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.

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.

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.

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.

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.

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.

Reduced plasma levels of angiotensin-(1-7) and renin activity in preeclamptic patients are associated with the angiotensin I- converting enzyme deletion/deletion genotype.

The relationship between preeclampsia and the renin-angiotensin system (RAS) is poorly understood. Angiotensin I-converting enzyme (ACE) is a key RAS component and plays an important role in blood pressure homeostasis by generating angiotensin II (Ang II) and inactivating the vasodilator angiotensin-(1-7) (Ang-(1-7)). ACE (I/D) polymorphism is characterized by the insertion (I) or deletion (D) of a 287-bp fragment, leading to changes in ACE activity. In the present study, ACE (I/D) polymorphism was correlated with plasma Ang-(1-7) levels and several RAS components in both preeclamptic (N = 20) and normotensive pregnant women (N = 20). The percentage of the ACE DD genotype (60%) in the preeclamptic group was higher than that for the control group (35%); however, this percentage was not statistically significant (Fisher exact test = 2.86, d.f. = 2, P = 0.260). The highest plasma ACE activity was observed in the ACE DD preeclamptic women (58.1 +/- 5.06 vs 27.6 +/- 3.25 nmol Hip-His Leu(-1) min(-1) mL(-1) in DD control patients; P = 0.0005). Plasma renin activity was markedly reduced in preeclampsia (0.81 +/- 0.2 vs 3.43 +/- 0.8 ng Ang I mL plasma(-1) h(-1) in DD normotensive patients; P = 0.0012). A reduced plasma level of Ang-(1-7) was also observed in preeclamptic women (15.6 +/- 1.3 vs 22.7 +/- 2.5 pg/mL in the DD control group; P = 0.0146). In contrast, plasma Ang II levels were unchanged in preeclamptic patients. The selective changes in the RAS described in the present study suggest that the ACE DD genotype may be used as a marker for susceptibility to preeclampsia.

Isolation and characterization of microsatellite DNA in the piracema fish Prochilodus lineatus (Characiformes).

We described five novel microsatellite loci for the piracema fish species Prochilodus lineatus (Characiformes), endemic to South America and of extreme importance to both commercial and artisanal fisheries across its occurrence area. A primary, unenriched genomic library was constructed and radioactively screened for repetitive motifs. Positive clones were automatically sequenced and based on the design of new primers, polymerase chain reaction assays were carried out to determine optimum reaction and electrophoretic conditions for each characterized locus. We evaluated its usefulness in population genetic studies by determining Hardy-Weinberg equilibrium, FIS and a jackknife estimate of the number of alleles for a sample of fish caught below the Funil Hydroelectric Power Plant dam (N = 95), Grande River, Brazil. The number of alleles varied from 3 to 21 and expected heterozygosities ranged from 0.58 to 0.91. Two of five loci were in Hardy-Weinberg equilibrium. Jackknife estimates of the number of alleles were higher than the observed number of alleles for three loci and could provide a measure of sampling bias. These markers should provide important tools for the determination of genetic structure, stock delimitation and reservoir fish management in the Grande River as well as to improve hatchery practices for environmental mitigation measures and to help sustain fisheries in the river.

Functional characterization and epitope analysis of a recombinant dermonecrotic protein from Loxosceles intermedia spider.

In the present study the recombinant form (recLiD1) of a dermonecrotic protein present in the Brazilian brown spider Loxosceles intermedia venom was expressed in Escherichia coli cells and purified by reversed-phase HPLC using a C8 Vydac column. About 25.8mg of purified recLiD1 was produced from a litre of bacterial culture. SDS/PAGE and immunoblot analysis of the recombinant protein revealed an apparent molecular weight of 32-35kDa. The later result was confirmed by mass spectrometry (32,758Da). recLiD1 displayed dermonecrotic and platelet aggregation activities which were qualitatively similar to that displayed by the crude L. intermedia venom. However, very low sphingomyelinase D enzymatic activity and complement-dependent haemolytic activities were observed. recLiD1 immunized BALB/c mice developed an antibody response. Anti-recLiD1 antibodies recognized L. intermedia venom in an indirect enzyme-linked immunosorbent assay (ELISA) and cross-reacted with crude venoms from L. intermedia, L. gaucho and L. laeta. An in vivo protection assay carried out 5 weeks after the end of the immunization protocol showed that 75% of the vaccinated mice could resist the challenge by 2.5LD(50) of L. intermedia venom. To characterize epitopes associated with protective antibodies, we prepare sets of immobilized synthetic 15 mer overlapping peptides covering the complete amino acid sequences of the recLiD1. Antibodies revealed one antigenic region in the N-terminal part of the toxin. The amino acid sequence of this epitope was found in several dermonecrotic proteins and some of its residues have been implicated with the active site of the toxin.

