Structural and electronic properties of 4H-cyclopenta[2,1-b,3;4-b′]dithiophene S-oxide (BTO) derivatives with an S, S=O, O, SiH2, or BH2 bridge: semi-empirical and DFT study

In this paper, we theoretically studied the geometries, stabilities, and the electronic and thermodynamic properties of 4H-cyclopenta[2,1-b,3;4-b']dithiopene S-oxide derivatives (BTO-X, with X = BH(2), SiH(2), S, S=O, or O) using semi-empirical methods, ab initio methods, and density functional theory. The geometries and thermodynamic parameters calculated by PM3 were in good agreement with those calculated with B3LYP/6-31 G*. The band gap calculated using B3LYP/6-31 G* ranged from 3.94 eV (BTO-O) to 3.16 eV (BTO-B). The absorption λ(max) calculated using B3LYP/6-31 G* was shifted to longer wavelengths when X = BH(2), SiH(2), or S=O (due to their electron-withdrawing effects) and to shorter wavelengths for BTO-S and BTO-O as compared to the λ(max) for the thiophene S-oxide (2TO) dimer. The changes in ΔH°, ΔS°, and ΔG° calculated using both semi-empirical and DFT methods were quite similar.     

Four isoflavanones from the stem bark of Platycelphium voënse

From the stem bark of Platycelphium voënse (Leguminosae) four new isoflavanones were isolated and characterized as (S)-5,7-dihydroxy-2′,4′-dimethoxy-3′-(3″-methylbut-2″-enyl)-isoflavanone (trivial name platyisoflavanone A), (±)-5,7,2′-trihydroxy-4′-methoxy-3′-(3″-methylbut-2″-enyl)-isoflavanone (platyisoflavanone B), 5,7-dihydroxy-4′-methoxy-2″-(2?-hydroxyisopropyl)-dihydrofurano-[4″,5″:3′,2′]-isoflavanone (platyisoflavanone C) and 5,7,2′,3″-tetrahydroxy-2″,2″-dimethyldihydropyrano-[5″,6″:3′,4′]-isoflavanone (platyisoflavanone D). In addition, the known isoflavanones, sophoraisoflavanone A and glyasperin F; the isoflavone, formononetin; two flavones, kumatakenin and isokaempferide; as well as two triterpenes, betulin and β-amyrin were identified. The structures were elucidated on the basis of spectroscopic evidence. Platyisoflavanone A showed antibacterial activity against Mycobacterium tuberculosis in the microplate alamar blue assay (MABA) with MIC = 23.7 μM, but also showed cytotoxicity (IC₅₀ = 21.1 μM) in the vero cell test.     

The RtcB RNA ligase is an essential component of the metazoan unfolded protein response

RNA ligation can regulate RNA function by altering RNA sequence, structure and coding potential. For example, the function of XBP1 in mediating the unfolded protein response requires RNA ligation, as does the maturation of some tRNAs. Here, we describe a novel in vivo model in Caenorhabditis elegans for the conserved RNA ligase RtcB and show that RtcB ligates the xbp‐1 mRNA during the IRE‐1 branch of the unfolded protein response. Without RtcB, protein stress results in the accumulation of unligated xbp‐1 mRNA fragments, defects in the unfolded protein response, and decreased lifespan. RtcB also ligates endogenous pre‐tRNA halves, and RtcB mutants have defects in growth and lifespan that can be bypassed by expression of pre‐spliced tRNAs. In addition, animals that lack RtcB have defects that are independent of tRNA maturation and the unfolded protein response. Thus, RNA ligation by RtcB is required for the function of multiple endogenous target RNAs including both xbp‐1 and tRNAs. RtcB is uniquely capable of performing these ligation functions, and RNA ligation by RtcB mediates multiple essential processes in vivo.     

Nitric oxide-mediated tumoricidal activity of murine microglial cells

Experimental metastases in the brain of mice are infiltrated by microglia, and parabiosis experiments of green fluorescent protein (GFP(+)) and GFP(-) mice revealed that these microglia are derived from circulating monocytes (GFP(+), F4/80(+), and CD68(+)). These findings raised the question as to whether microglia (specialized macrophages) possess tumoricidal activity. C8-B4 murine microglia cells were incubated in vitro in medium (control) or in medium containing both lipopolysaccharide and interferon-γ. Control microglia were not tumoricidal against a number of murine and human tumor cells, whereas lipopolysaccharide/interferon-γ-activated microglia lysed murine and human tumor cells by release of nitric oxide. Parallel experiments with murine peritoneal macrophages produced identical results. Neither activated microglia nor activated macrophages lysed nontumorigenic murine or human cells. Collectively, these data demonstrate that brain metastasis-associated microglia are derived from circulating mononuclear cells and exhibit selective and specific tumoricidal activity.     

