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Allen, Sara; Shea, John M.; Felmet, Tara; Gadra, Jessica; Dehn, Paula F.

doi:10.1023/a:1017585308255

Cited by 60 publications

(13 citation statements)

References 5 publications

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“…The lysis solution has been demonstrated to effectively separate protein thiols and nonprotein thiols in attached cells by extracting nonprotein thiols into the supernatant. 18 The supernatant of the bottom four rows was transferred to a different 96-well plate and treated with the GUALY’s reagent derivatizing solution for nonprotein thiols determination. The remaining protein precipitates in the bottom four rows were thoroughly washed to remove nonprotein thiols followed by addition of the GUALY’s reagent derivatizing solution before protein thiols were determined.…”

Section: Results and Discussionmentioning

confidence: 99%

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Rapid and Thiol-Specific High-Throughput Assay for Simultaneous Relative Quantification of Total Thiols, Protein Thiols, and Nonprotein Thiols in Cells

Yang

Guan

2014

Anal. Chem.

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Thiol groups in biological molecules play a significant role in various physiological functions and pathological conditions. Thiols are divided into two major groups: protein thiols and nonprotein thiols. Numerous methods have been reported for thiol assays. Most of these methods have been developed for glutathione, the principal nonprotein thiol, despite the fact that cellular protein thiols are more abundant than glutathione. Further, these methods usually involve a process of biological sample preparation followed by a separation method, and they are time-consuming. We reported previously a series of thiol-specific fluorogenic benzofurazan sulfides. These nonfluorescent benzofurazan sulfides react rapidly and specifically with a thiol to form a strong fluorescent thiol adduct. The rapid reaction, thiol-specific and fluorogenic nature of the sulfides successfully yielded an application of one of the sulfides for relative quantitation of total thiols in live cells through fluorescence microscopy. In this work, we employed the same compound to develop the first high-throughput method for simultaneous monitoring of protein thiols, nonprotein thiols, and total thiols in cells in a 96-well plate on a fluorescence microplate reader at λex = 430 nm and λem = 520 nm, respectively. The method is rapid and sensitive, and has been validated by an HPLC thiol assay method. The method can detect thiols with cell concentrations as low as 500 cells/well. We also demonstrated that the method can readily monitor changes in cellular thiol levels. Although the method cannot provide an absolute quantification for thiols because fluorescence intensity of different thiol adducts varies, it provides an accurate measurement of relative quantification, relative to the control. The method will be a valuable tool in thiol-related biomedical/pharmaceutical research.

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Section: Results and Discussionmentioning

confidence: 99%

“…The cell lysis solution was a solution of 5% (w/v) sulfosalicylic acid in deionized water containing 0.1% (v/v) Triton X-100. 18 The 3% (w/v) sulfosalicylic acid solution was prepared by dissolving sulfosalicylic acid in deionized water. N -Ethylmaleimide (NEM) was prepared as a 5 mM stock solution in deionized water.…”

Section: Experimental Sectionmentioning

confidence: 99%

Rapid and Thiol-Specific High-Throughput Assay for Simultaneous Relative Quantification of Total Thiols, Protein Thiols, and Nonprotein Thiols in Cells

Yang

Guan

2014

Anal. Chem.

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“…The synthesis of TBOP is presented as Electronic Supplementary Material (ESM). Sulfosalicylic acid (SSA) cell lysis solution was prepared as a 5% (w/v) solution in deionized water containing 0.1% (v/v) Triton X-100 [31]. TBOP’s derivatizing solution was prepared from its stock solution (1 mM in acetonitrile) as a 0.1 mM solution in phosphate buffer (0.45 M, pH 7.9) containing 1.8% SDS.…”

Section: Methodsmentioning

confidence: 99%

Non-protein thiol imaging and quantification in live cells with a novel benzofurazan sulfide triphenylphosphonium fluorogenic compound

