Effect of oleic and linoleic acids on the inflammatory phase of wound healing in rats

Inflammation is a crucial step for the wound healing process. The effect of linoleic and oleic acids on the inflammatory response of the skin during the healing process and on the release of pro-inflammatory cytokines by rat neutrophils in vitro was investigated. A wound in the dorsal surface of adult rats was performed and fatty acids were then topically administered. Both oleic and linoleic acids increased the wound healing tissue mass. The total protein and DNA contents of the wounds were increased by the treatment with linoleic acid. The treatments with oleic and linoleic acids did not affect vascular permeability. However, the number of neutrophils in the wounded area and air pouches was increased and the thickness of the necrotic cell layer edge around the wound was decreased. A dose-dependent increase in vascular endothelial growth factor-alpha (VEGF-alpha) and interleukin-1 beta (IL-1 beta) by neutrophils incubated in the presence of oleic and linoleic acid was observed. Oleic acid was able to stimulate also the production of cytokine-induced neutrophil chemoattractant in inflammation 2 alphalbeta (CINC-2 alpha/beta). This pro-inflammatory effect of oleic and linoleic acids may speed up the wound healing process. Copyright (c) 2007 John Wiley & Sons, Ltd.

Moderate exercise improves leucocyte function and decreases inflammation in diabetes

P>The genesis and progression of diabetes occur due in part to an uncontrolled inflammation profile with insulin resistance, increased serum levels of free fatty acids (FFA), proinflammatory cytokines and leucocyte dysfunction. In this study, an investigation was made of the effect of a 3-week moderate exercise regimen on a treadmill (60% of VO(2max), 30 min/day, 6 days a week) on inflammatory markers and leucocyte functions in diabetic rats. The exercise decreased serum levels of tumour necrosis factor (TNF)-alpha (6%), cytokine-induced neutrophil chemotactic factor 2 alpha/beta (CINC-2 alpha/beta) (9%), interleukin (IL)-1 beta (34%), IL-6 (86%), C-reactive protein (CRP) (41%) and FFA (40%) in diabetic rats when compared with sedentary diabetic animals. Exercise also attenuated the increased responsiveness of leucocytes from diabetics when compared to controls, diminishing the reactive oxygen species (ROS) release by neutrophils (21%) and macrophages (28%). Exercise did not change neutrophil migration and the proportion of neutrophils and macrophages in necrosis (loss of plasma membrane integrity) and apoptosis (DNA fragmentation). Serum activities of creatine kinase (CK) and lactate dehydrogenase (LDH) were not modified in the conditions studied. Therefore, physical training did not alter the integrity of muscle cells. We conclude that moderate physical exercise has marked anti-inflammatory effects on diabetic rats. This may be an efficient strategy to protect diabetics against microorganism infection, insulin resistance and vascular complications.

beta-ADRENERGIC ACTIVITY PRESERVES GLUT4 PROTEIN IN GLYCOLYTIC FIBERS IN FASTING

Glucose transporter 4 (GLUT4) expression in adipose tissue decreases during fasting. In skeletal muscle, we hypothesized that GLUT4 expression might be maintained in a beta-adrenergic-dependent way to ensure energy disposal for contractile function. Herein we investigate beta-blockade or beta-stimulation effects on GLUT4 expression in oxidative (soleus) and glycolytic [extensor digitorum longus (EDL)] muscles of fasted rats. Fasting increased GLUT4 mRNA in soleus (24%) and EDL (40%) but the protein content increased only in soleus (30%). beta 1-beta 2-, and beta 1-beta 2-beta 3-blockade decreased (20-30%) GLUT4 mRNA content in both muscles, although GLUT4 protein decreased only in EDL. When mRNA and GLUT4 protein regulations were discrepant, changes in the mRNA poly(A) tail length were detected, indicating a posttranscriptional modulation of gene expression. These results show that beta-adrenergic activity regulates GLUT4 gene expression in skeletal muscle during fasting, highlighting its participation in preservation of GLUT4 protein in glycolytic muscle. Muscle Nerve 40: 847-854, 2009

Short-chain fatty acids stimulate the migration of neutrophils to inflammatory sites

