Histochemical analysis of glycoconjugates in the skin of a catfish (arius tenuispinis, day).

A histochemical study using conventional carbohydrate histochemistry (periodic-acid staining including diastase controls, alcian blue staining at pH 1 and 2.5) as well as using a battery of 14 fluorescein isothiocyanate (FITC)-labelled lectins to identify glycoconjugates present in 10 different areas of the skin of a catfish (Arius tenuispinis) was carried out. The lectins used were: mannose-binding lectins (Con A, LCA and PSA), galactose-binding lectins (PNA, RCA), N-acetylgalactosamine-binding lectins (DBA, SBA, SJA and GSL I), N-acetylglucosamine-binding lectins (WGA and WGAs), fucose-binding lectins (UEA) and lectins which bind to complex carbohydrate configurations (PHA E, PHA L). Conventional glycoconjugate staining (PAS staining, alcian blue at pH 1 and 2.5) showed that the mucous goblet cells contain a considerable amount of glycoconjugates in all locations of the skin, whereas the other unicellular gland type, the club cells, lacked these glycoconjugates. The glycoproteins found in goblet cells are neutral and therefore stain magenta when subjected to PAS staining. Alcian blue staining indicating acid glycoproteins was distinctly positive at pH 1, but gave only a comparable staining at pH 2.5. The mucus of the goblet cells therefore also contains acid glycoproteins rich in sulphate groups. Using FITC-labelled lectins, the carbohydrate composition of the glycoproteins of goblet cells could be more fully characterized. A distinct staining of the mucus of goblet cells was found with the mannose-binding lectins LCA and PSA; the galactosamine-binding lectins DBA, SBA and GLS I; the glucosamine-binding lectin WGA; and PHA E which stains glycoproteins with complex carbohydrate configurations. No reaction occurred with the fucose-binding lectin UEA and the sialic acid-specific lectin SNA. In addition, the galactose-binding lectins PNA and RCA showed only a weak or completely negative staining of the mucus in the goblet cells. The specificity of the lectin staining could be proved by inhibiting binding of the lectins by competitive inhibition with the corresponding sugars. From these data, we can conclude that the mucus produced by the epidermal goblet cells of A. tenuispinis is rich in mannose, N-acetylgalactosamine and N-acetylglucosamine residues.

Prostanoid synthesis in whole blood cells from fish of the Arabian Gulf.

The ability to synthesise prostaglandins and thromboxane from 14C-labelled arachidonic acid was investigated in 11 species of fish from the Arabian Gulf. Cyclooxygenase activity was assessed in washed whole blood cells. Arachidonic acid and its metabolites were extracted and separated on silicic acid columns and thin layer chromatography (silica gel G). Total capacity to convert [14C]arachidonic acid to prostanoids varied from 1 to 35% among the 11 fish species studied. Gray shark (Chiloscyllium griseum) blood cells had the highest capacity (37 +/- 0.4%) to convert arachidonate into prostanoids and two species of catfish (Arius bilineatus and A. thalassinus) exhibited greater than 10% capacity to convert [14C]arachidonate into prostanoids. The major prostanoid synthesised by the two catfish (A. bilineatus and A thalassinus) was 6-keto PGF1 alpha, a stable metabolite of prostacyclin, PGI2. In contrast, A. teunispinis synthesised thromboxane B2, a stable metabolite of thromboxane A2. Thromboxane B2 (TXB2) was the major product synthesised by all three species of shark studied (Chil. griseum, Carcharhinus plumbeus, Carch. melanopterus), with 6-keto PGF1 alpha a minor product. Other fish studied showed a varied pattern of prostanoid synthesis. The synthesis of these prostanoids was almost completely blocked by preincubation of the whole blood cells from catfish and shark with indomethacin (0.5 microM) suggesting the involvement of cyclooxygenase-mediated prostanoid synthesis.

Effects of aqueous extract of onion on the liver and lung of rats.

The toxic effects of oral and intraperitoneal administration of onion extracts on lung and liver tissue of rats were investigated. Oral or intraperitoneal administration of low doses of onion (50 mg/kg) to rats had little effect on lung and liver tissues when compared to control animals. In contrast, administration of high doses of onion (500 mg/kg) resulted in apparent histological changes in lung and liver tissues of rats. Intraperitoneal administration of the high dose of onion was more damaging to lung and liver tissue than oral administration and resulted in a 25% rate of mortality in this treatment group. These results suggest that low doses of onion are nontoxic and may be administered with few ill effects.

Purification of a toxic factor from Arabian Gulf catfish epidermal secretions.

