Effects of selective prostaglandin EP2 and EP4 receptor agonists on bone resorption and formation in fetal rat organ cultures

Prostaglandin E2 (PGE2) can stimulate bone resorption and formation through receptors which activate adenylyl cyclase. We examined the effects of selective EP2 and EP4 agonists (EP2A and EP4A) on the release of previously incorporated 45Ca from fetal rat long bones and the incorporation of [3H]-proline or [3H]-thymidine (TdR) in fetal rat calvaria to assess the relative effects of these selective agonists on bone formation and resorption. Only EP4A was effective in increasing 45Ca release. Both agonists increased [3H]-TdR incorporation and [3H]-proline incorporation into calvariae, particularly in the presence of cortisol.     

Species diversity of the genus Osmundea (Ceramiales,Rhodophyta) in the Macaronesian region

Species diversity within the genus Osmundea in the Macaronesian region was explored by conducting a comprehensive sampling in the Azores, the Canary, and the Madeira archipelagos. Toward identification, all specimens were first observed alive to verify the absence of corps en cerise, a diagnostic character for the genus and morphometric data were measured (thallus length and width, first‐order branches length and width, branchlets length and width, cortical cell length and width in surface view, cortical cell length and width in transverse section). Specimens were sequenced for COI‐5P (39 specimens) and three species delimitation methods (Generalized Mixed Yule Coalescent, Automatic Barcode Gap Discovery method, and Poisson Tree Processes) were used to assess the threshold between infra‐ and interspecific relationships. Subsequently, one or several sequences of plastid‐encoded large subunit of RuBisCO (21 specimens) per delimited species were generated to assess the phylogenetic relationships among Macaronesian Osmundea. Moreover, for each delineated species, vegetative and reproductive anatomy was thoroughly documented and, when possible, specimens were either assigned to existing taxa or described as novel species. This integrative approach has provided data for (i) the presence of O. oederi, O. pinnatifida, and O. truncata in Macaronesia; (ii) the proposal of two novel species, O. prudhommevanreinei sp. nov. and O. silvae sp. nov.; and (iii) evidence of an additional species referred as “Osmundea sp.1,” which is a sister taxon of O. hybrida.     

Glucocorticoids Induce G1 as Well as S-Phase Lengthening in Normal Human Stimulated Lymphocytes: Differential Effects on Cell Cycle Regulatory Proteins

In order to analyze dexamethasone effects on peripheral blood lymphocyte proliferation, we defined various experimental conditions: dexamethasone introduced (i) at the time of phytohemagglutinin stimulation, (ii) 48 h after the beginning of phytohemagglutinin stimulation, and (iii) on unstimulated lymphocytes. In stimulated lymphocytes, we observed an early G1 accumulation (P< 0.005), a delayed increase in the duration of S-phase (P< 0.03), and a consequent increase in cell-cycle duration. The expression of several cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors (CKIs) was modified. Cyclin D3, CDK4, and CDK6 involved in G1-phase control were significantly decreased under dexamethasone treatment whatever the level of stimulation of lymphocytes (stimulated or unstimulated PBL). Cyclin E and CDK2, acting in G1/S-phase transition and S-phase regulation, decreased in stimulated lymphocytes before any modification of S-phase (P< 0.002). The expression of CKIs, mainly of p27^Kip1, appeared to vary with the degree of cell stimulation: a decrease was observed on treated unstimulated lymphocytes, while p27^Kip1increased in dexamethasone-treated cells during stimulation. Our results indicate sequential modifications of the cell-cycle regulation by dexamethasone starting with an action on G1 followed by S-phase control modifications. The protein analysis pinpoints the major complexes concerned: CDK4 and CDK6/cyclin D are mainly involved in G1-phase modifications, while CDK2 and its partner, cyclin E, might be specifically involved in the lengthening of S-phase. The variations observed for p27^Kip1might amplify the functional effects of dexamethasone on kinasic complexes.     

Mapping the interactions between a RUN domain from DENND5/Rab6IP1 and sorting nexin 1

Eukaryotic cells have developed a diverse repertoire of Rab GTPases to regulate vesicle trafficking pathways. Together with their effector proteins, Rabs mediate various aspects of vesicle formation, tethering, docking and fusion, but details of the biological roles elicited by effectors are largely unknown. Human Rab6 is involved in the trafficking of vesicles at the level of Golgi via interactions with numerous effector proteins. We have previously determined the crystal structure of Rab6 in complex with DENND5, alternatively called Rab6IP1, which comprises two RUN domains (RUN1 and RUN2) separated by a PLAT domain. The structure of Rab6/RUN1-PLAT (Rab6/R1P) revealed the molecular basis for Golgi recruitment of DENND5 via the RUN1 domain, but the functional role of the RUN2 domain has not been well characterized. Here we show that a soluble DENND5 construct encompassing the RUN2 domain binds to the N-terminal region of sorting nexin 1 by surface plasmon resonance analyses.     

