Obtenção de oligossacarídeos N-ligados às glicoproteínas dos líquens Sticta tomentosa e Sticta damaecornis

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

Oxidation of melatonin and its catabolites, N-1-acetyl-N (2)-formyl-5-methoxykynuramine and N-1-acetyl-5-methoxykynuramine, by activated leukocytes

N-1-acetyl-N-2-formyl-5-methoxykynuramine (AFMK) and N-1-acetyl-5-methoxykynuramine (AMK), two melatonin catabolites, have been described as potent antioxidants. We aimed to follow the kinetics of AFMK and AMK formation when melatonin is oxidized by phorbol myristate acetate (PMA) and lipopolysaccharide (LPS)-activated leukocytes. An HPLC-based method was used for AFMK and AMK determination in neutrophil and peripheral blood mononuclear cell cultures supernatants. Samples were separated isocratically on a C18 reverse-phase column using acetonitrile/H2O (25:75) as the mobile phase. AFMK was detected by fluorescence (excitation 340 nm and emission 460 nm) and AMK by UV-VIS absorbance (254 nm). Activation of neutrophils and mononuclear cells with PMA produces larger amounts of AFMK than activation with LPS, probably due to the lower levels of reactive oxygen species formation and myeloperoxidase (MPO) degranulation that occurs when cells are stimulated with LPS. The concentration of AMK found in the supernatant was about 5-10% (from 18-hr cultures) compared with AFMK. This result may reflect its reactivity. Indeed AMK, but not AFMK, is easily oxidized by activated neutrophils in a MPO and hydrogen peroxide-dependent reaction. In conclusion, we defined a simple procedure for the determination of AFMK and AMK in biological samples and demonstrated the capacity of leukocytes to oxidize melatonin and AMK.

In vivo CYP2E1 phenotyping as a new potential biomarker of occupational and experimental exposure to benzene

Assessing CYP2E1 phenotype in vivo may be important to predict individual susceptibility to those chemicals, including benzene, which are metabolically activated by this isoenzyme. Chlorzoxazone (CHZ), a specific CYP2E1 substrate, is readily hydroxylated to 6-OH-chlorzoxazone (6-OH-CHZ) by liver CYP2E1 and the metabolic ratio 6-OH-CHZ/CHZ in serum (MR) is a specific and sensitive biomarker of CYP2E1 activity in vivo in humans. We used this MR as a potential biomarker of effect in benzene-treated rats and, also, in humans occupationally exposed to low levels of benzene. Male Sprague-Dawley rats (375-400 g b.w.) were treated i.p. for 3 days with either a 0.5 ml solution of benzene (5 mmol/kg b.w.) in corn oil, or 0.5 ml corn oil alone. Twenty-four hours after the last injection, a polyethylene glycol (PEG) solution of CHZ (20 mg/kg b.w.) was injected i.p. in both treated and control animals. After 2, 5.10,15, 20,30,45, 60, 90, 120, 180, and 240 min from injection, 0.2 ml blood was taken from the tip tail and stored at -20 degrees C until analysis. A modified reverse phase HPLC method using a 5 mu m Ultrasphere C18 column equipped with a direct-connection ODS guard column, was used to measure CHZ and its metabolite 6-OH-CHZ in serum. No statistically significant difference in the MR was observed, at any sampling time, between benzene-treated and control rats. The concentration-versus-time area under the curve (AUC), however, was lower (p < 0.05, Mann-Whitney test), whereas the systemic clearance was higher (p < 0.05) in treated than in control rats. Eleven petrochemical workers occupationally exposed to low levels of airborne benzene (mean +/- SD, 25.0 +/- 24.4 mu g/m(3)) and 13 non-exposed controls from the same factory (mean +/- SD, 6.7 +/- 4.0 mu g/m(3)) signed an informed consent form and were administered 500 mg CHZ p.o. Two hours later a venous blood sample was taken for CHZ and 6-OH-CHZ measurements. Despite exposed subjects showed significantly higher levels of t,t-MA and S-PMA, two biomarkers of exposure to benzene, than non-exposed workers, no difference in the MR mean values +/- SD was found between exposed (0.59 +/- 0.29) and non-exposed (0.57 +/- 0.23) subjects. So, benzene was found to modify CHZ disposition, but not CYP2E1 phenotype in benzene-treated rats, nor in workers exposed to benzene, probably due to the levels of exposure being too low. (C) 2009 Elsevier B.V. All rights reserved.

