Jsowerbya, new genus of Muricidae (Mollusca: Gastropoda) from the Eocene of the Paris (France) and Hampshire (England) basins with a phylogenetic assessment of its Ocenebrine versus Ergalataxine affinities

Jsowerbya, nov. gen. (Gastropoda: Muricidae) includes three species from the Eocene of the Paris and Hampshire basins. It increases the number of extinct muricid genera, which curiously represent a very small fraction of the described genera. The species of Jsowerbya, often mistaken for the muricopsine genus Muricopsis, possess a unique combination of characters shared with the subfamilies Ocenebrinae and Ergalataxinae. A cladistic analysis, based on structural homologies of the spiral sculpture, however, suggests that Jsowerbya is closely related to the Ocenebrinae. Thus, Jsowerbya is here regarded as one the most basal Ocenebrinae.     

Accumulation of pollutants in fish

The amount of radioactivity which derived from 14C-labeled pollutants was determined in liver, kidney, intestine, blood, muscle and gills of carp, exposed for 6, 24 and 72 hr to high external concentrations of urea, methanol, atrazine and PCP. The results allowed one to calculate roughly the uptake rate for these compounds. It was low for urea (0.055 micrograms/g per hr), higher for methanol (0.12) and atrazine (0.16) and highest for PCP (1.5). The bioaccumulation factors (BFs) were determined for the different substances and organs. They correlated with the hydrophilic-lipophilic nature of the chemicals. The more lipophilic the substances the more accumulation occurred in the liver. PCP accumulated the most. BF was 300-400 in most tissues except muscle where it was quite low. The BF was 3-4 for atrazine in liver, kidney and intestine, but just 1 in blood, muscle and gills. There is some evidence that the BF for methanol equals 1 in liver, kidney, gills and intestine. It is less than 1 in blood and muscle. Urea was equally distributed in all organs and in the external medium.     

Use of cyanuric chloride-activated paper for detection of subpicogram quantities of specific DNA sequences and its application to linked restriction fragment length polymorphism analysis in a Duchenne muscular dystrophy affected family

Conditions for the optimal use of cyanuric chloride-activated (CCA) paper in Southern transfer hybridization experiments of genomic DNA were investigated. They depend critically on pH and ionic strength during transfer and on the composition of the hybridization solution. Simplified hybridization conditions using a SSC/dextran sulfate system at 65 degrees C without sodium dodecyl sulfate and the complex Denhardt's solution are applied. CCA paper allows repeated use in hybridization experiments. Under optimized conditions CCA paper allows a more sensitive detection of single-copy gene sequences in the subpicogram range than do nylon membranes. Application of these transfer and hybridization conditions with our newly developed CCA paper to carrier determination and prediction of the healthy male haplotype demonstrates its usefulness for prenatal counseling of a Duchenne muscular dystrophy family.     

Salt‐enhanced cultivation as a morphology engineering tool for filamentous actinomycetes: Increased production of labyrinthopeptin A1 in Actinomadura namibiensis

Salt‐enhanced cultivation as a morphology engineering tool for the filamentous actinomycete Actinomadura namibiensis was evaluated in 500‐mL shaking flasks (working volume 100 mL) with the aim of increasing the concentration of the pharmaceutically interesting peptide labyrinthopeptin A1. Among the inorganic salts added to a complex production medium, the addition of (NH₄)₂SO₄ led to the highest amount of labyrinthopeptin A1 production. By using 50 mM (NH₄)₂SO₄, the labyrinthopeptin A1 concentration increased up to sevenfold compared to the non‐supplemented control, resulting in 325 mg L^?1 labyrinthopeptin A1 after 10 days of cultivation. The performance of other ammonium‐ and sulfate‐containing salts (e.g., NH₄Cl, K₂SO₄) was much lower than the performance of (NH₄)₂SO₄. A positive correlation between the uptake of glycerol as one of the main carbon sources and nongrowth‐associated labyrinthopeptin productivity was found. The change in the cell morphology of A. namibiensis in conjunction with increased osmolality by the addition of 50 mM (NH₄)₂SO₄, was quantified by image analysis. A. namibiensis always developed a heterogeneous morphology with pellets and loose mycelia present simultaneously. In contrast to the non‐supplemented control, the morphology of (NH₄)₂SO₄‐supplemented cultures was characterized by smaller and circular pellets that were more stable against disintegration in the stationary production phase.     

