Stepwise Reconstitution of Interphase Microtubule Dynamics in Permeabilized Cells and Comparison to Dynamic Mechanisms in Intact Cells

Microtubules in permeabilized cells are devoid of dynamic activity and are insensitive to depolymerizing drugs such as nocodazole. Using this model system we have established conditions for stepwise reconstitution of microtubule dynamics in permeabilized interphase cells when supplemented with various cell extracts. When permeabilized cells are supplemented with mammalian cell extracts in the presence of protein phosphatase inhibitors, microtubules become sensitive to nocodazole. Depolymerization induced by nocodazole proceeds from microtubule plus ends, whereas microtubule minus ends remain inactive. Such nocodazole-sensitive microtubules do not exhibit subunit turnover. By contrast, when permeabilized cells are supplemented with Xenopus egg extracts, microtubules actively turn over. This involves continuous creation of free microtubule minus ends through microtubule fragmentation. Newly created minus ends apparently serve as sites of microtubule depolymerization, while net microtubule polymerization occurs at microtubule plus ends. We provide evidence that similar microtubule fragmentation and minus end–directed disassembly occur at the whole-cell level in intact cells. These data suggest that microtubule dynamics resembling dynamics observed in vivo can be reconstituted in permeabilized cells. This model system should provide means for in vitro assays to identify molecules important in regulating microtubule dynamics. Furthermore, our data support recent work suggesting that microtubule treadmilling is an important mechanism of microtubule turnover.     

Post-translationally modified 14-3-3 isoforms and inhibition of protein kinase C

This report compares the ability of individual members of the 14-3-3 protein family to inhibit particular protein kinase C (PKC) isoforms. We also show that two of these 14-3-3 isoforms ( and ) specific to mammalian and avian brain arein vivo post-translationally modified forms of and respectively. The presence of this modification enhances the activity of 14-3-3 as an inhibitor of protein kinase C nearly two fold.A method for analysing isoforms of 14-3-3 on acid-urea gels is also described. This permits the complete separation of all major isoforms and their unequivocal identification by a range of isoform specific antisera. The activity of recombinant 14-3-3 and isoforms renatured by a novel method after separation by reverse phase HPLC are compared. The effects of diacylglycerol and the phorbol ester, PMA (phorbol 12-myristate 13 acetate) on the inhibition suggest that one of the sites of interaction of 14-3-3 may be the cysteine-rich (C1) domain in PKC.     

Key drivers of effectiveness in small marine protected areas

Marine Protected Areas (MPAs) are a key management tool for the conservation of biodiversity and restoration of marine communities. While large, well-designed and enforced MPAs have been found to be effective, results from small MPAs vary. The Hawkesbury Shelf, a coastal bioregion in New South Wales, Australia, has ten small, near-shore MPAs known as Aquatic Reserves with a variety of protection levels from full no-take to partial protection. This study assessed the effectiveness of these MPAs and analysed how MPA age, size, protection level, wave exposure, habitat complexity, and large canopy-forming algal cover affected fish, invertebrate and benthic communities. We found aspect, protection level, complexity and algal canopy to be important predictors of communities in these MPAs. Most MPAs, however, were not effective in meeting their goals. Only full no-take protection (three out of ten MPAs) had a significant impact on fish assemblages. One no-take MPA—Cabbage Tree Bay—which is naturally sheltered from wave action and benefits from an active local community providing informal enforcement, accounted for most of the increased richness of large fish and increased biomass of targeted fish species. Our findings suggest that small MPAs can enhance biodiversity and biomass on a local scale but only if they have full no-take protection, are in sheltered locations with complex habitat, and have positive community involvement to engender support and stewardship. These results provide a baseline for robust assessment of the effectiveness of small MPAs and inform future management decisions and small MPA design in other locations.     

Impact of subchronic exposure to triclosan and/or fluoride on estrogenic activity in immature female rats: The expression pattern of calbindin‐D9k and estrogen receptor α genes

This study explored the influence of triclosan (TCS) in the absence and presence of sodium fluoride (NaF) on estrogenic activity and thyroid function of adolescent female rats. The results indicated that the individual exposure to TCS evoked a significant decline in T₃ and T₄ but the levels of estradiol, FSH, and LH were significantly elevated beside marked up regulation of calbindin‐D9k and estrogen α mRNA expression. On the other hand, the single exposure to NaF causes insignificant changes in thyroid hormones, but evoked a trend toward an increase in both estradiol and LH levels. No significant differences in the TSH level were recorded among the experimental groups. The joint exposure to TCS and NaF induced a significant improvement in thyroid and reproductive hormone levels. Overall, these findings revealed that exposure to TCS resulted in significant endocrine and reproductive alterations in immature female rats, while TCS + NaF coexposure resulted in lessening most effects.     

