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Expression and partial characterization of an elastase from Chromobacterium violaceum.

Chromobacterium violaceum was recovered at necropsy from the lungs, liver, spleen, and an interscapular abscess of a Chinese red panda (strain 98-9187) [J. Vet. Diagn. Invest. 12 (2000) 177]. As the lungs exhibited extensive, necrotizing lesions harboring bacterial aggregates, we sought to determine whether C. violaceum produced an elastase that might in part account for these lesions. The C. violaceum type strain (ATCC 12472(T)) and strain 98-9187 were shown to exhibit elastolytic activity by elastin Congo red and elastin nutrient agar assays. The activity was isolated from the periplasmic fraction and was present throughout the growth cycle. Activity increased markedly in late logarithmic phase growth. In elastin-limiting medium, activity rapidly decreased in early stationary phase indicating a tight regulation of yield. The activity was optimal at neutral pH and was sensitive to the metalloproteinase inhibitors EDTA and 1,10-phenanthroline. Activity was restored upon addition of zinc indicating the enzyme is a zinc metalloproteinase. A band corresponding to purified elastase activity was present at approximately 30kDa in a denaturing polyacrylamide gel.

Animals↗

Basal cell carcinoma with hyaline inclusions.

A 69 yr old man had a 4 mm basal cell carcinoma completely excised from the chin. Numerous hyaline cytoplasmic inclusions were contained within the tumor cells. The inclusions stained intensely red with Masson's trichrome, and immunocytochemically there was prominent rim labelling for keratins (bovine, callus and AE1/3) and muscle-specific actin, the latter more faintly decorating the centre of some inclusions. The inclusions were negative for antibodies to cytokeratin Cam5.2, epithelial membrane antigen (EMA), vimentin, S100, neurofilaments, glial fibrillary acidic protein (GFAP) and carcinoembryonic antigen (CEA) and there was no post Congo red apple green birefringence to indicate amyloid. Ultrastructure indicated the inclusions were composed of proteinaceous material surrounded by a defined rim of tonofilaments in cells showing no degenerative features. The findings suggested aberrant tumor cell keratinization. Familiarization with this rare variant of a common cutaneous carcinoma will alleviate diagnostic difficulties that may arise, particularly in superficial tumor curettage.

Aged↗

The use of fluorochromes in the cytochemical characterization of some phytoflagellates.

Sixteen fluorochromes were tested for the cytochemical characterization of two dinoflagellates (Amphidinuim carterae, Prorocentrum micans) and one chlorophycean flagellate (Dunaliella tertiolecta). Depending on the fluorochrome used, various cellular components (including the plasma membrane, thecal plates, pusule, trichocysts, nucleus, lipid bodies and vacuoles) were revealed. The different colours obtained from single or double fluorochrome staining enabled the differentiation and identification of most cellular components. Protoplasmic staining with Fluorescein diacetate suggested the occurrence of esterases in the three phytoflagellates. Rhodamine B, Neutral Red, FluoroBora P and Nile Blue revealed extensive occurrence of lipoid bodies in A. carterae, but Nile Blue showed considerable difference from the other stains in the inclusion size and intracellular location of these bodies. Chlortetracycline binding, and its inhibition by the Ca2+ionophore A23187, indicated that the plasma membrane, pusule system and trichocysts contain sites of Ca2+ binding. Calcofluor White ST proved superior to Congo Red and Lucifer Yellow in elucidating structural details of the thecal plates of P. micans. While Acridine Orange revealed the presence of surface-coat acidic polysaccharides, the fluoresceinated lectins established their glycoconjugate nature in all the three flagellates. Possible mechanisms of fluorochrome uptake are discussed.

Animals↗

Albumin protects human red blood cells against Abeta25-35-induced lysis more effectively than ApoE.

Inhibition of the lysis of human red blood cells (RBCs) exposed to amyloid peptide Abeta25-35 is an model for screening natural and synthetic substances potentially protective against amyloid damage. In this system, human serum and a component, namely apolipoprotein E (apoE), completely prevent RBC lysis. This report demonstrates that albumin, another serum component, is 8-fold more protective: a concentration of 12.5 microg/ml protects RBCs against 20 microM-Abeta25-35, and prevents the formation of fibrillar Abeta25-35 aggregates stainable by Congo Red. The biological relevance of these findings is suggested by the following: (1) a large fraction ( approximately 90%) of circulating Abeta1-42 is bound to albumin; (2) albumin immunoreactivity is present in brain amyloid plaques; and (3) incubation of Abeta with albumin rapidly decreases detectable levels of free Abeta suggesting epitope masking. The results add new and important functional consequences to the amyloid-albumin relationship and imply that experimental systems investigating Abeta cytotoxicity should consider the protective interaction of albumin.

