The third P-loop domain in cytoplasmic dynein heavy chain is essential for dynein motor function and ATP-sensitive microtubule binding

Sequence comparisons and structural analyses show that the dynein heavy chain motor subunit is related to the AAA family of chaperone-like ATPases. The core structure of the dynein motor unit derives from the assembly of six AAA domains into a hexameric ring. In dynein, the first four AAA domains contain consensus nucleotide triphosphate-binding motifs, or P-loops. The recent structural models of dynein heavy chain have fostered the hypothesis that the energy derived from hydrolysis at P-loop 1 acts through adjacent P-loop domains to effect changes in the attachment state of the microtubule-binding domain. However, to date, the functional significance of the P-loop domains adjacent to the ATP hydrolytic site has not been demonstrated. Our results provide a mutational analysis of P-loop function within the first and third AAA domains of the Drosophila cytoplasmic dynein heavy chain. Here we report the first evidence that P-loop-3 function is essential for dynein function. Significantly, our results further show that P-loop-3 function is required for the ATP-induced release of the dynein complex from microtubules. Mutation of P-loop-3 blocks ATP-mediated release of dynein from microtubules, but does not appear to block ATP binding and hydrolysis at P-loop 1. Combined with the recent recognition that dynein belongs to the family of AAA ATPases, the observations support current models in which the multiple AAA domains of the dynein heavy chain interact to support the translocation of the dynein motor down the microtubule lattice.     

Provider knowledge about emergency contraception in Ghana

In 1996, the Ministry of Health in Ghana included emergency contraception (EC) in its newly issued National Reproductive Health Service Policy and Standards. A short survey was conducted in the summer of 1997 to evaluate health providers' knowledge of EC. Of the 325 providers interviewed, about one-third (34%) had heard of EC. No provider had sufficient knowledge to prescribe EC correctly. A well-coordinated training programme for providers will have to precede successful introduction of EC in Ghana. Moreover, a dedicated product may be critical for the successful introduction of EC in a country like Ghana, where provider knowledge is low.     

1-Thio-beta-D-galactofuranosides: synthesis and evaluation as beta-D- galactofuranosidase inhibitors

Beta-D-galactofuranosidase is a good chemotherapeutic target for the design of inhibitors, since beta-D-galactofuranose is a constituent of important parasite glycoconjugates but is not present in the host mammals. With this aim, we have synthesized for the first time alkyl, benzyl and aryl 1-thio-beta-D-galactofuranosides by condensation of penta-O-benzoyl-alpha,beta-D-galactofuranose with the corresponding thiols, in the presence of SnCl4as catalyst. The complete chemical and spectroscopical characterization of these compounds showed that the reaction was stereoselective. Debenzoylation with sodium methoxide afforded the beta-S-galactofuranosides in high yield. The thioglycosides were tested as inhibitors of the beta-D- galactofuranosidase of Penicillium fellutanum, using for the first time 4-nitrophenyl-beta-D-galactofuranoside as chromogenic substrate. The 4- aminophenyl-1-thio-beta-D-galactofuranoside, obtained by catalytic hydrogenation of the nitrophenyl derivative, was the best inhibitor being then an adequate ligand for the preparation of an affinity phase aimed at the isolation of beta-d-galactofuranosidases from different sources. Also the inhibitory activity of d-galactono-1, 4-lactone was shown.      

Synthesis and antiproliferating activity of iron chelators of hydroxyamino-1,3,5-triazine family

We synthesized and evaluated new specific tridentate iron(III) chelators of 2,6-bis[hydroxyamino]-1,3,5-triazine (BHT) family for use in iron deprivation cancer therapy. Physical properties of BHT chelators are easily customizable allowing easy penetration through cellular membranes. Antiproliferative activity of new BHT chelators was studied on MDA-MB-231 and MiaPaCa cells and compared to a clinically available new oral iron chelator, deferasirox (DFX). The antiproliferative activity of new chelators was found to correlate with iron(III) chelation ability and some of analogs showed substantially higher antiproliferative activity than DFX.     

Single Particle Fluorescence Burst Analysis of Epsin Induced Membrane Fission

Vital cellular processes, from cell growth to synaptic transmission, rely on membrane-bounded carriers and vesicles to transport molecular cargo to and from specific intracellular compartments throughout the cell. Compartment-specific proteins are required for the final step, membrane fission, which releases the transport carrier from the intracellular compartment. The role of fission proteins, especially at intracellular locations and in non-neuronal cells, while informed by the dynamin-1 paradigm, remains to be resolved. In this study, we introduce a highly sensitive approach for the identification and analysis of membrane fission machinery, called burst analysis spectroscopy (BAS). BAS is a single particle, free-solution approach, well suited for quantitative measurements of membrane dynamics. Here, we use BAS to analyze membrane fission induced by the potent, fission-active ENTH domain of epsin. Using this method, we obtained temperature-dependent, time-resolved measurements of liposome size and concentration changes, even at sub-micromolar concentration of the epsin ENTH domain. We also uncovered, at 37°C, fission activity for the full-length epsin protein, supporting the argument that the membrane-fission activity observed with the ENTH domain represents a native function of the full-length epsin protein.     