Integration of BpMADS4 on various linkage groups improves the utilization of the rapid cycle breeding system in apple

Rapid cycle breeding in apple is a new approach for the rapid introgression of agronomically relevant traits (e.g. disease resistances) from wild apple species into domestic apple cultivars (Malus × domestica Borkh.). This technique drastically shortens the long‐lasting juvenile phase of apple. The utilization of early‐flowering apple lines overexpressing the BpMADS4 gene of the European silver birch (Betula pendula Roth.) in hybridization resulted in one breeding cycle per year. Aiming for the selection of non‐transgenic null segregants at the end of the breeding process, the flower‐inducing transgene and the gene of interest (e.g. resistance gene) that will be introgressed by hybridization need to be located on different chromosomes. To improve the flexibility of the existing approach in apple, this study was focused on the development and characterization of eleven additional BpMADS4 overexpressing lines of four different apple cultivars. In nine lines, the flowering gene was mapped to different linkage groups. The differences in introgressed T‐DNA sequences and plant genome deletions post‐transformation highlighted the unique molecular character of each line. However, transgenic lines demonstrated no significant differences in flower organ development and pollen functionality compared with non‐transgenic plants. Hybridization studies using pollen from the fire blight‐resistant wild species accession Malus fusca MAL0045 and the apple scab‐resistant cultivar ‘Regia’ indicated that BpMADS4 introgression had no significant effect on the breeding value of each transgenic line.     

Glycosylation of Skp1 Promotes Formation of Skp1–Cullin-1–F-box Protein Complexes in Dictyostelium

