Production of Transgenic Kidney Bean Shoots by Electroporation of Intact Cells

We obtained transformed bean shoots by electroporation of intact bean cells with the plasmid pDPG165 containing bar gene conferring herbicide resistance to plants. Transformed shoots were selected from electroporated callus on herbicide containing media. Data of molecular analysis (PCR and Southern blotting) confirmed the insertion of bar gene in the genome of herbicide resistant shoots. Detailed procedures for obtaining regenerative bean callus, optimization of electroporation of intact cells and transgenic shoots are given.     

The Relationship between the Traits of the Two-Row Spike,the High Number of Thillers,and the High Regeneration Capacity in in vitro Culture in Barley

In barley (Hordeum vulgare L.), the traits of the two-row spike, the high rate of plant tillering, and the high capacity for multiple plant regeneration (MPR) in a callus culture of immature embryos were shown to correlate. When a dihaploid line (DH) obtained from cv. Golden Promise (two-row spike, high number of thillers) was crossed to a DH line from cv. Bruce (six-row spike, low number of thillers), the two-row trait dominated the F₁ generation, whereas, in F₂, the segregation ratio was 3 : 1. From F₃ progeny, we isolated the families comprising two-row homo- and heterozygotes and six-row homozygotes. In an F₃ hybrid population, the two-row plants manifested higher tillering and MPR rates as compared to the six-row plants. The correlation between the traits of the two-row spike, the high tillering, and the high MPR capacity may depend on the pleiotropic V gene, which controls the general mechanisms of meristem functioning essential for the development of these three traits.     

The Weight of Evidence Does Not Support the Listing of Styrene as “Reasonably Anticipated to be a Human Carcinogen” in NTP's Twelfth Report on Carcinogens

Styrene was listed as “reasonably anticipated to be a human carcinogen” in the twelfth edition of the National Toxicology Program's Report on Carcinogens based on what we contend are erroneous findings of limited evidence of carcinogenicity in humans, sufficient evidence of carcinogenicity in experimental animals, and supporting mechanistic data. The epidemiology studies show no consistent increased incidence of, or mortality from, any type of cancer. In animal studies, increased incidence rates of mostly benign tumors have been observed only in certain strains of one species (mice) and at one tissue site (lung). The lack of concordance of tumor incidence and tumor type among animals (even within the same species) and humans indicates that there has been no particular cancer consistently observed among all available studies. The only plausible mechanism for styrene-induced carcinogenesis—a non-genotoxic mode of action that is specific to the mouse lung—is not relevant to humans. As a whole, the evidence does not support the characterization of styrene as “reasonably anticipated to be a human carcinogen,” and styrene should not be listed in the Report on Carcinogens.     

Long-term exclosure of sheep-grazing from an ancient wood: Vegetation change after a sixty-year experiment

Question: An ancient woodland site with a long history of coppicing and heavy grazing was protected from domesticated stock in 1955. Results of a vegetation-monitoring experiment were subsequently published in 1983. This study followed up the original research to investigate whether observed trends were as predicted. These included a shift in tree species composition in favour of shade-tolerant species, beech (Fagus sylvatica) and rowan (Sorbus aucuparia), at the expense of light-demanding birch (Betula spp.) and oak (Quercus petraea agg.), and progress towards a typical woodland ground flora. Location: Peak District National Park, United Kingdom. Methods: The mixed oak–birch woodland was re-surveyed in 2011. Two enclosures (1955 and 1980s) and an unenclosed control area were investigated. Overstorey structure and composition was assessed by measuring canopy openness and the girths of all trees and saplings. Herb layer species composition was also recorded in 28 vegetation plots. Results: We demonstrated a progressive decline in the number of mature oaks and birch in the old enclosure although they still regenerated successfully. Only a few individuals of beech and rowan appeared. Herb layer species composition differed between the subareas but since the 1980s, the temporal change in the old enclosure was negligible. The new enclosure followed a similar pattern in both canopy and herb layer as observed in the early years in the old enclosure. However, the control subarea had no regeneration of woody species and limited ground flora. Conclusions: After nearly 60 years, the replacement of light-demanding dominants by shade-tolerant trees was still limited, probably by low pH and stable light conditions. The findings are pertinent to the impacts of large herbivore grazing (domestic stock or wild) on woodland dynamics.     

MKP-1 coordinates ordered macrophage-phenotype transitions essential for stem cell-dependent tissue repair

Re-establishing tissue homoeostasis in response to injury requires infiltration of inflammatory cells and activation of resident stem cells. However, full tissue recovery also requires that the inflammation is resolved. While it is known that disturbing the interactions between inflammatory cells and tissue resident cells prevents successful healing, the molecular mechanisms underlying the paracrine interactions between these cell types are practically unknown. Here, and in a recent study, we provide mechanistic evidence that macrophages control stem cell-dependent tissue repair. In particular, we found that the temporal spacing of the pro- to anti-inflammatory macrophage polarization switch is controlled by the balance of p38 MAPK (termed here p38) and the MAPK phosphatase MKP-1 during the muscle healing process. Moreover, we demonstrate a new function for MKP-1-regulated p38 signaling in deactivating macrophages during inflammation resolution after injury. Specifically, at advanced stages of regeneration, MKP-1 loss caused an unscheduled “exhaustion-like” state in muscle macrophages, in which neither pro- nor anti-inflammatory cytokines are expressed despite persistent tissue damage, leading to dysregulated reparation by the tissue stem cells. Mechanistically, we demonstrate that p38 and MKP-1 control the AKT pathway through a miR-21-dependent PTEN regulation. Importantly, both genetic and pharmacological interference with the individual components of this pathway restored inflammation-dependent tissue homeostasis in MKP-1-deficient mice and delayed inflammation resolution and tissue repair dysregulation in wild-type mice. Because the process of tolerance to bacterial infection involves a progressive attenuation of pro-inflammatory gene expression, we discuss here the potential similarities between the mechanisms underlying inflammation resolution during tissue repair and those controlling endotoxin tolerance.     

How good are comparative models in the understanding of protein dynamics?

The 3D structure of a protein is essential to understand protein dynamics. If experimentally determined structure is unavailable, comparative models could be used to infer dynamics. However, the effectiveness of comparative models, compared to experimental structures, in inferring dynamics is not clear. To address this, we compared dynamics features of ~800 comparative models with their crystal structures using normal mode analysis. Average similarity in magnitude, direction, and correlation of residue motions is >0.8 (where value 1 is identical) indicating that the dynamics of models and crystal structures are highly similar. Accuracy of 3D structure and dynamics is significantly higher for models built on multiple and/or high sequence identity templates (>40%). Three-dimensional (3D) structure and residue fluctuations of models are closer to that of crystal structures than to templates (TM score 0.9 vs 0.7 and square inner product 0.92 vs 0.88). Furthermore, long-range molecular dynamics simulations on comparative models of RNase 1 and Angiogenin showed significant differences in the conformational sampling of conserved active-site residues that characterize differences in their activity levels. Similar analyses on two EGFR kinase variant models highlight the effect of mutations on the functional state-specific αC helix motions and these results corroborate with the previous experimental observations. Thus, our study adds confidence to the use of comparative models in understanding protein dynamics.     

NF-κB Signaling Negatively Regulates Osteoblast Dedifferentiation during Zebrafish Bone Regeneration

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