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PubMed1991

Relationship between the iron regulated outer membrane proteins and the outer membrane proteins of in vivo grown Pasteurella multocida.

The SDS-PAGE patterns of the outer membrane protein (OMP) extracts of Pasteurella multocida strain P1059, grown under iron-restricted, iron-replete and in vivo conditions, were examined. The results showed that the iron-regulated outer membrane proteins (IROMPs) with molecular masses of 76 kDa, 84 kDa, and 94 kDa were expressed by bacteria grown in iron-restricted media. They were also expressed by in vivo grown P. multocida. Convalescent-phase sera, obtained from turkeys which had survived pasteurellosis, contained antibodies that reacted intensly with th three IROMPs. This indicated that these proteins were expressed in vivo. Bacteria expressing the IROMPs showed greater binding to Congo Red when compared to cells not expressing IROMPs. Cells expressing the IROMPs or its OMP extracts grown in iron-restricted media also showed greater binding to 59Fe-pasteurella siderophore (multocidin) when compared to bacteria or its extracts not expressing IROMPs. Convalescent-phase sera, which contained antibodies against the IROMPs, blocked this specific 59Fe-multocidin binding to IROMPs. Autoradiography was used to determine which of these IROMPs functioned as a receptor for the iron-multocidin complex. The results suggested that these three IROMPs have specific epitopes for binding to the iron multocidin complex.

Animals↗
PubMed2000

Amyloid-like inclusions in Huntington's disease.

Huntington's disease is a progressive, autosomal dominantly inherited, neurodegenerative disease that is characterized by involuntary movements (chorea), cognitive decline and psychiatric manifestations. This is one of a number of late-onset neurodegenerative disorders caused by expanded glutamine repeats, with a likely similar biochemical basis. Immunohistochemical studies on Huntington's disease tissue, using antibodies raised to the N-terminal region of huntingtin (adjacent to the repeat) and ubiquitin, have recently identified neuronal inclusions within densely stained neuronal nuclei, peri-nuclear and within dystrophic neuritic processes. However, the functional significance of inclusions is unknown. It has been suggested that the disease-causing mechanism in Huntington's disease (and the other polyglutamine disorders) is the ability of polyglutamine to undergo a conformational change that can lead to the formation of very stable anti-parallel beta-sheets; more specifically, amyloid structures. We examined, using Congo Red staining and both polarizing and confocal microscopy, post mortem human brain tissue from five Huntington's disease cases, two Alzheimer's disease cases and two normal controls. Brains from five transgenic mice (R6/2)(12) expressing exon 1 of the human huntingtin gene with expanded polyglutamine, and five littermate controls, were also examined by the same techniques. We have shown that some inclusions in Huntington's disease brain tissue possess an amyloid-like structure, suggesting parallels with other amyloid-associated diseases such as Alzheimer's and prion diseases.

Alzheimer Disease↗
PubMedDate not supplied

Proximal gastric vagotomy. A comparative study between the standard technique and the extended technique associated with denervation of the greater curvature.

Forty-six patients in the postoperative period of proximal gastric vagotomy (PGV) for duodenal ulcer (DU) were studied comparatively to verify whether the dividing of the gastroepiploic nerves (Rosati's maneuver) can reduce or not the occurrence of recurrent ulcer as it was proposed. Twenty-one patients who underwent PGV associated with Rosati's maneuver (PGV-R) were compared to 25 after standard PGV (PGV-S), according to several criteria: (1) clinical evaluation; (2) pre and postoperative basal and pentagastrin-stimulated gastric acidity; (3) postoperative basal and pentagastrin-stimulated serum pepsinogen; (4) postoperative basal and sham feeding-stimulated serum gastrin; (5) postoperative endoscopy; (6) endoscopic Congo red test. Both groups were similar (P greater than 0.05) as to age, sex, levels of preoperative gastric acidity and had a 24.4 month average follow-up (12 to 58 months). There has been no significant difference between the techniques studied as to clinical, secretory, morphological or hormonal gastric tests, although PGV-R proved more effective in reducing basal gastric acidity than PGV-S (P less than 0.05). We concluded that Rosati's maneuver does not improve the results obtained with PGV, although it provided greater reduction of basal gastric acidity than PGV-S.

