The metallo-beta-lactamase GOB is a mono-Zn(II) enzyme with a novel active site

Metallo-beta-lactamases (MbetaLs) are zinc-dependent enzymes able to hydrolyze and inactivate most beta-lactam antibiotics. The large diversity of active site structures and metal content among MbetaLs from different sources has limited the design of a pan-MbetaL inhibitor. Here we report the biochemical and biophysical characterization of a novel MbetaL, GOB-18, from a clinical isolate of a Gram-negative opportunistic pathogen, Elizabethkingia meningoseptica. Different spectroscopic techniques, three-dimensional modeling, and mutagenesis experiments, reveal that the Zn(II) ion is bound to Asp120, His121, His263, and a solvent molecule, i.e. in the canonical Zn2 site of dinuclear MbetaLs. Contrasting all other related MbetaLs, GOB-18 is fully active against a broad range of beta-lactam substrates using a single Zn(II) ion in this site. These data further enlarge the structural diversity of MbetaLs.     

Loss of proline-rich tyrosine kinase 2 function induces spreading and motility of epithelial prostate cells

Although prostate carcinoma is an aggressive cancer preferentially metastasizing to the bones, many prostate tumors remain localized and confined to the prostate indefinitely. Prediction of the behavior of anatomically localized and moderately differentiated prostate tumors remains difficult because of lack of prognostic markers. Cell motility is an important step in the progression of epithelial tumor toward invasive metastatic carcinomas and changes in the expression and function of adhesion molecules contribute to the acquisition of a more malignant phenotype. Proline-rich tyrosine kinase 2 (Pyk2) is implicated in regulating the organization of actin cytoskeleton, a process critical for cell migration, mitosis, and tumor metastasis. In this report, we investigated whether Pyk2 played a role in the acquisition of an aggressive phenotype in prostate cell. Data reported here demonstrate that loss of Pyk2 kinase function results in induction of cell motility and migration in EPN cells, a line of non-transformed epithelial cells derived from human normal prostate tissue. Changes in motility and migration of prostate cells were associated with changes in the expression of several proteins involved in cell adhesion and reorganization of actin cytoskeleton. Ablation of Pyk2 kinase activity caused a dramatic decrease of the expression of E-cadherin and IRS1 and an increase of the expression of alpha5-integrin. In addition, a massive reorganization of actin cytoskeleton was observed. Our data indicate that Pyk2 plays a central role in the mechanism that regulate cell-cell and cell-substrate interaction and lack of its kinase activity induces prostate cells to acquire a malignant, migrating phenotype.     

PML regulates p53 stability by sequestering Mdm2 to the nucleolus

The promyelocytic leukaemia (PML) tumour-suppressor protein potentiates p53 function by regulating post-translational modifications, such as CBP-dependent acetylation and Chk2-dependent phosphorylation, in the PML-Nuclear Body (NB). PML was recently shown to interact with the p53 ubiquitin-ligase Mdm2 (refs 4-6); however, the mechanism by which PML regulates Mdm2 remains unclear. Here, we show that PML enhances p53 stability by sequestering Mdm2 to the nucleolus. We found that after DNA damage, PML and Mdm2 accumulate in the nucleolus in an Arf-independent manner. In addition, we found that the nucleolar localization of PML is dependent on ATR activation and phosphorylation of PML by ATR. Notably, in Pml(-/-) cells, sequestration of Mdm2 to the nucleolus was impaired, as well as p53 stabilization and the induction of apoptosis. Furthermore, we demonstrate that PML physically associates with the nucleolar protein L11, and that L11 knockdown impairs the ability of PML to localize to nucleoli after DNA damage. These findings demonstrate an unexpected role of PML in the nucleolar network for tumour suppression.     

Endocytosis and cancer

Eukaryotic cells use endocytosis to internalise plasma membrane, surface receptors and their ligands, viruses and various extracellular soluble molecules. Endocytosis has been regarded as a long-term mechanism of signal attenuation via receptor clearance from the cell surface. However, additional, and quite unexpected, functions for endocytosis have emerged, which, together with its attenuation function, project a central role for this process in cellular homeostasis and control of proliferation. Subversion of endocytic control is thus predicted to play a causative role in hyperproliferative conditions, first and foremost cancer.     

A novel design for an instrumented stairway

Kinetics during stair ambulation is currently studied via either the use of sensing elements embedded in the steps of the stairway or simple rigid blocks of different height positioned on top of existing force platforms, typically embedded in a walkway for gait analysis. Neither of these approaches is truly satisfactory for gait analysis laboratories. The first one is expensive and requires setting up a dedicated space. The second approach is limited by the number of platforms utilized in the laboratory for evaluating level walking. This communication proposes a novel design, referred to as "interlaced stairway", that allows one to measure ground reaction force and position of the center of pressure (CoP) for four foot contacts during stair ambulation using only two force platforms embedded in a walkway. Accuracy and precision of the CoP estimates and natural frequency of the stairway structure were derived from experimental data. Test results indicate that the interlaced stairway structure does not appreciably reduce the quality of the measures gathered by the existing force platforms. Specifically, the estimated CoP coordinates show good agreement with the horizontal coordinates of the geometric center of the calibration object utilized to assess accuracy and precision of the CoP estimates (max difference < 6 mm). The natural frequency of the stairway structure is lower than the one for the unloaded force platform but higher than the frequency components of interest in stair ambulation analysis.     

