Ned B. Egen

Rate and quantity of delivery of venom from honeybee stings

To determine the rate and completeness of delivery of venom from honeybee stings, European bees were collected at the entrance of a hive and studied with the use of two laboratory models. In one model bees were induced to sting the shaved skin of anesthetized rabbits. The stings were removed from the skin at various time intervals after autotomization, and residual venom was assayed with a hemolytic method. In the other model the bees were induced to sting preweighed filter paper disks, which were weighed again after removal of the sting at various intervals. Results of both experiments were in agreement, showing that at least 90% of the venom sac contents were delivered within 20 seconds and that venom delivery was complete within 1 minute. The data suggest that a bee sting must be removed within a few seconds after autotomization to prevent anaphylaxis in an allergic person. The extensive variation found in the amount of venom delivered at each time point may explain inconsistencies in relationships among reactions to field stings, sting challenge testing, venom skin tests and RAST.

Isolation of monoclonal antibodies to phencyclidine from ascites fluid by preparative isoelectric focusing in the Rotofor

A monoclonal antibody to phencyclidine was developed, produced in mouse ascites fluid, and purified. The purification used only preparative-scale isoelectric focusing in the Rotofor and dialysis. In 4 h, 25% (4 mg) of the antibody from 10 ml of ascites fluid was purified to homogeneity while 63% of the total antibody was recovered.

Effects of constriction bands on rattlesnake venom absorption: A pharmacokinetic study

STUDY OBJECTIVE To determine whether the use of a constriction band alters systemic absorption of rattlesnake venom in pigs and whether constriction band use alters local swelling. DESIGN Using a crossover design, five pigs were studied with and without the use of a constriction band. 125I-Labeled Western Diamondback rattlesnake (Crotalus atrox) venom was injected subcutaneously into one foreleg. The protocol was repeated using the opposite foreleg six days later. The constriction band was applied at the time of injection and removed four hours later. Plasma radioactivity and leg circumference were measured serially. RESULTS Maximum plasma venom concentration and area under the venom concentration-time curve were compared in trials with and without constriction band. Within the initial four hours, application of a constriction band decreased maximum plasma venom concentration by 25% and area under the venom concentration-time curve by 33% (P less than .05). After the constriction band removal at four hours, maximum plasma venom concentration and the area under the venom concentration-time curve were not significantly different between groups. Application of a constriction band did not result in a statistically significant increase in maximum leg circumference as compared with trials without a constriction band. CONCLUSION The use of a constriction band was effective in reducing venom absorption while it was in place (reduced area under the venom concentration-time curve and maximum plasma venom concentration in the cuffed group), and constriction band removal did not result in a significant increase in maximum plasma venom concentration. Leg swelling was not affected by constriction band use. Because constriction band use delayed venom absorption without causing increased swelling, it may prove to be a useful first aid measure in human beings.

Ciguateric Fishes, Ciguatoxin (CTX) and Ciguatera Poisoning

AbstractCiguatera poisoning is caused by the eating of certain tropical and semi-pelagic marine fishes or by a few species of gastropods. In the case of the fishes, these have fed upon either carnivorous or benthonic algal feeders, which have accumulated the toxin through the food chain. The basic organisms involved are dinoflagellates, principally Gambierdiscus toxicus. The toxin, ciguatera toxin (CTX) is C55H76O18 and appears to favor a rather selective choice in voltage - dependent Na+ channels in nerve and muscle cells, and in synaptic terminals. In this respect, the channel(s) are different from those so far identified for tetrodotoxin, maitotoxin, and the other marine toxins for which specific channels have been implicated.Although the poisoning has been known for over 200 years, it has been during only most recent years that it has become a public health problem. While most cases occur in the Pacific Ocean and Carribean Sea, the poisoning is now been reported from numerous parts of the world, and w...

Isolation by preparative isoelectric focusing of a direct acting fibrinolytic enzyme from the venom of Agkistrodon contortrix contortrix (southern copperhead)

Fibrolase, a blood clot-lysing enzyme, was isolated from the venom of the snake Agkistrodon contortrix contortrix using preparative scale isoelectric focusing in the recycling isoelectric focusing (RIEF) apparatus. Two sequential purifications, beginning with 1.0 g of whole, dried venom, were employed. A pH 6-8 range gradient effected the first separation. While 100% of the enzyme was recovered in three fractions, 43% (one fraction) had 70% purity. The second run was a refractionation of three, pooled fractions from the first run, in a 0.7 pH range gradient. Of the fibrolase in the venom, 63% was recovered in four fractions. One of these represented 29% of venom fibrolase, with 97% purity. Gel filtration chromatography removed most of the remaining, higher molecular weight contaminants of the RIEF-purified enzyme.