SCFAs (short-chain fatty acids) are produced by anaerobic bacterial fermentation. Increased concentrations of these fatty acids are observed in inflammatory conditions, such as periodontal disease, and at sites of anaerobic infection. In the present study, the effect of the SCFAs acetate, propionate and butyrate on neutrophil chemotaxis and migration was investigated. Experiments were carried out in rats and in vitro. The following parameters were measured: rolling, adherence, expression of adhesion molecules in neutrophils (L-selectin and beta 2 integrin), transmigration, air pouch influx of neutrophils and production of cytokines [CINC-2 alpha beta (cytokine-induced neutrophil chemoattractant-2 alpha beta), IL-1 beta (interleukin-1 beta), MIP-1 alpha (macrophage inflammatory protein-1 alpha) and TNF-alpha (tumour necrosis factor-alpha)]. SCFAs induced in vivo neutrophil migration and increased the release of CINC-2 alpha beta into the air pouch. These fatty acids increased the number of rolling and adhered cells as evaluated by intravital microscopy. SCFA treatment increased L-selectin expression on the neutrophil surface and L-selectin mRNA levels, but had no effect on the expression of beta 2 integrin. Propionate and butyrate also increased in vitro transmigration of neutrophils. These results indicate that SCFAs produced by anaerobic bacteria raise neutrophil migration through increased L-selectin expression on neutrophils and CINC-2 alpha beta release.

Bj-PRO-5a, a natural angiotensin-converting enzyme inhibitor, promotes vasodilatation mediated by both bradykinin B(2) and M1 muscarinic acetylcholine receptors

Bradykinin-potentiating peptides (BPPs) or proline-rich oligopeptides (PROs) isolated from the venom glands of Bothrops jararaca (Bj) were the first natural inhibitors of the angiotensin-converting enzyme (ACE) described. Bj-PRO-5a (< EKWAP), a member of this structurally related peptide family, was essential for the development of captopril, the first site-directed ACE inhibitor used for the treatment of human hypertension. Nowadays, more Bj-PROs have been identified with higher ACE inhibition potency compared to Bj-PRO-5a. However, despite its modest inhibitory effect of ACE inhibition, Bj-PRO-5a reveals strong bradykinin-potentiating activity, suggesting the participation of other mechanisms for this peptide. In the present study, we have shown that Bj-PRO-5a induced nitric oxide (NO) production depended on muscarinic acetylcholine receptor M1 subtype (mAchR-M1) and bradykinin B(2) receptor activation, as measured by a chemiluminescence assay using a NO analyzer. Intravital microscopy based on transillumination of mice cremaster muscle also showed that both bradykinin B(2) receptor and mAchR-M1 contributed to the vasodilatation induced by Bj-PRO-5a. Moreover, Bj-PRO-5a-mediated vasodilatation was completely blocked in the presence of a NO synthase inhibitor. The importance of this work lies in the definition of novel targets for Bj-PRO-5a in addition to ACE, the structural model for captopril development. (C) 2011 Elsevier Inc. All rights reserved.

Involvement of sensory nerves and TRPV1 receptors in the rat airway inflammatory response to two environment pollutants: diesel exhaust particles (DEP) and 1,2-naphthoquinone (1,2-NQ)

The environmental chemical 1,2-naphthoquinone (1,2-NQ) is implicated in the exacerbation of airways diseases induced by exposure to diesel exhaust particles (DEP), which involves a neurogenic-mediated mechanism. Plasma extravasation in trachea, main bronchus and lung was measured as the local (125)I-bovine albumin accumulation. RT-PCR quantification of TRPV1 and tachykinin (NK(1) and NK(2)) receptor gene expression were investigated in main bronchus. Intratracheal injection of DEP (1 and 5 mg/kg) or 1,2-NQ (35 and 100 nmol/kg) caused oedema in trachea and bronchus. 1,2-NQ markedly increased the DEP-induced responses in the rat airways in an additive rather than synergistic manner. This effect that was significantly reduced by L-732,138, an NK(1) receptor antagonist, and in a lesser extent by SR48968, an NK(2) antagonist. Neonatal capsaicin treatment also markedly reduced DEP and 1,2-NQ-induced oedema. Exposure to pollutants increased the TRPV1, NK(1) and NK(2) receptors gene expression in bronchus, an effect was partially suppressed by capsaicin treatment. In conclusion, our results are consistent with the hypothesis that DEP-induced airways oedema is highly influenced by increased ambient levels of 1,2-NQ and takes place by neurogenic mechanisms involving up-regulation of TRPV1 and tachykinin receptors.

