Genomic insights into the Ixodes scapularis tick vector of Lyme disease.

Ticks transmit more pathogens to humans and animals than any other arthropod. We describe the 2.1 Gbp nuclear genome of the tick, Ixodes scapularis (Say), which vectors pathogens that cause Lyme disease, human granulocytic anaplasmosis, babesiosis and other diseases. The large genome reflects accumulation of repetitive DNA, new lineages of retro-transposons, and gene architecture patterns resembling ancient metazoans rather than pancrustaceans. Annotation of scaffolds representing ∼57% of the genome, reveals 20,486 protein-coding genes and expansions of gene families associated with tick-host interactions. We report insights from genome analyses into parasitic processes unique to ticks, including host 'questing', prolonged feeding, cuticle synthesis, blood meal concentration, novel methods of haemoglobin digestion, haem detoxification, vitellogenesis and prolonged off-host survival. We identify proteins associated with the agent of human granulocytic anaplasmosis, an emerging disease, and the encephalitis-causing Langat virus, and a population structure correlated to life-history traits and transmission of the Lyme disease agent.

No Accumulation of Transposable Elements in Asexual Arthropods.

Transposable elements (TEs) and other repetitive DNA can accumulate in the absence of recombination, a process contributing to the degeneration of Y-chromosomes and other nonrecombining genome portions. A similar accumulation of repetitive DNA is expected for asexually reproducing species, given their entire genome is effectively nonrecombining. We tested this expectation by comparing the whole-genome TE loads of five asexual arthropod lineages and their sexual relatives, including asexual and sexual lineages of crustaceans (Daphnia water fleas), insects (Leptopilina wasps), and mites (Oribatida). Surprisingly, there was no evidence for increased TE load in genomes of asexual as compared to sexual lineages, neither for all classes of repetitive elements combined nor for specific TE families. Our study therefore suggests that nonrecombining genomes do not accumulate TEs like nonrecombining genomic regions of sexual lineages. Even if a slight but undetected increase of TEs were caused by asexual reproduction, it appears to be negligible compared to variance between species caused by processes unrelated to reproductive mode. It remains to be determined if molecular mechanisms underlying genome regulation in asexuals hamper TE activity. Alternatively, the differences in TE dynamics between nonrecombining genomes in asexual lineages versus nonrecombining genome portions in sexual species might stem from selection for benign TEs in asexual lineages because of the lack of genetic conflict between TEs and their hosts and/or because asexual lineages may only arise from sexual ancestors with particularly low TE loads.

Targeted ablation of gonadotropin dependent endocrine tumors in transgenic mice through their luteinizing hormone receptor (LHR)

Gonadal somatic cell and adrenocortical endocrine tumors are rare. The incidence of adrenocortical carcinomas is only 1-2/1000000 a year. However, they are aggressive, especially in adulthood and currently surgery is the only curative treatment. Cytotoxic agents are in use in advanced cancers, but side effects and multidrug resistance are often problems. Thus there is a need for novel curative treatment methods. In contrast, ovarian granulosa cell tumors and testicular Leydig cell tumors are usually benign, especially at a younger age. The aim of the present thesis was to study a novel targeted treatment method through luteinizing hormone/chorionic gonadotropin receptor (LHCGR) in a transgenic mouse tumor model. The cytotoxic agent was lytic peptide Hecate-CGbeta conjugate where 23 amino acid Hecate, a synthetic form of honeybee venom melittin, was conjugated to 15 amino acid fragment of human chorionic gonadotropin β subunit. Lytic peptides are known to act only on negatively charged cells, such as bacteria and cancer cells and hereby, due to hCGbeta fragment, the conjugate is able to bind directly to LHCGR bearing cancer cells, saving the healthy ones. The experiments were carried out in inhibin-alpha-Simian Virus 40-T-antigen transgenic mice that are known to express LHCGR-bearing gonadal tumors, namely Leydig and granulosa cell tumors by 100% penetrance. If the mice are gonadectomized prepubertally they form adrenocortical tumors instead. Transgenic and wild type mice were treated for three consecutive weeks with control vehicle, Hecate or Hecate-CGbeta conjugate. GnRH antagonist or estradiol was given to a group of mice with or without Hecate-CGbeta conjugate to analyze the additive role of gonadotropin blockage in adrenocortical tumor treatment efficacy. Hecate-CGbeta conjugate was able to diminish the gonadal and adrenal tumor size effectively in males. No treatment related side effects were found. Gonadotropin blockage through GnRH antagonist was the best treatment in female adrenal tumors. The mode of cell death by Hecate-CGbeta conjugate was proven to be through necrosis. LHCGR and GATA-4 were co-expressed in tumors, where the treatment down-regulated their expression simultaneously, suggesting their possible use as tumor markers. In conclusion, the present thesis showed that Hecate-CGbeta conjugate targets its action selectively through LHCGR and selectively kills the LHCGR bearing tumor cells. It works both in gonadal somatic and in ectopic LHCGR bearing adrenal tumors. These results establish a more general principle that receptors expressed ectopically in malignant cells can be exploited in targeted cytotoxic therapies without affecting the normal healthy cells.

