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PubMed2001

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↗
PubMedDate not supplied

Congo red binding of Escherichia coli isolated from the cloacae of psittacine birds.

Binding of Congo red dye by Escherichia coli is associated with the pathogenicity of the organism. The purpose of the present study was to determine the incidence of Congo red binding exhibited by E. coli isolated from the cloacae of psittacine birds, to examine the association between the Congo red status of the E. coli isolates and the health status of birds, and to assess the potential value of Congo red binding as a screening test for identifying pathogenic strains of E. coli isolated form the cloacae of psittacine birds. Escherichia coli was isolated from the cloacae of 120/435 (28%) psittacine birds; 17/120 (14%) of the E. coli isolates bound the dye (Congo red-positive) and 103/120 (86%) did not bind the dye (Congo red-negative). All of the Congo red-positive isolates were recovered from subjectively abnormal birds, whereas Congo red-negative isolates were recovered from both subjectively normal (71/120 [59%]) and abnormal (32/120 [27%]) birds.

Animals↗
PubMed1995

Kinetics of Congo-red binding by haemin-limited and haemin-excess cells of Porphyromonas gingivalis W50.

The binding of Congo red to P. gingivalis W50 grown in a chemostat under haemin-limitation and haemin-excess was quantified. Congo red bound to both haemin-excess and haemin-limited cells with similar capacity and affinity. Binding of Congo red was greater than for ferri- (haemin) or ferroprotoporphyrin IX (haem), and was not influenced by redox potential at low added ligand concentrations. Both haemin-limited and haemin-excess cells showed positive co-operativity towards Congo red binding. Pre-exposure of haemin-limited and haemin-excess cells to sub-saturating concentrations of ferriprotoporphyrin IX did not affect Congo red binding, whereas pre-exposure of haemin-excess cells to ferroprotoporphyrin IX increased binding. Iron protoporphyrin IX binding was enhanced after exposure of both haemin-excess and haemin-limited cells to Congo red, especially under reducing conditions. These results confirm that Congo red binding cannot be used as an indirect measure of haemin binding, nor can Congo red be used to inhibit haemin binding to P. gingivalis.

Journal Article↗
PubMed2003

FT-Raman spectroscopy as diagnostic tool of Congo red binding to amyloids.

Chorion is the major component of silkmoth eggshell. More than 95% of its dry mass consists of the A and B families of low molecular weight structural proteins, which have remarkable mechanical and chemical properties protecting the oocyte and developing embryo from environmental hazards. We present data from FT-Raman spectroscopy of silkmoth chorion and amyloid-like fibrils formed from peptide analogues of chorion proteins, both unstained and stained by Congo red. The results show that FT-Raman spectroscopy is not a straightforward diagnostic tool for the specific interactions of Congo red with amyloids: a dilute aqueous solution of the Congo red dye at pH 5.5 and a thin solid film of the dye cast from this solution exhibit the same "diagnostic" Raman shifts relative to the neat Congo red dry powder as do amyloid fibrils formed from peptide analogues of chorion proteins stained by Congo red. An important consequence of this finding is that these shifts of the Raman active modes of Congo red are probably due to the formation of supramolecular dye aggregates in the presence of water. Therefore, this is not an appropriate diagnostic test for Congo red binding to amyloids.

Animals↗
PubMed2004

Role of actin filaments in targeting of Crimean Congo hemorrhagic fever virus nucleocapsid protein to perinuclear regions of mammalian cells.

