Reduction of ICAM-1 expression by carbon monoxide via soluble guanylate cyclase activation accounts for modulation of neutrophil migration

Previously, it was demonstrated that the heme/heme oxygenase (HO)/carbon monoxide (CO) pathway inhibits neutrophil recruitment during the inflammatory response. Herein, we addressed whether the inhibitory effect of the HO pathway on neutrophil adhesion and migration involves the reduction of intracellular adhesion molecule type (ICAM)-1 and beta(2)-integrin expression. Mice pretreated with a specific inhibitor of inducible HO (HO-1), zinc protoporphyrin (ZnPP) IX, exhibit enhanced neutrophil adhesion and migration induced by intraperitoneal injection of Escherichia coli lipopolysaccharide (LPS). These findings are associated with an increase in ICAM-1 expression on mesentery venular endothelium. In accordance, HO-1 inhibition did not enhance LPS-induced neutrophil migration and adhesion in ICAM-1-deficient mice. Furthermore, the treatment with a CO donor (dimanganese decacarbonyl, DMDC) that inhibits adhesion and migration of the neutrophils, reduced LPS-induced ICAM-1 expression. Moreover, neither DMDC nor ZnPP IX treatments changed LPS-induced beta(2)-integrin expression on neutrophils. The effect of CO on ICAM-1 expression seems to be dependent on soluble guanylate cyclase (sGC) activation, since 1H-(1,2,4)oxadiazolo (4,3-a)quinoxalin-1-one (sGC inhibitor) prevented the observed CO effects. Finally, it was observed that the nitric oxide (NO) anti-inflammatory effects on ICAM-1 expression appear to be indirectly mediated by HO-1 activation, since the inhibition of HO-1 prevented the inhibitory effect of the NO donor (S-nitroso-N-acetylpenicillamine) on LPS-induced ICAM-1 expression. Taken together, these results suggest that CO inhibits ICAM-1 expression on endothelium by a mechanism dependent on sGC activation. Thus, our findings identify the HO-1/CO/guanosine 3`5`-cyclic monophosphate pathway as a potential target for the development of novel pharmacotherapy to control neutrophil migration in inflammatory diseases.

Degeneration of dystrophic or injured skeletal muscles induces high expression of Galectin-1

Muscle degenerative diseases such as Duchenne Muscular Dystrophy are incurable and treatment options are still restrained. Understanding the mechanisms and factors responsible for muscle degeneration and regeneration will facilitate the development of novel therapeutics. Several recent studies have demonstrated that Galectin-1 (Gal-1), a carbohydrate-binding protein, induces myoblast differentiation and fusion in vitro, suggesting a potential role for this mammalian lectin in muscle regenerative processes in vivo. However, the expression and localization of Gal-1 in vivo during muscle injury and repair are unclear. We report the expression and localization of Gal-1 during degenerative-regenerative processes in vivo using two models of muscular dystrophy and muscle injury. Gal-1 expression increased significantly during muscle degeneration in the murine mdx and in the canine Golden Retriever Muscular Dystrophy animal models. Compulsory exercise of mdx mouse, which intensifies degeneration, also resulted in sustained Gal-1 levels. Furthermore, muscle injury of wild-type C57BL/6 mice, induced by BaCl(2) treatment, also resulted in a marked increase in Gal-1 levels. Increased Gal-1 levels appeared to localize both inside and outside the muscle fibers with significant extracellular Gal-1 colocalized with infiltrating CD45(+) leukocytes. By contrast, regenerating muscle tissue showed a marked decrease in Gal-1 to baseline levels. These results demonstrate significant regulation of Gal-1 expression in vivo and suggest a potential role for Gal-1 in muscle homeostasis and repair.

Bone Remodeling Adjacent to Morse Cone-Connection Implants with Platform Switch: A Fluorescence Study in the Dog Mandible