Altered reactivity of gastric fundus smooth muscle in the mouse with targeted disruption of the kinin B(1) receptor gene

Relaxing action of sodium nitroprusside (SNP) was significantly reduced in the stomach fundus of mice lacking the kinin B(1) receptor (B(1)(-/-)). Increased basal cGMP accumulation was correlated with attenuated SNP induced dose-dependent relaxation in B(1)(-/-) when compared with wild type (WT) control mice. These responses to SNP were completely blocked by the guanylate cyclase inhibitor ODQ(10 mu M). It was also found that Ca(2+)-dependent, constitutive nitric oxide synthase (cNOS) activity was unchanged but the Ca(2+)-independent inducible NOS (iNOS) activity was greater in B(1)(-/-) mice than in WT animals. Zaprinast (100 mu M), a specific phosphodiesterase inhibitor, increased the nitrergic relaxations and the accumulation of the basal as well as the SNP-stimulated cGMP in WT but not in B(1)(-/-) stomach fundus. From these findings it is concluded that the inhibited phosphodiesterase activity and high level of cGMP reduced the resting muscle tone, impairing the relaxant responses of the stomach in B(1)(-/-) mice. In addition, it can be suggested that functional B(2) receptor might be involved in the NO compensatory mechanism associated with the deficiency of kinin B(1) receptor in the gastric tissue of the transgenic mice. (C) 2009 Elsevier Inc. All rights reserved.

Deiodinase-mediated thyroid hormone inactivation minimizes thyroid hormone signaling in the early development of fetal skeleton

Thyroid hormone (TH) plays a key role on post-natal bone development and metabolism, while its relevance during fetal bone development is uncertain. To Study this, pregnant once were made hypothyroid and fetuses harvested at embryonic days (E) 12.5, 14.5, 16.5 and 18.5. Despite a marked reduction in fetal tissue concentration of both T4 and T3, bone development, as assessed at the distal epiphyseal growth plate of the femur and vertebra, was largely preserved Lip to E16.5. Only at E18.5, the hypothyroid fetuses exhibited a reduction in femoral type I and type X collagen and osteocalcin mRNA levels, in the length and area of the proliferative and hypertrophic zones, in the number of chondrocytes per proliferative column, and in the number of hypertrophic chondrocyres, in addition to a slight delay in endochondral and intramembranous ossification. This Suggests that LIP to E 16.5, thyroid hormone signaling in bone is kept to a minimum. In fact, measuring the expression level of the activating and inactivating iodothyronine deiodinases (D2 and D3) helped understand how this is achieved. D3 mRNA was readily detected as early as E14.5 and its expression decreased markedly (similar to 10-fold) at E18.5, and even more at 14 days after birth (P14). In contrast. D2 mRNA expression increased significantly by E18.5 and markedly (similar to 2.5-fold) by P14. The reciprocal expression levels of D2 and D3 genes during early bone development along with the absence of a hypothyroidism-induced bone phenotype at this time Suggest that coordinated reciprocal deiodinase expression keeps thyroid hormone signaling in bone to very low levels at this early stage of bone development. (c) 2008 Elsevier Inc. All rights reserved.

Expansion of CD4(+) CD25(+) Foxp3(+) T cells by bone marrow-derived dendritic cells

Dendritic cells (DCs) are the most important antigen-presenting cells of the immune system and have a crucial role in T-lymphocyte activation and adaptive immunity initiation. However, DCs have also been implicated in maintaining immunological tolerance. In this study, we evaluated changes in the CD4(+) CD25(+) Foxp3(+) T-cell population after co-culture of lymph node cells from BALB/c mice with syngeneic bone marrow-derived DCs. Our results showed an increase in CD4(+) CD25(+) Foxp3(+) T cells after co-culture which occurred regardless of the activation state of DCs and the presence of allogeneic apoptotic cells; however, it was greater when DCs were immature and were pulsed with the alloantigen. Interestingly, syngeneic apoptotic thymocytes were not as efficient as allogeneic apoptotic cells in expanding the CD4(+) CD25(+) Foxp3(+) T-cell population. In all experimental settings, DCs produced high amounts of transforming growth factor (TGF)-beta. The presence of allogeneic apoptotic cells induced interleukin (IL)-2 production in immature and mature DC cultures. This cytokine was also detected in the supernatants under all experimental conditions and enhanced when immature DCs were pulsed with the alloantigen. CD4(+) CD25(+) Foxp3(+) T-cell expansion during co-culture of lymph node cells with DCs strongly suggested that the presence of alloantigen enhanced the number of regulatory T cells (Tregs) in vitro. Our data also suggest a role for both TGF-beta and IL-2 in the augmentation of the CD4(+) CD25(+) Foxp3(+) population.

