Novas propriedades do SKTI (Inibidor de tripsina de soja): inibição para elastase neutrofílica humana e efeitos no processo de injúria pulmonar aguda

Seeds from legumes including the Glycine max are known to be a rich source of protease inhibitors. The soybean Kunitz trypsin inhibitor (SKTI) has been well characterised and has been found to exhibit many biological activities. However its effects on inflammatory diseases have not been studied to date. In this study, SKTI was purified from a commercial soy fraction, enriched with this inhibitor, using anion exchange chromatography Resource Q column. The purified protein was able to inhibit human neutrophil elastase (HNE) and bovine trypsin. . Purified SKTI inhibited HNE with an IC50 value of 8 µg (0.3 nM). At this concentration SKTI showed neither cytotoxic nor haemolytic effects on human blood cell populations. SKTI showed no deleterious effects on organs, blood cells or the hepatic enzymes alanine amine transferase (ALT) and aspartate amino transferase (AST) in mice model of acute systemic toxicity. Human neutrophils incubated with SKTI released less HNE than control neutrophils when stimulated with PAF or fMLP (83.1% and 70% respectively). These results showed that SKTI affected both pathways of elastase release by PAF and fMLP stimuli, suggesting that SKTI is an antagonist of PAF/fMLP receptors. In an in vivo mouse model of acute lung injury, induced by LPS from E. coli, SKTI significantly suppressed the inflammatory effects caused by elastase in a dose dependent manner. Histological sections stained by hematoxylin/eosin confirmed this reduction in inflammation process. These results showed that SKTI could be used as a potential pharmacological agent for the therapy of many inflammatory diseases

Adsorção de selênio em latossolos

A retenção de Se pelos colóides do solo constitui importante processo para a manutenção da sanidade ambiental. A informação sobre a adsorção de Se em solos altamente intemperizados é restrita e existem poucos padrões quantitativos disponíveis para a definição de estratégias de remediação de áreas contaminadas. Quantidades crescentes de Se (5, 10, 25, 50, 100 e 250 mg L-1), na forma de Na2SeO3, foram adicionadas a amostras de dez Latossolos brasileiros [três Latossolos Vermelho-Amarelos (LVA-1, LVA-2 e LVA-3), dois Latossolos Vermelhos (LV-1 e LV-2), um Latossolo Vermelho eutroférrico (LVef), um Latossolo Vermelho acriférrico (LVwf), dois Latossolos Amarelos (LA-1 e LA-2) e um Latossolo Amarelo acriférrico (LAwf)]. Isotermas de adsorção foram construídas e foi verificado o ajuste dos resultados experimentais aos modelos de Langmuir e de Freundlich. A equação de Langmuir ajustou melhor os resultados de adsorção de Se do que a isoterma de Freundlich. Todas as isotermas apresentaram o formato tipo-L (exponencial), com exceção daquelas obtidas para o LVA-1 e para o LVA-2, que apresentaram comportamento tipo-C (linear). Valores de adsorção máxima (Ads máx), estimada pelo modelo de Langmuir, variaram de 135 (LVA-3) a 2.245 mg kg-1 (LA-1), enquanto os coeficientes de afinidade (K L) estiveram entre 0,002 (LVA-2) e 0,326 (LVA-3). A constante de afinidade estimada pelo modelo de Freundlich (Kf) variou de 13,7 (LVA-2) a 180,1 (LAwf). A adsorção máxima de Se foi mais elevada no LVef e nos Latossolos ácricos (LAwf e LVwf), enquanto os maiores valores de Kf foram encontrados no LV-2, LVef, LVA-3 e LVwf. Não houve correlação entre os atributos dos solos e as constantes de Langmuir. Valores de Kf correlacionaram-se com os teores de argila (r = 0,42*) e com a capacidade de troca de ânions (r = 0,64*).

