Optical probing of sodium dynamics in neurons and astrocytes.

Changes in intracellular Na(+) concentration underlie essential neurobiological processes, but few reliable tools exist for their measurement. Here we characterize a new synthetic Na(+)-sensitive fluorescent dye, Asante Natrium Green (ANG), with unique properties. This indicator was excitable in the visible spectrum and by two-photon illumination, suffered little photobleaching and located to the cytosol were it remained for long durations without noticeable unwanted effects on basic cell properties. When used in brain tissue, ANG yielded a bright fluorescent signal during physiological Na(+) responses both in neurons and astrocytes. Synchronous electrophysiological and fluorometric recordings showed that ANG produced accurate Na(+) measurement in situ. This new Na(+) indicator opens innovative ways of probing neuronal circuits.

A role for altered TLR gene expression in association with increased expression of CD200R in the induction of mucosal tissue CD4(+) Treg in aged mice following gavage with a liver extract along with intramuscular monophosphoryl lipid A (MPLA) injection.

Previous studies showed a fetal sheep liver extract (FSLE), in association with LPS, injected into aged (>20 months) mice reversed the altered polarization (increased IL-4 and IL-10 with decreased IL-2 and IFN-gamma) in cytokine production seen from ConA stimulated lymphoid cells of those mice. Aged mice show a >60% decline in numbers and suppressive function of both CD4(+)CD25(+)Foxp3(+)Treg and so-called Tr3 (CD4(+)TGFbeta(+)). Their number/function is restored to levels seen in control (8-week-old) mice by FSLE. We have reported at length on the ability of a novel pair of immunoregulatory molecules, members of the TREM family, namely CD200:CD200R, to control development of dendritic cells (DCs) which themselves regulate production of Foxp3(+) Treg. The latter express a distinct subset of TLRs which control their function. We report that a feature of the altered Treg expression following combined treatment with FSLE and monophosphoryl lipid A, MPLA (a bioactive component of lipid A of LPS) is the altered gene expression both of distinct subsets of TLRs and of CD200Rs. We speculate that this may represent one of the mechanisms by which FSLE and MPLA alter immunity in aged mice.

Faster oxygen uptake kinetics during recovery is related to better repeated sprinting ability.

This study was designed to test the hypothesis that subjects having faster oxygen uptake (VO(2)) kinetics during off-transients to exercises of severe intensity would obtain the smallest decrement score during a repeated sprint test. Twelve male soccer players completed a graded test, two severe-intensity exercises, followed by 6 min of passive recovery, and a repeated sprint test, consisting of seven 30-m sprints alternating with 20 s of active recovery. The relative decrease in score during the repeated sprint test was positively correlated with time constants of the primary phase for the VO(2) off-kinetics (r = 0.85; p < 0.001) and negatively correlated with the VO(2) peak (r = -0.83; p < 0.001). These results strengthen the link found between VO(2) kinetics and the ability to maintain sprint performance during repeated sprints.

Induction of CTL in vivo by major histocompatibility complex class I-peptide complexes covalently associated on the cell surface.

The identification of endogenously produced antigenic peptides presented by MHC class I molecules has opened the way to peptide-based strategies for CTL induction in vivo. Here we demonstrate that the induction in vivo of CTL directed against naturally processed antigens can be triggered by injection of syngeneic cells expressing covalent major histocompatibility complex class I-peptide complexes. In the model system used, the induction of HLA-Cw3 specific cytotoxic T lymphocytes (CTL) in mice by cell surface-associated, covalent H-2Kd (Kd)-Cw3 peptide complexes was investigated. The Kd-restricted Cw3 peptide 170-179 (RYLKNGKETL), which mimics the major natural epitope recognized by Cw3-specific CTL in H-2d mice, was converted to a photoreactive derivative by replacing Arg-170 with N-beta-(4-azidosalicyloyl)-L-2,3-diaminopropionic acid. This peptide derivative was equivalent to the parental Cw3 peptide in terms of binding to Kd molecules and recognition by Cw3-specific CTL clones and could be cross-linked efficiently and selectively to Kd molecules on the surface of Con A-stimulated spleen cells from H-2d mice. Photocross-linking prevented the rapid dissociation of Kd-peptide derivative complexes that takes place under physiological conditions. Cultures of spleen cells or peritoneal exudate cells from mice inoculated i.p. with peptide-pulsed and photocross-linked cells developed a strong CTL response following antigenic stimulation in vitro. The cultured cells efficiently lysed not only target cells sensitized with the Cw3 170-179 peptide but also target cells transfected with the Cw3 gene. Moreover, their TCR preferentially expressed V beta 10 and J alpha pHDS58 segments as well as conserved junctional sequences, as has been observed previously in Cw3-specific CTL responses. In contrast, no Cw3-specific CTL response could be obtained in cultures derived from mice injected with Con A-stimulated spleen cells pulsed with the peptide derivative without photocross-linking.

