Computational redesign of a mononuclear zinc metalloenzyme for organophosphate hydrolysis

The ability to redesign enzymes to catalyze noncognate chemical transformations would have wide-ranging applications. We developed a computational method for repurposing the reactivity of metalloenzyme active site functional groups to catalyze new reactions. Using this method, we engineered a zinc-containing mouse adenosine deaminase to catalyze the hydrolysis of a model organophosphate with a catalytic efficiency (k(cat)/K(m)) of ~10(4) M(-1) s(-1) after directed evolution. In the high-resolution crystal structure of the enzyme, all but one of the designed residues adopt the designed conformation. The designed enzyme efficiently catalyzes the hydrolysis of the R(P) isomer of a coumarinyl analog of the nerve agent cyclosarin, and it shows marked substrate selectivity for coumarinyl leaving groups. Computational redesign of native enzyme active sites complements directed evolution methods and offers a general approach for exploring their untapped catalytic potential for new reactivities.     

Essential role of phosphatidylinositol 3-kinase in insulin-induced activation and phosphorylation of the cGMP-inhibited cAMP phosphodiesterase in rat adipocytes studies using the selective inhibitor wortmannin

Incubation of rat adipocytes with wortmannin, a potent and selective phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor, completely blocked the antilipolytic action of insulin (IC₅₀≈ 100 nM), the insulin-induced activation and phosphorylation of cGMP-inhibited cAMP phosphodiesterase (cGI-PDE) as well as the activation of the insulin-stimulated cGI-PDE kinase (IC₅₀≈ 10–30 nM). No direct effects of the inhibitor on the insulin-stimulated cGI-PDE kinase, the cGI-PDE and the hormone-sensitive lipase were observed. These data suggest that activation of PI 3-kinase upstream of the insulin-stimulated cGI-PDE kinase in the antilipolytic insulin signalchain has an essential role for insulin-induced cGI-PDE activation/ phosphorylation and anti-lipolysis.     

Chaotic gene regulatory networks can be robust against mutations and noise

Robustness to mutations and noise has been shown to evolve through stabilizing selection for optimal phenotypes in model gene regulatory networks. The ability to evolve robust mutants is known to depend on the network architecture. How do the dynamical properties and state-space structures of networks with high and low robustness differ? Does selection operate on the global dynamical behavior of the networks? What kind of state-space structures are favored by selection? We provide damage propagation analysis and an extensive statistical analysis of state spaces of these model networks to show that the change in their dynamical properties due to stabilizing selection for optimal phenotypes is minor. Most notably, the networks that are most robust to both mutations and noise are highly chaotic. Certain properties of chaotic networks, such as being able to produce large attractor basins, can be useful for maintaining a stable gene-expression pattern. Our findings indicate that conventional measures of stability, such as damage propagation, do not provide much information about robustness to mutations or noise in model gene regulatory networks.     

Cell structural changes in the needles of Norway spruce exposed to long-term ozone and drought

Effects of ozone and/or drought on Norway spruce needles werestudied using light microscopy and electron microscopy. Saplingswere exposed to ozone in open-top chambers during 1992–1995and also to drought in the late summers of 1993–1995.Samples from current and previous year needles were collectedfive times during 1995. Ozone increased the numbers of peroxisomesand mitochondria, which suggests that defence mechanisms againstoxidative stress were active. The results from peroxisomes suggestthat the oxidative stress was more pronounced in the upper sideof the needles, and those from mitochondria that defence wasmore active in the younger needle generation. Possibly due tothe good nitrogen status and the active defence, no ozone-specificchloroplast alterations were seen. At the end of the season,older needles from ozone treatments had smaller central vacuolescompared with other needles. Cytoplasmic vacuoles around thenucleus were increased by ozone in the beginning of the experiment,and did not increase towards the end of the season as in thecontrols. These results from vacuoles may indicate that ozoneaffected the osmotic properties of the cells. Decreased numberand underdevelopment of sclerenchyma cells and proliferationof tonoplast were related to nutrient imbalance, which was enhancedby drought. Larger vascular cylinders and more effective starchaccumulation before and after the drought periods compensatedfor the reduced water status. Numbers of peroxisomes and mitochondriawere increased in the drought-exposed needles before the onsetof drought treatments of the study year, i.e. these changeswere memory effects. Interactions between ozone and droughtwere few.     

