Oocyte-directed depletion of connexin43 using the Cre-LoxP system leads to subfertility in female mice

A T-DNA insertion mutant of FUSCA3 (fus3-T) in Arabidopsis thaliana exhibits several of the expected deleterious effects on seed development, but not the formation of brown seeds, a colouration which results from the accumulation of large amounts of anthocyanin. A detailed phenotypic comparison between fus3-T and a known splice point mutant (fus3-3) revealed that the seeds from both mutants do not enter dormancy and can be rescued at an immature stage. Without rescue, mature fus3-3 seeds are non-viable, whereas those of fus3-T suffer only a slight loss in their germinability. A series of comparisons between the two mutants uncovered differences with respect to conditional lethality, in histological and sub-cellular features, and in the relative amounts of various storage compounds and metabolites present, leading to a further dissection of developmental processes in seeds and a partial reinterpretation of the complex seed phenotype. FUS3 function is now known to be restricted to the acquisition of embryo-dependent seed dormancy, the determination of cotyledonary cell identity, and the synthesis and accumulation of storage compounds. Based on DNA binding studies, a model is presented which can explain the differences between the mutant alleles. The fus3-T lesion is responsible for loss of function only, while the fus3-3 mutation induces various pleiotropic effects conditioned by a truncation gene product causing severe mis-differentiation.     

From endosymbionts to host communities: factors determining the reproductive success of arthropod vectors

Elucidating the factors determining reproductive success has challenged scientists since Darwin, but the exact pathways that shape the evolution of life history traits by connecting extrinsic (e.g., landscape structure) and intrinsic (e.g., female’s age and endosymbionts) factors and reproductive success have rarely been studied. Here we collected female fleas from wild rodents in plots differing in their densities and proportions of the most dominant rodent species. We then combined path analysis and model selection approaches to explore the network of effects, ranging from micro to macroscales, determining the reproductive success of these fleas. Our results suggest that female reproductive success is directly and positively associated with their infection by Mycoplasma bacteria and their own body mass, and with the rodent species size and total density. In addition, we found evidence for indirect effects of rodent sex and rodent community diversity on female reproductive success. These results highlight the importance of exploring interrelated factors across organization scales while studying the reproductive success of wild organisms, and they have implications for the control of vector-borne diseases.     

Inhibition of pectin methyl esterase activity by green tea catechins

Pectin methyl esterases (PMEs) and their endogenous inhibitors are involved in the regulation of many processes in plant physiology, ranging from tissue growth and fruit ripening to parasitic plant haustorial formation and host invasion. Thus, control of PME activity is critical for enhancing our understanding of plant physiological processes and regulation. Here, we report on the identification of epigallocatechin gallate (EGCG), a green tea component, as a natural inhibitor for pectin methyl esterases. In a gel assay for PME activity, EGCG blocked esterase activity of pure PME as well as PME extracts from citrus and from parasitic plants. Fluorometric tests were used to determine the IC50 for a synthetic substrate. Molecular docking analysis of PME and EGCG suggests close interaction of EGCG with the catalytic cleft of PME. Inhibition of PME by the green tea compound, EGCG, provides the means to study the diverse roles of PMEs in cell wall metabolism and plant development. In addition, this study introduces the use of EGCG as natural product to be used in the food industry and agriculture.     

Reconstitution of the signal recognition particle of the halophilic archaeon Haloferax volcanii

The signal recognition particle (SRP) is a ribonucleoprotein complex involved in the recognition and targeting of nascent extracytoplasmic proteins in all three domains of life. In Archaea, SRP contains 7S RNA like its eukaryal counterpart, yet only includes two of the six protein subunits found in the eukaryal complex. To further our understanding of the archaeal SRP, 7S RNA, SRP19 and SRP54 of the halophilic archaeon Haloferax volcanii have been expressed and purified, and used to reconstitute the ternary SRP complex. The availability of SRP components from a haloarchaeon offers insight into the structure, assembly and function of this ribonucleoprotein complex at saturating salt conditions. While the amino acid sequences of H.volcanii SRP19 and SRP54 are modified presumably as an adaptation to their saline surroundings, the interactions between these halophilic SRP components and SRP RNA appear conserved, with the possibility of a few exceptions. Indeed, the H.volcanii SRP can assemble in the absence of high salt. As reported with other archaeal SRPs, the limited binding of H.volcanii SRP54 to SRP RNA is enhanced in the presence of SRP19. Finally, immunolocalization reveals that H.volcanii SRP54 is found in the cytosolic fraction, where it is associated with the ribosomal fraction of the cell.     