Identification of immunogenic proteins from Paracoccidioides brasiliensis antigenic fractions F0, FII and FIII.

Paracoccidioides brasiliensis causes a chronic granulomatous mycosis prevalent in South America, and cell-mediated immunity is the principal mode of protection against this fungal infection. In this context, one of the strategies to discover proteins that are target of an effective immune response against P. brasiliensis is the partial sequencing of cDNA from an expression library previously screened with immunoglobulins (Ig) to generate antigen sequence tags (AST). In the present work, a P. brasiliensis yeast cDNA expression library was screened with affinity chromatography-purified IgG from rabbit sera immunized with P. brasiliensis antigenic fractions (F0, FII or FIII) or from paracoccidioidomycosis (PCM) patient sera by indirect ELISA. From 119 clones selected by the immunoscreening procedure, 40% were recognized by IgG from PCM patients, 25% were recognized by anti-F0, 8% were selected by anti-FII and 11% recognized by FIII specific antibodies. The remaining clones presented cross-reaction to all anti-sera tested. The AST homologies with previously reported sequences in the nonredundant GenBank at NCBI revealed high significant homology to fungal proteins of known function. One of them matched calcineurin B of Neurospora crassa with 35% identity and 55% similarity in amino acid sequence. We also identified an AST homologous to a Kinesin like protein from Ustilagus maydis and other fungi with 86% identity and 91% similarity. On the other hand, the vast majority of selected cDNA clones are new genes and represent 60% of the total. Prediction of transmembrane regions with the prediction transmembrane protein topology with a hidden markov model (TMHMM) revealed consensus sequences representing structural membrane segments in 28 encoded proteins.

Molecular characterization of a neutralizing murine monoclonal antibody against Tityus serrulatus scorpion venom.

Monoclonal antibodies (mAbs) against Tityus serrulatus venom were obtained by the fusion of SP2/0 murine myeloma cells and spleen cells from BALB/c mice immunized with a toxic fraction (TstFG50) of the Tityus venom (this G50 chromatography fraction represents most of the toxicity of the crude venom) conjugated to bovine serum albumin (BSA) with glutaraldehyde. From the initial screening of over 200 hybridoma fusion wells, a panel of 9 anti-TstFG50 secreting hybridomas was established. The capacity of mAbs to neutralize the TstFG50 toxic fraction toxic was determined by in vitro neutralization assays and by inhibition of the binding of 125I-TsVII to its site on rat brain synaptosomes. Only mAbTs1 neutralized 50% of the toxic effects produced by scorpion venom and showed 35% inhibition of the binding of 125I-TsVII at 10(-7) M. To map the epitope recognized by the protective mAbTs1, we prepared a comprehensive series of overlapping 15-mer synthetic peptides covering the amino acid sequences of the four Tityus proteins. MAbTs1 reacted with peptide 26 of TsIV (KKSKDKKADSGYSYW), peptide 30 of TsVII (KKGSSGYSAWPASYS) and peptide 31 of TsNTxP (KKGSSGYSAWPASYS). MAbTs1 was not reactive with any peptide from TsII. The N-terminal lysine residue from the epitope was found to be critical for mAbTs1 binding. The epitope was positioned on the available three-dimensional structure of TsVII together with the recently identified residues from the pharmacophore of beta-scorpion toxins. The neutralizing properties of mAbTs1 might be explained by spatial vicinity of epitope residues with pharmacophore residues.

Characterization of the venom from the Brazilian Brown Spider Loxosceles similis Moenkhaus, 1898 (Araneae, Sicariidae).

Accidents caused by brown spiders (Loxosceles genus) are frequent in Brazil and are associated with dermonecrotic lesions and, eventually, systemic reactions that may be lethal. The major species implicated with human envenoming have been: L. intermedia, L. gaucho and L. laeta. In this study we characterized the venom from Loxosceles similis, a species of spider normally found inside caves. L. similis venom was characterized by two-dimensional gel electrophoresis and enzymatic activity (dermonecrosis and haemolysis). The lethal dose to mice and the capacity of commercial anti-serum to neutralize this venom were also analysed. The cross-reactivity with anti-venoms against L. intermedia, L. laeta and L. gaucho were studied. Our results showed that this venom was able to induce severe dermonecrotic lesions and showed the presence of the bacteria Clostridium septicum in association with the fangs. In addition, we have cloned the DNA coding for a dermonecrotic protein (LsD1), using the genomic DNA of L. similis. The deduced amino acid sequence showed a toxin of approximately 31.2 kDa with an estimated pI of 7.37 and sequence similar to LiD1, a protein from the dermonecrotic family of Loxosceles intermedia spider venom, a synanthropic species of medical importance.

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.

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.

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.

A toxin from the spider Phoneutria nigriventer that blocks calcium channels coupled to exocytosis.

