Expression cassette and plasmid construction for Yeast Surface Display in Saccharomyces cerevisiae

Objectives

Develop a Cell Surface Display system in Saccharomyces cerevisiae, based on the construction of an expression cassette for pYES2 plasmid.

Results

The construction of an expression cassette containing the α-factor signal peptide and the C-terminal portion of the α-agglutinin protein was made and its sequence inserted into a plasmid named pYES2/gDαAgglutinin. The construction allows surface display of bovine herpesvirus type 5 (BoHV-5) glycoprotein D (gD) on S. cerevisiae BY4741 strain. Recombinant protein expression was confirmed by dot blot, and indirect immunofluorescence using monoclonal anti-histidine antibodies and polyclonal antibodies from mice experimentally vaccinated with a recombinant gD.

Conclusions

These results demonstrate that the approach and plasmid used represent not only an effective system for immobilizing proteins on the yeast cell surface, as well as a platform for immunobiologicals development.

     

Deletion of CD73 increases exercise power in mice

Ecto-5′-nucleotidase or CD73 is the main source of extracellular adenosine involved in the activation of adenosine A_2A receptors, responsible for the ergogenic effects of caffeine. We now investigated the role of CD73 in exercise by comparing female wild-type (WT) and CD73 knockout (KO) mice in a treadmill-graded test to evaluate running power, oxygen uptake (V̇O₂), and respiratory exchange ratio (RER) — the gold standards characterizing physical performance. Spontaneous locomotion in the open field and submaximal running power and V̇O₂ in the treadmill were similar between CD73-KO and WT mice; V̇O₂max also demonstrated equivalent aerobic power, but CD73-KO mice displayed a 43.7 ± 4.2% larger critical power (large effect size, P < 0.05) and 3.8 ± 0.4% increase of maximum RER (small effect size, P < 0.05). Thus, KO of CD73 was ergogenic; i.e., it increased physical performance.     

Epilithic biofilms provide large amounts of nitrogen to tropical mountain landscapes

We show that epilithic biofilms are a relevant nitrogen (N) source in a rocky mountain range in Brazil. During different seasons, we quantified nitrate, ammonium, dissolved organic N (DON) and total dissolved N (TDN) leached by a simulated short rain event. We quantified the epilithic autotrophic biomass by taxonomic groups and its correlation with leached N. We hypothesized that leached N would be correlated to heterocystous cyanobacteria biomass since they are more efficient N₂ fixers. We estimated a landscape N supply of 8.5 kg.ha⁻¹.year⁻¹ considering the mean precipitation in the region. TDN in leachate was mainly composed of DON (83.8% ± 22%), followed by nitrate (12.1% ± 3%) and ammonium (5% ± 5%). The autotrophic epilithic community was mainly composed of non-heterocystous (Gloeocapsopsis) and heterocystous cyanobacteria (Scytonema and Stigonema), except for a site more commonly affected by fire events that showed a dominance of Chlorophyta. Biogeochemical upscaling was facilitated by the fact that N leaching was not different among sites or related to autotrophic epilithic biomass or assemblage composition. In conclusion, the capacity of epilithic biofilms to provide N to surrounding systems is an ecosystem service that underscores the necessity to conserve them and their habitats.     

Ecosystem dynamics based on plankton functional types for global ocean biogeochemistry models

Ecosystem processes are important determinants of the biogeochemistry of the ocean, and they can be profoundly affected by changes in climate. Ocean models currently express ecosystem processes through empirically derived parameterizations that tightly link key geochemical tracers to ocean physics. The explicit inclusion of ecosystem processes in models will permit ecological changes to be taken into account, and will allow us to address several important questions, including the causes of observed glacial–interglacial changes in atmospheric trace gases and aerosols, and how the oceanic uptake of CO₂ is likely to change in the future. There is an urgent need to assess our mechanistic understanding of the environmental factors that exert control over marine ecosystems, and to represent their natural complexity based on theoretical understanding. We present a prototype design for a Dynamic Green Ocean Model (DGOM) based on the identification of (a) key plankton functional types that need to be simulated explicitly to capture important biogeochemical processes in the ocean; (b) key processes controlling the growth and mortality of these functional types and hence their interactions; and (c) sources of information necessary to parameterize each of these processes within a modeling framework. We also develop a strategy for model evaluation, based on simulation of both past and present mean state and variability, and identify potential sources of validation data for each. Finally, we present a DGOM-based strategy for addressing key questions in ocean biogeochemistry. This paper thus presents ongoing work in ocean biogeochemical modeling, which, it is hoped will motivate international collaborations to improve our understanding of the role of the ocean in the climate system.     

A study of the fish community structure in a tidal creek.