O₂ sensing in diverse protozoa depends on the prolyl 4 hydroxylation of Skp1 and modification of the resulting hydroxyproline with a series of five sugars. In yeast, plants, and animals, Skp1 is associated with F-box proteins. The Skp1–F-box protein heterodimer can, for many F-box proteins, dock onto cullin-1 en route to assembly of the Skp1–cullin-1–F-box protein–Rbx1 subcomplex of E3^SCFUb ligases. E3^SCFUb ligases conjugate Lys48-polyubiquitin chains onto targets bound to the substrate receptor domains of F-box proteins, preparing them for recognition by the 26S proteasome. In the social amoeba Dictyostelium, we found that O₂ availability was rate-limiting for the hydroxylation of newly synthesized Skp1. To investigate the effect of reduced hydroxylation, we analyzed knockout mutants of the Skp1 prolyl hydroxylase and each of the Skp1 glycosyltransferases. Proteomic analysis of co-immunoprecipitates showed that wild-type cells able to fully glycosylate Skp1 had a greater abundance of an SCF complex containing the cullin-1 homolog CulE and FbxD, a newly described WD40-type F-box protein, than the complexes that predominate in cells defective in Skp1 hydroxylation or glycosylation. Similarly, the previously described FbxA–Skp1CulA complex was also more abundant in glycosylation-competent cells. The CulE interactome also included higher levels of proteasomal regulatory particles when Skp1 was glycosylated, suggesting increased activity consistent with greater association with F-box proteins. Finally, the interactome of FLAG-FbxD was modified when it harbored an F-box mutation that compromised Skp1 binding, consistent with an effect on the abundance of potential substrate proteins. We propose that O₂-dependent posttranslational glycosylation of Skp1 promotes association with F-box proteins and their engagement in functional E3^SCFUb ligases that regulate O₂-dependent developmental progression.Timely protein degradation is a cornerstone of cell cycling and the regulation of numerous physiological and developmental processes. Eukaryotes have evolved an extensive array of polyubiquitination enzymes to tag proteins on a protein-by-protein basis as a recognition marker for degradation in the 26S proteasome. The cullin-RING ubiquitin ligases (CRLs)¹ are a prominent subgroup of these enzymes (1) and consist of an E3 architecture that includes a substrate receptor, an adaptor (in most cases), the cullin scaffold, the RING protein, and an exchangeable E2 ubiquitin donor that has been charged with ubiquitin (Ub) by an E1 enzyme. The first discovered and still prototypic example is the CRL1 class (2), also referred to as SCF on account of the names of its founding subunits, Skp1, cullin-1, and F-box proteins (FBPs). The CRL1 (or SCF) complexes utilize FBPs as substrate receptors, Skp1 as the adaptor linking the FBP to the N-terminal region of cullin-1 (Cul1), and Rbx1 as the RING protein that tethers the E2 Ub donor to the Cul1 C-terminal region (see Fig. 2B). CRL1s can be activated by neddylation of Cul1 by a Nedd8-specific E2, which mobilizes Rbx1 to afford rotational flexibility of the E2 and displaces the inhibitor Cand1, permitting docking of the Skp1–FBP heterodimer (3–5). Deneddylation mediated by the eight-subunit COP9 signalosome is required for in vivo activity, suggesting that Cand1 serves as a substrate exchange factor to allow for re-equilibration of SCF complexes from preexisting subunits. Each reaction cycle requires the exchange of a new E2-Ub and typically assembles a K48-linked polyUb chain that is recognized by the proteasome. Substrate specificity is conferred by FBPs, a gene family that numbers 69 in humans, 20 in budding yeast, 300 in Caenorhabditis elegans, and ∼800 in Arabidopsis. Some characterized FBPs can recognize perhaps a dozen or more substrates, and the coding of recognition and the meaning of their control by the same FBP is under intense investigation (6). Recognition is often activated by posttranslational modification of the substrate (often phosphorylation). Regulation of SCF Ub ligases has centered on the neddylation cycle, which potentially influences all seven known CRLs. Regulation of Skp1, investigated in this paper, would be specific to CRLs possessing Skp1, which include CRL1 and possibly the minor class CRL7 (7).Open in a separate windowFig. 2.Skp1 modification pathway and global analysis of Skp1 interactions. A, Skp1 is sequentially modified by the indicated enzymes (in blue), resulting in the formation of a pentasaccharide at Pro143. B, model of the SCF complex in the context of the overall E3 Ub ligase, from studies in yeast, plants, and animals. Catalysis involves transfer of Ub from an exchangeable Ub-E2 conjugate to the substrate. Removal of Nedd8 by the COP9 signalosome facilitates binding of Cand1 to Cul1, which inhibits binding of Skp1 to Cul1. C, D, vegetative (growth stage) cells were filter-lysed, and a cytosolic fraction prepared via ultracentrifugation was chromatographed on a Superose 12 gel filtration column. Fractions were analyzed via Western blotting (representative examples are shown in C) followed by densitometry (D). The elution position of free Skp1 from a separate trial is indicated.The basic SCF model is thought to be widespread among eukaryotes but has been extensively studied only in fungi/yeasts, plants, and animals. The broad phylogeny represented by protists includes many benign and pathogenic unicellular organisms of great economic, health, and environmental impact. Emerging evidence reveals that Skp1 in some of these groups is subject to a novel form of prolyl 4(trans)-hydroxylation and complex glycosylation (8). The roles of these Skp1 modifications have been most studied in the social amoeba Dictyostelium, which undergoes a starvation-induced developmental program during which individual amoebae chemotactically aggregate into an initial mound that then elongates into a migratory slug. Under appropriate conditions, the slug reorganizes to form a fruiting body consisting of a ball of spores supported by a vertical cellular stalk. The slug-to-fruit switch, referred to as culmination, and sporulation are regulated by checkpoints that are sensitive to multiple factors, including O₂ (9–11). Functional studies of Dictyostelium Skp1 hydroxylation and glycosylation reveal roles in regulating the O₂ dependence of culmination and sporulation (12–14). For example, wild-type (wt) cells require 7% to 10% O₂ and phyA^– requires 18% to 21% O₂ in order to achieve 50% spore formation (a quantitative measure of fruiting body formation), whereas glycosylation mutants exhibit a complex pattern of intermediate requirements (13). In addition, at 21% O₂, phyA^– cells require an additional 3 to 4 h to complete development relative to their wt counterparts (14). In the apicomplexan Toxoplasma gondii, PhyA is also required for Skp1 glycosylation, and phyA^– parasites are deficient in proliferation, especially at low O₂ (15).The idea that O₂ availability is rate limiting for Skp1 modification was originally based on the observation that the Dictyostelium phyA^– phenotype mimics that of wt cells in low O₂ (9). However, the majority of Skp1 is hydroxylated and glycosylated in wt cells even at low O₂ levels where culmination is blocked or delayed. Further analysis of a submerged development model, in which terminal development depended on an atmosphere of 70% to 100% O₂ in order to overcome the diffusion barrier posed by the water layer, showed that at atmospheric O₂ levels of 5% to 21% where sporulation was blocked, unmodified Skp1 accumulated to a higher level than at permissive O₂ levels (10). As Skp1 modifications are thought to be irreversible, this likely resulted from slow hydroxylation of newly synthesized Skp1. To address this in a more physiological setting, we investigated nascent Skp1 directly using metabolic labeling with [³⁵S]Met/Cys and verified that the rate of hydroxylation of newly synthesized Skp1 polypeptide was indeed inversely proportional to O₂ levels, which makes PhyA-mediated hydroxylation of Skp1 an excellent candidate for the primary O₂ sensor for culmination.These modifications of Skp1 are of interest as a novel mechanism regulating the SCF ligase. Previously, we showed that hydroxylation and glycosylation of Dictyostelium Skp1 affect its conformation and promote binding to a soluble FBP, guinea pig Fbs1, in studies of purified proteins (16). Here we show that Dictyostelium Skp1 is indeed a subunit of a canonical SCF complex, as expected. The significance of undermodified Skp1 was examined via interactome analysis of Skp1 isoforms that accumulate in modification pathway mutants. Our findings revealed a lower abundance of SCF complexes than in wt cells, suggesting that Skp1 modification may promote SCF assembly and E3^SCFUb ligase activities that control timely turnover of select proteins involved in developmental progression.     

