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Force-field parametrization and molecular dynamics simulations of Congo red.

Congo red, a diazo dye widely used in medical diagnosis, is known to form supramolecular systems in solution. Such a supramolecular system may interact with various proteins. In order to examine the nature of such complexes empirical force field parameters for the Congo red molecule were developed. The parametrization of bonding terms closely followed the methodology used in the development of the charmm22 force field, except for the calculation of charges. Point charges were calculated from a fit to a quantum mechanically derived electrostatic potential using the CHELP-BOW method. Obtained parameters were tested in a series of molecular dynamics simulations of both a single molecule and a micelle composed of Congo red molecules. It is shown that newly developed parameters define a stable minimum on the hypersurface of the potential energy and crystal and ab initio geometries and rotational barriers are well reproduced. Furthermore, rotations around C-N bonds are similar to torsional vibrations observed in crystals of diphenyl-diazene, which confirms that the flexibility of the molecule is correct. Comparison of results obtained from micelles molecular dynamics simulations with experimental data shows that the thermal dependence of micelle creation is well reproduced.

Biphenyl Compounds↗

Comparison of Sirius red and Congo red as stains for amyloid in animal tissues.

Sirius red and Congo red were compared for specificity and sensitivity of amyloid staining in animal and human material. Previously described advantages of Sirius red as an amyloid dye were confirmed, as well as its disadvantage of lack of ultraviolet fluorescence. Two further disadvantages of Sirius red were discovered, both relating to animal material: (a) its unexpectedly weak staining of early experimentally induced amyloid deposits and (b) frequent uncontrollable nonspecific staining of fibrous tissues. It is therefore concluded that, overall, Congo red used by the improved alkaline technique of Puchtler, Sweat and Levine (1962) remains the best single method for demonstration of amyloid in both human and animal tissues.

Amyloid↗

Inhibition of formation of protease-resistant prion protein by Trypan Blue, Sirius Red and other Congo Red analogs.

Five compounds related to Congo Red were found to inhibit generation of protease-resistant prion protein in a cell-free system. In this assay Trypan Blue, Evans Blue, Sirius Red F3B, Primuline and Thioflavin-S were all more inhibitory than Congo Red itself. In scrapie-infected mouse neuroblastoma cells one compound, Sirius Red F3B, was capable of blocking the formation of protease-resistant prion protein to a similar extent as Congo Red; however, the other four compounds were less effective. Some of these compounds should be considered for testing in TSE disease models in live animals.

Animals↗

Binding of the protease-sensitive form of PrP (prion protein) to sulfated glycosaminoglycan and congo red [corrected].

Congo red and certain sulfated glycans are potent inhibitors of protease-resistant PrP accumulation in scrapie-infected cells. One hypothesis is that these inhibitors act by blocking the association between protease-resistant PrP and sulfated glycosaminoglycans or proteoglycans (e.g., heparan sulfate proteoglycan) that is observed in amyloid plaques of scrapie-infected brain tissue. Accordingly, we have investigated whether the apparent precursor of protease-resistant PrP, protease-sensitive PrP, binds to Congo red and heparin, a highly sulfated glycosaminoglycan with an inhibitory potency like that of heparan sulfate. Protease-sensitive PrP released from the surface of mouse neuroblastoma cells bound to heparin-agarose and Congo red-glass beads. Sucrose density gradient fractionation provided evidence that at least some of the PrP capable of binding heparin-agarose was monomeric. Free Congo red blocked PrP binding to heparin and vice versa, suggesting that these ligands share a common binding site. The relative efficacies of pentosan polysulfate, Congo red, heparin, and chondroitin sulfate in blocking PrP binding to heparin-agarose corresponded with their previously demonstrated potencies in inhibiting protease-resistant PrP accumulation. These results are consistent with the idea that sulfated glycans and Congo red inhibit protease-resistant PrP accumulation by interfering with the interaction of PrP with an endogenous glycosaminoglycan or proteoglycan.

Animals↗

"Congo" red: out of Africa?

CONTEXT: Congo red is the essential histologic stain for demonstrating the presence of amyloidosis in fixed tissues. To the best of my knowledge, nothing has been written about why the stain is named "Congo." OBJECTIVE: To understand the etymology and history of the Congo red histologic stain. DESIGN: Primary sources were consulted extensively, including 19th-century corporate documents, newspapers, legal briefs, patents, memoirs, and scientific papers. SETTING: Sources were obtained from multiple university libraries and German corporate archives. RESULTS: To Europeans in 1885, the word Congo evoked exotic images of far-off central Africa known as The Dark Continent. The African Congo was also a political flashpoint during the Age of Colonialism. "Congo" red was introduced in Berlin in 1885 as the first of the economically lucrative direct textile dyes. A patent on Congo red was filed by the AGFA Corporation of Berlin 3 weeks after the conclusion of the well-publicized Berlin West Africa Conference. During these important diplomatic talks, German Chancellor Otto von Bismarck presided over a discussion of free trade issues in the Congo River basin. A challenge to AGFA's Congo red patent led to a precedent-setting decision in intellectual property law. CONCLUSIONS: The Congo red stain was named "Congo" for marketing purposes by a German textile dyestuff company in 1885, reflecting geopolitical current events of that time.

