Development of tacrine clusters as positively cooperative systems for the inhibition of acetylcholinesterase

The synthesis of four tetra-tacrine clusters where the tacrine binding units are attached to a central scaffold via linkers of variable lengths is described. The multivalent inhibition potencies for the tacrine clusters were investigated for the inhibition of acetylcholinesterase. Two of the tacrine clusters displayed a small but significant multivalent inhibition potency in which the binding affinity of each of the tacrine binding units increased up to 3.2 times when they are connected to the central scaffold.     

Draft genome sequence of Bizionia argentinensis, isolated from Antarctic surface water

A psychrotolerant marine bacterial strain, designated JUB59(T), was isolated from Antarctic surface seawater and classified as a new species of the genus Bizionia. Here, we present the first draft genome sequence for this genus, which suggests interesting features such as UV resistance, hydrolytic exoenzymes, and nitrogen metabolism.     

Studies on reduction of S-nitrosoglutathione by human carbonyl reductases 1 and 3

Human carbonyl reductases 1 and 3 (CBR1 and CBR3) are monomeric NADPH-dependent enzymes of the short-chain dehydrogenase/reductase superfamily. Despite 72% identity in primary structure they exhibit substantial differences in substrate specificity. Recently, the endogenous low molecular weight S-nitrosothiol S-nitrosoglutathione (GSNO) has been added to the broad substrate spectrum of CBR1. The current study initially addressed whether CBR3 could equally reduce GSNO which was not the case. Neither the introduction of residues which contribute to glutathione binding in CBR1, i.e. K106Q and S97V/D98A, nor the exchange C143S, which prevents a theoretical disulfide bond with C227 in CBR3, could engender activity towards GSNO. However, exchanging amino acids 236-244 in CBR3 to correspond to CBR1 was sufficient to engender catalytic activity towards GSNO. Catalytic efficiency was further improved by the exchanges Q142M, C143S, P230W and H270S. Hence, the same residues previously reported as important for reduction of carbonyl compounds appear to be key to CBR1-mediated reduction of GSNO. Furthermore, for CBR1-mediated reduction of GSNO, considerable substrate inhibition at concentrations >5 K(m) was observed. Treatment of CBR1 with GSNO followed by removal of low molecular weight compounds decreased the GSNO reducing activity, suggesting a covalent modification. Treatment with dithiothreitol, but not with ascorbic acid, could rescue the activity, indicating S-glutathionylation rather than S-nitrosation as the underlying mechanism. As C227 has previously been identified as the reactive cysteine in CBR1, the variant CBR1 C227S was generated, which, in comparison to the wild-type protein, displayed a similar k(cat), but a 30-fold higher K(m), and did not show substrate inhibition. Collectively, the results clearly argue for a physiological role of CBR1, but not for CBR3, in GSNO reduction and thus ultimately in regulation of NO signaling. Furthermore, at higher concentrations, GSNO appears to work as a suicide inhibitor for CBR1, probably through glutathionylation of C227.     

A genetic map of Xenopus tropicalis

We present a genetic map for Xenopus tropicalis, consisting of 2886 Simple Sequence Length Polymorphism (SSLP) markers. Using a bioinformatics-based strategy, we identified unique SSLPs within the X. tropicalis genome. Scaffolds from X. tropicalis genome assembly 2.0 (JGI) were scanned for Simple Sequence Repeats (SSRs); unique SSRs were then tested for amplification and polymorphisms using DNA from inbred Nigerian and Ivory Coast individuals. Thus identified, the SSLPs were genotyped against a mapping cross panel of DNA samples from 190 F2 individuals. Nearly 4000 SSLPs were genotyped, yielding a 2886-marker genetic map consisting of 10 major linkage groups between 73 and 132 cM in length, and 4 smaller linkage groups between 7 and 40 cM. The total effective size of the map is 1658 cM, and the average intermarker distance for each linkage group ranged from 0.27 to 0.75 cM. Fluorescence In Situ Hybridization (FISH) was carried out using probes for genes located on mapped scaffolds to assign linkage groups to chromosomes. Comparisons of this map with the X. tropicalis genome Assembly 4.1 (JGI) indicate that the map provides representation of a minimum of 66% of the X. tropicalis genome, incorporating 758 of the approximately 1300 scaffolds over 100,000 bp. The genetic map and SSLP marker database constitute an essential resource for genetic and genomic analyses in X. tropicalis.     

