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PubMed1995

Enhanced secretion through the Shigella flexneri Mxi-Spa translocon leads to assembly of extracellular proteins into macromolecular structures.

Genes required for entry of Shigella flexneri into epithelial cells in vitro are clustered in two adjacent loci, one of which encodes secretory proteins, the IpaA-D proteins, and the other their dedicated secretion apparatus, the Mxi-Spa translocon. Ipa secretion, which is induced upon contact of bacteria with epithelial cells, is prevented during growth in vitro. Here, we show that ipaB and ipaD mutations lead to enhanced secretion of a set of about 15 proteins. These extracellular proteins and some Ipas associate in organized structures consisting of extended sheets. Growth of the wild-type strain in the presence of Congo red is shown to induce protein secretion through the Mxi-Spa translocon. Cultures grown to stationary phase in the presence of Congo red contain extracellular filaments whose composition and morphology are similar to those produced by the hypersecreting ipaB and ipaD mutants.

Antigens, Bacterial↗
PubMed2000

Secondary cutaneous amyloidosis in disseminated superficial porokeratosis: a case report.

Disseminated superficial porokeratosis (DSP) is a rare cause of secondary cutaneous amyloidosis. An 83-year-old male patient showed an increase in both size and number of DSP lesions after contracting pulmonary tuberculosis. The DSP lesions of the patient consisted of numerous annular eruptions on both sun-exposed and sun-protected areas, which occurred over a period of 20 years. Multiple skin biopsies were taken from normal or lesional/sun-exposed or sun-protected skin samples. Histopathologic examination included routine H&E stains, Congo red stains, thioflavin-T stains and anticytokeratin antibodies (AE1, AE3). And the results were as follows; 1) Positive staining with Congo red and thioflavin-T indicated an amyloid nature for the deposits, 2) confinement of the amyloid deposition just below the lesional epidermis (while sparing the neighboring uninvolved or distant normal skin) indicated some role of the lesional epidermis, and 3) positive staining with AE3 further indicated an epidermal origin-type II epithelial keratin-of the amyloid. We present a case of DSP with a local amyloid deposit, characterized by association of positive familial background, severe pruritus and pulmonary tuberculosis.

Aged↗
PubMed1993

pH profile of esophagus in patients with inlet patch of heterotopic gastric mucosa after tetragastrin stimulation. An endoscopic approach.

In a series of 1771 endoscopic examinations from 1988 to 1990, we observed 62 cases (3.3%) of inlet patch of heterotopic gastric mucosa (IPHGM) in the upper esophagus. Ten of the IPHGM patients complained of throat discomfort or globus sensation, and these symptoms were relieved by histamine H2-antagonists, suggesting that these symptoms could be caused by acid secretion from IPHGM. Acidity under tetragastrin stimulation was measured in the esophagus and compared with the pool of gastric juice and saliva under direct vision by a newly devised endoscopic method. Congo red staining was also carried out after pH measurement. Significant pH reduction at IPHGM was observed in three cases, and black coloration with Congo red staining in the IPHGM was observed in four cases. These findings suggest that IPHGM is a mucosal change with latent acid secretion.

Adult↗
PubMed1971

Chemical classes of amyloid substance.

Concentrates of amyloid substance derived from organs of 10 human patients representing a variety of clinical entities were characterized according to their amino acid compositions, their electrophoretic constituents mobile in urea-starch gel at pH 3 and their stability with respect to the binding of Congo red in the pH interval 9-12.5. The analyses revealed the existence of two major classes of amyloid substance. Material from one class, embracing those cases that had a chronic inflammatory disease as the principal associated process (type A amyloidosis), had a similar amino acid composition and was distinguished by the presence in the amyloid substance of a large amount of an electrophoretically well-defined group of low-molecular-weight proteins with an unusual amino acid composition (amyloid protein A); type A amyloid substance lost the typical binding of Congo red dye at pH 11.5 or lower. Concentrates of amyloid substance in the other class (type B amyloidosis), represented by a case of multiple myeloma, 3 other cases of neoplastic disease and a case of primary cardiac amyloidosis, were distinguished by the presence of an electrophoretically more heterogeneous group of protein constituents with different mobilities and with apparently higher molecular weights than that of protein A, by an amino acid composition that is clearly different from that of the first class, and by retention of the typical Congo red-binding property at pH 12 or higher. The major constituent, protein A, of amyloid substances of the first class is clearly different from ordinary fragments of immunoglobulins in size, electrophoretic mobility, and amino acid composition.

