Bronchoconstriction in nonhuman primates: a species comparison

Bronchoconstriction is a characteristic symptom of various chronic obstructive respiratory diseases such as chronic obstructive pulmonary disease and asthma. Precision-cut lung slices (PCLS) are a suitable ex vivo model to study physiological mechanisms of bronchoconstriction in different species. In the present study, we established an ex vivo model of bronchoconstriction in nonhuman primates (NHPs). PCLS prepared from common marmosets, cynomolgus macaques, rhesus macaques, and anubis baboons were stimulated with increasing concentrations of representative bronchoconstrictors: methacholine, histamine, serotonin, leukotriene D? (LTD?), U46619, and endothelin-1. Alterations in the airway caliber were measured and compared with previously published data from rodents, guinea pigs, and humans. Methacholine induced maximal airway constriction, varying between 74 and 88% in all NHP species, whereas serotonin was ineffective. Histamine induced maximal bronchoconstriction of 77 to 90% in rhesus macaques, cynomolgus macaques, and baboons and a lesser constriction of 53% in marmosets. LTD? was ineffective in marmosets and rhesus macaques but induced a maximum constriction of 44 to 49% in cynomolgus macaques and baboons. U46619 and endothelin-1 caused airway constriction in all NHP species, with maximum constrictions of 65 to 91% and 70 to 81%, respectively. In conclusion, PCLS from NHPs represent a valuable ex vivo model for studying bronchoconstriction. All NHPs respond to mediators relevant to human airway disorders such as methacholine, histamine, U46619, and endothelin-1 and are insensitive to the rodent mast cell product serotonin. Only PCLS from cynomolgus macaques and baboons, however, responded also to leukotrienes, suggesting that among all compared species, these two NHPs resemble the human airway mechanisms best.     

Aryl 1,4-diazepane compounds as potent and selective CB2 agonists: optimization of drug-like properties and target independent parameters

A high throughput screening campaign identified aryl 1,4-diazepane compounds as potent and selective cannabinoid receptor 2 agonists as compared to cannabinoid receptor 1. This class of compounds suffered from poor drug-like parameters as well as low microsomal stability and poor solubility. Structure-activity relationships are described with a focus on improving the drug-like parameters resulting in compounds with improved solubility and permeability.     

Disrupted‐in‐schizophrenia 1 functional polymorphisms and D2/D3 receptor availability: A [11C]‐(+)‐PHNO imaging study

The disrupted‐in‐schizophrenia 1 (DISC1) protein has been implicated in a range of biological mechanisms underlying chronic mental disorders such as schizophrenia. Schizophrenia is associated with abnormal striatal dopamine signalling, and all antipsychotic drugs block striatal dopamine 2/3 receptors (D_2/3Rs). Importantly, the DISC1 protein directly interacts and forms a protein complex with the dopamine D₂ receptor (D₂R) that inhibits agonist‐induced D₂R internalisation. Moreover, animal studies have found large striatal increases in the proportion of D₂R receptors in a high affinity state (D₂^highR) in DISC1 rodent models. Here, we investigated the relationship between the three most common polymorphisms altering the amino‐acid sequence of the DISC1 protein (Ser704Cys (rs821616), Leu607Phe (rs6675281) and Arg264Gln (rs3738401)) and striatal D_2/3R availability in 41 healthy human volunteers, using [¹¹C]‐(+)‐PHNO positron emission tomography. We found no association between DISC1 polymorphisms and D_2/3R availability in the striatum and D₂R availability in the caudate and putamen. Therefore, despite a direct interaction between DISC1 and the D₂R, none of its main functional polymorphisms impact striatal D_2/3R binding potential, suggesting DISC1 variants act through other mechanisms.     

Iron-Sulfur Cluster-dependent Catalysis of Chlorophyllide a Oxidoreductase from Roseobacter denitrificans

Bacteriochlorophyll a biosynthesis requires the stereo- and regiospecific two electron reduction of the C7-C8 double bond of chlorophyllide a by the nitrogenase-like multisubunit metalloenzyme, chlorophyllide a oxidoreductase (COR). ATP-dependent COR catalysis requires interaction of the protein subcomplex (BchX)₂ with the catalytic (BchY/BchZ)₂ protein to facilitate substrate reduction via two redox active iron-sulfur centers. The ternary COR enzyme holocomplex comprising subunits BchX, BchY, and BchZ from the purple bacterium Roseobacter denitrificans was trapped in the presence of the ATP transition state analog ADP·AlF₄⁻. Electron paramagnetic resonance experiments revealed a [4Fe-4S] cluster of subcomplex (BchX)₂. A second [4Fe-4S] cluster was identified on (BchY/BchZ)₂. Mutagenesis experiments indicated that the latter is ligated by four cysteines, which is in contrast to the three cysteine/one aspartate ligation pattern of the closely related dark-operative protochlorophyllide a oxidoreductase (DPOR). In subsequent mutagenesis experiments a DPOR-like aspartate ligation pattern was implemented for the catalytic [4Fe-4S] cluster of COR. Artificial cluster formation for this inactive COR variant was demonstrated spectroscopically. A series of chemically modified substrate molecules with altered substituents on the individual pyrrole rings and the isocyclic ring were tested as COR substrates. The COR enzyme was still able to reduce the B ring of substrates carrying modified substituents on ring systems A, C, and E. However, substrates with a modification of the distantly located propionate side chain were not accepted. A tentative substrate binding mode was concluded in analogy to the related DPOR system.     

