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A Congo red-magnesium oxalate agar medium was developed to detect expression of virulence-associated calcium dependency and Congo red absorption in Yersinia enterocolitica. Of the 157 pathogenic serotypes tested, 119 (75.8%) were positive; 98% of nonpathogenic serotypes and strains of three other Yersinia species were negative.
The endoscopic Congo red-methylene blue test was performed on 51 tumors of the large intestine. Results revealed that colorectal adenocarcinomas and adenomas bleached the Congo red and methylene blue sprayed over their surface and so appeared in sharp contrast to the bluish red mucosa of unaffected areas. No bleaching of the dyes was observed on the surface of non-neoplastic polyps. Moderately differentiated adenocarcinomas, submucosal or advanced cancers, and adenomas with severe atypia bleached the dyes most frequently. Thus this test facilitates early detection of colorectal tumors.
Congo red (CR) is a histologic dye that binds to many amyloid proteins because of their extensive beta-sheet structure. The absorbance spectrum of the dye changes upon binding to amyloid. This spectral change has previously been exploited to develop a method to study the interaction of CR with fibrillar beta-sheet insulin fibrils, a model amyloid protein. The amyloid beta-peptide (Abeta) is an amyloid protein which is deposited in the brains of Alzheimer's disease victims. Abeta is toxic to neurons in vitro in a manner that is highly dependent on the assembly of this peptide into beta-sheet fibrils. The CR-insulin assay has been applied as a means of studying the aggregation of Abeta, despite the fact that the CR-insulin procedure was never adequately developed for this purpose. In this study, we modify our original CR-insulin assay specifically for the purpose of quantifying Abeta aggregation and discuss the reasons why application of the CR-insulin method is not valid for this purpose. The CR-Abeta method is equally simple and retains the advantages of speed and lack of necessity for specialized instrumentation or expensive/radioactive reagents. Furthermore, this method can directly provide quantitation of aggregated Abeta in absolute terms (i.e., microg/ml).
Five pathologists estimated with point counting the amount of amyloid in heart muscle autopsy samples from 19 patients who suffered from amyloidosis. The serial sections had been stained with 5 different methods. The different variation sources involved in a quantitative study on amyloid and the interstain reproducibility between the results given by these 5 stains were analyzed. The volume fractions of amyloid after different stains were subjected to correlation analysis. In our test system about a third (34%) of the variation was due to the point counting method. Sirius red had the lowest and Congo red the highest variation due to point counting, since Sirius red gave the most intense staining and highest volume fractions for amyloid. The volume fractions were lowest in Congo red. About 40% of the total variation was composed of the field variation. This variation includes the variation due to variation between fields, field selection and subjective intraobserver variation. Sirius red showed the smallest field variation in respect to the total variation. Congo red, on the other hand, had the highest variation. The variation caused by pure interobserver variation is presented by the difference between total variation and the calculated field variation. This was largest with Sirius red and smallest with Congo red. The pure interobserver variation accounted for about 26% of the total variation. The high reproducibility with Sirius stain is most probably due to the high volume fractions of positively stained material after this stain, not to low interobserver variation. Congo red seems to have low interobserver variation. The senior pathologists were more reliable in measuring the amyloid deposits than the junior pathologists as suggested by interstain reproducibility and variation.(ABSTRACT TRUNCATED AT 250 WORDS)
Ability to detect pigmented and nonpigmented Pasteurella pestis is essential in plague research, and is currently dependent on use of the synthetic hemin-agar of Jackson and Burrows. We have devised a new differential medium for this purpose, containing Congo red dye and common, commercially available laboratory media. The ease and simplicity of preparation make the Congo red-agar a practical routine laboratory tool in plague research. These findings, possibly indicating a common binding site for hematin and Congo red, should be useful in efforts to determine the chemical nature of a bacterial component associated with high virulence in P. pestis.