Albumins↗

Characterization of the Aeromonas hydrophila group isolated from retail foods of animal origin.

During a recent survey of retail fresh foods of animal origin (fish and seafood, raw milk, poultry, and red meats) for organisms of the Aeromonas hydrophila group, we isolated representative strains from the various foods. In this study, we sought to characterize these isolates for biochemical properties and virulence-associated factors and to compare the food isolates with clinical isolates. We identified all food and clinical isolates as A. hydrophila and found that all isolates were typical in their biochemical reactions. Examination of the isolates for various virulence-associated factors indicated that most food and clinical isolates were serum resistant, beta-hemolytic, cytotoxin positive (against Y1 adrenal cells), hemagglutinin positive, Congo red positive, elastase positive, and staphylolysin positive. Mouse 50% lethal doses were log10 8 to 9 CFU for most isolates. All isolates had biotypes identical to those of enterotoxin-positive strains. The public health significance of these organisms in foods is not known at present, although their widespread occurrence and ability to grow competitively in foods kept at 5 degrees C represents a potential hazard.

Aeromonas↗

Binding of endostatin to phosphatidylserine-containing membranes and formation of amyloid-like fibers.

Endostatin, the 20-kDa C-terminal NC1 domain of collagen XVIII, is an endogenous inhibitor of tumor angiogenesis and tumor growth. A major problem in reconciling the many reported in vitro effects of endostatin is the lack of a high-affinity receptor, and a search for the latter continues. In accordance with the above, the molecular mechanisms of action of endostatin remain elusive. We show here that endostatin binds to membranes containing acidic phospholipids, phosphatidylserine (PS) or phosphatidylglycerol (PG). More specifically, a red shift in the fluorescence emission of Trp of endostatin in the presence of liposomes containing these anionic lipids was evident, revealing the average environment of Trps to become less hydrophobic. This shift was not observed for phosphatidylcholine (PC) liposomes, demonstrating the acidic lipid to be required. Quenching by endostatin of the fluorescence of a pyrene-labeled phospholipid analogue in PS containing membranes was seen, while there was no effect for PC liposomes. Resonance energy transfer from the Trp residues of endostatin to a dansyl-labeled phospholipid further confirmed the association of endostatin with PS-containing membranes, whereas there was no binding to PC liposomes. Intriguingly, the association of endostatin with PS-containing liposomes triggered the formation of fibers, with Congo red staining producing green birefringence characteristic for amyloid. Lipid was incorporated into these fibers, as shown by staining when a trace amount (X = 0.02) of fluorescent phospholipid analogues was present in the liposomes. No fiber formation was seen when endostatin was added to liposomes composed of PC only. Because PS has been reported to be exposed in the outer surface of the plasma membrane of cancer cells and vascular endothelial cells, our results suggest that this lipid could represent a target for endostatin in the cancer cell surface and tumors, thus suggesting a novel mechanism of its action. More specifically, analogous to a number of other cytotoxic proteins interacting with negatively charged lipids, PS-triggered fiber formation by endostatin on the surface of cancer cells would impair the permeability barrier function of the plasma membrane, resulting in cell death.

Amyloid↗

[The evaluation of an anti-leprosy program in Uélés (1975-1989)].

Foperda, who took over and continued the work initiated in 1924 by the Congo Red Cross, is responsible for the programme of fight against leprosy in the Ueles region (Zaire). Important results have been obtained since 1975 in the framework of a specialised medical service. These results are exposed and discussed herebelow. The evolution of the endemic leprosy in the region under survey allows to contemplate new trends for the pursuit of the programme in the coming years (controlled integration within polyvalent health services).

Democratic Republic of the Congo↗

Amyloid filaments in inclusion body myositis. Novel findings provide insight into nature of filaments.

