Structural analysis of the heterodimeric type IIS restriction endonuclease R.BspD6I acting as a complex between a monomeric site-specific nickase and a catalytic subunit

The heterodimeric restriction endonuclease R.BspD6I from Bacillus species D6 recognizes a pseudosymmetric sequence and cuts both DNA strands outside the recognition sequence. The large subunit, Nt.BspD6I, acts as a type IIS site-specific monomeric nicking endonuclease. The isolated small subunit, ss.BspD6I, does not bind DNA and is not catalytically active. We solved the crystal structures of Nt.BspD6I and ss.BspD6I at high resolution. Nt.BspD6I consists of three domains, two of which exhibit structural similarity to the recognition and cleavage domains of FokI. ss.BspD6I has a fold similar to that of the cleavage domain of Nt.BspD6I, each containing a PD-(D/E)XK motif and a histidine as an additional putative catalytic residue. In contrast to the DNA-bound FokI structure, in which the cleavage domain is rotated away from the DNA, the crystal structure of Nt.BspD6I shows the recognition and cleavage domains in favorable orientations for interactions with DNA. Docking models of complexes of Nt.BspD6I and R.BspD6I with cognate DNA were constructed on the basis of structural similarity to individual domains of FokI, R.BpuJI and HindIII. A three-helix bundle forming an interdomain linker in Nt.BspD6I acts as a rigid spacer adjusting the orientations of the spatially separated domains to match the distance between the recognition and cleavage sites accurately.     

The resuscitation-promoting factors of Mycobacterium tuberculosis are required for virulence and resuscitation from dormancy but are collectively dispensable for growth in vitro

Mycobacterium tuberculosis contains five resuscitation-promoting factor (Rpf)-like proteins, RpfA-E, that are implicated in resuscitation of this organism from dormancy via a mechanism involving hydrolysis of the peptidoglycan by Rpfs and partnering proteins. In this study, the rpfA-E genes were shown to be collectively dispensable for growth of M. tuberculosis in broth culture. The defect in resuscitation of multiple mutants from a 'non-culturable' state induced by starvation under anoxia was reversed by genetic complementation or addition of culture filtrate from wild-type organisms confirming that the phenotype was associated with rpf-like gene loss and that the 'non-culturable' cells of the mutant strains were viable. Other phenotypes uncovered by sequential deletion mutagenesis revealed a functional differentiation within this protein family. The quintuple mutant and its parent that retained only rpfD displayed delayed colony formation and hypersensitivity to detergent, effects not observed for mutants retaining only rpfE or rpfB. Furthermore, mutants retaining rpfD or rpfE were highly attenuated for growth in mice with the latter persisting better than the former in late-stage infection. In conjunction, these results are indicative of a hierarchy in terms of function and/or potency with the Rpf family, with RpfB and RpfE ranking above RpfD.     

Tropomyosin 4 defines novel filaments in skeletal muscle associated with muscle remodelling/regeneration in normal and diseased muscle

The organisation of structural proteins in muscle into highly ordered sarcomeres occurs during development, regeneration and focal repair of skeletal muscle fibers. The involvement of cytoskeletal proteins in this process has been documented, with nonmuscle gamma-actin found to play a role in sarcomere assembly during muscle differentiation and also shown to be up-regulated in dystrophic muscles which undergo regeneration and repair [Lloyd et al.,2004; Hanft et al.,2006]. Here, we show that a cytoskeletal tropomyosin (Tm), Tm4, defines actin filaments in two novel compartments in muscle fibers: a Z-line associated cytoskeleton (Z-LAC), similar to a structure we have reported previously [Kee et al.,2004], and longitudinal filaments that are orientated parallel to the sarcomeric apparatus, present during myofiber growth and repair/regeneration. Tm4 is upregulated in paradigms of muscle repair including induced regeneration and focal repair and in muscle diseases with repair/regeneration features, muscular dystrophy and nemaline myopathy. Longitudinal Tm4-defined filaments also are present in diseased muscle. Transition of the Tm4-defined filaments from a longitudinal to a Z-LAC orientation is observed during the course of muscle regeneration. This Tm4-defined cytoskeleton is a marker of growth and repair/regeneration in response to injury, disease state and stress in skeletal muscle.     

Development of transformation vectors based upon a modified plant alpha-tubulin gene as the selectable marker

A plant transformation and selection system has been developed utilizing a modified tubulin gene as a selectable marker. The vector constructs carrying a mutant alpha-tubulin gene from goosegrass conferring resistance to dinitroaniline herbicides were created for transformation of monocotyledonous and dicotyledonous plants. These constructs contained beta- and/or mutant alpha-tubulin genes driven either by ubiquitin or CaMV 35S promoter. The constructs were used for biolistic transformation of finger millet and soybean or for Agrobacterium-mediated transformation of flax and tobacco. Trifluralin, the main representative of dinitroaniline herbicides, was used as a selective agent in experiments to select transgenic cells, tissues and plantlets. Selective concentrations of trifluralin estimated for each species were as follows: 10 microM for Eleusine coracana, Glycine max, Nicotiana plumbaginifolia and Nicotiana sylvestris; 3 microM for Linum usitatissimum. PCR and Southern blotting analyses of transformed lines with a specific probe to nptII, alpha-tubulin or beta-tubulin genes were performed to confirm the transgenic nature of regenerated plants. Band specific for the mutant alpha-tubulin gene was identified in transformed plant lines. Results confirmed the stable integration of the mutant tubulin gene into the plant genomes. The present study clearly demonstrates the use of a plant mutant tubulin as a selective gene for plant transformation.     

