Substrate-induced double sided H-bond network as a means of domain closure in 3-phosphoglycerate kinase

Closure of the two domains of 3-phosphoglycerate kinase, upon substrate binding, is essential for the enzyme function. The available crystal structures cannot provide sufficient information about the mechanism of substrate assisted domain closure and about the requirement of only one or both substrates, since lattice forces may hinder the large scale domain movements. In this study the known X-ray data, obtained for the open and closed conformations, were probed by solution small-angle X-ray scattering experiments. The results prove that binding of both substrates is essential for domain closure. Molecular graphical analysis, indeed, reveals formation of a double-sided H-bond network, which affects substantially the shape of the main molecular hinge at beta-strand L, under the concerted action of both substrates.     

Yeast kinesin-8 depolymerizes microtubules in a length-dependent manner

The microtubule cytoskeleton and the mitotic spindle are highly dynamic structures, yet their sizes are remarkably constant, thus indicating that the growth and shrinkage of their constituent microtubules are finely balanced. This balance is achieved, in part, through kinesin-8 proteins (such as Kip3p in budding yeast and KLP67A in Drosophila) that destabilize microtubules. Here, we directly demonstrate that Kip3p destabilizes microtubules by depolymerizing them--accounting for the effects of kinesin-8 perturbations on microtubule and spindle length observed in fungi and metazoan cells. Furthermore, using single-molecule microscopy assays, we show that Kip3p has several properties that distinguish it from other depolymerizing kinesins, such as the kinesin-13 MCAK. First, Kip3p disassembles microtubules exclusively at the plus end and second, remarkably, Kip3p depolymerizes longer microtubules faster than shorter ones. These properties are consequences of Kip3p being a highly processive, plus-end-directed motor, both in vitro and in vivo. Length-dependent depolymerization provides a new mechanism for controlling the lengths of subcellular structures.     

Targeting the lateral interactions of transmembrane domain 5 of Epstein-Barr virus latent membrane protein 1

The lateral transmembrane protein-protein interaction has been regarded as "undruggable" despite its importance in many biological processes. The homo-trimerization of transmembrane domain 5 (TMD-5) of latent membrane protein 1 (LMP-1) is critical for the constitutive oncogenic activation of the Epstein-Barr virus (EBV). Herein, we report a small molecule agent, NSC 259242 (compound 1), to be a TMD-5 self-association disruptor. Both the positively charged acetimidamide functional groups and the stilbene backbone of compound 1 are essential for its inhibitory activity. Furthermore, cell-based assays revealed that compound 1 inhibits full-length LMP-1 signaling in EBV infected B cells. These studies demonstrated a new strategy for identifying small molecule disruptors for investigating transmembrane protein-protein interactions.     

Characterization of the PEG layer of sterically stabilized liposomes: a SAXS study

Synchrotron small-angle X-ray scattering analysis of the bilayer structure of a pharmacologically relevant sterically stabilized liposome system is presented. Describing the electron density profile of the bilayer with the superposition of Gaussian functions, the contribution of the poly(ethylene glycol) (PEG) layers to the total electron density was identified. The changes in the thickness of the PEG layer as well as the distribution of the PEG chains among the outer and inner leaflets of the bilayers were followed by changing the molar ratio of the PEG-lipid and the molar weight of the PEG molecule.     

Characterization of binding mode of imatinib to human α1-acid glycoprotein

Imatinib (IMT) is a selective tyrosine kinase inhibitor, used in the treatment of chronic myeloid leukemia and gastrointestinal stromal tumors. Its strong plasma protein binding was found to belong to the F1*S genetic variant of α(1)-acid glycoprotein (AGP). In this work, comparative AGP binding studies were performed with IMT fragment molecules to reveal which parts of the molecule are important in the high-affinity interaction provoking specific spectral changes. Molecular modeling calculations indicated that IMT docked into the X-ray structure of AGP/F1 adopts a bent, compact conformation. This binding mode is similar to those found in its complexes with some low-affinity kinases and a quinone reductase, being strikingly different from the extended conformation of IMT in its high-affinity kinase targets.     

Draft Genome Sequence Determination for Cystic Fibrosis and Chronic Granulomatous Disease Burkholderia multivorans Isolates

Burkholderia multivorans is a Gram-negative bacterium and a member of the Burkholderia cepacia complex, which is frequently associated with respiratory infections in people with cystic fibrosis (CF) and chronic granulomatous disease (CGD). We are reporting the genome sequences of 4 B. multivorans strains, 2 from CF patients and 2 from CGD patients.     

