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PubMed2005

Analysis of correlated domain motions in IgG light chain reveals possible mechanisms of immunological signal transduction.

It was shown experimentally that binding of a micelle composed of Congo red molecules to immunological complexes leads to the enhanced stability of the latter, and simultaneously prevents binding of a complement molecule (C1q). The dye binds in a cavity created by the removal of N-terminal polypeptide chain, as observed experimentally in a model system-immunoglobulin G (IgG) light chain dimer. Molecular Dynamics (MD) simulations of three forms of IgG light chain dimer, with and without the dye, were performed to investigate the role of N-terminal fragment and self-assembled ligand in coupling between V and C domains. Root-mean-square distance (RMSD) time profiles show that removal of N-terminal fragment leads to destabilization of V domain. A micelle composed of four self-assembled dye molecules stabilizes and fixes the domain. Analysis of root-mean-square fluctuation (RMSF) values and dynamic cross-correlation matrices (DCCM) reveals that removal of N-terminal fragment results in complete decoupling between V and C domains. Binding of self-assembled Congo red molecules improves the coupling, albeit slightly. The disruption of a small beta-sheet composed of N- and C-terminal fragments of the domain (NC sheet) is the most likely reason for the decoupling. Self-assembled ligand, bound in the place originally occupied by N-terminal fragment, is not able to take over the function of the beta-sheet. Lack of correlation of motions between residues in V and C domains denotes that light chain-Congo red complexes have hampered ability to transmit conformational changes between domains. This is a likely explanation of the lack of complement binding by immunological complexes, which bind Congo red, and supports the idea that the NC sheet is the key structural fragment taking part in immunological signal transduction.

Computer Simulation↗
PubMed1999

Histochemically reactive zinc in plaques of the Swedish mutant beta-amyloid precursor protein transgenic mice.

Endogenous metals such as zinc may contribute to beta-amyloid (Abeta) aggregation and hence the plaque formation. In the present study, we examined brains of four Swedish mutant amyloid precursor protein (APP) transgenic mice at 12 months of age for histochemically reactive zinc in the plaques. Here, we report that all the Congo red (+) mature plaques contained chelatable zinc, as demonstrated by staining with the zinc-specific fluorescent dye 6-methoxy-8-quinolyl-para-toluenesulfonamide (TSQ). On the other hand, Congo red (-) preamyloid Abeta deposits were not stained with TSQ. Interestingly, although cerebellum contained similar degree of preamyloid Abeta deposits as cerebral cortex, it was completely devoid of Congo red- or TSQ-stained mature plaques. Although zinc from plaques was only slowly and partially removed by a specific zinc remover, dithizone, treatment of brain sections with heparinase-III, which degrades heparan sulfate proteoglycan (HSPG), another major constituent of plaques, greatly fastened the zinc removal with dithizone. The present study has demonstrated the presence of histochemically reactive zinc in plaques, but not preamyloid Abeta deposits, of the Swedish mutant APP transgenic mice. Because preamyloid Abeta deposits fail to develop into congophilic plaques in cerebellum where synaptic vesicle zinc is deficient, the synaptic zinc may be a necessary element in the plaque formation. In holding zinc inside plaques, HSPG may contribute in addition to Abeta.

Aminoquinolines↗
PubMed2001

In vitro affinity of 99Tcm-labelled N2S2 conjugates of chrysamine G for amyloid deposits of systemic amyloidosis.

To date, systemic amyloidosis is diagnosed histologically in vitro using Congo red staining or in vivo using iodine-123 serum amyloid P component (123I-SAP) scintigraphy. 99Tcm-labelled derivatives of chrysamine G (CG), a lipophilic analogue of Congo red, were synthesized as potential tracer agents for direct and quantitative scintigraphic evaluation of amyloid deposits. To determine the affinity of 99Tcm-MAMA-CG, 99Tcm-Me4MAMA-CG and 99Tcm-MAMA-CG diethyl ester for amyloid, in vitro autoradiography was performed on sections of human kidney biopsy cylinders from kidneys with amyloid deposits (types AA, Alambda and Akappa) or control kidney tissue after incubation with the respective tracer agents. The binding of 99Tcm-MAMA-CG and its tetramethyl derivative was higher to kidney biopsy material with amyloid deposits of the AA, Alambda or Akappa type compared with control kidney tissue. This higher binding was prevented by the presence of 10 microM Congo red in the incubation medium. The diethyl ester of 9Tcm-MAMA-CG did not demonstrate increased binding to Congo red-positive kidney tissue. In conclusion, 99Tcm-MAMA-CG and 99Tcm-Me4MAMA-CG localize specifically to amyloid deposits in human kidney tissue, suggesting that these tracer agents may be applicable as specific targeting agents for diagnostic purposes in clinical amyloidosis.

