Exploring the proteomes of the venoms of the Peruvian pit vipers Bothrops atrox, B. barnetti and B. pictus

We report the comparative proteomic characterization of the venoms of Bothrops atrox, B. barnetti and B. pictus. The venoms were subjected to RP-HPLC and the resulting fractions analyzed by SDS-PAGE. The proteins were cut from the gels, digested with trypsin and identified via peptide mass fingerprint and manual sequencing of selected peptides by MALDI-TOF/TOF mass spectrometry. Around 20-25 proteins were identified belonging to only 6-7 protein families. Metalloproteinases of the classes P-I and P-III were the most abundant proteins in all venoms (58-74% based on peak area A214 nm), followed by phospholipases-A(2) (6.4-14%), disintegrins (3.2-9%) and serine proteinases (7-11%), and some of these proteins occurred in several isoforms. In contrast cysteine-rich secretory proteins and L-amino acid oxidases appeared only as single isoforms and were found only in B. atrox and B. barnetti. C-type lectins were also detected in all venoms but at low levels (~ 5%). Furthermore, the venoms contain variable numbers of peptides (<3 kDa) and non-protein compounds which were not considered in this work. The protein composition of the investigated Bothrops species is in agreement with their pharmacological and pathological effects.     

Hemolytic activity of venoms from snakes of the genera Bothrop, Lachesis, Crotalus, and Micrurus (Serpentes: Viperidae and Elapidae]

Hemolytic activity of eight Peruvian snake venoms from the families Viperidae and Elapidae (Bothrops atrox, B. pictus, B. hyoprorus, B. bilineatus, B. neuwedii, Lachesis m. muta, Crotalus d. terrificus, Micrurus tschudi), and three Brazilian viperids (B. jararacussu, B. alternatus and C. d. collilineatus) is described. None of the venoms caused direct lysis on washed human erythrocytes. However, all of them caused indirect hemolysis provided that the incubation medium contains an exogenous source of lecithin. Venom of Micrurus tschudi was the most hemolytic (HD50 2.8 ug/ml) while that of B. bilineatus was the least (HD50 681.3 ug/ml). Only six of eleven venoms showed parallel curves of hemolytic activity, and the HD50 varied from 198 to 681 ug/ml and the following decreasing order of hemolytic activity was obtained: L. muta, C. d. terrificus, C. d. collilineatus, B. hyoprorus, B. bilineatus, B. alternatus.     

Stimulation of sugar uptake in cultured fibroblasts by epidermal growth factor (EGF) and EGF-binding arginine esterase.

Mouse epidermal growth factor causes a rapid increase in 2-deoxyglucose uptake in stationary phase mouse (3T3) cells or human fibroblasts. Maximum effect is approximately two fold over control levels for 3T3 cells and about 50% over controls for human fibroblasts. Maximum effect on 3T3 cells is seen about two hours after addition of 10 ng/ml EGF to the culture medium. Stimulation is easily measureable within the first fifteen minutes after addition of the hormone and may be detected at hormone concentrations as low as 0.1 ng/ml. The EGF-binding arginine esterase found associated with EGF in the mouse submaxillary gland causes an enhancement of the EGF effect. In serum-free medium, the EGF effect is still readily observed, but no enhancement by the esterase is seen. SV40 virus-transformed 3T3 cells show no effect on deoxyglucose uptake after addition of 10 ng/ml EGF to the culture medium, but a response may be demonstrated after these cells are incubated for 12 hours or more in serumless medium. EFG stimulates transport of 3-O-methylglucose in stationary phase 3T3 and human fibroblasts but no EGF stimulation of alpha-amino-isobutyrate uptake in 3T3 cells is seen under conditions is reproted to inhibit intracellular degradation of human EGF by human fibroblasts, does not diminish the EGF effect on deoxyglucose uptake in human fibroblasts.     

