Successful xenogeneic germ cell transplantation from Jundia catfish (Rhamdia quelen) into adult Nile tilapia (Oreochromis niloticus) testes.

Fish germ cell transplantation presents several important potential applications for aquaculture, including the preservation of germplasm from endangered fish species with high genetic and commercial values. Using this technique in studies developed in our laboratory with adult male Nile tilapias (Oreochromis niloticus), all the necessary procedures were successfully established, allowing the production of functional sperm and healthy progeny approximately 2months after allogeneic transplantation. In the present study, we evaluated the viability of the adult Nile tilapia testis to generate sperm after xenogeneic transplant of germ cells from sexually mature Jundia catfish (Rhamdia quelen) that belong to a different taxonomic order. Therefore, in order to investigate at different time-periods post-transplantation, the presence and development of donor PKH26 labeled catfish germ cells were followed in the tilapia seminiferous tubules. From 7 to 20days post-transplantation, only PKH26 labeled spermatogonia were observed, whereas spermatocytes at different stages of development were found at 70days. Germ cell transplantation success and progression of spermatogenesis were indicated by the presence of labeled PKH26 spermatids and sperm on days 90 and 120 post-transplantation, respectively. Confirming the presence of the catfish genetic material in the tilapia testis, all recipient tilapias evaluated (n=8) showed the genetic markers evaluated. Therefore, we demonstrated for the first time that the adult Nile tilapia testis offers the functional conditions for development of spermatogenesis with sperm production from a fish species belonging to a different order, which provides an important new venue for aquaculture advancement.

General characterization of Tityus fasciolatus scorpion venom. Molecular identification of toxins and localization of linear B-cell epitopes.

This communication describes the general characteristics of the venom from the Brazilian scorpion Tityus fasciolatus, which is an endemic species found in the central Brazil (States of Goiás and Minas Gerais), being responsible for sting accidents in this area. The soluble venom obtained from this scorpion is toxic to mice being the LD50 is 2.984 mg/kg (subcutaneally). SDS-PAGE of the soluble venom resulted in 10 fractions ranged in size from 6 to 10-80 kDa. Sheep were employed for anti-T. fasciolatus venom serum production. Western blotting analysis showed that most of these venom proteins are immunogenic. T. fasciolatus anti-venom revealed consistent cross-reactivity with venom antigens from Tityus serrulatus. Using known primers for T. serrulatus toxins, we have identified three toxins sequences from T. fasciolatus venom. Linear epitopes of these toxins were localized and fifty-five overlapping pentadecapeptides covering complete amino acid sequence of the three toxins were synthesized in cellulose membrane (spot-synthesis technique). The epitopes were located on the 3D structures and some important residues for structure/function were identified.

Evolution of alternative methodologies of scorpion antivenoms production.

Scorpionism represents a serious public health problem resulting in the death of children and debilitated individuals. Scorpion sting treatment employs various strategies including the use of specific medicines such as antiserum, especially for patients with severe symptoms. In 1909 Charles Todd described the production of an antiserum against the venom of the scorpion Buthus quinquestriatus. Based on Todd's work, researchers worldwide began producing antiserum using the same approach i.e., immunization of horses with crude venom as antigen. Despite achieving satisfactory results using this approach, researchers in this field have developed alternative approaches for the production of scorpion antivenom serum. In this review, we describe the work published by experts in toxinology to the development of scorpion venom antiserum. Methods and results describing the use of specific antigens, detoxified venom or toxins, purified toxins and or venom fractions, native toxoids, recombinant toxins, synthetic peptides, monoclonal and recombinant antibodies, and alternative animal models are presented.

Identification and characterization of B-cell epitopes of 3FTx and PLA(2) toxins from Micrurus corallinus snake venom.