HDACi phenylbutyrate increases bystander killing of HSV-tk transfected glioma cells

Malignant glioma patients have a dismal prognosis with an urgent need of new treatment modalities. Previously developed gene therapies for brain tumors showed promising results in experimental animal models, but failed in clinical trials due to low transfection rates and insufficient expression of the transgene in tumor cells, as well as low bystander killing effects. We have previously shown that the histone deacetylase inhibitor 4-phenylbutyrate (4-PB) enhances gap junction communication between glioma cells in culture. In this study, we demonstrate an activation of recombinant HSV-tk gene expression, and a dramatic enhancement of gap junction-mediated bystander killing effect by administration of the HSV-tk prodrug ganciclovir together with 4-PB. These findings that 4-PB potentiates "suicide gene" expression as well as enhances gap junctional communication and bystander killing of tumor cells justify further testing of this paradigm as an adjunct to suicide gene therapy of malignant gliomas.     

Dose responses of gibberellins

To determine the response type, published data for the most widely used bioassays for gibberellins have been analyzed by means of a computer program for estimating sensitivity parameters, or by interpolation. The dose response data are almost uniformly subsensitive, i. e. more than an 81-fold increase in external gibberellin concentration is required for a change from 10 to 90% of maximal response (S₉₀/S₁₀). Data for the interaction of gibberellins with artificial membranes are, in contrast, markedly ultrasensitive (S₉₀/S₁₀± 10). This difference further strengthens the view that lipid structures do not function as receptors for gibberellins. Most of the subsensitive dose responses for gibberellins can be quite precisely represented by cooperative isotherms. However, available data are insufficiently detailed for an unequivocal choice among cooperative, multiphasic or more complex kinetics.     

Trait variations of ground flora species disentangle the effects of global change and altered land‐use in Swedish forests during 20 years

Northern forest ecosystems are exposed to a range of anthropogenic processes including global warming, atmospheric deposition, and changing land‐use. The vegetation of northern forests is composed of species with several functional traits related to these processes, whose effects may be difficult to disentangle. Here, we combined analyses of spatio‐temporal dynamics and functional traits of ground flora species, including morphological characteristics, responses to macro‐ and microclimate, soil conditions, and disturbance. Based on data from the Swedish National Forest Inventory, we compared changes in occurrence of a large number of ground flora species during a 20‐year period (1994–2013) in boreal and temperate Sweden respectively. Our results show that a majority of the common ground flora species have changed their overall frequency. Comparisons of functional traits between increasing and declining species, and of trends in mean trait values of sample plots, indicate that current floristic changes are caused by combined effects of climate warming, nitrogen deposition and changing land‐use. Changes and their relations with plant traits were generally larger in temperate southern Sweden. Nutrient‐demanding species with mesotrophic morphology were favored by ongoing eutrophication due to nitrogen deposition in the temperate zone, while dwarf shrubs with low demands on nitrogen decreased in frequency. An increase of species with less northern and less eastern distribution limits was also restricted to temperate Sweden, and indicates effects of a moister and milder macroclimate. A trend toward dense plantation forests is mirrored by a decrease of light‐demanding species in both vegetation zones, and a decrease of grassland species in the temperate zone. Although denser tree canopies may buffer effects of a warmer climate and of nitrogen deposition to some extent, traits related to these processes were weakly correlated in the group of species with changing frequency. Hence, our results indicate specific effects of these often confounded anthropogenic processes.     

A mis-expression study of factors affecting Drosophila PNS cell identity

Drosophila PNS sense organs arise from single sensory organ precursor (SOP) cells through a series of asymmetric divisions. In a mis-expression screen for factors affecting PNS development, we identified string and dappled as being important for the proper formation of adult external sensory (ES) organs. string is a G2 regulator. dappled has no described function but is implicated in tumorigenesis. The mis-expression effect from string was analysed using timed over expression during adult ES-organ and, for comparison, embryonic Chordotonal (Ch) organ formation. Surprisingly, string mis-expression prior to SOP division gave the greatest effect in both systems. In adult ES-organs, this lead to cell fate transformations producing structural cells, whilst in the embryo organs were lost, hence differences within the lineages exist. Mis-expression of dappled, lead to loss and duplications of entire organs in both systems, potentially affecting SOP specification, in addition to affecting neuronal guidance.