Bax-Induced Apoptosis in Leber's Congenital Amaurosis: A Dual Role in Rod and Cone Degeneration

Pathogenesis in the Rpe65^−/− mouse model of Leber''s congenital amaurosis (LCA) is characterized by a slow and progressive degeneration of the rod photoreceptors. On the opposite, cones degenerate rapidly at early ages. Retinal degeneration in Rpe65 ^−/− mice, showing a null mutation in the gene encoding the retinal pigment epithelium 65-kDa protein (Rpe65), was previously reported to depend on continuous activation of a residual transduction cascade by unliganded opsin. However, the mechanisms of apoptotic signals triggered by abnormal phototransduction remain elusive. We previously reported that activation of a Bcl-2-dependent pathway was associated with apoptosis of rod photoreceptors in Rpe65^−/− mice during the course of the disease. In this study we first assessed whether activation of Bcl-2-mediated apoptotic pathway was dependent on constitutive activation of the visual cascade through opsin apoprotein. We then challenged the direct role of pro-apoptotic Bax protein in triggering apoptosis of rod and cone photoreceptors.Quantitative PCR analysis showed that increased expression of pro-apoptotic Bax and decreased level of anti-apoptotic Bcl-2 were restored in Rpe65^−/−/Gnat1^−/− mice lacking the Gnat1 gene encoding rod transducin. Moreover, photoreceptor apoptosis was prevented as assessed by TUNEL assay. These data indicate that abnormal activity of opsin apoprotein induces retinal cell apoptosis through the Bcl-2-mediated pathway. Following immunohistological and real-time PCR analyses, we further observed that decreased expression of rod genes in Rpe65-deficient mice was rescued in Rpe65^−/−/Bax^−/− mice. Histological and TUNEL studies confirmed that rod cell demise and apoptosis in diseased Rpe65^−/− mice were dependent on Bax-induced pathway. Surprisingly, early loss of cones was not prevented in Rpe65^−/−/Bax^−/− mice, indicating that pro-apoptotic Bax was not involved in the pathogenesis of cone cell death in Rpe65-deficient mice.This is the first report, to our knowledge, that a single genetic mutation can trigger two independent apoptotic pathways in rod and cone photoreceptors in Rpe65-dependent LCA disease. These results highlight the necessity to investigate and understand the specific death signaling pathways committed in rods and cones to develop effective therapeutic approaches to treat RP diseases.     

A fluorescent ligand-binding alternative using Tag-lite® technology

G-protein-coupled receptors (GPCRs) are crucial cell surface receptors that transmit signals from a wide range of extracellular ligands. Indeed, 40% to 50% of all marketed drugs are thought to modulate GPCR activity, making them the major class of targets in the drug discovery process. Binding assays are widely used to identify high-affinity, selective, and potent GPCR drugs. In this field, the use of radiolabeled ligands has remained so far the gold-standard method. Here the authors report a less hazardous alternative for high-throughput screening (HTS) applications by the setup of a nonradioactive fluorescence-based technology named Tag-lite(?). Selective binding of various fluorescent ligands, either peptidic or not, covering a large panel of GPCRs from different classes is illustrated, particularly for chemokine (CXCR4), opioid (δ, μ, and κ), and cholecystokinin (CCK1 and CCK2) receptors. Affinity constants of well-known pharmacological agents of numerous GPCRs are in line with values published in the literature. The authors clearly demonstrate that the Tag-lite binding assay format can be successfully and reproducibly applied by using different cellular materials such as transient or stable recombinant cells lines expressing SNAP-tagged GPCR. Such fluorescent-based binding assays can be performed with adherent cells or cells in suspension, in 96- or 384-well plates. Altogether, this new technology offers great advantages in terms of flexibility, rapidity, and user-friendliness; allows easy miniaturization; and makes it completely suitable for HTS applications.     

Clubroot resistance QTL are modulated by nitrogen input in <Emphasis Type="Italic">Brassica napus</Emphasis>

Key message

Nitrogen levels can modulate the effectiveness of clubroot resistance in an isolate- and host-specific manner. While the same QTL were detected under high and low nitrogen, their effects were altered.

Abstract

Clubroot, caused by Plasmodiophora brassicae, is one of the most damaging diseases of oilseed rape and is known to be affected by nitrogen fertilization. However, the genetic factors involved in clubroot resistance have not been characterized under nitrogen-limiting conditions. This study aimed to assess the variability of clubroot resistance under different nitrogen levels and to characterize the impact of nitrogen supply on genetic resistance factors. Linkage analyses and a genome-wide association study were conducted to detect QTL for clubroot resistance and evaluate their sensitivity to nitrogen. The clubroot response of a set of 92 diverse oilseed rape accessions and 108 lines derived from a cross between ‘Darmor-bzh’ (resistant) and ‘Yudal’ (susceptible) was studied in the greenhouse under high- and low-nitrogen conditions, following inoculation with the P. brassicae isolates eH and K92-16. Resistance to each isolate was controlled by a major QTL and a few small-effects QTL. While the same QTL were detected under both high and low nitrogen, their effects were altered. Clubroot resistance to isolate eH, but not K92-16, was greater under a low-N supply versus a high-N supply. New sources of resistance were found among the oilseed rape accessions under both low and high-N conditions. The results are discussed relative to the literature and from a crop improvement perspective.

