scholarly journals Binding of Fab-horseradish peroxidase conjugates by charge and not by immunospecificity.

1985 ◽  
Vol 33 (1) ◽  
pp. 55-58 ◽  
Author(s):  
R M Pino
Keyword(s):  
Horseradish Peroxidase ◽  
Isoelectric Focusing ◽  
Collagen Fibers ◽  
Enzyme Digestion ◽  
Nonspecific Binding ◽  
Anionic Sites ◽  
Bruch's Membrane ◽  
Bruch’S Membrane ◽  
Type Viii ◽  
Positively Charged

The influence of horseradish peroxidase (HRP) charge on Fab-HRP conjugates was investigated. Rabbit nonimmune Fab coupled via periodate or glutaraldehyde to Sigma HRP type VI (pI greater than 10) were cationic (positively charged) as determined by analytical isoelectric focusing. These conjugates and HRP type VI alone stippled the basal laminae and collagen fibers in Bruch's membrane of the rat eye in a pattern identical to anionic (negative) sites. Binding was not present after the anionic sites were removed by enzyme digestion prior to immunolabeling or when HRP type VIII (anionic with pI 3.6) was used in an Fab-HRP conjugate or in an unbound form. These results indicate that anionic HRPs should be used in Fab-HRP preparations if a nonspecific binding to anionic sites is possible.

Vascular permeability in the rat eye to endogenous albumin and immunoglobulin G (IgG) examined by immunohistochemical methods.

1983 ◽  
Vol 31 (3) ◽  
pp. 411-416 ◽  
Author(s):  
R M Pino ◽  
C L Thouron
Keyword(s):  
Horseradish Peroxidase ◽  
Vascular Permeability ◽  
Immunoglobulin G ◽  
Protein A ◽  
Bruch's Membrane ◽  
Bruch’S Membrane ◽  
Background Staining ◽  
Horseradish Peroxidase Method ◽  
High Degree ◽  
Endogenous Albumin

Vascular permeability in the rat retina and choroid was examined by localizing endogenous albumin (radius, 35 A) and immunoglobulin G (IgG; radius, 55 A) by immunohistochemistry. Three techniques were used: protein A-horseradish peroxidase, peroxidase-antiperoxidase, and avidin-horseradish peroxidase. The protein A-horseradish peroxidase method yielded the least amount of tissue background staining with a high degree of reaction product found in blood vessels. With this method albumin was identified in retinal capillaries, the choriocapillaris, larger choroidal vessels, and in the stroma of the choroid. Very low levels were found in Bruch's membrane. Reaction product due to IgG was also present intravascularly, but little reaction product was present around the large vessels of the choroid and none was identified in Bruch's membrane. Comparisons were made between these localizations and those of intravenously injected hemeprotein tracers of similar size.

scholarly journals Location and chemical composition of anionic sites in Bruch's membrane of the rat.

1982 ◽  
Vol 30 (3) ◽  
pp. 245-252 ◽  
Author(s):  
R M Pino ◽  
E Essner ◽  
L C Pino
Keyword(s):  
Chemical Composition ◽  
Heparan Sulfate ◽  
Basal Lamina ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Ruthenium Red ◽  
Morphological Evidence ◽  
Anionic Sites ◽  
Bruch's Membrane ◽  
Bruch’S Membrane

The location and chemical composition of anionic sites in Bruch's membrane (BM) were examined using cationic probe molecules demonstrable in electron microscopic preparations and tissue digestion with specific degradative enzymes. Ruthenium red and native lysozyme revealed densities distributed at regular intervals in two major components of BM: the basal laminae of the retinal pigment epithelium (RPE) and choriocapillary endothelium (EN). Staining was not observed with succinylated lysozyme (anionic). Colloidal iron also failed to stain BM components. Following crude heparinase treatment at 43 degrees C (specific for heparan sulfate) anionic sites in the RPE basal lamina were not demonstrable with either ruthenium red or native lysozyme. Sites in the EN basal lamina were not affected. Chondroitinase treatment removed almost all of the ruthenium red-positive material in the EN basal lamina; lysozyme binding here was markedly reduced. No changes were observed in the RPE basal lamina after chondroitinase digestion. There was no morphological evidence for site removal by either neuraminidase or leech hyaluronidase, although a detachment of the RPE from BM often occurred after incubation of eye tissue in the latter. Pronase E removed all stainable material. These findings indicate that anionic sites in BM consist to a large extent of chondroitin sulfates and heparan sulfate.

