Caffeic acid derivatives: in vitro and in vivo anti-inflammatory properties

Caffeic acid and some of its derivatives such as caffeic acid phenetyl ester (CAPE) and octyl caffeate are potent antioxidants which present important anti-inflammatory actions. The present study assessed the in vitro and in vivo effects of five caffeic acid derivatives (caffeic acid methyl, ethyl, butyl, octyl and benzyl esters) and compared their actions to those of CAPE. In the model of LPS-induced nitric oxide (NO) production in RAW 264.7 macrophages, the pre-incubation of all derivatives inhibited nitrite accumulation on the supernatant of stimulated cells, with mean IC₅₀ (μM) values of 21.0, 12.0, 8.4, 2.4, 10.7 and 4.80 for methyl, ethyl, butyl, octyl, benzyl and CAPE, respectively. The effects of caffeic acid derivatives seem to be related to the scavenging of NO, as the compounds prevented SNAP-derived nitrite accumulation and decreased iNOS expression. In addition, butyl, octyl and CAPE derivatives significantly inhibited LPS-induced iNOS expression in RAW 264.7 macrophages. Extending the in vitro results, we showed that the pre-treatment of mice with butyl, octyl and CAPE derivatives inhibited carrageenan-induced paw edema and prevented the increase in IL-1β levels in the mouse paw by 30, 24 and 36%, respectively. Butyl, octyl and CAPE derivatives also prevented carrageenan-induced neutrophil influx in the mouse paw by 28, 49 and 31%, respectively. Present results confirm and extend literature data, showing that caffeic acid derivatives exert in vitro and in vivo anti-inflammatory actions, being their actions mediated, at least in part by the scavenging of NO and their ability to modulate iNOS expression and probably that of other inflammatory mediators.     

Expression and purification of the recombinant SALT lectin from rice (Oryza sativa L.)

The SALT protein is a 14.5 kDa mannose-binding lectin, originally described as preferentially expressed in rice plant roots in response to NaCl stress. Recombinant SALT lectin was produced in Escherichia coli from a cDNA clone encoding protein. After isopropyl-beta-d-thiogalactopyranoside induction, the expression level achieved was 23% of the soluble protein. The recombinant agglutinin was purified by a single-step process by dialyses against a high concentrated salt solution. After purification, hemagglutination assays of rabbit erythrocytes revealed that the recombinant SALT protein is a potent agglutinin (0.078 microg ml(-1) minimal concentration). The purified recombinant lectin was also used for comparative estimation of native protein amounts in protein extracts from rice plants by Western blot assay.     

LIMK1 regulates Golgi dynamics, traffic of Golgi-derived vesicles, and process extension in primary cultured neurons

In this study, we examined the subcellular distribution and functions of LIMK1 in developing neurons. Confocal microscopy, subcellular fractionation, and expression of several epitope-tagged LIMK1 constructs revealed that LIMK1 is enriched in the Golgi apparatus and growth cones, with the LIM domain required for Golgi localization and the PDZ domain for its presence at neuritic tips. Overexpression of wild-type LIMK1 suppresses the formation of trans-Golgi derived tubules, and prevents cytochalasin D-induced Golgi fragmentation, whereas that of a kinase-defective mutant has the opposite effect. Transfection of wild-type LIMK1 accelerates axon formation and enhances the accumulation of Par3/Par6, insulin-like growth factor (IGF)1 receptors, and neural cell adhesion molecule (NCAM) at growth cones, while inhibiting the Golgi export of synaptophysin-containing vesicles. These effects were dependent on the Golgi localization of LIMK1, paralleled by an increase in cofilin phosphorylation and phalloidin staining in the region of the Golgi apparatus, and prevented by coexpression of constitutive active cofilin. The long-term overexpression of LIMK1 produces growth cone collapse and axon retraction, an effect that is dependent on its growth cone localization. Together, our results suggest an important role for LIMK1 in axon formation that is related with its ability to regulate Golgi dynamics, membrane traffic, and actin cytoskeletal organization.     