Adult↗
PubMed2003

Aspirin and diabetes: inhibition of amylin aggregation by nonsteroidal anti-inflammatory drugs.

Islet amyloid polypeptide (amylin), cosecreted with insulin by the pancreatic beta-cells, has an important role in the regulation of islet cell hormone homeoastasis. Deposition of beta-sheet polypeptide fibrils into amyloid deposits is considered to be central to the pathology of a number of amyloidogenic disorders, including type-2 diabetes. Amyloid deposits comprised of beta-sheet fibrillar amylin observed in type-2 diabetics are cytotoxic and may have a prominent role in causing beta-cell dysfunction. The amyloidogenic process may impair beta-cell function before cell death and replacement by amyloid. Preservation of beta-cell viability and insulin secretion is a major objective in diabetic care. Using circular dichroism and Congo red absorption techniques we found that clinically relevant doses of aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDS) prevented and also reversed the beta-sheet conformation of human amylin. The specific COX-2 inhibitors were less effective. The anti-inflammatory steroid prednisolone or the analgesic acetaminophen had no effect on amylin fibrillogenesis. This action of NSAIDS was similar to their inhibition of beta-sheet conformation of the Alzheimer protein, amyloid-beta. Aspirin, currently recommended for the prevention of cardiovascular complications in diabetic patients, may also ameliorate the disease process in diabetes by preserving the beta-cell function.

Amyloid↗
PubMedDate not supplied

Prion protein and the scrapie agent: in vitro studies in infected neuroblastoma cells.

The mouse neuroblastoma cell line N2a was persistently infected with the Chandler strain of the mouse scrapie agent. Although the infection did not spread to infect > 1% of the cells, clones were established that had from 50 to 100% infected cells. These clones expressed the abnormal protease-resistant form of prion protein (PrP), which is believed to mediate brain degeneration in animals with scrapie and bovine spongiform encephalopathy and in humans with kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome. With this in vitro system, Congo red and several sulfated polysaccharides, including heparin and pentosan polysulfate, were found to inhibit accumulation of protease-resistant PrP. These results and additional data confirming PrP binding to heparin suggested a possible role for sulfated glycosaminoglycans in the generation of protease-resistant PrP during scrapie infection. Accumulation of protease-resistant PrP was also blocked in vitro by expression of foreign PrP molecules, indicating that PrP from different species might compete for common substrates in this process. These results using scrapie-infected cell lines provide new opportunities for development of drugs capable of blocking the brain degeneration caused by scrapie and other transmissible spongiform encephalopathies.

Animals↗
PubMed2003

Glycation induces formation of amyloid cross-beta structure in albumin.

Amyloid fibrils are components of proteinaceous plaques that are associated with conformational diseases such as Alzheimer's disease, transmissible spongiform encephalopathies, and familial amyloidosis. Amyloid polypeptides share a specific quarternary structure element known as cross-beta structure. Commonly, fibrillar aggregates are modified by advanced glycation end products (AGE). In addition, AGE formation itself induces protein aggregation. Both amyloid proteins and protein-AGE adducts bind multiligand receptors, such as receptor for AGE, CD36, and scavenger receptors A and B type I, and the serine protease tissue-type plasminogen activator (tPA). Based on these observations, we hypothesized that glycation induces refolding of globular proteins, accompanied by formation of cross-beta structure. Using transmission electron microscopy, we demonstrate here that glycated albumin condensates into fibrous or amorphous aggregates. These aggregates bind to amyloid-specific dyes Congo red and thioflavin T and to tPA. In contrast to globular albumin, glycated albumin contains amino acid residues in beta-sheet conformation, as measured with circular dichroism spectropolarimetry. Moreover, it displays cross-beta structure, as determined with x-ray fiber diffraction. We conclude that glycation induces refolding of initially globular albumin into amyloid fibrils comprising cross-beta structure. This would explain how glycated ligands and amyloid ligands can bind to the same multiligand "cross-beta structure" receptors and to tPA.