Regional differences in WT-1 and Tcf21 expression during ventricular development: implications for myocardial compaction

Background

Morphological and functional differences of the right and left ventricle are apparent in the adult human heart. A differential contribution of cardiac fibroblasts and smooth muscle cells (populations of epicardium-derived cells) to each ventricle may account for part of the morphological-functional disparity. Here we studied the relation between epicardial derivatives and the development of compact ventricular myocardium.

Results

Wildtype and Wt1^CreERT2/+ reporter mice were used to study WT-1 expressing cells, and Tcf21^lacZ/+ reporter mice and PDGFRα^-/-;Tcf21^LacZ/+ mice to study the formation of the cardiac fibroblast population. After covering the heart, intramyocardial WT-1+ cells were first observed at the inner curvature, the right ventricular postero-lateral wall and left ventricular apical wall. Later, WT-1+ cells were present in the walls of both ventricles, but significantly more pronounced in the left ventricle. Tcf21-^LacZ + cells followed the same distribution pattern as WT-1+ cells but at later stages, indicating a timing difference between these cell populations. Within the right ventricle, WT-1+ and Tcf21-lacZ+ cell distribution was more pronounced in the posterior inlet part. A gradual increase in myocardial wall thickness was observed early in the left ventricle and at later stages in the right ventricle. PDGFRα^-/-;Tcf21^LacZ/+ mice showed deficient epicardium, diminished number of Tcf21-^LacZ + cells and reduced ventricular compaction.

Conclusions

During normal heart development, spatio-temporal differences in contribution of WT-1 and Tcf21-^LacZ + cells to right versus left ventricular myocardium occur parallel to myocardial thickening. These findings may relate to lateralized differences in ventricular (patho)morphology in humans.     

Vegetative axillary bud dormancy induced by shade and defoliation signals in the grasses

Vegetative axillary bud dormancy and outgrowth is regulated by several hormonal and environmental signals. In perennials, the dormancy induced by hormonal and environmental signals has been categorized as eco-, endo- or para-dormancy. Over the past several decades para-dormancy has primarily been investigated in eudicot annuals. Recently, we initiated a study using the monoculm phyB mutant (phyB-1) and the freely branching near isogenic wild type (WT) sorghum (Sorghum bicolor) to identify molecular mechanisms and signaling pathways regulating dormancy and outgrowth of axillary buds in the grasses. In a paper published in the January 2010 issue of Plant Cell and Environment, we reported the role of branching genes in the inhibition of bud outgrowth by phyB, shade and defoliation signals. Here we present a model that depicts the molecular mechanisms and pathways regulating axillary bud dormancy induced by shade and defoliation signals in the grasses.Key words: axillary bud, dormancy, shade, phytochrome, defoliation, shoot branching, teosinte branched1, MAX2, cell cycle, sorghumThe dormancy and outgrowth of axillary buds is regulated by several plant hormones such as auxin, cytokinins, abscisic acid and strigolactones, and by environmental factors such as light quality, quantity and duration as well as water, temperature and nutrient status.^1–3 Since the fate of an axillary bud is regulated by such diverse hormonal and environmental signals and their interactions, the type of dormancy induced varies. In perennials, three types of bud dormancy have been identified.^4,5 Dormancy mediated by factors within the bud is known as endo-dormancy; while dormancy induced by factors within the plant but outside the bud is called paradormancy or correlative inhibition; the best known example being apical dominance. Dormancy induced due to unfavorable environmental conditions is known as eco-dormancy. Although there is an indepth knowledge about para-dormancy in annuals,⁶ few studies have been conducted on eco-dormancy. Similarly, studies of endo-dormancy have largely been restricted to low-temperature mediated growth-cessation of axillary buds of perennial plants.^7,8 To understand the regulation of dormancy and outgrowth of axillary buds in monocots, we initiated a study on the molecular mechanisms inhibiting bud outgrowth by shade and defoliation signals in sorghum. Our results published in the January 2010 issue of Plant, Cell & Environment indicate that different types of dormancy may be induced in axillary buds of annual grasses by various signals and there may be overlapping and independent molecular mechanisms mediating induction of axillary bud dormancy.