Off line extraction of phenol from human urine sample with isoamyl alcohol and determination by HPLC

This method has been developed for extraction and determination of phenol in a urine sample by high performance liquid chromatography.After acid hydrolysis, the free phenol was extracted with isoamyl alcohol solvent, followed by back extraction with 0.5 mol.L-1 sodium hydroxide solution and analyzed by an isocratic HPLC Varian System, equipped with reverse-phase column (MicroPak-C-18). The mobile phase was acetonitrile in 0.01 mol.L-1 hydrochloric acid solution, (20:80 v/v), and at a now-rate of 1.0 mL.min-1. The chromatogram was monitored at 220 nm in room temperature. The identification was based on retention time and the quantification was performed by automatic peak-area determination, corrected for the external standards method.The recovery was higher than 99.5 % for phenol and reproducibility of method was shown to be 2.3% standard deviation and 5.6% coefficient of variance (n=20). The limit detection was 0.05 mgL(-1) and a range of 0.05 to 20.0 mgL(-1) of phenol for linearity.

Lipopolysaccharide infusion up-regulates hepcidin mRNA expression in equine liver

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

Effects of twenty-four hour transport or twenty-four hour feed and water deprivation on physiologic and performance responses of feeder cattle

The objective of this study was to compare the effects of 24-h road transport or 24-h feed and water deprivation on acute-phase and performance responses of feeder cattle. Angus x Hereford steers (n = 30) and heifers (n = 15) were ranked by gender and BW (217 +/- 3 kg initial BW; 185 +/- 2 d initial age) and randomly assigned to 15 pens on d -12 of the experiment (3 animals/pen; 2 steers and 1 heifer). Cattle were fed alfalfa-grass hay ad libitum and 2.3 kg/animal daily (DM basis) of a corn-based concentrate throughout the experiment (d -12 to 28). on d 0, pens were randomly assigned to 1 of 3 treatments: 1) transport for 24 h in a livestock trailer for 1,200 km (TRANS), 2) no transport but feed and water deprivation for 24 h (REST), or 3) no transport and full access to feed and water (CON). Treatments were concurrently applied from d 0 to d 1. Total DMI was evaluated daily from d -12 to d 28. Full BW was recorded before treatment application (d -1 and 0) and at the end of experiment (d 28 and 29). Blood samples were collected on d 0, 1, 4, 7, 10, 14, 21, and 28. Mean ADG was greater (P < 0.01) in CON vs. TRANS and REST cattle but similar (P = 0.46) between TRANS and REST cattle (1.27, 0.91, and 0.97 kg/d, respectively; SEM = 0.05). No treatment effects were detected for DMI (P >= 0.25), but CON had greater G: F vs. TRANS (P < 0.01) and REST cattle (P = 0.08) whereas G: F was similar (P = 0.21) between TRANS and REST cattle. Plasma cortisol concentrations were greater (P <= 0.05) in REST vs. CON and TRANS cattle on d 1, 7, 14, and 28 and also greater (P = 0.02) in TRANS vs. CON cattle on d 1. Serum NEFA concentrations were greater (P < 0.01) in REST and TRANS vs. CON cattle on d 1 and greater (P < 0.01) in REST vs. TRANS cattle on d 1. Plasma ceruloplasmin concentrations were greater (P = 0.04) in TRANS vs. CON cattle on d 1, greater (P = 0.05) in REST vs. CON on d 4, and greater (P <= 0.05) in REST vs. TRANS and CON on d 14. Plasma haptoglobin concentrations were greater (P < 0.01) in TRANS vs. CON and REST cattle on d 1 and greater (P <= 0.05) for REST vs. TRANS and CON cattle on d 7. In conclusion, 24-h transport and 24-h nutrient deprivation elicited acute-phase protein reactions and similarly reduced feedlot receiving performance of feeder cattle. These results suggest that feed and water deprivation are major contributors to the acute-phase response and reduced feedlot receiving performance detected in feeder cattle transported for long distances.

