W.B. Elliott

Comparison of the allergenicity and antigenicity of yellow jacket and hornet venoms

The immunologic properties of yellow jacket and hornet venoms were compared by measuring their reaction with rabbit antisera and human IgE and IgG antibodies. Anti-hornet venom rabbit serum showed precipitin bands unique to hornet venom and several bands crossreacting with yellow jacket venom. Anti-yellow jacket venom rabbit serum reacted with yellow jacket venom but failed to react with the hornet venoms. Most sera from patients who had had allergic reactions after vespid stings reacted with yellow jacket and hornet venoms in RAST analysis. A few sera reacted with only one of the venoms. RAST inhibition studies confirmed the crossreactivity of these IgE antibodies. The IgG antibody response of 14 patients was measured after yellow jacket venom immunotherapy. All had rising titers of yellow jacket venom-specific IgG. There was also an increase in the IgG antibody response measured with hornet venom in the majority of patients. The rise was significant with yellow hornet venom (p less than 0.02) but failed to reach significance with bald-faced hornet venom (p greater than 0.05). In IgG radioimmunoassay inhibition studies using yellow jacket venom-coupled discs, yellow jacket venom was considerably more potent than hornet venom. These studies indicate major crossreactivity between yellow jacket and hornet venoms. In this group of patients, yellow jacket venom appeared to be the primary allergen.

Dimerization constants of water-soluble porphyrins in aqueous alkali

Abstract Spectrophotometric analysis of changes in absorption spectra on dilution of different 2,4-disubstituted derivatives of deuteroporphyrin yielded dimerization constants (KD) for each porphyrin in aqueous alkali. The KD values appear to be related to the hydrophobic/hydrophilic interactions of the system such that KD for proto- > meso- > deutero- > hemato- > coproporphyrin. The effects of alcohol, temperature, and ionic strength on the KD were examined. A simple approach to the graphic analysis of the dilution curves is presented for use when absorbance readings at A100 and A0 cannot be reliably determined, and the use of soluble porphyrins as model systems for studying hydrophobic/hydrophilic interactions in aqueous media is discussed.

Isolation of a phospholipase a from Agkistrodon piscivorus venom.

Abstract 1. 1. A protein with phospholipase A (phosphatide acyl-hydrolase, EC 3.1.1.4) activity has been isolated from Agkistrodon piscivorus venom by a combination of isoelectric precipitation and boiling, hydroxylapatite column chromatography and preparative acrylamide electrophoresis. 2. 2. Sedimentation equilibrium and gel filtration studies indicate the isolated phospholipase A has a molecular weight of about 14 000 ± 500 . 3. 3. Activity assays using hydroxamate formation indicate the enzyme can hydrolyze an acyl ester linkage of either l -α-phosphatidyl choline (synthetic, dipalmitoyl) or of phosphatidyl ethanolamine although the rate and extent of each reaction is different. 4. 4. The Agkistrodon piscovirus phospholipase A is active in the presence of ether and sodium deoxycholate but Ca2+, classically considered an absolute requirement for venom phospholipase A activity, does not appear to be necessary.

Comparison of biochemical and immunologic properties of venoms from four hornet species

Abstract Venoms of two American hornets, Vespula maculata (bald-faced hornet) and Vespula arenaria (yellow hornet), and two Old World hornets, Vespa crabro and Vespa orientalis , were investigated for biochemical and immunologic properties. They were similar with regard to enzymatic activities (phospholipase A, phospholipase B, and hyaluronidase) and molecular composition. Analysis of radioallergosorbent test (RAST) results obtained from 30 vespid-sensitive patients suggests extensive cross-reactivity between the venoms of V. arenaria , V. maculata , and V. crabro , but the venom of V. orientalis seems to lack at least one important allergen. Rabbit antisera to each of the four venoms contain precipitating antibodies to all four venoms. Strong cross-reactivity was found between the venoms of V. maculata , V. arenaria , and V. crabro and between V. crabro and V. orientalis . V. orientalis venom cross-reacts weakly with the venoms of both V. maculata and V. arenaria . It is concluded therefore that diagnosis and immunotherapy with commercially available venoms of V. maculata and V. arenaria should be adequate for most patients sensitive to V. maculata , V. arenaria , or V. crabro but probably not for a significant proportion of those individuals primarily sensitive to V. orientalis .

