RESUMO
Levels of essential metals in human breast milk (HBM) have been determined by different analytical techniques, but there is few woks about human whey milk fractions. However, the current trend lies in metalloproteomic and identification of different metalloproteins. In this sense, native separative techniques (N-PAGE and SEC) coupled to ICP-MS provide us with valuable information. Besides it is necessary the development of new methodologies in order to determine with accuracy and precision the profile of such metals and metalloproteins in the different whey protein fractions of HBM. Thus, the aim of this work was to develop a new method for metals and metalloproteins determination by SEC-ICP-MS in whey protein fractions of HBM. Human whey fractions were obtained of HBM samples by ultracentrifugation. Then, protein fractions of whey milk were separated by SEC coupled to ICP-MS for metalloproteins and Mn, Co, Cu and Se quantification. Besides, protein profile of whey milk was determined by N-PAGE and computer assisted image analysis. SEC-ICP-MS results indicated that first and second protein fractions showed detectable levels of the Mn, Co, Cu, and Se. Protein profile determined by N-PAGE and image analysis showed that molecular weight of protein fractions ranged between 68,878-1,228.277â¯Da. In this work, metalloproteins were analyzed by SEC coupled to ICP-MS, with adequate sensitivity and accuracy. Our study has shown the presence of Mn, Co, Cu and Se bound to two protein fractions in whey milk of HBM. Metals levels analyzed were within the ranges reported in the literature.
Assuntos
Metaloproteínas/análise , Metais/análise , Micronutrientes/análise , Leite Humano/química , Adulto , Cromatografia em Gel/instrumentação , Cromatografia em Gel/métodos , Estudos de Viabilidade , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Espectrometria de Massas/instrumentação , Espectrometria de Massas/métodos , Eletroforese em Gel de Poliacrilamida Nativa/instrumentação , Eletroforese em Gel de Poliacrilamida Nativa/métodos , Sensibilidade e Especificidade , Proteínas do Soro do Leite/análiseRESUMO
Bioremediation is a strategy to mitigate environmental impacts of hazardous pollutants from anthropogenic sources. Natural byproducts, including agroindustrial wastes (AW) can be used to induce enzyme biosynthesis, leading up to enhancement of pollutants degradation process. Therefore, this study aimed to evaluate the use of cupuaçu, Theobroma grandiflorum AW as Pycnoporus sanguineus Laccase (Lac) inducer in order to promote 17-α-ethinylestradiol (EE2) bioremediation. The macro and micro-nutrients levels of cupuaçu AWs were evaluated in order to establish further correlations with enzymatic biosynthesis induction. The fungus was cultivated for 7 days in temperature of 28 ± 2 °C and agitation of 150 rpm. For bioremediation, Lac enzymatic extract was added to EE2 solution (10 µg mL-1) and the percentage of removal was evaluated by HPLC after 1-24 hr of reaction. At optimized conditions, the enzyme extract production was remarkably enhanced by adding only 1% (w/v) of cupuaçu AW. Lac activity reached 1642 U mL-1 on the 6th day of culture, which was higher than positive control (511 U mL-1). 86% of EE2 removal was reached after 4 hr, and after 8 hr of reaction, 96.5% was removed. Analysis by direct infusion in MS-ESI-TOF exhibited intermediary compounds formed by radical hydroxilation.
Assuntos
Biodegradação Ambiental , Cacau/metabolismo , Poluentes Ambientais/metabolismo , Etinilestradiol/metabolismo , Lacase/biossíntese , Pycnoporus/enzimologia , Meios de Cultura/química , Indução Enzimática , Proteínas Fúngicas/análise , Eletroforese em Gel de Poliacrilamida Nativa , Açúcares/análise , TemperaturaRESUMO
Background: Defense-related anti-oxidative response is a vital defense mechanism of plants against pathogen invasion. Ralstonia solanacearum is an important phytopathogen. Bacterial wilt caused by R. solanacearum is the most destructive disease and causes severe losses in patchouli, an important aromatic and medicinal plant in Southeast Asia. The present study evaluated the defense response of patchouli inoculated with virulent R. solanacearum. Results: Results showed that the basic enzymatic activities differed not only between the leaves and stems but also between the upper and lower parts of the same organ of patchouli. POD, SOD, PPO, and PAL enzymatic activities were significantly elevated in leaves and stems from patchouli inoculated with R. solanacearum compared to those in control. The variation magnitude and rate of POD, PPO, and PAL activities were more obvious than those of SOD in patchouli inoculated with R. solanacearum. PAGE isoenzymatic analysis showed that there were one new POD band and two new SOD bands elicited, and at least two isoformic POD bands and two SOD bands were observably intensified compared to the corresponding control. Conclusion: Our results suggest that not only defense-related enzymatic activities were elevated but also the new isoenzymatic isoforms were induced in patchouli inoculated with R. solanacearum.
