43 results on '"Knoop FC"'
Search Results
2. Antimicrobial and cytolytic properties of the frog skin peptide, kassinatuerin-1 and its L- and D-lysine-substituted derivatives.
- Author
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Conlon JM, Abraham B, Galadari S, Knoop FC, Sonnevend A, and Pál T
- Subjects
- Amphibian Proteins chemistry, Amphibian Proteins genetics, Animals, Anti-Infective Agents chemistry, Anura, Candida albicans drug effects, Candida albicans growth & development, Cell Line, Erythrocytes drug effects, Escherichia coli drug effects, Escherichia coli growth & development, Fibroblasts drug effects, Hemolysis drug effects, Humans, Peptides chemistry, Peptides genetics, Protein Structure, Secondary, Staphylococcus aureus drug effects, Staphylococcus aureus growth & development, Structure-Activity Relationship, Amino Acid Substitution, Amphibian Proteins pharmacology, Anti-Infective Agents pharmacology, Peptides pharmacology, Skin chemistry
- Abstract
Kassinatuerin-1, a 21-amino-acid C-terminally alpha-amidated peptide first isolated from the skin of the African frog Kassina senegalensis, adopts an amphipathic alpha-helical conformation in a membrane-mimetic solvent (50% trifluoroethanol) and shows broad-spectrum antimicrobial activity. However, its therapeutic potential is limited by its relatively high cytolytic activity against mammalian cells. The antimicrobial and cytolytic properties of a peptide are determined by an interaction between cationicity, hydrophobicity, alpha-helicity and amphipathicity. Replacement of the C-terminal alpha-amide group in kassinatuerin-1 by carboxylic acid decreased both cationicity and alpha-helicity, resulting in an analog with decreased potency against Escherichia coli (4-fold) and Staphylococcus aureus (16-fold). Low cytolytic activities against human erythrocytes (LD50>400 microM) and L929 fibroblasts (LD50=105 microM) were also observed. Increasing cationicity, while maintaining amphipathic alpha-helical character, by progressively substituting Gly7, Ser18, and Asp19 on the hydrophilic face of the alpha-helix with L-lysine, increased antimicrobial potency against S. aureus and Candida albicans (up to 4-fold) but also increased hemolytic and cytolytic activities. In contrast, analogs with d-lysine at positions 7, 18 and 19 retained activity against Gram-negative bacteria but displayed reduced hemolytic and cytolytic activities. For example, the carboxylic acid derivative of [D-Lys7, D-Lys18, D-Lys19]kassinatuerin-1 was active (minimum inhibitory concentration (MIC)=6-12.5 microM) against a range of strongly antibiotic-resistant strains of E. coli but showed no detectable hemolytic activity at 400 microM and was 4-fold less cytolyic than kassinatuerin-1. However, the reduction in alpha-helicity produced by the D-amino acid substitutions resulted in analogs with reduced potencies against Gram-positive bacteria and against C. albicans.
- Published
- 2005
- Full Text
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3. Antimicrobial properties of the frog skin peptide, ranatuerin-1 and its [Lys-8]-substituted analog.
- Author
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Sonnevend A, Knoop FC, Patel M, Pál T, Soto AM, and Conlon JM
- Subjects
- Amino Acid Substitution, Amphibian Proteins, Animals, Anti-Bacterial Agents chemistry, Antimicrobial Cationic Peptides, Circular Dichroism, Microbial Sensitivity Tests, Peptides, Proteins isolation & purification, Rana catesbeiana metabolism, Structure-Activity Relationship, Anti-Bacterial Agents pharmacology, Lysine chemistry, Proteins chemistry, Proteins pharmacology, Rana catesbeiana anatomy & histology, Skin chemistry
- Abstract
The predicted conformation of ranatuerin-1 (SMLSVLKNLG(10)KVGLGFVACK(20)INK QC), an antimicrobial peptide first isolated from the skin of the bullfrog Rana catesbeiana, comprises three structural domains: alpha-helix (residues 1-8), beta-sheet (residues 11-16) and beta-turn (residues 20-25). Circular dichroism studies confirm significant alpha-helical character in 50% trifluoroethanol. Replacement of Cys-19 and Cys-25 by serine resulted only in decreased antimicrobial potency but deletion of either the cyclic heptapeptide region [residues (19-25)] or the N-terminal domain [residues (1-8)] produced inactive analogs. Substitution of the glycine residues in the central domain of the [Ser-19, Ser-25] analog by lysine produced inactive peptides despite increased alpha-helical content and cationicity. The substitution Asn-8-->Lys gave a ranatuerin-1 analog with increased alpha-helicity and cationicity and increased potency against a range of Gram-positive and Gram-negative bacteria and against C. albicans but only a small increase (21%) in hemolytic activity. In contrast, increasing alpha-helicity and hydrophobicity by the substitution Asn-22-->Ala resulted in a 3.5-fold increase in hemolytic activity. Effects on antimicrobial potencies of substitutions of neutral amino acids at positions 4, 18, 22, and 24 by lysine were less marked. Strains of pathogenic E. coli from different groups showed varying degrees of sensitivity to ranatuerin-1 (MIC between 5 and 40 microM) but [Lys-8] ranatuerin-1 showed increased potency (between 2- and 8-fold; P < 0.01) against all strains. The data demonstrate that [Lys-8] ranatuerin-1 shows potential as a candidate for drug development.
- Published
- 2004
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4. Characterization of novel antimicrobial peptides from the skins of frogs of the Rana esculenta complex.
- Author
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Ali MF, Knoop FC, Vaudry H, and Conlon JM
- Subjects
- Amino Acid Sequence, Animals, Anti-Bacterial Agents isolation & purification, Anti-Bacterial Agents pharmacology, Antimicrobial Cationic Peptides analysis, Antimicrobial Cationic Peptides isolation & purification, Candida albicans drug effects, Chromatography, High Pressure Liquid, Escherichia coli drug effects, Microbial Sensitivity Tests, Molecular Sequence Data, Molecular Weight, Protein Isoforms chemistry, Protein Isoforms isolation & purification, Protein Isoforms pharmacology, Proteins isolation & purification, Proteins pharmacology, Rana esculenta, Sequence Homology, Amino Acid, Skin metabolism, Spectrometry, Mass, Electrospray Ionization, Staphylococcus aureus drug effects, Amphibian Proteins, Anti-Bacterial Agents chemistry, Antimicrobial Cationic Peptides chemistry, Antimicrobial Cationic Peptides pharmacology, Proteins chemistry, Skin chemistry
- Abstract
Rana esculenta is a hybridogenetic hybrid between Rana ridibunda and Rana lessonae and so is best considered as a complex of interbreeding species rather than a discrete single species. In this study, antimicrobial peptides were isolated from a pooled extract of the skins of specimens of the R. esculenta complex collected in the wild. In addition to several peptides belonging to the brevinin and esculentin families that have been previously isolated from skin secretions of a single specimen of R. esculenta, three newly described members of the brevinin-2 family (brevinin-2Ei, brevinin-2Ej, and brevinin-2Ek) and one member of the temporin family (temporin-1Ec) were purified and characterized. In addition, three structurally related peptides with no sequence similarity with antimicrobial peptides isolated from other species of ranid frogs, that potently and selectively inhibit the growth of the Gram-positive bacterium Escherichia coli (minimal inhibitory concentration (MIC<5 microM)), were identified. These peptides show limited amino acid sequence similarity to the homologous exon gene products that encode the N-terminal flanking peptides of preprocaerulein, preproxenopsin, and preprolevitide and so have been termed caerulein precursor-related fragments (CPRF-Ea, CPRF-Eb, and CPRF-Ec). The data suggest that there may be considerable polymorphism among specimens from different populations of the R. esculenta complex. It is proposed that the distribution and amino acid sequences of skin antimicrobial peptides may be useful markers for taxonomic classification of particular sub-populations and for an understanding of phylogenetic interrelationships.
- Published
- 2003
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5. Antimicrobial peptides and protease inhibitors in the skin secretions of the crawfish frog, Rana areolata.
