Adsorption of 5′-AMP and catalytic synthesis of 5′-ADP onto phosphate surfaces: Correlation to solid matrix structures

A non-enzymatic formation of 5-ADP starting from phosphorylation of 5-AMP in the presence of either calcium phosphate or calcium pyrophosphate precipitates is reported. This reaction is taken as a model for the study of heterogeneous catalysis of transphosphorylation in prebiotic conditions. Experiments were performed in completely aqueous media and in media containing dimethyl sulfoxide (Me₂S0), to simulate periods of dehydration in primitive aquatic environments. It has been observed that the nucleotide is adsorbed onto both calcium phosphate and calcium pyrophosphate in accordance with Langmuir isotherms. Adsorptive capacity and affinity of the precipitates for nucleotide are changed by the presence of Me₂SO, suggesting that the interaction between biomonomers and surfaces can be modulated by the degree of hydration of the anionic components of these compounds. In completely aqueous environments, formation of 5-ADP from 5-AMP adsorbed on precipitates of calcium phosphate and calcium pyrophosphate is very small. However, in the presence of 60% Me₂SO this synthesis increases by factors of 3 and 6 for surfaces of calcium phosphate and calcium pyrophosphate, respectively, and follows first-order kinetics. Determinations of free energy changes show that phosphorylation of 5-AMP adsorbed to these precipitates is thermodynamically favorable. Depending on the precipitation time of the samples and the composition of the medium, structural analysis of these precipitates by electron and X-ray diffraction shows changes in their cristallinity grade. It is proposed that these changes are responsible for the modulation of the quantity of adsorbed nucleotides to the surface of solid matrices as well as the catalytic activity of the precipitates.Abbreviations 5-AMP 5-adenosine monophosphate - 5-ADP 5-adenosine diphosphate - BTP l,3-bis[tris(hydroxymethyl)-methylamino]propane - CTEM conventional transmission electron microscopy - Tris tris(hydroxymethyl)aminomethane - Pi (H₂PO ₄ ^– /HPO ₄ ^2– ) orthophosphate - Pi.Ca calcium phosphate - PPi (H₃P₂O ₇ ^– /H₂P₂O ₇ ^2– ) pyrophosphate - PPi.Ca calcium pyrophosphate - EGTA [ethylenebis(oxyethylene)nitrilo]tetraacetic acid This work has been submitted to the Department of Biochemistry, Institute of Biomedical Sciences, UFRJ, by A.C.T. in partial fulfillment of requirements for the MS degree.     

Temperature sensitive influenza A virus genome replication results from low thermal stability of polymerase-cRNA complexes

Background

Although many vaccinia virus proteins have been identified and studied in detail, only a few studies have attempted a comprehensive survey of the protein composition of the vaccinia virion. These projects have identified the major proteins of the vaccinia virion, but little has been accomplished to identify the unknown or less abundant proteins. Obtaining a detailed knowledge of the viral proteome of vaccinia virus will be important for advancing our understanding of orthopoxvirus biology, and should facilitate the development of effective antiviral drugs and formulation of vaccines.

Results

In order to accomplish this task, purified vaccinia virions were fractionated into a soluble protein enriched fraction (membrane proteins and lateral bodies) and an insoluble protein enriched fraction (virion cores). Each of these fractions was subjected to further fractionation by either sodium dodecyl sulfate-polyacrylamide gel electophoresis, or by reverse phase high performance liquid chromatography. The soluble and insoluble fractions were also analyzed directly with no further separation. The samples were prepared for mass spectrometry analysis by digestion with trypsin. Tryptic digests were analyzed by using either a matrix assisted laser desorption ionization time of flight tandem mass spectrometer, a quadrupole ion trap mass spectrometer, or a quadrupole-time of flight mass spectrometer (the latter two instruments were equipped with electrospray ionization sources). Proteins were identified by searching uninterpreted tandem mass spectra against a vaccinia virus protein database created by our lab and a non-redundant protein database.

Conclusion

Sixty three vaccinia proteins were identified in the virion particle. The total number of peptides found for each protein ranged from 1 to 62, and the sequence coverage of the proteins ranged from 8.2% to 94.9%. Interestingly, two vaccinia open reading frames were confirmed as being expressed as novel proteins: E6R and L3L.     

