Adaptation of the bicinchoninic acid protein assay for use with microtiter plates and sucrose gradient fractions

Redinbaugh, Margaret G.; Turley, Rickie B.

doi:10.1016/0003-2697(86)90091-6

Cited by 434 publications

(149 citation statements)

References 8 publications

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“…Aliquots were stored at (708C until use. An aliquot was used to determine the protein content by the bicinchoninic acid protein quantification method according to Redinbaugh & Turley (1986).…”

Section: Methodsmentioning

confidence: 99%

The role of selected cytochrome P450 enzymes on the bioactivation of aflatoxin B1 by duck liver microsomes

Diaz¹,

Murcia²,

Cepeda³

et al. 2010

Avian Pathology

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A study was conducted to determine the cytochrome (CYP) P450 enzymes responsible for the bioactivation of aflatoxin B1 (AFB1) into its epoxide form (AFBO) in duck liver microsomes. Six male and six female 6-week-old Pekin ducks were used. The biochemical toxicology strategies applied included the use of selective inhibitors, prototype substrate activity for specific human P450s, correlation between aflatoxin bioactivation and enzymatic activity of prototype substrates, and the expression of specific CYP450 enzymes using antibodies against human CYP450s. Enzymatic activity was detected for the duck orthologues CYP1A1/2, CYP2A6 and CYP3A4 but not for the CYP2D6 orthologue. Immunoreactive proteins for CYP1A1, CYP2A6 and CYP3A4 were also detected. Inhibition studies suggested that the duck turkey CYP2A6 orthologue and, to a lesser extent, the CYP1A1 orthologue are involved in the bioactivation of AFB1. Correlation studies, however, suggest that CYP3A4, CYP2A6 and CYP1A1/2 are all involved in AFBO formation. The finding that four CYP enzymes may be involved in AFB1 bioactivation in ducks could explain the high sensitivity of this species to AFB1. Further studies are needed to fully elucidate the phase I hepatic metabolism of AFB1 in ducks, the only poultry species that develops hepatic cancer from AFB1 exposure.

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Section: Methodsmentioning

confidence: 99%

The role of selected cytochrome P450 enzymes on the bioactivation of aflatoxin B1 by duck liver microsomes

Diaz¹,

Murcia²,

Cepeda³

et al. 2010

Avian Pathology

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“…Electron microscopy (13,28), determinations of citrate synthase activity (25,26), and protein (29) were carried out as described.…”

Section: Methodsmentioning

confidence: 99%

Glutathione is required for intestinal function.

Mårtensson

Jain²,

Meister³

1990

Proc. Natl. Acad. Sci. U.S.A.

257

167

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Glutathione (GSH) deficiency produced in mice by giving buthionine sulfoximine leads to severe degeneration of the epithelial cells of the jejunum and colon. This is prevented by giving GSH monoester (orally or i.p.) and also by giving GSH (orally, but not i.p.). The i.p. administration leads to high plasma levels of GSH but does not appreciably increase GSH levels in intestinal mucosa or pancreas. These and previous studies on lens, lung, lymphocytes, liver, heart, and skeletal muscle indicate that there is very little, if any, transport of intact GSH from plasma to these tissues. Cells can use extracellular GSH by a pathway involving its cleavage, uptake of products and intracellular GSH synthesis. Epithelial cells of the gastrointestinal tract may use this pathway and can also take up lumenal GSH (which arises partly from the bile) by a mechanism(s) that may involve transport of dipeptides or of GSH. It is suggested that biliary GSH normally functions in the protection of intestinal mucosa. Administration of GSH may be protective of the gastrointestinal epithelium and may also serve as a good source of cysteine moieties for intracellular GSH synthesis in the gastrointestinal tract and in other tissues. Administration of GSH delivery agents such as GSH esters is more effective than administration of GSH in increasing cellular and mitochondrial levels of GSH.Administration to experimental animals of L-buthionine-SR-sulfoximine (BSO), a transition-state inhibitor of yglutamylcysteine synthetase, leads to decreased synthesis of glutathione (GSH) in many tissues (1-4). Decreased cellular GSH levels and capacity for GSH synthesis sensitize cells to radiation and to certain drugs; these effects provide a potentially useful approach in cancer therapy (5-12). Administration of BSO offers several advantages over other methods of cellular GSH depletion, one of which is that it makes possible the selective decrease of cellular GSH for relatively long periods, thus facilitating study of the effects of GSH deficiency on cell structure and function. Administration of BSO to mice for 9-21 days, without application of stress, led to marked structural changes in skeletal muscle (13), lung (14), and epithelial cells ofnewborn lens (15); such effects were not found in heart (13) and liver (14). Mitochondrial damage is found in tissues affected by ; the mitochondrial levels of GSH in tissues markedly affected by GSH depletion (lung, muscle, lens) were -20%o of the untreated controls, whereas in heart and liver the mitochondrial GSH levels were >40% of the controls. Mitochondrial and other cellular damage was prevented by simultaneous administration of GSH monoester but not by administration of GSH. GSH monoesters can enter cells effectively and are converted to GSH intracellularly (16)(17)(18)(19). In contrast, GSH is poorly transported into cells.These studies were stimulated by the observation (13) that administration of BSO to 28-to 30-g mice for 9-21 days led to a marked decrease in weight gain and that simultaneous ...

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“…The protein concentration in all preparations was determined by the bicinchoninic acid method (BCA) in microtiter plates according to the method of REDINBAUGH & TURLEY 25 . Bovine serum albumin was used as a protein standard.…”

Section: Characterization Of the Excretion Secretion Productsmentioning

confidence: 99%

Excretion-secretion products and proteases from live Sporothrix schenckii yeast phase: immunological detection and cleavage of human IgG

Rosa

Gezuele

Calegari

et al. 2009

Rev. Inst. Med. trop. S. Paulo

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suMMARYAntigenic preparations from Sporothrix schenckii usually involve materials from mixed cultures of yeast and mycelia presenting cross-reactions with other deep mycoses. We have standardized pure yeast phase with high viability of the cells suitable to obtain specific excretion-secretion products without somatic contaminations. These excretion-secretion products were highly immunogenic and did not produce noticeable cross-reactions in either double immunodiffusion or Western blot. The antigenic preparation consists mainly of proteins with molecular weights between 40 and 70 kDa, some of them with proteolytic activity in mild acidic conditions. We also observed cathepsin-like activity at two days of culture and chymotrypsin-like activity at four days of culture consistent with the change in concentration of different secreted proteins. The proteases were able to cleave different subclasses of human IgG suggesting a sequential production of antigens and molecules that could interact and interfere with the immune response of the host.

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Adaptation of the bicinchoninic acid protein assay for use with microtiter plates and sucrose gradient fractions

Cited by 434 publications

References 8 publications

The role of selected cytochrome P450 enzymes on the bioactivation of aflatoxin B1 by duck liver microsomes

The role of selected cytochrome P450 enzymes on the bioactivation of aflatoxin B1 by duck liver microsomes

Glutathione is required for intestinal function.

Excretion-secretion products and proteases from live Sporothrix schenckii yeast phase: immunological detection and cleavage of human IgG

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