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PubMed2006

The use of the Congo red-related dye DBACR to recognize the heavy chain-derived abnormality of myeloma immunoglobulins.

INTRODUCTION: The aim of this study was to differentiate heavy and light chain-derived instability of monoclonal myeloma immunoglobulins by complexation of matched supramolecular dyes. These are composed of several micellar pieces of self-assembled dye molecules which may penetrate the protein interior of the binding locus with polypeptide chains. These dyes were used to elicit, by precipitation, the postulated higher aggregation tendency of the heavy chain derived from its higher hydrophobicity. MATERIALS AND METHODS: Agarose gel electrophoresis was used to create conditions for dye complexation and to reveal the precipitation. RESULTS: Congo red derivatives with aromatic ring substitutes, BACR and DBACR, of increased penetrating capability were chosen to provoke the precipitation of abnormal immunoglobulins by displacing association-prone polypeptide chains from the protein interior. CONCLUSIONS: The results of this study confirm the heavy chain-related propensity of some monoclonal immunoglobulins to aggregate and precipitate. The simplicity of the technique may improve clinical diagnosis and facilitate predictions of disease complications.

Antibodies, Neoplasm↗
PubMed1997

Secretion of Ipa proteins by Shigella flexneri: inducer molecules and kinetics of activation.

The type III Mxi-Spa secretion machinery of Shigella flexneri is responsible for secretion of Ipa proteins, which are involved in the entry of bacteria into epithelial cells. Ipa proteins accumulate within bacteria growing in laboratory media, and their secretion is activated upon contact of bacteria with eukaryotic cells. In this study, we have identified a group of chemical compounds, including Congo red, Evans blue, and direct orange, which are able to induce secretion of Ipa proteins by bacteria suspended in phosphate-buffered saline. Parameters of kinetics of activation of Ipa secretion by Congo red were determined by measuring by enzyme-linked immunosorbent assay the amount of IpaC secreted and by investigating the increase in susceptibility of Ipa proteins to proteinase K degradation. Ipa secretion occurred at 37 degrees C, was obtained with 5 to 10 microM Congo red, and was complete within 30 min. In addition, activation of Ipa secretion by Congo red was observed with bacteria harvested throughout the exponential phase of growth but not with bacteria in the stationary phase. The interactions of Congo red and Congo red-related compounds with the Mxi-Spa secretion apparatus might be specific hydrophobic interactions similar to those involved in binding of Congo red to amyloid proteins.

Antigens, Bacterial↗
PubMed1989

Morphology and distribution of plaque and related deposits in the brains of Alzheimer's disease and control cases. An immunohistochemical study using amyloid beta-protein antibody.

A monoclonal antibody (4D12/2/6) to a synthetic peptide consisting of residues 8-17 of the amyloid beta-protein of Alzheimer's disease was used in an immunohistochemical study to investigate the localization of beta-protein immunoreactivity in neuritic plaques in the brains of 20 cases with Alzheimer's disease and a similar number of nonAlzheimer controls. The morphology and distribution of immunoreactive plaque-like lesions and the sensitivity of immunostaining were assessed both with and without formic acid pretreatment of the sections, and these results were compared with those obtained using conventional Congo red and silver impregnation staining methods. Congo red and immunostaining without formic acid pretreatment mainly stained the core deposits of amyloid in compact plaques, whereas the silver stain could also detect numerous diffuse plaques. Immunostaining with formic acid pretreatment was the most sensitive technique, and this revealed many additional immunoreactive lesions which were impossible or difficult to detect with the other staining methods. These additional lesions included variable sized areas of faint granular staining with little evidence of amyloid deposition or degenerating neurites that are presumed to be very early stages in plaque development. Far fewer immunoreactive lesions were observed in the nonAlzheimer controls. It is concluded that an abundant presence of anti-beta-protein immunoreactive plaque lesions throughout the cortex and subcortical gray matter structures is typical of Alzheimer's disease even when only moderate numbers of plaques can be detected by Congo red or silver stain. This immunostaining procedure with a specific monoclonal antibody for beta-protein may be very useful for the postmortem diagnosis of Alzheimer's disease.