     

<i>Trichoderma</i> spp. fostering growth on <i>Capsicum chinense</i> Jacq. seedlings and antagonistic against <i>Meloidogyne incognita</i>

Fourteen native strains of Trichoderma spp. from wildand agricultural pathosystems in the state of Yucatan, Mexico, with growth-promoting ability of Capsicum chinense Jacq. seedlings were evaluated and antagonistic effect of their filtrate against second-stage juveniles (J2) of Meloidogyne incognita. The strains Th05-02 and Th27-08 showed the best significant effects on plant hight variable increments 55.57 and 47.62%, theTh07-04 with 29.48% more root length, theTh02-01 and Th07-04 isolates increased from 48.71 to 84.61% in volume radical and 53.40% of total dry biomass. Statistical analysis (p≤0.001) of Th43 and Th43-13-14 filtrates caused 100% mortality at 24 and 48h. In the test of reversibility to 24 h after replacing the filtrates Th43-13, Th43-14, TH09-06 and TH20-07 by sterile distilled water, the J2 did not recover their viability, so they were considered as the best potential strains of Trichoderma spp. with antagonistic capacity in J2 of M.incognita.     

Body size effects and rates of cytochrome b evolution in tube-nosed seabirds

Variation in rates of molecular evolution now appears to be widespread. The demonstration that body size is correlated with rates of molecular evolution suggests that physiological and ecological factors may be involved in molecular rate variation, but large-scale comparative studies are still lacking. Here, we use complete cytochrome b sequences from 85 species of tube-nosed seabirds (order Procellariiformes) and 5 outgroup species of penguins (order Sphenisciformes) to test for an association between body mass and rates of molecular evolution within the former avian order. Cladistic analysis of the 90 sequences estimates a phylogeny largely consistent with the traditional taxonomy of the Procellariiformes. The Diomedeidae, Procellariidae, and Pelecanoididae are monophyletic, while the Hydrobatidae are basal and paraphyletic. However, the two subfamilies within the Hydrobatidae (Hydrobatinae and Oceanitinae) are monophyletic. A likelihood ratio test detects significant deviation from clocklike evolution in our data. Using a sign test for an association between body mass and branch length in the seabird phylogeny, we find that larger taxa tend to have shorter terminal branch lengths than smaller taxa. This observation suggests that rates of mitochondrial DNA evolution are slower for larger taxa. Rate calibrations based on the fossil record reveal concordant body size effects. We interpret these results as evidence for a metabolic rate effect, as the species in this order exhibit large differences in metabolic rates, which are known to be highly correlated with body mass in this group. Our results support previous findings of body size effects and show that this effect can be significant even within a single avian order. This suggests that even lineage-specific molecular clocks may not be tenable if calibrations involve taxa with different metabolic rates.      

Chondrocalcin is internalized by chondrocytes and triggers cartilage destruction via an interleukin-1β-dependent pathway

Chondrocalcin is among the most highly synthesized polypeptides in cartilage. This protein is released from its parent molecule, type II pro-collagen, after secretion by chondrocytes. A participation of extracellular, isolated chondrocalcin in mineralization was proposed more than 25 years ago, but never demonstrated. Here, exogenous chondrocalcin was found to trigger MMP13 secretion and cartilage destruction ex vivo in human cartilage explants and did so by modulating the expression of interleukin-1β in primary chondrocyte cultures in vitro. Chondrocalcin was found internalized by chondrocytes. Uptake was found mediated by a single 18-mer peptide of chondrocalcin, which does not exhibit homology to any known cell-penetrating peptide. The isolated peptide, when artificially linked as a tetramer, inhibited gene expression regulation by chondrocalcin, suggesting a functional link between uptake and gene expression regulation. At the same time, the tetrameric peptide potentiated chondrocalcin uptake by chondrocytes, suggesting a cooperative mechanism of entry. The corresponding peptide from type I pro-collagen supported identical cell-penetration, suggesting that this property may be conserved among C-propeptides of fibrillar pro-collagens. Structural modeling localized this peptide to the tips of procollagen C-propeptide trimers. Our findings shed light on unexpected function and mechanism of action of these highly expressed proteins from vertebrates.     

SEGMENTATION APPROACHES THAT DIFFERENTIATE CONSUMPTION FREQUENCY FROM SENSORY PREFERENCE1

People can eat a food without having a strong preference for it, and people can prefer a food without eating it. Given this seeming disconnect between attitude and behavior, which type of measure or segment can best be used to profile or identify loyal consumer segments of a food, such as soy? This research compares a usage‐based method (heavy‐light‐nonusers) with a new attitude‐based method (seeker‐neutral‐avoider), and finds that the attitude‐based method differentiates purchase‐related intentions better than the usage‐based method. Implications for profiling consumer taste patterns and consumer segments are provided.