Pluripotency of Arabidopsis xylem pericycle underlies shoot regeneration from root and hypocotyl explants grown in vitro

We have established a detailed framework for the process of shoot regeneration from Arabidopsis root and hypocotyl explants grown in vitro . Using transgenic plant lines in which the GUS or GFP genes were fused to promoters of developmental genes ( WUS , CLV1 , CLV3 , STM , CUC1 , PLT1 , RCH1 , QC25 ), or to promoters of genes encoding indicators of the auxin response ( DR5 ) or transport ( PIN1 ), cytokinin (CK) response ( ARR5 ) or synthesis ( IPT5 ), or mitotic activity ( CYCB1 ), we showed that regenerated shoots originated directly or indirectly from the pericycle cells adjacent to xylem poles. In addition, shoot regeneration appeared to be partly similar to the formation of lateral root meristems (LRMs). During pre-culture on a 2, 4-dichlorophenoxyacetic acid (2, 4-D)-rich callus-inducing medium (CIM), xylem pericycle reactivation established outgrowths that were not true calli but had many characteristics of LRMs. Transfer to a CK-rich shoot-inducing medium (SIM) resulted in early LRM-like primordia changing to shoot meristems. Direct origin of shoots from the xylem pericycle occurred upon direct culture on CK-containing media without prior growth on CIM. Thus, it appeared that the xylem pericycle is more pluripotent than previously thought. This pluripotency was accompanied by the ability of pericycle derivatives to retain diploidy, even after several rounds of cell division. In contrast, the phloem pericycle did not display such developmental plasticity, and responded to CKs with only periclinal divisions. Such observations reinforce the view that the pericycle is an 'extended meristem' that comprises two types of cell populations. They also suggest that the founder cells for LRM initiation are not initially fully specified for this developmental pathway.     

Novel A-ring homodimeric C-3-carbamate analogues of 1alpha,25-dihydroxyvitamin D3: synthesis and preliminary biological evaluation

The synthesis of a new class of vitamin D3 analogues in which two units of 1alpha,25-dihydroxyvitamin D3 are linked at the C-3 position by a dicarbamate functionality of variable length is described. The analogues demonstrated no affinity for the vitamin D receptor and possessed no antiproliferative or transactivating properties.     

Starch-branching enzyme IIa is required for proper diurnal cycling of starch in leaves of maize

Starch-branching enzyme (SBE), a glucosyl transferase, is required for the highly regular pattern of α-1,6 bonds in the amylopectin component of starch. In the absence of SBEIIa, as shown previously in the sbe2a mutant of maize (Zea mays), leaf starch has drastically reduced branching and the leaves exhibit a severe senescence-like phenotype. Detailed characterization of the maize sbe2a mutant revealed that SBEIIa is the primary active branching enzyme in the leaf and that in its absence plant growth is affected. Both seedling and mature sbe2a mutant leaves do not properly degrade starch during the night, resulting in hyperaccumulation. In mature sbe2a leaves, starch hyperaccumulation is greatest in visibly senescing regions but also observed in green tissue and is correlated to a drastic reduction in photosynthesis within the leaf. Starch granules from sbe2a leaves observed via scanning electron microscopy and transmission electron microscopy analyses are larger, irregular, and amorphous as compared with the highly regular, discoid starch granules observed in wild-type leaves. This appears to trigger premature senescence, as shown by an increased expression of genes encoding proteins known to be involved in senescence and programmed cell death processes. Together, these results indicate that SBEIIa is required for the proper diurnal cycling of transitory starch within the leaf and suggest that SBEIIa is necessary in producing an amylopectin structure amenable to degradation by starch metabolism enzymes.     

Super-resolution optical DNA Mapping via DNA methyltransferase-directed click chemistry

We demonstrate an approach to optical DNA mapping, which enables near single-molecule characterization of whole bacteriophage genomes. Our approach uses a DNA methyltransferase enzyme to target labelling to specific sites and copper-catalysed azide-alkyne cycloaddition to couple a fluorophore to the DNA. We achieve a labelling efficiency of ∼70% with an average labelling density approaching one site every 500 bp. Such labelling density bridges the gap between the output of a typical DNA sequencing experiment and the long-range information derived from traditional optical DNA mapping. We lay the foundations for a wider-scale adoption of DNA mapping by screening 11 methyltransferases for their ability to direct sequence-specific DNA transalkylation; the first step of the DNA labelling process and by optimizing reaction conditions for fluorophore coupling via a click reaction. Three of 11 enzymes transalkylate DNA with the cofactor we tested (a readily prepared s-adenosyl-l-methionine analogue).     

