Purification and pore-forming activity of two hydrophobic polypeptides from the secretion of the Red Sea Moses sole (Pardachirus marmoratus).
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A new column chromatography procedure, based on ion exchange, chromatofocusing, and reverse phase high pressure liquid chromatography was employed to isolate the two main proteinaceous, toxic, cytolytic, pore-forming factors from the secretion of the Red Sea Moses sole Pardachirus marmoratus. Pardaxin I, comprising 10% of the gland secretion proteins, was shown to be 5-10 times more toxic, cytolytic, and active in membrane pore formation than pardaxin II (8% of gland secretion proteins). Gel electrophoresis, amino acid analysis, and NH2-terminal amino acid sequence reveals a high degree of homogeneity and resemblance between the two toxins. They are rich in aspartic acid, serine, glycine, and alanine and devoid of arginine, tyrosine, and tryptophan. Their NH2-terminal residue sequence was found to be NH2-Gly-Phe-Phe. Their hydrophobicity is evident from chromatographic behavior on a hydrophobic matrix, presence of 9 successive hydrophobic residues at the NH2 terminus, and a decrease in drop size during elution of active fractions during chromatographic purification. The minimal molecular weight of pardaxin I is about 3500 as determined by sodium dodecyl sulfate gel electrophoresis and amino acid analyses. It is composed of 35 amino acids and is free of carbohydrate and sialic acid residues. Mass spectrometry of the ethyl acetate extract of the gland secretion and purified toxin reveals the presence of sterols in the secretion but their absence in the purified toxins. Pardaxin I was iodinated without affecting its chemical and pore-forming properties. It binds to liposomes of different phospholipid compositions. In hyperpolarized unilamellar liposomes, pardaxin I produced a fast, nonspecific permeabilization and in multilamellar liposomes, a slow, cation-specific pore. It is suggested that pardaxins exert their effects due to their hydrophobic and pore-formation properties. (+info)
The action of pardaxin (PX), a toxin isolated from the secretion of the Red Sea flatfish, Pardachirus marmoratus, was studied on longitudinal muscle of guinea-pig ileum. Pardaxin contracted the ileum and subsequently abolished muscle contraction to 5-hydroxytryptamine (5-HT), but did not affect the responses to acetylcholine (ACh) and substance P(SP). Pardaxin-induced contraction was only partially suppressed by atropine and not affected by tetrodotoxin or morphine. Preparations desensitized to 5-HT or SP responded normally to pardaxin. Pardaxin-induced contractions were normal in K+-depolarizing Krebs Ringer solution and not affected by black widow spider venom. It is concluded that the pardaxin-induced muscle contractions are not mediated through the release of neurotransmitters and do not involve 5-HT, SP or ACh receptors, but are due to a direct action on the muscle contractile mechanism. (+info)
Transcription of vesicular stomatitis virus activated by pardaxin, a fish toxin that permeabilizes the virion membrane.
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The toxic protein, Pardaxin, of the Red Sea flatfish Pardachirus marmoratus readily induced transcription of vesicular stomatitis virus by making the virion membrane permeable to nucleoside triphosphates in the absence of nonionic detergents. Virion transcription was activated over a wide range of Pardaxin concentrations, but at optimal concentrations, the rate of transcription exceeded that induced by Triton X-100. The inhibitory effect of M protein was manifested for both Pardaxin-induced and Triton-induced transcription at high concentrations of vesicular stomatitis virions; however, unlike the Triton-induced reaction, the inhibitory effect of M protein was not reversed by polyglutamic acid added to the Pardaxin-induced transcription reaction. We propose that activation of virion transcription by Pardaxin resembles more closely intracellular transcription initiated by virion penetration than does detergent-activated transcription of vesicular stomatitis virus. (+info)
Pardaxin, a hydrophobic toxin of the Red Sea flatfish, disassembles the intact membrane of vesicular stomatitis virus.
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Reaction of vesicular stomatitis virus with pardaxin, the hydrophobic toxin of the Red Sea flatfish, resulted in a profound morphological change of many virions and dissociation of their membrane and nucleocapsid into components readily separable by density gradient centrifugation. The basic matrix protein and acidic pardaxin segregated largely with the high density nucleocapsid. The dissociated virion membrane formed lipoprotein vesicles which retained glycoprotein spikes and a certain amount of N protein but no appreciable amounts of other nucleocapsid proteins and little if any RNA. Iodination of the tyrosine residue of the glycoprotein tail fragment provided supporting evidence that the COOH terminus of the glycoprotein extends beyond the inner layer of the membrane into the interior of the virion. These data indicate that pardaxin may serve as a probe for studying the organization of viral membranes, and, hopefully, other biological membranes. (+info)
Conformations of peptide fragments comprising the amino-terminal, central, and carboxyl-terminal regions of a membrane-active polypeptide. Build-up of secondary structure in pardaxin.