Crimean-Congo hemorrhagic fever virus is the causative agent of a severe disease throughout Africa, Europe, and Asia. Like other members of the genus Nairovirus, the Crimean-Congo hemorrhagic fever virus contains three genomic RNA segments, the small (S), medium (M), and large (L) segments. The S segment encodes the viral nucleocapsid protein (NP), while the M and L segments encode the glycoproteins and the RNA-dependent RNA polymerase, respectively. In this study, the site of expression and assembly of Crimean-Congo hemorrhagic fever virus NP in mammalian cells have been investigated. It was found that the NP is localized in the perinuclear region of infected cells. By using the Semliki forest virus expression system, it was shown that the Crimean-Congo hemorrhagic fever virus NP is targeted to the perinuclear region of cells in the absence of native RNA segments and virally encoded glycoproteins. It was also demonstrated that the Crimean-Congo hemorrhagic fever virus NP was not expressed as a Golgi-membrane associated protein. By using Cytochalasin D, an agent that disrupts actin filaments, it was found that actin filaments are involved in targeting the viral NP to perinuclear regions. We also demonstrated that disruption of actin filaments reduced the assembly of infectious Crimean-Congo hemorrhagic fever virus up to 97%. Furthermore, we showed that the NP of Crimean-Congo hemorrhagic fever virus NP interacts with actin.

Actin Cytoskeleton↗
PubMed1997

Congo red reverses amyloid beta protein-induced cellular stress in astrocytes.

An amyloid-binding dye Congo red has been reported to prevent the neurotoxic effect of Alzheimer's amyloid beta protein (Abeta). In the present study, we investigated the effect of Congo red in cultured rat cortical astrocytes. Abeta (1 nM-10 microM) did not cause cell death, but potently inhibited the cellular redox activity as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. Congo red (0.2-20 microM), when added together with or prior to Abeta, significantly blocked Abeta-induced inhibition of redox activity. Furthermore, when Congo red was added after treatment with Abeta, the inhibited redox activity was restored to normal, indicating that Congo red can reverse Abeta-induced cellular stress. The reversing effect of Congo red cannot be explained by the inhibition of Abeta fibril formation and suggests a novel aspect of the interaction of Congo red with Abeta.

Amyloid beta-Peptides↗
PubMed1986

Characterization of Shigella flexneri sequences encoding congo red binding (crb): conservation of multiple crb sequences and role of IS1 in loss of the Crb+ phenotype.

The ability to bind Congo red (Crb+) is associated with virulence of Shigella flexneri and is encoded by a large, 220-kilobase plasmid. We cloned fragments of this plasmid to isolate the sequences encoding Congo red binding, to determine the degree of conservation of these sequences among S. flexneri strains, and to study the molecular basis for loss of the Crb+ phenotype. At least two separate BamHI fragments cloned into plasmid vectors encode Congo red binding in E. coli or S. flexneri. One Crb+ clone, pTKS2, contains a copy of IS1 adjacent to the crb sequences. IS1 appears to be responsible for deletions leading to loss of Congo red binding in this clone. In addition, this clone was found to integrate into the chromosome at relatively high frequency. Integration resulted in loss of the Crb+ phenotype. A second clone, pTKS15, which has only limited homology to pTKS2, also encodes Congo red binding. The Crb+ phenotype of transformants carrying pTKS15 was detected at 37 degrees C but not at 30 degrees C, and thus it resembles Congo red binding in wild-type S. flexneri. HindIII digests of plasmid DNA from 10 different S. flexneri strains were hybridized to both of these Crb+ clones and to an IS1 probe. More than one fragment hybridized to pTKS2 or pTKS15. In general, the sizes of these fragments were the same in S. flexneri strains of different serotypes, indicating conservation of these sequences. Three of five copies of IS1 were also found on the large S. flexneri plasmids. Two of the copies were on fragments of the same size in each strain. Analysis of Crb- derivatives of the 10 strains indicated that, although IS1 may be closely linked to crb sequences on the 220-kilobase plasmid, it is not responsible for the majority of deletions of this plasmid associated with loss of Congo red binding.

Base Sequence↗
PubMed1989

Congo red-mediated regulation of levels of Shigella flexneri 2a membrane proteins.