Sequencing type material resolves the identity and distribution of the generitype Lithophyllum incrustans,and related European species L. hibernicum and L. bathyporum (Corallinales,Rhodophyta)

DNA sequences from type material in the nongeniculate coralline genus Lithophyllum were used to unambiguously link some European species names to field‐collected specimens, thus providing a great advance over morpho‐anatomical identifi‐cation. In particular, sequence comparisons of rbcL, COI and psbA genes from field‐collected specimens allowed the following conclusion: the generitype species, L. incrustans, occurs mostly as subtidal rhodoliths and crusts on both Atlantic and Mediterranean coasts, and not as the common, NE Atlantic, epilithic, intertidal crust reported in the literature. The heterotypic type material of L. hibernicum was narrowed to one rhodolith belonging in Lithophyllum. As well as occurring as a subtidal rhodolith, L. hibernicum is a common, epilithic and epizoic crust in the intertidal zone from Ireland south to Mediterranean France. A set of four features distinguished L. incrustans from L. hibernicum, including epithallial cell diameter, pore canal shape of sporangial conceptacles and sporangium height and diameter. An rbcL sequence of the lectotype of Lithophyllum bathyporum, which was recently proposed to accommodate Atlantic intertidal collections of L. incrustans, corresponded to a distinct taxon hitherto known only from Brittany as the subtidal, bisporangial, lectotype, but also occurs intertidally in Atlantic Spain. Specimens from Ireland and France morpho‐anatomically identified as L. fasciculatum and a specimen from Cornwall likewise identified as L. duckerae were resolved as L. incrustans and L. hibernicum, respectively.     

Stable Carbon Isotope Discrimination Is under Genetic Control in the C4 Species Maize with Several Genomic Regions Influencing Trait Expression