The main goal of this work was to develop a strategy to identify B-cell epitopes on four different three finger toxins (3FTX) and one phospholipase A2 (PLA2) from Micrurus corallinus snake venom. 3FTx and PLA2 are highly abundant components in Elapidic venoms and are the major responsibles for the toxicity observed in envenomation by coral snakes. Overlapping peptides from the sequence of each toxin were prepared by SPOT method and three different anti-elapidic sera were used to map the epitopes. After immunogenicity analysis of the spot-reactive peptides by EPITOPIA, a computational method, nine sequences from the five toxins were chemically synthesized and antigenically and immunogenically characterized. All the peptides were used together as immunogens in rabbits, delivered with Freund's adjuvant for a first cycle of immunization and Montanide in the second. A good antibody response against individual synthetic peptides and M. corallinus venom was achieved. Anti-peptide IgGs were also cross-reactive against Micrurus frontalis and Micrurus lemniscatus crude venoms. In addition, anti-peptide IgGs inhibits the lethal and phospholipasic activities of M. corallinus crude venom. Our results provide a rational basis to the identification of neutralizing epitopes on coral snake toxins and show that their corresponding synthetic peptides could improve the generation of immuno-therapeutics. The use of synthetic peptide for immunization is a reasonable approach, since it enables poly-specificity, low risk of toxic effects and large scale production.

Molecular and functional characterization of metalloserrulases, new metalloproteases from the Tityus serrulatus venom gland.

Tityus serrulatus is a Brazilian scorpion species with great medical significance. While the effects of neurotoxins have been extensively studied, little is known about the proteases expressed in the venom gland of this arthropod. In this study, clones from a T. serrulatus (Ts) venom gland cDNA library were selected according to homology to proteases. The sequences were aligned in the database and classified by homology. Similarity and identity analyses of the sequences were carried out, and a phylogenetic tree was constructed with the sequences of other proteases. These cDNA sequences correspond to ten different metalloproteases, named metalloserrulases (TsMS). TsMS 1-9 belong to the metzincin family, which has three domains: signal peptide, propeptide, and metalloprotease domain; while TsMS 10 belongs to the gluzincin family. The proteolytic activity of the venom was inferred from the cleavage of fibrinogen, and the residues recognized by the proteases were determined by cleavage of a tripeptide library using a fluorescence resonance energy transfer assay. The Ts venom showed proteolytic activity on fibrinogen and preferential cleavage close to the basic residues K and R. Its activity could be inhibited by EDTA, indicating that the venom from this scorpion predominantly consists of metalloproteases.

ADP is a vasodilator component from Lasiodora sp. mygalomorph spider venom.

Members of the spider genus Lasiodora are widely distributed in Brazil, where they are commonly known as caranguejeiras. Lasiodora spider venom is slightly harmful to humans. The bite of this spider causes local pain, edema and erythema. However, Lasiodora sp. spider venom may be a source of important pharmacological tools. Our research group has described previously that Lasiodora sp. venom produces bradycardia in the isolated rat heart. In the present work, we sought to evaluate the vascular effect of Lasiodora sp. venom and to isolate the vasoactive compounds from the venom. The results showed that Lasiodora spider venom induced a concentration-dependent vasodilation in rat aortic rings, which was dependent on the presence of a functional endothelium and abolished by the nitric oxide synthase (NOS) inhibitor L-NAME. Western blot experiments revealed that the venom also increased endothelial NOS function by increasing phosphorylation of the Ser¹¹77 residue. Assay-directed fractionation isolated a vasoactive fraction from Lasiodora sp. venom. Mass spectrometry (MS) and nuclear magnetic resonance (NMR) assays identified a mixture of two compounds: adenosine diphosphate (ADP, approximately 90%) and adenosine monophosphate (AMP, approximately 10%). The vasodilator effects of Lasiodora sp. whole venom, as well as ADP, were significantly inhibited by suramin, which is a purinergic P2-receptor antagonist. Therefore, the results of the present work indicate that ADP is a main vasodilator component of Lasiodora sp. spider venom.