Role of connexin40 in the autoregulatory response of the afferent arteriole

Connexins in renal arterioles affect autoregulation of arteriolar tonus and renal blood flow and are believed to be involved in the transmission of the tubuloglomerular feedback (TGF) response across the cells of the juxtaglomerular apparatus. Connexin40 (Cx40) also plays a significant role in the regulation of renin secretion. We investigated the effect of deleting the Cx40 gene on autoregulation of afferent arteriolar diameter in response to acute changes in renal perfusion pressure. The experiments were performed using the isolated blood perfused juxtamedullary nephron preparation in kidneys obtained from wild-type or Cx40 knockout mice. Renal perfusion pressure was increased in steps from 75 to 155 mmHg, and the response in afferent arteriolar diameter was measured. Hereafter, a papillectomy was performed to inhibit TGF, and the pressure steps were repeated. Conduction of intercellular Ca(2+) changes in response to local electrical stimulation was examined in isolated interlobular arteries and afferent arterioles from wild-type or Cx40 knockout mice. Cx40 knockout mice had an impaired autoregulatory response to acute changes in renal perfusion pressure compared with wild-type mice. Inhibition of TGF by papillectomy significantly reduced autoregulation of afferent arteriolar diameter in wild-type mice. In Cx40 knockout mice, papillectomy did not affect the autoregulatory response, indicating that these mice have no functional TGF. Also, Cx40 knockout mice showed no conduction of intercellular Ca(2+) changes in response to local electrical stimulation of interlobular arteries, whereas the Ca(2+) response to norepinephrine was unaffected. These results suggest that Cx40 plays a significant role in the renal autoregulatory response of preglomerular resistance vessels.     

A model for mechanistic and system assessments of biochar effects on soils and crops and trade‐offs

We developed a biochar model within the Agricultural Production Systems sIMulator (APSIM) software that integrates biochar knowledge and enables simulation of biochar effects within cropping systems. The model has algorithms that mechanistically connect biochar to soil organic carbon (SOC), soil water, bulk density (BD), pH, cation exchange capacity, and organic and mineral nitrogen. Soil moisture (SW)–temperature–nitrogen limitations on the rate of biochar decomposition were included as well as biochar‐induced priming effect on SOC mineralization. The model has 10 parameters that capture the diversity of biochar types, 15 parameters that address biochar‐soil interactions and 4 constants. The range of values and their sensitivity is reported. The biochar model was connected to APSIM's maize and wheat crop models to investigate long‐term (30 years) biochar effects on US maize and Australia wheat in various soils. Results from this sensitivity analysis showed that the effect of biochar was the largest in a sandy soil (Australian wheat) and the smallest in clay loam soil (US maize). On average across cropping systems and soils the order of sensitivity and the magnitude of the response of biochar to various soil‐plant processes was (from high to low): SOC (11% to 86%) > N₂O emissions (?10% to 43%43%) > plant available water content (0.6% to 12.9%) > BD (?6.5% to ?1.7%) > pH (?0.8% to 6.3%) > net N mineralization (?19% to 10%) > CO₂ emissions (?2.0% to 4.3%) > water filled pore space (?3.7% to 3.4%) > grain yield (?3.3% to 1.8%) > biomass (?1.6% to 1.4%). Our analysis showed that biochar has a larger impact on environmental outcomes rather than agricultural production. The mechanistic model has the potential to optimize biochar application strategies to enhance environmental and agronomic outcomes but more work is needed to fill knowledge gaps identified in this work.     

Recognition of the Protein Kinase AVRPPHB SUSCEPTIBLE1 by the Disease Resistance Protein RESISTANCE TO PSEUDOMONAS SYRINGAE5 Is Dependent on S-Acylation and an Exposed Loop in AVRPPHB SUSCEPTIBLE1