Comparison of vespid venoms collected by electrostimulation and by venom sac extraction

Venoms of three vespid species, yellow jacket, bald-faced hornet, and yellow hornet, obtained by either electrostimulation of venom sac extraction were compared with regard to their enzymatic activity, antigenicity, and allergenicity. Phospholipase a, phospholipase B, and hyaluronidase enzymatic activities were present in all six preparations. The activity of venom sac extracts lay in the range found in different batches of venoms obtained by electrostimulation for each species. Analysis of sera from vespid-sensitive patients in the radioallergosorbent test (RAST) with discs coupled with either venom sac extracts or venoms obtained by electrostimulation showed a good correlation of the results within all three species (r = 0.95). In RAST inhibition the potency of venom sac extracts and venom obtained by electrostimulation was similar for each species. Analysis of rabbit antisera to the six preparations revealed similar patterns in immunoelectrophoresis and identity reactions between the major antigens within each species. Tissue protein contamination was detected in all venom sac extracts but not in venoms obtained by electrostimulation.

Immunologic comparison of phospholipases A present in Hymenoptera insect venoms.

A comparative study was made of the effect of anti-bee venom phospholipase A2 serum and anti-bald face hornet venom serum on the activity of phospholipase A2 present in yellow hornet, yellow jacket, bald face hornet, and honeybee venoms. Activity of phospholipase A2 present in the venoms was measured titrimetrically using egg yolk dispersion as the substrate. Antiserum against pure bee venom phospholipase A2 activity completely suppressed bee venom phospholipase A2 activity but failed to inhibit phospholipase A2 activity in yellow jacket, yellow hornet, and bald face hornet venoms. Anti-bald face hornet venom serum suppressed the activity of phospholipase A2 present in the four insect venoms with the inhibitory effect decreasing in the order: bald face hornet greater than yellow hornet greater than yellow jacket greater than honeybee. A combination of immunoelectrophoresis and immunodiffusion techniques gave indication of common antigenic sites in a component of yellow hornet venom and a component of bald face hornet venom. The results suggest that IgG antibodies against pure bee venom phospholipase A2 may not give any protection against the reaction(s) due to the phospholipase A2 in yellow hornet, yellow jacket, and bald face hornet venoms, while antibodies against bald face hornet venom phospholipase A2 may give some cross-protection.

Patterns of cytochrome oxidase inhibition by polycations.

Abstract1.The inhibition of cytochromec oxidase activity by three types of polycation (PL-3, PL-150 or 195, and salmine) § is described for three kinds of oxidase system: dispersed by Tween-80, detergent-free “soluble” oxidase, and particulate oxidase (submitochondrial particles).2.Salmine acts as a competitive inhibitor towards cytochromec in all three systems, with aKi between 1 and 4μM. PL-3 (low M.W. polylysine) acts as a non-competitive inhibitor of oxidation in all three systems, with aKi of between 10 μM (submitochondrial particles) and 100 μM (detergrent-free oxidase).3.PL-150 and PL-195, the high M.W. polylysines, act in three distinct ways, depending on the nature of the oxidase preparation: (a) as reversible competitive inhibitors, withKi of about 70 nM (with oxidase dispersed in Tween-80), (b) as stoichiometric inhibitors displaying pseudononcompetivie kinetics (with Keilin-Hartree submitochondrial particles), and (c) as “superstoichiometric” inhibitors, blocking up to 100 equivalents of oxidase, cytochromeaa3 (with detergent-free oxidase or with cholate-treated submitochondial particles).4.PL-195 also inhibits NADH and succinate oxidase activities in intact butc-deficient submitochondrial particles; sigmoidal inhibition curves can be observed in such systems. The rate of PL-195 binding was of the order of 106M−1 sec−1 and the true binding constant between 0.1 and 0.01 nM for the systems showing high affinity.5.High molecular weight polylysines may be useful in investigations of the topology and distribution of cytochromec binding sites on the mitochondrial membrane and in submitochondrial fragments and solubilized preparations. More than one oxidase molecule may be inhibited by one polymer molecule; submitochondrial fragments “opened” by cholate treatment may bind one polymer molecule to several surface sites; Keilin-Hartree particles, with a more “closed” configuration, sesm to bind one polymer molecule to a single site, suggesting a “ecrevice” structure.

