Propolis: lymphocyte proliferation and IFN-gamma production

We evaluated propolis influence on polyclonal activation of lymphocytes by concanavalin A (Con A). The in vitro experiments showed that propolis decreases splenocyte proliferation both in the absence or presence of Con A. The supression in mitogen-induced splenocyte proliferation also occurred when mice were treated intraperitoneally with propolis for 3 days. An increased of IFN-gamma production in the culture supernatants of the same cells was, observed. A dual action of propolis on lymphocyte activation was proposed: it decreases splenocyte proliferation in the presence or absence of Con A and stimulates IFN-gamma production by spleen cells. These results are important to understand the immunomodulatory action of propolis on the host's specific and non-specific immunity. (C) 2003 Elsevier B.V. Ireland Ltd. All rights reserved.

Effect of interleukin-10 on the Paracoccidioides brasiliensis killing by gamma-interferon activated human neutrophils

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Down-modulation of lymphoproliferation and interferon-gamma production by beta-glucan derived from Saccharomyces cerevisiae

beta-glucan, one of the major cell wall components of Saccharomyces cerevisiae, has been found to enhance immune functions. This study investigated in vivo and in vitro effects of beta-glucan on lymphoproliferation and interferon-gamma (IFN-gamma) production by splenic cells from C57BL/6 female mice. All experiments were performed with particulate beta-glucan derived from S. cerevisiae. Data demonstrated that both, i.p administration of particulate beta-glucan (20 or 100 µg/animal) and in vitro stimulation of splenic cells (20 or 100 µg/ml of culture) decreased lymphoproliferation and IFN-gamma production induced by concanavalin A. These results suggest that beta-glucan can trigger a down-modulatory effect regulating a deleterious immune system hyperactivity in the presence of a strong stimulus.

The effect of propolis on CCL5 and IFN-gamma expression by peripheral blood mononuclear cells from leishmaniasis patients

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Co-60 gamma irradiation prevents Bothrops jararacussu venom neurotoxicity and myotoxicity in isolated mouse neuromuscular junction

The ability of gamma radiation from Co-60 (2000 Gy) to attenuate the toxic effects of Bothrops jararacussu venom was investigated on mouse neuromuscular preparations in vitro. A comparative study between the effects of native and irradiated venoms was performed on both phrenic-diaphragm (PD) and extensor digitorum longus (EDL) preparations by means of myographic, biochemical and morphological techniques. Native venom (10 and 20 mug/ml) induced a concentration-dependent paralysis of both directly and indirectly evoked contractions on PD preparations. At 20 mug/ml, it also caused a pronounced myotoxic effect on the EDL muscle preparation that was characterized by an increase of creatine kinase release and by several morphological changes of this preparation. By contrast, irradiated venom, even at concentrations as high as 40 mug/ml, induced neither paralyzing nor myotoxic effects. It was concluded that Co-60 gamma radiation is able to abolish both the paralyzing and the myotoxic effects of B. jararacussu venom on the mouse neuromuscular junction. These findings support the hypothesis that gamma radiation could be an important toot to improve antisera production by reducing toxicity while preserving immunogenicity. (C) 2002 Elsevier B.V. Ltd. All rights reserved.

The shielding effect of glycerol against protein ionization in electrospray mass spectrometry

Most commercial recombinant proteins used as molecular biology tools, as well as many academically made preparations, are generally maintained in the presence of high glycerol concentrations after purification to maintain their biological activity. The present study shows that larger proteins containing high concentrations of glycerol are not amenable to analysis using conventional electrospray ionization mass spectrometry (ESI-MS) interfaces. In this investigation the presence of 25% (v/v) glycerol suppressed the signals of Taq DNA polymerase molecules, while 1% (v/v) glycerol suppressed the signal of horse heart myoglobin. The signal suppression was probably caused by the interaction of glycerol molecules with the proteins to create a shielding effect that prevents the ionization of the basic and/or acidic groups in the amino acid side chains. To overcome this difficulty the glycerol concentration was decreased to 5% (v/v) by dialyzing the Taq polymerase solution against water, and the cone voltage in the ESI triple-quadrupole mass spectrometer was set at 80-130 V. This permitted observation of a mass spectrum that contained ions corresponding to protonation of up to 50% of the ionizable basic groups. In the absence of glycerol up to 85% of the basic groups of Taq polymerase became ionized, as observed in the mass spectrum at relatively low cone voltages. An explanation of these and other observations is proposed, based on strong interactions between the protein molecules and glycerol. For purposes of comparison similar experiments were performed on myoglobin, a small protein with 21 basic groups, whose ionization was apparently suppressed in the presence of 1% (v/v) glycerol, since no mass spectrum could be obtained even at high cone voltages. Copyright (C) 2003 John Wiley Sons, Ltd.