Adult↗
PubMed1998

Expression of iron binding proteins and hemin binding activity in the dental pathogen Actinobacillus actinomycetemcomitans.

Actinobacillus actinomycetemcomitans was found to express a polypeptide immunologically related to the Neisseria gonorrhoeae FbpA iron binding protein. In addition, the expression of hitB and hitC homologs was detected by Northern blot analysis. This periodontal pathogen also expresses a polypeptide homologous to the 31-kDa Haemophilus influenzae protein, which shows amino acid sequence homology with the FimA and YfeA proteins from Streptococcus parasanguis and Yersinia pestis, respectively. Both A. actinomycetemcomitans protein homologs were located within the periplasmic space, and their synthesis was regulated by the iron and hemin concentration of the culture medium. Southern and Western blot analysis together with molecular cloning revealed the presence of a Fur-like repressor, which may control the iron regulation of gene expression in this bacterium. Cultivation in the presence of hemin or Congo red revealed the ability of this organism to bind hemin. This binding activity was further confirmed by isolating Escherichia coli DH5 alpha clones that produced red and brown colonies on agar plates containing Congo red and hemin, respectively, after transformation with an A. actinomycetemcomitans gene library.

Aggregatibacter actinomycetemcomitans↗
PubMed1985

Production of monoclonal antibody against amyloid fibril protein and its immunohistochemical application.

An autopsy case of multiple myeloma (IgA-lambda) with extensive amyloid arthropathy of the systemic joints was described. Heavy deposits of amyloid (amyloidoma) were observed in the articular cavities of the joints. Furthermore, numerous amyloid deposits were found in the walls of small blood vessels in the general organs. Incubation of the paraffin sections of amyloid with potassium permanganate produced little loss of Congo red affinity and apple-green birefringence under polarized light. A crude preparation of amyloid protein was isolated from a frozen intra-articular mass, which had been obtained at necropsy, and injected into the footpads of BALB/c mice with complete Freund's adjuvant. The immune spleen cells were fused with myeloma cells (P3 X 63-Ag8.653) under the presence of polyethylene glycol. Hybridoma cell lines, producing supernatants which reacted not only with amyloid substances but also with normal human tissues, were omitted from the subjects of recloning, and one hybridoma cell line (Am-1) producing a specific monoclonal antibody (MAb) against the immunized amyloid substance was finally obtained. Using the indirect immunoperoxidase method, the specificity of MAb Am-1 was confirmed in the cryostat sections as well as in the formalin-fixed paraffin sections of various organs of the case from which the crude amyloid protein was obtained and used for immunization. Amyloid deposits in 25 cases with amyloidosis or amyloid deposits were examined with MAb Am-1, and 2 cases showed positive reactivity with Am-1. These 2 cases presented primary amyloidosis and focal amyloid deposits in the oral cavity, respectively; Congo red staining of amyloid in these cases showed resistance to treatment with potassium permanganate, as observed in the amyloid of the original case used for immunization. Trypsin treatment of the sections resulted in a loss of positive reactivity with MAb Am-1 in all these cases. This result indicates that Am-1 recognizes a substance composed of protein molecules. Furthermore, the immunohistochemical distribution of Am-1 positive reaction was consistent with the histological distribution of substance which could be stained by Congo red and showed apple-green birefringence under polarized light. These results have suggested that Am-1 reacts with a portion of amyloid protein which is resistant to treatment with potassium permanganate followed by Congo red staining.

Aged↗
PubMed1997

Granular-lattice (Avellino) corneal dystrophy in Japanese patients.

PURPOSE: To determine amyloid deposition in the corneas of granular corneal dystrophy in Japanese patients. METHODS: Eight Japanese patients (10 eyes) with a clinical diagnosis of granular corneal dystrophy were investigated clinically and histologically. Each specimen obtained at surgery was stained with hematoxylin-eosin, Masson trichrome or Mallory, and Congo red stain. Amyloid deposit was identified by birefringence and dichroism under cross-polarized light after staining with Congo red. RESULTS: Seven (70%) of the 10 corneal buttons (six of eight patients) had amyloid deposits, as shown by Congo red staining with birefringence and dichroism. Of the six amyloid-positive patients, two patients (who were siblings) showed discrete gray-white corneal deposits with additional linear deposits. This finding is typical of Avellino corneal dystrophy. The corneas of the remaining four patients showed the discrete deposits typical of granular dystrophy. Some of them showed a few whitish fusiform and stellate opacities in the mid stroma, suggestive of Avellino corneal dystrophy. CONCLUSION: The high frequency of amyloid deposits in Japanese patients with granular corneal dystrophy may be caused by an allelic heterogeneity of the gene.