- Author
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Ali MF, Lips KR, Knoop FC, Fritzsch B, Miller C, and Conlon JM
- Subjects
- Amino Acid Sequence, Animals, Anti-Infective Agents isolation & purification, Anti-Infective Agents pharmacology, Erythrocytes drug effects, Hemolysis drug effects, Humans, Molecular Sequence Data, Peptide Fragments chemistry, Peptide Fragments isolation & purification, Peptides isolation & purification, Peptides pharmacology, Protease Inhibitors pharmacology, Amphibian Proteins, Anti-Infective Agents analysis, Antimicrobial Cationic Peptides chemistry, Peptides metabolism, Protease Inhibitors analysis, Ranidae, Skin metabolism
- Abstract
The dorsal skin of the crawfish frog, Rana areolata, is associated with numerous prominent granular glands. Proteomic analysis of electrically stimulated skin secretions from these glands enabled the identification and characterization of eight peptides with antimicrobial and hemolytic activity belonging to the previously identified brevinin-1, temporin-1, palustrin-2, palustrin-3, esculentin-1 (two peptides), and ranatuerin-2 (two peptides) families. The primary structures of the peptides were consistent with a close phylogenetic relationship between R. areolata and the pickerel frog, Rana palustris. Three structurally related cationic, cysteine-containing peptides were identified that show sequence similarity to peptide Leucine-Arginine, a peptide with immunomodulatory and histamine-releasing properties from the skin of the northern leopard frog, Rana pipiens. The skin secretions contained a 61-amino-acid-residue peptide that inhibited porcine trypsin and possessed a 10-cysteine-residue motif that is characteristic of a protease inhibitor previously isolated from the parasitic nematode, Ascaris suum. A 48-amino-acid-residue protein containing eight cysteine residues in the whey acidic protein (WAP) motif, characteristic of elafin (skin-derived antileukoproteinase) and secretory leukocyte protease inhibitor, was also isolated. The data suggest that protease inhibitors in skin secretions may play a role complementary to cationic, amphipathic alpha-helical peptides in protecting anurans from invasions by microorganisms.
- Published
- 2002
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6. Antimicrobial peptides with atypical structural features from the skin of the Japanese brown frog Rana japonica.
- Author
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Isaacson T, Soto A, Iwamuro S, Knoop FC, and Conlon JM
- Subjects
- Amino Acid Sequence, Animals, Anti-Bacterial Agents chemistry, Antimicrobial Cationic Peptides, Circular Dichroism, Japan, Microbial Sensitivity Tests, Molecular Sequence Data, Peptides chemistry, Peptides isolation & purification, Protein Conformation, Proteins isolation & purification, Proteins pharmacology, Ranidae, Sequence Homology, Amino Acid, Anti-Bacterial Agents pharmacology, Escherichia coli drug effects, Peptides pharmacology, Skin chemistry, Staphylococcus aureus drug effects
- Abstract
Japonicin-1 (FFPIGVFCKIFKTC) and japonicin-2 (FGLPMLSILPKALCILLKRKC), two peptides with differential growth-inhibitory activity against the Gram-negative bacterium, Escherichia coli and the Gram-positive bacterium Staphylococcus aureus, were isolated from an extract of the skin of the Japanese brown frog Rana japonica. Both peptides show little amino acid sequence similarity to previously characterized antimicrobial peptides isolated from the skins of Ranid frogs. Circular dichroism studies, however, demonstrate that japonicin-2 adopts an alpha-helical conformation in 50% trifluoroethanol in common with many other cationic antimicrobial peptides synthesized in amphibian skin. Peptides belonging to the brevinin-1, brevinin-2, and tigerinin families, previously identified in the skins of Asian Ranid frogs, were not detected but a temporin-related peptide (ILPLVGNLLNDLL.NH(2); temporin-1Ja), that atypically bears no net positive charge, was isolated from the extract. The minimum inhibitory concentrations (MICs) of the peptides against E. coli were japonicin-1, 30 microM; japonicin-2, 12 microM; and temporin-1Ja > 100 microM. The MICs against S. aureus were japonicin-1, > 100 microM; japonicin-2, 20 microM; and temporin-1Ja, > 100 microM.
- Published
- 2002
- Full Text
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7. Antimicrobial peptides isolated from skin secretions of the diploid frog, Xenopus tropicalis (Pipidae).
- Author
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Ali MF, Soto A, Knoop FC, and Conlon JM
- Subjects
- Amino Acid Sequence, Animals, Anti-Bacterial Agents, Candida albicans drug effects, Circular Dichroism, Erythrocytes drug effects, Escherichia coli drug effects, Hemolysis, Humans, Mass Spectrometry, Microbial Sensitivity Tests, Molecular Sequence Data, Molecular Weight, Norepinephrine pharmacology, Oligopeptides chemistry, Oligopeptides pharmacology, Skin chemistry, Skin drug effects, Staphylococcus aureus drug effects, Xenopus Proteins chemistry, Xenopus Proteins pharmacology, Anti-Infective Agents isolation & purification, Oligopeptides isolation & purification, Skin metabolism, Xenopus metabolism, Xenopus Proteins isolation & purification
- Abstract
Seven peptides (XT-1-XT-7) with antimicrobial activity were isolated from norepinephrine-stimulated skin secretions of the diploid clawed frog, Xenopus tropicalis. Structural characterization of the peptides demonstrated that amino acid sequence similarity to antimicrobial peptides previously isolated from Xenopus laevis was low, suggesting that the species are not closely related phylogenetically. Peptides XT-5 and XT-3 are probably the orthologs of X. laevis peptide glycine-leucine amide (PGL(a)) and the N-terminal spacer region of prolevitide, respectively. XT-1, XT-6 and XT-7 show limited structural similarity to the spacer region of X. laevis procaeruleins and the paralogs XT-2 and XT-4 are similar to corresponding regions of proxenopsin. Orthologs of the magainins were not identified. The C-terminally alpha-amidated peptide XT-7 (GLLGPLLKIAAKVGSNLL.NH2) showed the lowest minimum inhibitory concentrations against reference microorganisms (Staphylococcus aureus 5 microM, Escherichia coli 5 microM, and Candida albicans 40 microM) and was also active against clinical isolates of methicillin-resistant S. aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus group C, Shigella sonnei, Pseudomonas aeruginosa and Enterobacter cloacae. The peptide was, however, hemolytic against human erythrocytes (50% lysis at 70 microM). Circular dichroism studies showed that XT-7 has a random structure in aqueous solution, pH 7.0 but adopts an alpha-helical conformation in the presence of 50% trifluoroethanol. Decreasing the cationicity of XT-7 either by replacement of the C-terminal CONH2 group by COOH or by deletion of the Lys(8) residue produced analogs with greatly (>10-fold) decreased antimicrobial potencies.
- Published
- 2001
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8. Pseudin-2: an antimicrobial peptide with low hemolytic activity from the skin of the paradoxical frog.
- Author
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Olson L 3rd, Soto AM, Knoop FC, and Conlon JM
- Subjects
- Amino Acid Sequence, Animals, Anti-Bacterial Agents, Anti-Infective Agents chemical synthesis, Anti-Infective Agents chemistry, Anti-Infective Agents isolation & purification, Antimicrobial Cationic Peptides chemical synthesis, Antimicrobial Cationic Peptides chemistry, Antimicrobial Cationic Peptides isolation & purification, Candida albicans drug effects, Cell Death drug effects, Chromatography, High Pressure Liquid, Circular Dichroism, Death Domain Receptor Signaling Adaptor Proteins, Erythrocytes drug effects, Escherichia coli drug effects, Humans, Intracellular Signaling Peptides and Proteins, Microbial Sensitivity Tests, Molecular Sequence Data, Peptides chemical synthesis, Peptides chemistry, Peptides isolation & purification, Protein Structure, Secondary, Proteins chemistry, Sequence Alignment, Staphylococcus aureus drug effects, Amphibian Proteins, Anti-Infective Agents pharmacology, Antimicrobial Cationic Peptides pharmacology, Anura, DNA-Binding Proteins, Hemolysis drug effects, Peptides pharmacology, Skin chemistry
- Abstract
Four structurally related peptides (pseudins 1-4) with antimicrobial activity were isolated from an extract of the skin of the paradoxical frog Pseudis paradoxa (Pseudidae). Pseudin-2 (GLNALKKVFQGIHEAIKLINNHVQ) was the most abundant peptide (22 nmol/g tissue) and also the most potent (minimum inhibitory concentrations, MIC = 2.5 microM against Escherichia coli, 80 microM against Staphylococcus aureus, and 130 microM against Candida albicans). The concentration of pseudin-2 producing 50% hemolysis of human erythrocytes was >300 microM. Circular dichroism studies showed that the pseudins belong to the class of cationic, amphipathic alpha-helical antimicrobial peptides but their amino acid sequences are not similar to any previously characterized peptides from frog skin. The pseudins do, however, show sequence similarity with a region at the C-terminus of DEFT, a death effector domain-containing protein expressed in mammalian testicular germ cells that is involved in the regulation of apoptosis., (Copyright 2001 Academic Press.)
- Published
- 2001
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9. Antimicrobial peptides from the skin of the Japanese mountain brown frog, Rana ornativentris.