Inclusion body myositis (IBM) represents a serious debilitating disease of muscle without identifiable cause or treatment. Muscle biopsy specimens have characteristic rimmed vacuoles, varying degrees of inflammation, and, most importantly, cytoplasmic and intranuclear filamentous inclusions of unknown composition. Fresh-frozen sections of muscle biopsy specimens from 24 IBM cases were stained with Congo red dye (pH, 10.5 to 11.0). Control biopsy specimens included polymyositis, dermatomyositis, hereditary vacuolar myopathies of unknown cause, acid maltase deficiency, distal myopathy, oculopharyngeal dystrophy, and chloroquine myopathy. Sections were also immunostained with antibody to transthyretin, human P component, and immunoglobulin light chains. In the vacuolated fibers in IBM, amyloidogenic green-birefringent deposits were seen. Some deposits were delicate and wispy appearing, and others were plaque-like. The size of deposits varied, measuring 1 x 2 to 8 microns, and rarely up to 20 microns in length. The number of amyloid-positive fibers correlated with the number of vacuolated fibers. Similar deposits were seen in one case of distal myopathy and one hereditary vacuolar myopathy. Other control cases were negative for amyloid deposits. Antibody staining for known amyloidogenic proteins was negative. This study demonstrates that the filaments in IBM share properties with amyloid proteins. The location implies that this amyloid material is formed intracellularly, rather than having a systemic derivation. The association of amyloid deposits with autophagic vacuoles in IBM raises the likely possibility that the filaments represent a modification of a normal protein within an acidic degradative vacuolar compartment. An alternative possibility, considering the shared properties of IBM filaments and prions (which include size and amyloidogenic properties), is that IBM represents a human prion disease.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

The neuropathology of Williams syndrome. Report of a 35-year-old man with presenile beta/A4 amyloid plaques and neurofibrillary tangles.

OBJECTIVE: To study neuropathologically Williams syndrome in a 35-year-old patient. METHODS: Sections from multiple regions of the brain were examined with luxol fast blue and hematoxylineosin staining, and selected sections were stained with the silver impregnation technique (Bielschowsky technique) and Congo red. In addition, immunohistochemistry with monoclonal antibodies against glial fibrillary acidic protein, beta/A4 amyloid, paired helical filaments, and phosphorylated tau protein was performed on cortical, hippocampal, amygdaloid, and basal ganglian sections. RESULTS: No specific macroscopic or microscopic abnormalities were recognized that are specific for Williams syndrome. The histopathologic examination did, however, demonstrate the presence of Alzheimer-type changes, including beta/A4 amyloid-containing senile plaques and scattered neurofibrillary tangles in neocortex and medial temporal lobe structures (entorhinal cortex, CA1 area of the hippocampus, and amygdala). Plaques were most numerous in the amygdala (7/mm2) and in the entorhinal cortex (4/mm2). Neurofibrillary tangles were less numerous (< 1/mm2), except in the hippocampus, where approximately 2/mm2 were found. CONCLUSIONS: To our knowledge, ours represents the first neuropathologic description of a patient with Williams syndrome. Although Williams syndrome is usually sporadic, familial cases have been reported along with candidate chromosomal loci. If our findings are confirmed in additional patients with Williams syndrome, they may provide clues to other factors that are important in the pathogenesis of senile plaques (with beta/A4 amyloid deposition) and neurofibrillary tangles.

Adult↗

Multifocal amyloidosis of the pediatric airway.

Amyloidosis of the upper aerodigestive tract is rare in the pediatric age group. We present the first reported case (to our knowledge) of multifocal primary upper airway amyloidosis in a child. An otherwise-healthy 15-year-old girl presented with hoarseness, nasal congestion, and odynophagia. Diagnostic evaluation included flexible nasopharyngoscopy, rigid nasal endoscopy, direct laryngoscopy, bronchoscopy, and biopsy. The results of Congo red staining of the specimen were characteristic of amyloid. The findings of an extensive immunologic and systemic evaluation were normal. We discuss the treatment and postoperative course of this patient, as well as the clinical and pathologic characteristics of amyloidosis, with particular reference to the otolaryngologic manifestations of amyloidosis.

Adolescent↗

Role of NaOH-extractable cell wall proteins Ccw5p, Ccw6p, Ccw7p and Ccw8p (members of the Pir protein family) in stability of the Saccharomyces cerevisiae cell wall.

