Time-resolved Monitoring of Enzyme Activity with Ultrafast Hyper-CEST Spectroscopy

We propose a method to dynamically monitor the progress of an enzymatic reaction using NMR of hyperpolarized ^(129)Xe in a host-guest system. It is based on a displacement assay originally designed for fluorescence experiments that exploits the competitive binding of the enzymatic product on the one hand and a reporter dye on the other hand to a supramolecular host. Recently, this assay has been successfully transferred to NMR, using xenon as a reporter, cucurbit[6]uril as supramolecular host, and Hyper-CEST as detection technique. Its advantage is that the enzyme acts on the unmodified substrate and only the product is detected through immediate inclusion into the host. We here apply a method that drastically accelerates the acquisition of Hyper-CEST spectra in vitro using magnetic field gradients. This allows monitoring the dynamic progress of the conversion of lysine to cadaverine with a temporal resolution of ~30 s. Moreover, the method only requires to sample the very early onset of the reaction (<0.5 % of substrate conversion where the host itself is required only at μM concentrations) at comparatively low reaction rates, thus saving enzyme material and reducing NMR acquisition time. The obtained value for the specific activity agrees well with previously published results from fluorescence assays. We furthermore outline how the Hyper-CEST results correlate with xenon T_2 measurements performed during the enzymatic reaction. This suggests that ultrafast Hyper-CEST spectroscopy can be used for dynamically monitoring enzymatic activity with NMR

Collapse of a semiflexible polymer in poor solvent

We investigate the dynamics and the pathways of the collapse of a single, semiflexible polymer in a poor solvent via 3-D Brownian Dynamics simulations. Earlier work indicates that the condensation of semiflexible polymers generically proceeds via a cascade through metastable racquet-shaped, long-lived intermediates towards the stable torus state. We investigate the rate of decay of uncollapsed states, analyze the preferential pathways of condensation, and describe likelihood and lifespan of the different metastable states. The simulation are performed with a bead-stiff spring model with excluded volume interaction and exponentially decaying attractive potential. The semiflexible chain collapse is studied as functions of the three relevant length scales of the phenomenon, i.e., the total chain length $L$, the persistence length $L_p$ and the condensation length $L_0 = \sqrt{k_B T L_p/u_0}$, where $u_0$ is a measure of the attractive potential per unit length. Two dimensionless ratios, $L/L_p$ and $L_0/L_p$, suffice to describe the decay rate of uncollapsed states, which appears to scale as $(L/L_p)^{1/3} (L_0/L_p)$. The condensation sequence is described in terms of the time series of the well separated energy levels associated with each metastable collapsed state. The collapsed states are described quantitatively through the spatial correlation of tangent vectors along the chain. We also compare the results obtained with a locally inextensible bead-rod chain and with a phantom bead-spring model. Finally, we show preliminary results on the effects of steady shear flow on the kinetics of collapse.Comment: 9 pages, 8 figure

Time-resolved Monitoring of Enzyme Activity with Ultrafast Hyper-CEST Spectroscopy

We propose a method to dynamically monitor the progress of an enzymatic reaction using NMR of hyperpolarized ^(129)Xe in a host-guest system. It is based on a displacement assay originally designed for fluorescence experiments that exploits the competitive binding of the enzymatic product on the one hand and a reporter dye on the other hand to a supramolecular host. Recently, this assay has been successfully transferred to NMR, using xenon as a reporter, cucurbit[6]uril as supramolecular host, and Hyper-CEST as detection technique. Its advantage is that the enzyme acts on the unmodified substrate and only the product is detected through immediate inclusion into the host. We here apply a method that drastically accelerates the acquisition of Hyper-CEST spectra in vitro using magnetic field gradients. This allows monitoring the dynamic progress of the conversion of lysine to cadaverine with a temporal resolution of ~30 s. Moreover, the method only requires to sample the very early onset of the reaction (<0.5 % of substrate conversion where the host itself is required only at μM concentrations) at comparatively low reaction rates, thus saving enzyme material and reducing NMR acquisition time. The obtained value for the specific activity agrees well with previously published results from fluorescence assays. We furthermore outline how the Hyper-CEST results correlate with xenon T_2 measurements performed during the enzymatic reaction. This suggests that ultrafast Hyper-CEST spectroscopy can be used for dynamically monitoring enzymatic activity with NMR

The response function of a sphere in a viscoelastic two-fluid medium

In order to address basic questions of importance to microrheology, we study the dynamics of a rigid sphere embedded in a model viscoelastic medium consisting of an elastic network permeated by a viscous fluid. We calculate the complete response of a single bead in this medium to an external force and compare the result to the commonly-accepted, generalized Stokes-Einstein relation (GSER). We find that our response function is well approximated by the GSER only within a particular frequency range determined by the material parameters of both the bead and the network. We then discuss the relevance of this result to recent experiments. Finally we discuss the approximations made in our solution of the response function by comparing our results to the exact solution for the response function of a bead in a viscous (Newtonian) fluid.Comment: 12 pages, 2 figure

Amphibious Seismic Survey Images Plate Interface at 1960 Chile Earthquake

The southern central Chilean margin at the site of the largest historically recorded earthquake in the Valdivia region, in 1960 (Mw = 9.5), is part of the 5000-km-long active subduction system whose geodynamic evolution is controversially debated and poorly understood. Covering the area between 36° and 40°S, the oceanic crust is segmented by prominent fracture zones. The offshore forearc and its onshore continuation show a complex image with segments of varying geophysical character, and several fault systems active during the past 24 m.y. In autumn 2001, the project SPOC was organized to study the Subduction Processes Off Chile, with a focus on the seismogenic coupling zone and the forearc. The acquired seismic data crossing the Chilean subduction system were gathered in a combined offshore-onshore survey and provide new insights into the lithospheric structure and evolution of active margins with insignificant frontal accretion