- Author
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Kim JB, Iwamuro S, Knoop FC, and Conlon JM
- Subjects
- Amino Acid Sequence, Animals, Chromatography, High Pressure Liquid, Inhibitory Concentration 50, Male, Molecular Sequence Data, Peptides chemistry, Protein Conformation, Ranidae, Sequence Homology, Amino Acid, Time Factors, Amphibian Proteins, Anti-Bacterial Agents chemistry, Anti-Bacterial Agents pharmacology, Antimicrobial Cationic Peptides chemistry, Skin metabolism
- Abstract
Six peptides with antimicrobial activity were isolated from an extract of freeze-dried skin of the Japanese mountain brown frog Rana ornativentris. Two structurally related peptides (brevinin-20a GLFNVFKGALKTAGKHVAGSLLNQLKCKVSGGC, 11 nmol/g dried tissue, and brevinin-20b GIFNVFKGALKTAGKHVAGSLLNQLKCKVSGEC, 170 nmol/g) belong to the brevinin-2 family, previously identified in Asian and European, but not North American, Ranid frogs. Four peptides (temporin-10a FLPLLASLFSRLL.NH2, 13 nmol/g; temporin-10b FLPLIGKILGTI L.NH2, 350 nmol/g; temporin-10c FLPLLASLFSRLF.NH2, 14 nmol/g; and temporin-10d FLPLLASLFSGLF.NH2, 8 nmol/g) are members of the temporin family first identified in the European common frog Rana temporaria but also found in the skins of North American Ranids. The brevinin-2 peptides showed broad-spectrum activity against the gram-positive bacterium, Staphylococcus aureus, the gram-negative bacterium, Escherichia coli and the yeast Candida albicans, whereas the temporins showed potent activity only against S. aureus. The brevinins and temporins belong to the class of cationic antimicrobial peptides that adopt an amphipathic alpha-helical conformation but it is significant that temporin-10d, which lacks a basic amino acid residue, is still active against S. aureus (minimum inhibitory concentration=13 microM compared with 2 microM for temporin-10a). This suggests that strong electrostatic interaction between the peptide and the negatively charged phospholipids of the cell membrane is not an absolute prerequisite for antimicrobial activity.
- Published
- 2001
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10. Multiple antimicrobial peptides and peptides related to bradykinin and neuromedin N isolated from skin secretions of the pickerel frog, Rana palustris.
- Author
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Basir YJ, Knoop FC, Dulka J, and Conlon JM
- Subjects
- Amino Acid Sequence, Animals, Anti-Bacterial Agents chemistry, Anti-Bacterial Agents pharmacology, Bradykinin chemistry, Chromatography, High Pressure Liquid, Electric Stimulation, Escherichia coli drug effects, Inhibitory Concentration 50, Mass Spectrometry, Molecular Sequence Data, Molecular Weight, Neurotensin chemistry, Peptide Fragments chemistry, Peptides chemistry, Peptides pharmacology, Skin chemistry, Anti-Bacterial Agents isolation & purification, Peptides isolation & purification, Ranidae metabolism, Skin metabolism
- Abstract
The skin secretions of the North American pickerel frog Rana palustris are toxic to both microorganisms and predators. A total of 22 peptides with differential growth-inhibitory activity towards bacteria and yeast were isolated from the electrostimulated secretions of R. palustris skin and were characterized structurally. Thirteen of the antimicrobial peptides belong to five of the known families previously identified in the skins of other species of Ranid frogs: brevinin-1 (3 peptides), esculentin-1 (2 peptides), esculentin-2 (1 peptide), ranatuerin-2 (6 peptides), and temporin (1 peptide). Nine peptides show little structural similarity towards other known antimicrobial peptides and so are classified in new families: palustrin-1 (4 peptides) with 27-28 amino acid residues and a cystine-bridged heptapeptide ring; palustrin-2 (3 peptides) with 31 amino acids and a cyclic heptapeptide region and palustrin-3 (2 peptides) with 48 amino acids and a cyclic hexapeptide region. Peptides belonging to the esculentin-1, esculentin-2 and palustrin-3 families are the most potent (minimal inhibitory concentrations approximately 1 microM against Escherichia coli) whereas peptides of the brevinin-1 and esculentin-2 families show the broadest spectrum of activity. As well as bradykinin that is identical to the human peptide, a further 4 peptides structurally related to [Leu(8)]bradykinin and two peptides related to neuromedin-N (the hexapeptide KKPYIL and a larger, cystine-containing form HLRRCGKKPYILMACS) were purified from the skin secretions.
- Published
- 2000
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11. Induction of synthesis of an antimicrobial peptide in the skin of the freeze-tolerant frog, Rana sylvatica, in response to environmental stimuli.
- Author
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Matutte B, Storey KB, Knoop FC, and Conlon JM
- Subjects
- Adaptation, Physiological, Amino Acid Sequence, Animals, Anti-Bacterial Agents isolation & purification, Anti-Bacterial Agents pharmacology, Antimicrobial Cationic Peptides isolation & purification, Antimicrobial Cationic Peptides pharmacology, Chromatography, High Pressure Liquid, Escherichia coli drug effects, Escherichia coli growth & development, Freezing, Male, Microbial Sensitivity Tests, Molecular Sequence Data, Sequence Alignment, Sequence Analysis, Protein, Staphylococcus aureus drug effects, Staphylococcus aureus growth & development, Temperature, Amphibian Proteins, Anti-Bacterial Agents biosynthesis, Antimicrobial Cationic Peptides biosynthesis, Ranidae metabolism, Skin metabolism
- Abstract
An extract of skin taken from specimens of the freeze-tolerant wood frog, Rana sylvatica, that were collected from cold (<7 degrees C) ponds and maintained at 5 degrees C lacked detectable antimicrobial activity. In contrast, an extract of skin taken from specimens maintained at 30 degrees C for 3 weeks under laboratory conditions contained a high concentration (approximately 4 nmol/g) of a single antimicrobial peptide of the brevinin-1 family (FLPVVAGLAAKVLPSIICAVTKKC). The peptide inhibited growth of Escherichia coli (minimum inhibitory concentration 45 microM) and Staphylococcus aureus (minimum inhibitory concentration 7 microM). The data suggest that synthesis of the peptide is induced when the animal is in an environment that promotes the growth of microorganisms consistent with a role in the animal's defense strategy.
- Published
- 2000
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12. Purification and characterization of antimicrobial and vasorelaxant peptides from skin extracts and skin secretions of the North American pig frog Rana grylio.
- Author
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Kim JB, Halverson T, Basir YJ, Dulka J, Knoop FC, Abel PW, and Conlon JM
- Subjects
- Amino Acid Sequence, Animals, Anti-Bacterial Agents, Anti-Infective Agents chemistry, Anti-Infective Agents isolation & purification, Hemolysis, Humans, Male, Molecular Sequence Data, North America, Peptides chemistry, Peptides isolation & purification, Rats, Rats, Sprague-Dawley, Skin metabolism, Tissue Extracts chemistry, Vasodilator Agents chemistry, Vasodilator Agents isolation & purification, Anti-Infective Agents pharmacology, Candida albicans drug effects, Escherichia coli drug effects, Peptides pharmacology, Ranidae metabolism, Skin chemistry, Staphylococcus aureus drug effects, Vasodilator Agents pharmacology
- Abstract
Eight peptides with differential growth-inhibitory activity against the gram-positive bacterium Staphylococcus aureus, the gram-negative bacterium Escherichia coli and the yeast, Candida albicans were isolated from an extract of the skin of the North American pig frog Rana grylio. The primary structures of these antimicrobial peptides were different from previously characterized antimicrobial peptides from Ranid frogs but on the basis of sequence similarities, the peptides may be classified as belonged to four previously characterized peptide families: the ranatuerin-1, ranatuerin-2 and ranalexin families, first identified in the North American bullfrog, Rana catesbeiana, and the temporin family first identified in the European common frog Rana temporaria. Peptides belonging to the brevinin-1, brevinin-2, esculentin-1, and esculentin-2 families, previously isolated from the skins of other species of Ranid frogs, were not identified in the extracts. The ranatuerin-1 and ranalexin peptides showed broadest spectrum of antimicrobial activity whereas the temporins were active only against S. aureus. Synthetic replicates of temporin-1Gb (SILPTIVSFLSKFL.NH(2)) and temporin-1Gd (FILPLIASFLSKFL.NH(2)) produced concentration-dependent relaxation of preconstricted vascular rings from the rat thoracic aorta (EC(50) = 2.4+/-0.1 microM for temporin-1Gb and 2.3+/-0.2 microM for temporin-1Gd). The antimicrobial peptides that were isolated in extracts of the skin R. grylio were present in the same molecular forms in electrically-stimulated skin secretions of the animal demonstrating that the peptides are stored in the granular glands of the skin in their fully processed forms.
- Published
- 2000
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13. Purification and characterization of antimicrobial peptides from the skin of the North American green frog Rana clamitans.