The recognition of pathogen effector proteins by plants is typically mediated by intracellular receptors belonging to the nucleotide-binding leucine-rich repeat (NLR) family. NLR proteins often detect pathogen effector proteins indirectly by detecting modification of their targets. How NLR proteins detect such modifications is poorly understood. To address these questions, we have been investigating the Arabidopsis (Arabidopsis thaliana) NLR protein RESISTANCE TO PSEUDOMONAS SYRINGAE5 (RPS5), which detects the Pseudomonas syringae effector protein Avirulence protein Pseudomonas phaseolicolaB (AvrPphB). AvrPphB is a cysteine protease that specifically targets a subfamily of receptor-like cytoplasmic kinases, including the Arabidopsis protein kinase AVRPPHB Susceptible1 (PBS1). RPS5 is activated by the cleavage of PBS1 at the apex of its activation loop. Here, we show that RPS5 activation requires that PBS1 be localized to the plasma membrane and that plasma membrane localization of PBS1 is mediated by amino-terminal S-acylation. We also describe the development of a high-throughput screen for mutations in PBS1 that block RPS5 activation, which uncovered four new pbs1 alleles, two of which blocked cleavage by AvrPphB. Lastly, we show that RPS5 distinguishes among closely related kinases by the amino acid sequence (SEMPH) within an exposed loop in the C-terminal one-third of PBS1. The SEMPH loop is located on the opposite side of PBS1 from the AvrPphB cleavage site, suggesting that RPS5 associates with the SEMPH loop while leaving the AvrPphB cleavage site exposed. These findings provide support for a model of NLR activation in which NLR proteins form a preactivation complex with effector targets and then sense a conformational change in the target induced by effector modification.Pathogen recognition by plants is mediated by both transmembrane cell surface receptors and intracellular receptors (Jones and Dangl, 2006). The latter receptors typically belong to the nucleotide-binding leucine-rich repeat (NLR) superfamily of proteins, which also play a central role in the innate immune systems of many animals, including humans (von Moltke et al., 2013). In plants, most NLR proteins detect pathogen “effector” proteins, which are proteins secreted by pathogens to promote virulence on susceptible hosts. The immune response activated by NLR proteins is thus referred to as effector-triggered immunity. In the majority of examples studied, effector-triggered immunity is accompanied by localized host cell death around the site of pathogen ingress, which is referred to as the hypersensitive response (HR; Goodman and Novacky, 1994).Several NLR proteins have been shown to detect pathogen effector proteins indirectly by detecting the modification of other host proteins mediated by the effectors (DeYoung and Innes, 2006). The best characterized examples of NLR proteins that employ indirect recognition mechanisms are the RESISTANCE TO PSEUDOMONAS MACULICOLA1 (RPM1) and RESISTANCE TO PSEUDOMONAS SYRINGAE2 (RPS2) proteins of Arabidopsis (Arabidopsis thaliana), which detect modification to the RPM1 INTERACTING4 (RIN4) protein (Mackey et al., 2002; Axtell and Staskawicz, 2003), the RESISTANCE TO PSEUDOMONAS SYRINGAE5 (RPS5) protein of Arabidopsis, which detects modification of the AVRPPHB SUSCEPTIBLE1 (PBS1) protein kinase (Ade et al., 2007), and the Pseudomonas resistance and fenthion sensitivity (Prf) protein of tomato (Solanum lycopersicum), which detects modification of the Pseudomonas syringae pv tomato resistance (Pto) protein kinase (Salmeron et al., 1996; Rathjen et al., 1999). Our group has focused on RPS5, which detects the effector protein Avirulence protein Pseudomonas phaseolicolaB (AvrPphB) from Pseudomonas syringae (Simonich and Innes, 1995). AvrPphB functions as a Cys protease (Zhu et al., 2004) and specifically targets a subclass of plant receptor-like cytoplasmic kinases that include PBS1 (Shao et al., 2003; Zhang et al., 2010). AvrPphB likely targets these kinases in order to suppress defense responses induced by cell surface-localized plant immune receptors such as FLAGELLIN SENSITIVE2 (FLS2; Zhang et al., 2010). PBS1 can be coimmunoprecipitated with FLS2, and mutation of PBS1 reduces FLS2-mediated production of hydrogen peroxide and callose deposits (Zhang et al., 2010), confirming that PBS1 functions in defense signaling.Cleavage of PBS1 by AvrPphB is both necessary and sufficient to activate RPS5 (Ade et al., 2007), and null mutations in PBS1 block RPS5 activation (Swiderski and Innes, 2001). Because AvrPphB can cleave multiple closely related kinases in Arabidopsis (Zhang et al., 2010), these observations indicate that RPS5 can distinguish among these kinases, with only PBS1 cleavage activating RPS5. The molecular basis for this specificity is unknown.One contributor to the specificity of RPS5 may be subcellular localization. RPS5 localizes to the plasma membrane (PM), and amino acid substitutions that displace RPS5 from the PM eliminate RPS5-mediated defense responses (Qi et al., 2012). PBS1 is also expected to localize to the PM, because fusion of the N-terminal 100 amino acids of PBS1 to GFP causes GFP to localize to the PM in both Arabidopsis and Nicotiana benthamiana (Takemoto et al., 2012). Consistent with this expectation, PBS1 and RPS5 can be coimmunoprecipitated when transiently overexpressed in N. benthamiana (Ade et al., 2007). Furthermore, AvrPphB is both myristoylated and palmitoylated upon entry into plant cells and localizes to the PM, with PM localization of AvrPphB being required for the activation of RPS5 (Dowen et al., 2009). Although these data all point to a PM localization for PBS1, full-length PBS1 protein has not yet been localized, nor has the functional significance of PBS1 localization been assessed relative to the activation of RPS5.In this study, we demonstrate that PBS1 is targeted to the PM via S-acylation at its N terminus and that PM localization is required for RPS5 activation. We also describe a high-throughput genetic screen for uncovering new mutations in PBS1 that block RPS5 activation, which uncovered four new pbs1 alleles. Lastly, we show that RPS5 distinguishes PBS1 from closely related kinases based on a specific loop in the C-terminal half of PBS1.     