Constituintes químicos e atividade antiedematogênica de Peltodon radicans (Lamiaceae)

Most of the snakebite incidents in the Amazon region involve Bothrops atrox, whose venom presents the most potent edematogenic and necrotic activities in the genus. This work describes the studies of isolation of the chemical constituents and antiedematogenic activity of the species Peltodon radicans (Lamiaceae), which is used in the treatment of snakebites and scorpion stings in the region. The extracts presented aliphatic hydrocarbons, 3beta-OH,beta-amirin (1), 3beta-OH,alpha-amirin (2), beta-sitosterol (3), stigmasterol (4), ursolic acid (5), 2alpha,3beta,19alpha-trihydroxy-urs-12-en-28-oic acid (tormentic acid, 6), methyl 3beta-hydroxy,28-methyl-ursolate (7), sitosterol-3-O-beta-D-glucopyranoside (8), and stigmasterol-3-O-beta-D-glucopyranoside (9). The flower extracts presented the higher antiedematogenic activity. This is the first report on the study of the flowers, stem, and roots of this plant.

Aplicação de quitosana como suporte para a imobilização de enzimas de interesse industrial

Chitosan, poly[β-(1-4)-linked-2-amino-2-deoxy-D-glucose], is the N-deacetylated product of chitin which is a major component of arthropod and crustacean shells such as lobsters, crabs, shrimps, and cuttlefishes. In addition, chitosan has many significant biological and chemical properties such as biodegradability, biocompatibility and bioactivity as well as polycationic properties. Thus, it has been widely used in many industrial and biomedical applications including wastewater treatment, chromatographic support, carriers for controlled drug delivery and enzyme immobilization. This review is an insight into the exploitation of utilization of chitosan based-supports in different geometrical configurations on the immobilization of enzymes by different protocols for further application in biotransformation reactions.

Comparison of wildlife and captivity rattlesnakes (Crotalus durissus terrificus) microbiota

The study evaluated and compared the aerobic microbiota from the oral cavity, cloaca and venom of Crotalus durissus terrificus snakes, recently caught from the wild and kept under quarantine (WQ), individual captivity (IC) and collective captivity (CC). Antimicrobial drug effectiveness on isolated agents also was assayed. From group I, II and III were isolated, respectively, 29 (63.04%), 38 (90.48%) and 21 (42.86%) microorganisms from the cloaca; 15 (32.61%), 3 (7.14%) and 25 (51.02%) microorganisms from the oral cavity; and, 2 (4.35%), 1 (2.38%) and 3 (6.12%) microorganisms from venom. The most frequent bacteria were Pseudomonas aeruginosa, Proteus vulgaris and Morganella morganii, with sensitivity to amikacin, gentamicin, norfloxacin, sulfazotrin and tobramycin. Snakes kept in semi-open captivity exhibited the fewest microorganisms in oral cavities, perhaps due to the environment in captivity, with different temperature gradients, running water, absence of daily handling, circulating air, possibility of moving around, daily cleaning, and sunlight access.

Efficacy of the d-phenothrin/pyriproxyfen association against mites in naturally co-infested rabbits

The aim of the present study was to evaluate the efficacy of the d-phenothrin/pyriproxyfen association against Psoroptes ovis, Cheyletiella parasitivorax, and Leporacarus gibbus infestations in naturally co-infested rabbits. Twenty crossbreed (New Zealand White x California) rabbits concurrently infested by the three mite species were randomly divided in two groups. All rabbits presented with hyperemia, erythema and formation of crusts in the ear canals caused by P. ovis. Infestations by both C. parasitivorax and L. gibbus were considered asymptomatic in all animals.Ten animals were treated with a 4.4% d-phenothrin and 0.148% pyriproxyfen spray formulation until have their body surface uniformly sprayed, including external ear canals. The other ten rabbits remained untreated, serving as control group. Observations were done on days +7, +14, +21, +28, and +35 post-treatment. The d-phenothrin/pyriproxyfen association showed 100% efficacy against the three mite species and was responsible for the remission of psoroptic mange lesions on treated animals. No signs of intoxication were observed. The results indicate that d-phenothrin/pyriproxyfen spray formulation in a single application is an effective and clinically safe option for the control of different mite infestations in rabbits.