The ability of Shigella spp. to bind Congo red from agar medium is generally correlated with their virulence properties. We used a metabolically active culture of Shigella flexneri 2a to determine the effect of Congo red on its membrane protein profiles. Virulent S. flexneri grown in the presence of Congo red at 37 degrees C showed increased levels of three proteins with Mrs of 43,000, 58,000, and 63,000 (43K, 58K, and 63K proteins) in the Sarkosyl-soluble membrane fractions. The observed phenomenon was temperature dependent. At 30 or 42 degrees C the protein levels remained unaffected by the presence of Congo red. Similar regulation of the levels of the 43K, 58K, and 63K membrane proteins was also observed with Shigella dysenteriae 1 and enteroinvasive Escherichia coli, but not with enteropathogenic E. coli. The cellular uptake of Congo red seemed to be essential, but not sufficient, for regulation. All three proteins reacted with human convalescent-phase sera in immunoblots of S. flexneri 2a Sarkosyl-soluble membrane fractions. Using the 43K-specific antiserum as the primary antibody, by indirect immunofluorescence studies, we detected an increase in the level of the 43K protein in S. flexneri which had invaded epithelial cells. These observations strongly indicate that the 43K, 58K, and 63K proteins are virulence associated. We propose that the observed regulatory effect of Congo red on membrane proteins of S. flexneri is mediated through induction. Since the same regulatory effect was also observed during the invasion of epithelial cells by S. flexneri, it is suggested that Congo red mimics some host tissue factor in vitro.

Adult↗
PubMed1989

Quantitative evaluation of congo red binding to amyloid-like proteins with a beta-pleated sheet conformation.

The binding of Congo red to several purified amyloid-like peptides having a beta-pleated sheet conformation was quantitatively examined. Congo red binds preferentially to the beta-pleated sheet conformation of both insulin fibrils and poly-L-lysine. Congo red does not bind nearly so well to poly-L-serine or polyglycine, despite the fact that these peptides also have a beta-pleated sheet conformation. Binding to insulin fibrils was saturable with an apparent Bmax of 2 moles of Congo red per mole of insulin fibrils and an apparent KD of 1.75 x 10(-7) M. Binding to beta-poly-L-lysine was similar but had a much higher apparent Bmax of 43. Binding of Congo red to beta-poly-L-lysine was pH dependent and appeared to be determined by the number of protonated lysine residues in the 250 amino acid peptide. We present a new hypothesis in which Congo red binds to amyloid-like proteins via bonds between the two negatively charged sulfonic acid groups of Congo red and two positively charged amino acid residues of two separate protein molecules which are properly oriented by virtue of the beta-pleated sheet conformation of the peptide backbone.

Amyloid↗
PubMed2004

Improved detection of amyloid in fat pad aspiration: an evaluation of Congo red stain by fluorescent microscopy.

Amyloid fat pad aspiration specimens for cases with a clinical suspicion of amyloid typically are stained with Congo red and examined by brightfield microscopy. Congophilia with apple-green birefringence by polarization microscopy (PM) is considered diagnostic for amyloid. Examination of Congo red-stained slides by fluorescent microscopy (FM) is considered by some to be a more sensitive detection method. In this study, we assessed the utility of this technique in cytopathology archival slides from abdominal fat pad aspirations previously stained with Congo red dye. Seventy-eight cases of abdominal fat pad aspirations collected during the last 5 yr and stained with the Congo red procedure were obtained from archival files. Additionally, 20 adipose tissue material slides prepared from the surgical pathology specimens were examined as controls. One representative smear was examined in each case using FM equipped with rhodamine excitation/absorption (540/570 nm) filters. Relevant clinical information was obtained in all cases. Twelve cases (15.4%) of the 78 fat pad aspiration cases were reported originally as positive by Congo red stain using polarization and apple-green birefringence as diagnostic criteria. On review, four cases were deemed unsatisfactory. By FM examination 29 of the 74 (39.2%) cases were reclassified as positive for amyloid. The results were confirmed by immunohistochemical stain for amyloid P protein and electron microscopy. A number of similar distinct fluorescence and immunohistochemical patterns were recognized in the positive cases. Minimally weak fluorescence in the adipose tissue was observed in the control cases. The use of FM in Congo red-stained fat pad smears can improve the detection of amyloid in cytology preparations.

Adipose Tissue↗
PubMed2001

A new method for assessing foliar uptake of fungicides using Congo Red as a tracer.