Aged↗
PubMed1964

A HISTOLOGICAL STUDY OF HYALINE DEPOSITS IN LARYNGEAL, AURAL, AND NASAL POLYPS AND THEIR DIFFERENTIATION FROM AMYLOID.

Sections of hyalinized laryngeal polyps stained with haematoxylin and eosin or Congo red can easily be confused with localized amyloid of the larynx. Several histological methods were applied to 61 specimens containing hyaline deposits or hyalinized tissues which included 36 laryngeal polyps. The results were compared with seven cases of localized amyloidosis of the upper respiratory tract and four cases of secondary amyloidosis. The hyaline deposits gave a pattern of results which were different from amyloid. They showed no metachromasia, low affinity for Congo red, weak birefringence in an unstained state, no birefringence if stained with Congo red, weak or negative fluorescence if stained with Thioflavin T, low resistance to pepsin digestion, positive phosphotungstic acid haematoxylin staining areas, and a high affinity for the picric acid component of van Gieson stain. Hyalinized collagen closely resembles amyloid, but it can be excluded by a van Gieson stain. No evidence of amyloid formation was found in areas of hyaline deposits in the larynx. This supports the view that localized amyloidosis of the larynx and hyalinized laryngeal polyps are different pathological entities.

Amyloid↗
PubMed2006

Topo-optical visualization reactions of carbohydrate-containing amyloid deposits in the respiratory tract.

Staining with Congo red according to is the most commonly used method for the demonstration of amyloid, but structures other than amyloid can give false-positive results. To overcome this problem, introduced an aqueous Congo red staining with gum arabic as the mounting medium, which we have used in this and previous publications. Most histochemical studies on amyloid deposits to date have concentrated on conventional methods including staining with thioflavine, sirius red, alcian blue, methyl and crystal violet. In this study, we used topo-optical reactions with thiazine dyes on both the light and polarization microscopic level to establish the structure, distribution and location of carbohydrate components that occur within amyloid deposits, especially in the respiratory tract. Topo-optical staining reactions for the qualitative analysis of carbohydrate components in amyloid deposits included (1) reactions that identify the carbohydrate residues, (2) reactions that detect sialic acids and, (3) methods that visualize glycosaminoglycans. In conclusion, a comparison of consecutive serial sections stained with Congo red, aldehyde bisulfite toluidine blue reaction, sialic acid-specific topo-optical reaction, toluidine blue topo-optical reaction and chemically intensified basophilic reaction showed correlative staining patterns and anisotropic effects, corresponding to a close pathomorphological relationship between amyloid fibrils, periodate reactive carbohydrates, including sialic acids, and glycosaminoglycans.

Adult↗
PubMed1996

Separation of scrapie prion infectivity from PrP amyloid polymers.

The prion protein (PrP) undergoes a profound conformational change when the cellular isoform (PrPC) is converted into the scrapie form (PrPSc). Limited proteolysis of PrPsc produces PrP 27-30 which readily polymerizes into amyloid. To study the structure of PrP amyloid, we employed organic solvents that perturb protein conformation. Hexafluoro-2-propanol (HFIP), which promotes alpha-helix formation, modified the ultrastructure of rod-shaped PrP amyloids; flattened ribbons with a more regular substructure were found. As the concentration of HFIP was increased, the beta-sheet content and proteinase K resistance of PrP 27-30 as well as prion infectivity diminished. HFIP reversibly decreased the binding of Congo red dye to the rods while inactivation of prion infectivity was irreversible. In contrast to 10% HFIP, 1,1,1-trifluoro-2-propanol (TFIP) did not inactivate prion infectivity but like HFIP, TFIP did alter the morphology of the rods and abolish Congo red binding. This study separates prion infectivity from the amyloid properties of PrP 27-30 and underscores the dependence of prion infectivity on PrPSc conformation. The results also demonstrate that the specific beta-sheet-rich structures required for prion infectivity can be differentiated from those needed for amyloid formation as determined by Congo red binding.

1-Propanol↗
PubMed2004

Decolorization of azo dyes under batch anaerobic and sequential anaerobic/aerobic conditions.

