A comparison of survival and repair of UV-induced DNA damage in cultured insect versus mammalian cells.

Survival and unscheduled DNA synthesis (UDS) were measured in a cultured insect cell line, TN-368, and a cultured mammalian cell line, V-79-4, following exposure to several fluences of ultraviolet light. TN-368 cells were approximately seven times more resistant to the lethal effects of UV than V-79 cells, as determined by colony formation. The amount of UDS per unit amount of DNA is about the same in both cell types 4 hr after 10-50 J/m2 UV irradiations.

Isolation and characterization of myrmexins, six isoforms of venom proteins with anti-inflammatory activity from the tropical ant, Pseudomyrmex triplarinus.

Venom from the tropical ant, Pseudomyrmex triplarinus, has anti-inflammatory properties demonstrated by the carrageenin-induced edema animal mode. A multi-protein complex that inhibits edema was isolated from the venom and was further characterized by high performance liquid chromatography (HPLC), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) and amino acid sequencing. Although the complex exhibited a single band in SDS-PAGE electrophoresis, six proteins (isoforms) were resolved and purified to homogeneity and were designated myrmexin I-VI. They have very similar molecular masses between 6998 and 7142 Da. Each myrmexin is a heterodimer consisting of a small subunit (SS1 or SS2 or SS3) disulfide-linked to a larger, quite structurally unrelated subunit (LS1 or LS2). Thus, the myrmexin complex consists of six isoforms of venom proteins: myrmexin I (SS1/LS2), myrmexin II (SS1/LS1), myrmexin III (SS2/LS2), myrmexin IV (SS3/LS2), myrmexin V (SS2/LS1), myrmexin VI (SS3/LS1). Subunit SS1 is highly homologous to SS2 (96% of identity) and SS3 (87% of identity) and LS1 is highly homologous to LS2 (79% of identity). Our study suggests that myrmexins may represent a new class of anti-inflammatory proteins.

Toxicity of the venom from Nasonia vitripennis (Hymenoptera: Pteromalidae) toward fly hosts, nontarget insects, different developmental stages, and cultured insect cells.

A venom preparation from Nasonia vitripennis, a wasp ectoparasitoid of fly pupae, was assayed for lethality in different stages of insects representing ten different orders and in cultured insect cells. In most cases, the motor activity of the injected insects remained completely normal for 1-2 days after the injection and displayed none of the symptoms of paralysis commonly reported for venoms of the Hymenoptera. A natural host, the flesh fly Sarcophaga bullata, was highly sensitive in the pupal stage (LD50 = 5.4 and 5.5 VRE/g for nondiapausing and diapausing pupae, respectively), the stage that is normally parasitized, and larvae and adults were as susceptible to the venom as the pupae. Adults of another fly host, Phaenicia sericata, were nearly as sensitive (LD50 = 6.5 VRE/g), but nonhost adult flies were more tolerant. Among the other orders tested, pupae of several species (Plodia interpunctella, Trichoplusia ni, Tenebrio molitor) were more susceptible to envenomation than larval or adult stages. In fact, the highest sensitivity observed in this study (LD50 = 0.58 VRE/g) was with pupae of the cabbage looper, T. ni, a species that is not a natural host. In contrast, the larvae (LD50 = 7.23 VRE/g) and adults (LD50 = 7.48) of T. ni were far less sensitive. Adults of Nasonia vitripennis were not sensitive to their own venom (LD50 = > 533 VRE/g), although adults of another hymenopteran, Apis mellifera, were suceptible (4.62 VRE/g). Adults of Lymantria dispar, Oncopeltus fasciatus, Aphis nerii, Euborellia annulipes, Diapheromera femorata, Blattella germanica, Periplaneta americana, and Reticulitermes flavipes demonstrated a high tolerance to Nasonia venom. When tested in vitro, the venom caused cultured Lepidoptera (TN-368) and Diptera (NIH SaPe4) cells to round up, swell, and eventually die. The LC50S were 0.0014 and 0.0010 VRE/microliters for TN-368 and SaPe4 cells, respectively. Cytotoxicity was observed within 10 min after exposure to LC99 levels of venom, with 100% cell mortality at 100 min for the NIH SaPe4 cells and 24 hr for TN-368 cells. It is possible that the venom component responsible for in vivo and in vitro activities may be different, but results from the cell culture work suggest that this method offers a promising assay for quickly screening venom samples. The high susceptibility of flies and pupae of other insects to the venom, as well as its novel (nonparalytic) action suggest that it may have considerable potential for development as a biopesticide.

Partial biochemical characterization of venom from the ant, Pseudomyrmex triplarinus.

Venom from the ant, Pseudomyrmex triplarinus, contains 12 proteins with mol. wts of > 100,000-4200, and they constitute 41.5% of the dry weight. In comparison with published data on ant, wasp, and bee venoms, whole venom has intense phospholipase activity and intermediate hemolytic activity. Four major proteins were isolated and purified by low pressure chromatography. The most abundant protein had a mol. wt of 4200 and weak hemolytic activity. The second most common protein was 20,400 and had phospholipase A2 activity. The other two major proteins had mol. wts of 24,500 and 14,100 and both exhibited phospholipase and direct hemolytic activities. There are eight minor proteins (> 100,000-40,000), each present at about 1% or less of the total protein. Assayed as a mixture, they had hyaluronidase activity. Seventeen free amino acids were detected with aspartic acid, glutamic acid, and proline together making up 72% of the total mass of amino acids. Glycerol was present at a concentration of 3.1% of the dry weight and the venom was devoid of lipids.

