Vav GEFs are required for β2 integrin-dependent functions of neutrophils

Integrin regulation of neutrophils is essential for appropriate adhesion and transmigration into tissues. Vav proteins are Rho family guanine nucleotide exchange factors that become tyrosine phosphorylated in response to adhesion. Using Vav1/Vav3-deficient neutrophils (Vav1/3ko), we show that Vav proteins are required for multiple β2 integrin-dependent functions, including sustained adhesion, spreading, and complement-mediated phagocytosis. These defects are not attributable to a lack of initial β2 activation as Vav1/3ko neutrophils undergo chemoattractant-induced arrest on intercellular adhesion molecule-1 under flow. Accordingly, in vivo, Vav1/3ko leukocytes arrest on venular endothelium yet are unable to sustain adherence. Thus, Vav proteins are specifically required for stable adhesion. β2-induced activation of Cdc42, Rac1, and RhoA is defective in Vav1/3ko neutrophils, and phosphorylation of Pyk2, paxillin, and Akt is also significantly reduced. In contrast, Vav proteins are largely dispensable for G protein-coupled receptor–induced signaling events and chemotaxis. Thus, Vav proteins play an essential role coupling β2 to Rho GTPases and regulating multiple integrin-induced events important in leukocyte adhesion and phagocytosis

PKA and Epac cooperate to augment bradykinin-induced interleukin-8 release from human airway smooth muscle cells

Background: Airway smooth muscle contributes to the pathogenesis of pulmonary diseases by secreting inflammatory mediators such as interleukin-8 (IL-8). IL-8 production is in part regulated via activation of G(q)-and G(s)-coupled receptors. Here we study the role of the cyclic AMP (cAMP) effectors protein kinase A (PKA) and exchange proteins directly activated by cAMP (Epac1 and Epac2) in the bradykinin-induced IL-8 release from a human airway smooth muscle cell line and the underlying molecular mechanisms of this response.Methods: IL-8 release was assessed via ELISA under basal condition and after stimulation with bradykinin alone or in combination with fenoterol, the Epac activators 8-pCPT-2'-O-Me-cAMP and Sp-8-pCPT-2'-O-Me-cAMPS, the PKA activator 6-Bnz-cAMP and the cGMP analog 8-pCPT-2'-O-Me-cGMP. Where indicated, cells were pre-incubated with the pharmacological inhibitors Clostridium difficile toxin B-1470 (GTPases), U0126 (extracellular signal-regulated kinases ERK1/2) and Rp-8-CPT-cAMPS (PKA). The specificity of the cyclic nucleotide analogs was confirmed by measuring phosphorylation of the PKA substrate vasodilator-stimulated phosphoprotein. GTP-loading of Rap1 and Rap2 was evaluated via pull-down technique. Expression of Rap1, Rap2, Epac1 and Epac2 was assessed via western blot. Downregulation of Epac protein expression was achieved by siRNA. Unpaired or paired two-tailed Student's t test was used.Results: The beta(2)-agonist fenoterol augmented release of IL-8 by bradykinin. The PKA activator 6-Bnz-cAMP and the Epac activator 8-pCPT-2'-O-Me-cAMP significantly increased bradykinin-induced IL-8 release. The hydrolysis-resistant Epac activator Sp-8-pCPT-2'-O-Me-cAMPS mimicked the effects of 8-pCPT-2'-O-Me-cAMP, whereas the negative control 8-pCPT-2'-O-Me-cGMP did not. Fenoterol, forskolin and 6-Bnz-cAMP induced VASP phosphorylation, which was diminished by the PKA inhibitor Rp-8-CPT-cAMPS. 6-Bnz-cAMP and 8-pCPT-2'-O-Me-cAMP induced GTP-loading of Rap1, but not of Rap2. Treatment of the cells with toxin B-1470 and U0126 significantly reduced bradykinin-induced IL-8 release alone or in combination with the activators of PKA and Epac. Interestingly, inhibition of PKA by Rp-8-CPT-cAMPS and silencing of Epac1 and Epac2 expression by specific siRNAs largely decreased activation of Rap1 and the augmentation of bradykinin-induced IL-8 release by both PKA and Epac.Conclusion: Collectively, our data suggest that PKA, Epac1 and Epac2 act in concert to modulate inflammatory properties of airway smooth muscle via signaling to the Ras-like GTPase Rap1 and to ERK1/2.</p

PRELIMINARY STUDY TO TEST ROOIBOS (Aspalathus linearis) AS A NATURAL ANTIOXIDANT APPLIED TO OSTRICH MEAT PATTIES AND ITALIAN TYPE SALAMI