Amyloid↗
PubMed2000

Intracellular processing and toxicity of the truncated androgen receptor: nuclear congophilia-associated cell death.

The pathogenesis of the selective motor neuron death in spinal bulbar muscular atrophy (SBMA) is not fully understood. Similar to observations with other mutant polyglutamine (poly Q) expanded proteins, truncated androgen receptor (AR) with expanded poly Q tract cause intracellular aggregates; however, the precise relationship between aggregates and disease pathogenesis is unresolved. In order to have a better understanding of the cellular processing and toxicity of the mutant AR, we focused on a short N-terminal portion of AR containing normal or expanded poly Q repeats, and have carried out biochemical, immunocytochemical, cytochemical and ultrastructural studies of BHK cells at different intervals after transfection. In cells expressing mutant truncated AR, using an anti-AR N-terminal antibody, we observed no immune staining in the nucleus and identified immune negative aggregates surrounded by immunopositive material in the cytoplasm. Congo red staining identified a component of aggregates with a beta-pleated secondary structure in both cytosol and nucleus, while electron microscopy revealed a fibrillary-granular material as the ultrastructural correlate. In addition, acid phosphatase staining and ubiquitin immunocytochemistry demonstrated that in transfected cells, both lysosomal and nonlysosomal degradation systems are actively involved in handling the mutant truncated AR. The temporal relationship of nuclear congophilia to a subsequent massive cell death suggests that entry of proteolytic cleavage products into the nucleus, perhaps the expanded poly Q stretch itself, may play an important role in cell toxicity.

Animals↗
PubMed2002

Structure-based design and study of non-amyloidogenic, double N-methylated IAPP amyloid core sequences as inhibitors of IAPP amyloid formation and cytotoxicity.

Pancreatic amyloid is formed by the aggregation of the 37-residue islet amyloid polypeptide (IAPP) in type II diabetes patients and is cytotoxic. Pancreatic amyloid deposits are found in more than 95 % of type II diabetes patients and their formation is strongly associated with disease progression. IAPP amyloid forms via a conformational transition of soluble IAPP into aggregated beta-sheets. We recently identified IAPP(22-27) (NFGAIL) as a minimum length sequence sufficient to self-associate into beta-sheet-containing amyloid fibrils. Here, we have used the NFGAIL model of the IAPP amyloid core as a structural template to design non-amyloidogenic derivatives of amyloidogenic sequences of IAPP that are able to interact with the native sequences and inhibit amyloid formation. The design of the derivatives was based on a simple, structure-based minimalistic and selective N-methylation approach. Accordingly, a minimum number of two amide bonds on the same side of the beta-strand of the amyloid core was N-methylated. This was expected to eliminate the two intermolecular backbone NH to CO hydrogen bonds which are critical for the extension of the beta-sheet dimers into multimers and amyloid. Other beta-strand "contact sides" remained intact allowing for the derivatives to interact with the native sequences. Double N-methylated derivatives of amyloidogenic and cytotoxic partial IAPP sequences generated included F(N-Me)GA(N-Me)IL, NF(N-Me)GA(N-Me)IL, SNNF(N-Me)GA(N-Me)IL, and SNNF(N-Me)GA(N-Me)ILSS and were found to be devoid of beta-sheet structure, amyloidogenicity and cytotoxicity according to Fourier transform-infrared spectroscopy (FT-IR), Congo red (CR) staining, electron microscopy (EM), and cell viability tests. The derivatives were able to interact with the native sequences and inhibit amyloid formation as shown by circular dichroism spectroscopy (CD), FT-IR and EM. Moreover, SNNF(N-Me)GA(N-Me)ILSS inhibited cytotoxicity of SNNFGAILSS and is thus the first reported inhibitor of IAPP amyloid formation and cytotoxicity. Our results demonstrate the validity of the design approach for IAPP and suggest that it may find application in understanding the structural features of amyloid formation and in the development of inhibitors of amyloid formation and cytotoxicity of other amyloidogenic polypeptides as well.

