Coexpression, copurification, crystallization and preliminary X-ray analysis of a complex of ARL2-GTP and PDE delta

The small GTPase ARL2 (from Mus musculus) and an effector protein, the δ subunit of human cGMP phosphodiesterase (hPDE δ), were coexpressed and copurified from Escherichia coli as a stable complex. Coexpression significantly increased the otherwise low yield of PDE δ production in E. coli. The complex, which contains ARL2 in the activated GTP-bound form, was crystallized in two forms. The first belongs to the monoclinic space group P21, with unit-cell parameters a = 48.1, b = 45.7, c = 74.7 Å, β = 94.0° and one complex (39 kDa) in the asymmetric unit. Cryocooled crystals diffract to 2.3 Å using synchrotron radiation. The micro-focused X-­ray beam at beamline ID13 (ESRF) allowed the use of very small crystals, which helped to overcome twinning and enabled the identification of a molecular-replacement solution. The second form recrystallized from the first one after several months. These crystals belong to the orthorhombic space group P212121, with unit-cell parameters a = 44.5, b = 65.4, c = 104.4 Å and one complex in the asymmetric unit. They diffracted to 1.8 Å using synchrotron radiation

The chlorine nuclear quadrupole coupling tensor in chlorotrifluoroethylene

The 273 hyperfine components from 56 rotational transitions of chlorotrifluoroethylene were measured with a Fourier transform microwave spectrometer. A global least-squares fit was made to the rotational constants, quartic distortion constants, and 35Cl quadrupole coupling constants; the RMS deviation of the fit was 1.6 kHz. It is shown that such high-resolution measurements enable [chi]ab to be determined without the near degeneracies usually necessary with less precise data. The principle tensor quadrupole coupling constants are x[chi]zz = -77.46(10), xxx = 38.85(10), and [chi]yy = xcc = 38.614(3) MHz. The values are comparable to other vinyl chlorides, removing some anomalies from previous studies.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27550/1/0000594.pd

Origin of Hysteresis Observed During Fatigue of Ceramic-Matrix Composites

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66076/1/j.1151-2916.1990.tb05239.x.pd

Stress-corrosion mechanisms in silicate glasses

The present review is intended to revisit the advances and debates in the comprehension of the mechanisms of subcritical crack propagation in silicate glasses almost a century after its initial developments. Glass has inspired the initial insights of Griffith into the origin of brittleness and the ensuing development of modern fracture mechanics. Yet, through the decades the real nature of the fundamental mechanisms of crack propagation in glass has escaped a clear comprehension which could gather general agreement on subtle problems such as the role of plasticity, the role of the glass composition, the environmental condition at the crack tip and its relation to the complex mechanisms of corrosion and leaching. The different processes are analysed here with a special focus on their relevant space and time scales in order to question their domain of action and their contribution in both the kinetic laws and the energetic aspects.Comment: Invited review article - 34 pages Accepted for publication in J. Phys. D: Appl. Phy

Structure of a Classical MHC Class I Molecule That Binds “Non-Classical” Ligands

The chicken MHC YF1*7.1 X-ray structures reveal that this protein binds lipids and thus represents a "hybrid" class I complex with features of classical as well as non-classical MHC molecules

Surface Localization of Glucosylceramide during Cryptococcus neoformans Infection Allows Targeting as a Potential Antifungal

Cryptococcus neoformans (Cn) is a significant human pathogen that, despite current treatments, continues to have a high morbidity rate especially in sub-Saharan Africa. The need for more tolerable and specific therapies has been clearly shown. In the search for novel drug targets, the gene for glucosylceramide synthase (GCS1) was deleted in Cn, resulting in a strain (Δgcs1) that does not produce glucosylceramide (GlcCer) and is avirulent in mouse models of infection. To understand the biology behind the connection between virulence and GlcCer, the production and localization of GlcCer must be characterized in conditions that are prohibitive to the growth of Δgcs1 (neutral pH and high CO2). These prohibitive conditions are physiologically similar to those found in the extracellular spaces of the lung during infection. Here, using immunofluorescence, we have shown that GlcCer localization to the cell surface is significantly increased during growth in these conditions and during infection. We further seek to exploit this localization by treatment with Cerezyme (Cz), a recombinant enzyme that metabolizes GlcCer, as a potential treatment for Cn. Cz treatment was found to reduce the amount of GlcCer in vitro, in cultures, and in Cn cells inhabiting the mouse lung. Treatment with Cz induced a membrane integrity defect in wild type Cn cells similar to Δgcs1. Cz treatment also reduced the in vitro growth of Cn in a dose and condition dependent manner. Finally, Cz treatment was shown to have a protective effect on survival in mice infected with Cn. Taken together, these studies have established the legitimacy of targeting the GlcCer and other related sphingolipid systems in the development of novel therapeutics

