TLR2-dependent mast cell activation contributes to the control of Mycobacterium tuberculosis infection

Mast Cells (MCs) express toll-like receptor 2 (TLR2), a receptor known to be triggered by several major mycobacterial ligands and involved in resistance against Mycobacterium tuberculosis (MTB) infection. This study investigated whether adoptive transfer of TLR2 positive MCs (TLR2(+/+)) corrects the increased susceptibility of TLR2(-/-) mice to MTB infection. TLR2(-/-) mice displayed increased mycobacterial burden, diminished myeloid cell recruitment and proinflammatory cytokine production accompanied by defective granuloma formation. The reconstitution of these mice with TLR2(+/+) MCs, but not TLR2(-/-), confers better control of the infection, promotes the normalization of myeloid cell recruitment associated with reestablishment of the granuloma formation. In addition, adoptive transfer of TLR2(+/+) MC to TLR2(-/-) mice resulted in regulation of the pulmonary levels of IL-beta, IL-6, TNF-alpha, enhanced Th1 response and activated CD8(+) T cell homing to the lungs. Our results suggest that activation of MCs via TLR2 is required to compensate the defect in protective immunity and inability of TLR2(-/-) mice to control MTB infection. (C) 2009 Elsevier Masson SAS. All rights reserved.

Suramin inhibits macrophage activation by human group IIA phospholipase A(2), but does not affect bactericidal activity of the enzyme

Suramin is a polysulphonated napthylurea antiprotozoal and anthelminitic drug, which also presents inhibitory activity against a broad range of enzymes. Here we evaluate the effect of suramin on the hydrolytic and biological activities of secreted human group IIA phospholipase A(2) (hsPLA(2)GIIA). The hsPLA(2)GIIA was expressed in E. coli, and refolded from inclusion bodies. The hydrolytic activity of the recombinant enzyme was measured using mixed dioleoylphosphatidylcholine/dioleoylphosphatidylglycerol (DOPC/DOPG) liposomes. The activation of macrophage cell line RAW 264.7 by hsPLA(2) GIIA was monitored by NO release, and bactericidal activity against Micrococcus luteus was evaluated by colony counting and by flow cytometry using the fluorescent probe Sytox Green. The hydrolytic activity of the hsPLA(2) GIIA was inhibited by a concentration of 100 nM suramin and the activation of macrophages by hsPLA(2) GIIA was abolished at protein/suramin molar ratios where the hydrolytic activity of the enzyme was inhibited. In contrast, both the bactericidal activity of hsPLA(2) GIIA against Micrococcus luteus and permeabilization of the bacterial inner membrane were unaffected by suramin concentrations up to 50 mu M. These results demonstrate that suramin selectively inhibits the activity of the hsPLA(2) GIIA against macrophages, whilst leaving the anti-bacterial function unchanged.

Chronic Treatment with Quercetin does not Inhibit Angiotensin-Converting Enzyme In Vivo or In Vitro

The precise mechanisms explaining the anti-hypertensive effects produced by quercetin are not fully known. Here, we tested the hypothesis that chronic quercetin treatment inhibits the angiotensin-converting enzyme (ACE). We examined whether quercetin treatment for 14 days reduces in vivo responses to angiotensin I or enhances the responses to bradykinin in anaesthetised rats. We measured the changes in systemic arterial pressure induced by angiotensin I in doses of 0.03-10 mu g/kg, by angiotensin II in doses of 0.01-3 mu g/kg, and to bradykinin in doses of 0.03-10 mu g/kg in anaesthetised rats pre-treated with vehicle (controls), or daily quercetin 10 mg/kg intraperitoneally for 14 days, or a single i.v. dose of captopril 2 mg/kg. Plasma ACE activity was determined by a fluorometric method. Plasma quercetin concentrations were assessed by high performance liquid chromatography. Quercetin treatment induced no significant changes in the hypertensive responses to angiotensin I and angiotensin II, as well in the hypotensive responses to bradykinin (all p > 0.05). Conversely, as expected, a single dose of captopril inhibited the hypertensive responses to angiotensin I and potentiated the bradykinin responses (all p < 0.01), while no change was found in the vascular responses to angiotensin II (all p > 0.05). In addition, although we found significant amounts of quercetin in plasma samples (mean = 206 ng/mL), no significant differences were found in plasma ACE activity in rats treated with quercetin compared with those found in the control group (50 +/- 6 his-leu nmol/min/mL and 40 +/- 7 his-leu nmol/min/mL, respectively; p > 0.05). These findings provide strong evidence indicating that quercetin does not inhibit ACE in vivo or in vitro and indicate that other mechanisms are probably involved in the antihypertensive and protective cardiovascular effects associated with quercetin.