Africa↗

Clinical significance of Congo red test.

Clinical significance of Congo red test was studied, especially on its relation to the liver function and amyloidosis. No significant relationship was observed between Congo red test and Indocyanin green test in cases of various liver diseases except liver cirrhosis. Histological studies also revealed that there were no pathologic features specifically affecting Congo red index (CRI), even though CRI in cases of various liver diseases tended to be increased as compared with other diseases. As a cause of increased CRI which was seen most remarkably in cases of liver cirrhosis, obstructive change of the liver blood flow followed by the hepatic tissue damage is suspected. The concept that Congo red test is a kind of the hepatic excretory function test does not have, from our data, enough evidence. The present study confirmed our previous data on the fate of Congo red injected intravenously, which indicated that this test is one of the best tests available for RES function and this test is related mainly to Kupffer's cell rather than to the so-called hepatic excretory function. There were no cases which showed notable decrease of CRI in experimental amyloidosis and in clinical amyloidosis, and it can be said that Congo red test is not the best test for the diagnosis of amyloidosis.

Acute Disease↗

The congo red stain revisited.

The Congo red stain has undergone several modifications since it was first used by Bennhold in 1922 in order to increase the specificity for staining amyloid. Most of the laboratories in the United States use the method of Puchtler which uses alkaline Congo red solution. Some of the variables associated with the procedure were investigated by us. Our results showed the following: (1) amyloid showed green birefringence at all levels between 4 to 12 mu thick sections with better visualization of small deposits with increased thickness. Best results were obtained with 8 mu thick sections; (2) omission of the pretreatment with alkaline alcoholic solution of sodium chloride (NaCl) did not affect the sensitivity of the method; (3) the use of polar mounting media had no effect on amyloid and collagen birefringence; (4) 50 percent saturation of the Congo red staining solution with NaCl caused strong staining of collagen, elastic fibers and eosinophilic granules. In addition, collagen showed green birefringence and dichroism and its differentiation from amyloid became difficult; and (5) using the staining solution fully saturated with NaCl, no positive staining was seen with tissues other than amyloid. Collagen and elastic fibers showed red fluorescence which was of less intensity than amyloid. It is our conclusion that the method of Puchtler for detecting amyloid gives better results if the staining solution is fully saturated with NaCl. The pretreatment step may be deleted without compromising the quality of staining. Improved staining of amyloid enhances the specificity of green birefringence, dichroism, and red fluorescence.

Amyloid↗

Is Congo red an amyloid-specific dye?

Congo red (CR) binding, monitored by characteristic yellow-green birefringence under crossed polarization has been used as a diagnostic test for the presence of amyloid in tissue sections for several decades. This assay is also widely used for the characterization of in vitro amyloid fibrils. In order to probe the structural specificity of Congo red binding to amyloid fibrils we have used an induced circular dichroism (CD) assay. Amyloid fibrils from insulin and the variable domain of Ig light chain demonstrate induced CD spectra upon binding to Congo red. Surprisingly, the native conformations of insulin and Ig light chain also induced Congo red circular dichroism, but with different spectral shapes than those from fibrils. In fact, a wide variety of native proteins exhibited induced CR circular dichroism indicating that CR bound to representative proteins from different classes of secondary structure such as alpha (citrate synthase), alpha + beta (lysozyme), beta (concavalin A), and parallel beta-helical proteins (pectate lyase). Partially folded intermediates of apomyoglobin induced different Congo red CD bands than the corresponding native conformation, however, no induced CD bands were observed with unfolded protein. Congo red was also found to induce oligomerization of native proteins, as demonstrated by covalent cross-linking and small angle x-ray scattering. Our data suggest that Congo red is sandwiched between two protein molecules causing protein oligomerization. The fact that Congo red binds to native, partially folded conformations and amyloid fibrils of several proteins shows that it must be used with caution as a diagnostic test for the presence of amyloid fibrils in vitro.

Amyloid↗

Selective staining of eosinophils and their immature precursors in tissue sections and autoradiographs with Congo red.

The use of Congo red as an elective stain for eosinophilic granulocytes and their precursors in tissue sections and autoradiographs is demonstrated and discussed. The 0.5% alcoholic Congo red solution of Highman, normally used for the detection of amyloid, may also be used with only minor changes. This simple method may aid in the diagnosis of special hematological problems and facilitates the recognition of eosinophil granulocytes as well as proliferating and nonproliferating myelocytes in autoradiographs from paraffin sections.

Animals↗

Spectroscopic studies of Congo Red binding to RNA polymerase.