Abcc6 deficiency in the mouse leads to calcification of collagen fibers in Bruch's membrane

2012 ◽  
Vol 104 ◽  
pp. 59-64 ◽  
Author(s):  
Theo G.M.F. Gorgels ◽  
Peter Teeling ◽  
Johannes D. Meeldijk ◽  
Suzan T.M. Nillesen ◽  
Allard C. van der Wal ◽  
...  
Keyword(s):  
Collagen Fibers ◽  
Bruch's Membrane ◽  
Bruch’S Membrane

scholarly journals FHL-1 interacts with human RPE cells through the α5β1 integrin and confers protection against oxidative stress

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Rawshan Choudhury ◽  
Nadhim Bayatti ◽  
Richard Scharff ◽  
Ewa Szula ◽  
Viranga Tilakaratna ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Fate ◽  
Retinal Pigment ◽  
Factor H ◽  
Age Related Macular Degeneration ◽  
Bruch's Membrane ◽  
Bruch’S Membrane ◽  
Rpe Cells ◽  
Age Related

AbstractRetinal pigment epithelial (RPE) cells that underlie the neurosensory retina are essential for the maintenance of photoreceptor cells and hence vision. Interactions between the RPE and their basement membrane, i.e. the inner layer of Bruch’s membrane, are essential for RPE cell health and function, but the signals induced by Bruch’s membrane engagement, and their contributions to RPE cell fate determination remain poorly defined. Here, we studied the functional role of the soluble complement regulator and component of Bruch’s membrane, Factor H-like protein 1 (FHL-1). Human primary RPE cells adhered to FHL-1 in a manner that was eliminated by either mutagenesis of the integrin-binding RGD motif in FHL-1 or by using competing antibodies directed against the α5 and β1 integrin subunits. These short-term experiments reveal an immediate protein-integrin interaction that were obtained from primary RPE cells and replicated using the hTERT-RPE1 cell line. Separate, longer term experiments utilising RNAseq analysis of hTERT-RPE1 cells bound to FHL-1, showed an increased expression of the heat-shock protein genes HSPA6, CRYAB, HSPA1A and HSPA1B when compared to cells bound to fibronectin (FN) or laminin (LA). Pathway analysis implicated changes in EIF2 signalling, the unfolded protein response, and mineralocorticoid receptor signalling as putative pathways. Subsequent cell survival assays using H2O2 to induce oxidative stress-induced cell death suggest hTERT-RPE1 cells had significantly greater protection when bound to FHL-1 or LA compared to plastic or FN. These data show a non-canonical role of FHL-1 in protecting RPE cells against oxidative stress and identifies a novel interaction that has implications for ocular diseases such as age-related macular degeneration.

Biological Multilayer Films Assembled by Redox Polymer and HRP on Screen-Printed Carbon Electrode

2012 ◽  
Vol 571 ◽  
pp. 56-59
Author(s):  
Yu Fang Sha ◽  
Mei Zhao ◽  
Ming Quan Yang ◽  
Hai Xin Bai ◽  
Man Zhao
Keyword(s):  
Horseradish Peroxidase ◽  
Electrostatic Interaction ◽  
Electrode Surface ◽  
Multilayer Films ◽  
Carbon Electrode ◽  
Layer By Layer ◽  
Redox Polymer ◽  
Screen Printed Carbon Electrode ◽  
Positively Charged ◽  
Screen Printed

Biological multilayer films of redox polymer and horseradish peroxidase (HRP) were successfully assembled on a screen-printed carbon electrode using layer-by-layer (LBL) assembled method based on the electrostatic interaction. The screen-printed carbon electrode surface was modified by the positively charged redox polymer, and the negatively charged HRP by LBL method.

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