Newly Isolated Lactobacilli strains from Algerian Human Vaginal Microbiota: Lactobacillus fermentum Strains Relevant Probiotic’s Candidates

Probiotics and Antimicrobial Proteins - Lactobacilli strains are considered as a preventive means for treatment of vaginal infections or post-antibiotic treatment to repopulate the vaginal mucosa....     

DNA of a circular minichromosome linearized by restriction enzymes or other reagents is resistant to further cleavage: an influence of chromatin topology on the accessibility of DNA

The accessibility of DNA in chromatin is an essential factor in regulating its activities. We studied the accessibility of the DNA in a ∼170 kb circular minichromosome to DNA-cleaving reagents using pulsed-field gel electrophoresis and fibre-fluorescence in situ hybridization on combed DNA molecules. Only one of several potential sites in the minichromosome DNA was accessible to restriction enzymes in permeabilized cells, and in growing cells only a single site at an essentially random position was cut by poisoned topoisomerase II, neocarzinostatin and γ-radiation, which have multiple potential cleavage sites; further sites were then inaccessible in the linearized minichromosomes. Sequential exposure to combinations of these reagents also resulted in cleavage at only a single site. Minichromosome DNA containing single-strand breaks created by a nicking endonuclease to relax any unconstrained superhelicity was also cut at only a single position by a restriction enzyme. Further sites became accessible after ≥95% of histones H2A, H2B and H1, and most non-histone proteins were extracted. These observations suggest that a global rearrangement of the three-dimensional packing and interactions of nucleosomes occurs when a circular minichromosome is linearized and results in its DNA becoming inaccessible to probes.     

Wnt and RUNX2 mediate cartilage breakdown by osteoarthritis synovial fibroblast‐derived ADAMTS‐7 and ‐12

Failure of therapeutic approaches for the treatment of osteoarthritis (OA) based on the inhibition of metalloproteinases, might be because of their constitutive expression in homeostasis, together with their network complexity. The knowledge of this network would contribute to selective target pathological conditions. In this sense, blockade of mediators produced by neighbouring joint cells, such as synovial fibroblasts (SF), would prevent cartilage damage. Thus, we studied the contribution of ADAMTS‐7 and ‐12 from SF to cartilage oligomeric matrix protein (COMP) degradation, and the signalling pathways involved in their expression. We report for the first time in SF, the involvement of ERK‐Runx2 axis and Wnt/β‐catenin signalling in ADAMTS‐12 and ADAMTS‐7 expressions, respectively, with the subsequent consequences in COMP degradation from cartilage extracellular matrix. After stimulation with IL‐1β or fibronectin fragments, we showed that ERK inhibition decreased Runx2 activation and ADAMTS‐12 expression in OA‐SF, also reducing Fn‐fs‐induced COMP degradation. Blockage of Wnt signalling by DKK1 reduced ADAMTS‐7 and COMP degradation in OA‐SF as well. In addition, Wnt7B expression was induced by IL‐1β and by itself, also increasing ADAMTS‐7. Our results could contribute to the development of disease‐modifying OA drugs targeting ADAMTS‐7 and ‐12 for the prevention of extracellular matrix components degradation like COMP.     

Disulfide and amide-bridged cyclic peptide analogues of the VEGF₈₁₋₉₁ fragment: synthesis, conformational analysis and biological evaluation

The design, synthesis, conformational studies and binding affinity for VEGFR-1 receptors of a collection of linear and cyclic peptide analogues of the β-hairpin fragment VEGF(81-91) are described. Cyclic 11-mer peptide derivatives were prepared from linear precursors with conveniently located Cys, Asp or Dap residues, by the formation of disulfide and amide bridges, using solid-phase synthesis. Molecular modelling studies indicated a tendency to be structured around the central β-turn of the VEGF(81-91) β-hairpin in most synthesized cyclic compounds. This structural behavior was confirmed by NMR conformational analysis. The NHCO cyclic derivative 7 showed significant affinity for VEGFR-1, slightly higher than the native linear fragment, thus supporting the design of mimics of this fragment as a valid approach to disrupt the VEGF/VEGFR-1 interaction.