Crystal structure of the IL-22/IL-22R1 complex and its implications for the IL-22 signaling mechanism

Interleukin-22 (IL-22) is a member of the interleukin-10 cytokine family, which is involved in anti-microbial defenses, tissue damage protection and repair, and acute phase responses. Its signaling mechanism involves the sequential binding of IL-22 to interleukin-22 receptor 1 (IL-22R1), and of this dimer to interleukin-10 receptor 2 (IL-10R2) extracellular domain. We report a 1.9 A crystal structure of the IL-22/IL-22R1 complex, revealing crucial interacting residues at the IL-22/IL-22R1 interface. Functional importance of key residues was confirmed by site-directed mutagenesis and functional studies. Based on the X-ray structure of the binary complex, we discuss a molecular basis of the IL-22/IL-22R1 recognition by IL-10R2.

The binding of synthetic triiodo L-thyronine analogs to human transthyretin: Molecular basis of cooperative and non-cooperative ligand recognition

Transthyretin (TTR) is a tetrameric beta-sheet-rich transporter protein directly involved in human amyloid diseases. Several classes of small molecules can bind to TTR delaying its amyloid fibril formation, thus being promising drug candidates to treat TTR amyloidoses. In the present study, we characterized the interactions of the synthetic triiodo L-thyronine analogs and thyroid hormone nuclear receptor TR beta-selecfive agonists GC-1 and GC-24 with the wild type and V30M variant of human transthyretin (TTR). To achieve this aim, we conducted in vitro TTR acid-mediated aggregation and isothermal titration calorimetry experiments and determined the TTR:GC-1 and TTR:GC-24 crystal structures. Our data indicate that both GC-1 and GC-24 bind to TTR in a non-cooperative manner and are good inhibitors of TTR aggregation, with dissociation constants for both hormone binding sites (HBS) in the low micromolar range. Analysis of the crystal structures of TTRwt:GC-1(24) complexes and their comparison with the TTRwt X-ray structure bound to its natural ligand thyroxine (T4) suggests, at the molecular level, the basis for the cooperative process displayed by T4 and the non-cooperative process provoked by both GC-1 and GC-24 during binding to TTR. (C) 2010 Elsevier Inc. All rights reserved.

Crystal structure of the (Mg,Fe)[UO2(P,As)O-4](2) center dot 10H(2)O solid solution - A novel mineral variety of saleeite

The crystal structure of a novel variety {[(Mg0.81Fe0.19)(H2O)(6)](H2O)(4)}{(UO2)[(P0.67As0.33)O-4]}(2) of the mineral saleeite is determined using X-ray diffraction (Bruker Smart diffractometer, lambda MoK alpha, graphite monochromator, 2 theta(max) = 56.62 degrees, R = 0.0321 for 2317 reflections, T = 100 K). The main crystal data are as follows: a = 6.952(6) angstrom, b = 19.865(5) , angstrom, c = 6.969(2) angstrom, beta = 90.806(4)degrees, space group P12(l)/n1, Z = 2, and P-calcd = 3.34 g/cm(3). It is shown that the structure is formed by alternating (along the [010] direction) anionic layers, which are composed of uranium bipyramids and T(P,As) tetrahedra, and cation layers consisting of M(Mg, Fe) octahedra and water molecules, which are joined through a system of asymmetric hydrogen bonds. The hydrogen atoms are located, the scheme of hydrogen bonds is established, and their geometric characteristics are calculated.