Effect of a 1-hour single bout of moderate-intensity exercise on fat oxidation kinetics.

The present study aimed to examine the effects of a prior 1-hour continuous exercise bout (CONT) at an intensity (Fat(max)) that elicits the maximal fat oxidation (MFO) on the fat oxidation kinetics during a subsequent submaximal incremental test (IncrC). Twenty moderately trained subjects (9 men and 11 women) performed a graded test on a treadmill (Incr), with 3-minute stages and 1-km.h(-1) increments. Fat oxidation was measured using indirect calorimetry and plotted as a function of exercise intensity. A mathematical model (SIN) including 3 independent variables (dilatation, symmetry, and translation) was used to characterize the shape of fat oxidation kinetics and to determine Fat(max) and MFO. On a second visit, the subjects performed CONT at Fat(max) followed by IncrC. After CONT performed at 57% +/- 3% (means +/- SE) maximal oxygen uptake (Vo(2max)), the respiratory exchange ratio during IncrC was lower at every stage compared with Incr (P < .05). Fat(max) (56.4% +/- 2.3% vs 51.5% +/- 2.4% Vo(2max), P = .013), MFO (0.50 +/- 0.03 vs 0.40 +/- 0.03 g.min(-1), P < .001), and fat oxidation rates from 35% to 70% Vo(2max) (P < .05) were significantly greater during IncrC compared with Incr. However, dilatation and translation were not significantly different (P > .05), whereas symmetry tended to be greater in IncrC (P = .096). This study showed that the prior 1-hour continuous moderate-intensity exercise bout increased Fat(max), MFO, and fat oxidation rates over a wide range of intensities during the postexercise incremental test. Moreover, the shape of the postexercise fat oxidation kinetics tended to have a rightward asymmetry.

Concrete Pavement Mixture Design and Analysis (MDA): Application of a Portable X-Ray Fluorescence Technique to Assess Concrete Mix Proportions , TPF-5(205), 2012

Any transportation infrastructure system is inherently concerned with durability and performance issues. The proportioning and uniformity control of concrete mixtures are critical factors that directly affect the longevity and performance of the portland cement concrete pavement systems. At present, the only means available to monitor mix proportions of any given batch are to track batch tickets created at the batch plant. However, this does not take into account potential errors in loading materials into storage silos, calibration errors, and addition of water after dispatch. Therefore, there is a need for a rapid, cost-effective, and reliable field test that estimates the proportions of as-delivered concrete mixtures. In addition, performance based specifications will be more easily implemented if there is a way to readily demonstrate whether any given batch is similar to the proportions already accepted based on laboratory performance testing. The goal of the present research project is to investigate the potential use of a portable x-ray fluorescence (XRF) technique to assess the proportions of concrete mixtures as they are delivered. Tests were conducted on the raw materials, paste and mortar samples using a portable XRF device. There is a reasonable correlation between the actual and calculated mix proportions of the paste samples, but data on mortar samples was less reliable.

A fluorescent peptide substrate for the surface metalloprotease of Leishmania.

A fluorescent oligopeptide substrate for the promastigote surface protease (PSP) of Leishmania was designed using the data reported for the substrate specificity of the enzyme (Bouvier, J., Schneider, P., Etges, R. J., and Bordier, C. 1990. Biochemistry 29, 10113-10119). The indole fluorescence of the tryptophan residue was efficiently quenched through resonance energy transfer by an N-terminal dansyl group located five amino acid residues away. The heptapeptide, dansyl-A-Y-L-K-K-W-V-NH2, was cleaved by PSP between the tyrosine and leucine residues with a kcat/Km ratio of 8.8 x 10(6) M-1sec-1. Hydrolysis by the enzyme results in a time-dependent increase of fluorescence intensity of 3.7-fold. Assays can be designed based on the tryptophan fluorescence at 360 nm or by individual product analyses using thin-layer chromatography. The synthetic substrate is readily cleaved by the metalloprotease at the surface of fixed promastigotes. The specificity and sensitivity of such internally quenched fluorescent peptide substrate will facilitate the identification of novel inhibitors for the enzyme and aid in detailed studies on its enzymology.