Adult↗
PubMed1998

The crystal structure of a 3D domain-swapped dimer of RNase A at a 2.1-A resolution.

The dimer of bovine pancreatic ribonuclease A (RNase A) discovered by Crestfield, Stein, and Moore in 1962 has been crystallized and its structure determined and refined to a 2.1-A resolution. The dimer is 3D domain-swapped. The N-terminal helix (residues 1-15) of each subunit is swapped into the major domain (residues 23-124) of the other subunit. The dimer of bull seminal ribonuclease (BS-RNase) is also known to be domain-swapped, but the relationship of the subunits within the two dimers is strikingly different. In the RNase A dimer, the 3-stranded beta sheets of the two subunits are hydrogen-bonded at their edges to form a continuous 6-stranded sheet across the dimer interface; in the BS-RNase dimer, it is instead the two helices that abut. Whereas the BS-RNase dimer has 2-fold molecular symmetry, the two subunits of the RNase A dimer are related by a rotation of approximately 160 degrees. Taken together, these structures show that intersubunit adhesion comes mainly from the swapped helical domain binding to the other subunit in the "closed interface" but that the overall architecture of the domain-swapped oligomer depends on the interactions in the second type of interface, the "open interface." The RNase A dimer crystals take up the dye Congo Red, but the structure of a Congo Red-stained crystal reveals no bound dye molecule. Dimer formation is inhibited by excess amounts of the swapped helical domain. The possible implications for amyloid formation are discussed.

Animals↗
PubMed1984

Medullary carcinoma of the thyroid in fine-needle aspiration biopsies.

A study of fine-needle aspiration biopsies (FNAB) in six cases of medullary carcinoma of thyroid (MCT) revealed that the presence of spindle cells in addition to the more usually recognized polygonal cells is a common feature and a significant clue to the diagnosis. Both spindle and polygonal cells contain cytoplasmic granules that stain positively for calcitonin by the immunoperoxidase technic. Amyloid, when present, can be identified presumptively in the Papanicolaou stain. This can be confirmed by restaining with Congo red. Both immunoperoxidase staining for calcitonin and amyloid staining for Congo red work well on the decolorized Papanicolaou-stained smears.

Adult↗
PubMed2000

A novel family of cell wall-related proteins regulated differently during the yeast life cycle.

The Saccharomyces cerevisiae Ygr189c, Yel040w, and Ylr213c gene products show significant homologies among themselves and with various bacterial beta-glucanases and eukaryotic endotransglycosidases. Deletion of the corresponding genes, either individually or in combination, did not produce a lethal phenotype. However, the removal of YGR189c and YEL040w, but not YLR213c, caused additive sensitivity to compounds that interfere with cell wall construction, such as Congo red and Calcofluor White, and overexpression of YEL040w led to resistance to these compounds. These genes were renamed CRH1 and CRH2, respectively, for Congo red hypersensitive. By site-directed mutagenesis we found that the putative glycosidase domain of CRH1 was critical for its function in complementing hypersensitivity to the inhibitors. The involvement of CRH1 and CRH2 in the development of cell wall architecture was clearly shown, since the alkali-soluble glucan fraction in the crh1Delta crh2Delta strain was almost twice the level in the wild-type. Interestingly, the three genes were subject to different patterns of transcriptional regulation. CRH1 and YLR213c (renamed CRR1, for CRH related) were found to be cell cycle regulated and also expressed under sporulation conditions, whereas CRH2 expression did not vary during the mitotic cycle. Crh1 and Crh2 are localized at the cell surface, particularly in chitin-rich areas. Consistent with the observed expression patterns, Crh1-green fluorescent protein was found at the incipient bud site, around the septum area in later stages of budding, and in ascospore envelopes. Crh2 was found to localize mainly at the bud neck throughout the whole budding cycle, in mating projections and zygotes, but not in ascospores. These data suggest that the members of this family of putative glycosidases might exert a common role in cell wall organization at different stages of the yeast life cycle.

Alleles↗
PubMed2002

Spa32 regulates a switch in substrate specificity of the type III secreton of Shigella flexneri from needle components to Ipa proteins.