Prediction of glycosylphosphatidylinositol-anchored proteins in Arabidopsis. A genomic analysis

Glycosylphosphatidylinositol (GPI) anchoring of proteins provides a potential mechanism for targeting to the plant plasma membrane and cell wall. However, relatively few such proteins have been identified. Here, we develop a procedure for database analysis to identify GPI-anchored proteins (GAP) based on their possession of common features. In a comprehensive search of the annotated Arabidopsis genome, we identified 167 novel putative GAP in addition to the 43 previously described candidates. Many of these 210 proteins show similarity to characterized cell surface proteins. The predicted GAP include homologs of beta-1,3-glucanases (16), metallo- and aspartyl proteases (13), glycerophosphodiesterases (6), phytocyanins (25), multi-copper oxidases (2), extensins (6), plasma membrane receptors (19), and lipid-transfer-proteins (18). Classical arabinogalactan (AG) proteins (13), AG peptides (9), fasciclin-like proteins (20), COBRA and 10 homologs, and novel potential signaling peptides that we name GAPEPs (8) were also identified. A further 34 proteins of unknown function were predicted to be GPI anchored. A surprising finding was that over 40% of the proteins identified here have probable AG glycosylation modules, suggesting that AG glycosylation of cell surface proteins is widespread. This analysis shows that GPI anchoring is likely to be a major modification in plants that is used to target a specific subset of proteins to the cell surface for extracellular matrix remodeling and signaling.     

Genetic algorithm-based optimization of hydrophobicity tables

SUMMARY: The genomic abundance and pharmacological importance of membrane proteins have fueled efforts to identify them based solely on sequence information. Previous methods based on the physicochemical principle of a sliding window of hydrophobicity (hydropathy analysis) have been replaced by approaches based on hidden Markov models or neural networks which prevail due to their probabilistic orientation. In the current study, an optimization of the hydrophobicity tables used in hydropathy analysis is performed using a genetic algorithm. As such, the approach can be viewed as a synthesis between the physicochemically and statistically based methods. The resulting hydrophobicity tables lead to significant improvement in the prediction accuracy of hydropathy analysis. Furthermore, since hydropathy analysis is less dependent on the basis set of membrane proteins is used to hone the statistically based methods, as well as being faster, it may be valuable in the analysis of new genomes. Finally, the values obtained for each of the amino acids in the new hydrophobicity tables are discussed.     

Frozen-thawed human blood monocytes respond reproducibly to activation stimuli: Implications for screening of BRMs

The purpose of this study was to identify the optimal freezing conditions for human blood monocytes to allow their recovery and use forin vitro screening of activation stimuli. Human monocytes separated from buffy coats of healthy blood donors were suspended at a density of 1 × 10⁷ cells/ml in freezing medium consisting of 70% medium: 20% fetal bovine serum: 10% DMSO frozen in a stepdown freezer, and stored at –180°C. Monocytes were thawed at different times up to 4 months later. Viability was >90%. Fresh monocytes from different donors and frozen monocytes thawed at different times were incubated with different concentrations of lipopolysaccharide, muramyl tripeptide, muramyl dipeptide, or lipopeptide. Tumoricidal activity and IL-1 production of fresh monocytes varied greatly among the 5 different preparations. In contrast, the frozen monocytes (thawed at different times) produced uniform levels of antitumor activity and IL-1 production. These results show that monocytes recovered from frozen storage maintain their ability to respond to activation stimuli in a uniform and reproducible manner. Thus, the use of frozen-thawed monocytes is recommended for screening of macrophage activating agents.