Facilitation and predation structure a grassland detrital food web: the responses of soil nematodes to isopod processing of litter

1. Detritus can support successive consumers, whose interactions may be structured by changes in the condition of their shared resource. One model of such species interactions is a processing chain, in which consumers feeding on the resource in a less processed state change the resource condition for subsequent consumers. 2. In a series of experiments, the hypothesis was tested that a common detritivore, the terrestrial isopod Porcellio scaber, affects soil nematodes through the processing of plant litter. Different detrital resources were added to soil from a California coastal prairie in order to simulate litter processing by the detritivore. Treatments that included only whole grass litter corresponded to detrital food webs lacking detritivores, while treatments that included mixtures of P. scaber faeces and grass litter corresponded to different densities or feeding rates of P. scaber. 3. Simulated litter processing by P. scaber increased the abundance of bacterivorous nematodes by between 32% and 202% after 24-44 days in laboratory experiments, but had no effect on fungivorous or predaceous nematodes. 4. In a subsequent field experiment, however, fungivorous nematodes were suppressed by isopod litter processing while bacterivores showed no response. Instead, P. scaber processing of litter increased the abundance of predaceous nematodes in the field experiment by 176%. 5. When simulated litter processing of litter was crossed in laboratory experiments with predaceous nematode addition (comparable to the response of predators in the field experiment), the abundance of bacterivores was increased by isopod processing of litter (by an average of 122%), but suppressed by elevated densities of predaceous nematodes (by an average of 41%). 6. This suggests that litter processing by P. scaber facilitates the bacterial channel of the soil food web, but that predaceous nematodes suppress the response of bacterivores in the field. Processing chain interactions may, therefore, be important in understanding the relative importance of bacterial and fungal channels in the soil food web, while top-down effects of predators determine the resulting changes in population abundance and biomass.     

Novel pyrrolidine melanin-concentrating hormone receptor 1 antagonists with reduced hERG inhibition

We discovered novel pyrrolidine MCHR1 antagonist 1 possessing moderate potency. Profiling of pyrrolidine 1 demonstrated that it was an inhibitor of the hERG channel. Investigation of the structure-activity relationship of this class of pyrrolidines allowed us to optimize the MCHR1 potency and decrease the hERG inhibition. Increasing the acidity of the amide proton by converting the benzamide in lead 1 to an anilide provided single digit nanomolar MCHR1 antagonists while replacing the dimethoxyphenyl ring of 1 with alkyl groups possessing increased polarity dramatically reduced the hERG inhibition.     

Plasmid DNA fermentation strain and process‐specific effects on vector yield,quality, and transgene expression

Industrial plasmid DNA manufacturing processes are needed to meet the quality, economy, and scale requirements projected for future commercial products. We report development of a modified plasmid fermentation copy number induction profile that increases gene vaccination/therapy vector yields up to 2,600 mg/L. We determined that, in contrast to recombinant protein production, secretion of the metabolic byproduct acetate into the media had only a minor negative effect on plasmid replication. We also investigated the impact of differences in epigenetic dcm methylase‐directed cytosine methylation on plasmid production, transgene expression, and immunogenicity. While Escherichia coli plasmid production yield and quality are unaffected, dcm− versions of CMV and CMV‐HTLV‐I R promoter plasmids had increased transgene expression in human cells. Surprisingly, despite improved expression, dcm− plasmid is less immunogenic. Our results demonstrate that it is critical to lock the plasmid methylation pattern (i.e., production strain) early in product development and that dcm− strains may be superior for gene therapy applications wherein reduced immunogenicity is desirable and for in vitro transient transfection applications such as AAV production where improved expression is beneficial. Biotechnol. Bioeng. 2011;108: 354–363. © 2010 Wiley Periodicals, Inc.     