- Author
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Halverson T, Basir YJ, Knoop FC, and Conlon JM
- Subjects
- Amino Acid Sequence, Amphibian Proteins, Animals, Anti-Bacterial Agents, Anti-Infective Agents chemistry, Anti-Infective Agents pharmacology, Antimicrobial Cationic Peptides, Candida albicans drug effects, Chromatography, Gel, Chromatography, High Pressure Liquid, Escherichia coli drug effects, Microbial Sensitivity Tests, Molecular Sequence Data, Oligopeptides chemistry, Oligopeptides pharmacology, Peptides, Peptides, Cyclic chemistry, Peptides, Cyclic isolation & purification, Peptides, Cyclic pharmacology, Proteins chemistry, Proteins pharmacology, Ranidae, Sequence Homology, Amino Acid, Staphylococcus aureus drug effects, Anti-Infective Agents isolation & purification, Oligopeptides isolation & purification, Proteins isolation & purification, Skin chemistry
- Abstract
Ten peptides with differential growth-inhibitory activity against the gram-positive bacterium, Staphylococcus aureus, the gram-negative bacterium, Escherichia coli, and the yeast Candida albicans were isolated from an extract of the skin of a North American frog, the green frog Rana clamitans. Ranatuerin-1C (SMLSVLKNLGKVGLGLVACKINKQC), ranalexin-1Ca (FLGGLMKAFPALICAVTKKC), ranalexin-1Cb (FLGGLMKAFPAIICAVTKKC), ranatuerin-2Ca (GLFLDTLKGAAKDVAGKLLEGLKCKIAGC KP), and ranatuerin-2Cb (GLFLDTLKGLAGKLLQGLKCIKAGCKP), are members of three previously characterized families of antimicrobial peptides, first identified in the North American bullfrog Rana catesbeiana. In addition, five structurally related peptides (temporin-1Ca, -1Cb, -1Cc, -1Cd, and -1Ce), comprising 13 amino acid residues and containing a C-terminally alpha-amidated residue, belong to the temporin family first identified in the European common frog Rana temporaria. Peptides belonging to the brevinin-1, brevinin-2, esculentin-1, and esculentin-2 families, previously isolated from the skins of Asian and European Ranid frogs, were not identified in the extract. The data support the hypothesis that the distribution and amino acid sequences of the skin antimicrobial peptides are valuable tools in the identification and classification of Ranid frogs.
- Published
- 2000
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14. Kassinatuerin-1: a peptide with broad-spectrum antimicrobial activity isolated from the skin of the hyperoliid frog, Kassina senegalensis.
- Author
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Mattute B, Knoop FC, and Conlon JM
- Subjects
- Amino Acid Sequence, Amphibian Proteins, Animals, Anti-Bacterial Agents chemistry, Anti-Bacterial Agents isolation & purification, Molecular Sequence Data, Peptides chemical synthesis, Peptides pharmacology, Sequence Homology, Amino Acid, Staphylococcus aureus drug effects, Anti-Bacterial Agents pharmacology, Anura, Proteins pharmacology, Skin chemistry
- Abstract
Kassinatuerin-1 (GFMKYIGPLI(10)PHAVKAISDL(20)I.NH(2)) was isolated in high yield (75 nmol/g) from an extract of the skin of a Hyperoliid frog, the African running frog Kassina senegalensis and its sequence was confirmed by total synthesis. The peptide inhibited growth of the gram-negative bacterium Escherichia coli (minimum inhibitory concentration, MIC = 4 microM), the gram-positive bacterium Staphylococcus aureus (MIC = 8 microM), and the yeast Candida albicans (MIC = 70 microM). A structurally related peptide, kassinatuerin-2 (FIQYLAPLI(10)PHAVKAISDL(20)I.NH(2)) was also isolated in high yield (96 nmol/g) from the extract but was devoid of antimicrobial activity against these microrganisms. Kassinatuerin-1 may be classified with other linear, cationic antimicrobial peptides that can potentially adopt an amphipathic alpha-helical conformation but it contains almost no amino acid sequence identity with previously characterized bioactive peptides from frog skin., (Copyright 2000 Academic Press.)
- Published
- 2000
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15. Peptides with antimicrobial activity from four different families isolated from the skins of the North American frogs Rana luteiventris, Rana berlandieri and Rana pipiens.
- Author
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Goraya J, Wang Y, Li Z, O'Flaherty M, Knoop FC, Platz JE, and Conlon JM
- Subjects
- Amino Acid Sequence, Animals, Anti-Bacterial Agents, Anti-Infective Agents chemistry, Candida albicans drug effects, Escherichia coli drug effects, Female, Male, Molecular Sequence Data, Peptides chemistry, Rana pipiens metabolism, Sequence Homology, Amino Acid, Species Specificity, Staphylococcus aureus drug effects, Amphibian Proteins, Anti-Infective Agents isolation & purification, Anti-Infective Agents pharmacology, Antimicrobial Cationic Peptides, Peptides isolation & purification, Peptides pharmacology, Ranidae metabolism, Skin chemistry
- Abstract
The skins of frogs of the genus Rana synthesize a complex array of antimicrobial peptides that may be grouped into eight families on the basis of structural similarity. A total of 24 peptides with differential growth-inhibitory activity towards the Gram-positive bacterium Staphylococcus aureus, the Gram-negative bacterium Escherichia coli and the yeast Candida albicans were isolated from extracts of the skins of three closely related North American frogs, Rana luteiventris (spotted frog), Rana berlandieri (Rio Grande leopard frog) and Rana pipiens (Northern leopard frog). Structural characterization of the antimicrobial peptides demonstrated that they belonged to four of the known families: the brevinin-1 family, first identified in skin of the Asian frog Rana porosa brevipoda; the esculentin-2 family, first identified in the European frog Rana esculenta; the ranatuerin-2 family, first identified in the North American bullfrog Rana catesbeiana; and the temporin family, first identified in the European frog Rana temporaria. Peptides belonging to the brevinin-2, ranalexin, esculentin-1 and ranatuerin-1 families were not identified in the extracts. Despite the close phylogenetic relationship between the various species of Ranid frogs, the distribution and amino-acid sequences of the antimicrobial peptides produced by each species are highly variable and species-specific, suggesting that they may be valuable in taxonomic classification and molecular phylogenetic analysis.
- Published
- 2000
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16. Peptides with antimicrobial activity of the brevinin-1 family isolated from skin secretions of the southern leopard frog, Rana sphenocephala.
- Author
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Conlon JM, Halverson T, Dulka J, Platz JE, and Knoop FC
- Subjects
- Amino Acid Sequence, Animals, Anti-Bacterial Agents pharmacology, Escherichia coli drug effects, Microbial Sensitivity Tests, Molecular Sequence Data, Peptides metabolism, Peptides pharmacology, Ranidae, Sequence Homology, Amino Acid, Amphibian Proteins, Anti-Bacterial Agents isolation & purification, Antimicrobial Cationic Peptides, Peptides isolation & purification, Skin metabolism
- Abstract
Three peptides with growth-inhibitory activity towards the gram-negative bacterium Eschericia coli were isolated from electrically stimulated secretions from the skin of the southern leopard frog, Rana sphenocephala. Structural characterization demonstrated that the peptides [brevinin-1Sa, minimum inhibitory concentration (MIC) = 55 microM; brevinin-1Sb, MIC = 17 microM; brevinin-1Sc, MIC = 14 microM] represent new members of the brevinin-1 family of antimicrobial peptides, previously isolated from several other species of frogs of the genus Rana. Their high concentration in skin secretions and extreme variability in amino acid sequence suggest that the brevinin family of peptides may be of value as molecular markers for the identification and taxonomic classification of Ranid frogs.
- Published
- 1999
- Full Text
- View/download PDF
17. Ranatuerin 1T: an antimicrobial peptide isolated from the skin of the frog, Rana temporaria.
- Author
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Goraya J, Knoop FC, and Conlon JM
- Subjects
- Amino Acid Sequence, Amphibian Proteins, Animals, Antimicrobial Cationic Peptides, Dose-Response Relationship, Drug, Female, Male, Microbial Sensitivity Tests, Molecular Sequence Data, Peptides, Sequence Homology, Amino Acid, Anti-Infective Agents isolation & purification, Proteins isolation & purification, Rana temporaria, Skin chemistry
- Abstract
A peptide, termed ranatuerin 1T, with growth-inhibiting activity toward Staphylococcus aureus, was isolated from an extract of the skin of the European brown frog, Rana temporaria. The primary structure of the peptide was established as: GLLSGLKKVG10 KHVAKNVAVS20LMDSLKCKIS30GDC. In common with other anti-microbial peptides from Ranid frogs, (e.g., ranalexin, ranatuerins, gaegurins, brevinins, esculetins, rugosins), ranatuerin IT contains an intramolecular disulfide bridge forming a heptapeptide ring but there is little structural similarity outside this cyclic region. The minimum inhibitory concentration (MIC) of ranatuerin 1T was 120 microM against the Gram-positive bacterium S. aureus and 40 microM against the Gram-negative bacterium Escherichia coli, but the peptide was not active against the yeast Candida albicans.