The Saccharomyces cerevisiae cell wall contains more than 20 identified mannoproteins. Some of them can be released from the wall by hot SDS/mercaptoethanol treatment and are, therefore, considered as disulphide-linked or non-covalently attached to wall structural components. A number of covalently linked cell wall proteins are released after SDS extraction. They can be divided into these extractable by glucanases and those which can be released with 30 mM NaOH. The SDS-extractable proteins either possess enzymatic activities or are homologues of enzymes, mainly glucanases. Nothing is known, however, about the function of covalently linked proteins. In order to investigate the role of NaOH-extractable cell wall proteins, genes encoding all four identified members of this family of Pir proteins, CCW5, CCW6, CCW7 and CCW8, were disrupted and the phenotype of the mutants obtained was examined. They grew somewhat more slowly, were larger and irregularly shaped, and showed pronounced susceptibility to cell wall synthesis inhibitors like Calcofluor white and Congo red. In addition, the triple and the quadruple deletants had a decreased mating ability. All these properties were more obvious the more of these genes were disrupted, indicating that probably all members of this protein family are at least functionally equivalent in the cell wall.

Base Sequence↗

Fine-needle aspiration cytology of amyloid associated with nonneoplastic and malignant lesions.

To assess the value of fine-needle aspiration (FNA) cytology for the diagnosis of amyloid, we retrospectively studied all FNA cases diagnosed as having amyloid during a 6-yr period (1990-1996). FNA was performed on both superficial and deep locations. A total of 6 cases containing amyloid was studied, including primary medullary thyroid carcinoma, metastatic medullary thyroid carcinoma to a vertebrae, multiple myeloma, squamous-cell carcinoma of the lung metastatic to a hilar lymph node, primary pulmonary amyloid, and amyloid tumor in a vertebral body in a patient with primary systemic amyloidosis. Despite the location or disease association, the cytologic appearance of amyloid in all cases was similar. On Diff-Quik stain, amyloid appeared as amorphous, irregular, waxy basophilic to metachromatic clumps of material. Papanicolaou stain revealed cyanophilic to organophilic clumps of material with occasional prominent fissures. In all 6 cases, amyloid was confirmed by Congo red stain and in 3 cases by a thioflavin T stain. In 4 of the 6 cases (67%), amyloid was associated with an underlying malignancy. In 3 cases malignant cells were admixed with the amyloid, and in another case malignancy was present at a distant site. We conclude that FNA biopsy is a helpful initial procedure for the evaluation of patients with amyloid deposits. The clinical implications of amyloid found in any particular body site include both benign and malignant conditions. The presence of an associated neoplasm must be especially considered in the differential diagnosis of amyloid deposits.

Adult↗

Microfollicular thyroid carcinoma with amyloid rich stroma, resembling the medullary carcinoma of the thyroid (MCT).

A human thyroid tumor is described which, on light microscopy, exhibited the features of medullary carcinoma of the thyroid (MCT). The cells were arranged in solid lobules or trabeculae, and the stroma was abundant and gave positive reaction for amyloid as assessed by congo red, crystal violet and thioflavin T stains. However, there was microfollicular differentiation in certain portions of the tumor even where the tumor was invasively growing. On electron microscopy, numerous microfollicles were identified even where unsuspected by light microscopy. There were junctional complexes, the cells possessed well developed rough endoplasmic reticulum, prominent Golgi and numerous dense bodies obviously derived from the Golgi vesicles, which were of the same morphology as "secretory" granules described previously by some authors in MCT. However, we find this kind of cytology typically in microfollicular thyroid carcinomas. Presence of about 100 A thick fibrils in the stroma was consistent with histochemically positive amyloid. The biochemical data were compatible with the differentiated follicular cell origin of the tissue. The homogenate contained poorly iodinated thyroglobulin, and thyroid peroxidase activity. Calcitonin was undetectable by a sensitive radioimmunoassay. It is concluded that this tumor was follicular thyroid carcinoma with amyloid rich stroma. The presence of amyloid and dense bodies with homogenous electron dense contents is insufficient for making conclusions about histogenesis of thyroid tumors. The so called MCT with amyloid stroma probably represents a heterogenous group of thyroid tumors, at least some of them derived from follicular epithelium.

Adenocarcinoma↗

Amyloid stroma in a tubular carcinoma of palatal salivary gland: a histochemical and ultrastructural study.

Amyloid as defined by positive Congo red green birefringence and STB red birefringence was observed in the stroma of a tubular carcinoma of minor salivary gland origin. The amyloid exhibited additional histochemical characteristics of immunamyloid, viz., positive DMAB-nitrate and DC-reactions and spontaneous autofluorescence. Electron microscopic studies of the epithelial tumor cells displayed large amounts of cytoplasmic microfilaments similar to those present in the extracellular amyloid masses. It is suggested that the amyloid is derived from spontaneous assembly or polymerization of cytoplasmic microfibrils of the dying tumor cells as has been proposed for the amyloid associated with the calcifying epithelial odontogenic tumor (CEOT).