Determination of glutamic acid decarboxylase 65 peptides presented by the type I diabetes-associated HLA-DQ8 class II molecule identifies an immunogenic peptide motif

Particular HLA class II allelic sequences are associated with susceptibility to type I diabetes. To understand the mechanism, knowledge of the molecular nature of the specific TCR/peptide/class II interactions involved in the disease process is required. To this end, we have introduced the diabetes-associated human class II HLA-DQ8 allele (DQA1*0301/DQB1*0302) as a transgene into mice and analyzed T cell responses restricted by this molecule to an important Ag in human diabetes, human glutamic acid decarboxylase 65. Hybridomas were used to determine the particular peptides from this Ag presented by HLA-DQ8 to T cells and to map the core minimal epitopes required for T cell stimulation. Analysis of these core epitopes reveals a motif and relevant features for peptides that are immunogenic to T cells when presented by HLA-DQ8. The major immunogenic epitopes of glutamic acid decarboxylase 65 do not contain a negatively charged residue that binds in the P9 pocket of the HLA-DQ8 molecule. PBMC from HLA-DQ8+ diabetic and nondiabetic individuals respond to these peptides, confirming that the mouse model is a useful tool to define epitopes of autoantigens that are processed by human APC and recognized by human T cells.     

Genetic and Pathogenic Characterization of Different Stagonospora sp. Isolated from Bindweed

Genetic variation among 38 isolates of Stagonospora sp. and 10 isolates of Septoria sp. from bindweed was studied using (a) restriction fragment length plymorphism (RFLP) analysis of the internal transcribed spacer (ITS) region, and (b) random amplified polymorphic DNA (RAPD) PCR analysis. RFLP analysis revealed three types of fragment patterns among the isolates. A total of 26 distinct groups, based on common fragment patterns, were identified using cluster analysis of the RAPD-PCR data. When the grouping results of the two methods were compared, the fragment pattern types and clusters were generally in agreement. The degree of pathogenicity of six genetically characterized isolates of Stagonospora sp. was assessed on three ecotypes of field bindweed (Convolvulus arvensis). Disease symptoms were observed with all isolates on all ecotypes, but only Stagonospora convolvuli strain LA39, a potential biocontrol agent, showed a high degree of pathogenicity on all ecotypes. A mixture of two Stagonospora sp. enhanced the mean necrotic leaf area on bindweed from 33.9 and 39.0% (when applied alone) to 64.9% applied together at the same final concentration of 5 X 106 spores ml -1 . Molecular methods were used to identify the two pathogens. Both were present on the same plant when applied together, but never found in the same lesion.     

Evolution of dispersal in spatially and temporally variable environments: The importance of life cycles

Spatiotemporal variability of the environment is bound to affect the evolution of dispersal, and yet model predictions strongly differ on this particular effect. Recent studies on the evolution of local adaptation have shown that the life cycle chosen to model the selective effects of spatiotemporal variability of the environment is a critical factor determining evolutionary outcomes. Here, we investigate the effect of the order of events in the life cycle on the evolution of unconditional dispersal in a spatially heterogeneous, temporally varying landscape. Our results show that the occurrence of intermediate singular strategies and disruptive selection are conditioned by the temporal autocorrelation of the environment and by the life cycle. Life cycles with dispersal of adults versus dispersal of juveniles, local versus global density regulation, give radically different evolutionary outcomes that include selection for total philopatry, evolutionary bistability, selection for intermediate stable states, and evolutionary branching points. Our results highlight the importance of accounting for life‐cycle specifics when predicting the effects of the environment on evolutionarily selected trait values, such as dispersal, as well as the need to check the robustness of model conclusions against modifications of the life cycle.     

Molecular basis for substrate recognition and septum cleavage by AtlA,the major N-acetylglucosaminidase of Enterococcus faecalis

The cleavage of septal peptidoglycan at the end of cell division facilitates the separation of the two daughter cells. The hydrolases involved in this process (called autolysins) are potentially lethal enzymes that can cause cell death; their activity, therefore, must be tightly controlled during cell growth. In Enterococcus faecalis, the N-acetylglucosaminidase AtlA plays a predominant role in cell separation. atlA mutants form long cell chains and are significantly less virulent in the zebrafish model of infection. The attenuated virulence of atlA mutants is underpinned by a limited dissemination of bacterial chains in the host organism and a more efficient uptake by phagocytes that clear the infection. AtlA has structural homologs in other important pathogens, such as Listeria monocytogenes and Salmonella typhimurium, and therefore represents an attractive model to design new inhibitors of bacterial pathogenesis. Here, we provide a 1.45 Å crystal structure of the E. faecalis AtlA catalytic domain that reveals a closed conformation of a conserved β-hairpin and a complex network of hydrogen bonds that bring two catalytic residues to the ideal distance for an inverting mechanism. Based on the model of the AtlA–substrate complex, we identify key residues critical for substrate recognition and septum cleavage during bacterial growth. We propose that this work will provide useful information for the rational design of specific inhibitors targeting this enterococcal virulence factor and its orthologs in other pathogens.     

Allencotyla pricei sp. n. (Microcotyloidea: Heteraxinidae) from the gills of the pile surfperch, Damalichthys vacca (Girard), in southern California

Allencotyla pricei sp. n. (Monogenea: Heteraxinidae) from the gills of pile surfperch, Damalichthys vacca (Girard), from Redondo Beach, California is described. This species is distinguished primarily by the prebifurcal position of the genital atrium and vagina, and by possessing an elongate esophagus. The systematic arrangement of microcotyloid forms characterized by an asymmetric haptor is discussed.