Amino Acid Sequence↗
PubMed1998

Age-related congophilic inclusions in the brains of apolipoprotein E-deficient mice.

The hippocampal region of apolipoprotein E-deficient mice of varying ages was examined for any morphological changes by light and electron microscopy. Unusual periodic acid-Schiff-positive granules were seen in the hippocampal area of these animals as early as the fourth week of life and their numbers increased gradually with age. These granules were never found in control C57BL/6J (B6) mice before six months-of-age and their numbers were invariable low. They were strongly congophilic when stained with a modified Congo Red technique and reacted with a monoclonal antibody specific to amino acids 17-24 and 35-43 of the beta-amyloid peptide. The immunostaining of these granules with the beta-amyloid peptide was lost after specific adsorption with the appropriate synthetic peptide. These granules were identified ultrastructurally as non-membrane-bound fibrillogranular material in the cytoplasm of protoplasmic astrocytes. The data indicate that an amyloid-like protein accumulates in the protoplasmic astrocytes of the hippocampus of apolipoprotein E-deficient mice, especially in the brains of old animals.

Aging↗
PubMed1976

Experience with the sodium sulphate-Alcian Blue stain for amyloid in cardiac pathology.

The sodium sulphate-Alcian Blue (SAB) method, which stains amyloid green, was evaluated in 220 hearts from elderly patients. The technique proved superior to the Congo red, crystal violet, and thioflavine T methods used either singly or as a battery for the demonstration of cardiac amyloid. Amyloid was easily identified under the X3 objective, even in small amounts. A few non-amyloid components stained varying shades of green but were easily distinguished on morphological grounds. No false positive or equivocal reactions occurred, and in particular elastic laminae and paravascular connective tissue were not tinctorially confused with amyloid. The SAB stain is technically simple and consistently reproducible, and no special light source is required for examination. An additional advantage in cardiac pathology is the simultaneous demonstration of any fibrosis, basophilic myofibre degeneration, tissue mast cells and mucoid degeneration of valves present.

Aged↗
PubMed2001

Analysis of the minimal amyloid-forming fragment of the islet amyloid polypeptide. An experimental support for the key role of the phenylalanine residue in amyloid formation.

The development of type II diabetes was shown to be associated with the formation of amyloid fibrils consisted of the islet amyloid polypeptide (IAPP or amylin). Recently, a short functional hexapeptide fragment of IAPP (NH(2)-NFGAIL-COOH) was found to form fibrils that are very similar to those formed by the full-length polypeptide. To better understand the specific role of the residues that compose the fragment, we performed a systematic alanine scan of the IAPP "basic amyloidogenic units." Turbidity assay experiments demonstrated that the wild-type peptide and the Asn(1) --> Ala and Gly(3) --> Ala peptides had the highest rate of aggregate formation, whereas the Phe(2) --> Ala peptide did not form any detectable aggregates. Dynamic light-scattering experiments demonstrated that all peptides except the Phe(2) --> Ala form large multimeric structures. Electron microscopy and Congo red staining confirmed that the structures formed by the various peptides are indeed amyloid fibrils. Taken together, the results of our study provide clear experimental evidence for the key role of phenylalanine residue in amyloid formation by IAPP. In contrast, glycine, a residue that was suggested to facilitate amyloid formation in other systems, has only a minor role, if any, in this case. Our results are discussed in the context of the remarkable occurrence of aromatic residues in short functional fragments and potent inhibitors of amyloid-related polypeptides. We hypothesize that pi-pi interactions may play a significant role in the molecular recognition and self-assembly processes that lead to amyloid formation.

Alanine↗
PubMed1989

Two simple methods for quantifying low-affinity dye-substrate binding.