The phage T4 restriction endoribonuclease RegB: a cyclizing enzyme that requires two histidines to be fully active

The regB gene, from the bacteriophage T4, codes for an endoribonuclease that controls the expression of a number of phage early genes. The RegB protein cleaves its mRNA substrates with an almost absolute specificity in the middle of the tertranucleotide GGAG, making it a unique well-defined restriction endoribonuclease. This striking protein has no homology to any known RNase and its catalytic mechanism has never been investigated. Here, we show, using ³¹P nuclear magnetic resonance (NMR), that RegB produces a cyclic 2′,3′-phosphodiester product. In order to determine the residues crucial for its activity, we prepared all the histidine-to- alanine point mutants of RegB. The activity of these mutants was characterized both in vivo and in vitro. In addition, their binding capability was quantified by surface plasmon resonance and their structural integrity was probed by ¹H/¹⁵N NMR correlation spectroscopy. The results obtained show that only the H48A and the H68A substitutions significantly reduce RegB activity without changing its ability to bind the substrate or affecting its overall structure. Altogether, our results define RegB as a new cyclizing RNase and present His48 and His68 as potent catalytic residues. The effect of the in vivo selected R52L mutation is also described and discussed.     

Cell surface hydrophobicity and slime production of Staphylococcus epidermidis Brazilian isolates

The cell surface hydrophobicity of 60 isolates and three reference strains of Staphylococcus epidermidis was assayed by means of bacterial aggregation in liquid broth, phosphate-buffered saline, and in ammonium sulfate, as well as by affinity of the bacteria to n-hexadecane and polystyrene surfaces. In order to better characterize the isolates, the influence of bacterial growth time and enzyme treatment on cell hydrophobicity and the analysis of the slime production were also investigated. The strains presented the following profiles when assayed by the ammonium sulfate aggregation test (SAT): SAT < 1M, SAT 1M - <2M, SAT 2M - <4M, and SAT >or=4M. When SAT < 1M, the strains showed positive results for most of the cell surface hydrophobicity tests. None of the strains belonging to the groups with SAT >or= 1M showed spontaneous aggregation (SA), auto-aggregation (AA), or glass adherence, albeit 32 (62.7%) strains were polystyrene adherent and 42 (82.3%) presented weak adherence to n-hexadecane (>20%). The best correlation of the results was found among the AA and glass adherence tests (100%), followed by SA/ glass adherence (98%) and SA/ AA test (98%). The polystyrene adherence test and microbial adherence to n-hexadecane test (MATH) showed 78% correlation. Proteinase K treatment reduced bacterial adherence to polystyrene, but did not influence the SAT values. Three distinct groups of strains were distinguished by the polystyrene micromethod and glass tube adherence assay: 0.0-0.4 O.D. group, including non-glass adherent isolates; 0.5-0.7 O.D. group, including strains with variable profiles (adherent or non-adherent); and 0.8-1.3 O.D. group, composed of glass-adherent strains. Evaluation by a single method seemed not to reliably determine the surface hydrophobicity characteristics of S. epidermidis clinical isolates. Auto-aggregation properties of the strains that adhered to glass seemed related to slime expression, rather than cell surface hydrophobicity. Data also suggested involvement of protein components in adherence to polystyrene, but not in auto-aggregation properties assayed by SAT.     