Shaping immune responses through the activation of dendritic cells–P2 receptors

Dendritic cells (DCs) activate and shape the adaptive immune response by capturing antigens, migrating to peripheral lymphoid organs where naïve T cells reside, expressing high levels of MHC and costimulatory molecules and secreting cytokines and chemokines. DCs are endowed with a high degree of functional plasticity and their functions are tightly regulated. Besides initiating adaptive immune responses, DCs play a key role in maintaining peripheral tolerance toward self-antigens. On the basis of the information gathered from the tissue where they reside, DCs adjust their functional activity to ensure that protective immunity is favoured while unwanted or exaggerated immune responses are prevented. A wide variety of signals from neighbouring cells affecting DC functional activity have been described. Here we will discuss the complex role of extracellular nucleotides in the regulation of DC function and the role of P2 receptors as possible tools to manipulate immune responses

Incidence and survival of childhood bone cancer in northern England and the West Midlands, 1981–2002

There is a paucity of population-based studies examining incidence and survival trends in childhood bone tumours. We used high quality data from four population-based registries in England. Incidence patterns and trends were described using Poisson regression. Survival trends were analysed using Cox regression. There were 374 cases of childhood (ages 0–14 years) bone tumours (206 osteosarcomas, 144 Ewing sarcomas, 16 chondrosarcomas, 8 other bone tumours) registered in the period 1981–2002. Overall incidence (per million person years) rates were 2.63 (95% confidence interval (CI) 2.27–2.99) for osteosarcoma, 1.90 (1.58–2.21) for Ewing sarcoma and 0.21 (0.11–0.31) for chondrosarcoma. Incidence of Ewing sarcoma declined at an average rate of 3.1% (95% CI 0.6–5.6) per annum (P=0.04), which may be due to tumour reclassification, but there was no change in osteosarcoma incidence. Survival showed marked improvement over the 20 years (1981–2000) for Ewing sarcoma (hazard ratio (HR) per annum=0.95 95% CI 0.91–0.99; P=0.02). However, no improvement was seen for osteosarcoma patients (HR per annum=1.02 95% CI 0.98–1.05; P=0.35) over this time period. Reasons for failure to improve survival including potential delays in diagnosis, accrual to trials, adherence to therapy and lack of improvement in treatment strategies all need to be considered

SOCS2 Influences LPS Induced Human Monocyte-Derived Dendritic Cell Maturation

Dendritic cells (DCs) are highly specific antigen presenting cells, which link innate and adaptive immune responses and participate in protecting hosts from invading pathogens. DCs can be generated in vitro by culturing human monocytes with GM-CSF and IL-4 followed by LPS induced DC maturation. We set out to study the suppressor of cytokine signaling (SOCS) proteins during maturation and activation of human monocyte-derived DCs from peripheral blood in vitro. We found that the expression of SOCS2 mRNA and protein is dramatically up-regulated during DC maturation. Silencing of SOCS2 using siRNA, inhibited DC maturation as evidenced by a decreased expression of maturation markers such as CD83, co-stimulatory molecules CD40, CD86 and HLA-DR. Furthermore, silencing of SOCS2 decreased LPS induced activation of MAP kinases (SAKP/JNK, p38, ERK), IRF3, decreased the translocation of the NF-κB transcription factor and reduced downstream gene mRNA expression. These results suggest a role for SOCS2 in the MyD88-dependent and -independent TLR4 signaling pathways. In conclusion, our results demonstrate that SOCS2 is required for appropriate TLR4 signaling in maturating human DCs via both the MyD88-dependent and -independent signaling pathway

The Arabidopsis cytochrome P450 CYP86A1 encodes a fatty acid ω-hydroxylase involved in suberin monomer biosynthesis

The lipophilic biopolyester suberin forms important boundaries to protect the plant from its surrounding environment or to separate different tissues within the plant. In roots, suberin can be found in the cell walls of the endodermis and the hypodermis or periderm. Apoplastic barriers composed of suberin accomplish the challenge to restrict water and nutrient loss and prevent the invasion of pathogens. Despite the physiological importance of suberin and the knowledge of the suberin composition of many plants, very little is known about its biosynthesis and the genes involved. Here, a detailed analysis of the Arabidopsis aliphatic suberin in roots at different developmental stages is presented. This study demonstrates some variability in suberin amount and composition along the root axis and indicates the importance of ω-hydroxylation for suberin biosynthesis. Using reverse genetics, the cytochrome P450 fatty acid ω-hydroxylase CYP86A1 (At5g58860) has been identified as a key enzyme for aliphatic root suberin biosynthesis in Arabidopsis. The corresponding horst mutants show a substantial reduction in ω-hydroxyacids with a chain length <C20, demonstrating that CYP86A1 functions as a hydroxylase of root suberized tissue. Detailed expression studies revealed a strong root specificity and a localized expression in the root endodermis. Transgenic expression of CYP86A1 fused to GFP distributed CYP86A1 to the endoplasmic reticulum, indicating that suberin monomer biosynthesis takes place in this sub-cellular compartment before intermediates are exported in the apoplast