Scientific challenges of bioethanol production in Brazil

Bioethanol (fuel alcohol) has been produced by industrial alcoholic fermentation processes in Brazil since the beginning of the twentieth century. Currently, 432 mills and distilleries crush about 625 million tons of sugarcane per crop, producing about 27 billion liters of ethanol and 38.7 million tons of sugar. The production of bioethanol from sugarcane represents a major large-scale technology capable of producing biofuel efficiently and economically, providing viable substitutes to gasoline. The combination of immobilization of CO₂ by sugarcane crops by photosynthesis into biomass together with alcoholic fermentation of this biomass has allowed production of a clean and high-quality liquid fuel that contains 93% of the original energy found in sugar. Over the last 30 years, several innovations have been introduced to Brazilian alcohol distilleries resulting in the improvement of plant efficiency and economic competitiveness. Currently, the main scientific challenges are to develop new technologies for bioethanol production from first and second generation feedstocks that exhibit positive energy balances and appropriately meet environmental sustainability criteria. This review focuses on these aspects and provides special emphasis on the selection of new yeast strains, genetic breeding, and recombinant DNA technology, as applied to bioethanol production processes.     

Computer-manipulated stimuli as a research tool in Mozambique tilapia <Emphasis Type="Italic">Oreochromis mossambicus</Emphasis>

Multimodal communication is essential in social interactions in cichlid fish, including conspecifics’ recognition, agonistic interactions and courtship behaviour. Computer-manipulated image stimuli and sound playback offer powerful tools to assess the relative relevance of visual and acoustic stimuli in fish behavioural studies, but these techniques require validation for each taxon. The aim of the present study was to investigate whether Mozambique tilapia Oreochromis mossambicus responds to computer-manipulated visual stimuli and acoustic playback. Six experiments were conducted: computer animation playback, video playback, interaction with a mirror, presentation of a live male in a jar alone and combined with courting sound playback or with white noise playback. Individual agonistic interactions (lateral displays, up and down swimming, butting) and courting behaviours (tilting leading, digging) were tallied for each experiment. Our results suggest that non-interactive computer-manipulated visual stimuli is not a suitable tool in behavioural research with Mozambique tilapia. In contrast, interaction with a live male in a jar seems to remain the best visual research instrument inducing significant strong behavioural responses. Although none or only a few agonistic interactions were observed towards video playbacks or computer animations, such interactions significantly increased towards a male in jar and were modulated by courtship sound playback, suggesting the additional relevance of sound playback as a tool in behavioural research with Mozambique tilapia, including the study of multimodal signalling.     

Testing Darwin's transoceanic dispersal hypothesis for the inland nettle family (Urticaceae)

Dispersal is a fundamental ecological process, yet demonstrating the occurrence and importance of long‐distance dispersal (LDD) remains difficult, having rarely been examined for widespread, non‐coastal plants. To address this issue, we integrated phylogenetic, molecular dating, biogeographical, ecological, seed biology and oceanographic data for the inland Urticaceae. We found that Urticaceae originated in Eurasia c. 69 Ma, followed by ≥ 92 LDD events between landmasses. Under experimental conditions, seeds of many Urticaceae floated for > 220 days, and remained viable after 10 months in seawater, long enough for most detected LDD events, according to oceanographic current modelling. Ecological traits analyses indicated that preferences for disturbed habitats might facilitate LDD. Nearly half of all LDD events involved dioecious taxa, so population establishment in dioecious Urticaceae requires multiple seeds, or occasional selfing. Our work shows that seawater LDD played an important role in shaping the geographical distributions of Urticaceae, providing empirical evidence for Darwin's transoceanic dispersal hypothesis.     

The first phylogenetic study of Mesembrinellidae (Diptera: Oestroidea) based on molecular data: clades and congruence with morphological characters

The Mesembrinellidae (Diptera: Oestroidea) comprise a small group of strictly Neotropical calyptrate flies, with 36 described species. The group has often been treated as a subfamily of Calliphoridae, but there is growing evidence that it corresponds to a distinct Oestroidea lineage. Internal relationships have so far been addressed based only on morphology, with results lacking resolution and support. This is the first molecular phylogeny for the group, which is based on the analyses of 80 terminal taxa (22 mesembrinellid and 28 outgroup species) and 5 molecular markers (ITS2, 28S, COI, COII and 16S). Maximum‐parsimony, maximum‐likelihood and Bayesian inference methods were used, the latter two with partitioning strategies considering codon position and secondary structure information. Results corroborate the Mesembrinellidae as a monophyletic lineage inside Oestroidea. Three clades were consistently recovered: (1) (Laneella + Mesembrinella patriciae); (2) (Mesembrinella (excluding M. patriciae)  + Eumesembrinella); and (3) (Huascaromusca + Giovanella). Re‐examination of the female reproductive tract of M. patriciae revealed a Laneela‐type spermatheca, which corroborates the position of the species recovered in the molecular phylogenetic analyses. Mesembrinella and Huascaromusca are in all cases paraphyletic with regards to Eumesembrinella and Giovanella, respectively. These latter two genera should, thus, be seen as subjective junior synonyms.     