Síntese de zeólitas tipo ton utilizando um sal de imidazólio como agente direcionador de estrutura

This work reports the synthesis of zeolites with different compositions (pure silica, Si/Ti and Si/Al), via hydroxide and fluoride medium using the cation 1-butyl-3- methylimidazolium as structure directing agent. Initially, the cation was synthesized in chloride form and used for the synthesis in hydroxide medium. An anion-exchange (Cl- for OH-) was required for the synthesis in fluoride medium. Different reactants were used for the formation of gels synthesis, resulting in the crystallization of MFI and TON phases, the latter predominant in many compositions. The cation and synthesized zeolites obtained were characterized by different techniques such as NMR, TG/DTG, XRD, SEM, N2 adsorption and desorption, DRS and EPMA. Besides characterizing the cation and zeolites, the mother liquor of hydroxide synthesis was characterized and it was possible to observe a modification of the cation in the synthesis conditions employed. The materials synthesized in this work can be applied in catalytic reactions and adsorption

DNA and heparin chaperone the refolding of purified recombinant replication protein A subunit 1 from Leishmania amazonensis

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

Purification and characterization of the exopolygalacturonase produced by Aspergillus giganteus in submerged cultures

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

Structural and functional characteristics of selected soft wheat starches

Starches from eight soft wheat samples (two parent lines and six offspring) were isolated; relationships between their structures and properties were examined. Branch chain-length distributions of amylopectins were determined by using high-performance anion exchange chromatography equipped with an amyloglucosidase reactor and a pulsed amperometric detector (HPAEC-ENZ-PAD). Results showed that the average chain length of the eight samples varied at DP 25.6-26.9. Starch samples of lines 02, 60, 63, 95, and 114 consisted of amylopectins with more long chains (DP greater than or equal to 37) and longer average chain length (DP 26.2-26.9) than that of other samples. These starch samples of longer branch chain length displayed higher gelatinization temperatures (55.3-56.5degreesC) than that of other samples (54.4-54.9degreesC) and higher peak viscosity (110-131 RVU) and lower pasting temperature (86.3-87.6degreesC) than others (83-100 RVU and 88.2-88.9degreesC, respectively). The M-w of amylopectins, determined by using high-performance size exclusion chromatography equipped with multiangle laser-light scattering and refractive index detectors (HPSEC-MALLS-RI), were similar for all samples (6.17 x 10(8) to 6.97 x 10(8)). There were no significant differences in amylose and phosphorus contents between samples. These results indicated that physical properties of wheat starch were affected by the branch-chain length of amylopectin.

1,3-dipolar cycloaddition of CS2 to the coordinated azide in the cyclopalladated [Pd(bzan)(mu-N-3)](2). Crystal and molecular structure of di(mu-N,S-1,2,3,4-thiatriazole-5-thiolate)bis[(benzylideneaniline-C-2,N)palladium(II)]

The compound [Pd(bzan)(mu -N-3)](2) 1, bzan = benzylideneaniline, was prepared from [Pd(bzan) (mu -OOCCH3)](2) by an anion exchange reaction. The 1,3-dipolar cycloaddition of carbon disulfide to the bridged coordinated azide in the cyclometallated compound I was investigated. The species resulting from this reaction, di(mu -N,S-1,2,3,4-thiatriazol-5-thiolate)bis[(benzylideneaniline)palladium(II)] 2, was characterized by IR spectroscopy and X-ray diffraction. The compound 2 is a dimer containing two [Pd(benzylideneaniline)] moieties connected by two vicinal bridging N,S-1,2,3,4-thiatriazole-5-thiolate anions in a square-planar coordination geometry for the palladium atoms.

Voltammetric sensor for sodium nitroprusside determination in biological fluids using films of poly-L-lysine

Sodium nitroprusside (NP), a commercial vasodilator, can be pre-concentrated on vitreous carbon electrode modified by films of 97.5%: 2.5% Poly-L-lysine (PLL): glutaraldehyde (GA). This coating gives acceptable anion exchange properties whilst giving the required improvement of adhesion to the glassy carbon electrode surface. Linear response range and detection limit on nitroprusside in B-R buffer pH 4.0, were 1 x 10(-6) to 2 x 10-(5) mol L-1 and 1 x 10(-7) mol L-1, respectively. The repeatability of the proposed sensor, evaluated in term of relative standard deviation, was measured as 4.1% for 10 experiments. The voltammetric sensor was directly applied to determination of nitroprusside in human plasma and urine samples and the average recovery for these samples was around 95-97% without any pre treatment.