Biochemical and immunological characteristics of Peruvian Loxosceles laeta spider venom: neutralization of its toxic effects by anti-loxoscelic antivenoms.

This manuscript describes the general biochemical properties and immunological characteristics of Peruvian spider Loxosceles laeta venom (PLlv), which is responsible for the largest number of accidents involving venomous animals in Peru. In this work, we observed that the venom of this spider is more lethal to mice when compared with L. laeta venom from Brazil (BLlv). The LD50 of PLlv was 1.213 mg/kg when the venom was intradermally injected. The venom displayed sphingomyelinase activity and produced dermonecrotic, hemorrhagic and edema effects in rabbits. 2-D SDS-PAGE separation of the soluble venoms resulted in a protein profile ranging from 20 to 205 kDa. Anti-PLlv and anti-BLlv sera produced in rabbits and assayed by ELISA showed that rabbit antibodies cross-reacted with PLlv and BLlv and also with other Brazilian Loxosceles venoms. Western blotting analysis showed that bands corresponding to 25-35 kDa are the proteins best recognized in every Loxosceles spp venoms analyzed. The immunized rabbits displayed protective effect after challenge with PLlv and BLlv. In vitro assays with horse anti-loxoscelic antivenoms produced in Brazil and Peru demonstrated that these commercial antivenoms were efficient to inhibit the sphingomyelinase activity of PLlv and BLlv.

Evaluation of genetic diversity and population structure in a commercially important freshwater fish Prochilodus costatus (Characiformes, Prochilodontidae) using complex hypervariable repeats.

We used complex hypervariable repeats to evaluate the genetic diversity and structure of Prochilodus costatus (Characiformes), an ecologically and economically important species endemic to the São Francisco River basin. Hydroelectric dams along the river have led to population fragmentation, which can limit gene flow. Restocking from hatcheries has been used to repopulate declining populations. To determine how fragmentation and hatchery supplementation affect P. costatus population structure, we studied populations from three sites up and downstream of the Gafanhoto Dam (Pará River, State of Minas Gerais). High levels of genetic diversity were found within populations (0.926 to 0.873); the three populations showed significant differentiation (F(ST) = 0.16), suggesting that populations from the three sites were affected by fragmentation of the river and by hatchery contributions. These results will be useful for developing a management and conservation plan for fish species in this area.

General biochemical and immunological characteristics of the venom from Peruvian scorpion Hadruroides lunatus.

This communication describes the general biochemical properties and some immunological characteristics of the venom from the Peruvian scorpion Hadruroides lunatus, which is the most medically relevant species in Peru. The soluble venom of this scorpion is toxic to mice, the LD50 determined was 0.1 mg/kg and 21.55 mg/kg when the venom was injected intracranial or intraperitoneally, respectively. The soluble venom displayed proteolytic, hyaluronidasic, phospholipasic and cardiotoxic activities. High performance liquid chromatography of the soluble venom resulted in the separation of 20 fractions. Two peptides with phospholipasic activity were isolated to homogeneity and their molecular masses determined by mass spectrometry (MALDI TOF). Anti-H. lunatus venom sera were produced in rabbits. Western blotting analysis showed that most of the protein content of this venom is immunogenic. H. lunatus anti-venom displayed consistent cross-reactivity with venom antigens from the new World-scorpions Tityus serrulatus and Centruroides sculpturatus venoms; however, a weaker reactivity was observed against the venom antigens from the old World-scorpion Androctonus australis Hector.

Evaluation of Brazilian biotechnology patent activity from 1975 to 2010.