- Published
- 1999
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18. Antimicrobial peptides of the brevinin-2 family isolated from gastric tissue of the frog, Rana esculenta.
- Author
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Wang Y, Knoop FC, Remy-Jouet I, Delarue C, Vaudry H, and Conlon JM
- Subjects
- Amino Acid Sequence, Animals, Anti-Bacterial Agents pharmacology, Escherichia coli drug effects, Molecular Sequence Data, Peptides pharmacology, Ranidae, Skin chemistry, Amphibian Proteins, Anti-Bacterial Agents chemistry, Antimicrobial Cationic Peptides, Peptides chemistry, Rana esculenta, Stomach chemistry
- Abstract
Four structurally related peptides with potent growth-inhibitory activity towards Escherichia coli were isolated from an extract of the stomach of the European green frog Rana esculenta, and were identified as members of the brevinin-2 family. Two peptides, termed brevinin-2Eg (GIMDTLKNLA10 KTAGKGALQS20 LLNHASCK LS30GQC) and brevinin-2Eh (GIMDTLKNLA10 KTAGKGALQS20 LLNHASCKL S30 KQC) have not been described previously. One peptide is identical to brevinin-2Ec, previously isolated from R. esculenta skin secretions, and one peptide is identical to brevinin-2Ef whose structure has been deduced from a cloned cDNA prepared from a R. esculenta skin cDNA library. The data demonstrate that certain peptides of the brevinin-2 family, like the magainins in the toad, Xenopus laevis, may play an important role in protecting the gastrointestinal tract of Ranid frogs against microbial invasion.
- Published
- 1998
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- View/download PDF
19. Ranatuerins: antimicrobial peptides isolated from the skin of the American bullfrog, Rana catesbeiana.
- Author
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Goraya J, Knoop FC, and Conlon JM
- Subjects
- Amino Acid Sequence, Animals, Anti-Bacterial Agents pharmacology, Molecular Sequence Data, Proteins pharmacology, Sequence Alignment, Skin metabolism, Staphylococcus aureus drug effects, Anti-Bacterial Agents isolation & purification, Proteins isolation & purification, Rana catesbeiana metabolism
- Abstract
Nine peptides, termed ranatuerins 1-9, with antimicrobial activity towards Staphylococcus aureus, were isolated from an extract of the skin of the adult American bullfrog, Rana catesbeiana. In common with other cytolytic peptides from Ranid frogs, (e.g. ranalexin, gaegurins, brevinins), ranatuerins 1 and 4 contain an intramolecular disulfide bridge forming a heptapeptide ring whereas in ranatuerins 2 and 3 the disulfide bridge forms a hexapeptide ring. The structurally related ranatuerins 5-9 comprise 12 - 14 amino acids and show sequence similarity towards the hemolytic peptides A1 and B9 previously isolated from the skin of Rana esculenta. Of the peptides purified, ranatuerin 1 (SMLSVLKNLGKVGLG FVACKINKQC) showed the broadest spectrum of antimicrobial action with inhibitory activity against S. aureus, Escherichia coli and Candida albicans., (Copyright 1998 Academic Press.)
- Published
- 1998
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20. Chlamydia pneumoniae and occlusive vascular disease: identification and characterization.
- Author
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Pai J, Knoop FC, Hunter WJ 3rd, and Agrawal DK
- Subjects
- Blotting, Southern methods, Chlamydia Infections diagnosis, Humans, Immunohistochemistry methods, Polymerase Chain Reaction methods, Arterial Occlusive Diseases microbiology, Chlamydia Infections microbiology, Chlamydophila pneumoniae isolation & purification
- Abstract
Chlamydia pneumoniae, a respiratory pathogen, has been associated with occlusive vascular disease, including atherosclerosis and intimal hyperplasia, through seroepidemiologic studies. Furthermore, using immunohistochemistry (IHC), polymerase chain reaction (PCR), transmission electron microscopy (TEM), and in situ hybridization, this association has been reconfirmed by detecting this organism in atherosclerotic vascular tissue. This review summarizes and critically analyzes these findings and also discusses various mechanisms of how Chlamydia pneumoniae could be involved in the pathogenesis of occlusive vascular disease. Although more studies are needed to reproduce these results and, possibly, uncover a mechanism, the current literature fails to include detailed methodologies for studying Chlamydia pneumoniae. Therefore, to provide a general standard, we have also outlined specific protocols for IHC, PCR, and TEM. These protocols incorporate essential components from various studies and are presented in a concise and easily adaptable format.
- Published
- 1998
- Full Text
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21. Impact of anaerobic growth conditions on toxic shock syndrome toxin-i production by Staphylococcus aureus.
- Author
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Lungren M, Chaisilwattana P, Knoop FC, and Monif GR
- Abstract
Objective: The impact of anaerobic growth conditions on the Staphylococcus aureus toxic shock syndrome toxin-1 (TSST-1) production was studied., Methods: Ten strains of S. aureus derived from patients with toxic shock syndrome (TSS), 10 isolates of S. aureus, and documented TSST-1-producing strains recovered from patients with either staphylococcal septicemia or staphylococcal nongenital abscesses were grown under aerobic and anaerobic conditions. The bacterial growth was measured using optical density (OD) determinations at 520 nm. The toxin production was assayed using the TS-RPLA latex agglutination test., Results: Both TSS and non-TSS strains of S. aureus grown under aerobic and anaerobic conditions exhibited comparable OD patterns of growth, and the levels of toxin production remained constant during the logarithmic phase. Toxin titers developed during the logarithmic growth phase and peaked after 24 h of incubation. When stationary-phase isolates grown initially under aerobic conditions were subjected to strict anaerobic conditions, subsequent toxin titers, compared with isolates grown in the continued presence of oxygen, were depressed 2-fold, peaking at a later time., Conclusions: TSST-1 production is diminished under continued anaerobic conditions.
- Published
- 1996
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22. Clostridium difficile: clinical disease and diagnosis.
- Author
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Knoop FC, Owens M, and Crocker IC
- Subjects
- Animals, Clostridioides difficile isolation & purification, Clostridioides difficile pathogenicity, Humans, Immunoenzyme Techniques, Latex Fixation Tests, Polymerase Chain Reaction, Enterocolitis, Pseudomembranous diagnosis, Enterocolitis, Pseudomembranous epidemiology, Enterocolitis, Pseudomembranous microbiology, Enterocolitis, Pseudomembranous pathology
- Abstract
Clostridium difficile is an opportunistic pathogen that causes a spectrum of disease ranging from antibiotic-associated diarrhea to pseudomembranous colitis. Although the disease was first described in 1893, the etiologic agent was not isolated and identified until 1978. Since clinical and pathological features of C. difficile-associated disease are not easily distinguished from those of other gastrointestinal diseases, including ulcerative colitis, chronic inflammatory bowel disease, and Crohn's disease, diagnostic methods have relied on either isolation and identification of the microorganism or direct detection of bacterial antigens or toxins in stool specimens. The current review focuses on the sensitivity, specificity, and practical use of several diagnostic tests, including methods for culture of the etiologic agent, cellular cytotoxicity assays, latex agglutination tests, enzyme immunoassay systems, counterimmunoelectrophoresis, fluorescent-antibody assays, and polymerase chain reactions.
- Published
- 1993
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23. Pharmacologic action of Escherichia coli heat-stable (STa) enterotoxin.
- Author
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Knoop FC and Owens M
- Subjects
- Amino Acid Sequence, Animals, Bacterial Toxins genetics, Bacterial Toxins isolation & purification, Calcium metabolism, Cyclic GMP metabolism, Diarrhea etiology, Enterotoxins genetics, Enterotoxins isolation & purification, Escherichia coli physiology, Escherichia coli Proteins, Humans, Intestinal Secretions drug effects, Molecular Sequence Data, Phosphatidylinositols metabolism, Protein Kinases metabolism, Bacterial Toxins pharmacology, Enterotoxins pharmacology
- Abstract
Escherichia coli produces a heat-stable (STa) enterotoxin that belongs to a family of peptides that mediate several diarrheal diseases, including traveler's diarrhea and epidemic diarrhea in infants and newborns. The STa enterotoxin consists of 18 or 19 amino acids and is encoded by genes specified on a transposon. Intestinal secretion is induced by specific binding to high affinity receptors that reside on the brush border cell membrane of the small intestine. Receptor activation by STa enterotoxin induces a sequence of events that culminate in the release of fluid and electrolytes into the intestinal lumen. These events include the stimulation of particulate guanylate cyclae and subsequent increase of intracellular cyclic GMP, involvement of particulate protein kinase, elevation of intracellular calcium, and activation of the phosphatidylinositol pathway. The release of archidonic acid and production of prostaglandins and/or leukotrienes have also been implicated in the action of STa. Evidence indicates that the STa enterotoxin receptor may be a single multifunctional membrane protein.