Protein Phosphorylation and APP Metabolism

Numerous lines of evidence place signal transduction cascades at the core of many processes having a direct role in neurodegeneration and associated disorders. Key players include neurotransmitters, growth factors, cytokines, hormones, and even binding and targeting proteins. Indeed, abnormal phosphorylation of key control proteins has been detected in many cases and is thought to underlie the associated cellular dysfunctions. Several signaling cascades have been implicated, affecting processes as varied as protein processing, protein expression, and subcellular protein localization, among others. The Alzheimer's amyloid precursor protein (APP) is a phosphoprotein, with well-defined phosphorylation sites but whose function is not clearly understood. The factors and pathways regulating the processing of APP have been particularly elusive, both in normal ageing and the Alzheimer's disease (AD) condition. Not surprisingly, the physiological function(s) of the protein remain(s) to be elucidated, although many hypotheses have been advanced. Nonetheless, considerable data has accumulated over the last decade, placing APP in key positions to be modulated both directly and indirectly by phosphorylation and phosphorylation-dependent events. The pathological end product of APP processing is the main proteinaceous component of the hallmark senile plaques found in the brains of AD patients, that is, a toxic peptide termed A. In this minireview we address the importance of phosphorylation and signal transduction cascades in relation to APP processing and A production. The possible use of the identified molecular alterations as therapeutic targets is also addressed.     

Differential regulation of the release of tumor necrosis factor-alpha and of eicosanoids by mast cells in rat airways after antigen challenge

BACKGROUND: Rat trachea display a differential topographical distribution of connective tissue mast cells (CTMC) and mucosal mast cells (MMC) that may imply regional differences in the release of allergic mediators such as tumor necrosis factor-alpha (TNF-alpha) and eicosanoids. AIM: To evaluate the role of CTMC and MMC for release of TNF-alpha and eicosanoids after allergenic challenge in distinct segments of rat trachea. MATERIALS AND METHODS: Proximal trachea (PT) and distal trachea (DT) from ovalbumin (OVA)-sensitized rats, treated or not with compound 48/80 (48/80) or dexamethasone, were incubated in culture medium. After OVA challenge, aliquots were collected to study release of TNF-alpha and eicosanoids. RESULTS: Release of TNF-alpha by PT upon OVA challenge peaked at 90 min and decayed at 6 and 24 h. Release from DT peaked at 30-90 min and decayed 6 and 24 h later. When CTMC were depleted with 48/80, OVA challenge exacerbated the TNF-alpha release by PT at all time intervals, while DT exacerbated TNF-alpha levels 6 and 24 h later only. Dexamethasone reduced TNF-alpha production after 90 min of OVA challenge in PT and at 3 and 6h in DT. OVA challenge increased prostaglandin D2) in DT and leukotriene B4 in both segments but did not modify prostaglandin E2 and leukotriene C4 release. CONCLUSION: OVA challenge induces TNF-alpha release from MMC, which is negatively regulated by CTMC. The profile of TNF-alpha and eicosanoids depends on the time after OVA challenge and of the tracheal segment considered.     

Antiplatelet properties of novel N-substituted-phenyl-1,2,3-triazole-4-acylhydrazone derivatives

This paper describes the design, synthesis and pharmacological evaluation of new N-acylhydrazone (NAH) compounds, belonging to the N-substituted-phenyl-1,2,3-triazole-4-acylhydrazone class (2a-p). Classical heteroaromatic ring bioisosterism strategies were applied to the previously reported N-phenylpyrazolyl-4-acylhydrazone derivative 1, elected as lead-compound due to its important anti-aggregating profile on arachidonic acid induced platelet aggregation (IC(50)=24+/-0.5 micro M), from which emerge this new series 2. These new compounds 2a-p were readily synthesized, characterized and tested on platelet aggregation assays induced by collagen (5 micro g/mL), ADP (5 micro M) and arachidonic acid (100 micro M) in rabbit citrated platelet-rich plasma. Compounds 2b, 2d, and 2h were found to be the most potent, exhibiting a significant antiplatelet activity on arachidonic acid- and collagen-induced platelet aggregation. In addition, these new antiplatelet agents are free of gastric ulcerogenic effect and presented discrete anti-inflammatory and analgesic properties. The N-para-chlorophenyltriazolyl-4-acylhydrazone compound 2h produced the highest inhibitory effect on collagen (IC(50)=21.6+/-0.4 micro M) and arachidonic acid-induced platelet aggregation (IC(50)=2.2+/-0.06 micro M), suggesting that the nature of the substituent on the phenyl ring of the N-heteroaromatic system of NAH moiety may be an important structural requirement for the improvement of antiplatelet activity, in comparison with lead-series 1.