Homocysteine metabolism in peripheral blood mononuclear cells: evidence for cystathionine beta-synthase activity in resting state

Activated peripheral blood mononuclear cells (PBMC) release homocysteine and possess cystathionine β-synthase (CBS) activity; however, it was thought that there is no CBS in resting state. Previously, we found that nickel decreased intracellular homocysteine concentration in un-stimulated (e.g. resting) PBMC, suggesting that resting PBMC might also have active homocysteine metabolism. Here, we demonstrated that un-stimulated PBMC synthesize (incorporate L-[methyl-14C]methionine to DNA, lipids and proteins), release (increase extracellular homocysteine), and metabolize homocysteine. Intracellular homocysteine concentration varied with incubation time, depending on extracellular concentrations of methionine, homocysteine, and glutathione. Methionine synthase activity was constant and independent of thiol concentrations. In Western blot, CBS protein was clearly identified in freshly isolated PBMC. CBS protein level and activity increased with incubation time, upon stimulation, and similar to intracellular homocysteine, depending on intra- and extracellular homocysteine and glutathione concentrations. According to our knowledge, this is the first evidence that certifies homocysteine metabolism and regulatory role of CBS activity to keep balanced intracellular homocysteine level in resting PBMC. Homocysteine, released by PBMC, in turn can modulate its functions contributing to the development of hyperhomocysteinemia-induced diseases.     

Quantification of extraradical soil mycelium and ectomycorrhizas of Boletus edulis in a Scots pine forest with variable sporocarp productivity

The availability of most edible ectomycorrhizal mushrooms depends on their natural fructification. Sporocarp formation of these fungi is linked to habitat characteristics and climate conditions, but these data alone do not explain all the trends of fungal fruiting and dynamics. It could be hypothesized that the amount of soil mycelia could also be related to the production of carpophores. Soil samples (five cylinders of 250 cm³ per plot) were taken monthly, from September to November, in five fenced permanent plots (5 × 5 m) in Pinar Grande (Soria, Spain), a Pinus sylvestris stand situated in the north of the Sistema Ibérico mountain range. Plots were chosen to establish a gradient of Boletus edulis productivity from 0 to 38.5 kg/ha year, according to the mean fresh weight of sporocarps collected during the last 10 years. B. edulis ectomycorrhizal root tips were identified in each soil sample according to its morphology and counted. DNA extractions were performed with the PowerSoil^TM DNA Isolation Kit and quantification of extraradical soil mycelium by real-time polymerase chain reaction using specific primers and a TaqMan? probe. The concentration of soil mycelium of B. edulis (mg mycelium/g soil) did not differ significantly between plots (p = 0.1397), and sampling time (p = 0.7643) within the fructification period. The number of mycorrhizal short roots per soil volume showed significant differences between the plots (p = 0.0050) and the three sampling times (p < 0.0001). No significant correlation between the number of mycorrhizas and the productivity of the plot (kg of B. edulis/ha year) was detected (p = 0.615). A statistically significant positive correlation (p = 0.0481) was detected between the concentration of mycelia of B. edulis in the soil samples and the presence of short roots mycorrhizal with B. edulis in these samples. The productivity of the plots, in terms of sporocarps produced during the last 10 years, was not correlated either with the concentration of soil mycelium or with the presence or abundance of ectomycorrhizas.     

HR-pQCT-based homogenised finite element models provide quantitative predictions of experimental vertebral body stiffness and strength with the same accuracy as μFE models

This study validated two different high-resolution peripheral quantitative computer tomography (HR-pQCT)-based finite element (FE) approaches, enhanced homogenised continuum-level (hFE) and micro-finite element (μFE) models, by comparing them with compression test results of vertebral body sections. Thirty-five vertebral body sections were prepared by removing endplates and posterior elements, scanned with HR-pQCT and tested in compression up to failure. Linear hFE and μFE models were created from segmented and grey-level CT images, and apparent model stiffness values were compared with experimental stiffness as well as strength results. Experimental and numerical apparent elastic properties based on grey-level/segmented CT images (N=35) correlated well for μFE (r2=0.748/0.842) and hFE models (r2=0.741/0.864). Vertebral section stiffness values from the linear μFE/hFE models estimated experimental ultimate apparent strength very well (r2=0.920/0.927). Calibrated hFE models were able to predict quantitatively apparent stiffness with the same accuracy as μFE models. However, hFE models needed no back-calculation of a tissue modulus or any kind of fitting and were computationally much cheaper.     

The Initial Draining Lymph Node Primes the Bulk of the CD8 T Cell Response and Influences Memory T Cell Trafficking after a Systemic Viral Infection

Lymphocytic choriomeningitis virus (LCMV) causes a systemic infection in mice with virus replication occurring in both peripheral tissues and secondary lymphoid organs. Because of the rapid systemic dissemination of the virus, the secondary lymphoid organs responsible for the induction of the LCMV-specific CD8 T cell response are poorly defined. We show that the mediastinal lymph node (MedLN) serves as the primary draining lymph node following LCMV infection. In addition, we demonstrate that the MedLN is responsible for priming the majority of the virus-specific CD8 T cell response. Following resolution of the acute infection, the draining MedLN exhibits characteristics of a reactive lymph node including an increased presence of germinal center B cells and increased cellularity for up to 60 days post-infection. Furthermore, the reactive MedLN harbors an increased frequency of CD62L⁻ effector memory CD8 T cells as compared to the non-draining lymph nodes. The accumulation of LCMV-specific CD62L⁻ memory CD8 T cells in the MedLN is independent of residual antigen and is not a unique feature of the MedLN as footpad infection with LCMV leads to a similar increase of virus-specific CD62L⁻ effector memory CD8 T cells in the draining popliteal lymph node. Our results indicate that CD62L⁻ effector memory CD8 T cells are granted preferential access into the draining lymph nodes for an extended time following resolution of an infection.