- Published
- 1992
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24. The effect of Escherichia coli heat-stable enterotoxin on protein kinase activity.
- Author
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Knoop FC, Martig RJ, and Boehm WJ
- Subjects
- Animals, Calmodulin metabolism, Cell Membrane metabolism, Cyclic AMP metabolism, Cyclic GMP metabolism, Dose-Response Relationship, Drug, Drug Stability, Electrophoresis, Polyacrylamide Gel, Hot Temperature, Intestines cytology, Intestines enzymology, Intestines ultrastructure, Intracellular Fluid metabolism, Membrane Proteins metabolism, Mice, Phosphorylation, Rats, Enterotoxins pharmacology, Escherichia coli, Protein Kinases metabolism
- Abstract
Isolated rat enterocytes were incubated with E. coli heat-stable enterotoxin or buffer alone and the protein kinase activity and cyclic GMP level determined on the particulate fraction or cytosol, respectively. In the control cells, particulate protein kinase activity and cyclic GMP concentration were at a maximum after 20 sec and 1 min of incubation, respectively. In heat-stable enterotoxin-treated cells the particulate protein kinase activity was significantly increased (P less than 0.05) after 20 sec of incubation, but decreased (P less than 0.05) after 30 sec, 1 min and 2 min, when compared to the control reaction. During this time period the concentration of intracellular cyclic GMP increased 10-fold. The effect of heat-stable enterotoxin on particulate protein kinase activity and cyclic GMP concentration was dose-dependent. Analysis of radioactive membrane phosphorylation products indicate a role for phosphoproteins with a mol. wt of 25,000 and 120,000. These results suggest that the action of heat-stable enterotoxin may involve an effect on protein kinase.
- Published
- 1990
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25. Bacillus pumilus in the induction of clindamycin-associated enterocolitis in guinea pigs.
- Author
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Brophy PF and Knoop FC
- Subjects
- Animals, Bacillus isolation & purification, Cells, Cultured, Feces microbiology, Guinea Pigs, Hot Temperature, Hydrogen-Ion Concentration, Intestinal Mucosa microbiology, Molecular Weight, Peptide Hydrolases pharmacology, Bacillus pathogenicity, Bacterial Toxins pharmacology, Clindamycin adverse effects, Enterocolitis, Pseudomembranous etiology
- Abstract
Antibiotic-associated enterocolitis was induced in guinea pigs by the intraperitoneal injection of clindamycin. The colonic and cecal mucosa and feces of acutely ill animals were cultured under aerobic and anaerobic conditions on 5% sheep blood agar plates and on a selective and differential medium for Clostridium difficile. All morphologically distinct colony types were isolated in pure culture and identified. A sterile cell-free filtrate of each isolate was tested for ability to induce morphological changes in cultured monolayers of mouse adrenal cells. The filtrate of a predominant isolate, Bacillus pumilus, induced an alteration of cellular morphology; the sterile filtrate of other isolates were unreactive. Toxin contained in cell-free filtrates of B. pumilus caused a syndrome identical to clindamycin-associated enterocolitis when injected intracecally into guinea pigs. The toxin had a molecular weight of 6,500 daltons as determined by molecular sieve chromatography and was inactivated with pronase, lipase, and trypsin. The minimal inhibitory concentrations of clindamycin and vancomycin for B. pumilus were 50 micrograms/ml and less than or equal to 0.4 micrograms/ml, respectively.
- Published
- 1982
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26. Effect of cholera enterotoxin on calcium uptake and cyclic AMP accumulation in rat basophilic leukemia cells.
- Author
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Knoop FC and Thomas DD
- Subjects
- Animals, Basophils drug effects, Cells, Cultured, Cyclic GMP metabolism, Rats, Basophils metabolism, Calcium metabolism, Cyclic AMP metabolism, Enterotoxins pharmacology, Leukemia, Experimental metabolism
- Abstract
Cholera enterotoxin (CT), at an optimal concentration of 2.38 X 10(-10) M, stimulated calcium uptake (P less than 0.01) and cyclic AMP accumulation (P less than 0.02) in cultured rat basophilic leukemia cells. No significant effect of CT on calcium release or cyclic GMP accumulation was detected. Pharmacologic and chemical agents which block calcium uptake or prostaglandin synthesis antagonized the effect of CT.
- Published
- 1984
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27. Stimulation of calcium uptake and cyclic GMP synthesis in rat basophilic leukemia cells by Escherichia coli heat-stable enterotoxin.
- Author
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Knoop FC and Thomas DD
- Subjects
- Animals, Cells, Cultured, Dose-Response Relationship, Drug, Drug Stability, Hot Temperature, Rats, Stimulation, Chemical, Time Factors, Calcium metabolism, Cyclic GMP biosynthesis, Enterotoxins pharmacology, Escherichia coli, Leukemia, Experimental metabolism
- Abstract
The effect of Escherichia coli heat-stable (ST) enterotoxin on calcium and cyclic nucleotide metabolism in rat basophilic leukemia cell cultures was investigated. Addition of ST enterotoxin to rat basophilic leukemia cell cultures resulted in dose- and time-dependent stimulation of calcium uptake and elevation of the intracellular cyclic GMP (cGMP) concentration. The effect of ST enterotoxin on calcium uptake (P less than 0.02) and cGMP synthesis (P less than 0.02) was demonstrated after 5 and 30 min of incubation at 37 degrees C, respectively. In further studies ST enterotoxin did not enhance calcium release or the intracellular concentration of cyclic AMP. The stimulation of calcium uptake and cGMP synthesis by ST enterotoxin was inhibited by pharmacological and chemical agents which block cellular calcium entry and prostaglandin synthesis. These results demonstrate that ST enterotoxin induces calcium uptake and cGMP synthesis in rat basophilic leukemia cell cultures.
- Published
- 1983
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28. In vitro attachment of Treponema hyodysenteriae to mammalian epithelial cells.
- Author
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Knoop FC, Schrank GD, and Ferraro FM
- Subjects
- Adrenal Glands, Animals, Cell Line, Cells, Cultured ultrastructure, Colon, Epithelium, Mice, Swine, Temperature, Cells, Cultured microbiology, Treponema
- Abstract
The interaction between Treponema hyodysenteriae and isolated swine intestinal epithelial cells or mouse adrenal cells in culture was examined. Studies were performed in which treponemes were incubated with each type of anomal cell in an atmosphere of 5% CO2 in air. Coincubation was terminated at various time intervals, and the percentage of treponemal attachment evaluated by light microscopy. The extent of attachment was dependent on both incubation time and temperature. The mechanism of attachment to the animal cell surface was examined by scanning electron microscopy. Interaction of the parasite with the host cell did not appear to alter cellular morphology or result in changes of the cell surface at the site of attachment. Preference for a cellular site of attachment was not found.
- Published
- 1979
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29. The interaction of cholera toxin subunit A with cultured adrenal cells.
- Author
-
Knoop FC
- Subjects
- Animals, Cattle, Cell Membrane metabolism, Cells, Cultured, Cholera Toxin isolation & purification, Chromatography, Gel, Cytosol metabolism, Electrophoresis, Polyacrylamide Gel, Iodine Radioisotopes, Isotope Labeling, Mice, Molecular Weight, Peptides metabolism, Serum Albumin, Bovine metabolism, Serum Albumin, Radio-Iodinated metabolism, Adrenal Glands cytology, Cholera Toxin metabolism
- Abstract
Subunits A and B of cholera enterotoxin were isolated by chromatography on a Bio-Gel P-60 column in the presence of 4% formic acid. The purity and biological activity of the isolated subunits was assessed by polyacrylamide disc gel electrophoresis and mouse adrenal cell assay, respectively. The specific uptake of isolated 125I-labeled subunits A and B, peptides A1 and A2 and bovine serum albumin (BSA) by cultured adrenal cells was investigated. The results indicate that iodosubunit A, or peptide A1 or A2, traverses the plasma membrane and is released to the cell cytosol. A significant portion of bound iodosubunits A or B was associated with the plasma membrane, suggesting the presence of specific membrane receptors. The biological acitivity of subunit A was determined by the mouse adrenal cell assay. The purified subunit caused a characteristic cellular change from epithelioid to rounded morphology. A 30-fold higher concentration of subunit A (on a mole/mole basis) as compared with native toxin was required for a maximum morphologic response. These results extend previous observations related to the bioactivity of subunit A of the cholera enterotoxin molecule.
