) and BVDV (50, 51, and 53 with EC₅₀ = 1.5, 0.8, and 1.0 μM, respectively). The last compounds inhibited at low micromolar concentrations the NS5B RdRp of BVDV and also of HCV, the latter sharing structural similarity with the former. The considered compounds represent attractive leads for the development of antiviral agents against poxviruses, pestiviruses and even HCV, which are important human and veterinary pathogens.     

Non-additive effects of RBP4, ESR1 and IGF2 polymorphisms on litter size at different parities in a Chinese-European porcine line

Background

The aim of this work was to study the effects on litter size of variants of the porcine genes RBP4, ESR1 and IGF2, currently used in genetic tests for different purposes. Moreover, we investigated a possible effect of the interaction between RBP4-MspI and ESR1-PvuII polymorphisms. The IGF2-intron3-G3072A polymorphism is actually used to select lean growth, but other possible effects of this polymorphism on reproductive traits need to be evaluated.

Methods

Detection of polymorphisms in the genomic and cDNA sequences of RBP4 gene was carried out. RBP4-MspI and IGF2-intron3-G3072A were genotyped in a hyperprolific Chinese-European line (Tai-Zumu) and three new RBP4 polymorphisms were genotyped in different pig breeds. A bivariate animal model was implemented in association analyses considering the number of piglets born alive at early (NBA₁₂) and later parities (NBA₃₊ ) as different traits. A joint analysis of RBP4-MspI and ESR1-PvuII was performed to test their possible interaction. In the IGF2 analysis, paternal or maternal imprinting effects were also considered.

Results

Four different RBP4 haplotypes were detected (TGAC, GGAG, GAAG and GATG) in different pig breeds and wild boars. A significant interaction effect between RBP4-MspI and ESR1-PvuII polymorphisms of 0.61 ± 0.29 piglets was detected on NBA₃₊. The IGF2 analysis revealed a significant increase on NBA₃₊ of 0.74 ± 0.37 piglets for the paternally inherited allele A.

Conclusions

All the analyzed pig and wild boar populations shared one of the four detected RBP4 haplotypes. This suggests an ancestral origin of the quoted haplotype. The joint use of RBP4-MspI and ESR1-PvuII polymorphisms could be implemented to select for higher prolificacy in the Tai-Zumu line. In this population, the paternal allele IGF2-intron3-3072A increased litter size from the third parity. The non-additive effects on litter size reported here should be tested before implementation in other pig breeding schemes.     

Voltage‐dependent κ‐opioid modulation of action potential waveform‐elicited calcium currents in neurohypophysial terminals

Release of neurotransmitter is activated by the influx of calcium. Inhibition of Ca²⁺ channels results in less calcium influx into the terminals and presumably a reduction in transmitter release. In the neurohypophysis (NH), Ca²⁺ channel kinetics, and the associated Ca²⁺ influx, is primarily controlled by membrane voltage and can be modulated, in a voltage‐dependent manner, by G‐protein subunits interacting with voltage‐gated calcium channels (VGCCs). In this series of experiments we test whether the κ‐ and µ‐opioid inhibition of Ca²⁺ currents in NH terminals is voltage‐dependent. Voltage‐dependent relief of G‐protein inhibition of VGCC can be achieved with either a depolarizing square pre‐pulse or by action potential waveforms. Both protocols were tested in the presence and absence of opioid agonists targeting the κ‐ and µ‐receptors in neurohypophysial terminals. The κ‐opioid VGCC inhibition is relieved by such pre‐pulses, suggesting that this receptor is involved in a voltage‐dependent membrane delimited pathway. In contrast, µ‐opioid inhibition of VGCC is not relieved by such pre‐pulses, indicating a voltage‐independent diffusible second‐messenger signaling pathway. Furthermore, relief of κ‐opioid inhibition during a physiologic action potential (AP) burst stimulation indicates the possibility of activity‐dependent modulation in vivo. Differences in the facilitation of Ca²⁺ channels due to specific G‐protein modulation during a burst of APs may contribute to the fine‐tuning of Ca²⁺‐dependent neuropeptide release in other CNS terminals, as well. J. Cell. Physiol. 225: 223–232, 2010. © 2010 Wiley‐Liss, Inc.     