The analysis of patent activity is one methodology used for technological monitoring. In this paper, the activity of biotechnology-related patents in Brazil were analyzed through 30 International Patent Classification (IPC) codes published by the Organization for Economic Cooperation and Development (OECD). We developed a program to analyse the dynamics of the major patent applicants, countries and IPC codes extracted from the Brazilian Patent Office (INPI) database. We also identified Brazilian patent applicants who tried to expand protection abroad via the Patent Cooperation Treaty (PCT). We had access to all patents published online at the INPI from 1975 to July 2010, including 9,791 biotechnology patent applications in Brazil, and 163 PCTs published online at World Intellectual Property Organization (WIPO) from 1997 to December 2010. To our knowledge, there are no other online reports of biotechnology patents previous to the years analyzed here. Most of the biotechnology patents filed in the INPI (10.9%) concerned measuring or testing processes involving nucleic acids. The second and third places belonged to patents involving agro-technologies (recombinant DNA technology for plant cells and new flowering plants, i.e. angiosperms, or processes for obtaining them, and reproduction of flowering plants by tissue culture techniques). The majority of patents (87.2%) were filed by nonresidents, with USA being responsible for 51.7% of all biotechnology patents deposited in Brazil. Analyzing the resident applicants per region, we found a hub in the southeast region of Brazil. Among the resident applicants for biotechnology patents filed in the INPI, 43.5% were from São Paulo, 18.3% were from Rio de Janeiro, and 9.7% were from Minas Gerais. Pfizer, Novartis, and Sanofi were the largest applicants in Brazil, with 339, 288, and 245 biotechnology patents filed, respectively. For residents, the largest applicant was the governmental institution FIOCRUZ (Oswaldo Cruz Foundation), which filed 69 biotechnology patents within the period analyzed. The first biotechnology patent applications via PCT were submitted by Brazilians in 1997, with 3 from UFMG (university), 2 from individuals, and 1 from EMBRAPA (research institute).

Cutaneous loxoscelism caused by Loxosceles similis venom and neutralization capacity of its specific antivenom.

Members of the spider genus Loxosceles pose a marked health risk to humans because of the seriousness of the necrotic and systemic effects of their bite, known as loxoscelism. The recent confirmation of Loxosceles similis in residences of Belo Horizonte in Minas Gerais Province, Brazil increases the local potential risk of loxoscelism at higher levels. The first characterization of the venom from this species showed that its main biological effects had a similar intensity as other species (e.g. Loxosceles intermedia, Loxosceles laeta, and Loxosceles gaucho). Therefore, we wished to further analyse the biological activity of the L. similis venom as well as the capacity of anti-L. similis-venom serum to reduce dermonecrotic effects to rabbit skin. Histological analysis of rabbit skin 2, 4 and 8h after intradermal injection of L. similis venom demonstrated a dense inflammatory infiltrate, edema, degeneration and necrosis of the skin muscle, dissociation of collagen fibers, and disruption of reticular fibers. Importantly, pre-incubation of the venom with anti-L. similis-venom serum significantly decreased all of these effects. Anti-L. similis antivenom generated antibodies that were strongly reactive to L. similis venom and capable of neutralizing the dermonecrotic effects in rabbits caused by this venom. Moreover, the antivenom significantly reduced the sphingomyelinase activity of L. similis crude venom. Venoms produced by male and female spiders were equally reactive towards anti-L. similis and anti-L. intermedia antivenoms, but female venom induced larger lesions on rabbits. In contrast, female venom acted as an immunization enhancer and protected animals from L. similis envenomation to a greater degree than male venom. In conclusion, the results shown in this study for L. similis antivenom merits a more in depth study of its properties, which may become a valuable tool against loxoscelism.

Histopathological characterization of experimentally induced cutaneous loxoscelism in rabbits inoculated with Loxosceles similis venom