Enzymatic and structural characterization of a basic phospholipase A(2) from the sea anemone Condylactis gigantea

This work aimed at the isolation and structural/functional characterization of a phospholipase A(2) (CgPLA(2)) from the extract of the anemone Condylactis gigantea. CgPLA2 was isolated with a high purity level through three chromatographic steps, showing pT8.6 and molecular weights of 14,500 and 29,000 for the monomer and dimer, respectively. CgPLA2 showed a high catalytic activity upon fluorescent phospholipids inducing no direct hemolytic activity. This enzyme, which is Ca2+-dependent, showed a lower stability against temperature and pH variations when compared with snake venom enzymes. The enzymatic activity was significantly reduced or completely abolished after chemical modification of CgPLA2 with BPB. Its cDNA was then obtained, with 357 base pairs which codified for a mature protein of 119 amino acid residues. A comparative analysis of the primary structure of CgPLA2 revealed 84%, 61%, 43% and 42% similarity to the PLA2s from Adamsia carciniopados, Nematostella vectensis, Vipera russelli russelli and Both raps jararacussu, respectively. (C) 2010 Elsevier Masson SAS. All rights reserved.

Effects of temperature-dependent viscosity variation on entropy generation, heat and fluid flow through a porous-saturated duct of rectangular cross-section

Effect of temperature-dependent viscosity on fully developed forced convection in a duct of rectangular cross-section occupied by a fluid-saturated porous medium is investigated analytically. The Darcy flow model is applied and the viscosity-temperature relation is assumed to be an inverse-linear one. The case of uniform heat flux on the walls, i.e. the H boundary condition in the terminology of Kays and Crawford, is treated. For the case of a fluid whose viscosity decreases with temperature, it is found that the effect of the variation is to increase the Nusselt number for heated walls. Having found the velocity and the temperature distribution, the second law of thermodynamics is invoked to find the local and average entropy generation rate. Expressions for the entropy generation rate, the Bejan number, the heat transfer irreversibility, and the fluid flow irreversibility are presented in terms of the Brinkman number, the Péclet number, the viscosity variation number, the dimensionless wall heat flux, and the aspect ratio (width to height ratio). These expressions let a parametric study of the problem based on which it is observed that the entropy generated due to flow in a duct of square cross-section is more than those of rectangular counterparts while increasing the aspect ratio decreases the entropy generation rate similar to what previously reported for the clear flow case.

Enzyme deactivation in fixed bed reactors with michaelis-menten kinetics

Rate expression for enzyme poisoning which are consistent with a Michaelis-Menten main reaction are used to analyze the performance of a fixed bed reactor containing immobilized enzyme. When enzyme deactivation results from the irreversible bonding of a product molecule to an existing substrate-enzyme complex, it is shown that minimum enzyme activity can occur in the interior of the bed, well away from the ends. This suggests that bed sectioning techniques may enable direct evaluation of fundamental poisoning mechanisms.

Enzyme immobilization in porous solid supports - penetration of immobilized enzyme

The process of enzyme immobilization under the diffusion-controlled regime (i.e., fast attachment of enzyme compared to its diffusion) is modeled and theoretically solved in this article. Simple and compact solutions for the penetration depth of immobilized enzyme and the bulk enzyme concentration versus time are presented. Furthermore, the conditions for the validity of our solutions are also given in this article so that researchers can discover when the theoretical solutions can be applied to their systems.