Aged↗
PubMed1999

Designing conditions for in vitro formation of amyloid protofilaments and fibrils.

We have been able to convert a small alpha/beta protein, acylphosphatase, from its soluble and native form into insoluble amyloid fibrils of the type observed in a range of pathological conditions. This was achieved by allowing slow growth in a solution containing moderate concentrations of trifluoroethanol. When analyzed with electron microscopy, the protein aggregate present in the sample after long incubation times consisted of extended, unbranched filaments of 30-50 A in width that assemble subsequently into higher order structures. This fibrillar material possesses extensive beta-sheet structure as revealed by far-UV CD and IR spectroscopy. Furthermore, the fibrils exhibit Congo red birefringence, increased fluorescence with thioflavine T and cause a red-shift of the Congo red absorption spectrum. All of these characteristics are typical of amyloid fibrils. The results indicate that formation of amyloid occurs when the native fold of a protein is destabilized under conditions in which noncovalent interactions, and in particular hydrogen bonding, within the polypeptide chain remain favorable. We suggest that amyloid formation is not restricted to a small number of protein sequences but is a property common to many, if not all, natural polypeptide chains under appropriate conditions.

Acid Anhydride Hydrolases↗
PubMed1984

Diagnosis of early gastric cancers in the upper part of the stomach by the endoscopic Congo red-methylene blue test.

The clinicopathological features of the early gastric cancers in the upper part of the stomach, and the accuracy of their diagnosis by routine endoscopic examination were examined, and the accuracy of diagnosis of minute and flat cancers in the upper portion of the stomach by routine endoscopic examination and by the endoscopic Congo red-methylene blue test developed in our clinic were compared. Early cancers in the upper part of the stomach occurred in the elderly patient, and grossly elevated types were frequent. Endoscopically they were most difficult to diagnose by a single routine endoscopic examination, because they were frequently overlooked, and adequate biopsy was difficult in some cases. A correct diagnosis of minute and flat cancers in the upper part of the stomach by routine examination was made in only 27.3% and 25.0% of the cases, respectively. But with the Congo red-methylene blue test the diagnostic rate was raised significantly to 75.0% and 83.3%, respectively. In this test, Congo red and methylene blue sprayed on the surface of the tumor were bleached white 2 to 5 minutes after application, in sharp contrast to the unaffected mucosa.

Adenocarcinoma↗
PubMed2001

A simple alternative method for rapid detection of slime produced by Staphylococcus epidermidis isolates in bacterial keratitis.

BACKGROUND & OBJECTIVES: Slime is a known virulence factor of Staphylococcus epidermidis. The conventional Christensen's method for detection of slime in the laboratory takes at least 48 h. We, therefore, tried to evaluate the efficacy of the Congo red agar method as a routine procedure for detecting slime among isolates from corneal ulcers. METHODS: A total of 244 isolates from corneal ulcers were identified as S. epidermidis by the standard procedures. Slime was detected both by the conventional Christensen's method as well as by the Congo red agar method. RESULTS: Ninety two (37.7%) isolates were positive and 86 (35.2%) were negative for slime by both the techniques. Fifty four (22.1%) isolates were positive in Congo red agar, but negative by Christensen's method; whereas only 12 (4.9%) were negative by Congo red but positive by Christensen's method. Detection of slime by Congo red agar method was rapid i.e., all the 146 strains were positive within 24 h of incubation. On the other hand, Christensen's method had a delayed response; 42.3 per cent (44/104) strains being negative during the first 24 h of incubation. INTERPRETATION & CONCLUSION: Our results suggested that culture on Congo red agar was a sensitive and rapid test for detecting slime. This might help in the quick identification in a routine laboratory of slime positive isolates in bacterial keratitis.

Bacteriological Techniques↗
PubMed2002

Screening for amyloid in subcutaneous fat tissue of Egyptian patients with rheumatoid arthritis: clinical and laboratory characteristics.