Label-free DNA detection based on modified conducting polypyrrole films at microelectrodes

A label-free electrochemical detection method for DNA hybridization based on electrostatic modulation of the ion-exchange kinetics of a polypyrrole film deposited at microelectrodes is reported. Synthetic single-stranded 27-mer oligonucleotides (probe) have been immobilized at 2,5-bis(2-thienyl)-N-(3-phosphorylpropyl)pyrrole film formed by electropolymerization on the previously formed polypyrrole layer. The 27- or 18-mer target oligonucleotides were monitored via the electrochemically driven anion exchange of the inner polypyrrole film. The performance of the miniaturized DNA biosensor system was studied in respect to selectivity, sensitivity, reproducibility, and regeneration of the sensor. Control experiments were performed with a noncomplementary target of 27-mer DNA and 12 base-pair mismatched 18-mer sequences, respectively, and did not show any unspecific binding. Under optimized experimental conditions, the label-free electrochemical biosensor enabled the detection limits of 0.16 and 3.5 fmol for the 18- and 2 7-mer DNA strand, respectively. Furthermore, we demonstrate reusability of the electrochemical DNA biosensor after successful recovery of up to 100% of the original signal by regenerating the DNA label-free electrode with 50 mM HCl at room temperature.

Purification and characterization of pectin methylesterase from acerola (Malpighia glabra L.)

The enzyme pectinmethylesterase (PME) from acerola was extracted and purified by gel anion-exchange chromatography (Q Sepharose) and filtration on Sephadex G-100. The results showed two different PME isoforms (PME1 and PME2), with molecular masses of 25.10 and 5.20 kDa, respectively. PMEI specific activity increased by 9.63% after 60 min incubation at 98 degrees C, while PME2 retained 66% of its specific activity under the same conditions. The K-m values of PMEI, PME2 and concentrated PME were 0.94, 0.08 and 0.08mg mL(-1), respectively. The V-max value of PMEI, PME2 and concentrated were 204.08, 2, 158.73 and 2.92 mu mol min(-1) mg(-1) protein, respectively. (c) 2007 Society of Chemical Industry.

Purification and characterization of two beta-glucosidases from the thermophilic fungus Thermoascus aurantiacus

beta-Glucosidase from the fungus Thermoascus aurantiacus grown oil semi-solid fermentation medium (using ground corncob as substrate) was partially purified in 5 steps - ultrafiltration, ethanol precipitation, gel filtration and 2 anion exchange chromatography runs, and characterized. After the first anion exchange chromatography, beta-glucosidase activity was eluted in 3 peaks (Gl-1, Gl-2, Gl-3). Only the Gl-2 and Gl-3 fractions were adsorbed on the gel matrix. Gl-2 and Gl-3 exhibited optimum pH at 4.5 and 4.0, respectively. The temperature optimum of both glucosidases was at 75-80 degreesC. The pH stability of Gl-2 (4.0-9.0) was higher than Gl-3 (5.5-8.5); both enzyme activities showed similar patterns of thermostability. Under conditions of denaturing gel chromatography the molar mass of Gl-2 and Gl-3 was 175 and 157 kDa, respectively. Using 4-nitrophenyl beta-D-glucopyranoside as substrate, K-m, values of 1.17 +/- 0.35 and 1.38 +/- 0.86 mmol/L were determined for Gl-2 and Gl-3, respectively. Both enzymes were inhibited by Ag+ and stimulated by Ca2+.

Partial purification and characterization of lysine-ketoglutarate reductase in normal and opaque-2 maize endosperms

