Previously it was reported that supplementation of insect cell culture with Lonomia obliqua hemolymph could extend culture longevity (Maranga et al. Biotechnol. Prog. 2003, 19, 58-63). In this work the anti-apoptotic properties of this hemolymph in Spodoptera frugiperda (Sf-9) cell culture were investigated. The presence or absence of apoptotic cells was characterized by light microscopy, flow cytometry, and agarose gel electrophoresis. Hemolymph was fractionated by several ion exchange and gel filtration chromatographic steps for identification of the compounds responsible for this effect. Fractions exhibiting a potent anti-apoptotic effect were isolated and tested in cell culture. A protein of about 51 kDa was identified, isolated, and tested for apoptosis inhibition. Addition of this purified protein to Sf-9 cultures was able to prevent apoptosis induced by nutrient depletion as well as by potent apoptosis chemical inducers such as Actinomycin D. This work confirms that the enhanced culture longevity obtained by supplementation with L. obliqua hemolymph is due to the presence of potent anti-apoptotic factors.

The benefits of insect cell culture medium supplementation with hemolymph of Lonomia obliqua were investigated. The addition of hemolymph to the medium induced high levels of cell growth, and the viability was maintained for longer periods. The maximum cell yield increased almost 3-fold after hemolymph supplementation. Cultures in their stationary phase were rescued through hemolymph supplementation, also reaching high cell concentrations. These actions were much dependent on the concentration of hemolymph; low hemolymph concentration had a positive effect in cell growth, whereas high hemolymph concentration showed a deleterious effect. Fractionation of hemolymph by gel filtration chromatography showed the presence of three factors with different activity in insect cell culture: an potential anti-apoptotic factor, a growth-promoting factor, and an enzyme that hydrolyzes sucrose. Addition of hemolymph to the medium induced high levels of glucose production. The sucrose to glucose conversion was also linearly dependent upon the hemolymph concentration. Therefore, we conclude that cell growth and longevity can be increased by supplementation of the culture medium with hemolymph.

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Potent antiviral activity against measles, influenza and polio viruses was observed in the hemolymph of Lonomia obliqua. The antiviral protein responsible for this activity was isolated, purified by gel filtration chromatography using a gel filtration column system (Superdex 75) and further fractionated using a Resource-Q ion exchange column system. Experiments with the purified protein led to a 157-fold reduction (from 3.3+/-1.25 x 10(7) to 2.1+/-1.5 x 10(5) TCID(50)mL(-1)) in measles virus production and a 61-fold reduction (from 2.8+/-1.08 x 10(9) to 4.58+/-1.42 x 10(7)mL(-1)) in polio virus production. Heating and freezing seem to have no influence over its antiviral activity. Also, the protein does not display virucidal activity and does not act on receptors on the cell membrane. The observations suggest an intracellular mechanism of action and that the protein may act as a constitutive agent that affects the innate antiviral immune response.

Gene expression in animal cells allows large scale production of proteins used for either structure and function studies or therapeutic purposes. Maximizing recombinant protein production is necessary to optimize cell growth and protein expression. Some studies have demonstrated the presence of pharmacologically active substances in insect hemolymph. In this work, we have identified and purified a protein from Lonomia obliqua hemolymph able to increase the production of the rabies virus glycoprotein, expressed in Drosophila melanogaster S2 cells, by about 59%.

The control of viral infections, mainly those caused by influenza viruses, is of great interest in Public Health. Several studies have shown the presence of active properties in the hemolymph of arthropods, some of which are of interest for the development of new pharmacological drugs. Recently, we have demonstrated the existence of a potent antiviral property in the hemolymph of Lonomia obliqua caterpillars. The aim of this study was to produce an antiviral protein in a baculovirus/Sf9 cell system. The resulting bacmid contains the sequence coding for the antiviral protein previously described by our group. Total RNA from L. obliqua caterpillars was extracted with Trizol and used in the reverse transcription assay with oligo(d)T primer followed by polymerase chain reactions (RT-PCR) with specific primers for the cDNA coding for the antiviral protein, based on the sequence deposited in the GenBank database. Restriction sites were inserted in the cDNA for ligation in the donor plasmid pFastBac1™. The recombinant plasmid was selected in Escherichia coli DH5α and subsequently used in the transformation of E. coli DH10Bac for the construction of the recombinant bacmid. This bacmid was used for the expression of the antiviral protein in the baculovirus/Sf9 cell system. After identifying the protein by western blot, activity tests were performed, showing that the purified recombinant protein was able to significantly reduce viral replication (about 4 logs). Studies on the optimization of the expression system for the production of this antiviral protein in insect cells are in progress.

The authors observed an injury caused by the sting of a false tocandira ant in the hand of an amateur fisherman and they describe the clinical findings and the evolution of the envenoming, which presented an acute and violent pain, cold sweating, nausea, a vomiting episode, malaise, tachycardia and left axillary's lymphadenopathy. About three hours after the accident, still feeling intense pain in the place of the sting, he presented an episode of great amount of blood in the feces with no history of digestive, hematological or vascular problems. The intense pain decreased after eight hours, but the place stayed moderately painful for about 24 hours. In that moment, he presented small grade of local edema and erythema. The authors still present the folkloric, pharmacological and clinical aspects related to the tocandiras stings, a very interesting family of ants, which presents the largest and more venomous ants of the world.

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