Envenomation by Loxosceles bites is characterized by dermonecrotic and/or systemic features that lead to several clinical signs and symptoms called loxoscelism. Dermonecrotic lesions are preceded by thrombosis of the dermal plexus. Recent studies show that atheromatous plaque is prone to thrombosis due to endothelial cell apoptosis. To the best of our knowledge, there are no reports of microscopic dermal lesion and endothelial cell apoptosis induced by Loxosceles similis venom in the literature. Thus, the aim of the present study is to describe histological lesions induced by L. similis venom in rabbit skin and to elucidate whether apoptosis of endothelial cells is involved in the pathogenesis of loxoscelism. Forty male rabbits were split into two groups: the control group (intradermally injected with 50 µL of PBS) and the experimental group (intradermally injected with 0.5 µg of L. similis crude venom diluted in 50 µL of PBS). After 2, 4, 6 and 8 hours of injection, skin fragments were collected and processed for paraffin or methacrylate embedding. Sections of 5 µm thick were stained by HE, PAS or submitted to TUNEL reaction. Microscopically, severe edema, diffuse heterophilic inflammatory infiltrate, perivascular heterophilic infiltrate, thrombosis, fibrinoid necrosis of arteriolar wall and cutaneous muscle necrosis were observed. Two hours after venom injection, endothelial cells with apoptosis morphology were evidenced in the dermal plexus. Apoptosis was confirmed by TUNEL reaction. It seems that endothelial cell apoptosis and its consequent desquamation is an important factor that induces thrombosis and culminates in dermonecrosis, which is characteristic of cutaneous loxoscelism.

Mimotopes of mutalysin-II from Lachesis muta snake venom induce hemorrhage inhibitory antibodies upon vaccination of rabbits.

Mutalysin-II (mut-II) from Lachesis muta snake venom is an endopeptidase with hemorrhagic activity. A mAb against mutalysin-II that neutralized the hemorrhagic effect was produced previously. To identify the mAb epitopes, sets of 15-mer overlapping peptides covering the mut-II amino acid sequence were synthesized using the SPOT method and tested but failed to react with the mAb. Using a phage-display approach seventeen clones reactive with mAb were identified. Additional immunoassays with the peptides and mAb identified the QCTMDQGRLRCR, TCATDQGRLRCT, HCFHDQGRVRCA, HCTMDQGRLRCR and SCMLDQGRSRCR sequences as possible epitopes. Immunization of rabbits with these peptides induced antibodies that recognize mut-II and protected against the hemorrhagic effects of Lachesis venom.

Molecular cloning and expression of epsilon toxin from Clostridium perfringens type D and tests of animal immunization.

Epsilon toxin produced by Clostridium perfringens types B and D causes enterotoxemia in sheep, goats and calves. Enterotoxemia can cause acute or superacute disease, with sudden death of the affected animal. It provokes huge economic losses when large numbers of livestock are affected. Therapeutic intervention is challenging, because the disease progresses very rapidly. However, it can be prevented by immunization with specific immunogenic vaccines. We cloned the etx gene, encoding epsilon toxin, into vector pET-11a; recombinant epsilon toxin (rec-epsilon) was expressed in inclusion bodies and was used for animal immunization. Serum protection was evaluated and cross-serum neutralization tests were used to characterize the recombinant toxin. To analyze the potency of the toxin (as an antigen), rabbits were immunized with 50, 100 or 200 microg recombinant toxin, using aluminum hydroxide gel as an adjuvant. Titers of 10, 30 and 40 IU/mL were obtained, respectively. These titers were higher than the minimum level required by the European Pharmacopoeia (5 IU/mL) and by the USA Code of Federal Regulation (2 IU/mL). This rec-epsilon is a good candidate for vaccine production against enterotoxemia caused by epsilon toxin of C. perfringens type D.

A protective immune response against lethal, dermonecrotic and hemorrhagic effects of Loxosceles intermedia venom elicited by a 27-residue peptide.

Antibodies raised against recombinant Loxosceles intermedia dermonecrotic protein isoform 1 (rLiD1) display neutralizing capacity for the L. intermedia whole venom. We previously found that an immunodominant continuous B-cell epitope, recognized by these antibodies corresponds to a region of the protein known to be involved in the active site. In this study, we extend previous work by preparing a 27-residue synthetic replica of this epitope ((25)NLGANSIETDVSFDDNANPEYTYHGIP(51)) and using it as an immunogen in mice and rabbits. The immunization process induced antibodies that protected mice from a lethal dose of L. intermedia crude venom and rabbits against the dermonecrotic effects of rLiD1. An Ala scan of the epitope indicated that 4 residues, E44, Y45, T46 and Y47, are essential (over 70% decrease in binding upon replacement with alanine) for antibody recognition. The possible mechanisms of neutralization are discussed in light of these findings.