OBJECTIVE: To screen for amyloid and to assess associated clinical and laboratory characteristics in Egyptian patients with rheumatoid arthritis (RA). METHODS: Abdominal subcutaneous fat aspirates were consecutively collected from 112 patients (103 women, nine men) having RA for five years or more. To detect amyloid, fat smears were stained with Congo red and the concentration of amyloid A protein in fat tissue was measured. Clinical, radiological, and laboratory characteristics of the patients were assessed. RESULTS: Amyloid was detected in eight (7%) of the fat smears stained with Congo red. Compared with the Congo red stain, the sensitivity for detecting amyloid by measurement of amyloid A protein in fat tissue was 75% and the specificity was 100%. The amount of amyloid found was small for both methods. The median disease duration of the eight amyloid patients was significantly longer (17 years) than that of the non-amyloid patients (10 years). Bronchopulmonary disease and constipation were more common, whereas proteinuria and chronic renal insufficiency were not. The number of swollen joints and the number of red blood cells were significantly lower in the amyloid group. CONCLUSIONS: Quantification of amyloid A protein and staining with Congo red are strongly concordant methods of screening for amyloid in fat tissue. The prevalence of amyloid in Egyptian patients with RA is 7%. Proteinuria is not a discriminating feature, whereas long disease duration, constipation, bronchopulmonary symptoms, and a moderate to low number of red blood cells may help to identify the arthritic patients with amyloid.

Adipose Tissue↗
PubMed1997

Kinetics of Cellulose Digestion by Fibrobacter succinogenes S85.

Growing cultures of Fibrobacter succinogenes S85 digested cellulose at a rapid rate, but nongrowing cells and cell extracts did not have detectable crystalline cellulase activity. Cells that had been growing exponentially on cellobiose initiated cellulose digestion and succinate production immediately, and cellulose-dependent succinate production could be used as an index of enzyme activity against crystalline cellulose. Cells incubated with cellulose never produced detectable cellobiose, and cells that were preincubated for a short time with thiocellobiose lost their ability to digest cellulose (competitive inhibition [K(infi)] of only 0.2 mg/ml or 0.56 mM). Based on these results, the crystalline cellulases of F. succinogenes were very sensitive to feedback inhibition. Different cellulose sources bound different amounts of Congo red, and the binding capacity was HCl-regenerated cellulose > ball-milled cellulose > Sigmacel > Avicel > filter paper. Congo red binding capacity was highly correlated with the maximum rates of metabolism of cellulose digestion and inversely related to K(infm). Congo red (250 (mu)g/ml) did not inhibit the growth of F. succinogenes S85 on cellobiose, but this concentration of Congo red inhibited the rate of ball-milled cellulose digestion. A Lineweaver-Burk plot of ball-milled cellulose digestion rate versus the amount of cellulose indicated that Congo red was a competitive inhibitor of cellulose digestion (K(infi) was 250 (mu)g/ml).

Journal Article↗
PubMed1992

Comparison of minimally invasive methods of parietal cell vagotomy in a porcine model.

BACKGROUND: Parietal cell vagotomy (PCV) is an accepted therapy for peptic ulcer disease. Traditional operative PCV results may be obtained laparoscopically or endoscopically. The purpose of this study was to quantitatively evaluate newer methods of PCV. METHODS: Variations in PCV, gastroscopic chemoneurolysis and laparoscopic photoneurolysis, were evaluated in 20 to 25 kg pigs. Traditional operative PCV was performed by laparotomy and served as the operative control. With a posterior truncal vagotomy, a PCV was performed laparoscopically by an anterior seromyotomy with either operative division of the neurovascular bundles with titanium clips or with a defocused CO2 laser. Transesophageal gastroscopic PCV was performed by transmucosal injections of the chemoneurolytic agents, 0.75% cobaltous chloride or 0.1% benzalkonium chloride. Adequacy of PCV at the time of operation was assessed by endoscopic Congo red testing. Two weeks later, repeat Congo red testing was performed by open gastrotomy. Quantitation of completeness of PCV and statistical comparison was determined by photographing the pentagastrin-stimulated gastric mucosa 5 minutes after Congo red application and subsequent comparison of innervated area versus total gastric mucosal area by a computer-driven digitized area-calculation program. RESULTS: All PCV techniques studied produced significant acid-secretory reduction, and both laparoscopic and gastroscopic PCV denervated the parietal cells in a manner comparable with operative PCV. Laser photoneurolysis could only be accomplished by producing full-thickness necrosis of the gastric wall. Submucosal injection of cobaltous chloride produced granulomatous nodules with foreign body crystals. Unlike operative and laparoscopic PCV, transmucosal gastroscopic benzalkonium PCV did not produce gross gastroparesis. CONCLUSIONS: All evaluated laparoscopic and endoscopic PCV techniques effectively denervated the parietal cells of the porcine stomach when evaluated at 2 weeks after treatment. Gastroscopic submucosal injection of benzalkonium chloride produced effective denervation with no evident histologic tissue changes and suggestive evidence of normal gastric emptying. Further evaluation with more extended periods of observation of these new techniques of performing PCV appear warranted.