Babesia bovis: expression of adhesion molecules in bovine umbilical endothelial cells stimulated with plasma from infected cattle

Ten male, 12-month-old Jersey with intact spleens, serologically and parasitologically free from Babesia were housed individually in an arthropod-free isolation system from birth and throughout entire experiment. The animals were randomly divided into two groups. Five animals (group A) were intravenously inoculated with 6.6 X10(7) red blood cells parasitized with pathogenic sample of Babesia bovis (passage 7 BboUFV-1), for the subsequent "ex vivo" determination of the expression of adhesion molecules. Five non-inoculated animals (group B) were used as the negative control. The expression of the adhesion molecules ICAM-1, VCAM, PECAM-1 E-selectin and thrombospondin (TSP) was measured in bovine umbilical vein endothelial cells (BUVECs). The endothelial cells stimulated with a pool of plasma from animals infected with the BboUFV-1 7th passage sample had a much more intense immunostaining of ICAM-1, VCAM, PECAM-1 E-selectin and TSP, compared to the cells which did not received the stimulus. The results suggest that proinflammatory cytokines released in the acute phase of babesiosis may be involved in the expression of adhesion molecules thereby implicating them in the pathophysiology of babesiosis caused by B. bovis.

Muscarinic toxins : characterization of adrenoceptor binding and applicability of membrane anchoring via glycosylphosphatidylinositol tail

Adrenoceptors (ARs), G-protein coupled receptors (GPCRs) at the plasma membrane, respond to endogenous catecholamines noradrenaline and adrenaline. These receptors mediate several important physiological functions being especially important in the cardiovascular system and in the regulation of smooth muscle contraction. Impairments in the function of these receptors can thus lead to severe diseases and disorders such as to cardiovascular diseases and benign prostatic hyperplasia. The Eastern green mamba (Dendroaspis angusticeps) venom has been shown to contain toxins that can antagonize the functions of GPCRs. The most well-known are muscarinic toxins (MTs) targeting muscarinic acetylcholine receptors (mAChRs) with high affinity and selectivity. However, some reports have indicated that these toxins might also act on the α1- and α2-ARs which can be divided into various subtypes; the α1-ARs to α1A-, α1B- and α1D-ARs and α2-ARs to α2A-, α2B- and α2C-ARs. In this thesis, the interaction of four common MTs (MT1, MT3, MT7 and MTα) with the adrenoceptors was characterized. It was also evaluated whether these toxins could be anchored to the plasma membrane via glycosylphosphatidylinositol (GPI) tail. Results of this thesis reveal that muscarinic toxins are targeting several α-adrenoceptor subtypes in addition to their previously identified target receptors, mAChRs. MTα was found to interact with high affinity and selectivity with the α2B-AR whereas MT7 confirmed its selectivity for the M1 mAChR. Unlike MTα and MT7, MT1 and MT3 have a broad range of target receptors among the α-ARs. All the MTs characterized were found to behave as non-competitive antagonists of receptor action. The interaction between MTα and the α2B-AR was studied more closely and it was observed that the second extracellular loop of the receptor functions as a structural entity enabling toxin binding. The binding of MTα to the α2B-AR appears to be rather complex and probably involves dimerized receptor. Anchoring MTs to the plasma membrane did not interfere with their pharmacological profile; all the GPI-anchored toxins created retained their ability to block their target receptors. This thesis shows that muscarinic toxins are able to target several subtypes of α-ARs and mAChRs. These toxins offer thus a possibility to create new subtype specific ligands for the α-AR subtypes. Membrane anchored MTs on the other hand could be used to block α-AR and mAChR actions in disease conditions such as in hypertension and in gastrointestinal and urinary bladder disorders in a cell-specific manner and to study the physiological functions of ARs and mAChRs in vivo in model organisms.

Leaf damage in a mangrove swamp at Sepetiba Bay, Rio de Janeiro, Brazil

Leaf damage to Rhizophora mangle L., Avicennia schaueriana Stapf. & Leechman, and Laguncularia racemosa L. was studied in a two hectare mangrove swamp site in Sepetiba Bay. Seventeen arthropod morphospecies were identified as being responsible for the damage, and their species diversity was highest on A. schaueriana, followed by R. mangle and L. racemosa. Damage in terms of relative area was greatest in L. racemosa. Almost 9% of mangrove canopy leaf area demonstrated some damage. Loss of leaf area to herbivory was 12.1%, 8.3% and 6.2% in L. racemosa, A. schaueriana and R. mangle respectively. L. racemosa and A. schaueriana showed high percentages of leaf-margin damage in terms of the total damaged leaf area (82.2% and 56.3% respectively), while the most important type of damage in R. mangle was necrosis, representing 58.1% of the total damaged leaf area.

