Fingerprinting of cell lines by directed amplification of minisatellite-region DNA (DAMD)

The development of in vitro propagation of cells has been an extraordinary technical advance for several biological studies. The correct identification of the cell line used, however, is crucial, as a mistaken identity or the presence of another contaminating cell may lead to invalid and/or erroneous conclusions. We report here the application of a DNA fingerprinting procedure (directed amplification of minisatellite-region DNA), developed by Heath et al. [Nucleic Acids Research (1993) 21: 5782-5785], to the characterization of cell lines. Genomic DNA of cells in culture was extracted and amplified by PCR in the presence of VNTR core sequences, and the amplicons were separated by agarose gel electrophoresis. After image capture with a digital camera, the banding profiles obtained were analyzed using a software (AnaGel) specially developed for the storage and analysis of electrophoretic fingerprints. The fingerprints are useful for construction of a data base for identification of cell lines by comparison to reference profiles as well as comparison of similar lines from different sources and periodic follow-up of cells in culture

Crotoxin, the major toxin from the rattlesnake Crotalus durissus terrificus, inhibits 3H-choline uptake in guinea pig ileum

We examined the effect of crotoxin, the neurotoxic complex from the venom of the South American rattlesnake Crotalus durissus terrificus, on the uptake of 3H-choline in minces of smooth muscle myenteric plexus from guinea pig ileum. In the concentration range used (0.03-1 ÁM) and up to 10 min of treatment, crotoxin decreased 3H-choline uptake by 50-75 percent compared to control. This inhibition was time dependent and did not seem to be associated with the disruption of the neuronal membrane, because at least for the first 20 min of tissue exposure to the toxin (up to 1 ÁM) the levels of lactate dehydrogenase (LDH) released into the supernatant were similar to those of controls. Higher concentrations of crotoxin or more extensive incubation times with this toxin resulted in elevation of LDH activity detected in the assay supernatant. The inhibitory effect of crotoxin on 3H-choline uptake seems to be associated with its phospholipase activity since the equimolar substitution of Sr2+ for Ca2+ in the incubation medium or the modification of the toxin with p-bromophenacyl bromide substantially decreased this effect. Our results show that crotoxin inhibits 3H-choline uptake with high affinity (EC25 = 10 +/- 5 nM). We suggest that this inhibition could explain, at least in part, the blocking effect of crotoxin on neurotransmission

Binding sites and actions of Tx1, a neurotoxin from the venom of the spider Phoneutria nigriventer, in guinea pig ileum

Tx1, a neurotoxin isolated from the venom of the South American spider Phoneutria nigriventer, produces tail elevation, behavioral excitation and spastic paralysis of the hind limbs after intracerebroventricular injection in mice. Since Tx1 contracts isolated guinea pig ileum, we have investigated the effect of this toxin on acetylcholine release, as well as its binding to myenteric plexus-longitudinal muscle membranes from the guinea pig ileum. [125I]-Tx1 binds specifically and with high affinity (Kd = 0.36 + or - 0.02 nM) to a single, non-interacting (nH = 1.1), low capacity (Bmax 1.1 pmol/mg protein) binding site. In competition experiments using several compounds (including ion channel ligands), only PhTx2 and PhTx3 competed with [125I]-Tx1 for specific binding sites (K0.5 apparent = 7.50 x 10-4 g/l and 1.85 x 10-5 g/l, respectively). PhTx2 and PhTx3, fractions from P. nigriventer venom, contain toxins acting on sodium and calcium channels, respectively. However, the neurotoxin PhTx2-6, one of the isoforms found in the PhTx2 pool, did not affect [125I]-Tx1 binding. Tx1 reduced the [3H]-ACh release evoked by the PhTx2 pool by 33 per cent, but did not affect basal or KCl-induced [3H]-ACh release. Based on these results, as well as on the homology of Tx1 with toxins acting on calcium channels (w-Aga IA and IB) and its competition with [125I]-w-Cono GVIA in the central nervous system, we suggest that the target site for Tx1 may be calcium channels.

Induction of neutralizing antibodies in mice immunized with scorpion toxins detoxified by liposomal entrapment

The possibility of producing neutralizing antibodies against the lethal effects of scorpion toxins was evaluated in the mouse model by immunization with an immunogen devoid of toxicity. A toxic fraction (5 mg) from the venom of the scorpion Tityus serrulatus was entrapped in sphingomyelin-cholesterol liposomes. The liposomes were treated for 1 h at 37 degrees Celsius with a 1 per cent (w/w) trypsin solution in 0.2 M sodium carbonate buffer, pH 8.3. This treatment led to a strong reduction in venom toxicity. Immunization was performed as follows: mice were injected sc with 20 mug of the liposome-entrapped toxic fraction on days 1 and 21 and a final injection (20 mug) was administered ip on day 36. After injection of the immunogen, all mice developed an IgG response which was shown to be specific for the toxic antigen. The antibodies were measured 10 days after the end of the immunization protocol. In an in vitro neutralization assay we observed that pre-incubation of a lethal dose of the toxic fraction with immune serum strongly reduced its toxicity. In vivo protection assays showed that mice with anti-toxin antibodies could resist the challenge with the toxic fraction, which killed, 30 min after injection, all non-immune control mice.