Snake population venomics and antivenomics of Bothrops atrox: Paedomorphism along its transamazonian dispersal and implications of geographic venom variability on snakebite management

We describe two geographically differentiated venom phenotypes across the wide distribution range of Bothrops atrox, from the Colombian Magdalena Medio Valley through Puerto Ayacucho and El Paují, in the Venezuelan States of Amazonas and Orinoquia, respectively, and S?o Bento in the Brazilian State of Maranh?o. Colombian and Venezuelan venoms show an ontogenetic toxin profile phenotype whereas Brazilian venoms exhibit paedomorphic phenotypes. Venoms from each of the 16 localities sampled contain both population-specific toxins and proteins shared by neighboring B. atrox populations. Mapping the molecular similarity between conspecific populations onto a physical map of B. atrox range provides clues for tracing dispersal routes that account for the current biogeographic distribution of the species. The proteomic pattern is consistent with a model of southeast and southwest dispersal and allopatric fragmentation northern of the Amazon Basin, and trans-Amazonian expansion through the Andean Corridor and across the Amazon river between Monte Alegre and Santarém. An antivenomic approach applied to assess the efficacy towards B. atrox venoms of two antivenoms raised in Costa Rica and Brazil using Bothrops venoms different than B. atrox in the immunization mixtures showed that both antivenoms immunodepleted very efficiently the major toxins (PIII-SVMPs, serine proteinases, CRISP, LAO) of paedomorphic venoms from Puerto Ayacucho (Venezuelan Amazonia) through S?o Bento, but had impaired reactivity towards PLA(2) and P-I SVMP molecules abundantly present in ontogenetic venoms. The degree of immunodepletion achieved suggests that each of these antivenoms may be effective against envenomations by paedomorphic, and some ontogenetic, B. atrox venoms.     

Reproductive studies of Brahman cattle IV. Luteinizing hormone levels in ovariectomized Brahman, Brahman × Hereford and Hereford cows following a 20 mg dose of Estradiol-17β

Luteinizing Hormone (LH) levels were quantitated by radioimmunoassay (RIA) in six mature, long-term ovariectomized cows each of Brahman (B), Brahman × Hereford (B×H) and Hereford (H) breeding following an in-tramuscular injection of 20 mg of Estradiol-17_β (E) suspended in corn oil. After E administration all cows were bled via coccygeal venipuncture every two hours from 0–8 hours post-injection, every hour from 9–24 hours post-injection, concluding with bleedings every two hours from 26–36 hours post-injection. An LH surge was observed in 5/6 B cows, 6/6 B×H cows and 6/6 H cows. Basal LH levels (mean of first eight data points of each breed type) did not differ (P>.10) between B (3.5 ng/ml), B×H (2.4 ng/ml) and H (2.4 ng/ml). Elapsed time from E injection to peak LH value varied significantly (P<.05) between B, B×H and H, respectively (27.8 hrs, 23.8 hrs, 22.2 hrs). Peak LH values also varied between breed (B, 20.2 ng/ml; B×H, 36.0 ng/ml; H, 113.2 ng/ml: P<.005). The area under the LH curve differed significantly between B, B×H and H (P<.05), however, the duration of the LH surge was not different between breeds; B (13.2 hrs), B×H (16.2 hrs) and H (15.3 hrs). Overall significant period effects (P<.05), breed effects (P<.10) and period × breed interactions (P<.05) were found. In summary, B are less reactive to a 20 mg dose of E than are B×H or H using the following criteria: time to peak LH value, peak LH value and area under the LH curve. These data strongly indicate inherent differences between breeds regarding estrogen feedback mechanisms at the hypophysial-hypothalamic axis.     

Lipid monolayers: action of phospholipase A of Crotalus atrox and Naja naja venoms on phosphatidyl choline and phosphatidal choline

The activity of phospholipase A on phosphatidyl choline and phosphatidal choline spread as monolayers on phosphate buffers containing snake venom (Crotalus atrox or Naja naja) was studied by measuring the fall of surface potential as a function of time, pH, film pressure, temperature, and concentrations of phosphate and venom. At 25 degrees C, pH 7.0, and 0.2 micrograms of venom per ml, optimal activity was observed with both venoms on both substrates at 12 dynes/cm film pressure on 0.04 m phosphate. Under these conditions, the pH optimum for C. atrox was broad (6.6-7.4) and that for N. naja was sharp (8.0) for the action on phosphatidyl choline, whereas both venoms had a sharp optimum at pH 8.0 in their action on phosphatidal choline. The optimal temperature with phosphatidyl choline was 27.5 degrees C for N. naja and 40 degrees C for C. atrox. In line with studies of phospholipase A activity in bulk phase in ether, phosphatidal choline was attacked much more slowly than phosphatidyl choline by C. atrox. Under conditions where both venoms had equal activity on phosphatidyl choline, C. atrox was only half as active as N. naja on phosphatidal choline. The studies suggest that the linkage of the hydrophobic chains in glycerophosphatides may affect their interaction with proteins.     