Batch anaerobic and sequential anaerobic upflow anaerobic sludge blanket (UASB)/aerobic continuous stirred tank reactor (CSTR) were used to determine the color and COD removals under anaerobic/aerobic conditions. Two azo dyes namely "Reactive Black 5 (RB 5)," "Congo Red (CR)," and glucose as a carbon source were used for synthetic wastewater. The course of the decolorization process approximates to first order and zero order kinetics with respect to dye concentration for RB 5 and Congo Red azo dyes, respectively, in batch conditions. The decolorization kinetic constant (K0) values increased from 3.6 to 11.8 mg(L h)(-1) as increases in dye concentrations from 200 to 3200 mg L(-1) for CR. Increases in dye concentrations from 0 to 3200 mg L(-1) reduce the decolorization rate constant (k1) values from 0.0141 to 0.0019 h(-1) in batch studies performed with RB 5. Decolorization was achieved effectively under test conditions but ultimate decolorization of azo dyes was not observed at all dye concentrations in batch assay conditions. Dye concentrations of 100 mg L(-1) and 3000 mg L(-1) of glucose-COD containing basal medium were used for continuous studies. The effect of organic loadings and HRT, on the color removal efficiencies and methane gas productions were monitored. 94.1-45.4% COD and 79-73% color removal efficiencies were obtained at an organic system during decolorization of Reactive Black 5. 92.3-77.0% COD and 95.3-92.2% decolorization efficiencies were achieved at a organic loading rate of 1.03-6.65 kg (m3 day)(-1) and a HRT of 3.54-0.49 for Congo Red treatment. The results of this study showed that, although decolorization continued, COD removal efficiencies and methane gas production were depressed at high organic loadings under anaerobic conditions. Furthermore, VFA accumulation, alkalinity consumption, and methane gas percentage were monitored at organic loading as high as 2.49-4.74 kg (m3 day)(-1) and 24.60-30.62 kg (m3 day)(-1), respectively, through the decolorization of RB 5 and CR dyes in the UASB reactor.

Bacteria, Aerobic↗
PubMed1988

Cell surface properties correlated with cohesion in Myxococcus xanthus.

The gliding behavior of Myxococcus xanthus cells is controlled by two multigene systems, A and S, which encode information for adventurous and social behaviors, respectively. The S system can be genetically disrupted through mutation, such as a dsp mutation, or phenotypically disrupted by treating cells with the diazo dye Congo red (Arnold and Shimkets, J. Bacteriol. 170:5765-5770, 1988). One of the functions controlled by the S system is cell agglutination. Immediately after the induction of agglutination, wild-type cells begin to form aggregates, and within 30 min the cells are packed side-to-side in clumps containing thousands of cells. Changes in the cohesive properties of S+ cells are correlated with changes in the topology of the cell surface observed by electron microscopy. Two types of cell-associated appendages were observed on wild-type cells: thin filaments (ca. 5 nm in diameter), which have been called fimbriae or pili, at one cell pole, and thick, flaccid filaments (ca. 50 nm in diameter), referred to as fibrils, at both the sides and tips of cells. Cohesion was correlated with the secretion of the thick fibrils, which coat the cell surface and form an extracellular matrix in which the cells are interconnected. Several lines of evidence suggest that these thick fibrils are involved in cohesion. First, Dsp cells were unable to agglutinate or secrete this extracellular material. Second, wild-type cells which were treated with Congo red neither agglutinated nor secreted the extracellular fibrils. Finally, removal of the Congo red from wild-type cells restored cohesion and also restored production of the thick fibrils. Attempts to estimate the efficiency with which two cells cohered following collision suggested that under optimal conditions, one in three collisions resulted in stable contact. The collision efficiency decreased linearly as the cell density increased, suggesting a cell density-dependent regulation of cohesion. Some aspects of gliding behavior can be explained in terms of an inducer and an inhibitor of S motility.

Cell Communication↗
PubMed2001

Presence of icaA and icaD genes and slime production in a collection of staphylococcal strains from catheter-associated infections.