The Arabian Gulf catfish, Arius bilineatus (Valenciennes) secretes a proteinaceous epidermal secretion when threatened or injured. A toxic factor has been isolated and purified from the crude extract (crude skin toxin) of these secretions by a combination of gel filtration on Sephacryl S-300 and preparative discontinuous polyacrylamide gel electrophoresis. The purified skin toxin has a molecular weight of 39,000 Da and an isoelectric point (pI) of 5.45. Injection of the purified skin toxin into rabbits i.v. and determination of the LD50 indicated that the protein had been purified approximately 30 fold by these procedures. Injection of the purified skin toxin into rabbits caused agitation, convulsions and death within 5 min. Analysis of plasma levels of lactate dehydrogenase, glutamate-oxaloacetate transaminase and glutamate pyruvate transaminase in injected rabbits indicated that the skin toxin caused cardiac and liver damage to the animals.

Detection of 8-epi prostaglandin F2alpha in an extract of epidermal secretion of the catfish from the Arabian Gulf.

We provide herein mass spectrometric evidence for the existence of F2 isoprostanes in an epidermal secretion of the catfish from the Arabian Gulf. The compounds were extracted, purified by thin layer chromatography (TLC) and derivatized into the pentafluorobenzyl trimethylsilyl (PFB-TMSi) derivatives and quantified using SIM-negative ion gas chromatography-mass spectrometry based on the addition of tetradeuterated 8-epi PGF2alpha as internal standard prior to sample workup. Freshly collected secretions from the male catfish contained 5.63 and 4.17 ng/ml (two determinations) and from the female catfish 3.60 and 3.81 ng/ml (two determinations) of free 8-epi PGF2alpha. The level of these compounds increases approximately 8-fold upon storage of the extract at -20 degrees. In view of the potent reported actions of F2 isoprostanes on vasoconstriction and as thromboxane receptor antagonists, the presence of isoprostanes in the catfish secretions may contribute to the reported ulcer healing properties of these secretions.

Multiple vasoactive factors in epidermal secretions of the Arabian Gulf catfish, Arius bilineatus (Valenciennes).

1. The Arabian Gulf catfish produces proteinaceous epidermal secretions when threatened or injured. 2. The soluble fraction of the catfish epidermal secretions (SES) has vasoconstrictor activities on sheep renal arteries, which can be inhibited by several antagonists, including atropine, indomethacin, prazosin, and verapamil. 3. Mepyramine, yohimbine, and ketanserin have negligible effects on SES-induced contraction. 4. SES exhibits a significant tachyphylaxis upon addition of a second (8.4% reduction) and third (47% reduction) dose of SES to the organ bath, which can be partially prevented by addition of a fresh arterial section prior to each addition. 5. A vasoconstricting activity has been partially purified from SES by gel filtration on Fractogel HW-65(F) and appears to be a protein with a pI near 7.3. This activity is affected only by verapamil and prazosin.

Histopathological evidence of protective action of garlic against collagen and arachidonic acid toxicity in rabbits.

Soluble rat tail tendon collagen produced respiratory distress, agitation, convulsions and finally death in rabbits when infused intravenously (i.v.) in lethal doses. Similar observations were noted when a lethal dose of arachidonic acid (unsaturated essential fatty acid) was infused. These agents caused thrombocytopenia, indicative of in vivo platelet aggregation, hypotension and increased levels of thromboxane (TX) B2 (a stable metabolite of TXA2) in the plasma. Histopathological examination of lung, heart and liver tissue indicated that the lungs and livers of treated animals were adversely affected, while heart tissues appeared to be normal. Histopathological examination of lung and liver tissues of animals pretreated with garlic, then treated with a lethal dose of collagen or arachidonic acid showed a significant reduction in the damage observed compared to animals not pretreated with garlic.

Platelet activating factors (AGEPC) from epidermal secretions of the Arabian Gulf catfish, Arius bilineatus, which stimulate wound healing.

High levels of platelet activating factor (PAF) activity were demonstrated by platelet aggregation and serotonin release assays to be present in fright induced epidermal secretions of the Arabian Gulf catfish, Arius bilineatus (Valenciennes, 1840). The PAF activity was purified by thin-layer chromatography. Mass spectral analysis combined with chemical and enzymatic modification of the purified PAF and inhibitor studies indicated that PAF activity was due to the presence of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine (AGEPC) molecules. The total AGEPC concentration in the epidermal secretions based on PAF assays was 8 x 10(8) M, well above the threshold level for platelet activation which is near 5 x 10(-11) M. Thus, stimulated epidermal secretory cells of Arius bilineatus supply platelet activating molecules at physiologically high concentrations to sites of injury.

Antithrombotic activity of garlic: its inhibition of the synthesis of thromboxane-B2 during infusion of arachidonic acid and collagen in rabbits.

