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.     

Oxalic acid production and its role in pathogenesis of Sclerotinia sclerotiorum

Abstract Sunflower plants were inoculated with a virulent isolate of Sclerotinia sclerotiorum and with the same isolate nutritionally conditioned to produce small amounts of oxalic acid. The preconditioned isolate behaved as hypovirulent. Tomato plants were inoculated with four S. sclerotiorum isolates of increasing virulence. A close correlation among disease severity, accumulation of oxalic acid, decrease in pH and inhibition of polyphenoloxidase in both infected host tissues was demonstrated. Oxalic acid production as an important factor of virulence in S. sclerotiorum is emphasized and its effect on the phenolic metabolism of the host via inhibition of polyphenoloxidase is suggested.     

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.     

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.     

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.     

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.     

Unfolding studies of tissue transglutaminase

Activation of tissue transglutaminase by calcium involves a conformational change which allows exposition of the active site to the substrate via movements of domains 3 and 4 that lead to an increase of the inter-domain distance. The inhibitor GTP counteracts these changes. Here we investigate the possible existence of non-native conformational states still compatible with the enzyme activity produced by chemical and thermal perturbations. The results indicate that chemical denaturation is reversible at low guanidine concentrations but irreversible at high concentrations of guanidine. Indeed, at low guanidine concentrations tissue TG-ase exists in a non-native state which is still affected by the ligands as in the native form. In contrast, thermal unfolding is always irreversible, with aggregation and protein self-crosslinkage in the presence of calcium. DSC thermograms of the native protein in the absence of ligands consist of two partly overlapped transitions, which weaken in the presence of calcium and merge together and strengthen in the presence of GTP. Overall, the present work shows, for the first time, the reversible denaturation of a TG-ase isoenzyme and suggests the possibility that also in in vivo, the enzyme may acquire non-native conformations relevant to its patho-physiological functions.     

Long term bed rest with and without vibration exercise countermeasures: effects on human muscle protein dysregulation

The present investigation, the first in the field, was aimed at analyzing differentially, on individual samples, the effects of 55 days of horizontal bed rest, a model for microgravity, on myosin heavy and myosin light chain isoforms distribution (by SDS) and on the proteome (by 2-D DIGE and MS) in the vastus lateralis (VL), a mixed type II/I (～50:50%) head of the quadriceps and in the calf soleus (SOL), a predominantly slow (～35:65%) twitch muscle. Two separate studies were performed on six subjects without (BR) and six with resistive vibration exercise (RVE) countermeasures, respectively. Both VL and SOL underwent in BR decrements of ～15% in cross-sectional area and of ～22% in maximal torque that were prevented by RVE. Myosin heavy chain distribution showed increased type I and decreased type IIA in BR both in VL and in SOL, the opposite with RVE. A substantial downregulation of proteins involved in aerobic metabolism characterized both in SOL and VL in BR. RVE reversed the pattern more in VL than in SOL, whereas proteins involved in anaerobic glycolysis were upregulated. Proteins from the Z-disk region and from costamers were differently dysregulated during bed rest (both BR and RVE), particularly in VL.     

IL-1α and Complement Cooperate in Triggering Local Neutrophilic Inflammation in Response to Adenovirus and Eliminating Virus-Containing Cells

Inflammation is a highly coordinated host response to infection, injury, or cell stress. In most instances, the inflammatory response is pro-survival and is aimed at restoring physiological tissue homeostasis and eliminating invading pathogens, although exuberant inflammation can lead to tissue damage and death. Intravascular injection of adenovirus (Ad) results in virus accumulation in resident tissue macrophages that trigger activation of CXCL1 and CXCL2 chemokines via the IL-1α-IL-1RI signaling pathway. However, the mechanistic role and functional significance of this pathway in orchestrating cellular inflammatory responses to the virus in vivo remain unclear. Resident metallophilic macrophages expressing macrophage receptor with collagenous structure (MARCO⁺) in the splenic marginal zone (MZ) play the principal role in trapping Ad from the blood. Here we show that intravascular Ad administration leads to the rapid recruitment of Ly-6G⁺7/4⁺ polymorphonuclear leukocytes (PMNs) in the splenic MZ, the anatomical compartment that remains free of PMNs when these cells are purged from the bone marrow via a non-inflammatory stimulus. Furthermore, PMN recruitment in the splenic MZ resulted in elimination of virus-containing cells. IL-1α-IL-1RI signaling is only partially responsible for PMN recruitment in the MZ and requires CXCR2, but not CXCR1 signaling. We further found reduced recruitment of PMNs in the splenic MZ in complement C3-deficient mice, and that pre-treatment of IL-1α-deficient, but not wild-type mice, with complement inhibitor CR2-Crry (inhibits all complement pathways at C3 activation) or CR2-fH (inhibits only the alternative complement activation pathway) prior to Ad infection, abrogates PMN recruitment to the MZ and prevents elimination of MARCO⁺ macrophages from the spleen. Collectively, our study reveals a non-redundant role of the molecular factors of innate immunity – the chemokine-activating IL-1α-IL-1RI-CXCR2 axis and complement – in orchestrating local inflammation and functional cooperation of PMNs and resident macrophages in the splenic MZ, which collectively contribute to limiting disseminated pathogen spread via elimination of virus-containing cells.