Type III secretion systems (TTSS) are essential virulence determinants of many gram-negative bacteria and serve, upon physical contact with target cells, to translocate bacterial proteins directly across eukaryotic cell membranes. The Shigella TTSS is encoded by the mxi/spa loci located on its virulence plasmid. By electron microscopy secretons are visualized as tripartite with an external needle, a transmembrane domain, and a cytoplasmic bulb. In the present study, we generated a Shigella spa32 mutant and studied its phenotype. The spa32 gene shows low sequence homology to Salmonella TTSS1 invJ/spaN and to flagellar fliK. The spa32 mutant, like the wild-type strain, secreted the Ipas and IpgD, which are normally secreted via the TTSS, at low levels into the growth medium. However, unlike the wild-type strain, the spa32 mutant could neither be induced to secrete the Ipas and IpgD instantaneously upon addition of Congo red nor penetrate HeLa cells in vitro. Additionally, the Spa32 protein is secreted in large amounts by the TTSS during exponential growth but not upon Congo red induction. Interestingly, electron microscopy analysis of the spa32 mutant revealed that the needle of its secretons were up to 10 times longer than those of the wild type. In addition, in the absence of induction, the spa32 mutant secreted normal levels of MxiI but a large excess of MxiH. Taken together, our data indicate that the spa32 mutant presents a novel phenotype and that the primary defect of the mutant may be its inability to regulate or control secretion of MxiH.

Adhesins, Bacterial↗
PubMed2005

Aggregation and porin-like channel activity of a beta sheet peptide.

Beta sheet peptides (e.g., amyloid beta) are known to form ion channels in lipid bilayers possibly through aggregation, though the channel structure is not clear. We have recently reported that a short beta sheet peptide, (xSxG)(6), forms porin-like voltage-gated channels in lipid bilayers [Thundimadathil et al. (2005) Biochem. Biophys. Res. Commun. 330, 585-590]. To account for the porin-like activity, oligomerization of the peptide into a beta barrel-like structure was proposed. In this work, peptide aggregation in aqueous and membrane environments and a detailed study of channel properties were performed to gain insight into the mechanism of channel formation. The complex nature of the channel was revealed by kinetic analysis and the occurrence of interconverting multiple conductance states. Ion channels were inhibited by Congo red, suggesting that the peptide aggregates are the active channel species. Peptide aggregation and fibril formation in water were confirmed by electron microscopy (EM) and Congo red binding studies. Furthermore, oligomeric structures in association with lipid bilayers were detected. Circular dichroism of peptide-incorporated liposomes and peptide-lipid binding studies using EM suggest a lipid-induced beta sheet aggregation. Gel electrophoresis of peptide-incorporated liposomes showed dimeric and multimeric structures. Taken together, this work indicates insertion of (xSxG)(6) as oligomers into the lipid bilayer, followed by rearrangement into a beta barrel-like pore structure. A large peptide pore comprising several individual beta sheets or smaller beta sheet aggregates is expected to have a complex behavior in membranes. A dyad repeat sequence and the presence of glycine, serine, and hydrophobic residues in a repeated pattern in this peptide may be providing a favorable condition for the formation of a beta barrel-like structure in lipid bilayers.

Amino Acid Sequence↗
PubMed2006

Formation of complex films with water-soluble CTAB molecules.

This communication reports the formation of complex Langmuir monolayer at the air-water interface with the water-soluble N-cetyl N,N,N-trimethyl ammonium bromide (CTAB) molecules when interacted with the stearic acid (SA) molecules. The reaction kinetics of the formation of the CTAB-SA complex was monitored by observing the surface pressure versus time graph. Multilayered LB films of this complex doped with Congo red was successfully formed onto a quartz substrate. UV-Vis absorption and steady-state fluorescence spectroscopic characteristics of this doped LB films confirms the successful incorporation of Congo red molecules in to the CTAB-SA complex films.

Absorption↗
PubMed1979

Relation of intestinal metaplasia to the acid-secreting area.

The distribution of intestinal metaplasia in relation to the acid-secreting are was investigated in 66 patients not suffering from gastroduodenal diseases. Studies were made via the endoscopic Congo Red test in combination with the methylene blue staining method. In this test, the acid-secreting area is identified as a "discolored" area in which Congo Red turns from red to blue-black, and areas of intestinal metaplasia were observed as areas staining blue with methylen blue. A close relationship was found between the acid-secreting area and the distribution of intestinal metaplasia; in general, intestinal metaplasia tended to be more extensive and more frequent when the acid-secreting area was small. It was also found that intestinal metaplasia increased with age. However, even in patients who were over 40 years of age, intestinal metaplasia was seen in 12.5% only, if the acid-secreting area was large. Hence, it appears likely that development of intestinal metaplasia is more closely related to the acid-secreting function than to aging.