To further demonstrate the association of phenotype and virulence of Congo red Escherichia coli (CREC), experiments were designed to reproduce airsacculitis and colisepticemia in 1-day-old chicks via aerosol exposure. In eight separate experiments in which a total of 462 chicks were exposed to CREC, the mortality rate was 4.11% and the morbidity rate was 13.4%, with both the dead and diseased chicks showing lesions of fibrinous airsacculitis, pericarditis, and perihepatitis. In contrast, when the corresponding non-CREC derivatives (the same E. coli strains, but not expressing the CR phenotype) were used as the aerosol inoculum in five additional experiments using 284 chicks, no chicks died and no lesions were observed. These results clearly show a strong correlation between the expression of the CR phenotype and virulence in avian E. coli.
In this study, we examined the relationship between the virulence of Shigella flexneri 2a and the ability of strains of S. flexneri 2a to absorb Congo red. Spontaneous nonpigmented (i.e., unable to bind Congo red [Pcr-]) derivatives of a virulent, pigmented (Pcr+) strain of S. flexneri 2a were isolated and assayed for virulence as determined by their ability to invade epithelial cells. All Pcr- mutants examined lost the ability to invade epithelial cells and were thus avirulent. Agarose gel electrophoresis of plasmid DNA from these avirulent, Pcr- mutants showed that the majority of these strains had lost a plasmid band corresponding to a virulence-associated plasmid, pSf2a140. In many of the mutants, concomitant loss of pigmentation, virulence, and pSf2a140 was accompanied by the appearance of a new plasmid, smaller than pSf2a140. We believe these new plasmids to be deletion derivatives of pSf2a140 and that loss of pigmentation and loss of virulence are associated with deletions in pSf2a140. We transduced Pcr- mutants to Pcr+ and isolated transductants which suppressed the Pcr- phenotype. None of the Pcr+ transductants regained the ability to invade epithelial cells. Several suppressors of the Pcr- phenotype were identified as mutations in cell wall biosynthesis. These results support our belief that although pigmentation is usually associated with virulence, genetic determinants unrelated to virulence can also affect the ability of the cell to bind Congo red. Therefore, the ability of S. flexneri 2a to bind Congo red does not necessarily imply the ability to invade epithelial cells. However, loss of ability to bind Congo red is accompanied by loss of virulence.
An elderly patient who had nephrotic syndrome for many years was found to have mesangial expansion and thickening of the glomerular capillary basement membranes (GBMs) on light microscopy. Electron microscopy (EM) showed tubular structures measuring 24 to 27 nm in the mesangium and the subepithelial areas. Congo red staining for amyloid was negative. This case represents another instance of congo red-negative amyloidosis-like glomerulopathy.
The interaction of the direct dye Congo red with intact beta-D-glucans provides the basis for a rapid and sensitive assay system for bacterial strains possessing beta-(1 leads to 4),(1 leads to 3)-D-glucanohydrolase, beta-(1 leads to 4)-D-glucanohydrolase, and beta-(1 leads to 3)-D-glucanohydrolase activities. A close correspondence was observed between cellulolytic activity and beta-(1 leads to 4)-D-glucanohydrolase and beta-(1 leads to 4),(1 leads to 3)-D-glucanohydrolase activities in isolates from the bovine rumen. Many of these isolates also possessed beta-(1 leads to 3)-D-glucanohydrolase activity, and this characteristic may have taxonomic significance.
Congo red binding by virulent A-layer-containing (A+) and avirulent A-layer-deficient (A-) strains of Aeromonas salmonicida was examined. Congo red binding to A+ cells was enhanced by salt and thus hydrophobically driven, but at low Congo red concentrations binding was salt independent. Congo red was bound by A+ cells by a kinetically distinct mechanism (Kd, 0.25 microM) which was absent in A- isogenic strains. Purified A-layer protein ("A protein") protein A also bound Congo red with similar affinity (Kd, 0.40 microM). Congo red binding was structurally specific; it was not influenced by a wide variety of compounds including amino acids and nucleotides and only weakly inhibited by structurally similar dyes. However, protoporphyrin IX and hemin were strong competitive inhibitors of Congo red binding. Protoporphyrin and hemin were bound only by A+ strains (KdS of 0.41 and 0.63 microM, respectively). Furthermore, binding of these porphyrins was strongly inhibited by Congo red but weakly inhibited by hematoporphyrin. Purified A protein also bound protoporphyrin IX and hemin with affinities similar to those of A+ cells (KdS of 0.94 and 0.41 microM, respectively.