Purpose: The aim of this study was to evaluate, through fluorescence analysis, the effect that different interimplant distances, after prosthetic restoration, will have on bone remodeling in submerged and nonsubmerged implants restored with a ""platform switch."" Materials and Methods: Fifty-six Ankylos implants were placed 1.5 mm subcrestally in seven dogs. The implants were placed so that two fixed prostheses, with three interimplant contacts separated by 1-mm, 2-mm, and 3-mm distances, could be fabricated for each side of the mandible. The sides and the positions of the groups were selected randomly. To better evaluate bone remodeling, calcein green was injected 3 days before placement of the prostheses at 12 weeks postimplantation. At 3 days before sacrifice (8 weeks postloading), alizarin red was injected. The amounts of remodeled bone within the different interimplant areas were compared statistically before and after loading in submerged and nonsubmerged implants. Results: Statistically significant differences existed in the percentage of remodeled bone seen in the different regions. Mean percentages of remodeled bone in the submerged and nonsubmerged groups, respectively, were as follows: for the 1-mm distance, 23.0% +/- 0.05% and 23.1% +/- 0.03% preloading and 27.0% +/- 0.03% and 25.2% +/- 0.04% postloading, for the 2-mm distance, 18.2% +/- 0.05% and 18.1% +/- 0.04% preloading and 21.3% +/- 0.07% and 19.9% +/- 0.03% postloading, for the 3-mm distance, 18.3% +/- 0.03% and 18.3% +/- 0.03% preloading and 18.8% +/- 0.04% and 19.8% +/- 0.04% postloading, for distal-extension regions, 16.6% +/- 0.02% and 17.4% +/- 0.04% preloading and 17.0% +/- 0.04% and 18.4% +/- 0.04% postloading. Conclusions: Based upon this animal study, loading increases bone formation for submerged or nonsubmerged implants, and the interimplant distance of 1 mm appears to result in more pronounced bone remodeling than the 2-mm or 3-mm distances in implants with a ""platform switch."" INT J ORAL MAXILLOFAC IMPLANTS 2009;24:257-266

Are the magnetic fields of millisecond pulsars similar to 10(8) G?

It is generally assumed that the magnetic fields of millisecond pulsars (MSPs) are similar to 10(8) G. We argue that this may not be true and the fields may be appreciably greater. We present six evidences for this: (1) The similar to 10(8)G field estimate is based on magnetic dipole emission losses which is shown to be questionable; (2) The MSPs in low mass X-ray binaries (LMXBs) are claimed to have < 10(11) G on the basis of a Rayleygh-Taylor instability accretion argument. We show that the accretion argument is questionable and the upper limit 10(11) G may be much higher; (3) Low magnetic field neutron stars have difficulty being produced in LMXBs; (4) MSPs may still be accreting indicating a much higher magnetic field; (5) The data that predict similar to 10(8) G for MSPs also predict ages on the order of, and greater than, ten billion years, which is much greater than normal pulsars. If the predicted ages are wrong, most likely the predicted similar to 10(8) G fields of MSPs are wrong; (6) When magnetic fields are measured directly with cyclotron lines in X-ray binaries, fields a parts per thousand << 10(8) G are indicated. Other scenarios should be investigated. One such scenario is the following. Over 85% of MSPs are confirmed members of a binary. It is possible that all MSPs are in large separation binaries having magnetic fields > 10(8) G with their magnetic dipole emission being balanced by low level accretion from their companions.

Lifetime Overproduction of Circulating Angiotensin-(1-7) Attenuates Deoxycorticosterone Acetate-Salt Hypertension-Induced Cardiac Dysfunction and Remodeling

We evaluated the development of arterial hypertension, cardiac function, and collagen deposition, as well as the level of components of the renin-angiotensin system in the heart of transgenic rats that overexpress an angiotensin (Ang)-(1-7)-producing fusion protein, TGR(A1-7)3292 (TG), which induces a lifetime increase in circulating levels of this peptide. After 30 days of the induction of the deoxycorticosterone acetate (DOCA)-salt hypertension model, DOCA-TG rats were hypertensive but presented a lower systolic arterial pressure in comparison with DOCA-Sprague-Dawley (SD) rats. In contrast to DOCA-SD rats that presented left ventricle (LV) hypertrophy and diastolic dysfunction, DOCA-TG rats did not develop cardiac hypertrophy or changes in ventricular function. In addition, DOCA-TG rats showed attenuation in mRNA expression for collagen type I and III compared with the increased levels of DOCA-SD rats. Ang II plasma and LV levels were reduced in SD and TG hypertensive rats in comparison with normotensive animals. DOCA-TG rats presented a reduction in plasma Ang-(1-7) levels; however, there was a great increase in Ang-(1-7) (approximate to 3-fold) accompanied by a decrease in mRNA expression of both angiotensin-converting enzyme and angiotensin-converting enzyme 2 in the LV. The mRNA expression of Mas and Ang II type 1 receptors in the LV was not significantly changed in DOCA-SD or DOCA-TG rats. This study showed that TG rats with increased circulating levels of Ang-(1-7) are protected against cardiac dysfunction and fibrosis and also present an attenuated increase in blood pressure after DOCA-salt hypertension. In addition, DOCA-TG rats showed an important local increase in Ang-(1-7) levels in the LV, which might have contributed to the attenuation of cardiac dysfunction and prefibrotic lesions. (Hypertension. 2010;55:889-896.)