Effective Tityus serrulatus anti-venom produced using the Ts1 component.

Scorpion stings are a public health problem in Brazil, with most incidents involving the species Tityus serrulatus. Some T. serrulatus toxins may act as immunogens for the production of a specific anti-venom, but many of the component toxins remain poorly characterized. Here, we describe the immunological characteristics of the toxin Ts1 (also known as TsVII and Ts-gamma) and evaluate production of neutralizing antibodies against the crude venom of T. serrulatus. Recombinant Ts1 with one copy (Ts1(1)) or two copies in tandem (Ts1(2)) was expressed in BL21 (DE3) cells. Rabbits and mice were immunized with the recombinant proteins (inclusion bodies) and then tested for production of neutralizing antibodies. Neutralization assays showed that anti-Ts1(1) and anti-Ts1(2) protected animals challenged with T. serrulatus crude venom and native Ts1. Thus, Ts1 could be used in a mixed "cocktail" of immunogens for T. serrulatus anti-venom production.

Expression and characterization of LTx2, a neurotoxin from Lasiodora sp. effecting on calcium channels.

Here, we described the expression and characterization of the recombinant toxin LTx2, which was previously isolated from the venomous cDNA library of a Brazilian spider, Lasiodora sp. (Mygalomorphae, Theraphosidae). The recombinant toxin found in the soluble and insoluble fractions was purified by reverse phase high-performance liquid chromatography (HPLC). Ca2+ imaging analysis revealed that the recombinant LTx2 acts on calcium channels of BC3H1 cells, blocking L-type calcium channels.

Heterologous expression of an insecticidal gene from the armed spider (Phoneutria nigriventer)

Insect-pests are global problems that cause severe damage to crop plants, and their control is commonly based on chemical insecticides. However, negative effects of pesticides on the environment and human health emphasize the necessity to develop alternative methods for insect-pest control. In the present study, a gene coding for the insecticidal peptide TX4(6-1) of the Brazilian armed spider (Phoneutria nigriventer) was cloned in fusion with maltose binding protein (MBP) and expressed in Escherichia coli. The affinity purified protein MBP-GlyTX4 was cleaved with the Xa factor and used for a bioassay against Spodoptera frugiperda and rabbit immunization. Five micrograms GlyTX4 protein injected into the hemocoel of larvae and abdominal cavity of adults produced trembling and uncoordinated movements immediately after injection and all adult insects died after 12h. After two days, larvae became paralyzed and the epidermal color changed to dark brown. Furthermore, the development stage was prolonged for two weeks. Alternatively, slices of maize leaves were imbibed with 15 micrograms of the recombinant protein cleaved with the Xa factor and used as diet for larvae. In this experiment, all larvae died in about 30 minutes. Polyclonal antibodies anti-MBP-GlyTX4 were effective for recognizing MBP and GlyTX4 in whole cell extract from E. coli expressing the recombinant protein.

The Loxtox protein family in Loxosceles intermedia (Mello-Leitão) venom.

We isolated cDNA sequences coding for dermonecrotic/sphingomyelinases factor proteins from the brown spider Loxosceles intermedia, here named Loxtox proteins. The amino acid sequences based on cloned cDNA of several Loxtox proteins revealed at least six distinct groups of proteins expressed in the venom gland. The level of similarity among the toxins varied from 99% to 55%. The finding of several isoforms of Loxtox in the venom of this spider may reflect an evolutionary adaptation for different prey types and reinforces the idea of an efficient mutational mechanism in the venom gland of spiders.