In plants with C₄ photosynthesis, physiological mechanisms underlying variation in stable carbon isotope discrimination (Δ13C) are largely unknown, and genetic components influencing Δ13C have not been described. We analyzed a maize (Zea mays) introgression library derived from two elite parents to investigate whether Δ13C is under genetic control in this C₄ species. High-density genotyping with the Illumina MaizeSNP50 Bead Chip was used for a detailed structural characterization of 89 introgression lines. Phenotypic analyses were conducted in the field and in the greenhouse for kernel Δ13C as well as plant developmental and photosynthesis-related traits. Highly heritable significant genetic variation for Δ13C was detected under field and greenhouse conditions. For several introgression library lines, Δ13C values consistently differed from the recurrent parent within and across the two phenotyping platforms. Δ13C was significantly associated with 22 out of 164 analyzed genomic regions, indicating a complex genetic architecture of Δ13C. The five genomic regions with the largest effects were located on chromosomes 1, 2, 6, 7, and 9 and explained 55% of the phenotypic variation for Δ13C. Plant development stage had no effect on Δ13C expression, as phenotypic as well as genotypic correlations between Δ13C, flowering time, and plant height were not significant. To our knowledge, this is the first study demonstrating Δ13C to be under polygenic control in the C₄ species maize.During photosynthesis, plants use light energy to convert atmospheric CO₂ and water into carbohydrates. For the element carbon, there are two stable isotopes, ¹²C and ¹³C. Due to the physical and chemical properties of photosynthetic CO₂ fixation, plants are depleted in ¹³C compared with atmospheric CO₂. In C₃ plants, this discrimination of stable carbon isotopes (Δ13C) has long been used to detect genetic differences of water use efficiency and has been shown to be an accurate predictor for yield under drought (Rebetzke et al., 2002). As Δ13C is linearly related to the ratio of intercellular to atmospheric CO₂ partial pressure (Farquhar et al., 1982), stomatal closure under drought stress is associated with reduced Δ13C. For C₄ plants, our knowledge about the mechanisms underlying Δ13C and about its association with water use efficiency is much more limited. Differences in Δ13C between genotypes of C₄ species have been reported, among others, for sorghum (Sorghum bicolor; Hubick et al., 1990) and maize (Zea mays; Monneveux et al., 2007). However, comprehensive studies analyzing the inheritance of Δ13C have not been performed to date.In C₃ plants, the important steps of CO₂ uptake include the diffusion of atmospheric CO₂ through the boundary layer and the stomata. Subsequently, CO₂ is taken up by the cell and enters the chloroplast, where carboxylation by Rubisco takes place. During photosynthetic carbon fixation, the strongest fractionation of carbon isotopes occurs during the carboxylation reaction of Rubisco (Roeske and O’Leary, 1984). A theoretical model of Δ13C in C₃ photosynthesis has been described by Farquhar et al. (1982), in which Δ13C depends linearly on the ratio of intercellular to ambient partial pressure of CO₂ (p_i p_a−1), and thus provides an indication of stomatal conductance and photosynthetic capacity. Additionally, the model includes the dependency of Δ13C on the fractionation of carbon isotopes during CO₂ diffusion in the air and on the enzymatic properties of the Rubisco enzyme.For rice (Oryza sativa), tomato (Solanum lycopersicum), and wheat (Triticum aestivum), it has been shown that genetic variation for Δ13C is quantitative, genotype-by-environment interaction is small, and the trait heritability is high (Condon and Richards, 1992; Rebetzke et al., 2002; Comstock et al., 2005; Impa et al., 2005). Quantitative trait loci (QTL) for Δ13C have been mapped (Handley et al., 1994; Price et al., 2002; Rebetzke et al., 2008), and in the model plant Arabidopsis (Arabidopsis thaliana), four genes have been identified that are associated with Δ13C. Two are involved in stomatal patterning and thus influence stomatal conductance (Masle et al., 2005; Nilson and Assmann, 2010), and one of them influences photosynthetic capacity as well (Masle et al., 2005). One gene plays a role in cuticular wax composition and is also associated with stomatal conductance (Lü et al., 2012), whereas the fourth gene encodes a cellulose synthase subunit, and mutations in this gene lead to decreased Δ13C. Presumably, this is the result of a decreased cell turgor due to a decreased water transport capacity of the xylem (Liang et al., 2010).For C₄ plants, our knowledge about the genetic mechanisms and physiological processes underlying Δ13C is much more limited, due to the more complex mechanism of CO₂ fixation. The first carboxylation step in C₄ plants takes place in mesophyll cells, in which CO₂ is fixed by phosphoenolpyruvate carboxylase (PEPC). During this reaction, combined with the fractionation of carbon isotopes during HCO₃⁻ formation, carbon is actually enriched in ¹³C (Farquhar, 1983). The C₄ organic acid formed by PEPC is transported to the bundle sheath cells, where CO₂ is released to be fixed by Rubisco in the second step. However, a fraction of CO₂ released in the bundle sheath can diffuse back to the mesophyll cells. The proportion of carbon fixed by PEPC that subsequently leaks out of the bundle sheath cells is termed leakiness (ϕ) and reduces the opportunity of Rubisco to discriminate against ¹³C in C₄ plants. According to the theoretical model by Farquhar (1983), Δ13C and p_i p_a−1 are also linearly related in C₄ plants, but the regression slope is determined by ϕ. Consequently, there can be a positive or a negative correlation of Δ13C and p_i p_a−1 depending on ϕ (Hubick et al., 1990). Regarding the entire fixation process, discrimination against ¹³C in C₄ plants is not as strong as in C₃ plants, and so far there have been few studies reporting a genetic variation of Δ13C in C₄ plants. In sorghum, small but significant differences in Δ13C have been observed among 12 cultivars (Hubick et al., 1990), and similar to C₃ plants, Δ13C has been shown to be correlated with transpiration efficiency (Henderson et al., 1998). Additionally, it has been shown for maize and sugarcane (Saccharum officinarum) that stress conditions lead to an increase in Δ13C (Bowman et al., 1989; Meinzer et al., 1994; Ranjith et al., 1995; Buchmann et al., 1996). Experiments under drought and under well-watered conditions showed higher values for Δ13C in drought-tolerant maize hybrids than in susceptible checks (Monneveux et al., 2007).The use of Δ13C as an indirect trait in breeding for drought tolerance in C₄ species would be highly beneficial, given a stable trait expression and high heritability similar to that in C₃ plants. To assess whether Δ13C can also be used in C₄ plants as an indirect selection trait for drought-tolerant lines, it needs to be shown that Δ13C is under genetic control, although the physiology and molecular mechanisms of Δ13C are not yet fully understood. In this study, we used an introgression library (IL; Eshed and Zamir, 1994) derived from two elite parents to analyze the genetic variation in Δ13C under well-watered conditions. ILs have been successfully used in genetics to identify QTL for various qualitatively and quantitatively inherited traits. An IL is a defined set of nearly isogenic inbred lines derived from repeated backcrosses with one of the parents (recurrent parent [RP]) and marker-assisted selection for single fragments (Supplemental Fig. S1). Ideally, each IL line carries a single chromosome fragment of a donor parent (DP) in the genetic background of an RP. Taken together, the different segments cover the whole donor genome, allowing estimation of the effects of single donor fragments in an otherwise identical genetic background (Eshed and Zamir, 1994). The RP of the IL under investigation originates from southeastern Europe and is an elite inbred line of the maize dent pool. As DP, we chose an unrelated maize line representative of the European flint pool. The IL (IL_01–IL_89) was genotyped using the Illumina MaizeSNP50 Bead Chip (Ganal et al., 2011) carrying 56,110 single-nucleotide polymorphism (SNP) markers.Kernel Δ13C of 77 IL lines was measured in the field and in the greenhouse (Δ13C is genetically controlled in the C₄ species maize. Our specific goals were (1) to characterize the genetic architecture of Δ13C (i.e. to determine the number of genomic regions associated with Δ13C), (2) to localize genomic regions influencing Δ13C, and (3) to assess the extent to which genotypic variation in Δ13C might be the result of differences in plant development.