Cleavage of the urokinase receptor (uPAR) on oral cancer cells : regulation by transforming growth factor - beta 1 (TGF-beta 1) and potential effects on migration and invasion

Background: Urokinase plasminogen activator (uPA) receptor (uPAR) is up-regulated at the invasive tumour front of human oral squamous cell carcinoma (OSCC), indicating a role for uPAR in tumour progression. We previously observed elevated expression of uPAR at the tumour-stroma interface in a mouse model for OSCC, which was associated with increased proteolytic activity. The tumour microenvironment regulated uPAR expression, as well as its glycosylation and cleavage. Both full-length- and cleaved uPAR (uPAR (II-III)) are involved in highly regulated processes such as cell signalling, proliferation, migration, stem cell mobilization and invasion. The aim of the current study was to analyse tumour associated factors and their effect on uPAR cleavage, and the potential implications for cell proliferation, migration and invasion. Methods: Mouse uPAR was stably overexpressed in the mouse OSCC cell line AT84. The ratio of full-length versus cleaved uPAR as analysed by Western blotting and its regulation was assessed by addition of different protease inhibitors and transforming growth factor - beta 1 (TGF-beta 1). The role of uPAR cleavage in cell proliferation and migration was analysed using real- time cell analysis and invasion was assessed using the myoma invasion model. Results: We found that when uPAR was overexpressed a proportion of the receptor was cleaved, thus the cells presented both full-length uPAR and uPAR (II-III). Cleavage was mainly performed by serine proteases and urokinase plasminogen activator (uPA) in particular. When the OSCC cells were stimulated with TGF-beta 1, the production of the uPA inhibitor PAI-1 was increased, resulting in a reduction of uPAR cleavage. By inhibiting cleavage of uPAR, cell migration was reduced, and by inhibiting uPA activity, invasion was reduced. We could also show that medium containing soluble uPAR (suPAR), and cleaved soluble uPAR (suPAR (II-III)), induced migration in OSCC cells with low endogenous levels of uPAR. Conclusions: These results show that soluble factors in the tumour microenvironment, such as TGF-beta 1, PAI-1 and uPA, can influence the ratio of full length and uPAR (II-III) and thereby potentially effect cell migration and invasion. Resolving how uPAR cleavage is controlled is therefore vital for understanding how OSCC progresses and potentially provides new targets for therapy.Peer reviewe

The diterpenoid alkaloid noroxoaconitine is a Mapkap kinase 5 (MK5/PRAK) inhibitor

The mitogen-activated protein kinase-activated protein kinase MK5 is ubiquitously expressed in vertebrates and is implicated in cell proliferation, cytoskeletal remodeling, and anxiety behavior. This makes MK5 an attractive drug target. We tested several diterpenoid alkaloids for their ability to suppress MK5 kinase activity. We identified noroxoaconitine as an ATP competitor that inhibited the catalytic activity of MK5 in vitro (IC50 = 37.5 μM; Ki = 0.675 μM) and prevented PKA-induced nuclear export of MK5, a process that depends on kinase active MK5. MK5 is closely related to MK2 and MK3, and noroxoaconitine inhibited MK3- and MK5- but not MK2-mediated phosphorylation of the common substrate Hsp27. Molecular docking of noroxoaconitine into the ATP binding sites indicated that noroxoaconitine binds more strongly to MK5 than to MK3. Noroxoaconitine and derivatives may help in elucidating the precise biological functions of MK5 and may prove to have therapeutic values