Protective effect of methylcellulose and other polymers on insect cells subjected to laminar shear stress.

The relative sensitivity of two insect cell lines to laminar shear stress was determined, and the protective effect of polymers added to the growth media of two insect cell lines, Trichoplusia ni (TN-368) and Spodoptera frugiperda (SF-9), was evaluated. TN-368 and SF-9 cells were found to be equally sensitive to laminar shear stress. Methylcellulose [0.5% (w/v) Dow E4M Methocel] and dextran [4.5% (w/v)] increased the resistance of suspended cells to lysis due to laminar shear stress by factors of up to 76 and 28, respectively, compared to cells in media without additives. It was observed that the protective effect of Pluronic F-68 was concentration-dependent: 0.2% and 0.3% (w/v) F-68 increased the resistance of SF-9 cells to shear stress by factors of 15 and 42, respectively. However, increasing the concentration to 0.5% did not significantly increase the cells' resistance compared to 0.3% (w/v). F-68 at 0.2% only increased the resistance of TN-368 cells by a factor of 6. It is believed that the protection is a result of the polymer adsorbing to the cell membrane. None of the polymer additives tested had a significant effect on SF-9 or TN-368 growth rate.

Expression of three recombinant proteins using baculovirus vectors in 23 insect cell lines.

Recombinant Autographa california baculoviruses expressing genes for pseudorabies virus glycoprotein (gp50T), human plasminogen (HPg), and beta-galactosidase (beta-gal) were used to infect 23 cell lines or strains. The objectives were to compare amounts of recombinant proteins expressed in the cell lines, compare yields from clones and parent lines, investigate the effects of long-term culture in serum-free medium on production, and determine if some lines yield gp50T with different glycosylation patterns. For HPg, IZD-MB0503 had the highest yield and four other lines (IPLB-TN-R2, IPLB-SF-1254, IPLB-LdEIta, and CM-1) had levels above that of SF-9 cells. For gp50T, four lines (IPLB-HvT1, IPLB-SF21AE, IPLB-SF21AE-15, and IPLB-SF-1254) had higher amounts than SF-9 cells. Some lines yielded gp50T with molecular mass about 1000 daltons larger than that from SF-9 cells, which suggests increased oligosaccharide processing. Equally high levels of beta-gal were expressed in three lines (SF-9, IZD-MB0503, and BCIRL-PX2-HNV3). The major conclusion is that no single cell line produced highest yields for all three recombinant proteins. Four lines were cultured in serum-free medium for 31-34 passages and then infected with the three recombinant viruses. For most cell line-recombinant combinations, the yields in serum-free medium were equal to or better than those in serum-supplemented medium. Medium composition had a much stronger effect on foreign gene expression than on susceptibility of cells to wild-type virus.

Effect of an experimental systemic compound, CGA-184699, on life stages of the cat flea (Siphonaptera: Pulicidae).

The experimental drug CGA-184699 (N-[2,5-dichloro-4-(1,1,2,3,3,3-hexafluoropropoxy)-phenylaminoc arbonyl]-2,6-difluorobenzamide) was evaluated for efficacy against cat fleas, Ctenocephalides felis (Bouché), held in flea cages on cats. When administered orally, the compound acts systemically and prevents development of the next generation of fleas. There was no effect on adults, but eggs from adults that fed on treated cats had reduced viability. Most larvae that emerged from surviving eggs died. Within the first 2 wk after treatment of cats with CGA-184699, most death of progeny occurred in the egg stage, but as time passed, more eggs hatched but larval mortality prevented development to adults. A single oral dose of CGA-184699 virtually eliminated the next generation of adult fleas for a period of 44 d.

Inhibition of human platelet aggregation and secretion by ant venom and a compound isolated from venom.

Venom from the tropical ant, Pseudomyrmex triplarinus, has activity against rheumatoid arthritis. Since platelets are involved in inflammatory responses, they were employed to study the effects of venom on prostaglandin-dependent human platelet aggregation and secretion. The assay is very sensitive and uses microliter volumes, which makes it useful as a screen during isolation and characterization of venom components. Whole venom inhibited arachidonic acid- and U46619-induced platelet aggregation with IC50s of 45 and 39 micrograms/ml, respectively. This suggested that venom prevented the action of prostaglandins. Pure venom was fractionated by gel filtration and at least three materials with antiplatelet activity were detected. The smallest component (factor F) was most active and was purified by additional molecular filtration and characterized by UV absorbance, thin-layer chromatography, nuclear magnetic resonance spectra, and activity to platelets. Factor F was identified as adenosine, which is known to stimulate platelet adenylate cyclase and has not been previously reported to be a component of insect venom.

Evaluation of a single oral dose of lufenuron to control flea infestations in dogs.

A single dose of lufenuron was administered to dogs to test its efficacy in controlling cat flea (Ctenocephalides felis) infestations for at least 30 days. Efficacy measurements revealed marked differences in the reproduction capability of fleas collected from dogs in the treatment vs the control group. Essentially, all of the eggs collected from dogs treated with lufenuron were unable to develop into normal adult fleas. Conversely, in the control group, 68.6% of the flea eggs developed into normal adult progeny.