Strategies for identifying and predicting islet autoantigen T-cell epitopes in insulin-dependent diabetes mellitus

T cells recognize peptide epitopes bound to major histocompatibility complex molecules. Human T-cell epitopes have diagnostic and therapeutic applications in autoimmune diseases. However, their accurate definition within an autoantigen by T-cell bioassay, usually proliferation, involves many costly peptides and a large amount of blood, We have therefore developed a strategy to predict T-cell epitopes and applied it to tyrosine phosphatase IA-2, an autoantigen in IDDM, and HLA-DR4(*0401). First, the binding of synthetic overlapping peptides encompassing IA-2 was measured directly to purified DR4. Secondly, a large amount of HLA-DR4 binding data were analysed by alignment using a genetic algorithm and were used to train an artificial neural network to predict the affinity of binding. This bioinformatic prediction method was then validated experimentally and used to predict DR4 binding peptides in IA-2. The binding set encompassed 85% of experimentally determined T-cell epitopes. Both the experimental and bioinformatic methods had high negative predictive values, 92% and 95%, indicating that this strategy of combining experimental results with computer modelling should lead to a significant reduction in the amount of blood and the number of peptides required to define T-cell epitopes in humans.

The critical power function is dependent on the duration of the predictive exercise tests chosen

The linear relationship between work accomplished (W-lim) and time to exhaustion (t(lim)) can be described by the equation: W-lim = a + CP.t(lim). Critical power (CP) is the slope of this line and is thought to represent a maximum rate of ATP synthesis without exhaustion, presumably an inherent characteristic of the aerobic energy system. The present investigation determined whether the choice of predictive tests would elicit significant differences in the estimated CP. Ten female physical education students completed, in random order and on consecutive days, five art-out predictive tests at preselected constant-power outputs. Predictive tests were performed on an electrically-braked cycle ergometer and power loadings were individually chosen so as to induce fatigue within approximately 1-10 mins. CP was derived by fitting the linear W-lim-t(lim) regression and calculated three ways: 1) using the first, third and fifth W-lim-t(lim) coordinates (I-135), 2) using coordinates from the three highest power outputs (I-123; mean t(lim) = 68-193 s) and 3) using coordinates from the lowest power outputs (I-345; mean t(lim) = 193-485 s). Repeated measures ANOVA revealed that CPI123 (201.0 +/- 37.9W) > CPI135 (176.1 +/- 27.6W) > CPI345 (164.0 +/- 22.8W) (P < 0.05). When the three sets of data were used to fit the hyperbolic Power-t(lim) regression, statistically significant differences between each CP were also found (P < 0.05). The shorter the predictive trials, the greater the slope of the W-lim-t(lim) regression; possibly because of the greater influence of 'aerobic inertia' on these trials. This may explain why CP has failed to represent a maximal, sustainable work rate. The present findings suggest that if CP is to represent the highest power output that an individual can maintain for a very long time without fatigue then CP should be calculated over a range of predictive tests in which the influence of aerobic inertia is minimised.

Morphine has a dual concentration-dependent effect on K+-evoked substance P release from rat peripheral airways

Our previous investigations of possible lung mechanisms underlying the effectiveness of nebulized morphine for the relief of dyspnoea, have shown a high density of non-conventional opioid binding sites in rat airways with similar binding characteristics (opioid alkaloid-sensitive, opioid peptide-insensitive) to that of putative mu(3)-opioid receptors on immune cells. To investigate whether these lung opioid binding sites are functional receptors, this study was designed to determine (using superfusion) whether morphine modulates the K+-evoked release of the pro-inflammatory neuropeptide, substance P (SP), from rat peripheral airways. Importantly, K+-evoked SP release was Ca2+-dependent, consistent with vesicular release. Submicromolar concentrations of morphine (1 and 200 nM) inhibited K+-evoked SP release from rat peripheral airways in a naloxone (1 mu M) reversible manner. By contrast, 1 mu M morphine enhanced K+-evoked SP release and this effect was not reversed by 1 mu M naloxone. However, 100 mu M naloxone not only antagonized the facilitatory effect of 1 mu M morphine on K+-evoked SP release from rat peripheral airways but it inhibited release to a similar extent as 200 nM morphine. It is possible that these latter effects are mediated by non-conventional opioid receptors located on mast cells, activation of which causes naloxone-reversible histamine release that in turn augments the release of SP from sensory nerve terminals in the peripheral airways. Clearly, further studies are required to investigate this possibility. (C) 1997 Academic Press Limited.

