Restriction of retinoic acid activity by Cyp26b1 is required for proper timing and patterning of osteogenesis during zebrafish development

Skeletal syndromes are among the most common birth defects. Vertebrate skeletogenesis involves two major cell types: cartilage-forming chondrocytes and bone-forming osteoblasts. In vitro, both are under the control of retinoic acid (RA), but its exact in vivo effects remained elusive. Here, based on the positional cloning of the dolphin mutation, we have studied the role of the RA-oxidizing enzyme Cyp26b1 during cartilage and bone development in zebrafish. cyp26b1 is expressed in condensing chondrocytes as well as in osteoblasts and their precursors. cyp26b1 mutants and RA-treated wild-type fish display a reduction in midline cartilage and the hyperossification of facial and axial bones, leading to fusions of vertebral primordia, a defect not previously described in the context of RA signaling. Fusions of cervical vertebrae were also obtained by treating mouse fetuses with the specific Cyp26 inhibitor R115866. Together with data on the expression of osteoblast markers, our results indicate that temporal and spatial restriction of RA signaling by Cyp26 enzymes is required to attenuate osteoblast maturation and/or activity in vivo. cyp26b1 mutants may serve as a model to study the etiology of human vertebral disorders such as Klippel-Feil anomaly.     

Application of the Percolation Theory to Random Networks of Biochemical Reactions

Percolation Theory is used to get statements about random networks. It can be seen that the probability distribution that vertices belong to a finite cluster in dependence on the mean catalytic activity C = K/N has an analogy to phase transition of second kind.     

Presence and Characterization of a Mosaic Genomic Island Which Distinguishes Sorbitol-Fermenting Enterohemorrhagic Escherichia coli O157:H? from E. coli O157:H7

A mosaic genomic island comprising Shigella resistance locus (SRL) sequences flanked by segments of Escherichia coli O157:H7 strain EDL933 O islands 43, 81, and 82 was identified in sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:H⁻ strain 493/89. This mosaic island is absent from strain EDL933. PCR targeting the SRL-related sequence is a useful tool to distinguish SF EHEC O157:H⁻ from EHEC O157:H7.     

Phosphomannose-isomerase (<Emphasis Type="Italic">pmi</Emphasis>) gene as a selectable marker for <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of rapeseed

Nodal segments (4 ± 1 mm long) of Hibiscus moscheutos (hardy hibiscus) were excised from in vitro proliferating microshoots and utilized to evaluate initial factors involved in bulk alginate encapsulation. The factors evaluated were; storage vessel type, volume and multiple rinse effects of CaCl₂ solutions, and sodium alginate concentrations (2.5, 2.75, 3.0 or 3.25%) for bulk alginate encapsulation. Results indicate that vessels utilized for bulk alginate encapsulation should have a lower base area (L × W) to height ratio to reduce the amount of alginate matrix shrinkage resulting in exposure of nodal segments. Increased volumes and multiple 50 mM CaCl₂ solution rinses did not have an effect on alginate solidification. Shoot length, root number, and root length significantly decreased in a linear manner from nodal explants stored for 4 weeks with increasing concentrations of sodium alginate. This research suggests an innovative technique for alginate encapsulation of H. moscheutos utilizing bulk methods as an alternative to single bead alginate encapsulation.     

Adeno-associated virus type 2 capsids with externalized VP1/VP2 trafficking domains are generated prior to passage through the cytoplasm and are maintained until uncoating occurs in the nucleus