Renal biopsies from seven patients with Congo red-negative amyloid-like fibrillary glomerulopathy (FGP) were examined by protein A gold immuno-electron microscopy. Ultrastructurally, the fibrils in all cases exhibited positive immunostaining for IgG, both Ig light chains, C3, and amyloid P component (AP), but did not show positive immunostaining for glomerular basement membrane (GBM)-associated proteins (collagen type IV and heparan-sulfate proteoglycans) or microfibril-associated proteins (fibronectin and fibrillin). In a triple-label study, AP and IgG were colocalized along the same fibril, whereas the gold probes for the detection of collagen type IV were absent. The results suggest that the fibrils are comprised of polyclonal IgG and C3 that bind AP. AP was immunolocalized sparsely but regularly along the lamina rara interna of normal GBM. AP was absent in the fibrils in a case of diabetic glomerulopathy, was scattered randomly without specificity for the electron-dense deposits in the GBM of membranous glomerulopathy, and lined up regularly along the fibrils in amyloid deposits. FGP is an entity in which the fibrils bind AP but lack the beta-pleated sheet structure necessary for Congo red staining that is typical of amyloid.
The traditional way of identifying amyloid in tissue sections has been staining with Congo red and demonstration of green birefringence under crossed polarizers. The original method of Congo red staining, described by Bennhold in 1922, has undergone several modifications to improve its sensitivity, specificity, and reliability. The most common modification is the alkaline Congo red method described by Puchtler and co-workers in 1962. Specificity is improved by using freshly prepared stain and a staining solution fully saturated with sodium chloride. Amyloid proteins can be further distinguished by autoclaving or by treating the tissue with potassium permanganate or alkaline guanidine. Autoclaving the tissues at 120 C for 30 min causes protein AA to lose its affinity for Congo red. Prolongation of autoclaving to 120 min abolishes the Congophilia of protein AL, but prealbumin-related amyloid shows little or no change. Treatment of the tissue with potassium permanganate causes protein AA and B2-microglobulin amyloid to lose their affinity to Congo red. Protein AA fails to stain with Congo red after treatment with alkaline guanidine for 1 min and protein AL and systemic senile amyloid protein (SSA) after 2 hr. Familial amyloid protein (FAP), prealbumin type, can stand 2 hr of alkaline guanidine treatment without losing its ability to stain with Congo red. Other methods of detection of amyloid include fluorescent stains, e.g., thioflavin T or S, and metachromatic stains such as crystal violet. Immunofluorescence and immunoperoxidase methods are used to identify and classify amyloid proteins in tissues. Antibodies against the P component, proteins AA and AL and FAP have been used with great precision. Due to cross-reactivity, these methods do not differentiate between some types of familial and senile systemic amyloidosis.
Bands that have cellulolytic activity are visualized after polyacrylamide gel electrophoresis by laying the slab gel on top of a thin sheet of 2% agar containing 0.1% carboxymethylcellulose. After a suitable incubation time, zones of carboxymethylcellulose hydrolysis are revealed by staining the agar replica with Congo red.
Three cases of amyloidosis-like glomerulopathy are presented in which renal amyloidosis was initially diagnosed on the basis of ultrastructural findings, despite negative Congo red staining. The histologic and immunofluorescence findings and, on careful examination, ultrastructural features of this amyloidosis-like glomerulopathy all serve to distinguish it from true amyloidosis. The clinical behavior suggests that it is a primary glomerulopathy since, with time, no other systems become involved.