Age-related changes in cerebellar phosphatase-1 reduce Na,K-ATPase activity

We evaluated whether changes in protein content and activity of PP-1 and PP-2A were the mechanism underneath the basal age-related reduction in alpha(2/3)-Na,K-ATPase activity in rats cerebella and whether this occurred through the cyclic GMP-PKG pathway. PP1 activity, but not its expression, increased with age, whereas PP-2 was not changed. The activity Of ot2/3-Na,K-ATPase varied with age. and there was a negative association between the PP-1 and alpha(2/3)-Na,K-ATPase activities. In young rats, the inhibition of PP-1 and PP-2A by okadaic acid (OA) increased in a dose-dependent manner alpha(1)- and alpha(2/3)-Na,K-ATPase, but had no effect on Mg-ATPase activity. A direct stimulation of PKG with 8-Br-cyclic GMP did not surmount the effect of OA. This analogue of cyclic GMP inhibited PP-1 activity only, indicating that at least part of the increase in alpha(1)- and alpha(2/3)-Na,K-ATPase activity induced by OA was mediated by the cyclic GMP-PKG-PP-1 cascade. Taking into account that PP1 inhibition increased alpha(2/3)-Na,K-ATPase activity, we propose that an age-related increase in PP-1 activity due to a decrease in cyclic GMP-PKG modulation plays a role for the age-related reduction of alpha(2/3)-Na,K-ATPase activity in rat cerebellum. (C) 2007 Elsevier Inc. All rights reserved.

PAS-1, an Ascaris suum Protein, Modulates Allergic Airway Inflammation via CD8(+) gamma delta TCR(+) and CD4(+) CD25(+) FoxP3(+) T Cells

We have previously demonstrated that PAS-1, a 200 kDa protein from Ascaris suum, has a potent immunomodulatory effect on humoral and cell-mediated responses induced by APAS-3 (an allergenic protein from A. suum) or unrelated antigens. In this study, we investigated the mechanisms by which PAS-1 is able to induce this effect on an allergic airway inflammation induced by OVA in mice. C57BL/6 mice were adoptively transferred on day 0 with seven different PAS-1-primed cell populations: PAS-1-primed CD19(+) or B220(+) or CD3(+) or CD4(+) or CD8(+) or CD4(+) CD25) or CD4(+) CD25(+) lymphocytes. These mice were immunized twice with OVA and alum by intraperitoneal route (days 0 and 7) and challenged twice by intranasal route (days 14 and 21). Two days after the last challenge, the airway inflammation was evaluated by antibody levels, cellular migration, eosinophil peroxidase levels, cytokine and eotaxin production, and pulmonary mechanical parameters. Among the adoptively transferred primed lymphocytes, only CD4(+) CD25(+), CD8(+) or the combination of both T cells impaired the production of total IgE and OVA-specific IgE and IgG1 antibodies, eosinophilic airway inflammation, Th2-type cytokines (IL-4, IL-5 and IL-13), eotaxin release and airway hyperreactivity. Moreover, airway recruited cells from CD4(+) CD25(+) and CD8(+) T-cell recipient secreted more IL-10/TGF-beta and IFN-gamma, respectively. Moreover, we found that PAS-1 expands significantly the number of CD4(+) CD25(+) FoxP3(+) and CD8(+) gamma delta TCR(+) cells. In conclusion, these findings demonstrate that the immunomodulatory effect of PAS-1 is mediated by these T-cell subsets.

Angiotensin type 1 receptor mediates thyroid hormone-induced cardiomyocyte hypertrophy through the Akt/GSK-3 beta/mTOR signaling pathway