Animals↗
PubMed1989

[Chromoendoscopic study of the gastric mucosa].

Chromoendoscopy is an intravital endoscopic mucosa dyeing method. Chromoendoscopical methods suitable for examination of gastric mucosa are described including the steps of premedication as well as the steps of Congo-red and methylene-blue tests. The Congo-red test is capable of estimating the extent of acid-secreting area of gastric mucosa. Large acid secreting areas were found in four-fifth of duodenal ulcer patients with the Congo-red test. An equal ratio of small and large acid-secreting areas was found in gastric ulcer patients. The extent of acid-secreting area decreased parallely with the increase of mucosal atrophy in chronic gastritis patients. Methylene-blue test is suitable for detecting mucosal areas containing intestinal metaplasia and cancer. Intestinal metaplasia was detected histologically in 80% of cases containing mucosal areas stained by methylene-blue test. According to the authors' observations chromoendoscopy is a time consumptive additional method, which requires careful premedication and accurate endoscopic technique. Chromoendoscopy is suitable to detect the accurate places of mucosal biopsies therefore it seems to be a valuable element of morpho-functional diagnostic methods.

Congo Red↗
PubMed1989

New method for detecting slime production by coagulase negative staphylococci.

An alternative method for detecting the production of slime by coagulase negative staphylococci was compared with the routinely used Christensen method on 124 isolates of coagulase negative staphylococci from carriage sites, blood cultures, and infected peritoneal dialysis fluids. The alternative method requires the use of a specially prepared solid medium--brain heart infusion broth, supplemented with 5% sucrose, and Congo red stain. Of the 124 tests, there was complete agreement between methods in 107 and only one strain was clearly negative by Christensen's method while positive on Congo red agar. The Congo red method is rapid, sensitive, and reproducible and has the advantage that colonies remain viable on the medium. It is also not subject ot interbatch variation of media which sometimes affects the reproducibility of the Christensen method.

Bacteriological Techniques↗
PubMed1994

Postoperative recurrent ulcer.

Figure 2 summarizes my evaluation of most patients with postoperative recurrent ulcers. First, serum gastrin and calcium levels should be determined in all patients. At the initial endoscopy, any silk sutures should be removed, and gastric ulcers biopsied. The presence of gastric ulcers or evidence of delayed gastric emptying should influence the surgical procedure. Ideally, a Congo red test should be performed at the initial endoscopy. The only caveat is that excessive sedation may invalidate the test results. If the initial diagnostic endoscopy does not include a Congo red test and serum gastrin and calcium levels are normal, it is reasonable to proceed with medical management without vagotomy testing. I believe that if medical management fails and operative treatment is planned, all patients should undergo vagotomy testing. If the Congo red test is definitively positive, sham feeding is not necessary. On the other hand, if the Congo red test suggests hyposecretion, I believe that sham feeding is mandatory prior to proceeding with operation. Conversely, sham feeding tests may be falsely negative in postoperative patients. Therefore, many of our patients undergo both Congo red testing and sham feeding prior to reoperation. Finally, if delayed gastric emptying is believed to be part of the clinical syndrome, a radionuclide scan should be performed to assess gastric emptying. It can be either a solid-meal study or an acetanilidoiminodiacetic acid scan, which measures bile reflux as well as determines emptying of bile from the fasted stomach (see introductory article in this issue).

Humans↗
PubMed2000

Amyloid-like protein structure in mammalian ocular lenses.