Neutralizing capacity of a new monovalent anti-Bothrops atrox antivenom: comparison with two commercial antivenoms

Three horse-derived antivenoms were tested for their ability to neutralize lethal, hemorrhagic, edema-forming, defibrinating and myotoxic activities induced by the venom of Bothrops atrox from Antioquia and Chocó (Colombia). The following antivenoms were used: a) polyvalent (crotaline) antivenom produced by Instituto Clodomiro Picado (Costa Rica), b) monovalent antibothropic antivenom produced by Instituto Nacional de Salud-INS (Bogotá), and c) a new monovalent anti-B. atrox antivenom produced with the venom of B. atrox from Antioquia and Chocó. The three antivenoms neutralized all toxic activities tested albeit with different potencies. The new monovalent anti-B. atrox antivenom showed the highest neutralizing ability against edema-forming and defibrinating effects of B. atrox venom (41 ± 2 and 100 ± 32 µl antivenom/mg venom, respectively), suggesting that it should be useful in the treatment of B. atrox envenomation in Antioquia and Chocó

Atherosclerosis and acute arterial thrombosis in rabbits: a model using balloon desendothelization without dietary intervention

Acute thrombosis can be induced in rabbits by a triggering protocol using Russell's viper venom and histamine given after 8 months of a 1% cholesterol diet and balloon desendothelization. In the present study, we tested the hypothesis that aortic desendothelization performed 4 months before the triggering protocol without a high cholesterol diet is a highly effective and less expensive way of producing arterial atherosclerosis and thrombosis. Nineteen male New Zealand white rabbits on a normal diet were studied. The control group (N = 9) received no intervention during the 4-month observation period, while the other group (N = 10) was submitted to aortic balloon desendothelization using a 4F Fogarty catheter. At the end of this period, all animals were killed 48 h after receiving the first dose of the triggering treatment. Eight of 10 rabbits (80%) in the balloon-trauma group presented platelet-rich arterial thrombosis while none of the animals in the control group had thrombus formation (P<0.01). Thus, this model, using balloon desendothelization without dietary manipulation, induces arterial atherosclerosis and thrombosis and may provide possibilities to test new therapeutic approaches

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 37oC with a 1% (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 µg of the liposome-entrapped toxic fraction on days 1 and 21 and a final injection (20 µg) 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

Fibrinolytic action on fresh human clots of whole body extracts and two semipurified fractions from Lonomia achelous caterpillar

The severe bleeding diathesis produced by intoxication with the venom of Lonomia achelous caterpillars is characterized by prolonged bleeding from superficial skin wounds as well as massive hemorrhage into body cavities. The aim of the present study was to evaluate the effect of the crude venom and its fibrinolytic fractions on in vitro lysis of whole blood clots. Venom fractions with fibrinolytic activity were obtained by gel filtration chromatography on Sephadex G75 using imidazole buffer, pH 7.4, at a flow rate of 24 ml/h. Four peaks with fibrinolytic activity were obtained by this method. The highest activity was found in the first two peaks (both peaks were used for the experiments). The results show that the caterpillar venom degraded the preformed clots at a slower rate than plasmin. In addition, plasma protease inhibitors of the fibrinolytic system (a2-antiplasmin, a2-macroglobulin, PAI, etc.) only weakly inhibited the lytic effect of the caterpillar venom. These characteristics, as well as the pattern of fibrinogen degradation products, the delay period on fibrin plate lysis and amidolytic activity on chromogenic substrate, reported previously, indicate that the caterpillar enzymes are different from plasmin and trypsin.

A single-step purification of bothropstoxin-1

Bothrops venoms are complex mixtures of components with a wide range of biological activities. Among these substances, myotoxins have been investigated by several groups. Bothropstoxin-1 (Bthtx-1) is a phospholipase A2-like basic myotoxin from Bothrops jararacussu. The purification of this component involves two chromatographic steps. Although providing a pure material, the association of these two steps is time consuming and a single-step method using high performance chromatography media would be useful. In the present study, we describe a single-step purification method for Bthtx-1. Bothrops jararacussu venom was dissolved in 1 ml buffer. After centrifugation, the supernatant was injected into a Resource-S cation exchange column connected to an FPLC system and eluted with a linear salt gradient. The complete procedure took 20 min, representing a considerable time gain when compared to a previously described method (Homsi-Brandenburgo MI et al. (1988) Toxicon, 26: 615-627). Bthtx-1 purity and identity, assessed by SDS-PAGE and N-terminal sequencing, resulted in a single band with a molecular mass of about 14 kDa and the expected sequence of the first 5 residues, S-L-F-E-L. Although the amount of protein purified after each run is lower than in the previously described method, we believe that this method may be useful for small-scale purifications.