Evanguedes Kalapothakis

A New and Fast Technique to Generate Offspring after Germ Cells Transplantation in Adult Fish: The Nile Tilapia ( Oreochromis niloticus ) Model

Background Germ cell transplantation results in fertile recipients and is the only available approach to functionally investigate the spermatogonial stem cell biology in mammals and probably in other vertebrates. In the current study, we describe a novel non-surgical methodology for efficient spermatogonial transplantation into the testes of adult tilapia (O. niloticus), in which endogenous spermatogenesis had been depleted with the cytostatic drug busulfan. Methodology/Principal Findings Using two different tilapia strains, the production of fertile spermatozoa with donor characteristics was demonstrated in adult recipient, which also sired progeny with the donor genotype. Also, after cryopreservation tilapia spermatogonial cells were able to differentiate to spermatozoa in the testes of recipient fishes. These findings indicate that injecting germ cells directly into adult testis facilitates and enable fast generation of donor spermatogenesis and offspring compared to previously described methods. Conclusion Therefore, a new suitable methodology for biotechnological investigations in aquaculture was established, with a high potential to improve the production of commercially valuable fish, generate transgenic animals and preserve endangered fish species.

Phoneutria nigriventer venom: a cocktail of toxins that affect ion channels.

Abstract1. We review the pharmacological actions of toxins present in the venom of the aggressive spider Phoneutria nigriventer.2. This venom is rich in toxins that affect ion channels and neurotransmitter release. Voltage-gated sodium, calcium, and potassium channels have been described as the main targets of these toxins.3. In addition to these classical actions Phoneutria toxins have also been shown to affect glutamate transporter.4. It is expected that molecular genetics in addition to biochemical, biophysical and pharmacological approaches will help to further define Phoneutria toxins and their mechanisms of action in the near future.

Expression of IFN-γ, TNF-α, IL-10 and TGF-β in lymph nodes associates with parasite load and clinical form of disease in dogs naturally infected with Leishmania (Leishmania) chagasi.

American visceral leishmaniasis is a zoonosis of the New World. Dogs are the main reservoir of the disease and there is much interest in the understanding of mechanisms implicated in protection against canine infection. Nevertheless, most studies in dogs have not been carried out in organs that are targets of infection. This work is first to report the profile of cytokines and parasite burdens, as determined by real-time PCR, in the lymph nodes of dogs naturally infected with Leishmania chagasi. With this purpose, 18 mongrel dogs were divided in three groups: control non-infected dogs (n=6) and naturally infected animals with L. chagasi, asymptomatic (n=6) and symptomatic (n=6). Parasite burden in lymph nodes was 73-fold greater in symptomatic than asymptomatic animals. Prescapular lymph nodes of asymptomatic dogs had the highest expression of IFN-gamma and TNF-alpha and low parasite burden, indicating that these cytokines play a role in protection against infection. Highest expression of IL-10 and TGF-beta and high parasite burden were observed in symptomatic dogs, suggesting a role for these cytokines in the progression of disease. Hence, the balance of expression of IFN-gamma and TNF-alpha (protective) and IL-10 and TGF-beta (disease progression) in lymph nodes determine parasite burden and clinical expression in naturally infected dogs.

Venom variability among several Tityus serrulatus specimens

Individual differences in venom composition among several Tityus serrulatus specimens collected in the same area were examined by enzyme-linked immunosorbent assay (ELISA). Polyclonal antibodies raised against whole venom and against the alpha-type (toxin IV-5 or Ts IV) and the beta-type toxin (toxin gamma or Ts VII) were used to study specific variations in the venom. The ELISA results indicated clear differences among the scorpion venoms examined. The lethality (LD50) determined by subcutaneous injections of pooled venom with the same characteristics showed an interesting correlation between the expression level of each component studied and the lethal effect of the venom. Among the groups analysed, the group with the highest concentration of alpha-type toxin showed the highest toxicity. The groups with the lowest level of toxicity were those with a low concentration of alpha-type toxin. The results show that the lethality of the venom varies from specimen to specimen and suggest that alpha-type toxin must be the major lethal component in the whole venom.

Phoneutria nigriventer toxin Tx3-1 blocks A-type K+ currents controlling Ca2+ oscillation frequency in GH3 cells.