Several studies have implicated the renin angiotensin system in the cardiac hypertrophy induced by thyroid hormone. However, whether Angiotensin type 1 receptor (AT(1)R) is critically required to the development of T(3)-induced cardiomyocyte hypertrophy as well as whether the intracellular mechanisms that are triggered by AT(1)R are able to contribute to this hypertrophy model is unknown. To address these questions, we employed a selective small interfering RNA (siRNA, 50 nM) or an AT(1)R blocker (Losartan, 1 mu M) to evaluate the specific role of this receptor in primary cultures of neonatal cardiomyocytes submitted to T(3) (10 nM) treatment. The cardiomyocytes transfected with the AT(1)R siRNA presented reduced mRNA (90%, P < 0.001) and protein (70%, P < 0.001) expression of AT(1)R. The AT(1)R silencing and the AT(1)R blockade totally prevented the T(3)-induced cardiomyocyte hypertrophy, as evidenced by lower mRNA expression of atrial natriuretic factor (66%, P < 0.01) and skeletal alpha-actin (170%, P < 0.01) as well as by reduction in protein synthesis (85%, P < 0.001). The cardiomyocytes treated with T(3) demonstrated a rapid activation of Akt/GSK-3 beta/mTOR signaling pathway, which was completely inhibited by the use of PI3K inhibitors (LY294002, 10 mu M and Wortmannin, 200 nM). In addition, we demonstrated that the AT(1)R mediated the T(3)-induced activation of Akt/GSK-3 beta/mTOR signaling pathway, since the AT(1)R silencing and the AT(1)R blockade attenuated or totally prevented the activation of this signaling pathway. We also reported that local Angiotensin I/II (Ang I/II) levels (120%, P < 0.05) and the AT(1)R expression (180%, P < 0.05) were rapidly increased by T(3) treatment. These data demonstrate for the first time that the AT(1)R is a critical mediator to the T(3)-induced cardiomyocyte hypertrophy as well as to the activation of Akt/GSK-3 beta/mTOR signaling pathway. These results represent a new insight into the mechanism of T(3)-induced cardiomyocyte hypertrophy, indicating that the Ang I/II-AT(1)R-Akt/GSK-3 beta/mTOR pathway corresponds to a potential mediator of the trophic effect exerted by T(3) in cardiomyocytes.

The Thyroid Hormone Receptor (TR) beta-Selective Agonist GC-1 Inhibits Proliferation But Induces Differentiation and TR beta mRNA Expression in Mouse and Rat Osteoblast-Like Cells

Previous studies showed anabolic effects of GC-1, a triiodothyronine (T3) analogue that is selective for both binding and activation functions of thyroid hormone receptor (TR) beta 1 over TR alpha 1, on bone tissue in vivo. The aim of this study was to investigate the responsiveness of rat (ROS17/2.8) and mouse (MC3T3-E1) osteoblast-like cells to GC-1. As expected, T3 inhibited cellular proliferation and stimulated mRNA expression of osteocalcin or alkaline phosphatase in both cell lineages. Whereas equimolar doses of T3 and GC-1 equally affected these parameters in ROS17/2.8 cells, the effects of GC-1 were more modest compared to those of T3 in MC3T3-E1 cells. Interestingly, we showed that there is higher expression of TR alpha 1 than TR beta 1 mRNA in rat (similar to 20-90%) and mouse (similar to 90-98%) cell lineages and that this difference is even higher in mouse cells, which highlights the importance of TR alpha 1 to bone physiology and may partially explain the modest effects of GC-1 in comparison with T3 in MC3T3-E1 cells. Nevertheless, we showed that TR beta 1 mRNA expression increases (similar to 2.8- to 4.3-fold) as osteoblastic cells undergo maturation, suggesting a key role of TR beta 1 in mediating T3 effects in the bone forming cells, especially in mature osteoblasts. It is noteworthy that T3 and GC-1 induced TR beta 1 mRNA expression to a similar extent in both cell lineages (similar to 2- to 4-fold), indicating that both ligands may modulate the responsiveness of osteoblasts to T3. Taken together, these data show that TR beta selective T3 analogues have the potential to directly induce the differentiation and activity of osteoblasts.

Dielectric Dispersion of Water in the Frequency Range from 10 mHz to 30 MHz

We investigate the dielectric dispersion of water, specially in the low-frequency range, by using the impedance spectroscopy technique. The frequency dependencies of the real R and imaginary Z parts of the impedance Could not be explained by means of the Usual description of the dielectric properties of the water as all insulating liquid containing ions. This is due to the incomplete knowledge of the parameters entering in the fundamental equations describing the evolution of the system, and oil the mechanisms regulating the exchange of charge of the cell with the external circuit. We propose a simple description of our experimental data based on the model of Debye, by invoking a dc conductivity of the cell, related to the nonblocking character of the electrodes. A discussion on the electric Circuits able to simulate the cell under investigation, based oil bulk and Surface elements, is also reported. We find that the simple circuit formed by a series of two parallels of resistance and capacitance is able to reproduce the experimental data concerning the real and imaginary part of the electrical impedance of the cell for frequency larger than 1 Hz. According to this description, one of the parallels takes into account the electrical properties of interface between the electrode and water, and the other of the bulk. For frequency lower than 1 Hz, a good agreement with the experimental data is obtained by simulating the electrical properties of the interface by means of the constant phase element.