The fish assemblage in a mangrove tidal creek was studied in monthly collections between August 1998 and July 1999 in the quadrature low tide and high tide, using a fyke net of 30 m x 1.5 m and meshes of 8 and 10 mm. Simultaneously with the biological collections, temperature, salinity, pH, water transparency, and current speed data were obtained. The collections resulted in the capture of 36,873 fish representing 22 families and 52 species. Immature individuals abounded (63.8%), and among the adult specimens there was a larger number of females. The species Anchoa parva, Harengula clupeola, Genidens genidens, and Bairdiella ronchus, contributed to 84.9% of the total capture of fish, however the contribution in mass was of 55.2%, indicating a predominance of small individuals. A smaller number of fish and species seem to occupy the area in the period between August and December, in comparison to the period between February and July, when the larger aggregations appear, mainly of the species Anchoa parva, Harengula clupeola, Opisthonema oglinum, Genidens genidens, Diapterus rhombeus, and Bairdiella ronchus. The environmental factors, pH, water transparency, and rainfall seem to have an influence in the structure of the observed biological patterns.     

Chance and necessity: land-snail faunas of Sao Miguel, Azores, compared with those of Madeira

The land-snail faunas of 23 sites from forest, disturbed andopen habitats were sampled on São Miguel, Azores archipelago.Collectively, the samples contained nearly all the known faunaof the island. Forest sites were the richest; while the numberof non-endemic species differed little between habitats, forestsheld more endemic species, many of which were widely distributed.Open habitats have no unique endemic species. These resultsare compared with similar data from Madeira. Although the twoislands are of the same size, and share the same Laurisilvaforest cover, their faunas differ in important aspects of diversity.While many non-endemic species are common to both islands, andoccur in similar numbers, endemic species are more numerouson Madeira, even though the numbers at individual forest sitesare, on average, fewer than on São Miguel. Many endemicsare confined to open, coastal habitats. These differences arediscussed in terms of the islands' histories, the range of habitatsavailable and the ease of dispersal from continental sources.Age, accidents of colonization and the range of natural habitatsavailable must have major effects on the faunas, but presentendemic faunal diversity is more a product of speciation andextinction than of immigration. (Received 13 May 2006; accepted 7 July 2006)     

Effects of taper on swimming force and swimmer performance after an experimental ten-week training program

The purpose of this research was to examine how an 11-day taper after an 8.5-week experimental training cycle affected lactate levels during maximal exercise, mean force, and performance in training swimmers, independent of shaving, psychological changes, and postcompetition effects. Fourteen competition swimmers with shaved legs and torsos were recruited from the S?o Paulo Aquatic Federation. The training cycle consisted of a basic training period (endurance and quality phases) of 8.5 weeks, with 5,800 m.d(-1) mean training volume and 6 d.wk(-1) frequency; and a taper period (TP) of 1.5 weeks' duration that incorporated a 48% reduction in weekly volume without altering intensity. Attained swimming force (SF) and maximal performance over 200-m maximal swim (Pmax) before and after taper were measured. After taper, SF and Pmax improved 3.6 and 1.6%, respectively (p < 0.05). There were positive correlations (p < 0.05) between SF and Pmax before (r = 0.86) and after (r = 0.83) the taper phase. Peak lactate concentrations after SF were unaltered before (6.79 +/- 1.2 mM) and after (7.15 +/- 1.8 mM) TP. Results showed that TP improved mean swimming velocity, but not in the same proportion as force after taper, suggesting that there are other factors influencing performance in faster swimming.     

Morphological events during the Trypanosoma cruzi cell cycle

The replication and segregation of organelles producing two identical daughter cells must be precisely controlled during the cell cycle progression of eukaryotes. In kinetoplastid flagellated protozoa, this includes the duplication of the single mitochondrion containing a network of DNA, known as the kinetoplast, and a flagellum that grows from a cytoplasmic basal body through the flagellar pocket compartment before emerging from the cell. Here, we show the morphological events and the timing of these events during the cell cycle of the epimastigote form of Trypanosoma cruzi, the protozoan parasite that causes Chagas' disease. DNA staining, flagellum labeling, bromodeoxyuridine incorporation, and ultra-thin serial sections show that nuclear replication takes 10% of the whole cell cycle time. In the middle of the G2 stage, the new flagellum emerges from the flagellar pocket and grows unattached to the cell body. While the new flagellum is still short, the kinetoplast segregates and mitosis occurs. The new flagellum reaches its final size during cytokinesis when a new cell body is formed. These precisely coordinated cell cycle events conserve the epimastigote morphology with a single nucleus, a single kinetoplast, and a single flagellum status of the interphasic cell.