Observations on the oxidoreduction of the two cytochromes b in cytochrome c-deficient mitochondria and submitochondrial particles

1. In cytochrome c depleted mitochondria cytochrome bT is reduced rapidly upon addition of ATP or slowly during state 4 respiration, but cytochrome bK is effectively reduced in such mitochondira respiring upon glutamate plus malate in all energy states. In mitochondria or in submitochondrial particles oxidized NADH or succinate, cytochromes bK and bT were always reduced and oxidized independently. 2. Difference spectra for the two b cytochromes were obtained in the presence of respiratory chain inhibitors. Reduced cytochrome bK in the presence of cyanide can be reoxidized by CoQ2. Cytochrome bT reduced in the presence of antimycin can be reoxidized by O2 if rotenone is added to an NADH-reduced sysem or malonate to a succinate-reduced system. There is no evidence for electron transfer between the two b cytochromes. 3. It is suggested that there is no electron transfer from cytochrome bT to cytochrome bK, but that a cytochrome bKbT dimer accepts electrons from the CoQ pool jointly with cytochrome c1 and another acceptor, perhaps the FeS centre. The major steady state species is b2K+b3T+, and a Q-loop occurs with reduction of CoQ by the fully reduced species b2K+b2T+. All proposed interactions between CoQ and Complex III are 2-electron processes and the change from 2-electrons to 1-electron transfer occurs within Complex III itself.

Structural and respiratory effects of Agkistrodon piscivorus phospholipase A on rat liver mitochondria

1. 1. Oxygen electrode tracings show that isolated Agkistrodon piscivorus phospholipase A alters mitochondrial respiration and phosphorylation in a manner identical to whole venom: at low concentrations it increases mitochondrial respiration in the absence of phosphate acceptor; at high concentrations it causes severe inhibition of electron transport; and, at intermediate concentrations it produces a stage of respiratory decline in which ADP acts as an inhibitor (phosphate acceptor inhibition). 2. 2. Electron microscope studies confirm that whole A. piscivorus venom and isolated phospholipase A produce identical morphological alterations in mitochondria and that structural disruption accompanies respiratory decline. 3. 3. Serum albumin can reverse the uncoupling caused either by whole A. piscivorus venom or its purified phospholipase A. 4. 4. Incubation in the presence of succinate, or succinate and ADP, protects mitochondria against the respiratory damage produced by phospholipase A; but incubation in the presence of glutamate-malate affords no protection. 5. 5. There is a remarkable similarity among the overall effects produced on mitochondria by venom phospholipase A, the classical uncouplers and oleic acid.

Immunologic and biochemical evaluation of the potency of whole insect body extracts

Recent studies have indicated that currently available whole body extracts have little potency and are ineffective for diagnosis and treatment of stinging insect allergy. Pure venom is a potent effective allergen but is difficult to obtain in sufficient quantities from all Hymenoptera species. In these studies, an attempt was made to prepare a potent whole body extract. Whole bee body extracts were prepared with different extraction periods and at cold and room temperatures. Potency was examined biochemically by measurements of phospholipase A (PLA) activity and immunologically by PLA and bee venom radioallergosorbent test (RAST) inhibition experiments and gel diffusion studies with the use of rabbit antisera. All extracts prepared in the laboratory had some potency, indicating that it is possible to make a whole body extract containing small quantities of PLA or bee venom. However, the potency of these extracts was minimal as compared with bee venom. Three commercial extracts were almost devoid of detectable immunologic activity. While further attempts may be made to prepare a potent whole body insect extract, these results suggest that it is necessary to obtain venom in relatively pure form for the diagnosis and treatment of stinging insect allergy.