Cathepsin X cleaves the beta 2 cytoplasmic tail of LFA-1 inducing the intermediate affinity form of LFA-1 and alpha-actinin-1 binding

The motility of T cells depends on the dynamic spatial regulation of integrin-mediated adhesion and de-adhesion. Cathepsin X, a cysteine protease, has been shown to regulate T-cell migration by interaction with lymphocyte function associated antigen-1 (LFA-1). LFA-1 adhesion to the ICAM-1 is controlled by the association of actin-binding proteins with the cytoplasmic tail of the beta(2) chain of LFA-1. Cleavage by cathepsin X of the amino acid residues S(769), E(768) and A(767) from the C-terminal of the beta(2) cytoplasmic tail of LFA-1 is shown to promote binding of the actin-binding protein alpha-actinin-1. Furthermore, cathepsin X overexpression reduced LFA-1 clustering and induced an intermediate affinity LFA-1 conformation that is known to associate with a-actinin-1. increased levels of intermediate affinity LFA-1 resulted in augmented cell spreading due to reduced attachment of T cells to the ICAM-1-coated surface. Gradual cleavage of LFA-1 by cathepsin X enables the transition between intermediate and high affinity LFA-1, an event that is crucial for effective T-cell migration.

1,4-Dioxane enhances properties and biocompatibility of polyanionic collagen for tissue engineering applications

Polyanionic collagen obtained from bovine pericardial tissue submitted to alkaline hydrolysis is an acellular matrix with strong potential in tissue engineering. However, increasing the carboxyl content reduces fibril formation and thermal stability compared to the native tissues. In the present work, we propose a chemical protocol based on the association of alkaline hydrolysis with 1,4-dioxane treatment to either attenuate or revert the drastic structural modifications promoted by alkaline treatments. For the characterization of the polyanionic membranes treated with 1,4-dioxane, we found that (1) scanning electron microscopy (SEM) shows a stronger reorientation and aggregation of collagen microfibrils; (2) histological evaluation reveals recovering of the alignment of collagen fibers and reassociation with elastic fibers; (3) differential scanning calorimetry (DSC) shows an increase in thermal stability; and (4) in biocompatibility assays there is a normal attachment, morphology and proliferation associated with high survival of the mouse fibroblast cell line NIH3T3 in reconstituted membranes, which behave as native membranes. Our conclusions reinforce the ability of 1,4-dioxane to enhance the properties of negatively charged polyanionic collagen associated with its potential use as biomaterials for grafting, cationic drug- or cell-delivery systems and for the coating of cardiovascular devices.

Deactivation of Ferrylmyoglobin by Vanillin as Affected by Vanillin Binding to beta-Lactoglobulin

Vanillin was found to be efficient as a deactivator of ferrylmyoglobin with a second-order rate constant of k(2) = S7 +/- 1 L mol(-1) s(-1) for reduction to metmyoglobin with Delta H(double dagger) = 58.3 +/- 0.3 kJ mol(-1) and Delta S(double dagger) = -14 +/- 1 J mol(-1) K(-1) in aqueous pH 7.4 solution at 25 degrees C. Binding to beta-lactoglobulin (AG) was found to affect the reactivity of vanillin at 25 degrees C only slightly to k(2) = 48 +/- 2 L mol(-1) s(-1) (Delta H(double dagger) = 68.4 +/- 0.4 kJ mol(-1) and Delta S(double dagger) = 17 +/- 1 J mol(-1) K(-1)) for deactivation of ferrylmyoglobin. Binding of vanillin to beta LG was found to have a binding stoichiometry vanillin/beta LG > 10 with K(A) = 6 x 10(2) L mol(-1) and an apparent total Delta H degrees of approximately -38 kJ mol(-1) and Delta S degrees = -S5.4 +/- 4J mol(-1) K(-1) at 25 degrees C and Delta C(p), (obs) = -1.02 kJ mol(-1) K(-1) indicative of increasing ordering in the complex, as determined by isothermal titration microcalorimetry. From tryptophan fluorescence quenching for beta LG by vanillin, approximately one vanillin was found to bind to each beta LG far stronger with K(A) = 5 x 10(4) L, mol(-1) and a Delta H degrees = 10.2 kJ mol(-1) and Delta S degrees = 55J mol(-1) K(-1) at 25 degrees C. The kinetic entropy/enthalpy compensation effect seen for vanillin reactivity by binding to beta LG is concluded to relate to the weakly bound vanillin oriented through hydrogen bonds on the beta LG surface with the phenolic group pointing toward the solvent, in effect making both Delta H(double dagger) and Delta S(double dagger) more positive. The more strongly bound vanillin capable of tryptophan quenching in the fiLG calyx seems less or nonreactive.