UV Light Reveals the Diversity of Jurassic Shell Colour Patterns: Examples from the Cordebugle Lagerst?tte (Calvados,France)

Viewed under UV light the diverse and exceptionally well-preserved molluscs from the Late Jurassic Cordebugle Konservat Lagerstätte (Calvados, Normandy, France) reveal fluorescent fossil shell colour patterns predating the oldest previously known instance of such patterns by 100 Myr. Evidently, residual colour patterns are observable in Mesozoic molluscs by application of this non-destructive method, provided the shells are not decalcified or recrystallized. Among 46 species which are assigned to twelve gastropod families and eight bivalve families, no less than 25 species yielded positive results. Out of nine colour pattern morphologies that have been distinguished six occur in gastropods and three in bivalves. The presence of these variant morphologies clearly indicates a significant pre-Cenozoic diversification of colour patterns, especially in gastropods. In addition, the occurrence of two distinct types of fluorescence highlights a major difference in the chemical composition of the pigments involved in colour pattern formation in gastropods. This discovery enables us to discriminate members of higher clades, i.e. the Vetigastropoda emitting red fluorescence from the Caenogastropoda and Heterobranchia emitting whitish-beige to yellow fluorescence. Consequently, fluorescent colour patterns may help to allocate part of the numerous enigmatic Mesozoic gastropod taxa to their correct systematic position.     

Capacity of Broadly Neutralizing Antibodies to Inhibit HIV-1 Cell-Cell Transmission Is Strain- and Epitope-Dependent

An increasing number of broadly neutralizing antibodies (bnAbs) are considered leads for HIV-1 vaccine development and novel therapeutics. Here, we systematically explored the capacity of bnAbs to neutralize HIV-1 prior to and post-CD4 engagement and to block HIV-1 cell-cell transmission. Cell-cell spread is known to promote a highly efficient infection with HIV-1 which can inflict dramatic losses in neutralization potency compared to free virus infection. Selection of bnAbs that are capable of suppressing HIV irrespective of the transmission mode therefore needs to be considered to ascertain their in vivo activity in therapeutic use and vaccines. Employing assay systems that allow for unambiguous discrimination between free virus and cell-cell transmission to T cells, we probed a panel of 16 bnAbs for their activity against 11 viruses from subtypes A, B and C during both transmission modes. Over a wide range of bnAb-virus combinations tested, inhibitory activity against HIV-1 cell-cell transmission was strongly decreased compared to free virus transmission. Activity loss varied considerably between virus strains and was inversely associated with neutralization of free virus spread for V1V2- and V3-directed bnAbs. In rare bnAb-virus combinations, inhibition for both transmission modes was comparable but no bnAb potently blocked cell-cell transmission across all probed virus strains. Mathematical analysis indicated an increased probability of bnAb resistance mutations to arise in cell-cell rather than free virus spread, further highlighting the need to block this pathway. Importantly, the capacity to efficiently neutralize prior to CD4 engagement correlated with the inhibition efficacy against free virus but not cell-cell transmitted virus. Pre-CD4 attachment activity proved strongest amongst CD4bs bnAbs and varied substantially for V3 and V1V2 loop bnAbs in a strain-dependent manner. In summary, bnAb activity against divergent viruses varied depending on the transmission mode and differed depending on the window of action during the entry process, underscoring that powerful combinations of bnAbs are needed for in vivo application.