Safety of new algorithms for premeal insulin boluses in high glycaemic index meals in persons with type 1 diabetes mellitus using insulin pumps

Aims: Consumption of glucose or foodstuffs with high glycaemic index (GI) in persons with type 1 diabetes mellitus (PWD1) is a hot topic in present diabetology. The aim of our pilot prospective study was to assess the efficiency of empirically suggested simple algorithms for premeal boluses in PWD1 using insulin pumps and continuous glucose monitoring (CGM). Methods: Six PWD1 (aged 46.2+/-15.09 y, diabetes duration 14.5+/-9.65 y, HbA1c/IFCC 6.3+/-1.59%, BMI 23.6+/-1.67 kg/m(2), mean+/-SD) on insulin pumps Paradigm 522/722 with RT-CGMS sensors (Medtronic MiniMed, Northridge, CA) underwent a 12-week CGM. In one week, subjects consumed 50g of carbohydrates in eleven alternative meals (rice squares, dark chocolate, white bread, honey, glucose, ravioli with meat and Eidam cheese, mashed potatoes with fish fingers, apricot dumplings with butter, spa waffles, spalta squares, and tomato soup with pasta) eaten for breakfasts, lunches, snacks and dinners in order to calculate their GI. The insulin boluses were adjusted according to empirically defined algorithms. Average glucose levels and daily insulin doses over three one-week periods (before testing, testing and after testing) were compared. Results: During the observational period, the weekly averages of glucose levels (9.1+/-2.33 mmol/l vs. 9.2+/-2.30 mmol/l vs. 9.0+/-2.43 mmol/l, respectively) and daily insulin doses (39.1+/- 8.14 IU/d vs. 39.7+/-10.7 IU/d vs. 38.6+/-9.97 IU/d, respectively) were similar. One-week consumption of high GI foodstuffs had only a negligeable effect on average glucose levels. Conclusion: The suggested algorithms for premeal insulin boluses appear to limit the risk of potential hyperglycaemia resulting from intake of high GI foodstuffs.     

Changes in the water level of Lake Peipsi and their reflection in a sediment core

A comprehensive study (chronological, lithological and geochemical) of an 8.5 m postglacial sediment sequence from Lake Peipsi was conducted to elucidate the effects of lake-level changes on the sedimentary environment and biogeochemical dynamics in a large lake. Four lithological units were distinguished in the sediment sequence studied: clayey silt, slightly laminated greyish carbonaceous gyttja, brownish-grey gyttja and dark gyttja. These units indicate that large shifts in sedimentation processes occurred in the past. The sediment data show that fluctuations in water depth had a profound impact on the lake environment, recorded as changes in the lithological composition, phosphorus content of sediments and composition of diatom assemblages. The corresponding changes are best reflected in sediments accumulated during phases of regression when the lake area was also smaller. As the water depth and area of the lake increased, wave-induced erosion and resedimentation smoothed or even disturbed the initial information. The increase of phosphorus content in the unconsolidated, high-porosity surface sediments (0.5 m) is most probably connected with active diffusion and matter exchange between the water-sediment pools.     

Molecular insight into mechanism of antimicrobial action of the beta-hairpin peptide arenicin: specific oligomerization in detergent micelles

Arenicins are 21-residue cationic antimicrobial peptides isolated from marine polychaeta Arenicola marina. The peptides exhibit potent broad-spectrum antimicrobial activity. In water solution arenicin-2 adopts a beta-hairpin conformation, stabilized by one disulfide and nine hydrogen bonds. To determine the propensity for the peptide oligomerization in membrane mimetic systems, the recombinant arenicin-2 was overexpressed as a fused form in Escherichia coli. The arenicin-2 oligomerization and intermolecular packing in membrane mimicking environment were investigated using high-resolution NMR spectroscopy. The present studies show that arenicin-2 preserves a beta-hairpin structure and forms asymmetric dimers upon incorporation into the dodecylphosphocholine micelle. Two monomers of arenicin-2 are aligned parallel to each other by the N-terminal strands of the beta-hairpin (CN upward arrow upward arrowNC type of association). Polyacrylamide gel electrophoresis analysis indicated that in environment of anionic SDS micelles the arenicin-2 might undergo further oligomerization and form tetramers. Our results afford further molecular insight into possible mechanism of antimicrobial action of arenicins.