Jelleines: a family of antimicrobial peptides from the Royal Jelly of honeybees (Apis mellifera)

Four antimicrobial peptides were purified from Royal Jelly of honeybees, by using reverse phase-HPLC and sequenced by using Q-Tof-MS/MS: PFKLSLHL-NH2 (Jelleine-I), TPFKLSLHL-NH2 (Jelleine-II), EPFKLSLHL-NH2 (Jelleine-III), and TPFKLSLH-NH2 (Jelleine-IV). The peptides were synthesized on-solid phase, purified and submitted to different biological assays: antimicrobial activity, mast cell degranulating activity and hemolysis. The Jelleines-I-III presented exclusively antimicrobial activities against yeast, Gram+ and Gram- bacteria; meanwhile, Jelleine-IV was not active in none of the assays performed. These peptides do not present any similarity with the other antimicrobial peptides from the honeybees; they are produced constitutively by the workers and secreted into Royal Jelly. (C) 2004 Elsevier B.V. All rights reserved.

Crotacetin, a novel snake venom C-type lectin homolog of convulxin, exhibits an unpredictable antimicrobial activity

Snake venom (sv) C-type lectins encompass a group of hemorrhagic toxins that are capable of interfering with blood stasis. A very well-studied svC-type lectin is the heterodimeric toxin, convulxin (CVX), from the venom of South American rattlesnake Crotalus durissus terrificus. CVX is able to activate platelets and induce their aggregation by acting via p62/GPVI collagen receptor. By using polymerase chain reaction homology screening, we have cloned several cDNA precursors of CVX subunit homologs. One of them, named crotacetin (CTC) beta-subunit, predicts a polypeptide with a topology very similar to the tridimensional conformations of other subunits of CVX-like snake toxins, as determined by computational analysis. Using gel permeation and reverse-phase high-performance liquid chromatography, CTC was purified from C. durissus venoms. CTC can be isolated from the venom of several C. durissus subspecies, but its quantitative predominance is in the venom of C. durissus cascavella. Functional analysis indicates that CTC induces platelet aggregation, and, importantly, exhibits an antimicrobial activity against Gram-positive and -negative bacteria, comparable with CVX.

Development of an HPLC-UV/Vis method for the determination of dyes in a gasoline sample employing different pre-treatments

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

The effect of alpha-tocopherol on the oxidative cleavage of beta-carotene

Two cleavage pathways of beta-carotene have been proposed, one by central cleavage and the other by random (excentric) cleavage. The central cleavage pathway involves the metabolism of beta-carotene at the central double bond (15, 15') to produce retinal by beta-carotene 15, 15'-dioxygenase (E.C.888990988). The random cleavage of beta-carotene produces beta-apo-carotenoids, but the mechanism is not clear. To understand the various mechanisms of beta-carotene cleavage, beta-carotene was incubated with the intestinal postmitochondrial fractions of 10-week-old male rats for 1 h and cleavage products of beta-carotene were analyzed using reverse-phase, high-performance liquid chromatography (HPLC). We also studied the effects of alpha-tocopherol and NAD(+)/NADH on beta-carotene cleavage. In addition to beta-carotene, we used retinal and beta-apo-14'-carotenoic acid as substrates in these incubations. Beta-apo-14'-carotenoic acid is the two-carbon longer homologue of retinoic acid. In the presence of alpha-tocopherol, beta-carotene was converted exclusively to retinal, whereas in the absence of alpha-tocopherol, both retinal and beta-apo-carotenoids were formed. Retinoic acid was produced from both retinal and beta-apo-14'-carotenoic acid incubations only in the presence of NAD(+). Our data suggest that in the presence of an antioxidant such as alpha-tocopherol, beta-carotene is converted exclusively to retinal by central cleavage. In the absence of an antioxidant, beta-carotene is cleaved randomly by enzyme-related radicals to produce beta-apo-carotenoids, and these beta-apo-carotenoids can be oxidized further to retinoic acid via retinal. (C) 2000 Elsevier B.V.