- Published
- 1978
- Full Text
- View/download PDF
30. Clindamycin-associated enterocolitis in guinea pigs: evidence for a bacterial toxin.
- Author
-
Knoop FC
- Subjects
- Adrenal Glands cytology, Animals, Bacterial Toxins pharmacology, Feces analysis, Guinea Pigs, Hot Temperature, Bacterial Toxins isolation & purification, Clindamycin adverse effects, Colitis chemically induced
- Abstract
Experimental enterocolitis was induced in guinea pigs by intraperitoneal injection of clindamycin. Specimens of feces were collected daily in phosphate-buffered saline (pH 7.0) and pooled every second day. The pooled samples were centrifuged to remove solids, and the supernatant was sterilized by membrane filtration. The sterile fecal supernatants were then dialyzed for 48 h against two 15-liter changes of phosphate-buffered saline and subsequently tested for toxicity in cultured monolayers of mouse adrenal cells. A filterable toxin(s) was found in the fecal supernatants on days 2, 4, and 6 postchallenge and not in pretreatment samples. The toxin(s) caused enterocolitis when administered orogastrically to healthy animals and altered the morphology of cultured mouse adrenal cells. The alteration of adrenal cell morphology was neutralized by specific antitoxin to Clostridium histolyticum.
- Published
- 1979
- Full Text
- View/download PDF
31. Investigation of a hemolysin produced by enteropathogenic Treponema hyodysenteriae.
- Author
-
Knoop FC
- Subjects
- Hemolysin Proteins isolation & purification, Molecular Weight, Pronase pharmacology, Temperature, Treponema growth & development, Treponema pathogenicity, Hemolysin Proteins biosynthesis, Treponema metabolism
- Abstract
A hemolysin produced by Treponema hyodysenteriae, the etiological agent of swine dysentery, was investigated. A virulent isolate (B204) was inoculated into a standard culture medium consisting of Trypticase soy broth without dextrose (BBL Microbiology Systems) supplemented with 10% fetal calf serum in an atmosphere of 70:30 deoxygenated H2-CO2. Sterile cell-free filtrates were prepared at 2-h intervals and assayed for hemolytic activity by using washed sheep erythrocytes. The maximum hemolytic titer was obtained during the early log phase of growth (4 h). A loss of hemolytic activity was observed when cell-free filtrates were stored at 23 and 4 degrees C. Storage at -20 or -80 degrees C after lyophilization resulted in retention of the hemolytic titer for periods of up to 30 days. Enzymatic inactivation of the hemolysin was accomplished with pronase, but not with deoxyribonuclease, ribonuclease, lipase, or trypsin. Addition of exogenous ribonucleic acid-core to the standard culture medium resulted in a dose-dependent increase in the amount of hemolysin produced. The hemolysin could be purified by acid and ammonium sulfate precipitation followed by ion exchange and molecular sieve chromatography. The molecular weight of the hemolysin was 68,000 when determined by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis.
- Published
- 1981
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32. Effect of chemical and pharmacological agents on the secretory activity induced by Escherichia coli heat-stable enterotoxin.
- Author
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Knoop FC and Abbey DM
- Subjects
- Animals, Aspirin pharmacology, Chlorpromazine pharmacology, Diphenoxylate pharmacology, Egtazic Acid pharmacology, Hydrocortisone pharmacology, Loperamide pharmacology, Methylprednisolone pharmacology, Mice, Phentolamine pharmacology, Propranolol pharmacology, Trifluoperazine pharmacology, Enterotoxins pharmacology, Escherichia coli analysis, Intestinal Mucosa metabolism
- Abstract
The effect of aspirin (ASP), chlorpromazine (CPZ), diphenoxylate (DP), ethylene glycol tetraacetate (EGTA), hydrocortisone (HC), loperamide (LPA), methylprednisolone (MP), phenotolamine mesylate (PTM), propranolol (PR), and trifluoperazine (TPZ) on the secretory activity induced by Escherichia coli heat-stable (ST) enterotoxin in infant mice was studied. LPA and DP, which are used therapeutically for diarrhea, did not inhibit the effect of ST enterotoxin; MP and HC, known inhibitors of cholera enterotoxin, and two adrenergic agents (PR and PTM) had no effect on ST-induced secretory activity. TPZ, EGTA, ASP, and CPZ caused a significant (P less than 0.05) decrease in the secretory activity induced by ST enterotoxin, CPZ, EGTA, and TPZ inhibited secretory activity induced by 8-bromoguanosine 3'5'-cyclic monophosphoric acid (8-BrcGMP), a cGMP analog.
- Published
- 1981
- Full Text
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33. The aggregational status of cholera enterotoxin fragment A following biochemical fractionation.
- Author
-
Knoop FC
- Subjects
- Biological Assay, Isoelectric Point, Macromolecular Substances, Molecular Weight, Peptides analysis, Structure-Activity Relationship, Cholera Toxin analysis
- Abstract
Aggregates of frabment A of Vibrio cholerae enterotoxin were revealed following isoelectric focusing in 8 M urea of molecular sieve chromatography in 4% (v/v) formic acid. These aggregates consisted of dimers which required the presence of 10 M urea, 1% sodium dodecyl sulfate (SDS), 2 mM ethylenediaminetetracetic acid (EDTA) and heat (60 degrees C for 1 h) for complete dissociation. All aggregates were homogeneous when tested by standard analytical and SDS polyacrylamide gel electrophoresis or immunodiffusion analysis. Aggregates of fragment A were biologically active in the mouse Y1 adrenal cell assay.
- Published
- 1978
34. Pediatric gastroenteritis associated with Aeromonas sobria and Campylobacter jejuni.
- Author
-
Dang HS, Clark RB, Knoop FC, Shuck KM, and Kimura LK
- Subjects
- Aeromonas isolation & purification, Campylobacter fetus isolation & purification, Feces microbiology, Female, Humans, Infant, Bacterial Infections microbiology, Campylobacter Infections microbiology, Gastroenteritis microbiology
- Published
- 1986
35. The effect of calcium and prostaglandin inhibitors on the intestinal fluid response to heat-stable enterotoxin of Escherichia coli.
- Author
-
Thomas DD and Knoop FC
- Subjects
- Animals, Calcium physiology, Cromolyn Sodium pharmacology, Diltiazem pharmacology, Dose-Response Relationship, Drug, Escherichia coli, Mice, Nifedipine pharmacology, Quinacrine pharmacology, Tolmetin analogs & derivatives, Tolmetin pharmacology, Calcium antagonists & inhibitors, Diarrhea physiopathology, Enterotoxins pharmacology, Intestinal Secretions drug effects, Prostaglandin Antagonists pharmacology
- Abstract
The role of calcium and prostaglandins in the intestinal fluid response to Escherichia coli heat-stable enterotoxin (ST) was investigated in infant mice. Drugs that inhibit calcium uptake-cromolyn sodium, diltiazem, and nifedipine-caused a significant (P less than 0.02, P less than 0.02, and P less than 0.01, respectively) decrease in the fluid response when administered with ST. The effect of cromolyn sodium and nifedipine was dose-dependent; both agents were effective when administered 30 min before toxin challenge. Inhibitors of prostaglandin synthesis-quinacrine hydrochloride and zomepirac sodium-caused a significant (P less than 0.05 and P less than 0.02, respectively) reduction in the fluid response when administered with or 30 min before ST. The fluid response to cyclic 8-bromoguanosine 3',5'-monophosphate or to nitroprusside, which directly activated guanylate cyclase, was not altered by inhibitors of calcium uptake or prostaglandin synthesis. These observations indicate that calcium and prostaglandins are involved in the initial events that result in an intestinal fluid response to E. coli ST.
- Published
- 1982
- Full Text
- View/download PDF
36. Experimental infection of rabbit ligated ileal loops with Treponema hyodysenteriae.
- Author
-
Knoop FC
- Subjects
- Animals, Humans, Ileum ultrastructure, Intestinal Mucosa pathology, Intestinal Mucosa ultrastructure, Intestinal Secretions analysis, Rabbits, Treponemal Infections physiopathology, Ileum pathology, Treponemal Infections pathology
- Abstract
An in vivo animal model was used to assess the enteropathogenicity of the etiological agent (Treponema hyodysenteriae) of swine dysentery. Multiple ligated ileal loops, prepared in New Zealand white rabbits, were challenged with either pathogenic (B78 and B204) or nonpathogenic (Pu) isolates of the organism. The pathogenic isolates induced the onset of intestinal fluid accumulation as early as 4 h, with maximal fluid induction at 18 h postchallenge. Gross lesions of the intestinal mucosa, observed in ileal loops of rabbits sacrificed 24 h postchallenge, were characteristic of swine dysentery. Both pathogenic isolates colonized the epithelial surface and eroded the mucosal barrier, as determined by histological and scanning electron microscopic observations. Intestinal fluid accumulation and erosion of the mucosal barrier were not observed in ileal loops exposed to the nonpathogenic isolate (Pu) or to either of the nonviable pathogenic (B78 and B204) isolates. The ability of pathogenic isolates to initiate and produce infection in rabbit ligated ileal loops, which closely resembles the disease in swine, provides a system with which to study experimental swine dysentery.