Plasmin potentiates synaptic N-methyl-D-aspartate receptor function in hippocampal neurons through activation of protease-activated receptor-1

Protease-activated receptor-1 (PAR1) is activated by a number of serine proteases, including plasmin. Both PAR1 and plasminogen, the precursor of plasmin, are expressed in the central nervous system. In this study we examined the effects of plasmin in astrocyte and neuronal cultures as well as in hippocampal slices. We find that plasmin evokes an increase in both phosphoinositide hydrolysis (EC(50) 64 nm) and Fura-2/AM fluorescence (195 +/- 6.7% above base line, EC(50) 65 nm) in cortical cultured murine astrocytes. Plasmin also activates extracellular signal-regulated kinase (ERK1/2) within cultured astrocytes. The plasmin-induced rise in intracellular Ca(2+) concentration ([Ca(2+)](i)) and the increase in phospho-ERK1/2 levels were diminished in PAR1(-/-) astrocytes and were blocked by 1 microm BMS-200261, a selective PAR1 antagonist. However, plasmin had no detectable effect on ERK1/2 or [Ca(2+)](i) signaling in primary cultured hippocampal neurons or in CA1 pyramidal cells in hippocampal slices. Plasmin (100-200 nm) application potentiated the N-methyl-D-aspartate (NMDA) receptor-dependent component of miniature excitatory postsynaptic currents recorded from CA1 pyramidal neurons but had no effect on alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate- or gamma-aminobutyric acid receptor-mediated synaptic currents. Plasmin also increased NMDA-induced whole cell receptor currents recorded from CA1 pyramidal cells (2.5 +/- 0.3-fold potentiation over control). This effect was blocked by BMS-200261 (1 microm; 1.02 +/- 0.09-fold potentiation over control). These data suggest that plasmin may serve as an endogenous PAR1 activator that can increase [Ca(2+)](i) in astrocytes and potentiate NMDA receptor synaptic currents in CA1 pyramidal neurons.     

Determination of safety distance limits for a human near a cellular base station antenna,adopting the IEEE standard or ICNIRP guidelines

This paper investigates the minimum distance for a human body in the near field of a cellular telephone base station antenna for which there is compliance with the IEEE or ICNIRP threshold values for radio frequency electromagnetic energy absorption in the human body. First, local maximum specific absorption rates (SARs), measured and averaged over volumes equivalent to 1 and to 10 g tissue within the trunk region of a physical, liquid filled shell phantom facing and irradiated by a typical GSM 900 base station antenna, were compared to corresponding calculated SAR values. The calculation used a homogeneous Visible Human body model in front of a simulated base station antenna of the same type. Both real and simulated base station antennas operated at 935 MHz. Antenna-body distances were between 1 and 65 cm. The agreement between measurements and calculations was excellent. This gave confidence in the subsequent calculated SAR values for the heterogeneous Visible Human model, for which each tissue was assigned the currently accepted values for permittivity and conductivity at 935 MHz. Calculated SAR values within the trunk of the body were found to be about double those for the homogeneous case. When the IEEE standard and the ICNIRP guidelines are both to be complied with, the local SAR averaged over 1 g tissue was found to be the determining parameter. Emitted power values from the antenna that produced the maximum SAR value over 1 g specified in the IEEE standard at the base station are less than those needed to reach the ICNIRP threshold specified for the local SAR averaged over 10 g. For the GSM base station antenna investigated here operating at 935 MHz with 40 W emitted power, the model indicates that the human body should not be closer to the antenna than 18 cm for controlled environment exposure, or about 95 cm for uncontrolled environment exposure. These safe distance limits are for SARs averaged over 1 g tissue. The corresponding safety distance limits under the ICNIRP guidelines for SAR taken over 10 g tissue are 5 cm for occupational exposure and about 75 cm for general-public exposure.     

Extent of intracellular pH changes during H+ extrusion by maize root-tip cells

³¹P-Nuclear-magnetic-resonance spectra of maize (Zea mays L.) root tips, that had been induced to extrude large amounts of H⁺ in response to fusicoccin (FC) in the presence of potassium salts, indicate that the cytoplasmic pH does not become higher than that of controls. In fact, the cytoplasmic pH may become slightly (approx. 0.1 pH unit) lower in cells extruding H⁺. Estimations of the buffer capacity of the cells show that without active intracellular pH regulation, H⁺ extrusion caused by FC would cause the intracellular pH to rise by at least 0.6 pH unit h^-1. Our results indicate that intracellular pH is tightly regulated even during extreme rates of acid extrusion, and that a rise in cytoplasmic pH is not the signal linking H⁺ extrusion with enhanced organic-acid synthesis or other intracellular responses to H⁺ pumping.Abbreviations FC fusicoccin - P_i inorganic phosphate - NMR nuclear magnetic resonance - chemical shift - MDP methylene diphosphonic acid     

Intestinal inflammation caused by magnesium deficiency alters basal and oxidative stress-induced intestinal function