PURPOSE: Over the past 30 years, extensive Raman and infrared spectroscopy studies determined that the major proteins in normal mammalian lenses exist predominantly as beta-pleated sheets in vivo. The present study examines the presence of amyloid protein supramolecular order of lens protein beta-pleated sheet arrays. METHODS: The classic amyloidophilic stains Congo red and thioflavine were used in situ to identify lens regions with amyloid protein structure using brightfield microscopy. Birefringence in Congo red stained lenses was determined using polarizing light microscopy. Thioflavine stained lenses were also examined by fluorescence microscopy to detect a diagnostic fluorescence red-shift indicative of the intercalation of thioflavine into beta-sheet amyloid protein structures. RESULTS: The major findings are 1) Congo red staining begins in the lens interior at an abrupt boundary coinciding with the extensive reorganization of cell biology and physical environment that occurs during normal lens fiber cell differentiation. 2) Apple-green birefringence in Congo red stained lenses co-localizes with amyloidophilic dye affinity. 3) Thioflavine stains the lens interior beginning at the same boundary. 4) A red shift in thioflavine fluorescence was detected in the lens interior that co-localizes with amyloidophilic dye affinities and birefringence in organelle-free lens fibers. CONCLUSIONS: The present data demonstrating amyloidophilic dye binding, in situ red shift in thioflavine fluorescence, and apple-green birefringence provide evidence that lens protein beta-sheet arrays are organized in an amyloid-like supramolecular order in interior fiber cells of mammalian ocular lenses. The inherent stability of amyloid-like protein structure may contribute to the long-term structural integrity and transparency of the lens.

Amyloid↗
PubMed2003

Local and long-range structural effects caused by the removal of the N-terminal polypeptide fragment from immunoglobulin L chain lambda.

The role of the N-terminal polypeptide fragment of the immunoglobulin l-chain in V domain packing stability, and the flexibility of the whole chain was approached by molecular dynamics simulation. The observations were supported by experimental analysis. The N-terminal polypeptide fragment appeared to be the low-stability packing element in the V domain. At moderately elevated temperature it may be replaced at its packing locus by Congo red and then removed by proteolysis. After removal of Congo red by adsorption to (diethylamino)ethyl (DEAE) cellulose, the stability of complete L chain and of L chain devoid of the N-terminal polypeptide fragment were compared. The results indicated that the N-terminal polypeptide fragment plays an essential role in the stability of the V domain. Its removal makes the domain accessible for ANS and Congo red dye binding without heating. The decreased domain stability was registered in particular as increased root mean square (RMS) fluctuation and higher susceptibility to proteolytic attack. The long-range effect was most clearly manifested at 340 K as independent V and C domain fluctuation in the l-chain devoid of the N-terminal polypeptide fragment. This is likely due to the lack of direct connections between the N- and C-termini of the V domain polypeptide. In a complete V domain the connection involves residues 8-12 and 106-110 in particular. Partial or complete disruption of this connection increases the freedom of V domain rotation, while its increased cohesion strengthens the coupling of the V and C domains, making the whole L chain less flexible.

Amino Acid Sequence↗
PubMed2006

Recycling of agricultural solid waste, coir pith: removal of anions, heavy metals, organics and dyes from water by adsorption onto ZnCl2 activated coir pith carbon.

The abundant lignocellulosic agricultural waste, coir pith is used to develop ZnCl(2) activated carbon and applied to the removal of toxic anions, heavy metals, organic compounds and dyes from water. Sorption of inorganic anions such as nitrate, thiocyanate, selenite, chromium(VI), vanadium(V), sulfate, molybdate, phosphate and heavy metals such as nickel(II) and mercury(II) has been studied. Removal of organics such as resorcinol, 4-nitrophenol, catechol, bisphenol A, 2-aminophenol, quinol, O-cresol, phenol and 2-chlorophenol has also been investigated. Uptake of acidic dyes such as acid brilliant blue, acid violet, basic dyes such as methylene blue, rhodamine B, direct dyes such as direct red 12B, congo red and reactive dyes such as procion red, procion orange were also examined to assess the possible use of the adsorbent for the treatment of contaminated ground water. Favorable conditions for maximum removal of all adsorbates at the adsorbate concentration of 20 mg/L were used. Results show that ZnCl(2) activated coir pith carbon is effective for the removal of toxic pollutants from water.