Effect of estrogen on vascular smooth muscle cells is dependent upon cellular phenotype

To investigate the growth-regulating action of estrogen on vascular smooth muscle cells (SMC), effects of beta-17-estradiol (beta-E-2) on phenotypic modulation and proliferation of rabbit aortic SMC were observed in vitro. At 10(-8) M, beta-E-2 significantly slowed the decrease in volume fraction of myofilaments (V(v)myo) of freshly dispersed SMCs in primary culture, indicating an inhibitory effect of beta-E-2 On spontaneous phenotypic modulation of SMC from a contractile to a synthetic phenotype. Freshly dispersed SMCs treated with beta-E-2 also had a relatively longer quiescent phase than control cells before intense proliferation occurred. This was in contrast to SMCs in passage 2-3 (synthetic state), where beta-E-2-treated cells replicated significantly faster than untreated cells. beta-E-2 also markedly enhanced the serum-induced DNA synthesis of synthetic SMCs in a concentration-dependent manner within physiological range (10(-10) to 10-8 M). These findings indicate that the growth-regulating effect of estrogen on vascular SMC is dependent on the cell's phenotypic stare. It delays the cell cycle re-entry of the contractile SMCs by retarding their phenotypic modulation however, once cells have modulated to the synthetic phenotype, it promotes their replication. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.

Chronic ethanol treatment leads to increased ornithine decarboxylase activity: Implications for a role of polyamines in ethanol dependence and withdrawal

Recent research has focused on the N-methyl-D-aspartate receptor system as a major site of ethanol action in the brain and specifically on compensatory changes in the expression of the polyamine-sensitive NR2B subunit. Therefore, we examined the effects of chronic ethanol treatment on polyamine homeostasis in the rat brain. Wistar rats were made dependent by ethanol vapor inhalation. This caused a rise in hippocampal ornithine decarboxylase (ODC) activity that was correlated with the appearance of physiological dependence. ODC activity returned to control levels within 3 days of ethanol withdrawal. Enzyme activity also increased in the cerebral cortex, striatum, and cerebellum of the ethanol-dependent rats. The concentration of the polyamines (putrescine, spermidine, and spermine) in the hippocampus was increased in ethanol-dependent rats. Injection of the ODC inhibitor, gamma-difluoromethylornithine (500 mg/kg) at the onset of withdrawal resulted in a significant reduction in the severity of withdrawal behaviors. The level of ODC activity and the severity of withdrawal behaviors were positively correlated. Perturbed polyamine homeostasis may represent an important molecular component in the initiation of ethanol withdrawal behaviors in the ethanol-dependent rat.

Incoherent interlayer transport and angular-dependent magnetoresistance oscillations in layered metals

The effect of incoherent interlayer transport on the interlayer resistance of a layered metal is considered. We find that for both quasi-one-dimensional and quasi-two-dimensional Fermi liquids the angular dependence of the magnetoresistance is essentially the same for coherent and incoherent transport. Consequently, the existence of a three-dimensional Fermi surface is not necessary to explain the oscillations in the magnetoresistance that are seen in many organic conductors as the field direction is varied. [S0031-9007(98)07660-1].

A pore-size-dependent equation of state for multilayer adsorption in cylindrical mesopores

The chemical potential of adsorbed film inside cylindrical mesopores is dependent on the attractive interactions between the adsorbed molecules and adsorbent, the curvature of gas/adsorbed phase interface, and surface tension. A state equation of the adsorbed film is proposed to take into account the above factors. Nitrogen adsorption on model adsorbents, MCM-41, which exhibit uniform cylindrical channels, are used to verify the theoretical analysis. The proposed theory is capable of describing the important features of adsorption processes in cylindrical mesopores. According to this theory, at a given relative pressure, the smaller the pore radius is, the thicker the adsorbed film will be. The thickening of adsorbed films in the pores as the vapor pressure increases inevitably causes an increase in the interface curvature, which consequently leads to capillary condensation. Besides, this study confirmed that the interface tension depends substantially on the interface curvature in small mesopores. A quantitative relationship between the condensation pressure and the pore radius can be derived from the state equation and used to predict the pore radius from a condensation pressure, or vice versa.