Leishmanicidal effect of Spiranthera odoratíssima (Rutaceae) and its isolated alkaloid skimmianine occurs by a nitric oxide dependent mechanism

Leishmaniasis is one of the neglected diseases. High cost, systemic toxicity, and diminished efficacy due to development of resistance by the parasites has a negative impact on the current treatment options. Thus, the search for a new, effective and safer anti-leishmanial drug becomes of paramount importance. Compounds derived from natural products may be a better and cheaper source in this regard. This study evaluated the in vitro anti-leishmanial activity of Spiranthera odoratíssima (Rutaceae) fractions and isolated compounds, using promastigote and amastigote forms of different Leishmania species. J774 A.1 macrophage was used as the parasite host cell for the in vitro assays. Evaluations of cytoxicity, nitric oxide (NO), interleukin-10 and in silico analysis were carried out. In vitro experiments showed that the fruit hexanic fraction (Fhf) and its alkaloid skimmianine (Skm) have a significant (P<0·001) effect against L. braziliensis. This anti-L. braziliensis activity of Fhf and Skm was due to increased production of NO and attenuation of IL-10 production in the macrophages at concentrations ranging from 1·6 to 40·0 μg/ml. The in silico assay demonstrated significant interaction between Skm and amino acid residues of NOS2. Skm is thus a promising drug candidate for L. braziliensis due to its potent immunomodulatory activity.     

Variation in Trill Characteristics in Tree Pipit Songs: Different Trills for Different Use?

Transmitting information about singer's quality is an important function of song in many bird species, and this information should be useful in territorial interactions. Fast trills, being physically demanding song structures, are particularly suitable candidates for signalling of quality or aggressive motivation. We have evaluated trill characteristics in songs within a population of the Tree Pipit, a common European songbird with no sexual dimorphism, in which song apparently plays a key role in territory defence as well as mate choice. Two types of relatively fast trills (each of them in multiple variants differing in complexity) were commonly observed in repertoires of Tree Pipit males. Trill rates significantly differed among individuals, suggesting that these song structures may carry information about male quality in this species. We tested by playback experiments whether both trill types are used in territorial encounters. Only one of the trill types was sung by males in response to playback, regardless on the trill type played to them. In an immediate response to playback, they increased the frequency of use of this trill, and also significantly increased the trill rate in comparison with spontaneous songs. This confirmed field observations, suggesting that this trill is important in male–male interactions. On the contrary, the use of the fastest, apparently more demanding, trill type actually decreased after the simulated territorial intrusion. We hypothesize that the latter one is more directed towards females, and that while performance of both trill types may reflect male quality, they are primarily used in different contexts.     

Barriers,rather than refugia,underlie the origin of diversity in toads endemic to the Brazilian Atlantic Forest