Abstract: GH3 cells present spontaneous Ca2+ action potentials and oscillations of intracellular Ca2+, which can be modified by altering the activity of K+ or Ca2+ channels. We took advantage of this spontaneous activity to screen for effects of a purified toxin (Tx3‐1) from the venom of Phoneutria nigriventer on ion channels. We report that Tx3‐1 increases the frequency of Ca2+ oscillations, as do two blockers of potassium channels, 4‐aminopyridine and charybdotoxin. Whole‐cell patch clamp experiments show that Tx3‐1 reversibly inhibits the A‐type K+ current (IA) but does not block other K+ currents (delayed‐rectifying, inward‐rectifying, and large‐conductance Ca2+‐sensitive) or Ca2+ channels (T and L type) in these cells. In addition, we describe the sequence of a full cDNA clone of Tx3‐1, which shows that Tx3‐1 has no homology to other known blockers of K+ channels and gives insights into the processing of this neurotoxin. We conclude that Tx3‐1 is a selective inhibitor of IA, which can be used to probe the role of this channel in the control of cellular function. Based on the effect of Tx3‐1, we suggest that IA is an important determinant of the frequency of Ca2+ oscillations in unstimulated GH3 cells.

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 ω‐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 ω‐agatoxin IVA, an antagonist of P/Q calcium channels. ω‐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 ω‐agatoxin IVA‐sensitive calcium channels, which are involved in controlling exocytosis in rat brain cortical synaptosomes.

Effects of 3-beta-diol, an androgen metabolite with intrinsic estrogen-like effects, in modulating the aquaporin-9 expression in the rat efferent ductules

BackgroundFluid homeostasis is critical for normal function of the male reproductive tract and aquaporins (AQP) play an important role in maintenance of this water and ion balance. Several AQPs have been identified in the male, but their regulation is not fully comprehended. Hormonal regulation of AQPs appears to be dependent on the steroid in the reproductive tract region. AQP9 displays unique hormonal regulation in the efferent ductules and epididymis, as it is regulated by both estrogen and dihydrotestosterone (DHT) in the efferent ductules, but only by DHT in the initial segment epididymis. Recent data have shown that a metabolite of DHT, 5-alpha-androstane-3-beta-17-beta-diol (3-beta-diol), once considered inactive, is also present in high concentrations in the male and indeed has biological activity. 3-beta-diol does not bind to the androgen receptor, but rather to estrogen receptors ER-alpha and ER-beta, with higher affinity for ER-beta. The existence of this estrogenic DHT metabolite has raised the possibility that estradiol may not be the only estrogen to play a major role in the male reproductive system. Considering that both ER-alpha and ER-beta are highly expressed in efferent ductules, we hypothesized that the DHT regulation of AQP9 could be due to the 3-beta-diol metabolite.MethodsTo test this hypothesis, adult male rats were submitted to surgical castration followed by estradiol, DHT or 3-beta-diol replacement. Changes in AQP9 expression in the efferent ductules were investigated by using immunohistochemistry and Western blotting assay.ResultsData show that, after castration, AQP9 expression was significantly reduced in the efferent ductules. 3-beta-diol injections restored AQP9 expression, similar to DHT and estradiol. The results were confirmed by Western blotting assay.ConclusionThis is the first evidence that 3-beta-diol has biological activity in the male reproductive tract and that this androgen metabolite has estrogen-like activity in the efferent ductules, whose major function is the reabsorption of luminal fluid.

Electrophysiological characterization and molecular identification of the Phoneutria nigriventer peptide toxin PnTx2-61

A cDNA with 403 nucleotides encoding the precursor of the toxin PnTx2‐6 was cloned and sequenced. Subsequent analysis revealed that the precursor begins with a signal peptide and a glutamate‐rich propeptide. The succeeding peptide confirmed the reported sequence of PnTx2‐6. The purified toxin exerted complex effects on Na+ current of frog skeletal muscle. There was a marked decrease of the inactivation kinetics, and a shift to hyperpolarizing potentials of both the Na+ conductance and the steady‐state inactivation voltage dependences, along with a reduction of the current amplitude. The concentration dependence of the modified current suggests a K D of 0.8 μM for the toxin–channel complex.

Purification, amino-acid sequence and partial characterization of two toxins with anti-insect activity from the venom of the South American scorpion Tityus bahiensis (Buthidae).

We report here the isolation by a two-step chromatographic procedure of two new toxins from the South American scorpion Tityus bahiensis. Their amino-acid sequences and some of their biological features were established. The two toxins have different biological properties. Toxin TbIT-I had almost no activity or pharmacological effects in vertebrate tissues whereas it was lethal to house flies (LD50 80.0 ng/house fly). In contrast, Tb2-II was active against both mammals (intracerebroventricular injection of 100 ng/mouse was lethal) and insects (LD50 40.0 ng/house fly). The amino-acid sequences of these toxins were established and found to be similar (60-95%) to previously described beta-toxins from the Tityus genus. Based on the available comparative information, this study attempts identify possible structure-function relationships that may be responsible for the differences in bioactivity displayed by these toxins.

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 45 Ca2+ 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 45 Ca2+ influx and they should be useful in studies involving Ca2+ -dependent processes.