Yang

Guan

2017

Anal Bioanal Chem

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Thiols (-SH) play various roles in biological systems. They are divided into protein thiols (PSH) and non-protein thiols (NPSH). Due to the significant roles thiols play in various physiological/pathological functions, numerous analytical methods have been developed for thiol assays. Most of these methods are developed for glutathione, the major form of NPSH. Majority of these methods require tissue/cell homogenization before analysis. Due to a lack of effective thiol specific fluorescent/fluorogenic reagents, methods for imaging and quantifying thiols in live cells are limited. Determination of an analyte in live cells can reveal information that cannot be revealed by analysis of cell homogenates. Previously, we reported a thiol specific thiol-sulfide exchange reaction. Based on this reaction, a benzofurazan sulfide thiol specific fluorogenic reagent was developed. The reagent was able to effectively image and quantify total thiols (PSH+NPSH) in live cells through fluorescence microscopy. The reagent was later named as GUALY’s reagent. Here we would like to report an extension of the work by synthesizing a novel benzofurazan sulfide triphenylphosphonium derivative [(((7,7′-thiobis(benzo[c][1,2,5]oxadiazole-4,4′-sulfonyl))bis(methylazanediyl))bis(butane-4,1-diyl))bis(triphenylphosphonium) (TBOP)]. Like GUALY’s reagent, TBOP is a thiol specific fluorogenic agent that is non-fluorescent but forms fluorescent thiol adducts in a thiol-specific fashion. Different than GUALY’s reagent, TBOP reacts only with NPSH but not with PSH. TBOP was effectively used to image and quantify NPSH in live cells using fluorescence microscopy. TBOP is a complementary reagent to GUALY’s reagent in determining the roles of PSH, NPSH, and total thiols in thiol-related physiological/pathological functions in live cells through fluorescence microscopy.

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“…GSH and GSSG intracellular levels were measured by method described by Allen et al with few modifications [54]. Briefly, cells were exposed to Tat alone or in combination with NAC and then lysed with Triton X-100.…”

Section: Methodsmentioning

confidence: 99%

The HIV-1 Transactivator Factor (Tat) Induces Enterocyte Apoptosis through a Redox-Mediated Mechanism

et al. 2011

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The intestinal mucosa is an important target of human immunodeficiency virus (HIV) infection. HIV virus induces CD4+ T cell loss and epithelial damage which results in increased intestinal permeability. The mechanisms involved in nutrient malabsorption and alterations of intestinal mucosal architecture are unknown. We previously demonstrated that HIV-1 transactivator factor (Tat) induces an enterotoxic effect on intestinal epithelial cells that could be responsible for HIV-associated diarrhea. Since oxidative stress is implicated in the pathogenesis and morbidity of HIV infection, we evaluated whether Tat induces apoptosis of human enterocytes through oxidative stress, and whether the antioxidant N-acetylcysteine (NAC) could prevent it. Caco-2 and HT29 cells or human intestinal mucosa specimens were exposed to Tat alone or combined with NAC. In an in-vitro cell model, Tat increased the generation of reactive oxygen species and decreased antioxidant defenses as judged by a reduction in catalase activity and a reduced (GSH)/oxidized (GSSG) glutathione ratio. Tat also induced cytochrome c release from mitochondria to cytosol, and caspase-3 activation. Rectal dialysis samples from HIV-infected patients were positive for the oxidative stress marker 8-hydroxy-2′-deoxyguanosine. GSH/GSSG imbalance and apoptosis occurred in jejunal specimens from HIV-positive patients at baseline and from HIV-negative specimens exposed to Tat. Experiments with neutralizing anti-Tat antibodies showed that these effects were direct and specific. Pre-treatment with NAC prevented Tat-induced apoptosis and restored the glutathione balance in both the in-vitro and the ex-vivo model. These findings indicate that oxidative stress is one of the mechanism involved in HIV-intestinal disease.

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Cited by 60 publications

References 5 publications

Rapid and Thiol-Specific High-Throughput Assay for Simultaneous Relative Quantification of Total Thiols, Protein Thiols, and Nonprotein Thiols in Cells

Rapid and Thiol-Specific High-Throughput Assay for Simultaneous Relative Quantification of Total Thiols, Protein Thiols, and Nonprotein Thiols in Cells

Non-protein thiol imaging and quantification in live cells with a novel benzofurazan sulfide triphenylphosphonium fluorogenic compound

The HIV-1 Transactivator Factor (Tat) Induces Enterocyte Apoptosis through a Redox-Mediated Mechanism

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