Application of liquid chromatography/electrospray ionization tandem mass spectrometry to the analysis of polyphenolic compounds from an infusion of Byrsonima crassa Niedenzu

A fast and reliable method, based on high-performance liquid chromatography coupled to electrospray ionization ion trap tandem mass spectrometry (HPLC/ESI-ITMS), was developed to investigate the infusion prepared from the leaves of Byrsonima crassa Niedenzu (Malpighiaceae), a native plant used in Brazil against gastric disorders. The use of on-line reverse-phase HPLC/ESI-ITMS allowed separation of three major classes of compounds and identification of over 20 very polar compounds characterized as galloylquinic acids, proanthocyanidins, and flavonoid glycosides, as well as the dimeric flavonoid amentoflavone and minor amounts of galloyl hexose and galloyl saccharose. This approach provided data that will allow establishment of a method for a future standardization of the infusion. Copyright (C) 2005 John Wiley & Sons, Ltd.

Determination of cocaine in samples of hair using the chromatographic column, ISRP-C-18

A method has been developed for the determination of the cocaine levels in samples of hair, using a chromatographic column internal surface reverse phase (ISRP)-C-18, for direct injection of the extracts of hair, without purification or derivation of the sample. The method allows monitoring an individual stopped for using or making cocaine. This method allowed the determination of levels of cocaine concentration in 75% of the analyzed samples of chemical dependents' hair, with cocaine detected at levels of 0.37-16.85 mug g(-1). In the other analyzed samples (25%), the drug was not detected, because the corresponding individuals told us that they consumed cocaine infrequently and in small amounts. The detection

Determination of aldehydes and ketones in fuel ethanol by high-performance liquid chromatography with electrochemical detection

A new methodology was developed for analysis of aldehydes and ketones in fuel ethanol by high-performance liquid chromatography (HPLC) coupled to electrochemical detection. The electrochemical oxidation of 5-hydroxymetkylfurfural, 2-furfuraldehyde, butyraldehyde, acetone and methyl ethyl ketone derivatized with 2,4-dinitrophenylhydrazine (DNPH) at glassy carbon electrode present a well defined wave at +0.94 V; +0.99 V; +1.29 V; +1.15 V and +1.18 V, respectively which are the basis for its determination on electrochemical defector. The carbonyl compounds derivatized were separated by a reverse-phase column under isocratic conditions with a mobile phase containing a binary mixture of methanol /LiClO4(aq) at a concentration of 1.0 x 10(-3) mol L-1 (80:20 v/v) and a flow-rate of 1.1 mL min(-1). The optimum potential for the electrochemical detection of aldehydes-DNPH and ketones-DNPH was +1.0 V vs. Ag/AgCl. The analytical curve of aldehydes-DNPH and ketones-DNPH presented linearity over the range 5.0 to 400.0 ng mL(-1), with detection limits of 1.7 to 2.0 ng mL(-1) and quantification limits from 5.0 to 6.2 ng mL(-1), using injection volume of 20 mu L. The proposed methodology was simple, low time-consuming (15 min/analysis) and presented analytical recovery higher than 95%.

Annexin-A1 gene expression during liver development and post-translation modification after experimental endotoxemia

Objective and design: We have previously reported a role for annexin-A1 in liver proliferation and tumorogenicity as well as its action as an acute phase protein in a model of endotoxemia in interleukin-6 null mice.Material and methods: In this study, we have investigated the analysis of the gene and protein expression in annexin-A1 null mice and the wild type livers during foetal and adult life, and in the presence of a proinflammatory stimulus.Results: The data indicate a link between the expression of the annexin-A1 as serine-phosphorylated-protein during early events of the inflammatory response and as tyrosine-phosphorylated-form at later time-points, during the resolution of inflammation.Conclusions: The study of annexin-A1 post-translation modification may promote a new annexin-A1 peptide discovery programme to treat specific pathologies.

Proteins of Leishmania (Viannia) shawi confer protection associated with Th1 immune response and memory generation

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