Physiological responses of Spartina alterniflora to varying environmental conditions in Virginia marshes

Physiological measurements were used to investigate the dependence of photosynthesis on light, temperature, and intercellular carbon dioxide (CO₂) levels in the C₄ marsh grass Spartina alterniflora. Functional relationships between these environmental variables and S. alterniflora physiological responses were then used to improve C₄-leaf photosynthesis models. Field studies were conducted in monocultures of S. alterniflora in Virginia, USA. On average, S. alterniflora exhibited lower light saturation values (~1000 μmol m⁻² s⁻¹) than observed in other C₄ plants. Maximum carbon assimilation rates and stomatal conductance to water vapor diffusion were 36 μmol (CO₂) m⁻² s⁻¹ and 200 mmol (H₂O) m⁻² s⁻¹, respectively. Analysis of assimilation-intercellular CO₂ and light response relationships were used to determine Arrhenius-type temperature functions for maximum rate of carboxylation (V _cmax), phosphoenolpyruvate carboxylase activity (V _pmax), and maximum electron transport rate (J _max). Maximum V _cmax values of 105 μmol m⁻² s⁻¹ were observed at the leaf temperature of 311 K. Optimum V _pmax values (80.6 μmol m⁻² s⁻¹) were observed at the foliage temperature of 308 K. The observed V _pmax values were lower than those in other C₄ plants, whereas V _cmax values were higher, and more representative of C₃ plants. Optimum J _max values reached 138 μmol (electrons) m⁻² s⁻¹ at the foliage temperature of 305 K. In addition, the estimated CO₂ compensation points were in the range of C₃ or C₃–C₄ intermediate plants, not those typical of C₄ plants. The present results indicate the possibility of a C₃–C₄ intermediate or C₄-like photosynthetic mechanism rather than the expected C₄-biochemical pathway in S. alterniflora under field conditions. In a scenario of atmospheric warming and increased atmospheric CO₂ concentrations, S. alterniflora will likely respond positively to both changes. Such responses will result in increased S. alterniflora productivity, which is uncharacteristic of C₄ plants.     

Dexamethasone modulates interleukin-12 production by inducing monocyte chemoattractant protein-1 in human dendritic cells

Glucocorticoids have long been used as first-line immunosuppressants, although their precise mechanism of action has not been fully elucidated yet. This study evaluated the gene and protein expression of monocyte chemoattractant protein-1 (MCP-1), and its relationship with interleukin-12 and interleukin-10 synthesis, in human monocyte-derived dendritic cells exposed to dexamethasone. Dendritic cells were differentiated in the presence or in the absence of dexamethasone and then activated by IFN-gamma+soluble CD40 ligand; the gene and protein expression of target cytokines was measured by real-time PCR and ELISA, respectively. Our results showed that dexamethasone-primed mature dendritic cells expressed low levels of interleukin-12, and, at the opposite, high levels of interleukin-10 and MCP-1. Transfection experiments confirmed the ability of dexamethasone to activate MCP-1 gene promoter. Dexamethasone increased also MCP-2, but not MCP-3 synthesis, and the gene expression of CC chemokine receptor-2 by mature dendritic cells. The addition of anti-MCP-1 blocking antibody depressed MCP-1 release, and increased interleukin-12 production in dexamethasone-treated dendritic cells, thus demonstrating that interleukin-12 downregulation is largely dependent on MCP-1 overexpression. Our findings suggest that the induction of MCP expression in human dendritic cells by dexamethasone, and the amplification of cell response via the upregulation of the chemokine cognate receptor, may be critical to inhibit type 1 T-helper-biased immune response and, possibly, to favor type 2 T-helper-skewed response.     

The role of geomagnetic cues in green turtle open sea navigation

Background

Laboratory and field experiments have provided evidence that sea turtles use geomagnetic cues to navigate in the open sea. For instance, green turtles (Chelonia mydas) displaced 100 km away from their nesting site were impaired in returning home when carrying a strong magnet glued on the head. However, the actual role of geomagnetic cues remains unclear, since magnetically treated green turtles can perform large scale (>2000 km) post-nesting migrations no differently from controls.

Methodology/Principal Findings

In the present homing experiment, 24 green turtles were displaced 200 km away from their nesting site on an oceanic island, and tracked, for the first time in this type of experiment, with Global Positioning System (GPS), which is able to provide much more frequent and accurate locations than previously used tracking methods. Eight turtles were magnetically treated for 24–48 h on the nesting beach prior to displacement, and another eight turtles had a magnet glued on the head at the release site. The last eight turtles were used as controls. Detailed analyses of water masses-related (i.e., current-corrected) homing paths showed that magnetically treated turtles were able to navigate toward their nesting site as efficiently as controls, but those carrying magnets were significantly impaired once they arrived within 50 km of home.

Conclusions/Significance

While green turtles do not seem to need geomagnetic cues to navigate far from the goal, these cues become necessary when turtles get closer to home. As the very last part of the homing trip (within a few kilometers of home) likely depends on non-magnetic cues, our results suggest that magnetic cues play a key role in sea turtle navigation at an intermediate scale by bridging the gap between large and small scale navigational processes, which both appear to depend on non-magnetic cues.