- Published
- 1979
- Full Text
- View/download PDF
37. Plesiomonas shigelloides overgrowth in the small intestine.
- Author
-
Penn RG, Giger DK, Knoop FC, and Preheim LC
- Subjects
- Achlorhydria complications, Aged, Chronic Disease, Diverticulum complications, Feces microbiology, Humans, Male, Microbial Sensitivity Tests, Protein Deficiency complications, Tetracycline pharmacology, Diarrhea etiology, Intestine, Small microbiology, Vibrionaceae growth & development
- Abstract
An 83-year-old male presented with chronic diarrhea and protein malnutrition associated with Plesiomonas shigelloides overgrowth in the small intestine. This overgrowth was related to achlorhydria and small-bowel diverticula. Tests for heat-labile and heat-stable enterotoxin were negative on the P. shigelloides isolated from both the small bowel aspirate (3 x 10(8) bacteria per ml) and the patient's stool. The patient responded satisfactorily to tetracycline, to which this unusual isolate was susceptible in vitro. The ability to correctly identify and determine the antimicrobial susceptibility of P. shigelloides in the setting of small-bowel overgrowth will help determine appropriate therapy.
- Published
- 1982
- Full Text
- View/download PDF
38. Physiochemical properties of a heat-stable enterotoxin produced by Escherichia coli of human origin.
- Author
-
Madsen GL and Knoop FC
- Subjects
- Chromatography, Gel, Electrophoresis, Polyacrylamide Gel, Hot Temperature, Humans, Isoelectric Point, Molecular Weight, Bacterial Toxins analysis, Enterotoxins analysis, Escherichia coli analysis
- Abstract
Concentrated cell-free filtrates, prepared from a human strain of enterotoxigenic Escherichia coli, were subjected to isoelectric focusing, molecular sieve chromatography, and polyacrylamide disc gel electrophoresis. Isoelectric focusing in a pH 3 to 5 gradient resulted in two biologically active peaks, I and II, that electrofocused at pI 1.5 and 3.8, respectively. Molecular sieve chromatography of the major enterotoxic peak (II) at pH 3.8 indicated a molecular weight of 2,500. Polyacrylamide disc gel electrophoresis of ST revealed a single protein band containing enterotoxic activity.
- Published
- 1980
- Full Text
- View/download PDF
39. Effect of chlorpromazine on the secretory activity of Escherichia coli heat-stable enterotoxin.
- Author
-
Abbey DM and Knoop FC
- Subjects
- Animals, Chlorpromazine administration & dosage, Dose-Response Relationship, Drug, Hot Temperature, Mice, Chlorpromazine pharmacology, Enterotoxins pharmacology, Escherichia coli, Intestinal Secretions drug effects
- Abstract
The effect of chlorpromazine on the net intestinal accumulation of fluid induced by Escherichia coli heat-stable (ST) enterotoxin in an infant mouse model was examined. Chlorpromazine, when administered with ST enterotoxin, caused a highly significant decrease in net intestinal fluid accumulation. The inhibition of ST activity was dose dependent with various concentrations of chlorpromazine (P less than 0.001). A significant inhibition of toxic activity was also observed when chlorpromazine was administered before (P less than 0.02) or after (P less than 0.05) ST enterotoxin challenge. No significant differences in fluid accumulation were observed between control mice treated with buffer alone and those treated with only chlorpromazine. These data indicate that chlorpromazine markedly decreases the net intestinal fluid accumulation induced by E. coli ST enterotoxin. Further studies on the potential use of chlorpromazine in both the prophylaxis and the treatment of diarrheal diseases appear warranted.
- Published
- 1979
- Full Text
- View/download PDF
40. Effect of heat-stable enterotoxin of Escherichia coli on cultured mammalian cells.
- Author
-
Thomas DD and Knoop FC
- Subjects
- Animals, Calcium Channel Blockers metabolism, Cell Line, Cells, Cultured, Dose-Response Relationship, Drug, Enterotoxins metabolism, Escherichia coli Proteins, Histamine Release drug effects, Humans, Prostaglandin Antagonists metabolism, Rats, Temperature, Bacterial Toxins, Enterotoxins pharmacology
- Abstract
The binding of biologically active 125I-labeled heat-stable enterotoxin (ST) of Escherichia coli with cultured mammalian cells was dose dependent and could be inhibited with low concentrations of unlabeled toxin or by neutralization with specific antiserum. There was positive cooperativity among cell binding sites. A single cultured cell bound approximately 4 X 10(4) molecules of ST; the dissociation constant was 1.33 X 10(-10) M. The specific binding of ST was partially inhibited by Pronase (Sigma Chemical Company, St. Louis, Missouri) and trypsin, but not by lipid- or carbohydrate-specific enzymes, simple sugars, or saccharides. Addition of ST to cultures of rat basophilic leukemia cells resulted in a dose-dependent secretion of histamine. Pharmacologic agents that inhibited calcium uptake or prostaglandin synthesis decreased the amount of histamine released. These data demonstrate the specific binding of ST by cultured cells and support the contention that calcium and prostaglandins may be important in the molecular mechanism(s) whereby ST activates guanylate cyclase.
- Published
- 1983
- Full Text
- View/download PDF
41. Effect of lodoxamide on the secretory response induced by Escherichia coli and Vibrio cholerae enterotoxins in infant mice.
- Author
-
Knoop FC and Thomas DD
- Subjects
- Animals, Bacterial Toxins isolation & purification, Biological Assay, Enterotoxins isolation & purification, Escherichia coli Proteins, Gastric Mucosa drug effects, Intestinal Mucosa drug effects, Mice, Nitriles, Oxamic Acid analogs & derivatives, Tromethamine pharmacology, Amino Acids pharmacology, Bacterial Toxins toxicity, Enterotoxins toxicity, Gastric Mucosa metabolism, Histamine H1 Antagonists pharmacology, Intestinal Mucosa metabolism, Oxamic Acid pharmacology, Tromethamine analogs & derivatives
- Abstract
The effect of lodoxamide tromethamine, a calcium antagonist, on intestinal fluid accumulation induced by Escherichia coli heat-stable (ST) and Vibrio cholerae (CT) enterotoxins in infant mice was investigated. The simultaneous administration of lodoxamide with ST or CT enterotoxin resulted in a significant (P less than 0.01) inhibition of the intestinal fluid response. A minimum concentration of 10(-7) or 10(-8)M lodoxamide caused an inhibition (P less than 0.01) of the ST- or CT-mediated fluid response, respectively. Treatment of infant mice with buffer or drug alone did not result in fluid accumulation. A significant inhibition of ST and CT enterotoxic activities was also observed when lodoxamide was administered 30 min before (P less than 0.02) or 30 min after (P less than 0.01) toxin challenge. These data suggest that calcium may be important in the ST- or CT-mediated induction of fluid accumulation. Further studies on the potential use of lodoxamide tromethamine in both the prophylaxis and treatment of diarrheal disease appear warranted.
- Published
- 1984
- Full Text
- View/download PDF
42. Inhibition of the secretory activity of Escherichia coli heat-stable enterotoxin by indomethacin.
- Author
-
Madsen GL and Knoop FC
- Subjects
- Animals, Biological Assay, Body Water metabolism, Dose-Response Relationship, Drug, Intestinal Mucosa metabolism, Mice, Bacterial Toxins antagonists & inhibitors, Enterotoxins antagonists & inhibitors, Escherichia coli, Indomethacin pharmacology
- Abstract
The effect of indomethacin on the net intestinal accumulation of fluid induced by Escherichia coli heat-stable (ST) enterotoxin in the infant mouse model was examined. Indomethacin, when administered with ST enterotoxin, caused a striking decrease in net intestinal fluid accumulation. This inhibition of ST activity was dose dependent with various concentrations of indomethacin (P less than 0.01). A significant inhibition of toxicity was also observed when indomethacin was given before (P less than 0.01) or after (P less than 0.02) ST enterotoxin challenge. No significant differences in fluid accumulation were observed between control mice treated with buffer alone and those challenged with only indomethacin. These data indicate that indomethacin markedly decreases the net intestinal fluid accumulation induced by E. coli ST enterotoxin. Further studies on the potential use of indomethacin in both the prophylaxis and the therapy of diarrheal diseases appear warranted.
- Published
- 1978
- Full Text
- View/download PDF
43. The effect of cholera toxin on the release of phosphate by intestinal cell membranes.
- Author
-
Knoop FC
- Subjects
- Adenosine Triphosphate metabolism, Animals, Cell Membrane metabolism, Ileum drug effects, Ileum metabolism, In Vitro Techniques, Intestinal Mucosa metabolism, Rabbits, Time Factors, Cell Membrane drug effects, Cholera Toxin pharmacology, Intestinal Mucosa drug effects, Phosphates metabolism
- Published
- 1979
- Full Text
- View/download PDF
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