Rabbits were given collagen and arachidonic acid intravenously. Blood pressure, platelet counts, plasma thromboxane-B2 (TXB2) and plasma 6-keto-prostaglandin F1 alpha, (6-keto-PGF1 alpha) were determined. Both thrombogenic agents, upon infusion of a lethal dose, caused thrombocytopenia, indicative of in vivo platelet aggregation and hypotension. These changes were associated with an increase in plasma levels of TXB2 and 6-keto-PGF1 alpha measured by radioimmunoassay (RIA). Pretreatment of rabbits with an aqueous extract of garlic (500 mgkg) provided protection from thrombocytopenia and hypotension. Thromboxane-B2 synthesis was significantly reduced in animals pretreated with garlic and then injected with a lethal dose of either collagen or arachidonic acid. The amount of TXB2 synthesized in these animals was not sufficient to induce thrombocytopenia or hypotension. All animals pretreated with garlic were well protected against the effects of collagen or arachidonate infusion, and no apparent symptoms were observed in these animals. These observations indicate that garlic may be beneficial in the prevention of thrombosis.

Diabetic ulcer healing preparations from the skin of the Arabian Gulf catfish (Arius bilineatus Val.): a novel and effective treatment.

A novel and simple treatment for healing of infected diabetic foot ulcers in uncontrolled diabetes mellitus patients was devised. The preparations which involve aqueous extracts from the skin of the Arabian Gulf catfish are enriched with different fractions from the same source and with catfish lipids. Eight patients with non-healing foot ulcers and two with wet gangrene were treated. It was found that the ulcers were completely healed. The gangrenic feet noticeably improved in 48 h. The treatment resulted in natural debridation of the necrotic tissues. The ulcer site was invaded by angiogenesis and granulation tissues. Sensation returned to the otherwise neuropathic extremity. The rate of growth of new tissues was proportional to the amount of healing material applied, and its effect ceased shortly after the interruption of the treatment. No. atrophy of the skin lesions was noted and no side-effects were detected.

Histopathological observations on organs from rabbits injected with the skin toxin of the Arabian Gulf catfish (Arius bilineatus, Valenciennes).

Toxicity of soluble protein extracts (skin toxin) from epidermal skin secretions of the catfish, Arius bilineatus (previously identified as Arius thalassinus), was examined in rabbits. Intravenous injections containing doses as low as 2 mg protein/kg body weight caused mortality in most animals tested. Histopathological examination of lung, heart, liver and kidney tissues of rabbits injected with skin toxin indicated that the lungs and livers of treated animals were adversely affected, while heart and kidney tissues appeared to be normal. Lethality of skin toxin was prevented by pretreatment of the rabbits with indomethacin. Histopathological examination of lung and liver tissues of indomethacin pretreated animals showed a significant reduction in the damage observed after injection of skin toxin.

Comparative biochemical and pharmacological properties of epidermal secretions from Ariid catfish of the Arabian Gulf.

1. Comparative biochemical and pharmacological studies on epidermal secretions from three species of Ariid catfish were carried out to determine whether they share common properties. 2. Protein compositions are generally similar, but show some differences in size and numbers of proteins. 3. Lipids also differ. However, seasonal and dietary changes in lipid compositions exceed the differences among species. 4. Enzymes in the secretions appear common to each fish species, though some differences are noted in relative activities. 5. Toxicity responses of rabbits to injections with secretions from each of the catfish species indicated that two were lethal, with similar lethal dose levels when injected intravenously. 6. Secretions from all three catfish induced changes in plasma enzyme levels indicative of heart and liver damage, and also induced changes in blood glucose and cholesterol levels.

A simple spectrophotometric method for the determination of thrombin activity.

The activity of thrombin in commercial preparations was measured by an easy, precise spectrophotometric method that allows determination of thrombin activity in dilute solutions more accurately than other methods hitherto used. The method can also be used to compare fibrinogen content in plasma samples.

A spectrophotometric method for the standardization of thromboplastin clot-promoting activity.

A simple spectrophotometric method for the quantitating of the clot promoting activity of thromboplastin in commercial preparations was developed. Thromboplastin activities were proportional to the maximum rate of clot formation. This was calculated from curves of absorption change during the coagulation of plasma coagulation control. The clot-promoting effects of thromboplastin in commercial preparations varied widely when assayed by this method.

Prostaglandin associated mortality following intravenous injection of catfish epidermal secretions in rabbits.

Toxicity of soluble protein extracts from epidermal gel secretions of the catfish, Arius thalassinus, was examined in rabbits. Intravenous injections containing doses as low as 2 mg protein/kg body weight caused mortality in all animals tested. An increase in plasma levels of thromboxane B2 (TXB2) and of 6-keto prostaglandin F1 alpha (6-keto PGF1 alpha) were observed following injections. Both the mortality and prostaglandin release were prevented by pretreatment of rabbits with either indomethacin or hydrocortisone. A similar indomethacin sensitive induction of prostaglandin release was noted following the in vitro treatment of arterial tissue sections with gel. Lethality appears to result from gel substances stimulating phospholipase activity to yield arachidonic acid, which is then metabolized to give toxic levels of prostaglandins.