Common features of parvovirus capsids are open pores at the fivefold symmetry axes that traverse the virion shell. Upon limited heat treatment in vitro, the pores can function as portals to externalize VP1/VP2 protein N-terminal sequences which harbor infection-relevant functional domains, such as a phospholipase A(2) catalytic domain. Here we show that adeno-associated virus type 2 (AAV2) also exposes its VP1/VP2 N termini in vivo during infection, presumably in the endosomal compartment. This conformational change is influenced by treatment with lysosomotropic reagents. While incubation of cells with bafilomycin A1 reduced exposure of VP1/VP2 N termini, incubation with chloroquine stimulated externalization transiently. N-terminally located basic amino acid clusters with nuclear localization activity also become exposed in this process and are accessible on the virus capsid when it enters the cytoplasm. This is an obligatory step in AAV2 infection. However, a direct role of these sequences in nuclear translocation of viral capsids could not be determined by microinjection of wild-type or mutant viruses. This suggests that further modifications of the capsid have to take place in a precytoplasmic entry step that prepares the virus for nuclear entry. Microinjection of several capsid-specific antibodies into the cell nucleus blocked AAV2 infection completely, supporting the conclusion that AAV2 capsids bring the infectious genome into the nucleus.     

Sustained and rapid chromosome movements are critical for chromosome pairing and meiotic progression in budding yeast

Telomere-led chromosome movements are a conserved feature of meiosis I (MI) prophase. Several roles have been proposed for such chromosome motion, including promoting homolog pairing and removing inappropriate chromosomal interactions. Here, we provide evidence in budding yeast that rapid chromosome movements affect homolog pairing and recombination. We found that csm4Δ strains, which are defective for telomere-led chromosome movements, show defects in homolog pairing as measured in a "one-dot/two-dot tetR-GFP" assay; however, pairing in csm4Δ eventually reaches near wild-type (WT) levels. Charged-to-alanine scanning mutagenesis of CSM4 yielded one allele, csm4-3, that confers a csm4Δ-like delay in meiotic prophase but promotes high spore viability. The meiotic delay in csm4-3 strains is essential for spore viability because a null mutation (rad17Δ) in the Rad17 checkpoint protein suppresses the delay but confers a severe spore viability defect. csm4-3 mutants show a general defect in chromosome motion but an intermediate defect in chromosome pairing. Chromosome velocity analysis in live cells showed that while average chromosome velocity was strongly reduced in csm4-3, chromosomes in this mutant displayed occasional rapid movements. Lastly, we observed that spo11 mutants displaying lower levels of meiosis-induced double-strand breaks showed higher spore viability in the presence of the csm4-3 mutation compared to csm4Δ. On the basis of these observations, we propose that during meiotic prophase the presence of occasional fast moving chromosomes over an extended period of time is sufficient to promote WT levels of recombination and high spore viability; however, sustained and rapid chromosome movements are required to prevent a checkpoint response and promote efficient meiotic progression.     

TLR5-dependent immunogenicity of a recombinant fusion protein containing an immunodominant epitope of malarial circumsporozoite protein and the FliC flagellin of Salmonella Typhimurium

Recently, we described the improved immunogenicity of new malaria vaccine candidates based on the expression of fusion proteins containing immunodominant epitopes of merozoites and Salmonella enterica serovar Typhimurium flagellin (FliC) protein as an innate immune agonist. Here, we tested whether a similar strategy, based on an immunodominant B-cell epitope from malaria sporozoites, could also generate immunogenic fusion polypeptides. A recombinant His6-tagged FliC protein containing the C-terminal repeat regions of the VK210 variant of Plasmodium vivax circumsporozoite (CS) protein was constructed. This recombinant protein was successfully expressed in Escherichia coli as soluble protein and was purified by affinity to Ni-agarose beads followed by ion exchange chromatography. A monoclonal antibody specific for the CS protein of P. vivax sporozoites (VK210) was able to recognise the purified protein. C57BL/6 mice subcutaneously immunised with the recombinant fusion protein in the absence of any conventional adjuvant developed protein-specific systemic antibody responses. However, in mice genetically deficient in expression of TLR5, this immune response was extremely low. These results extend our previous observations concerning the immunogenicity of these recombinant fusion proteins and provide evidence that the main mechanism responsible for this immune activation involves interactions with TLR5, which has not previously been demonstrated for any recombinant FliC fusion protein.