Partial purification and pharmacological characterization of a neurotoxic fraction isolet from the venom of the spider Lycosa erythrognatha

The effect of the venom of the spider Lycosa erythrognatha on the frog sciatic nerve was investigated with the single sucrose-gap method. Solutions containing the crude venom (40 µg protein/ml) markedly increased the duration of compound action potentials and caused the appearance of long-lasting depolarizing post-potentials. These effects were only partially (20 percent) reversded by extensive washsing with control solution. The active material was sensitive to proteolytic treatments with pronase or trypsin and was separated with 20 percent acetonitrile and 0.1 percent trifluoroacetic acid by reverse phase chromatography. The effect of this fraction (LycIV) on the post-potential amplitude was concentration-dependent, and was fitted with a quadratic hyperbola having a half maximal effect of 0.9 µg protein/ml. SDS-polyacrylamide gel electrophoresis of LycIV showed an enriched polypeptide band with apparent molecular weight of ~8 kDa. The observed effects were similar to those of toxins that inhibit sodium channel inactivation and different from the effects of potassium channel blockers. Pore formation or membrane disruption could be ruled out. It was concluded that the venom contains a neurotoxic polypeptide that alters the repolarization of action potentials, probably by inhibiting sodium channel inactivation

Immunization of horses with Crotalus durissus Terrificus (South American Rattlesnake) venom: a comparision of four different procedures

A comparative study was carried out on horses immunized with Crotalus durissus terrificus venom using four different inoculation procedures, which included the use Freund's adjuvant, A1(OH)3 and liposomes as adjuvants. The antibody titer was assessed by enzyme linked immunosorbent assay (ELISA) and the neutralizing potency by the neutralizing median effective dose (ED50). The inoculation schedule used in horses to obtain antivenom serum consisted of scinjections of a 7.5 mg venom starting dose in 5.0ml sterile saline emulsified with an equal volume sterile saline at 2-dayintervals. This immunization procedure, based in low doses of antigen (37.5mg/horse) emulsified with Freund's adjuvant, proceduced a more protective andsustained immune response whencompared with other procedures using A1(OH)3 and 5.0 mg/horse in liposome) or high (870.0 mg/horse in A1(OH)3 and 20.0 mg/horse in liposome) antigen doses. The ED50 values evaluated at the end of the procedure were 15.4 *l serum/20 gmouse when antigen was emulsified with Freund's adjuvant; 21.7 * serum/20 g mouse when 870,0 mg antigen/horse was emulsified with A1(OH)3 and 30.0 *l serum/20 g mouse when 50.0 mg antigen/hors was emulsified with a1(OH)3.When antigen was emulsified with liposome, the immune serum was ineffective against the lethal effects of C.d terrificus venom. The inoculation schedule used in horses to obtain hyperimmune serum consisted of reimmunization with sc booster injections of 7.5 mg venom in 5.0 ml sterile saline emulsified with an equal volume of Freund's incomplete adjuvant. One week later, 2.5 mg venom in 12.0 ml sterile saline was inoculated at 2-day intervals.This reimmunization schedule,based on low doses of antigen (15.0 mg/horse) emulsified with Freund's incomplete adjuvant or with saline, produced a protective andsustained immune response, regardless of the initial immunization procedure. The ED50 evaluatedfor each of the animals five days after the reimmunization period was never more than 20 * serum/20 g mouse. The liposome inoculation method employed a membrane-stabilized reverse phase evaporation preparation of sphingomyelin/cholesterol 2.5/1 (w/w) liposomes. This procedure permits incorporation of 1.0 mg protein per mg of phospholipid. This liposome inoculation method , which stimulates a rapid, sustained and protective immune response in mice and rabbits inoculated with both C.d. collineatus and C.d. terrificus, was not effective when horses were immunized with C.

Neutralization by homologous plasma of Crotalus durissus terrificus (South American rattlesnake) venom and crotoxin

A resistência de certos mamíferos à açäo tóxica de venenos de serpentes é bem conhecida, näo só na literatura especializada mas também popularmente. Essa resistência estende-se também a algumas serpentes venenosas e näo venenosas. O mecanismo responsável pelo fenômeno näo é único em todos os casos mas, em alguns, deve-se à presença de fatores antitóxicos no sangue circulante desses animais. Neste trabalho mostramos que o plasma de casvavel é capaz de neutralizar o efeito letal do veneno crotálico e de seu principal componente tóxico (crotoxina), em camundongos. O plasma crotálico inibe também a atividade fosfolipásica A2 da crotoxina in vitro, geralmente associada à sua toxicidade. A açäo neutralizante do plasma crotálico está associada á sua fraçäo alfa1-globulina, provavelmente devido à presença de um fator anti-tóxico na sua composiçäo