Both Staphylococcus epidermidis and Staphylococcus aureus are important causes of infections associated with catheters and other medical devices. It has recently been shown that not only S. epidermidis but also S. aureus can produce slime and carries the ica operon responsible for slime production. In the operon, coexpression of icaA and icaD is required for full slime synthesis. In this study, the presence of icaA and icaD was determined in a collection of 91 staphylococcal (68 S. epidermidis and 23 S. aureus) strains from intravenous catheter-associated infections, in 10 strains from the skin and mucosa of healthy volunteers, and in two reference strains by a PCR method. Slime-forming ability was tested on Congo red agar plates; 49% of S. epidermidis strains from catheters and, surprisingly, 61% of S. aureus strains were icaA and icaD positive and slime forming. All the saprophytic strains turned out to be negative for both icaA and icaD and also non-slime forming. Two S. aureus and one S. epidermidis strain from catheters, detected as icaA and icaD positive by PCR analysis and as slime forming (black colonies) at 24 h on Congo red agar, at 48 h exhibited tiny red spikes at the center of black colonies. The onset of these variants could not be ascribed to a mutagenic potential of Congo red, which, in the Ames test, was devoid of mutagenicity. PCR analysis showed that these red variants were negative for both icaA and icaD and even lacking the entire icaADBC operon. The data reported indicate an important role of ica genes as a virulence marker in staphylococcal infections from intravenous catheters.

Biofilms↗
PubMed1985

Gastric pseudopolyposis: a new clinical manifestation of type A gastritis.

Three cases of type A gastritis associated with multiple polypoid lesions of the body of the stomach are presented. The endoscopic congo-red test showed the red to black color changes of the sprayed congo-red only on the top of the polypoid lesions. The endoscopic methylene-blue test revealed no dye absorption on the antral mucosa or on the top of the polypoid lesions, while the dye was absorbed on the flat mucosa surrounding the top of the polypoid lesions. Histological examinations revealed well-preserved oxyntic glands on the top of the polypoid lesions. The flat mucosa showed marked atrophy or intestinal metaplasia. There have been several reports of experimental models of type A gastritis, where chief and parietal cells decreased in considerable number. These findings and the experimental models suggest that the polypoid lesions resulted from atrophic changes of type A gastritis. The top of the polypoid lesions is the residual oxyntic glands. The lesions, therefore, should be called "gastric pseudopolyposis." The spontaneous disappearance of the polypoid lesions seen in one case is thought to be a result of a further progression of the atrophic gastritis. A pronounced G-cell increase was noted in the antrum and lower body of the stomach. This is considered a secondary phenomenon following the marked reduction of acid secretion.

Aged↗
PubMed1997

Comparison of albumin binding capacities of three different reactive dye-derivatized poly(ethylene glycol dimethacrylate-hydroxyethyl methacrylate) microbeads.

Bovine serum albumin (BSA) adsorption onto dye-derivatized poly(ethylene glycol dimethacrylate-hydroxyethyl methacrylate) [poly(EGDMA-HEMA)] microbeads carrying three different reactive dye ligands (i.e. Congo Red. Cibacron Blue F3GA, and Alkali Blue 6B) was investigated. Swellable poly(EGDMA-HEMA) microbeads, in the size range of 150-200 microns, were produced by a modified suspension copolymerization of EGDMA and HEMA. The dyes were covalently attached to the microbeads. The maximum amounts of dye loadings were 14.5, 16.5, and 23.7 mumol g-1 for Congo Red, Cibacron Blue F3GA, and Alkali Blue 6B, respectively. The maximum BSA adsorption on the dye-derivatized microbeads from aqueous solutions containing different amounts of BSA were 90, 60.5, and 40 mg g-1 for the Congo Red, Cibacron Blue F3GA, and Alkali Blue 6B carrying microbeads, respectively. The maximum BSA adsorptions were observed at pH 6.0 in all cases. Desorption of albumin molecules were achieved by using 1.0 M NaSCN (pH 8.0). High desorption ratios (more than 85% of the adsorbed BSA) were observed in all cases. It was possible to reuse these novel sorbents without significant losses in the adsorption capacities.

Adsorption↗
PubMedDate not supplied

Development of small molecule probes for the beta-amyloid protein of Alzheimer's disease.

This study describes the synthesis and in vitro testing of small molecule probes that may eventually prove useful as markers of amyloid deposition in living patients. The prototype agent, Chrysamine G (CG), is a derivative of Congo red. CG binds synthetic beta-amyloid well in vitro, as does a fluorinated derivative. The mechanism of binding appears to be the same as Congo red--through a bidentate attachment spanning several amyloid peptide chains. CG is much more lipophilic than Congo red and crosses the blood-brain barrier in normal mice, achieving a brain/blood ratio over 10/1. There was no acute toxicity in mice at doses 10 times those used in the distribution studies. CG appears to be a relatively high affinity probe for beta-amyloid that appears to have low toxicity and can cross the blood-brain barrier. These characteristics are promising for development of in vivo amyloid probes similar to CG.

Alzheimer Disease↗