Mitochondrial DNA phylogeography and population history of the grey wolf canis lupus

The grey wolf (Canis lupus) and coyote (C. latrans) are highly mobile carnivores that disperse over great distances in search of territories and mates. Previous genetic studies have shown little geographical structure in either species. However, population genetic structure is also influenced by past isolation events and population fluctuations during glacial periods. In this study, control region sequence data from a worldwide sample of grey wolves and a more limited sample of coyotes were analysed. The results suggest that fluctuating population sizes during the late Pleistocene have left a genetic signature on levels of variation in both species. Genealogical measures of nucleotide diversity suggest that historical population sizes were much larger in both species and grey wolves were more numerous than coyotes. Currently, about 300 000 wolves and 7 million coyotes exist. In grey wolves, genetic diversity is greater than that predicted from census population size, reflecting recent historical population declines. By contrast, nucleotide diversity in coyotes is smaller than that predicted by census population size, reflecting a recent population expansion following the extirpation of wolves from much of North America. Both species show little partitioning of haplotypes on continental or regional scales. However, a statistical parsimony analysis indicates local genetic structure that suggests recent restricted gene flow.     

Differing Evolutionary Histories of the ACTN3*R577X Polymorphism among the Major Human Geographic Groups

It has been proposed that the functional ACTN3*R577X polymorphism might have evolved due to selection in Eurasian human populations. To test this possibility we surveyed all available population-based data for this polymorphism and performed a comprehensive evolutionary analysis of its genetic diversity, in order to assess the action of adaptive and random mechanisms on its variation across human geographical distribution. The derived 577X allele increases in frequency with distance from Africa, reaching the highest frequencies on the American continent. Positive selection, detected by an extended haplotype homozygosisty test, was consistent only with the Eurasian data, but simulations with neutral models could not fully explain the results found in the American continent. It is possible that particularities of Native American population structure could be responsible for the observed allele frequencies, which would have resulted from a complex interaction between selective and random factors.     

Screening of chitin deacetylase from Mucoralean strains (Zygomycetes) and its relationship to cell growth rate

Chitin deacetylase (CDA) is an enzyme that catalyzes the hydrolysis of acetamine groups of N-acetyl-d-glucosamine in chitin, converting it to chitosan in fungal cell walls. In the present study, the activity in batch culture of CDA from six Mucoralean strains, two of them wild type, isolated from dung of herbivores of Northeast Brazil, was screened. Among the strains tested, Cunninghamella bertholletiae IFM 46114 showed a high intracellular enzyme activity of 0.075 U/mg protein after 5 days of culture, and a wild-type strain of Mucor circinelloides showed a high intracellular enzyme activity of 0.060 U/mg protein, with only 2 days of culture, using N-acetylchitopentaose as substrate. This enzyme showed optimal activity at pH 4.5 in 25 mM glutamate-sodium buffer at 50°C, and was stable over 1 h preincubation at the same temperature. The kinetic parameters of CDA did not follow Michaelis-Menten kinetics, but rather Hill affinity distribution, showing probable allosteric behavior. The apparent K_HILL and V_max of CDA were 288±34 nmol/l and 0.08±0.01 U mg protein^–1 min^–1, respectively, using N-acetylchitopentaose as substrate at pH 4.5 at 50°C.     

Mitogenome of Coprophanaeus ensifer and phylogenetic analysis of the Scarabaeidae family (Coleoptera)

Several studies about the phylogenetic relationships of the Scarabaeinae subfamily (Coleoptera: Scarabaeidae) have been performed, but some phylogenetic uncertainties persist including the relationship and monophyly of different tribes and some genera. The aim of this study was to characterize the mitogenome of Coprophanaeus ensifer in order to establish its position within the Scarabaeidae family and to contribute to the resolution of some phylogenetic uncertainties. The mitogenome was sequenced on the Illumina HiSeq 4000, assembled using the Mitobim software and annotated in MITOS WebServer. The phylogenetic trees were reconstructed by Bayesian inference. The C. ensifer mitogenome is a molecule of 14,964 bp that contains the number and organization of the genes similar to those of most Coleoptera species. Phylogenetic reconstruction suggests monophyly of the tribe Phanaeini and supports the hypothesis that Coprini is a sister group of Phanaeini. The results also revealed the position of the tribe Oniticellini which is grouped with Onthophagini and Onitini. The geographic distribution of these species that form the most ancestral clade suggests with Scarabaeinae originated in Africa. Keywords: Dung beetle, mitochondrial genome, phylogenomics