Binding specificity of sea anemone toxins to Nav 1.1-1.6 sodium channels: unexpected contributions from differences in the IV/S3-S4 outer loop

Sea anemones are an important source of various biologically active peptides, and it is known that ATX-II from Anemonia sulcata slows sodium current inactivation. Using six different sodium channel genes (from Nav1.1 to Nav1.6), we investigated the differential selectivity of the toxins AFT-II (purified from Anthopleura fuscoviridis) and Bc-III (purified from Bunodosoma caissarum) and compared their effects with those recorded in the presence of ATX-II. Interestingly, ATX-II and AFT-II differ by only one amino acid (L36A) and Bc-III has 70% similarity. The three toxins induced a low voltage-activated persistent component primarily in the Nav1.3 and Nav1.6 channels. An analysis showed that the 18 dose-response curves only partially fit the hypothesized binding of Lys-37 (sea anemone toxin Anthopleurin B) to the Asp (or Glu) residue of the extracellular IV/S3-S4 loop in cardiac (or nervous) Na+ channels, thus suggesting the substantial contribution of some nearby amino acids that are different in the various channels. As these channels are atypically expressed in mammalian tissues, the data not only suggest that the toxicity is highly dependent on the channel type but also that these toxins and their various physiological effects should be considered prototype models for the design of new and specific pharmacological tools.     

Isolation, primary culture and morphological characterization of oenocytes from Aedes aegypti pupae

Oenocytes are ectodermic cells that participate in a number of critical physiological roles such as detoxification and lipid storage and metabolism in insects. In light of the lack of information on oenocytes from Aedes aegypti and the potential role of these cells in the biology of this major yellow fever and dengue vector, we developed a protocol to purify and maintain Ae. aegypti pupa oenocytes in primary culture. Ae. aegypti oenocytes were cultured as clustered and as isolated ovoid cells with a smooth surface. Our results demonstrate that these cells remain viable in cell culture for at least two months. We also investigated their morphology in vivo and in vitro using light, confocal, scanning and transmission electron microscopes. This work is the first successful attempt in isolating and maintaining Ae. aegypti oenocytes in culture, and a significant step towards understanding the role of this cell type in this important disease vector. The purification and the development of primary cultures of insect oenocytes will allow future studies of their metabolism in producing and secreting compounds.     

Body fat responses to a 1‐year combined exercise training program in male coronary artery disease patients

Objective:

To analyze the body fat (BF) content and distribution modifications in coronary artery disease (CAD) patients in response to a 1‐year combined aerobic and resistance exercise training (CET) program.

Design and Methods:

We followed two groups of CAD male patients for 12 months. One group consisted of 17 subjects (57 ± 12 years) who engaged in a CET program (CET group) and the other was a age‐matched control group of 10 subjects (58 ± 11 years). BF content and distribution were measured through dual energy X‐ray absorptiometry (DXA) at baseline and follow‐up.

Results:

We found no differences on body mass and BMI between baseline and end of follow‐up in both groups but, in CET group, we found significant reductions in all analyzed BF depots, including total BF (21.60 ± 6.00 vs. 20.32 ± 5.89 kg, P < 0.01), % total BF (27.8 ± 5.5 vs. 26.4 ± 5.4%, P < 0.05), trunk fat (12.54 ± 3.99 vs. 11.77 ± 4.01 kg, P < 0.05), % trunk fat (31.1 ± 6.9 and 29.2 ± 7.1%, P < 0.05), appendicular fat (8.22 ± 2.08 vs. 7.72 ± 2.037 kg, P < 0.01), % appendicular fat (25.7 ± 4.9 and 24.5 ± 4.9%, P < 0.05), and abdominal fat (2.95 ± 1.06 vs. 2.75 ± 1.10 kg, P < 0.05). Control group showed significant increase in appendicular fat (7.63 ± 1.92 vs. 8.10 ± 2.12 kg, P < 0.05).

Conclusions:

These results confirm the positive effect of CET on body composition of CAD patients, despite no changes in body mass or BMI. In this study, we observed no alterations on BF distribution meaning similar rate of fat loss in all analyzed BF depots. These results also alert for the limitations of BMI for tracking body composition changes.     

Antimicrobial peptides isolated from Phyllomedusa nordestina (Amphibia) alter the permeability of plasma membrane of Leishmania and Trypanosoma cruzi

Nature has provided inspiration for Drug Discovery studies and amphibian secretions have been used as a promising source of effective peptides which could be explored as novel drug prototypes for neglected parasitic diseases as Leishmaniasis and Chagas disease. In this study, we isolated four antimicrobial peptides (AMPs) from Phyllomedusa nordestina secretion, and studied their effectiveness against Leishmania (L.) infantum and Trypanosoma cruzi. The antiparasitic fractions were characterized by mass spectrometry and Edman degradation, leading to the identification of dermaseptins 1 and 4 and phylloseptins 7 and 8. T. cruzi trypomastigotes were susceptible to peptides, showing IC₅₀ values in the range concentration of 0.25–0.68 μM. Leishmania (L.) infantum showed susceptibility to phylloseptin 7, presenting an IC₅₀ value of 10 μM. Except for phylloseptin 7 which moderate showed cytotoxicity (IC₅₀ = 34 μM), the peptides induced no cellular damage to mammalian cells. The lack of mitochondrial oxidative activity of parasites detected by the MTT assay, suggested that peptides were leishmanicidal and trypanocidal. By using the fluorescent probe SYTOX® Green, dermaseptins 1 and 4 and phylloseptins 7 and 8 showed time-dependent plasma membrane permeabilization of T. cruzi; phylloseptin 7 also showed a similar effect in Leishmania parasites. The present study demonstrates for the first time that AMPs target the plasma membrane of Leishmania and T. cruzi, leading to cellular death. Considering the potential of amphibian peptides against protozoan parasites and the reduced mammalian toxicity, they may contribute as scaffolds for drug design studies.     