The aim of this study was to determine the effect of magnesium deficiency on small intestinal morphology and function. Rats were assigned to 4 groups and placed on magnesium sufficient or deficient diet for 1 or 3 weeks. Infiltration of neutrophils and mucosal injury were assessed in stained sections of small intestine. Magnesium deficiency alone induced a significant increase in neutrophil infiltration and increased vascular ICAM-1 expression, in the absence of changes in mucosal injury or expression of proinflammatory mediators. Magnesium deficiency was associated with hyposecretory epithelial cell responses and vascular macromolecular leak in the small intestine and lung, which was attributed partly to reduced expression of NOS-3. To determine the effect of hypomagnesmia on the intestinal responses to a known oxidative stress, groups of rats were randomized to either sham operation or superior mesenteric artery occlusion for 10 (non-injurious) or 30 (injurious) minutes followed by a 1- or 4-hour reperfusion period. In response to mesenteric ischemia/reperfusion, deficient rats showed exaggerated PMN influx, but similar mucosal injury. Intestinal ischemia in sufficient animals induced vascular macromolecular leak in the small intestine and lung at 4 hours of reperfusion, with levels similar to those observed in untreated deficient rats. Acute magnesium repletion of deficient rats 24 h before surgery attenuated the exaggerated inflammation in deficient rats. These data show that magnesium deficiency induced a subclinical inflammation in the small intestine in the absence of mucosal injury, but with significant functional changes in local and remote organs and increased sensitivity to oxidative stress. The opinions contained herein are those of the authors and are not to be construed as official policy or reflecting the views of the Department of Defense     

Synthesis and biophysical evaluation of minor-groove binding C-terminus modified pyrrole and imidazole triamide analogs of distamycin

Five polyamide derivatives with rationally modified C-terminus moieties were synthesized and their DNA binding specificity and affinity determined. A convergent approach was employed to synthesize polyamides containing an alkylaminopiperazine (4 and 5), a truncated piperazine (6), or an alkyldiamino-C-terminus moiety (7 and 8) with two specific objectives: to investigate the effects of number of potential cationic centers and steric bulk at the C-terminus. CD studies confirmed that compounds 4, 5, 7, and 8 bind in the minor groove of DNA. The alkylpiperazine containing compounds (4 and 5) showed only moderate binding to DNA with DeltaT(m) values of 2.8 and 8.3 degrees C with their cognate sequence, respectively. The alkyldiamino compounds (7 and 8) were more impressive producing a DeltaT(m) of >17 and >22 degrees C, respectively. Compound 6 (truncated piperazine) did not stabilize its cognate DNA sequence. Footprints were observed for all compounds (except compound 6) with their cognate DNA sequence using DNase I footprinting, with compound 7 producing a footprint of 0.1 microM at the expected 5'-ACGCGT-3' site. SPR analysis of compound 7 binding to 5'-ACGCGT-3', 5'-ACCGGT-3', and 5'-AAATTT-3' produced binding affinities of 2.2x10(6), 3.3x10(5), and 1x10(5)M(-1), respectively, indicating a preference for its cognate sequence of 5'-ACGCGT-3'. These results are in good agreement with the footprinting data. The results indicate that steric crowding at the C-terminus is important with respect to binding. However, the number of cationic centers within the molecule may also play a role. The alkyldiamino-containing compounds (7 and 8) warrant further investigation in the field of polyamide research.     

MicroRNA-binding is required for recruitment of human Argonaute 2 to stress granules and P-bodies

Argonaute proteins are the core components of the RNA-induced silencing complex, the central effector of the mammalian RNA interference pathway. In the cytoplasm, they associate with at least two types of cytoplasmic RNA granules; processing bodies and stress granules, which function in mRNA degradation and translational repression, respectively. The significance of Argonaute association with these RNA granules is not entirely clear but it is likely related to their activities within the RNAi pathway. Understanding what regulates targeting of Argonautes to RNA granules may provide clues as to their functions at these organelles. To this end, there are a number of conflicting reports that describe the role of small RNAs in targeting Argonaute proteins in mammalian cells. We employed quantitative microscopic analyses of human Argonaute 2 (hAgo2) mutants to study factors that govern localization of this RNA-binding protein to cytoplasmic RNA granules. We report, for the first time, that hAgo2 is recruited to stress granules as a consequence of its interaction with miRNAs. Moreover, loading of small RNAs onto hAgo2 is not required for its stability, suggesting that a pool of unloaded hAgo2 may exist for extended periods of time in the cytoplasm.