Adult↗
PubMed1992

Activity staining of endoglucanases in polyacrylamide gels.

The endoglucanases of Penicillium funiculosum were analyzed for the presence of multiple forms using a modified version of the Congo red method. Postelectrophoretic slab gels were directly incubated in a solution of carboxymethylcellulose for a period as short as 15 min and then the activities were visualized by staining with Congo red. Ten distinct bands of clearances were obtained indicating the presence of at least as many multiple forms.

Acrylic Resins↗
PubMed1996

Molecular cloning and characterization of nlpH, encoding a novel, surface-exposed, polymorphic, plasmid-encoded 33-kilodalton lipoprotein of Borrelia afzelii.

Borrelia burgdorferi sensu lato organisms, comprising B. burgdorferi sensu stricto, Borrelia afzelii, and Borrelia garinii, are tick-borne pathogens causing Lyme borreliosis in humans. To identify putative virulence determinants, a B. afzelii DNA library was screened for Congo red dye binding, a property associated with virulence in pathogenic bacteria. One clone was found to carry a 663-nucleotide-long open reading frame encoding a Congo red dye-binding protein with a calculated molecular mass of 25,660 Da. The amino acid sequence deduced from its nucleotide sequence was found to include a consensus bacterial lipidation site present at residues 15 to 18 (Leu-Ser-Gly-Cys). The lipoprotein nature was demonstrated by incorporation of radioactive palmitate; hence, this protein has been termed NlpH, for new lipoprotein H. NlpH is located on the surface of B. afzelii, and the nlpH gene is found on a circular plasmid. The nlpH gene is also found in B. burgdorferi sensu stricto and B. garinii. Immediately upstream of nlpH is located a smaller reading frame encoding a polypeptide containing the casein kinase II phosphorylation recognition sequence, (Ser/Thr)-X-Y-(Glu/Asp), repeated 10 times.

Amino Acid Sequence↗
PubMed1997

Involvement of a 70-kb plasmid of the epidemic Shigella dysenteriae type 1 (Dt66) strain in drug-resistance, lipopolysaccharide synthesis, and virulence.

The present work characterizes a 70 kb plasmid of the Indian epidemic isolate Shigella dysenteriae type 1 strain Dt66 in relation to virulence and drug-resistance characters. Curing of the plasmids of Dt66 strain by acriflavine (AF) yielded seven groups of AF-cured derivatives. One group of derivatives containing 5 plasmids (70-2.5 kb) as against 6 (120-2.5 kb) in the wild type showed resistance to chloramphenicol, pivmecillinam, streptomycin, and tetracycline (CmrPivrSmrTcr) but sensitivity for ampicillin and nalidixic acid (AmsNals). This derivative when used as donor in conjugation experiments with a plasmidless E. coli KL318 strain (AmsCmsPivsSmrTcsNalr) as recipient, transferred only its 70 kb plasmid into the recipient with concomitant transfer of the CmrPivr phenotype. Both the donor and the transconjugant exhibited a diffuse pattern of adherence and produced keratoconjunctivitis in guinea pigs. Transconjugants contained a 42 kDa lipopolysaccharide band which was absent in recipient. Moreover, both donor and transconjugants showed Congo red binding ability. The results suggest that the 70 kb plasmid of S. dysenteriae Dt66 strain encodes not only Cmr,Pivr character but also is associated with virulence-related characters such as Congo red binding, LPS-biosynthesis, HeLa cell adherence, and keratoconjunctivitis related to virulence.

Animals↗
PubMed1997

Alzheimer's amyloid-beta peptide inhibits sodium/calcium exchange measured in rat and human brain plasma membrane vesicles.