Binding with low-affinity ligands, such as histological dyes, can be difficult to quantitate owing to the dissociation of bound ligand with washing or the retention of nonspecifically bound ligand because of incomplete washing. The present report describes two simple, rapid methods of discriminating bound from free ligand without the need for washing steps. One method is based on the spectral changes induced in a dye ligand, Congo red, on binding to the "receptor" insulin fibrils. This method discriminates spectrophotometrically between bound and free ligand without requiring any physical separation of the two forms. No radioactive ligands are necessary, and, by using disposable cuvettes, the entire binding assay can be done in a single container without the need for transfers. The second method employs a non-traditional filtration approach that avoids the need for a washing step by measuring the decrease in concentration of the dye ligand in the filtrate rather than by applying the usual approach of measuring the absolute amount of ligand bound to the precipitated "receptor." Both methods show saturation of binding sites and give similar values for the KD and Bmax.

Congo Red↗
PubMed1998

Characterization of an amorphous deposit in the lamina propria in oral snuff users in the Sudan as collagen.

Histological and ultrastructural features of 25 oral snuff dipper's lesions with distinctive subepithelial hyaline deposits were investigated. Periodic acid-Schiff reaction with and without diastase digestion demonstrated the presence of glycogen and other carbohydrates, but histochemical stains for normal collagen, elastin and fibrin showed a weak variable reactivity of the deposit. Although in 7/25 cases the deposit was in close proximity to labial salivary glands and on occasions ducts were found within the deposit, the presence of mucin was not a consistent feature. Congo red staining and immunohistochemical investigation with an anti-amyloid antibody did not support the previous contention that such deposits were amyloid in nature. Immunohistochemically, collagen antibodies also provided negative results, but ultrastructural features of three biopsies studied suggest that the bulk of this deposit is made up of collagen, as typical cross-striated fibrils were found. The pathogenesis of this deposit could therefore be interpreted as over-production and/or reduced turnover of collagen by resident fibroblasts, which is further altered by the ingredients of toombak. The deposit does not appear to be a secretory product.

Adult↗
PubMed1987

Fine-needle aspiration biopsy from subcutaneous fat. An easy way to diagnose secondary amyloidosis.

Secondary amyloidosis is usually diagnosed by demonstrating amyloid deposits on histological sections by Congo red staining. An alternative method is a fine needle aspiration biopsy (FNAB) from subcutaneous fat which, in this study, was carried out on 301 patients. In order to test the efficiency of FNAB we analysed 146 patients from whom in addition to FNAB one or two histological samples including 125 oral and 65 rectal biopsies were available. FNAB proved very reliable for demonstrating secondary amyloidosis as estimated by the index of sensitivity (0.82). The corresponding figures for rectal and oral biopsy were 0.97 and 0.64 respectively. Although the rectal biopsy proved to be the best method, we strongly recommend FNAB from the subcutaneous fat as the preferred method for screening and diagnosing secondary amyloidosis.

Abdominal Muscles↗
PubMedDate not supplied

Enhancement of cellulase activity by clones selected from the combinatorial library of the cellulose-binding domain by cell surface engineering.

To improve the cellulolytic activity of a yeast strain displaying endoglucanase IotaIota (EG II) from Trichoderma reesei, a combinatorial library of the cellulose-binding domain (CBD) of EG II was constructed by using cell surface engineering. When EG II degrades celluloses, CBD binds to cellulose, and its catalytic domain cleaves the glycosidic bonds of cellulose. CBD had a flat face, composed of five amino acids for binding. It was supposed that the three hydrophobic amino acid residues of the five amino acid residues were essential for binding to cellulose. Therefore, by improving the two remaining amino acid residues, construction of mutants with a combinatorial library of the two amino acids in CBD was carried out and binding ability and hydrolysis activity were measured. In the first screening by halo assay using the Congo Red staining method, about 200 of the 2000 colonies formed clear halos, and then five colonies with the clearest halos were finally selected. In the second screening, the binding ability of the five mutants to phosphoric acid-swollen Avicel was measured. In addition, the measurement of hydrolysis activity toward carboxymethylcellulose (CMC) using the screened mutants was carried out. As a result, the mutated EG II exhibiting higher binding ability (1.5-fold) had higher hydrolysis activity (1.3-fold) compared to the parent EG II-displaying yeast cell, demonstrating that CBD has confirmatively some effect on the cellulase activity through its binding ability of the enzyme to cellulose.