Rooibos (Aspalathus linearis) is a South African fermented herbal tea which has been gaining attention in the market for its interesting functional properties among which is the protection from oxidative stress caused by superoxide, hydroxyl and peroxyl radical formation. It mainly acts as a chain-breaker, metal-chelator and also as a radical scavenger. Despite this antioxidant property, it has never been tested on meat products. Green rooibos is the unfermented form which possesses higher amounts of total polyphenols and flavonoids than the fermented form. An experimental was designed in order to test green rooibos antioxidant activity on refrigerated ostrich meat patties: one Control (0% green rooibos) and three Treatments (T1=2% dried leaves; T2=2% water extract; T3=2% freeze-dried extract) were studied. A second trial evaluated the antioxidant activity of increasing inclusion levels of fermented rooibos extract in the production of low-fat ostrich salami (Control=0%; T1=0.25%; T2=0.5%; T3=1% extract). Data on TBARS and weight losses were recorded and the rooibos antioxidant effect was tested by a one-way ANOVA. Green rooibos was effective in lowering the lipid oxidation rate in ostrich meat patties and fermented rooibos extract limited the TBARS content of ostrich salami at 15 days of ripening. Further studies are needed to test the lowest effective inclusion level, sensory qualities and economical sustainability of using this natural antioxidant

First evaluation of unfermented and fermented rooibos (Aspalathus linearis) in preventing lipid oxidation in meat products

This study consisted of two trials aiming to evaluate, for the first time, the antioxidant potential of rooibos in meat products. With this purpose, the first trial evaluated three unfermented (green) rooibos forms (dried leaves, water extract, freeze-dried extract) added at 2% inclusion level to ostrich meat patties on an 8-day shelf-life trial. A Control group without green rooibos inclusion was also considered. The second trial evaluated the addition of different concentrations (0%, 0.25%, 0.5% and 1%) of a fermented rooibos extract to nitrite-free ostrich salami. The 2% green rooibos inclusion considerably lowered the TBARS content of ostrich patties, in this way extending their shelf-life. The fermented form (0.5% and 1%) was also effective in delaying lipid oxidation in ostrich salami until 15 days of ripening. The antioxidant potential of both green and fermented forms of rooibos in meat products was confirmed, even if its effect on lipid oxidation requires further study and long-term effects are not yet fully understood

Stereoselectivity in the reduction of bicyclic tetramates

The reduction of bicyclic tetramates can be achieved with high levels of diastereocontrol, but small changes in the substitution of the bicyclic lactam system can lead to changes in the steric bias of the concave/convex system. The tetramates and pyroglutamates prepared in this work exhibited limited antibacterial activity. </p

Antibacterial mimics of natural products by side-chain functionalization of bicyclic tetramic acids

Tetramic acids with unsaturated acyl chains are widely found in natural products possessing a range of biological activities, and bicyclic tetramates represent a suitable scaffold to prepare simple mimics of such complex molecules. An efficient route to functionalize the C(6)-acyl group of a bicyclic tetramate was developed and utilized to prepare a small chemical library with a range of saturated and unsaturated side-chains. The analogues with lipophilic residues possessed highly potent antibacterial activity, which was selective for Gram-positive bacteria, and the best compound was 37-fold more potent than the cephalosporin C control and with an appropriate therapeutic window

Antibacterial mimics of natural products by side-chain functionalization of bicyclic tetramic acids

Tetramic acids with unsaturated acyl chains are widely found in natural products possessing a range of biological activities, and bicyclic tetramates represent a suitable scaffold to prepare simple mimics of such complex molecules. An efficient route to functionalize the C(6)-acyl group of a bicyclic tetramate was developed and utilized to prepare a small chemical library with a range of saturated and unsaturated side-chains. The analogues with lipophilic residues possessed highly potent antibacterial activity, which was selective for Gram-positive bacteria, and the best compound was 37-fold more potent than the cephalosporin C control and with an appropriate therapeutic window

Testing two NIRs instruments to predict chicken breast meat quality and exploiting machine learning approaches to discriminate among genotypes and presence of myopathies

To discriminate among three poultry meat types (hybrid broiler, hybrid broiler affected by breast myopathies, and slow-growing native breed), and to predict the proximate and the amino acid (AA) composition of breast meat, two NIRs (Near-Infrared) instruments operating between 850 and 2500 nm coupled with chemometric algorithms and Machine Learning (ML) approaches, were tested. The Partial Least Square Discriminant Analysis was performed for genotype identification, resulting in a Mathew Correlation Coefficient (MCC) ranging from 0.61 to 1.00, according to the spectra pretreatments and instrument adopted. The Partial Least Square Regression allowed reaching a high cross-validation determination coefficient (R2cv) for crude protein (0.98) and ether extract (0.99), while only three AA (aspartic acid, alanine and methionine) reached R2cv > 0.55. The latter predictions were successfully used to discriminate between genotypes using Factorial Discriminant Analysis, with an MCC ranging from 0.67 to 0.95. Overall, both tested NIRs instruments allowed to determine the chemical composition of fresh and freeze-dried chicken meat. In this sense, a significant improvement of NIRs data interpretability was achieved thanks to the use of ML algorithms, as it was possible to discriminate the chemical composition of meat depending on the genetic group and the presence of breast myopathies