Mn(II)-Mn(III)-Mn(IV) Redox Cycling Inhibits the Removal of Methylparaben and Acetaminophen Mediated by Horseradish Peroxidase: New Insights into the Mechanism

DNA methylation is a cell-type-specific epigenetic marker that is essential for transcriptional regulation, silencing of repetitive DNA and genomic imprinting. It is also responsible for the pathogenesis of many diseases, including cancers. Herein, we present a simple approach for quantifying global DNA methylation in ovarian cancer patient plasma samples based on a new class of biopolymer nanobeads. Our approach utilises the immune capture of target DNA and electrochemical quantification of global DNA methylation level within the targets in a three-step strategy that involves (i) initial preparation of target single-stranded DNA (ss-DNA) from the plasma of the patients’ samples, (ii) direct adsorption of polymer nanobeads on the surface of a bare screen-printed gold electrode (SPE-Au) followed by the immobilisation of 5-methylcytosine (5mC)-horseradish peroxidase (HRP) antibody, and (iii) immune capture of target ss-DNA onto the electrode-bound PHB/5mC-HRP antibody conjugates and their subsequent qualification using the hydrogen peroxide/horseradish peroxidase/hydroquinone (H2O2/HRP/HQ) redox cycling system. In the presence of methylated DNA, the enzymatically produced (in situ) metabolites, i.e., benzoquinone (BQ), binds irreversibly to cellular DNA resulting in the unstable formation of DNA adducts and induced oxidative DNA strand breakage. These events reduce the available BQ in the system to support the redox cycling process and sequel DNA saturation on the platform, subsequently causing high Coulombic repulsion between BQ and negatively charged nucleotide strands. Thus, the increase in methylation levels on the electrode surface is inversely proportional to the current response. The method could successfully detect as low as 5% methylation level. In addition, the assay showed good reproducibility (% RSD ≤ 5%) and specificity by analysing various levels of methylation in cell lines and plasma DNA samples from patients with ovarian cancer. We envision that our bioengineered polymer nanobeads with high surface modification versatility could be a useful alternative platform for the electrochemical detection of varying molecular biomarkers.

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Effects of Vincristine on Endothelial Microtubules in the Spinal Cord

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100090270 ◽

1976 ◽

Vol 34 ◽

pp. 58-59

Author(s):

John L. Beggs ◽

John D. Waggener ◽

Wanda Miller

Keyword(s):

Spinal Cord ◽

Biological Activity ◽

Horseradish Peroxidase ◽

Transport Mechanism ◽

Vasogenic Edema ◽

Vesicular Transport ◽

Close Proximity

Microtubules (MT) are versatile organelles participating in a wide variety of biological activity. MT involvement in the movement and transport of cytoplasmic components has been well documented. In the course of our study on trauma-induced vasogenic edema in the spinal cord we have concluded that endothelial vesicles contribute to the edema process. Using horseradish peroxidase as a vascular tracer, labeled endothelial vesicles were present in all situations expected if a vesicular transport mechanism was in operation. Frequently,labeled vesicles coalesced to form channels that appeared to traverse the endothelium. The presence of MT in close proximity to labeled vesicles sugg ested that MT may play a role in vesicular activity.

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Comparative strengths and weaknesses of peroxidase and colloidal gold in pre- and post-embedding immunolabeling techniques

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100129279 ◽

1987 ◽

Vol 45 ◽

pp. 1002-1005

Author(s):

D. R. Abrahamson ◽

P. L. St.John ◽

E. W. Perry

Keyword(s):

Electron Microscopy ◽

Horseradish Peroxidase ◽

Light Microscopy ◽

Colloidal Gold ◽

Light Microscope ◽

Ultrastructural Localization ◽

The Other ◽

Quantitative Studies ◽

Dense Suspension ◽

Antigen Localization

Antibodies coupled to tracers for electron microscopy have been instrumental in the ultrastructural localization of antigens within cells and tissues. Among the most popular tracers are horseradish peroxidase (HRP), an enzyme that yields an osmiophilic reaction product, and colloidal gold, an electron dense suspension of particles. Some advantages of IgG-HRP conjugates are that they are readily synthesized, relatively small, and the immunolabeling obtained in a given experiment can be evaluated in the light microscope. In contrast, colloidal gold conjugates are available in different size ranges and multiple labeling as well as quantitative studies can therefore be undertaken through particle counting. On the other hand, gold conjugates are generally larger than those of HRP but usually can not be visualized with light microscopy. Concern has been raised, however, that HRP reaction product, which is exquisitely sensitive when generated properly, may in some cases distribute to sites distant from the original binding of the conjugate and therefore result in spurious antigen localization.

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Unlabeled Antibody Immunocytochemistry Applied to Murine Mammary Tumor Cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100115572 ◽

1975 ◽

Vol 33 ◽

pp. 390-391

Author(s):

Wm. J. Arnold ◽

J. Russo ◽

H. D. Soule ◽

M. A. Rich

Keyword(s):

Horseradish Peroxidase ◽

Mammary Tumor ◽

Covalent Binding ◽

Petri Dish ◽

Gamma Chain ◽

Mammary Tumor Virus ◽

Mammary Tumor Cells ◽

Murine Mammary Tumor ◽

Tumor Virus ◽

Unlabeled Antibody

Our studies of mammary tumor virus have included the application of the unlabeled antibody enzyme method of Sternberger to mammary tumor derived mouse cells in culture and observation with an electron microscope. The method avoids the extravagance of covalent binding of indicator molecules (horseradish peroxidase) with precious antibody locator molecules by relying instead upon specific antibody-antigen linkages. Our reagents included: Primary Antibody, rabbit anti-murine mammary tumor virus (MuMTV) which was antiserum 113 AV-2; Secondary Antibody, goat anti-rabbit IgG gamma chain (Cappel Laboratories); andthe Indicator, rabbit anti-horseradish peroxidase - horseradish peroxidase complex (PAP) (Cappel Labs.). Dilutions and washes were made in 0.05 M Tris 0.15 M saline buffered to pH 7.4. Cell monolayers, after light fixation in glutaraldehyde, were incubated in place by a protocol adapted from Sternberger and Graham and Karnovsky, then embedded by our usual method for monolayers. Reagents were confined to specific areas by neoprene 0-rings (Parker Seal Co.) reducing the amount of reagent needed to 50 microliters, 1/6th of that required to wet a 35 mm petri dish.

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