Several lines of evidence suggest that amyloid deposition in the brain contributes to neuronal degeneration in Alzheimer's disease (AD). In the AD brain, diffuse plaques composed mostly of amorphous beta amyloid (Am-beta A) are inert, whereas compact plaques composed of beta amyloid fibrils (Fib-beta A) are associated with neurodegenerative changes. The effects of these two types of amyloid were tested on primary rat hippocampal neurons. Fib-beta A induced the formation of dystrophic neurites and caused neuronal cell death, whereas Am-beta A was not toxic. In addition, Fib-beta A caused synapse loss in the remaining viable neurons, whereas Am-beta A did not significantly affect synapse number. We also examined the effects of amylin, the primary constituent of the amyloid fibrils that form in the pancreas in adult-onset diabetes. Amylin was toxic to rat and human insulin-producing islet cells in the concentration range of fibril formation. The relative toxic potencies of amylin peptides of different species correlated with their fibril-forming capacity. Soluble amylin was not toxic. The amyloid fibril-binding dye Congo red inhibited the toxicity of both beta A and amylin. Congo red afforded protection against toxicity by a dual mechanism. When present during the phase of fibril polymerization, Congo red could inhibit fibril formation from some peptides. When added to preformed fibrils, Congo red bound to fibrils rendering them nontoxic. These results suggest that fibril formation is necessary for both beta A and amylin toxicity. Congo red appears to be a general inhibitor of amyloid fibril toxicity and may therefore be a useful prototype for drugs targeted to the amyloid pathology of AD and adult-onset diabetes.
Cells of Oocystis solitaria after treatment with inhibitors of a) microfibril orientation b) microfibril synthesis, and c) microfibril crystallization have been investigated with the freeze-fracturing technique. Changes in morphology of the E-face of the plasma membrane as a result of these treatments have been recorded. Terminal complexes are removed by cycloheximide, but not by colchicine or congo red treatments. Microfibril imprints are removed by congo red but not by colchicine or cycloheximide treatments. Congo red induces in addition the presence of increased numbers of paired, thickened, terminal complexes. Prolonged (4-6 h) treatment with congo red causes the insertion of a second set (oriented at 90 degrees to the first set) of parallel terminal complexes in the E-face. These results are discussed in relation to plasma membrane turnover.
Six current histological methods for demonstrating amyloid (crystal violet, thioflavine-T fluorescence, Congo-red staining and fluorescence, Sirius-red staining, and Congo- or Sirius-red birefringence) were applied in 25 cases of amyloidosis of various types and 47 pseudo-amyloid lesions. The results were compared and were correlated with those of ancillary histochemical tests and clinico-pathological data and each method's sensitivity and specificity for amyloid was evaluated. Thioflavine-T and, to a lesser degree, Congo-red fluorescence and Sirius-red staining proved very sensitive but not specific. Green birefringence with Congo or Sirius red was specific but not completely sensitive. The coexistence of Congo-red (and Sirius-red) staining and a positive DMAB-nitrite reaction occurred in all amyloid specimens and appeared specific for amyloid.
Congo red binds to the cell wall and inhibits the growth of yeast. In a screening for multicopy suppressor genes of Congo red hypersensitivity of erd1Delta mutant, we found that a previously uncharacterized gene, YBR005w, makes most of the Saccharomyces cerevisiae strains resistant to Congo red. This gene was named RCR1 (resistance to Congo red 1). An rcr1Delta null mutant showed an increased sensitivity to Congo red. RCR1 encodes a novel ER membrane protein with a single transmembrane domain. Molecular dissection suggested that the transmembrane domain and a part of the C-terminal polypeptide are sufficient for the activity. We examined the effect of RCR1 in various null mutants of genes related to the cell wall. The resistance of mutants to Congo red correlates with a reduction of chitin content. Multicopy RCR1 caused a significant decrease in the chitin content while the amount of alkali-soluble glucan did not change. The binding of Calcofluor white to the cell wall significantly decreased in these cells. Our results show that RCR1 regulates the chitin deposition and add firm genetic and biochemical evidences that the primary target of Congo red is chitin in S. cerevisiae.