Comparative protein studies of several Pythium species using isoelectric focusing

Buffer-soluble mycelial proteins of six species of Pythium were compared by isoelectric focusing. Species studied included P. aphanidermatum (P.a.), P. deliense (P.de.), P. myriotylum (P.m.), P. dissotocum (P.di.), P. violae (P.v.), P. ultimum var. ultimum (P.u.u.) and P.u. var. sporangiferum (P.u.s.). Each species had one or more distinct bands, in addition to many common bands of protein, when focused between pH 4-8. A higher concentration of proteins focused in the acidic region of broad pH range gels. Major protein bands were detected with Coomassie stain. Silver staining increased detection of proteins in low concentration (minor bands) in acidic, neutral, and alkaline regions. Other distinct bands of protein were identified for each species when protein samples were further resolved by focusing in the acidic region (pH 3-6). Several isolates each of P.a., P.u., and P.de., collected from various regions of North America, produced consistent protein banding patterns with minor variations. Although banding patterns of P. aphanidermatum differed slightly when isolates were cultured on different media, protein patterns were still specifically identifiable. No differences in protein bands were observed between P. u.u. and P. u.s. The utility of isoelectric focusing for the differentiation of species of Pythium is discussed.

Characterization of synthetic carrier ampholytes by repetitive scanning of the electric field during focusing

This paper reports the utilization of a potential gradient array detector for monitoring the dynamics of the electric field during isoelectric focusing. Transient and steady state electric field profiles are presented for synthetic carrier ampholyte mixtures with a wide (approximately 3-10) pH range. Two available commercial products (Ampholine and Pharmalyte) and a laboratory synthesized mixture (PEHA ampholytes) are compared. The formation of conductivity gaps and their migration toward the cathode in extended experiments (cathodic drift) can be visualized with this system.

Purification of lentil lectins using preparative electrophoresis

Electrophoresis presents an interesting alternative to chromatography for the purification of biological compounds. To document the performance of three preparative electrophoresis apparatus currently available, they were applied to the purification of lectins from lentil seeds which contain two isolectins usually purified by chromatography. Purification by electrophoresis consists of first isolating a mixture of the two isolectins and then separating them. For the first step, either of two free-flow electrophoresis apparatus were employed: the Elphor VaP 22, using field step electrophoresis and the Biostream using zone electrophoresis. To optimize the process, the Biostream was modified to a recycling mode. This required repositioning one dialysis membrane which separates an electrode from the separation chamber. This allowed the lentil extract to be desalted by electrodialysis directly in the apparatus prior to fractionation. A high concentration of lectins was collected at the cathode and acidic proteins were collected at the anode. The bulk of the extract was recycled until the whole volume was processed. In a second step the isolectins were separated by recycling isoelectric focusing in the recycling isoelectric focusing apparatus. The present work clearly demonstrates that electrophoretic methods provide lectins with higher purity than chromatographically purified commercial products.

Effects of preparatory procedures on the venom from a rattlesnake (Crotalus molossus molossus), as determined by isoelectric focusing

A study was made on the venom of a single specimen of the rattlesnake Crotalus molossus molossus, utilizing isoelectric focusing in polyacrylamide gels, to determine what effects various commonly employed preparative procedures might have on the chemical properties of the venom. The procedures included storage at several temperatures, freezing and thawing, evaporation, desiccation and lyophilization. Results indicated that the numbers and relative positions of the protein bands were constant with each procedure. It was concluded that the various procedures did not cause enzymatic degradation or major shifts in the pI of the bands. The significance of the findings is discussed.

Fluid Stabilization during Isoelectric Focusing in Cylindrical and Annular Columns

Abstract The interest in fluid stabilization of focusing columns results from the attractive prospect of preparative isoelectric focusing as an alternative to chromatographic separations for downstream processing of proteins and pep-tides. In isoelectric focusing there is a variation of buffer composition, pH, conductivity, and temperature along the separation axis. The disturbing effects of the latter two parameters can be minimized using solid support media or gels. However, gravity-dependent convection and gravity-independent electroosmosis hamper focusing in free fluids in a complex way that prevents the scaling up of this process while maintaining the exquisite resolution of the analytical scale. The present study discusses methods for minimizing these disturbing effects in annular and cylindrical focusing columns of varying cross-sectional areas and internal geometries.