Cetopsis varii,a new species of whale catfish (Siluriformes: Cetopsidae) from the Meta Basin,Colombia

Cetopsis varii sp. nov. Cetopsidae (Cetopsinae) is described from the Río Meta basin in eastern Colombia, at middle portions of the Río Orinoco basin. The new species differs from congeners by a combination of features: the absence of a humeral spot, presence of eye, conical teeth on vomer and dentary and rounded posterior nares, along with details of body, dorsal and caudal‐fin pigmentation. The osteology of the new species is investigated through high‐resolution X‐ray computed tomography and cleared and stained specimens. Data thus obtained are used to code the new species for character states utilized in a previous phylogenetic analysis of Cetopsidae. Analysis of the expanded matrix shows that C. varii is the sister group to C. orinoco.     

Micropropagation of Melissa officinalis L. through proliferation of axillary shoots

Multiple shoots were differentiated in cotyledonary nodes of 10 d old seedlings of Melissa officinalis, cultured on MS medium supplemented with BAP (0-4 mg/l). The production of shoots was further induced in subcultures of the original expiant, after the first harvest of shoots (stump), using similar conditions. The highest average number of shoots in the two inoculations was obtained with 2 mg/l of BAP: 24 axillary shoots per explant, 7 in the first inoculation and 17 in the second one. The maximum elongation of shoots was achieved with BAP at 0.2 mg/l, and higher concentrations of the hormone induced a decrease in their size. A range of BAP concentrations between 0.2–0.5 mg/l allowed the production of more shoots with a size suitable for rooting. Roots were induced in 30 d old shoots, transferred to MS medium individually supplemented with IBA or NAA (0–4 mg/l). Micropropagated plants were successfully transferred to soil.Abbreviations MS Murashige and Skoog (1962) medium - BAP 6-benzylaminopurine - IDA indole-3--butyric acid - NAA 1-naphthalene acetic acid - FAA formalin-acetic acid-alcohol     

Biochemical Analyses of Proteins from <Emphasis Type="Italic">Duttaphrynus melanostictus</Emphasis> (<Emphasis Type="Italic">Bufo melanostictus</Emphasis>) Skin Secretion: Soluble Protein Retrieval from a Viscous Matrix by Ion-Exchange Batch Sample Preparation

A crucial step in scientific analysis can be sample preparation, and its importance increases in the same rate as the sensitivity of the following employed/desired analytical technique does. The need to analyze complex, viscous matrices is not new, and diverse approaches have been employed, with different success rates depending on the intended molecules. Solid-phase extraction, for example, has been successfully used in sample preparation for organic molecules and peptides. However, due to the usual methodological conditions, biologically active proteins are not successfully retrieved by this technique, resulting in a low rate of protein identification reported for the viscous amphibian skin secretion. Here we describe an ion-exchange batch processing sample preparation technique that allows viscous or adhesive materials (as some amphibian skin secretions) to be further processed by classical liquid chromatography approaches. According to our protocol, samples were allowed to equilibrate with a specific resin that was washed with appropriated buffers in order to yield the soluble protein fraction. In order to show the efficiency of our methodology, we have compared our results to classically prepared skin secretion, i.e., by means of filtration and centrifugation. After batch sample preparation, we were able to obtain reproductive resolved protein chromatographic profiles, as revealed by SDS-PAGE, and retrieve some biological activities, namely, hydrolases belonging to serine peptidase family. Not only that, but also the unbound fraction was rich in low molecular mass molecules, such as alkaloids and steroids, making this sample preparation technique also suitable for the enrichment of such molecules.     

Morphological and biochemical characterization of the cutaneous poison glands in toads (<Emphasis Type="Italic">Rhinella marina</Emphasis> group) from different environments

Background

Amphibian defence against predators and microorganisms is directly related to cutaneous glands that produce a huge number of different toxins. These glands are distributed throughout the body but can form accumulations in specific regions. When grouped in low numbers, poison glands form structures similar to warts, quite common in the dorsal skin of bufonids (toads). When accumulated in large numbers, the glands constitute protuberant structures known as macroglands, among which the parotoids are the most common ones. This work aimed at the morphological and biochemical characterization of the poison glands composing different glandular accumulations in four species of toads belonging to group Rhinella marina (R. icterica, R. marina, R. schneideri and R. jimi). These species constitute a good model since they possess other glandular accumulations together with the dorsal warts and the parotoids and inhabit environments with different degrees of water availability.

Results

We have observed that the toads skin has three types of poison glands that can be differentiated from each other through the morphology and the chemical content of their secretion product. The distribution of these different glands throughout the body is peculiar to each toad species, except for the parotoids and the other macroglands, which are composed of an exclusive gland type that is usually different from that composing the dorsal warts. Each type of poison gland presents histochemical and biochemical peculiarities, mainly regarding protein components.

Conclusions

The distribution, morphology and chemical composition of the different types of poison glands, indicate that they may have different defensive functions in each toad species.