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The conformations of synthetic peptides of different lengths corresponding to the amino-terminal, central, and carboxyl-terminal regions of pardaxin (GFFALIPKIISSPLFKTLLSAVGSALSSSGEQE) have been studied by circular dichroism spectroscopy. The peptides GFFALIPKIISSPLF-OMe, GFFALIPKIISSPLFK-OMe corresponding to the amino-terminal region, as well as peptides KIISSPLFKTLLSAV and IISSPLFKTLLSAV corresponding to the central region of the toxin have a marked tendency to adopt helical conformation. Ordered conformation is also discernible in the 8- and 7-residue peptides GFFALIPK-OMe and IISSPLF-OMe. Peptides corresponding to the central segments KTLLSAV, LSAVGSAL, and the carboxyl-terminal segment SSSGEQE, however, exhibit very little secondary structure. The peptide segments that adopt ordered conformation show similar conformation when present in the entire toxin as suggested by proton magnetic resonance data (Zagorski, M. G., Norman, D. G., Barrow, C. J., Iwashita, T., Tachibana, K., and Patel, D. J. (1991) Biochemistry 30, 8009-8017). The observation that peptide segments corresponding to the amino-terminal and central regions of the toxin adopt ordered conformations compared to the carboxyl-terminal segment in isolation as well as in the toxin, indicates a role for these regions in initiating and maintaining ordered conformation of pardaxin. (+info)
A class of highly potent antibacterial peptides derived from pardaxin, a pore-forming peptide isolated from Moses sole fish Pardachirus marmoratus.
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Pardaxin, a 33-amino-acid pore-forming polypeptide toxin isolated from the Red Sea Moses sole Pardachirus marmoratus, has a helix-hinge-helix structure. This is a common structural motif found both in antibacterial peptides that can act selectively on bacterial membranes (e.g., cecropin), and in cytotoxic peptides that can lyse both mammalian and bacterial cells (e.g., melittin). Herein we show that pardaxin possesses a high antibacterial activity with a significantly reduced hemolytic activity towards human red blood cells (hRBC), compared with melittin. Its potency is comparable to that of other known native antibacterial peptides such as magainin, cecropins and dermaseptins. To determine the structural features responsible for the selective hemolytic and antibacterial activities, and the structural requirements for a high antibacterial activity, 8 truncated and modified pardaxin analogues were synthesized and structurally and functionally characterized. Each peptide was synthesized with a free carboxylate or amino group (i.e., aminated form) at its C-terminus. The aminated form of pardaxin has both high hemolytic and antibacterial activity. A truncated analogue, with 11 amino acids removed from the C-terminal domain, had dramatically reduced hemolytic activity. However, the aminated form of this analogue was significantly more potent that pardaxin against most bacteria tested, suggesting that the C-terminal tail of pardaxin is responsible for non-selective activity against erythrocytes and bacteria. Furthermore, a positive charge added to its N-terminus significantly increased its antibacterial activity and abolished its low hemolytic activity. The 22-amino-acid C-terminal domain and the short 11-amino-acid N-terminal domain were, in their aminated forms, active only against gram-positive bacteria. Secondary-structure determination using circular dichroism spectroscopy revealed that all the aminated analogues had 25-80% more alpha-helical content in 40% CF3CH2OH/water than their non-aminated forms. Using model phospholipid membranes it was found that all the analogues that were less hemolytic but had retained antibacterial activity could permeate acidicly charged phospholipid vesicles better than zwitterionic phospholipid vesicles, a property characteristics of all the native antibacterial peptides tested so far (e.g., cecropins, magainins and dermaseptins). Pardaxin and its analogues therefore represent a new class of antibacterial peptides that can serve as a basis for the design of therapeutic agents. Furthermore, negative-staining electron microscopy revealed that total inhibition of bacterial growth was due to total lysis of the bacterial wall. Therefore, it might be more difficult for bacteria to develop resistance to such a destructive mechanism, compared with the more specific mechanisms of the currently used antibiotics. (+info)
Weever fish stings: a report of two cases presenting to an accident and emergency department.
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Two patients are described who suffered weever fish stings and presented to an accident and emergency department. The characteristic symptoms and treatment are described. (+info)
Stingray injury.
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A case of stingray injury is reported. Local symptoms and signs include intense pain, oedema around the wound, erythema and petechiae. Systemic symptoms and signs include nausea and vomiting, muscle cramps, diaphoresis, syncope, headache, muscle fasciculations, and cardiac arrhythmias. Treatment aims to reverse local and systemic effects of the venom, alleviate pain, and prevent infection. Antitetanus prophylaxis is important. Treatment for anaphylaxis may be necessary. (+info)