Optimization and regeneration kinetics of lymphatic-specific photodynamic therapy in the mouse dermis.

Lymphatic vessels transport fluid, antigens, and immune cells to the lymph nodes to orchestrate adaptive immunity and maintain peripheral tolerance. Lymphangiogenesis has been associated with inflammation, cancer metastasis, autoimmunity, tolerance and transplant rejection, and thus, targeted lymphatic ablation is a potential therapeutic strategy for treating or preventing such events. Here we define conditions that lead to specific and local closure of the lymphatic vasculature using photodynamic therapy (PDT). Lymphatic-specific PDT was performed by irradiation of the photosensitizer verteporfin that effectively accumulates within collecting lymphatic vessels after local intradermal injection. We found that anti-lymphatic PDT induced necrosis of endothelial cells and pericytes, which preceded the functional occlusion of lymphatic collectors. This was specific to lymphatic vessels at low verteporfin dose, while higher doses also affected local blood vessels. In contrast, light dose (fluence) did not affect blood vessel perfusion, but did affect regeneration time of occluded lymphatic vessels. Lymphatic vessels eventually regenerated by recanalization of blocked collectors, with a characteristic hyperplasia of peri-lymphatic smooth muscle cells. The restoration of lymphatic function occurred with minimal remodeling of non-lymphatic tissue. Thus, anti-lymphatic PDT allows control of lymphatic ablation and regeneration by alteration of light fluence and photosensitizer dose.

Imaging exocytosis and recycling of synaptic-like microvesicles in astrocytes.

Optical imaging techniques are well suited for following the dynamics of physiological processes in living cells. Total internal reflection fluorescence (TIRF) microscopy based on evanescent wave illumination (EWi) allows spectacular, real-time visualization of individual vesicle movements, fusions, and retrievals at the cell surface (i.e., within 100 nm of the plasma membrane). TIRF microscopy is an ideal approach for studying the properties of exocytosis and recycling in cultured astrocytes, particularly because these cells have a rather flat surface and contain secretory vesicles with sparse distribution. Among all populations of secretory vesicles, we focus here on synaptic-like microvesicles (SLMVs). We illustrate how TIRF microscopy using EWi is useful to study exocytosis and recycling of SLMVs at the single-vesicle level and, when combined with epifluorescence illumination (EPIi), can provide detailed information on the kinetics of exocytosis, endocytosis, and re-acidification at the whole-cell level.

Photosynthesis and Rubisco kinetics in spring wheat and meadow fescue under conditions of simulated climate change with elevated CO[sub 2] and increased temperatures

Selostus: Kevätvehnän ja nurminadan fotosynteesi ja Rubisco-kinetiikka simuloidun ilmastonmuutoksen eli kohotetun hiilidioksidipitoisuuden ja kohotetun lämpötilan oloissa

Thrombin-sensitive dual fluorescence imaging and therapeutic agent for detection and treatment of synovial inflammation in murine rheumatoid arthritis.

We have developed a thrombin-sensitive polymeric photosensitizer prodrug (T-PS) to selectively image and eradicate inflammatory lesions in rheumatoid arthritis (RA). Thrombin is a serine protease up-regulated in synovial tissues of rheumatoid arthritis (RA) patients. T-PS consists of a polymeric backbone, to which multiple photosensitizer (PS) units are tethered via short thrombin-cleavable peptide linkers. Fluorescence emission and phototoxicity of the prodrug are efficiently quenched due to the interaction of neighboring photosensitizer units. The prodrug is passively delivered to the inflammation site via the enhanced permeability and retention (EPR) effect. Subsequent site-selective proteolytic cleavage of the peptide linkers restores its photoactivity by increasing the mutual distance between PS. Whole animal imaging in murine collagen-induced arthritis, an experimental model of RA revealed a dose-dependent fluorescence increase in arthritic paws after systemic prodrug injection. In addition, administration of T-PS resulted in much higher fluorescence selectivity for arthritic joints as compared to the free PS. Irradiation of the arthritic joints induced light dose dependent phototoxic effects such as apoptosis, vascular damage and local hemorrhage. Long-term observations showed complete regression of the latter. Irradiated non-arthritic tissues or non-irradiated arthritic tissues showed no histological effects after photodynamic therapy with T-PS. This illustrates that T-PS can localize inflammatory lesions with excellent selectivity and induce apoptosis and vascular shut down after irradiation.