Immunostimulating effects of muramyl dipeptide, glucosaminyl muramyl dipeptide and their synthetic derivatives in vitro

The effect of muramyldipeptide (MDP), glucosaminylmuramyldipeptide (GMDP) and their six synthetic derivatives on production of tumor necrosis factor (TNF), interleukin-1 (IL-1) and interleukin-2 (IL-2) by murine spleen cells in vitro was studied. MDP induced insignificant TNF production and did not stimulate production of IL-1 by the murine splenocytes within a 24-hour cultivation period whereas in combination with lipopolysaccharide (LPS) it induced significant production of both the cytokins. GMDP induced marked production of TNF (54 per cent cytotoxic index) and IL-1 (stimulation index 8). Addition of LPS in an amount of 10 ng/ml increased production of TNF by the murine splenocytes under the effect of GMDP but had no effect on production of IL-1. Neither MDP nor GMDP even in combination with LPS induced production of IL-2 by splenocytes of mice DVA/2 and C57B1/6 at activation for 24 hours. All the synthetic derivatives of MDP and GMDP except the MDP polymer activated TNF production by the murine spleen cells. GMDP lysine had the highest effect: 67 per cent cytotoxic index. In combination with LPS its cytotoxic index amounted to 87 per cent. The TNF activity was always higher when LPS in an amount of 10 ng/ml was added to the glycopeptides.     

Mycotoxins produced from fungi isolated from foodstuffs and soil: comparison of toxicity in fibroblasts and rat feeding tests.

Thirty-nine isolates of fungi obtained from foodstuffs and soil samples from various parts of the world have been identified. The isolates were grown on a solid rice medium, and extracts were prepared with 50% aqueous methanol. The extracts were examined for toxicity in the following systems: (i) cytotoxicity to cultured normal human diploid skin fibroblasts (proliferating and nonproliferating) and mouse fibroblasts; (ii) skin toxicity after topical application on rats; and (iii) rat feeding tests in which rats were examined for death, overt pathological effects including congestion and hemorrhage of tissues, weight loss, food refusal, and uterine growth. Sixteen culture extracts were highly toxic as indicated by death, congestion and hemorrhage of tissues, and net weight loss. One half of the isolates were highly cytotoxic (50% lethal concentration, 0.01 to 5 micrograms/ml) as indicated by the ability to cause death and disintegration of 3T3 Swiss mouse fibroblasts and human diploid skin fibroblasts during 3 to 4 days in culture. The remainder were moderately cytotoxic (50% lethal concentration, 5 to 250 micrograms/ml). Four culture extracts were highly toxic by some clinical criteria but did not cause congestion and hemorrhage of tissues and were weakly cytotoxic (50% lethal concentration, 250 to 5,000 micrograms/ml). Six culture extracts exhibited moderate toxicity (weight loss only) and low cytotoxicity (50% lethal concentration, 3,000 to 50,000 micrograms/ml). Four culture extracts caused uterine enlargement as the major clinical sign, suggesting the presence of zearalenone. Eleven culture extracts were weakly cytotoxic and caused no major clinical signs, except skin toxicity in two extracts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Insulin and epidermal growth factor stimulate glycolysis in quiescent 3T3 fibroblasts with no changes in key glycolytic enzyme activities

In order to study the effect of insulin and epidermal growth factor (EGF) on glycolysis in quiescent 3T3 fibroblasts and their mechanisms of action, lactic acid produced by cells and activities of key glycolytic enzymes in cell extracts were determined. Insulin increased lactic acid production; the maximal stimulation occurred at the concentrations above 250 ng/ml and the half-maximal dose was 50 ng/ml. This effect of insulin appeared as early as one hour, and lactic acid production in the presence of insulin linearly increased up to 4 h. The 24-h pretreatment with insulin exhibited no significant effect on the production by cells afterward incubated either with or without insulin. Lactic acid production decreased as the concentration of phloridzin increased. However, insulin stimulation of the production still remained in the presence of phloridzin. Parahydroxymercuribenzoate reduced production only by the equivalent of the increase due to insulin. EGF also increased lactic acid production; this effect occurred at 1 ng/ml and was maximal at 100 ng/ml. The activities of hexokinase, phosphofructokinase and pyruvate kinase in quiescent cells were not increased by insulin, and the affinities for substrates of these enzymes remained unaltered. These findings suggest that glucose uptake is a rate-limiting step in glycolysis in quiescent 3T3 fibroblasts and that the stimulatory effect of insulin on glycolysis is mediated by enhanced glucose entry.     