Analyzing glycerol-mediated protein oligomerization by electrospray ionization mass spectrometry

Glycerol is widely used as protein stabilizer, in both local and commercial preparations, so it has become necessary to develop methods for mass spectrometric analysis of protein preparations in the presence of glycerol. However, this stabilizing agent may cause signal suppression when present in high concentrations, and is also known to induce protein supercharging even at low concentrations. This work reports the,use of electrospray ionization (ESI) mass spectrometry to characterize glycerol-mediated protein oligomerization. this phenomenon seems to involve the formation of strong non-covalent interactions between protein and glycerol involving close contact between the monomers, leading to formation of protein oligomers adducted with glycerol molecules under the characteristic analytical conditions of the ESI interface. At high orders of oligomerization a lower number of glycerol molecules is required to maintain the high oligomeric states than for the dimers and trimers, and it is possible that for the higher oligomers the monomers become so close to one another that non-covalent bonds between the side chains of the amino acid residues in the proteins may be established. Copyright (C) 2005 John Wiley & Sons, Ltd.

Advantages of using nested collision induced dissociation/post-source decay with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: sequencing of novel peptides from wasp venom

We have examined the applicability of the 'nested' collision induced dissociation/post-source decay (CID/PSD) method to the sequencing of novel peptides from solitary wasps which have neurotoxic venom for paralyzing other insects. The CID/PSD spectrum of a ladder peptide derived from an exopeptidase digest was compared with that of the intact peptide. The mass peaks observed only in the CID/PSD spectrum of a ladder peptide were extracted as C-terminal fragment ions. Assignment of C-terminal fragment ions enabled calculation of N-terminal fragment masses, leading to differentiation between N-terminal fragment ions and internal fragment ions. This methodology allowed rapid and sensitive identification by removing ambiguity in the assignment of the fragment ions, and proved useful for sequencing unknown peptides, in particular those available as natural products with a limited supply. Copyright (C) 2000 John Wiley & Sons, Ltd.

Dynamics of glyphosate-induced conformational changes of Mycobacterium tuberculosis 5-enolpyruvylshikimate-3-phosphate synthase (EC 2.5.1.19) determined by hydrogen-deuterium exchange and electrospray mass spectrometry

The enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) catalyzes the reaction between shikimate 3-phosphate and phosphoenolpyruvate to form 5-enolpyruvylshikimate 3-phosphate, an intermediate in the shikimate pathway, which leads to the biosynthesis of aromatic amino acids. EPSPS exists in an open conformation in the absence of substrates and/or inhibitors and in a closed conformation when bound to the substrate and/or inhibitor. In the present report, the H/D exchange properties of EPSPS from Mycobacterium tuberculosis (Mt) were investigated for both enzyme conformations using ESI mass spectrometry and circular dichroism (CD). When the conformational changes identified by H/D exchanges were mapped on the 3-D structure, it was observed that the apoenzyme underwent extensive conformational changes due to glyphosate complexation, characterized by an increase in the content of alpha-helices from 40% to 57%, while the beta-sheet content decreased from 30% to 23%. These results indicate that the enzyme underwent a series of rearrangements of its secondary structure that were accompanied by a large decrease in solvent access to many different regions of the protein. This was attributed to the compaction of 71% of alpha-helices and 57% of beta-sheets as a consequence of glyphosate binding to the enzyme. Apparently, MtEPSPS undergoes a series of inhibitor-induced conformational changes, which seem to have caused synergistic effects in preventing solvent access to the core of molecule, especially in the cleft region. This may be part of the mechanism of inhibition of the enzyme, which is required to prevent the hydration of the substrate binding site and also to induce the cleft closure to avoid entrance of the substrates.

Mode of Peroxisome Proliferator-Activated Receptor gamma Activation by Luteolin

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Monitoring the positioning of short polycationic peptides in model lipid bilayers by combining hydrogen/deuterium exchange and electrospray ionization mass spectrometry

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Hydrogen/deuterium exchange mass spectrometry for characterizing phosphoenolpyruvate-induced structural transitions in Mycobacterium tuberculosis 5-enolpyruvylshikimate-3-phosphate synthase (EC 2.5.1.1)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Medium Chain Fatty Acids Are Selective Peroxisome Proliferator Activated Receptor (PPAR) gamma Activators and Pan-PPAR Partial Agonists

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Chemoenzymatic Synthesis of alpha-Hydroxy-beta-methyl-gamma-hydroxy Esters: Role of the Keto-Enol Equilibrium To Control the Stereoselective Hydrogenation in a Key Step

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The Xmnl polymorphic site 5 ' to the gene G gamma in a Brazilian patient with sickle cell anaemia - fetal haemoglobin concentration, haematology and clinical features

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)