Assuntos
Ralstonia solanacearum/patogenicidade , Pogostemon/enzimologia , Pogostemon/microbiologia , Fenilalanina Amônia-Liase/metabolismo , Superóxido Dismutase/metabolismo , Virulência , Catecol Oxidase/metabolismo , Peroxidase/metabolismo , Ralstonia solanacearum/fisiologia , Eletroforese em Gel de Poliacrilamida , Enzimas/imunologia , Enzimas/metabolismo , Eletroforese em Gel de Poliacrilamida Nativa , Pogostemon/imunologia , AntioxidantesRESUMO
Locusts are able to digest the cellulose of Gramineae plants, resulting in their being considered as major crop pests. To illustrate the mechanism involved in cellulose digestion, the cellulolytic activity and zymography in the gut contents of 16 locust species were determined using carboxymethyl cellulose (CMC) as substrate. The diversity of gut symbiotic bacteria was studied using denaturing gradient gel electrophoresis (DGGE). The results showed that high CMC activity was present in Acrididae gut fluid (mean 356.4 U/g proteins). Of the 5 locust species, Oxya chinensis had the highest diversity of intestinal symbiotic bacteria, characterized by the DGGE profile containing more than 20 bands of 16S rRNA. Klebsiella pneumoniae, in the gut of Locusta migratoria manilensis, was identified as the most abundant symbiotic bacterium by DNA sequencing, with a relative abundance of 19.74%. In comparison, Methylobacterium sp was the most dominant species in the Atractomorpha sinensis gut, with a relative abundance of 29.04%. The results indicated that the cellulolytic enzymes and gut microbial communities probably reflected their phylogenetic relationship with different locust species and associated feeding strategies.
Assuntos
Bactérias/metabolismo , Celulose/metabolismo , Sistema Digestório/microbiologia , Gafanhotos/microbiologia , Simbiose , Animais , Bactérias/genética , Sequência de Bases , Primers do DNA , Hidrólise , Eletroforese em Gel de Poliacrilamida Nativa , RNA Ribossômico 16S/genéticaRESUMO
Deletion of the yeast mitochondrial gene COX2 encoding subunit 2 (Cox2) of cytochrome c oxidase (CcO) results in loss of respiration (Δcox2 strain). Supekova et al. (2010) [1] transformed a Δcox2 strain with a vector expressing Cox2 with a mitochondrial targeting sequence (MTS) and the point mutation W56R (Cox2(W56R)), restoring respiratory growth. Here, the CcO carrying the allotopically-expressed Cox2(W56R) was characterized. Yeast mitochondria from the wild-type (WT) and the Δcox2+Cox2(W56R) strains were subjected to Blue Native electrophoresis. In-gel activity of CcO and spectroscopic quantitation of cytochromes revealed that only 60% of CcO is present in the complemented strain, and that less CcO is found associated in supercomplexes as compared to WT. CcOs from the WT and the mutant exhibited similar subunit composition, although activity was 20-25% lower in the enzyme containing Cox2(W56R) than in the one with Cox2(WT). Tandem mass spectrometry confirmed that W(56) was substituted by R(56) in Cox2(W56R). In addition, Cox2(W56R) exhibited the same N-terminus than Cox2(WT), indicating that the MTS of Oxa1 and the leader sequence of 15 residues were removed from Cox2(W56R) during maturation. Thus, Cox2(W56R) is identical to Cox2(WT) except for the point mutation W56R. Mitochondrial Cox1 synthesis is strongly reduced in Δcox2 mutants, but the Cox2(W56R) complemented strain led to full restoration of Cox1 synthesis. We conclude that the cytosol-synthesized Cox2(W56R) follows a rate-limiting process of import, maturation or assembly that yields lower steady-state levels of CcO. Still, the allotopically-expressed Cox2(W56R) restores CcO activity and allows mitochondrial Cox1 synthesis to advance at WT levels.