Adenocarcinoma↗

Gross and histological types of early gastric carcinomas in relation to the acid-secreting area.

The relation of gastric acid-secreting function to the gross and histological types and locations of carcinomas was examined in 53 patients with early gastric carcinomas. Studies were made by the endoscopic Congo red test developed in this clinic. Results indicated a close correlation between the gross and histological types of early gastric carcinoma and the extent of the acid-secreting area. In general, when the acid-secreting area was large, the carcinomas were ulcerated and histologically undifferentiated and they were chiefly located in nonacid-secreting areas adjacent to acid-secreting areas, or sometimes they were surrounded by acid-secreting areas. On the contrary, when there was little or no acid-secreting area the carcinomas were polyploid and histologically differentiated, and they were located in nonacid-secreting areas far from acid-secreting areas.

Adenocarcinoma↗

Gastric hamartomatous polyps in the absence of familial polyposis coli.

Hamartomatous polyps in the stomach have been described as gastric lesions of familial polyposis coli. Four cases of multiple gastric hamartomatous polyps not associated with polyposis coli were encountered. Histologically, all these polyps consisted of normal oxyntic glands with numerous cystic dilatations of the glandular lumens of various sizes. Endoscopically, the polyps were all located in the acid secreting area and had an acid secreting function, which was revealed by the endoscopic Congo red test.

Adult↗

A kindred with multiple endocrine neoplasia type 2A associated with pruritic skin lesions.

BACKGROUND: A kindred affected by multiple endocrine neoplasia type 2A (MEN 2A), associated with symmetric, bilateral, scapular pruritic skin lesions (PSL), is reported. METHODS: There were 21 members, including the propositus, in four generations. Screening of 10 family members showed a palpable thyroid tumor in 1 and positive results for a pentagastrin test in 7 others. Two of these patients had surgically confirmed pheochromocytoma. Two others had a biochemical diagnosis of pheochromocytoma but did not have surgery. RESULTS: Medullary thyroid carcinoma was confirmed in five patients and nodular C-cell hyperplasia in another. In five affected adults, PSL were observed in the interscapular region, crossing the midline in some. These lesions were characterized by hyperkeratosis and hyperpigmentation. In all the patients, the pruritus had been present long before the clinical or biochemical diagnosis. Skin biopsies were performed in two of these patients. No amyloid deposits were found (Congo red stain and electron microscopic examination were used). Two children (14 and 6 years old) in the fourth generation complained of scapular pruritus, although skin lesions were not apparent. CONCLUSIONS: Localized PSL must be sought when screening for MEN 2A.

Adolescent↗

Toxicity of various amyloid beta peptide species in cultured human blood-brain barrier endothelial cells: increased toxicity of dutch-type mutant.

The amyloid beta peptide (A beta) is the major component of the neuritic and cerebrovascular amyloid plaques that are one of the characteristic features of Alzheimer's disease (AD). This peptide has been shown to be toxic to several relevant cell types, including neurons, cerebrovascular smooth muscle cells, and endothelial cells. We have studied the toxic effects of both soluble and aggregated species of A beta(1-40) and the mutation A beta(1-40)Glu-->Gln(22), which is the major species deposited in the cerebrovascular blood vessels of victims of hereditary cerebral hemorrhage with amyloidosis, Dutch type. We find that aggregates of both peptides, as well as of A beta(1-42) and A beta(25-35), are toxic to cultured human cerebrovascular endothelial cells (hBEC) obtained from the brain of a victim of AD (at doses lower than those that are toxic to CNS neurons or leptomeningeal smooth muscle cells). Soluble A beta(1-40) Gln(22) is equally toxic to hBEC, whereas wild-type A beta(1-40) is toxic only at higher doses. This toxicity is seen at the lowest dose of A beta(1-40) Gln (22) used, 20 nM. The soluble A beta(1-40)Gln(22) aggregates on the surface of the cells, in contrast to A beta(1-40), and its toxicity can be blocked both by an inhibitor of free radical formation and by Congo red, which inhibits amyloid fibril formation. We discuss the possibility that the enhanced toxicity of A beta(1-40)Gln(22) is mediated by a A beta receptor on the endothelial cells.

Amyloid beta-Peptides↗