Umbilical cord-derived mesenchymal stromal cells modulate monocyte function to suppress T cell proliferation

Mesenchymal stromal cells (MSCs) may be derived from a variety of tissues, with human umbilical cord (UC) providing an abundant and noninvasive source. Human UC-MSCs share similar in vitro immunosuppressive properties as MSCs obtained from bone marrow and cord blood. However, the mechanisms and cellular interactions used by MSCs to control immune responses remain to be fully elucidated. In this paper, we report that suppression of mitogen-induced T cell proliferation by human UC-, bone marrow-, and cord blood-MSCs required monocytes. Removal of monocytes but not B cells from human adult PBMCs (PBMNCs) reduced the immunosuppressive effects of MSCs on T cell proliferation. There was rapid modulation of a number of cell surface molecules on monocytes when PBMCs or alloantigen-activated PBMNCs were cultured with UC-MSCs. Indomethacin treatment significantly inhibited the ability of UC-MSCs to suppress T cell proliferation, indicating an important role for PGE(2). Monocytes purified from UC-MSC coculture had significantly reduced accessory cell and allostimulatory function when tested in subsequent T cell proliferation assays, an effect mediated in part by UC-MSC PGE(2) production and enhanced by PBMNC alloactivation. Therefore, we identify monocytes as an essential intermediary through which UC-MSCs mediate their suppressive effects on T cell proliferation.     

On/off TLR signaling decides proinflammatory or tolerogenic dendritic cell maturation upon CD1d-mediated interaction with invariant NKT cells

Invariant NKT (iNKT) cells play an effector/adjuvant function during antimicrobial and antitumoral immunity and a regulatory role to induce immune tolerance and prevent autoimmunity. iNKT cells that differentially modulate adaptive immunity do not bear a unique phenotype and/or specific cytokine secretion profile, thus opening questions on how a single T cell subset can exert opposite immunological tasks. In this study, we show that iNKT cells perform their dual roles through a single mechanism of action relying on the cognate interaction with myeloid dendritic cells (DCs) and leading to opposite effects depending on the presence of other maturation stimuli simultaneously acting on DCs. The contact of murine purified iNKT cells with immature autologous DCs directly triggers the tolerogenic maturation of DCs, rendering them able to induce regulatory T cell differentiation and prevent autoimmune diabetes in vivo. Conversely, the interaction of the same purified iNKT cells with DCs, in the presence of simultaneous TLR4 stimulation, significantly enhances proinflammatory DC maturation and IL-12 secretion. The different iNKT cell effects are mediated through distinct mechanisms and activation of different molecular pathways within the DC: CD1d signaling and activation of the ERK1/2 pathway for the tolerogenic action, and CD40-CD40L interaction and NF-κB activation for the adjuvant effect. Our data suggest that the DC decision to undergo proinflammatory or tolerogenic maturation results from the integration of different signals received at the time of iNKT cell contact and could have important therapeutic implications for exploiting iNKT cell adjuvant/regulatory properties in autoimmune diseases, infections, and cancer.     

HDL and electronegative LDL exchange anti- and pro-inflammatory properties

Electronegative LDL [LDL(–)] is a minor modified LDL subfraction present in blood with inflammatory effects. One of the antiatherogenic properties of HDL is the inhibition of the deleterious effects of in vitro modified LDL. However, the effect of HDL on the inflammatory activity of LDL(–) isolated from plasma is unknown. We aimed to assess the putative protective role of HDL against the cytokine released induced in monocytes by LDL(–). Our results showed that LDL(–) cytokine release was inhibited when LDL(–) was coincubated with HDL and human monocytes and also when LDL(–) was preincubated with HDL and reisolated prior to cell incubation. The addition of apoliprotein (apo)AI instead of HDL reproduced the protective behavior of HDL. HDL preincubated with LDL(–) promoted greater cytokine release than native HDL. Incubation of LDL(–) with HDL decreased the electronegative charge, phospholipase C-like activity, susceptibility to aggregation and nonesterified fatty acid (NEFA) content of LDL(–), whereas these properties increased in HDL. NEFA content in LDL appeared to be related to cytokine production because NEFA-enriched LDL induced cytokine release. HDL, at least in part through apoAI, inhibits phospholipase-C activity and cytokine release in monocytes, thereby counteracting the inflammatory effect of LDL(–). In turn, HDL acquires these properties and becomes inflammatory.     

A pH-dependent dimer lock in spider silk protein

Spider dragline silk, one of the strongest polymers in nature, is composed of proteins termed major ampullate spidroin (MaSp) 1 and MaSp2. The N-terminal (NT) domain of MaSp1 produced by the nursery web spider Euprosthenops australis acts as a pH-sensitive relay, mediating spidroin assembly at around pH 6.3. Using amide hydrogen/deuterium exchange combined with mass spectrometry (MS), we detected pH-dependent changes in deuterium incorporation into the core of the NT domain, indicating global structural stabilization at low pH. The stabilizing effects were diminished or abolished at high ionic strength, or when the surface-exposed residues Asp40 and Glu84 had been exchanged with the corresponding amides. Nondenaturing electrospray ionization MS revealed the presence of dimers in the gas phase at pH values below—but not above—6.4, indicating a tight electrostatic association that is dependent on Asp40 and Glu84 at low pH. Results from analytical ultracentrifugation support these findings. Together, the data suggest a mechanism whereby lowering the pH to < 6.4 results in structural changes and alteration of charge-mediated interactions between subunits, thereby locking the spidroin NT dimer into a tight entity important for aggregation and silk formation.