Adsorption↗
PubMed2002

Subepithelial corneal amyloid deposits in a case of congenital glaucoma: a case report.

PURPOSE: To report a rare association of subepithelial amyloid deposits in a long-standing case of congenital glaucoma. METHODS: A 5-year-old girl was brought to the emergency service with complaints of bleeding from the left eye after injury to the eye with the door handle. Parents gave history of enlarged black portion of the eye since birth with increasing white opacity from the age of 6 months. On examination under general anesthesia, the left eye showed a limbal tear extending from 7 to 3.30 o'clock position and uveal prolapse. The right eye showed corneal edema, megalocornea, central white raised plaque, and high intraocular pressure. A diagnosis of bilateral congenital glaucoma with open globe injury, left eye, corneal scarring with degeneration, right eye was made and was advised enucleation. The enucleated eye was submitted for routine histologic examination. At a 3-year follow-up, there was an increase in the corneal scarring, with no significant improvement in vision. The child refused keratoplasty and was referred to low vision centre for visual rehabilitation. RESULTS: The enucleated eye was distorted and filled with blood. The cornea showed confluent pink homogenous wavy deposits in the subepithelial region. These deposits appeared brick red with Congo Red stain and showed apple green birefringence when viewed under polarized filters, confirming the amyloid nature of the deposits. There was loss of ganglion cell layer in the detached retina. CONCLUSION: The subepithelial amyloid deposits, presumably bilateral, in advanced case of congenital glaucoma were possibly secondary to the long-standing edema, scarring and vascularization. These secondary changes should be kept in mind as they could contribute to further deterioration in vision in advanced cases.

Amyloidosis↗
PubMed2004

[Treatment of pulmonary amyloidosis with dimethyl sulfoxide--a case report].

Metastatic lung tumor was suspected in a 52-year-old woman who showed multiple nodules on her chest radiographs. Conventional examinations did not define the diagnosis, and so a biopsy was performed using video-assisted thoracoscopic surgery. Pathological examination demonstrated deposits of amorphous materials which were stained red by Congo red staining, even after potassium permanganate treatment. Green birefringence was also observed in the deposits under a polarized light microscope. A diagnosis of localized pulmonary amyloidosis with AL type amyloid protein was made, and therapy with dimethyl sulfoxide (10 ml/day) was started. During the two-year therapy, little exacerbation on pulmonary nodules was observed. It was suggested that dimethyl sulfoxide inhibited the progression of the disease.

Amyloidosis↗
PubMed2006

[Histopathological diagnosis of amyloidosis].

For the diagnosis of amyloidosis, histological evidence of amyloid deposition is essential. Histologically, an amyloid deposit is stained orange red with Congo red and shows green birefringence under polarized light. When amyloidosis is clinically suspected, endoscopic biopsy of the stomach, duodenum or colon, or aspiration biopsy of abdominal fat is usually performed. If clinicians suspect amyloidosis, they should advise pathologists. Identification of the chemical type of amyloid is necessary with respect to treatment and prognosis. Immunohistochemical examination of amyloid in formalin-fixed, paraffin-embedded sections is simple to perform in most pathological laboratories. In Japan, almost all cases of systemic amyloidosis are classified as AL, AA, ATTR or Abeta2M amyloidosis, so the use of anti-immunoglobulin light chain, anti-amyloid A, anti-transthyretin and anti-beta2 microglobulin antibody is recommended for the classification of systemic amyloidosis. Formic acid pretreatment, which is often used for immunohistochemical detection of amyloidosis, is useful and easy for antigen retrieval. Amyloid deposits of AL amyloidosis are sometimes not immunostained well with commercial anti-immunoglobulin light chain antibody. Previously, we generated polyclonal antibodies against synthetic peptides corresponding to positions 118-134 of immunoglobulin lambda light chain and positions 116-133 of immunoglobulin kappa light chain. These antibodies are very useful for detecting AL amyloidosis because they react with amyloid deposits on formalin-fixed, paraffin-embedded specimens in almost all AL amyloidosis cases. Exact diagnosis and typing of amyloidosis are necessary for therapy.

Amyloid↗