Table I.

Overview of the experiments and experimental designs

The Shigella flexneri Type 3 Secretion System Is Required for Tyrosine Kinase-Dependent Protrusion Resolution,and Vacuole Escape during Bacterial Dissemination

Shigella flexneri is a human pathogen that triggers its own entry into intestinal cells and escapes primary vacuoles to gain access to the cytosolic compartment. As cytosolic and motile bacteria encounter the cell cortex, they spread from cell to cell through formation of membrane protrusions that resolve into secondary vacuoles in adjacent cells. Here, we examined the roles of the Type 3 Secretion System (T3SS) in S. flexneri dissemination in HT-29 intestinal cells infected with the serotype 2a strain 2457T. We generated a 2457T strain defective in the expression of MxiG, a central component of the T3SS needle apparatus. As expected, the ΔmxiG strain was severely affected in its ability to invade HT-29 cells, and expression of mxiG under the control of an arabinose inducible expression system (ΔmxiG/pmxiG) restored full infectivity. In this experimental system, removal of the inducer after the invasion steps (ΔmxiG/pmxiG (Ara withdrawal)) led to normal actin-based motility in the cytosol of HT-29 cells. However, the time spent in protrusions until vacuole formation was significantly increased. Moreover, the number of formed protrusions that failed to resolve into vacuoles was also increased. Accordingly, the ΔmxiG/pmxiG (Ara withdrawal) strain failed to trigger tyrosine phosphorylation in membrane protrusions, a signaling event that is required for the resolution of protrusions into vacuoles. Finally, the ΔmxiG/pmxiG (Ara withdrawal) strain failed to escape from the formed secondary vacuoles, as previously reported in non-intestinal cells. Thus, the T3SS system displays multiple roles in S. flexneri dissemination in intestinal cells, including the tyrosine kinase signaling-dependent resolution of membrane protrusions into secondary vacuoles, and the escape from the formed secondary vacuoles.     

Eosinophils and Megakaryocytes Support the Early Growth of Murine MOPC315 Myeloma Cells in Their Bone Marrow Niches

Multiple myeloma is a bone marrow plasma cell tumor which is supported by the external growth factors APRIL and IL-6, among others. Recently, we identified eosinophils and megakaryocytes to be functional components of the micro-environmental niches of benign bone marrow plasma cells and to be important local sources of these cytokines. Here, we investigated whether eosinophils and megakaryocytes also support the growth of tumor plasma cells in the MOPC315.BM model for multiple myeloma. As it was shown for benign plasma cells and multiple myeloma cells, IL-6 and APRIL also supported MOPC315.BM cell growth in vitro, IL-5 had no effect. Depletion of eosinophils in vivo by IL-5 blockade led to a reduction of the early myeloma load. Consistent with this, myeloma growth in early stages was retarded in eosinophil-deficient ΔdblGATA-1 mice. Late myeloma stages were unaffected, possibly due to megakaryocytes compensating for the loss of eosinophils, since megakaryocytes were found to be in contact with myeloma cells in vivo and supported myeloma growth in vitro. We conclude that eosinophils and megakaryocytes in the niches for benign bone marrow plasma cells support the growth of malignant plasma cells. Further investigations are required to test whether perturbation of these niches represents a potential strategy for the treatment of multiple myeloma.