Heparin inhibits melanosome uptake and inflammatory response coupled with phagocytosis through blocking PI3k/Akt and MEK/ERK signaling pathways in human epidermal keratinocytes

To gain insight for the role of mast cell‐produced heparin in the regulation of epidermal homeostasis and skin pigmentation, we have investigated the effect of heparin on melanosome uptake and proinflammatory responses in normal human epidermal keratinocytes (NHEKs). We quantified phagocytic activity of NHEKs with uptake of melanosomes or fluorescent microspheres. Heparin exhibited the inhibitory effect on keratinocyte phagocytosis through blocking PI3k/Akt and MEK/ERK signaling pathways. In fact, the heparin‐treated NHEKs showed impaired activation of Akt and ERK during phagocytosis, whereas PI3k and MEK inhibitors significantly suppressed melanosome uptake by NHEKs. In addition, the inflammation marker cycloxygenase‐2 (COX‐2) expression and prostaglandin E₂ (PGE₂) production were induced during phagocytosis, while these effects were downregulated in the presence of heparin. Our observations suggest that heparin may play an antiphagocytic and anti‐inflammation role in epidermis of human skin.     

Matrix metalloproteinase 2 and membrane type 1 matrix metalloproteinase co‐regulate axonal outgrowth of mouse retinal ganglion cells

Restoration of correct neural activity following central nervous system (CNS) damage requires the replacement of degenerated axons with newly outgrowing, functional axons. Unfortunately, spontaneous regeneration is largely lacking in the adult mammalian CNS. In order to establish successful regenerative therapies, an improved understanding of axonal outgrowth and the various molecules influencing it, is highly needed. Matrix metalloproteinases (MMPs) constitute a family of zinc‐dependent proteases that were sporadically reported to influence axon outgrowth. Using an ex vivo retinal explant model, we were able to show that broad‐spectrum MMP inhibition reduces axon outgrowth of mouse retinal ganglion cells (RGCs), implicating MMPs as beneficial factors in axonal regeneration. Additional studies, using more specific MMP inhibitors and MMP‐deficient mice, disclosed that both MMP‐2 and MT1‐MMP, but not MMP‐9, are involved in this process. Furthermore, administration of a novel antibody to MT1‐MMP that selectively blocks pro‐MMP‐2 activation revealed a functional co‐involvement of these proteinases in determining RGC axon outgrowth. Subsequent immunostainings showed expression of both MMP‐2 and MT1‐MMP in RGC axons and glial cells. Finally, results from combined inhibition of MMP‐2 and β1‐integrin were suggestive for a functional interaction between these molecules. Overall, our data indicate MMP‐2 and MT1‐MMP as promising axonal outgrowth‐promoting molecules.

     

Molecular and morphological aspects of annealing-induced stabilization of starch crystallites

A unique series of potato (mutant) starches with highly different amylopectin/amylose (AP/AM) ratios was annealed in excess water at stepwise increasing temperatures to increase the starch melting (or gelatinization) temperatures in aqueous suspensions. Small-angle X-ray scattering (SAXS) experiments revealed that the lamellar starch crystals gain stability upon annealing via thickening for high-AM starch, whereas the crystal surface energy decreases for AM-free starch. In starches with intermediate AP/AM ratio, both mechanisms occur, but the surface energy reduction mechanism prevails. Crystal thickening seems to be associated with the cocrystallization of AM with AP, leading to very disordered nanomorphologies for which a new SAXS data interpretation scheme needed to be developed. Annealing affects neither the crystal internal structure nor the spherulitic morphology on a micrometer length scale.     

Thrombophilia in mice expressing a tissue factor variant lacking its transmembrane and cytosolic domain

Mice with a targeted truncation in the gene encoding tissue factor of blood coagulation (TF) to eliminate the cytosolic domain and carrying a neo(R) cassette in intron 5 unexpectedly displayed severe spontaneous thrombosis in various vascular beds. Thrombosis was observed in heterozygous TF(+/neo) mice, causing death of over 50% of adults within 36 weeks of birth, and fulminantly exacerbating in pregnant females. Homozygous TF(neo/neo) mice were more severely affected and died within 7 weeks after birth. These TF(neo) mice primarily synthesized a mutant mRNA aberrantly spliced from exon 5 to neo(R), encoding an apparently non-vesicle-binding soluble TF lacking both the transmembrane and cytosolic domain, but still capable of blood coagulation induction. This severe thrombotic phenotype associated with the presence of a non-anchored soluble TF variant underscores the recently recognized significance of circulating TF for thrombus formation and development.     

Streptomyces lividans as host for heterologous protein production

Abstract Streptomycetes are Gram-positive soil bacteria with a differentiated morphology. They are considered interesting candidates for the production of heterologous proteins for several reasons, including their efficient secretion mechanism by which the secreted proteins are localized into the culture supernatant. In view of this potential, this review article describes different aspects of gene expression and regulation in Streptomyces , and summarizes and discusses results obtained using Streptomyces lividans as host for secretion of heterologus proteins of prokaryotic and eukaryotic origin.