Binding Sites↗
PubMed2003

Occurrence of ica genes for slime synthesis in a collection of Staphylococcus epidermidis strains from orthopedic prosthesis infections.

Staphylococcus epidermidis is a frequent pathogen in infections associated with orthopedic implants. We studied 123 S. epidermidis strains from infections related to orthopedic implants, as regards their ability to express a factor of virulence, namely the slime, an extracellular polysaccharide, which mediates adherence to implants and bacterial colonization. The slime-producing ability was determined by PCR detection of icaA and icaD genes responsible for slime synthesis, and by culture on Congo red agar plates in which slime-producing strains form black colonies, while nonslime-forming ones develop red colonies. 56% of the S. epidermidis isolates were icaA- icaD-positive and grew to become black colonies. In the evaluation of the distribution of slime-forming strains in different sites and types of implants, we found a slight, but not statistically significant, increase in slime-forming strains in total joint prostheses, where tissue compression near the articular faces can form niches in which bacteria crowd, sheltered by the slime. Our findings confirm the role of ica genes as a virulence marker in the pathogenesis of implant-associated orthopedic infections. However, they do not show the existence of a higher frequency of slime-positive strains in a specific type of implant.

Congo Red↗
PubMed1982

Mutagenicity of azo dyes in the Salmonella/activation test.

The detection of certain bis-azo biphenyl dyes (Trypan blue, Evans blue and Congo red) by a modification of the standard Ames test protocol is reported. Detection of a mutagenic response required both the inclusion of FMN in S-9 mix and use of a static pre-incubation step prior to addition of top-agar and pouring in the usual manner. Evidence is presented to substantiate the proposal that use of this protocol provides optimal conditions for the two stages of activation required for the production of mutagenic metabolites from these dyes. These are (i) initial flavin-mediated (hepatic) azo reduction resulting in the formation of proximate mutagenic metabolites followed by (ii) the oxidative metabolism by S-9 resulting in the formation of ultimately mutagenic products.

Animals↗
PubMed2000

Fibrils formed in vitro from alpha-synuclein and two mutant forms linked to Parkinson's disease are typical amyloid.

Two missense mutations in the gene encoding alpha-synuclein have been linked to rare, early-onset forms of Parkinson's disease (PD). These forms of PD, as well as the common idiopathic form, are characterized by the presence of cytoplasmic neuronal deposits, called Lewy bodies, in the affected region of the brain. Lewy bodies contain alpha-synuclein in a form that resembles fibrillar Abeta derived from Alzheimer's disease (AD) amyloid plaques. One of the mutant forms of alpha-synuclein (A53T) fibrillizes more rapidly in vitro than does the wild-type protein, suggesting that a correlation may exist between the rate of in vitro fibrillization and/or oligomerization and the progression of PD, analogous to the relationship between Abeta fibrillization in vitro and familial AD. In this paper, fibrils generated in vitro from alpha-synuclein, wild-type and both mutant forms, are shown to possess very similar features that are characteristic of amyloid fibrils, including a wound and predominantly unbranched morphology (demonstrated by atomic force and electron microscopies), distinctive dye-binding properties (Congo red and thioflavin T), and antiparallel beta-sheet structure (Fourier transform infrared spectroscopy and circular dichroism spectroscopy). alpha-Synuclein fibrils are relatively resistant to proteolysis, a property shared by fibrillar Abeta and the disease-associated fibrillar form of the prion protein. These data suggest that PD, like AD, is a brain amyloid disease that, unlike AD, is characterized by cytoplasmic amyloid (Lewy bodies). In addition to amyloid fibrils, a small oligomeric form of alpha-synuclein, which may be analogous to the Abeta protofibril, was observed prior to the appearance of fibrils. This species or a related one, rather than the fibril itself, may be responsible for neuronal death.

Alanine↗