In order to develop a new method for measuring foliar uptake of fungicides, Congo Red was selected as a tracer, and optimum procedures were established for washing, extracting and analyzing it and fungicides from leaf surfaces. Congo Red, a water-soluble dye, was not absorbed into cucumber or rice leaf, even in the presence of various surfactants, and was completely washable from leaf surfaces by aqueous acetonitrile solutions. Congo Red and fungicides in washings were quantified to calculate the amount of foliar uptake of the latter, by comparing the ratio to Congo Red. The optimum concentration of Congo Red in a formulation should be established in order to minimize its influence on fungicide uptake. Although Congo Red has proved to be useful with a conventional droplet application method, it will give more realistic and practical results with the spraying method used in the present study.

Acetonitriles↗
PubMed2006

Sensitivity and specificity of Congo red staining according to Romhányi. Comparison with Puchtler's or Bennhold's methods.

The sensitivity and specificity of various Congo red staining methods is very important in the diagnosis of amyloidosis. When using a less sensitive staining method, some true positive cases of amyloidosis remain undetected. A more highly specific method potentially detects more cases and reveals amyloidosis in an earlier stage of deposition. In this paper, the Congo red staining method according to Romhányi is discussed in comparison with Puchtler's and Bennhold's methods. Using Romhányi's technique, there is no alcoholic differentiation, and thus no dye molecules are washed off the amyloid filaments. The binding of the oriented dye molecules is optimal for polarization microscopy. With this method, the polar hydrophilic mounting medium, gum Arabic is used. Mounted in this carbohydrate-containing, hydrophilic medium, the Congo red molecules are oriented parallel to the surface of the amyloid filaments and the sign is linear positive, corresponding to an additive character of topo-optical staining reactions. Otherwise, the Congo red molecules are oriented perpendicular to the surface of collagen, reducing the intensity of birefringence and even inducing an inversion of the original sign of the collagen birefringence. With alcoholic differentiation, Congo red dye molecules are extracted and this decreases the birefringence of amyloid deposits, i.e. minimal amyloid deposits may be missed. Using the apolar hydrophobic mounting medium, Canada balsam, an axis-parallel arrangement of Congo red dye molecules on the surface of collagen fibers and amyloid will occur, resulting in an additive topo-optical reaction with a green polarization color and a false positive diagnosis of amyloidosis ("phantom amyloidosis").

Amyloid↗
PubMed2006

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↗
PubMed2002

Evaluation of gastric acid secretion at endoscopy with a modified Congo red test.

BACKGROUND: Markedly decreased or absent gastric acid production is associated with a number of clinically significant conditions, and identification of patients with hypo/achlorhydria may be important. However, current methods of assessing impaired acid secretion are unreliable, time-consuming, and/or complex. The aim of this prospective study was to evaluate a modified endoscopic Congo red test for the diagnosis of hypo/achlorhydria by correlation with a standard gastric acid secretory test. METHODS: One hundred six consecutive outpatients with or without dyspeptic symptoms referred for endoscopy were evaluated by using a modified endoscopic Congo red test and a standard test of gastric acid secretion. The modified endoscopic Congo red test suggested hypo/achlorhydria when there was no color shift or a shift of small extent (less than one third of fundic mucosa). Hypo/achlorhydria by the standard gastric acid secretory test was defined as a maximal acid output of less than 6.9 mmol/hour in men and 5.0 mmol/hour in women. RESULTS: The accuracy of the modified endoscopic Congo red test for the diagnosis of hypo/achlorhydria was 0.98 (95% CI [0.93, 0.99]). The sensitivity was 1.0 (95% CI [0.92, 1.00]) and specificity 0.96 (95% CI [0.88, 0.99]). All patients tolerated the modified endoscopic Congo red test well. CONCLUSION: The modified endoscopic Congo red is an accurate, simple, fast, inexpensive, and well-tolerated chromoendoscopic method for identification of patients with hypo/achlorhydria during routine upper endoscopy.

Achlorhydria↗
PubMed1995

Congo red binding by Porphyromonas gingivalis is mediated by a 66 kDa outer-membrane protein.