Lysine-ketoglutaratc reductase catalyzes the first step of lysine catabolism in maize (Zea mays L.) endosperm. The enzyme condenses L-lysine and α-ketoglutarate into saccharopine using NADPH as cofactor. It is endosperm-specific and has a temporal pattern of activity, increasing with the onset of kernel development, reaching a peak 20 to 25 days after pollination, and thereafter decreasing as the kernel approaches maturity. The enzyme was extracted from the developing maize endosperm and partially purified by ammonium-sulfate precipitation, anion-exchange chromatography on DEAE-cellulose, and affinity chromatography on Blue-Sepharose CL-6B. The preparation obtained from affinity chromatography was enriched 275-fold and had a specific activity of 411 nanomoles per minute per milligram protein. The native and denaturated enzyme is a 140 kilodalton protein as determined by polyacrylamide gel electrophoresis. The enzyme showed specificity for its substrates and was not inhibited by either aminoethyl-cysteine or glutamate. Steady-state product-inhibition studies revealed that saccharopine was a noncompetitive inhibitor with respect to α-ketoglutarate and a competitive inhibitor with respect to lysine. This is suggestive of a rapid equilibriumordered binding mechanism with a binding order of lysine, α-ketoglutarate, NADPH. The enzyme activity was investigated in two maize inbred lines with homozygous normal and opaque-2 endosperms. The pattern of lysine-ketoglutarate reductase activity is coordinated with the rate of zein accumulation during endosperm development. A coordinated regulation of enzyme activity and zein accumulation was observed in the opaque-2 endosperm as the activity and zein levels were two to three times lower than in the normal endosperm. Enzyme extracted from L1038 normal and opaque-2 20 days after pollination was partially purified by DEAE-cellulose chromatography. Both genotypes showed a similar elution pattern with a single activity peak eluted at approximately 0.2 molar KCL. The molecular weight and physical properties of the normal and opaque-2 enzymes were essentially the same. We suggest that the Opaque-2 gene, which is a transactivator of the 22 kilodalton zein genes, may be involved in the regulation of the lysine-ketoglutarate reductase gene in maize endosperm. In addition, the decreased reductase activity caused by the opaque-2 mutation may explain, at least in part, the elevated concentration of lysine found in the opaque-2 endosperm.

Níveis críticos de fósforo no solo e nas folhas para a implantação de Eucalyptus grandis Hill ex Maiden, em quatro tipos de solos

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

Mecanismos de absorção de aminoácidos e oligopeptídios. Controle e implicações na dietoterapia humana

The mechanisms involved in the absorption of amino acids and oligopeptides are reviewed regarding their implications in human feedings. Brush border and basolateral membranes are crossed by amino acids and di-tripeptides by passive (facilitated or simple diffusion) or active (Na + or H + co-transporters) pathways. Active Na +-dependent system occurs mainly at brush border and simple diffusion at basolateral, both membranes have the passive facilitated transport. Free-amino acids use either passive or active transport systems whereas di-tripeptides do mainly active (H + co-transporter). Brush border have distinctive transport system for amino acids and di-tripeptides. The former occurs mainly by active Na + dependency whereas the later is active H +-dependent with little affinity for tetra or higher peptides. Free amino acids are transported at different speed by saturable, competitive carriers with specificity for basic, acidic or neutral amino acids. Di and tripeptides have at least two carriers both electrogenic and H +-dependent. The basolateral membrane transport of amino acids is mostly by facilitated diffusion while for di-tripeptides it is an active anion exchange associated process. The main regulation of amino acids and di-tripeptide transport is the presence o substrate at the mucosal membrane with higher the substrate higher the absorption. Di and tripeptides are more efficiently absorbed than free amino acids which in turns are better absorbed than oligopeptides. So di-tripeptides result in better N-retention and is particularly useful in cases of lower intestinal absorption capacity. The non-absorbed peptides are digested and fermented by colonic bacteria resulting short-chain fatty acids, dicarboxylic acids, phenolic compounds and ammonia. Short-chain fatty acid provides energy for colonocytes and bacteria and the ammonia not fixed by bacteria returns to the liver for ureagenesis.

Purification and characterization of two β-glucosidases from the thermophilic fungus Thermoascus aurantiacus

β-Glucosidase from the fungus Thermoascus aurantiacus grown on semi-solid fermentation medium (using ground corncob as substrate) was partially purified in 5 steps-ultrafiltration, ethanol precipitation, gel filtration and 2 anion exchange chromatography runs, and characterized. After the first anion exchange chromatography, β-glucosidase activity was eluted in 3 peaks (Gl-1, Gl-2, Gl-3). Only the Gl-2 and Gl-3 fractions were adsorbed on the gel matrix. Gl-2 and Gl-3 exhibited optimum pH at 4.5 and 4.0, respectively. The temperature optimum of both glucosidases was at 75-80°C. The pH stability of Gl-2 (4.0-9.0) was higher than Gl-3 (5.5-8.5); both enzyme activities showed similar patterns of thermostability. Under conditions of denaturing gel chromatography the molar mass of Gl-2 and Gl-3 was 175 and 157 kDa, respectively. Using 4-nitrophenyl β-D-glucopyranoside as substrate, Km values of 1.17 ± 0.35 and 1.38 ± 0.86 mmol/L were determined for Gl-2 and Gl-3, respectively. Both enzymes were inhibited by Ag+ and stimulated by Ca2+.