In this study, we investigated the relative contribution of geographic barriers and Pleistocene refuges in the diversification of the Rhinella crucifer species complex, a group of endemic toads with a widespread distribution in the Brazilian Atlantic Forest (AF). We used intensive sampling and multilocus DNA sequence data to compare nucleotide diversity between refuge and nonrefuge areas, investigate regional demographic patterns, estimate demographic parameters related to genetic breaks and test refuge versus barrier scenarios of diversification using approximate Bayesian computation. We did not find higher levels of genetic diversity in putative refuge areas, either at regional or biome scale. Rather, the demographic history of the species complex supports regional differences with moderate population growth in the north and central regions and stability in southern AF. Genetic breaks were dated to the Plio–Pleistocene; however, our analyses rejected the role of refuges in creating a northern and central divergence, supporting a recent colonization scenario at a smaller scale within the central AF. Overall, our data rule out massive climatically driven fragmentation and large‐scale recolonization events for populations across the biome. We confirmed the importance of geographic barriers in creating main divergences and underscored the importance of searching for cryptic discontinuities in the landscape. Comparison of our results with those of other AF taxa indicates organismal specific responses to moderate shifts in habitat and that multiple refuges may constitute a more realistic model for diversification of Atlantic Forest biota.     

Alona iheringula Sinev & Kotov, 2004 (Crustacea,Anomopoda, Chydoridae,Aloninae): Life Cycle and DNA Barcode with Implications for the Taxonomy of the Aloninae Subfamily

Knowledge of reproductive rates and life cycle of the Cladocera species is essential for population dynamic studies, secondary production and food webs, as well as the management and preservation of aquatic ecosystems. The present study aimed to understand the life cycle and growth of Alona iheringula Kotov & Sinev, 2004 (Crustacea, Anomopoda, Chydoridae), a Neotropical species, as well as its DNA barcoding, providing new information on the Aloninae taxonomy. The specimens were collected in the dammed portion of the Cabo Verde River (21°26′05″ S and 46°10′57″ W), in the Furnas Reservoir, Minas Gerais State, Brazil. Forty neonates were observed individually two or three times a day under controlled temperature (25±1°C), photoperiod (12 h light/12 h dark) and feeding (Pseudokirchneriella subcapitata at a concentration of 10⁵ cells.mL⁻¹ and a mixed suspension of yeast and fish feed in equal proportion). Individual body growth was measured daily under optical microscope using a micrometric grid and 40× magnification. The species had a mean size of 413(±29) µm, a maximum size of 510 µm and reached maturity at 3.24(±0.69) days of age. Mean fecundity was 2 eggs per female per brood and the mean number of eggs produced per female during the entire life cycle was 47.6(±6.3) eggs per female. The embryonic development time was 1.79(±0.23) days and the maximum longevity was 54 days. The species had eight instars throughout its life cycle and four instars between neonate and primipara stage. The present study using molecular data (a 461 bp smaller COI fragment) demonstrated a deep divergence in the Aloninae subfamily.     

Study of tensiometric properties,microbiological and collagen content in nile tilapia skin submitted to different sterilization methods

Tissue bioengineering development is a global concern and different materials are studied and created to be safe, effective and with low cost. Nile Tilapia skin had shown its biological potential as covers for the burn wound. This study evaluates the tilapia skin histological, collagen properties and tensiometric resistance, after treatment by different sterilization methods. Tilapia skin samples were submitted to two sterilization processes: (1) chemical, which consisted in two 2% chlorhexidin baths, followed by sequential baths in increasing glycerol concentrations; and (2) radiation, when glycerolized skin samples were submitted to gamma radiation at 25, 30 and 50 kGy. Microscopic analyzes were performed through Haematoxylin–eosin and Picrosirius Red under polarized light. For tensiometric analysis, traction tests were performed. Glycerol treated skin presented a discrete collagen fibers disorganization within the deep dermis, while irradiated skin did not show any additional change. Throughout the steps of chemical sterilization, there was a higher proportion of collagen with red/yellow birefringence (type I) in the skin samples up to the first bath in chlorhexidin, when compared to samples after the first two glycerol baths (P < 0.005). However, there was no difference in relation to total collagen between groups. In irradiated skin, there was a larger total collagen preservation when using until 30 kGy (P < 0.005). Tensiometric evaluation did not show significant differences in relation to maximum load in the groups studied. We concluded that chemical and radiation (25 and 30 kGy) are efficient methods to sterilize Nile Tilapia skin without altering its microscopic or tensiometric characteristics.