James T. Barrett

Platelet aggregation and sphingomyelinase D activity of a purified toxin from the venom of Loxosceles reclusa.

A facile and quantitative assay for measuring the activity of sphingomyelinase D in recluse spider venom has been developed using L-alpha-[palmitoyl-1-14C]lysophosphatidylcholine as substrate. This assay avoids the problem of substrate insolubility that occurs when sphingomyelin and other insoluble lipids are used as substrates. This assay has been employed in gel filtration and isoelectric focusing isolation techniques to purify sphingomyelinase D from spider venom. The purified sphingomyelinase exhibits four active enzyme forms in isoelectric focusing with pI values of 8.7, 8.4, 8.2, and 7.8. Each active form when examined in SDS-polyacrylamide gel electrophoresis gave an estimated molecular weight of 32 000. The four active enzyme forms were immunologically cross-reactive with each other as demonstrated with radioimmune assays using an antiserum developed to one of the active forms. Each active form hydrolysed sphingomyelin to release choline and produce N-acylsphingosine phosphate. One of the active enzyme forms was characterized further in dermonecrosis and platelet aggregation measurements. This purified sphingomyelinase D was identified as a poisonous toxin that can developed typical dermonecrotic spider lesions when injected into experimental animals at levels expected to be delivered in a normal bite. Furthermore, the purified toxin acts to aggregate human blood platelets. The toxin-induced platelet aggregation has been related to serotonin release as aggregation occurs, and it has been shown to be inhibited by EDTA over the range of 0.6 yo 3.0 mM EDTA. It is suggested that spider-induced dermonecrosis could result in part from platelet aggregation at and near the site of envenomation.

Red blood cell lysis induced by the venom of the brown recluse spider: The role of sphingomyelinase D

Red blood cell lysis induced by the venom of Loxosceles reclusa, the brown recluse spider, may be related to the hemolytic anemia observed in several cases of spider envenomation. These investigations demonstrate that the venom of the brown recluse spider contains a calcium-dependent, heat-labile hemolysin of molecular weight approximately 19,000. The pH optimum for the hemolytic reaction was 7.1, and the optimum calcium concentration for venom-induced lysis was observed within the range of 6 to 10 mm. Sheep red blood cells were more susceptible to the spider hemolysin than human red blood cells, although both types exhibited appreciable lysis. Digestion of sheep red blood cell membranes with partially purified venom lysin resulted in degradation of the sphingomyelin component. However, reaction of the membranes with the venom lysin produced no release of water-soluble phosphate, and no free fatty acids were generated. These results indicate that the sphingomyelin-degrading activity of the venom is not a phospholipase C- or a phospholipase A2-type activity. Sphingomyelin was employed as substrate for the venom hemolysin, and the organic and aqueous fractions of the reaction mixtures were analyzed by thin-layer chromatography. Analysis of the organic fraction revealed a phosphate-containing product with the solubility and chromatographic characteristics of N-acylsphingosine phosphate (ceramide phosphate), and analysis of the aqueous fraction demonstrated the presence of choline. The isolation and identification of these products indicate that the sphingomyelin of the red cell membrane is hydrolyzed by a sphingomyelinase D-type activity expressed by the partially purified venom hemolysin. A close correspondence between the hemolytic and sphingomyelinase D activities was observed when the partially purified hemolysin was further characterized in polyacrylamide gel electrophoresis at pH 8.3 and pH 4.9. The hemolytic and sphingomyelinase activities were coincident within the electrophoretic pattern at both pHs. The results presented demonstrate conclusively a direct lytic action of brown recluse venom upon red blood cells and report for the first time the presence of sphingomyelinase D in spider venom.

Hyaluronidase and esterase activities of the venom of the poisonous brown recluse spider

Abstract Venom of Loxosceles reclusa free from impurities was expressed from venom glands collected by microdissection. Polyacrylamide gel electrophoresis of the venom at pH 8.3 demonstrated 7 or 8 major plus 3 or 4 minor components. Upon electrophoresis at pH 4.9 two major components plus 3 or 4 minor components were noted. Monophoretic hyaluronidase prepared by Sephadex gel filtration and electrophoresis at pH 8.3 exhibited optimum activity from pH 5.0 to 6.6. Sodium dodecyl sulfate gel electrophoresis of purified hyaluronidase revealed two components with estimated molecular weights of 33,000 and 63,000. The purified hyaluronidase exhibited activity against chondroitin sulfate, types A, B, and C at approximately 20–30% of that upon hyaluronic acid. The enzyme was inhibited 10–20% by the heavy metal ions, Fe+3 and Cu+2. Rabbit antivenom inhibited the spreading effect of whole venom in vivo and completely inhibited hyaluronidase in vitro. Incorporation of [14C]leucine into the spider venom led to the separation of hyaluronidase from the dermonecrotic activity of the venom. The venom demonstrated activity against carbobenzoxy- l -tyrosine-p-nitrophenyl ester and β-naphthylacetate which was inhibited approximately 65% by 2.5 × 10−3 m levels of EDTA and EGTA but not by 2.5 × 10−4 m o-phenanthroline. The esterase activity resisted concentrations of p-chloromercuribenzoate which totally inactivated papain. The venom appeared devoid of collagenase, dipeptidase, acetylcholinesterase, phosphodiesterase, ribonuclease A, and deoxyribonuclease.