The azo dye Congo Red has a high affinity for nucleotide-binding enzymes. We have studied the binding of Congo Red to RNA polymerase by circular dichroism (CD) and difference absorption spectroscopy, steady-state kinetics, and nitrocellulose filter-binding. Induced CD shows that a large number of Congo Red molecules bind to the holoenzyme. CD also demonstrates that the core enzyme at low ionic strengths has a distinctive Congo Red binding site which is not present in the holoenzyme, nor in the core enzyme at higher ionic strengths or in the presence of poly(dT). CD studies indicate that Congo Red can readily displace double-stranded polynucleotides (T7 DNA or poly[d(A-T)] from RNA polymerase. Single-stranded DNA (poly(dT) and T7 DNA in open complexes) is not displaced from RNA polymerase except at high Congo Red concentrations. Both kinetics and nitrocellulose filter-binding measurements support this conclusion. Difference spectra indicate that the bound Congo Red molecules undergo stacking. We postulate that RNA polymerase binds Congo Red in a region with which a segment of DNA normally interacts, and that Congo Red is a potent inhibitor because the stacked dye has a polyanionic character.

Binding Sites↗

Congo red and protein aggregation in neurodegenerative diseases.

Congo red is a commonly used histological dye for amyloid detection. The specificity of this staining results from Congo red's affinity for binding to fibril proteins enriched in beta-sheet conformation. Unexpectedly, recent investigations indicate that the dye also possesses the capacity to interfere with processes of protein misfolding and aggregation, stabilizing native protein monomers or partially folded intermediates, while reducing concentration of more toxic protein oligomers. Inhibitory effects of Congo red upon amyloid toxicity may also range from blockade of channel formation and interference with glycosaminoglycans binding or immune functions, to the modulation of gene expression. Particularly, Congo red exhibits ameliorative effect in models of neurodegenerative disorders, such as Alzheimer's, Parkinson's, Huntington's and prion diseases. Another interesting application of Congo red analogues is the development of imaging probes. Based on their small molecular size and penetrability through blood-brain barrier, Congo red congeners can be used for both antemortem and in vivo visualization and quantification of brain amyloids. Therefore, understanding mechanisms involved in dye-amyloidal fibril binding and inhibition of aggregation will provide instructive guides for the design of future compounds, potentially useful for monitoring and treating neurodegenerative diseases.

Amyloid beta-Peptides↗

Congo Red Absorption by Rhizobium leguminosarum.

Congo red absorption is generally considered a contraindication of Rhizobium. However, R. leguminosarum takes up the dye on yeast extract-mannitol agar. The uptake of congo red varies among strains of R. leguminosarum, as shown elsewhere with strains of R. trifolii and R. meliloti. Congo red absorption does not distinguish rhizobia from other bacteria, but may be useful as a strain marker.

Journal Article↗

Protein distorsion-derived mechanism of signal discrimination in monocytes revealed using Congo red to stain activated cells.

The supramolecular dye Congo red was used to check whether monocyte activation may be mediated by a torsion-dependent mechanism preventing transduction of weak random signals in cell contacts in a way corresponding to the discrimination mechanism found in complement fixation by immune complexes. Tight cell-cell contacts generating torsional effects may be expected to produce alteration of receptor structure, making them accessible for binding of supramolecular dyes. In this study, Congo red was used to observe the binding accessibility of (1) monocytes (human) induced by contact with cancer cells (HCV29T, human), (2) monocytes (mouse) stimulated by interaction with heat-aggregated IgG and (3) monocytes (mouse) activated by rosetting in the presence of an SRBC-anti-SRBC system. Microscopic studies confirmed the activation of monocytes manifested by their clustering and Congo red binding, but only tightly clustered cells appeared to attach the dye on the surface. Usually not the whole cell surface is found to be engaged in dye complexation. Staining occurs predominantly on the interfaces of reacting cells, making probable the suggestion that cell adhesion receptors are involved in dye binding. The cells in the central areas of tight clusters undergo accelerated death. In the presence of Congo red they are easily recognized as intensely fluorescent. The characteristic localization of dead cells in the central area of clusters indicates that death is not random but results from cell activation. The role of Congo red in this process remains to be clarified. The staining characteristics of monocytes after application of Congo red probably discloses the initial step in signal transduction generated by torsional movements in receptor proteins.

Animals↗

Differentiation of phase I and variant strains of Bordetella pertussis on Congo red media.

The addition of Congo red, Trypan blue or haemin to the growth medium allowed the differentiation of phase-I and variant strains of Bordetella pertussis. Phase-I strains produced red (CR+), blue or dark brown colonies on a modified cyclodextrin solid medium containing Congo red, Trypan blue or haemin, respectively, whereas variant (Vir- and phase IV) strains grew as pale (CR-) colonies. Spontaneous CR- variants were isolated and characterised and had a phenotype like that of Vir- or phenotypically modulated, C-mode strains in that they did not produce the haemolysin, haemagglutinin(s), histamine-sensitising factor (pertussis toxin), heat-labile toxin and two major envelope polypeptides associated with phase-I strains. Two such variants had reduced virulence for mice. CR+ strains, when grown on a high nicotinic acid medium to induce modulation, gave CR- colonies. Thus the CR+ phenotype is a characteristic of phase-I B. pertussis and its expression appears to be controlled in a manner similar to that of other phase I-related factors. CR- variants of B. parapertussis and B. bronchiseptica were also deficient in these factors. Four isolates of B. avium were CR-.

Bordetella pertussis↗