Retinyl acetate inhibits platelet-derived growth factor-induced Ca2+ signals in C3H 10T1/2 fibroblasts

Mitogenic stimulation of density-arrested C3H 10T1/2 mouse fibroblasts by serum or purified platelet-derived growth factor (PDGF) was potently inhibited by retinyl acetate (RAc; IC50 = 0.1 microgram/ml, 0.3 x 10(-6) M) when administered during the first 2 hours of mitogen exposure. This inhibitory effect of RAc coincided with a period early in the cell growth-division cycle when density-arrested C3H 10T1/2 cells stimulated by PDGF were found to require physiological levels of extracellular Ca2+ for the transition from G0 to G1 of the cell cycle. To determine if the inhibitory effect of RAc was mediated through alterations in the Ca2+ signaling pathway induced by mitogens, we examined Fura-2-loaded fibroblasts for changes in the Ca2+ response elicited by PDGF. Addition of PDGF (5 ng/ml) induced a transient increase in the [Ca2+]i that was not significantly effected by the extracellular Ca2+ concentration. Treatment of cells with RAc caused a concentration- and time-dependent inhibition of this PDGF-stimulated Ca2+ flux (IC50 = 0.45 microgram/ml or 1.5 x 10(-6) M; t1/2 = 15 min), whereas release of intracellularly stored Ca2+ by thrombin was unaffected by RAc (1.2 micrograms/ml, 4 x 10(-6) M). Treatment with RAc did not significantly affect PDGF binding to cell surface receptors or the generation of inositol phosphates. These results suggest that the mechanism by which RAc inhibits PDGF- or serum-induced mitogenesis is through modulation of the Ca2+ signal stimulated by PDGF, and thereby depriving the cell of a rise in intracellular Ca2+ necessary for progression through the cell cycle.     

Myotoxic phospholipases A(2) in bothrops snake venoms: effect of chemical modifications on the enzymatic and pharmacological properties of bothropstoxins from Bothrops jararacussu

Venoms from eight Bothrops spp. were fractionated by ion-exchange chromatography on CM-Sepharose at pH 8.0 for the purification of myotoxins. Chromatographic profiles showed differences regarding myotoxic components among these venoms. B. alternatus, B. atrox and B. jararaca venoms did not show the major basic myotoxic fractions identified in the other venoms. Polyacrylamide gel electrophoresis for basic proteins also showed distinct patterns for these toxins. In vivo, all the isolated myotoxins induced release of creatine kinase due to necrosis of muscle fibers, accompanied by polymorphonuclear cell infiltration, and edema in the mouse paw. In addition, the toxins showed cytotoxic and liposome-disrupting activities in vitro. B. jararacussu bothropstoxins-I (BthTX-I) and II (BthTX-II) were submitted to chemical modifications of: His, by 4-bromophenacyl bromide (BPB) or photooxidation by Rose Bengal (RB); Tyr, by 2-nitrobenzenesulphonyl fluoride (NBSF); and Trp, by o-nitrophenylsulphenyl chloride (NPSC). The myotoxic and cytotoxic activities of BthTX-I, a Lys49 PLA(2) homologue, after modification by BPB, RB, NBSF and NPSC, were reduced to 50%, 20%, 75%, 65% and 13%, 0.5%, 76%, 58%, respectively. However, the edema-inducing and liposome-disrupting activities were not significantly reduced by the above modifications. BPB-treated BthTX-II, an Asp49 PLA(2) homologue, lost most of its catalytic, indirect hemolytic, anticoagulant, myotoxic and cytotoxic activities. The edema-inducing and liposome-disrupting activities were reduced to 50% and 80%, respectively. Lethality caused by BthTX-I and -II was strongly reduced after treatment with BPB or RB, but only partially with NBSF or NPSC. BthTX-I and -II, both native or modified, migrated similarly in a charge-shift electrophoresis. Antibodies raised against BthTX-I or -II, B. asper Basp-II and the C-terminal 115-129 peptide from Basp-II did not show significant differences in their cross-reactivity with the modified toxins, except with RB photooxidized toxins.     