COMPOSITION OF SUPERCRITICAL CARBON DIOXIDE EXTRACTS OF PITANGA (EUGENIA UNIFLORA L.) LEAVES

The leaves of the Pitanga bush (Eugenia uniflora L.) are considered to be effective against many diseases. Extracts from Pitanga leaves have been found to show pronounced anti-inflammatory action and to have antimicrobial and antifungal activities, among other properties. In this work, extracts from Pitanga leaves were obtained by hydrodistillation and by extraction with supercritical carbon dioxide (SC-CO(2)) at three conditions of temperature and pressure. In the SC-CO(2) extractions also were collected the components that are lost with the CO(2) in the exit of the system using Porapak-Q polymer trap. All extracts were analyzed by gas chromatography-mass spectrometry (GC-MS). Thirty-nine compounds were found in the extracts and twenty-six were identified. The main components identified in the extracts in decreasing quantitative order were: curzerene, germacrene B, C(15)H(20)O(2) and beta-elemene for hydrodistillation; C(15)H(20)O(2) and curzerene for SC-CO(2) extracts and 3-hexen-1-ol, curzerene, C(15)H(20)O(2), beta-elemene and germacrene B for SC-CO(2) extracts captured in Porapak-Q. PRACTICAL APPLICATIONS The natural extracts are a potential source of compounds possessing biological activities. They can be used in foods, pharmaceutics and cosmetics. Pitanga is an exotic fruit from Brazil and extracts from its leaves have been used against many diseases in Brazilian folk medicine. Supercritical extraction is an interesting process for the production of natural extracts because it is a clean process and the knowledge of composition of extracts is crucial for the identification of the probable active components.

Thyroid hormone stimulates NO production via activation of the PI3K/Akt pathway in vascular myocytes

Aims Thyroid hormone (TH) rapidly relaxes vascular smooth muscle cells (VSMCs). However, the mechanisms involved in this effect remain unclear. We hypothesize that TH-induced rapid vascular relaxation is mediated by VSMC-derived nitric oxide (NO) production and is associated with the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signalling pathway. Methods and results NO levels were determined using a NO-specific fluorescent dye (DAF-2) and nitrite (NO(2)) levels. Expression of NO synthase (NOS) isoforms and proteins of the PI3K/Akt pathway was determined by both western blotting and immunocytochemistry. Myosin light chain (MLC) phosphorylation levels were also investigated by western blotting. Exposure of cultured VSMCs from rat thoracic aortas to triiodothyronine (T3) resulted in a significant decrease of MLC phosphorylation levels. T3 also induced a rapid increase in Akt phosphorylation and increased NO production in a dose-dependent manner (0.001-1 mu M). VSMCs stimulated with T3 for 30 min showed an increase in the expression of all three NOS isoforms and augmented NO production, effects that were prevented by inhibitors of PI3K. Vascular reactivity studies showed that vessels treated with T3 displayed a decreased response to phenylephrine, which was reversed by NOS inhibition. These data suggest that T3 treatment induces greater generation of NO both in aorta and VSMCs and that this phenomenon is endothelium independent. In addition, these findings show for the first time that the PI3K/Akt signalling pathway is involved in T3-induced NO production by VSMCs, which occurs with expressive participation of inducible and neuronal NOS. Conclusion Our data strongly indicate that T3 causes NO-dependent rapid relaxation of VSMC and that this effect is mediated by the PI3K/Akt signalling pathway.

A comparative study on the effects of the benzodiazepine midazolam and the dopamine agents, apomorphine and sulpiride, on rat behavior in the two-way avoidance test

In recent years. studies in behavioral pharmacology have shown the involvement of dopaminergic mechanisms in avoidance behavior as assessed by the two-way active avoidance test (CAR). Changes in dopaminergic transmission also occur in response to particularly threatening challenges. However, studies on the effects of benzodiazepine (BZD) drugs ill this test are still unclear. Given the interplay of dopamine and other neurotransmitters in the neurobiology of anxiety and schizophrenia the aim of this work was to evaluate the effects of systemic administration of midazolam, the dopaminergic agonist apomorphine, and the D(2) receptor antagonist sulpiride using the CAR, a test that shows good sensitivity to typical neuroleptic drugs. Whereas midazolam did not alter the avoidance response. apomorphine increased and sulpiride reduced them in this test. Escape was not affected by any drug treatments. Heightened avoidance was not associated with the increased motor activity caused by apomorphine. In contrast with the benzodiazepine midazolam, activation of post-synaptic D(2) receptors with apomorphine facilitates, whereas the D(2) receptor antagonism with sulpiride inhibited the acquisition of the avoidance behavior. Together, these results bring additional evidence for a role of D(2) mechanisms in the acquisition of the active avoidance. (C) 2009 Elsevier Inc. All rights reserved.