Ultrasound: A new immunosuppressant☆

Abstract The medical application of low-energy ultrasound as a diagnostic aid has developed in the absence of appropriate studies of its hazards. Mice given a 5-min exposure to a 2-MHz ultrasound wave at a maximum intensity of 8.9 mW/cm2 applied over the area of the spleen responded to an injection of sheep erythrocytes with hemagglutinin log2 titers of 5.9 ± 0.15 and hemolysin titers of 7.6 ± 0.14. Sham-treated controls developed hemagglutinin titers of 7.2 ± 0.24 and hemolysin titers of 8.7 ± 0.14. This immunosuppressive effect of ultrasound was dose dependent and was not observed when ultrasound energy was applied to the mouse thigh. Ultrasound is a more efficient suppressant when given in close temporal association with the antigen and is more influential upon the primary compared to the secondary response. Mercaptoethanol reduction and single radial immunofiffusion experiments revealed that a primary difference between the experimental and control sera was an IgM deficiency of the former. This was accompanied by a significant loss of IgG2a and IgG2b. This difference in immunoglobulin levels of ultrasound exposed compared to normal mice was reflected by the lowered number of direct antibody plaque-forming cells in the spleen of the treated animals.

Depression of phagocytosis by ultrasound

Abstract Swiss mice insonated with a 2 megaHertz (MHz) wave at an energy level of 8.9-milliwatts/square centimeter (mW/cm2) applied over the liver had an impaired ability to clear injected colloidal carbon from their blood compared to untreated mice. Mice evaluated immediately after treatment had a depressed phagocytic index (K) and clearance half-time ( T 1 2 ) but their phagocytic index corrected for liver and spleen weight (α) was normal. All three parameters deviated from normal if the clearance assay was conducted 48 or 72 hr after insonation, indicating a latency of the effect of ultrasound on the phagocytic system. The demonstration that peritoneal macrophages exposed in vivo to ultrasound also had depressed phagocytic abilities indicated that the reduced clearance of carbon from the blood of mice was not entirely due to damage to the liver.

Avian delayed-type hypersensitivity: Adaptation of the migration-inhibition assay

Abstract In this study White Leghorn cockerels were sensitized with Mycobacterium tuberculosis and/or bovine gammaglobulin (BGG). The migration of spleen cells from chickens with delayed dermal hypersensitivity to PPD was markedly inhibited in the presence of PPD but not by BGG. When birds were made doubly sensitive to mycobacteria and BGG, the migration of their spleen cells was inhibited by both antigens. Cells from animals immunized to produce high levels of circulating antibody to BGG were not inhibited in the presence of antigen. Sensitive spleen cells incubated with specific antigen elaborated a substance into the medium which inhibited the migration of normal cells. The results of these experiments indicate that the delayed hypersensitive response in birds parallels that of the mammal in that it is antigen specific, reproducible, independent of the antibody response, and transferable to normal cells with a soluble cell-free product.

Inactivation of complement by Loxosceles reclusa spider venom.

Zymosan depletion of serum complement in guinea pigs rendered them highly resistant to lesion by Loxosceles reclusa spider venom. Guinea pigs deficient in C4 of the complement system are as sensitive to the venom as normal guinea pigs. The injection of 35 micrograms of whole recluse venom intradermally into guinea pigs lowered their complement level by 35.7%. Brown recluse spider venom in concentrations as slight as 0.02 micrograms protein/ml can totally inactivate one CH50 of guinea pig complement in vitro. Bee, scorpion, and other spider venoms had no influence on the hemolytic titer of complement. Fractionation of recluse spider venom by Sephadex G-200 filtration separated the complement-inactivating property of the venom into three major regions which could be distinguished on the basis of heat stability as well as size. None was neutralized by antivenom. Polyacrylamide gel electrophoresis of venom resolved the complement inactivators into five fractions. Complement inactivated by whole venom or the Sephadex fractions could be restored to hemolytic activity by supplements of fresh serum but not by heat-inactivated serum, pure C3, pure C5, or C3 and C5 in combination.

Studies on a hemagglutinin from the lichen Parmelia michauxiana

Abstract A phytohemagglutinin which has activity against erythrocytes from several species has been isolated from the lichen, Parmelia michauxiana by Sephadex gel filtration. The agglutinin contained 80% hexose, combining D -glucose, D -galactose and D -mannitol in the ratio of 1.00:1.76:1.75. In the analytical ultracentrifuge the active material migrated as a single peak with an apparent sedimentation coefficient of 2.3 S. The activity of the agglutinin was independent of temperature between 4 and 37° but was markedly pH and ionic strength dependent. The agglutinin resisted autoclaving. It was not inhibited by any of several sugars, did not stimulate mitosis of leukocytes, and did not hemolyze erythrocytes in the presence of complement.

Serum amylase changes due to antipancreas serum.

Summary As determined by an in vitro and an in vivo test, guinea pig pancreas has been found to be antigenic. Its antiserum produced pancreas pathology and serum amylase increases when administered passively to normal guinea pigs.

Chemotactic activity of venom from the brown recluse spider (Loxosceles reclusa)

Abstract The incubation of whole Loxosceles reclusa venom with complement generated a chemotactic activity for guinea pig neutrophils which had been harvested from peritoneal exudate cells. Venom-guinea pig complement mixtures were 42% more chemotactic than the complement alone and venom-human complement mixtures were 55% more chemotactic than human complement alone. Venom plus diluent was no more chemotactic than diluent alone, although venom plus heat inactivated guinea pig serum was slightly chemotactic. Purified guinea pig or human C3 or C5 incubated with recluse spider venom was not chemotactic nor did venom alter the serologically active quantity of C3 or C5 in human serum. Both C4 deficient and properdin deficient sera were suitable substrates for the chemotaxigen in recluse spider venom.