Mycotoxins produced from fungi isolated from foodstuffs and soil: comparison of toxicity in fibroblasts and rat feeding tests

Thirty-nine isolates of fungi obtained from foodstuffs and soil samples from various parts of the world have been identified. The isolates were grown on a solid rice medium, and extracts were prepared with 50% aqueous methanol. The extracts were examined for toxicity in the following systems: (i) cytotoxicity to cultured normal human diploid skin fibroblasts (proliferating and nonproliferating) and mouse fibroblasts; (ii) skin toxicity after topical application on rats; and (iii) rat feeding tests in which rats were examined for death, overt pathological effects including congestion and hemorrhage of tissues, weight loss, food refusal, and uterine growth. Sixteen culture extracts were highly toxic as indicated by death, congestion and hemorrhage of tissues, and net weight loss. One half of the isolates were highly cytotoxic (50% lethal concentration, 0.01 to 5 micrograms/ml) as indicated by the ability to cause death and disintegration of 3T3 Swiss mouse fibroblasts and human diploid skin fibroblasts during 3 to 4 days in culture. The remainder were moderately cytotoxic (50% lethal concentration, 5 to 250 micrograms/ml). Four culture extracts were highly toxic by some clinical criteria but did not cause congestion and hemorrhage of tissues and were weakly cytotoxic (50% lethal concentration, 250 to 5,000 micrograms/ml). Six culture extracts exhibited moderate toxicity (weight loss only) and low cytotoxicity (50% lethal concentration, 3,000 to 50,000 micrograms/ml). Four culture extracts caused uterine enlargement as the major clinical sign, suggesting the presence of zearalenone. Eleven culture extracts were weakly cytotoxic and caused no major clinical signs, except skin toxicity in two extracts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Target-sensitive immunoliposomes as an efficient drug carrier for antiviral activity

We have shown previously that target-sensitive immunoliposomes composed of palmitoyl antibody stabilized phosphatidylethanolamine bilayers could be destabilized by binding to the target cells (Ho, R. J. Y., Rouse, B. T., and Huang, L., Biochemistry (1986) 25, 5500-5506). Target-sensitive immunoliposome-encapsulated and free cytotoxic drugs of nucleoside analogs cytosine-beta-D-arabinoside (AraC) or acycloguanosine (acyclovir, ACV) were compared for their antiviral efficacy and cell cytotoxicity. Target-insensitive immunoliposomes and nontargeted liposomes were also investigated. When the mouse fibroblast L929 cells were infected at low multiplicity with herpes simplex virus, AraC encapsulated in target-sensitive immunoliposomes composed of transphosphatidylated egg phosphatidylethanolamine effectively inhibited virus replication and had far less cell cytotoxicity than free drug. As a measure of cytotoxicity, the drug concentration required to inhibit 50% of [3H]thymidine incorporation from 6 to 42 h (CD50) was determined. For free AraC, this value was 0.3 ng/ml, whereas for target-sensitive immunoliposome-encapsulated AraC, the CD50 exceeded 1 microgram/ml. However, target-sensitive immunoliposome-encapsulated AraC was virus inhibitory (50% effective dose = ED50) at 1.8 ng/ml. A free drug concentration of at least 1000-fold greater was required for comparable antiviral activity. A similar phenomenon was observed when ACV was administered via target-sensitive immunoliposomes. The CD50 values of the free and target-sensitive immunoliposome-encapsulated ACV were 12.5 ng/ml and 1.4 micrograms/ml, respectively, whereas the ED50 values of the free and target-sensitive immunoliposome-encapsulated ACV were 1.1 and 125 ng/ml, respectively. Consequently, our results indicated the superiority of target-sensitive immunoliposomes at drug delivery, especially when drugs were cytotoxic to cells. The use of liposomes of the target-insensitive variety provided some enhancement of activity, but this was several-fold less than that observed with target-sensitive immunoliposomes. In addition, the nucleoside transport inhibitors, p-nitrothiobenzylinosine and dipyridamole, were shown to inhibit the liposome-mediated antiviral activity of AraC. This finding indicated that site-specific cytosolic delivery of nucleoside analogs by target-sensitive immunoliposomes involved a cellular nucleoside transport system. A mechanism of action is proposed.     