The RNA helicase DbpA exhibits a markedly different conformation in the ADP-bound state when compared with the ATP- or RNA-bound states

The motor enzymes that belong to the family of RNA helicases catalyze the strand separation of duplex RNA via ATP hydrolysis. Among these enzymes, Escherichia coli DbpA is a unique RNA helicase because it possesses ATPase-specific activity toward the peptidyl transferase center in 23 S ribosomal RNA. For this reason, it has been the subject of numerous biochemical and structure-function studies. The ATP-stimulated unwinding activity of DbpA toward specific and nonspecific RNA duplexes has been demonstrated. However, the underlying molecular and structural basis, which facilitates its helicase activities, is presently not known. We combined time-dependent limited proteolysis digestion, fluorescence spectroscopy, and three-dimensional structural homology modeling techniques to study the structural conformations of DbpA with respect to its binding to stoichiometric ratios of RNA and cofactors. We show that the conformational state of DbpA is markedly different in the ADP-bound state than in any other state (ATP- or RNA-bound). These results, together with structural homology studies, suggest that a hinge region located in the core domain of DbpA mediates such conformational changes.     

Structural basis for potent slow binding inhibition of human matrix metalloproteinase-2 (MMP-2)

The zinc-dependent gelatinases belong to the family of matrix metalloproteinases (MMPs), enzymes that have been shown to play a key role in angiogenesis and tumor metastasis. These enzymes are capable of hydrolyzing extracellular matrix (ECM) components under physiological conditions. Specific and selective inhibitors aimed at blocking their activity are highly sought for use as potential therapeutic agents. We report herein on a novel mode of inhibition of gelatinase A (MMP-2) by the recently characterized inhibitors 4-(4-phenoxphenylsulfonyl)butane-1,2-dithiol (inhibitor 1) and 5-(4-phenoxphenylsulfonyl) pentane-1,2-dithiol (inhibitor 2). These synthetic inhibitors are selective for MMP-2 and MMP-9. We show that the dithiolate moiety of these inhibitors chelates the catalytic zinc ion of MMP-2 via two sulfur atoms. This mode of binding results in alternation of the coordination number of the metal ion and the induction of conformational changes at the microenvironment of the catalytic zinc ion; a set of events that is likely to be at the root of the potent slow binding inhibition behavior exhibited by these inhibitors. This study demonstrates a distinct approach for the understanding of the structural mechanism governing the molecular interactions between potent inhibitors and catalytic sites of MMPs, which may aid in the design of effective inhibitors.     

Phospholipase D1 production of phosphatidic acid at the plasma membrane promotes exocytosis of large dense-core granules at a late stage

Substantial efforts have recently been made to demonstrate the importance of lipids and lipid-modifying enzymes in various membrane trafficking processes, including calcium-regulated exocytosis of hormones and neurotransmitters. Among bioactive lipids, phosphatidic acid (PA) is an attractive candidate to promote membrane fusion through its ability to change membrane topology. To date, however, the biosynthetic pathway, the dynamic location, and actual function of PA in secretory cells remain unknown. Using a short interference RNA strategy on chromaffin and PC12 cells, we demonstrate here that phospholipase D1 is activated in secretagogue-stimulated cells and that it produces PA at the plasma membrane at the secretory granule docking sites. We show that phospholipase D1 activation and PA production represent key events in the exocytotic progression. Membrane capacitance measurements indicate that reduction of endogenous PA impairs the formation of fusion-competent granules. Finally, we show that the PLD1 short interference RNA-mediated inhibition of exocytosis can be rescued by exogenous provision of a lipid that favors the transition of opposed bi-layer membranes to hemifused membranes having the outer leaflets fused. Our findings demonstrate that PA synthesis is required during exocytosis to facilitate a late event in the granule fusion pathway. We propose that the underlying mechanism is related to the ability of PA to alter membrane curvature and promote hemi-fusion.