Na+/Ca2+ exchange activity was measured by monitoring vesicular Ca2+ content after incubation in buffers containing 45Ca2+. When Na+-loaded vesicles were placed into Na+-free buffer, vesicular Ca2+ content increased rapidly and reached a plateau after two to three minutes. Only preaggregated amyloid-beta1-40 (Abeta1-40) and Abeta25-35 reduced vesicular Ca2+ content. Both peptides produced a maximal reduction in Ca2+ content of approximately 50%. The peptides reduced Ca2+ content with similar potency and half maximal effects were seen at less than 10 microM for Abeta25-35. Calcium-loaded vesicles mediate a rapid Ca2+/Ca2+ exchange, which also was inhibited by aggregated Abeta25-35. Aggregated Abeta25-35 did not affect the passive Ca2+ permeability of the vesicles. Aggregated Abeta25-35 reduced Ca2+ content in plasma membrane vesicles isolated from normal and Alzheimer's disease frontal cortex with less potency but the same efficacy as seen in rat brain. Aggregated Abeta25-35 did not produce nonspecific effects on vesicle morphology such as clumping or loss of intact vesicles. When placed in the buffer used to measure Ca2+ content, Congo Red at molar ratios of less than one blocked the inhibitory effect of preaggregated Abeta25-35. When added in equimolar concentrations to freshly dissolved and unaggregated Abeta25-35, Congo Red also was effective at blocking the inhibitory effect on Ca2+ content. In contrast, vitamin E (antioxidant) and N-tert-butyl-alpha-phenylnitrone (spin trapping agent) failed to block the inhibitory action of aggregated Abeta25-35. The exact mechanisms of Abeta-induced neurotoxicity in cell culture has yet to be solved. Accumulation of free radicals play a necessary role, but disruptions of Ca2+ homeostasis are also important. The data presented here are consistent with a proposed mechanism where aggregated Abeta peptides directly interact with hydrophobic surfaces of the exchanger protein and/or lipid bilayer and interfere with plasma membrane Ca2+ transport.

Alzheimer Disease↗
PubMed1991

Is the vagotomy complete?

Incomplete vagotomy is the single most common cause of ulcer recurrence. Completeness of vagotomy was assessed postoperatively in 17 patients using the congo red test. Various types of vagotomies included truncal vagotomy and gastrojejenostomy in 6, highly selective vagotomy in 5, truncal vagotomy and pyloroplasty in 3 and selective vagotomy with gastrojejenostomy in 3 cases. Congo red test was positive in 13 cases, with 9 of these showing evidence of incomplete vagotomy manifesting as erosions, duodenitis, stomal ulceration or ulcer recurrence.

Adolescent↗
PubMed1990

Role of plasmids in virulence-associated attributes and in O-antigen expression in Shigella dysenteriae type 1 strains.

The association of plasmids with virulence characters and O-antigen expression was studied in two virulent and seven avirulent mutant strains of Shigella dysenteriae type 1. Deletion of a 12-Mda segment from a 140-Mda plasmid in two smooth avirulent mutants made the derivatives avirulent in the Sereny test and non-invasive in HeLa cells. The mutants were unable to bind Congo red, and did not express the virulence marker antigen. Mutants completely lacking the 140-Mda plasmid also showed similar avirulent characters. However, rough mutants retained the ability to bind Congo red. Our results indicate that the essential gene(s) for virulence may be located on the 140-Mda plasmid, a small deletion from which may lead to avirulence. This deletion did not affect the protein antigen expression nor change their antigenicity. Analysis of lipopolysaccharide (LPS) patterns showed that strains containing the 6-Mda plasmid produced the complete LPS and were smooth, whereas strains containing either the 140- or a 4- or 2-Mda plasmid, in the absence of the 6-Mda plasmid, produced smaller amounts of O antigen and were rough. Western-blot analysis and crossed immuno-electrophoresis gave similar results. The 140-, 4-, or 2-Mda plasmid, in the absence of the 6-Mda plasmid, may code for non-specific galactosyl transferase-like activity which can add, non-specifically and at a reduced level, the galactose residue (the first sugar in the O antigen repeat unit) to the LPS core.(ABSTRACT TRUNCATED AT 250 WORDS)

Agglutination Tests↗
PubMed2003

[A case of psoriatic arthropathy complicated with HTLV-I carrier and secondary amyloidosis].

We report a case of psoriatic arthropathy complicated with HTLV-I carrier and secondary amyloidosis. She was a 45-year-old woman and was diagnosed as psoriatic arthropathy and HTLV-I carrier in December 1999. She was treated with combination therapy corticosteroid, salazosulfapyridine, methotrexate, cyclophosphamide, and gold sodium. However, her arthralgia and fever was not completely improved. In April in 2001, she was admitted to our hospital because of nausea, vomiting, and diarrhea. Her colon fiberscopic examination showed rubber, erosion, and multiple ulcers of the rectum through descending colon mucosa. Her biopsied specimens of the colon mucosa showed Congo Red dye stained amyloid deposits. Because the loss of stain for Congo Red dye after exposure to potassium permanganate, the deposits were amyloid A protein. This case is considered as a rare case of psoriatic arthropathy and HTLV-I carrier complicated with secondary amyloidosis in a relatively short period (approximately two years) after onset of psoriatic arthropathy.

Amyloidosis↗