Luteolytic activity of a synthetic prostaglandin and PGF2α in heifers

Two experiments involving 44 cycling heifers were conducted to evaluate the luteolytic activity of a synthetic prostaglandin, AY 24366, and PGF_2α. Activity was assessed by the decline in progesterone level of peripheral blood and occurrence of estrus. Progesterone concentrations of jugular blood plasma were quantified by radioimmunoassay. In the first experiment, 36 heifers were treated during diestrus with AY 24366 (A - 10mg intrauterine, B - 30mg intramuscular and C - 60mg im) or with PGF_2α (D - 5mg, iu, E - 15mg im and F - 30mg im). Mean progesterone 0, 24 and 48 hours after treatment were A - 6.33, 5.55 and 5.06; B - 6.35, 2.79 and 3.92; C - 5.23, 2.69 and 3.91; D - 5.19, 1.50 and 1.51; E - 4.69, 0.85 and 0.61; F - 6.66, 0.80 and 0.48 ng/ml. Standing estrus was observed in 1, 1, 1, 4, 5 and 6 females in groups A, B, C, D, E and F respectively within 72 hours of treatment. PGF_2α resulted in significantly (P<0.01) lower progesterone at 24 and 48 hours than AY 24366. However, im administration of the latter did significantly (P<0.05) lower progesterone at 24 hours. In the second trial six heifers were treated with either 120 or 180mg of AY 24366 im on day 12 of the cycle. Mean progesterone declined from 3.84 to 2.12 ng/ml (P<0.01) by 6 hours and to 1.59 ng/ml by 12 hours. Thereafter the decline was gradual and reached a level of 0.65 ng/ml at 72 hours. All six heifers showed standing estrus at 78±2 hours and were inseminated. Two in each group conceived. Doses of 15mg PGF_2α and 120mg AY 24366 were effective in causing luteal regression, however, the latter caused respiratory discomfort for 5 to 10 minutes post treatment.     

Luteolytic activity of a synthetic prostaglandin and PGF2alpha in heifers.

Two experiments involving 44 cycling heifers were conducted to evaluate the luteolytic activity of a synthetic prostaglandin, AY 24366, and PGF2alpha. Activity was assessed by the decline in progesterone level of peripheral blood and occurrence of estrus. Progesterone concentrations of jugular blood plasma were quantified by radioimmunoassay. In the first experiment, 36 heifers were treated during diestrus with AY 24366 (A-10mg intrauterine, B-30mg intramuscular and C-60mg im) or with PGF2alpha (D-5mg iu, E-15mg im and F-30mg im). Mean progesterone 0, 24 and 48 hours after treatment were A-6.33, 5.55 and 5.06; B-6.35, 2.79 and 3.92; C-5.23, 2.69 and 3.91; D-5.19, 1.50 and 1.51; E-4.69, 0.85 and 0.61; F-6.66, 0.80 and 0.48 ng/ml. Standing estrus was observed in 1, 1, 1, 4, 5 and 6 females in groups A, B, C, D, E and F respectively within 72 hours of treatment. PGF2alpha resulted in significantly (P less than 0.01) lower progesterone at 24 and 48 hours than AY 24366. However, in administration of the latter did significantly (P less than 0.05) lower progesterone at 24 hours. In the second trial six heifers were treated with either 120 or 180mg of AY 24366 im on day 12 of the cycle. Mean progesterone declined from 3.84 to 2.12 ng/ml (P less than 0.01) by 6 hours and to 1.59 ng/ml by 12 hours. Thereafter the decline was gradual and reached a level of 0.65 ng/ml at 72 hours. All six heifers showed standing estrus at 78 +/-2 hours and were inseminated. Two in each group conceived. Doses of 15mg PGF2alpha and 120mg AY 24366 were effective in causing luteal regression, however, the latter caused respiratory discomfort for 5 to 10 minutes post treatment.     

Snake venoms promote stress-induced senescence in human fibroblasts

Snake venoms are widely studied in terms of their systemic toxicity and proteolytic, hemotoxic, neurotoxic, and cytotoxic activities. However, little is known about snake-venom-mediated effects when used at low, noncytotoxic concentrations. In the current study, two human fibroblast cell lines of different origin, namely WI-38 fetal lung fibroblasts and BJ foreskin fibroblasts were used to investigate snake-venom-induced adaptive response at a relatively noncytotoxic concentration (0.01 µg/ml). The venoms of Indochinese spitting cobra ( Naja siamensis), western green mamba ( Dendroaspis viridis), forest cobra ( Naja melanoleuca), and southern copperhead ( Agkistrodon contortrix) were considered. Snake venoms promoted FOXO3a-mediated oxidative stress response and to a lesser extent DNA damage response, which lead to changes in cell cycle regulators both at messenger RNA and protein levels, limited cell proliferation and migration, and induced cellular senescence. Taken together, we have shown for the first time that selected snake venoms may also exert adverse effects when used at relatively noncytotoxic concentrations.     