Congo red was bound from solution by strains of Porphyromonas gingivalis including W50, HG189, HG184, NCTC 11834, Bg 381, WPH35, the slower brown pigmenting colonial variant W50/BR1, and the avirulent mutant W50/BE1, and by Porphyromonas endodontalis HG370 and Porphyromonas asaccharolytica B537. SDS-PAGE of whole cells of all species examined displayed a 66 kDa Congo-red-binding component which was also detected in the outer membranes of P. gingivalis W50 grown in the chemostat under both haemin limitation and haemin excess, and which corresponded to a Coomassie-blue-stained band of the same mobility. Pretreatment of haemin-excess batch-grown cells of P. gingivalis W50 with polymyxin B, which binds to lipid A, did not inhibit binding, whilst binding was enhanced in the presence of 2 M ammonium sulphate, suggesting the involvement of non-specific hydrophobic interactions. Binding was also reduced by pretreatment with trypsin and papain, and by 8-anilino-1-naphthalenesulphonic acid, which binds to hydrophobic amino acids. The 66 kDa binding component was sensitive to proteinase K digestion, and loss of Congo red staining of this band correlated with the quantitative reduction in Congo red binding by whole cells. These data, and our previous work, show that Congo red and iron protoporphyrin IX (haemin) are bound to different outer-membrane components, and that Congo red binding may be of little value as a marker to detect virulent strains of P. gingivalis or those expressing haemin-binding proteins.

Bacterial Outer Membrane Proteins↗
PubMed1985

Congo red agar, a differential medium for Aeromonas salmonicida, detects the presence of the cell surface protein array involved in virulence.

Strains of the fish pathogen Aeromonas salmonicida which possess the cell surface protein array known as the A-layer (A+) involved in virulence formed deep red colonies on tryptic soy agar containing 30 micrograms of Congo red per ml. These were readily distinguished from colorless or light orange colonies of avirulent mutants lacking A-layer (A-). The utility of Congo red agar for quantifying A+ and A- cells in the routine assessment of culture virulence was demonstrated. Intact A+ cells adsorbed Congo red, whereas A- mutants did not bind Congo red unless first permeabilized with EDTA. The dye-binding component of A+ cells was shown to be the 50,000-Mr A-protein component of the surface array. Purified A-protein avidly bound Congo red at a dye-to-protein molar ratio of about 30 by a nonspecific hydrophobic mechanism enhanced by high salt concentrations. Neither A+ nor A- cells adsorbed to Congo red-Sepharose columns at low salt concentrations. On the other hand, A+ (but not A-) cells were avidly bound at high salt concentrations.

Aeromonas↗
PubMed1988

Congo red binding and salt aggregation as indicators of virulence in Shigella species.

Smooth strains of Shigella dysenteriae type 1, Shigella flexneri, Shigella boydii, and Shigella sonnei which form pigmented colonies (Pcr+) on Congo red agar were virulent in the Sereny test. Smooth variants unable to bind Congo red (Pcr-) were avirulent. Measurements of dye uptake from solution showed that S. dysenteriae type 1 bound the most dye, followed in order of uptake by S. flexneri, S. boydii, and S. sonnei. Using the salt aggregation test (SAT) to determine cell surface hydrophobicity, we found the same order of species. The SAT could not, however, detect differences in surface properties between Pcr+ and Pcr- pairs of isogenic smooth strains. Enteroinvasive Escherichia coli strains used in the study showed SAT and Congo red-binding properties which were similar to those of the S. flexneri strains. A direct correlation was found between pigment-binding ability and the presence of the large 140-megadalton plasmid in S. flexneri, enteroinvasive E. coli, and S. boydii but not in S. dysenteriae type 1 or S. sonnei strains. Congo red interacted with outer membranes and outer membrane proteins of S. dysenteriae type 1 but not with lipopolysaccharides. However, rough mutants of Shigella species deficient in lipopolysaccharides bound Congo red and formed pigmented colonies, showing that dye binding as a virulence assay may be misinterpreted in such cases. There was complete correlation of the Pcr+ phenotype with virulence in the smooth strains in this study, suggesting that Congo red binding can be utilized as a quick and reliable alternative to the Sereny test.

Bacterial Adhesion↗
PubMed1994

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↗