1. The aim of the present experiments was to investigate the pharmacological action of a toxin from the spider Phoneutria nigriventer, Tx3-3, on the function of calcium channels that control exocytosis of synaptic vesicles. 2. Tx3-3, in confirmation of previous work, diminished the intracellular calcium increase induced by membrane depolarization with KCl (25 mM) in rat cerebrocortical synaptosomes. The toxin was very potent (IC50 0.9 nM) at inhibiting calcium channels that regulate calcium entry in synaptosomes. In addition, Tx3-3 blocked the exocytosis of synaptic vesicles, as measured with the fluorescent dye FM1-43. 3. Using omega-toxins that interact selectively with distinct neuronal calcium channels, we investigated whether the target of Tx3-3 overlaps with known channels that mediate exocytosis. The results indicate that the main population of voltage-sensitive calcium channels altered by Tx3-3 can also be inhibited by omega-agatoxin IVA, an antagonist of P/Q calcium channels. Omega-conotoxin GVIA, which inhibits N type calcium channels did not decrease significantly the entry of calcium or exocytosis of synaptic vesicles in depolarized synaptosomes. 4. It is concluded that Tx3-3 potently inhibits omega-agatoxin IVA-sensitive calcium channels, which are involved in controlling exocytosis in rat brain cortical synaptosomes.

PnTx3-6 a spider neurotoxin inhibits K+-evoked increase in [Ca2+](i) and Ca2+-dependent glutamate release in synaptosomes.

The present experiments investigated the effect of a neurotoxin purified from the venom of the spider Phoneutria nigriventer. This toxic component, P. nigriventer toxin 3-6 (PnTx3-6), abolished Ca(2+)-dependent glutamate release with an IC(50) of 74.4nM but did not alter Ca(2+)-independent secretion of glutamate when brain cortical synaptosomes were depolarized by KCl (33mM). This effect was most likely due to interference with the entry of calcium through voltage activated calcium channels (VACC), reducing the increase in the intrasynaptosomal free calcium induced by membrane depolarization with an IC(50) of 9.5nM. We compared the alterations induced by PnTx3-6 with the actions of toxins known to block calcium channels coupled to exocytosis. Our results indicate that PnTx3-6 inhibition of glutamate release and intrasynaptosomal calcium involves P/Q type calcium channels and this toxin can be a valuable tool in the investigation of calcium channels.

Phoneutria nigriventer toxins block tityustoxin-induced calcium influx in synaptosomes.

Neurotoxins can help the understanding of mechanisms involved in neurotransmission. We here report that two neurotoxin isoforms, Tx3-3 and Tx3-4 obtained from the venom of the spider Phoneutria nigriventer inhibited the 45Ca2+ influx in rat cortical synaptosomes induced by the scorpion venom tityustoxin. The IC50 for Tx3-3 and Tx3-4 were 0.32 and 7.9 nM, respectively. The neurotoxins Tx3-3 and Tx3-4 are very effective in inhibiting 45Ca2+ influx and they should be useful in studies involving Ca(2+)-dependent processes.

Inhibition of glutamate uptake by Tx3-4 is dependent on the redox state of cysteine residues.

Glutamate transporters are essential for the homeostasis of glutamate and normal function of glutamatergic synapses. Their function was shown to be regulated by redox agents and dimerizations that involves redox changes of cysteine residues. Peptide neurotoxins are also known to be rich in cysteine residues that contribute to their activity and stability. Among them is the toxin Tx3-4, from the spider Phoneutria nigriventer, which is able to inhibit glutamate uptake in rat hippocampal synaptosomes. Based on results obtained with manipulation of the redox state of cysteine residues in synaptosomes and in Tx3-4, we suggest that the effect of this toxin on glutamate uptake is due to interactions that involve cysteines both in the toxin and in the transporters.

Clonal composition of human adamantinomatous craniopharyngiomas and somatic mutation analyses of the patched (PTCH), Gsalpha and Gi2alpha genes.

Craniopharyngioma is the most common childhood tumor and thought to arise from embryonic remnants of Rathke's pouch. The paucity of published data on the molecular basis of these tumors prompted us to examine 22 adamantinomatous craniopharyngiomas looking for genetic abnormalities. Using the X-linked polymorphic androgen receptor gene as a tool for X-chromosome inactivating analysis, we found that a subset of craniopharyngiomas are monoclonal and therefore are probably due to acquired somatic genetic defects. Thus, we investigated these tumours for mutations within three candidate genes, Gsalpha, Gi2alpha and patched (PTCH). Using single stranded conformational polymorphism (SSCP), denaturing gradient gel electrophoresis and direct sequencing, the presence of somatic mutations in these genes could not be demonstrated in any tumor. Our data indicate that a subset of craniopharyngiomas are monoclonal and the mutations in the PTCH, Gsalpha, and Gi2alpha contribute little if any to craniopharyngioma development.