Developmental patterns of plasma dehydroepiandrosterone sulfate and testosterone in male pigs

The relationship between plasma levels of dehydroepiandrosterone sulfate (DHAS) and testosterone (T) was determined by radioimmunoassays in growing and adult pigs. Seven young males were bled at 2-weekly intervals between 1 and 47 weeks of age and two adult boars were cannulated for short-term studies. Plasma samples were extracted with methylene chloride and T was isolated by Celite chromatography. DHAS was assayed directly in the aqueous phase.Dehydroepiandrosterone occurred predominantly (89.7 ± 10.6%) as the sulfoconjugate in boar plasma (n = 50). Plasma DHAS was undetectable in castrated males (n = 2). At 1 week of age, mean levels (± S.D.) of DHAS and T were 5.0 ± 3.0 ng/ml and 0.15 ± 0.10 ng/ml, respectively; and they rose to small peaks of 16.0 ± 2.0 ng/ml and 0.63 ± 0.10 ng/ml at 3 weeks. At 7 weeks, the levels of DHAS and T increased gradually from 10.0 ± 6.7 and 0.11 ± 0.10 ng/ml to 27.0 ± 6.6 and 1.84 ± 0.61 ng/ml at 19 weeks. There followed a marked increase to 4.90 ± 3.30 ng/ml at 21 weeks for T and a less abrupt rise to 44.0 ± 9.3 ng/ml at 23 weeks for DHAS. The mean levels remained high from then onwards, fluctuating between 24.0 ± 8.7 and 54.5 ± 5.0 ng/ml for DHAS and between 1.73 ± 0.86 and 4.43 ± 1.26 ng/ml for T. Episodic fluctuations were noted in two boars during hourly collection for 24 h, with mean levels of 9.0 ± 4.9 and 50.0 ± 10.4 ng/ml for DHAS, and 1.76 ± 0.83 and 3.26 ± 0.63 ng/ml for T, respectively.For all ages of males, plasma DHAS and T levels were highly correlated (r = 0.95) with greater concentrations of DHAS in all samples. Although individual differences in steroid profiles were noted, concentrations for DHAS and T showed almost parallel increases at puberty and corresponding fluctuations in adult boars. It is suggested that plasma DHAS determinations provide a simple, sensitive assessment of androgen production in the male pig.     

Characterization of the snake venoms from seven Brazilian species of Bothrops by FPLC anion-exchange chromatography.

1. The elution profiles and the caseinolytic, myotoxic, coagulant and hemorrhagic activities of the venoms of seven Bothrops species fractionated on a Mono-Q FPLC column were analyzed. 2. Each venom separated into 16-20 peaks, with good reproducibility and the activities were concentrated in virtually discrete regions of the chromatogram. 3. There is a considerable overlap of active proteins in the different species venoms and our results indicate that a venom pool with the species B. jararaca, B. jararacussu, B. moojeni, B. neuwiedi and B. atrox venoms would contain the major active proteins determined in the seven species.     

A Mycobacterium ulcerans toxin,mycolactone, induces apoptosis in primary human keratinocytes and in HaCaT cells

The pathogenicity of Mycobacterium ulcerans (Buruli ulcer) depends on cytotoxic effect of its exotoxin mycolactone. Since epidermis represents a barrier against infectious agents and balanced apoptosis is essential in epidermal homeostasis, we explored if mycolactone A/B induces apoptosis on two human keratinocyte populations, stem cells (KSC) and transit amplifying cells (TAC), and on human keratinocyte line, HaCaT. Treatment of TAC with 1 and 10 ng/ml mycolactone-induced 60 and 90% apoptosis. KSC were more resistant than TAC: 50 and 75% of cells underwent apoptosis after 10 and 100 ng/ml toxin-treatment. Higher doses (1000 ng/ml) induced about 30% apoptosis on HaCaT. In contrast, mycolactone A/B was devoid of toxicity neither on human hepatoma HuH7 nor on human embryonic kidney HEK 293 T cell lines. In conclusion, mycolactone induces apoptosis in human keratinocytes, thus contributing to Buruli ulcer lesions development.