Inteins in pathogenic fungi: a phylogenetic tool and perspectives for therapeutic applications

Inteins or "internal proteins" are coding sequences that are transcribed and translated with flanking sequences (exteins). After translation, the inteins are excised by an autocatalytic process and the host protein assumes its normal conformation and develops its expected function. These parasitic genetic elements have been found in important, conserved proteins in all three domains of life. Most of the eukaryotic inteins are present in the fungi kingdom and the PRP8 intein is one of the most widespread inteins, occurring in important pathogens such as Cryptococcus neoformans (varieties grubii and neoformans), Cryptococcus gattii, Histoplasma capsulatum and Paracoccidioides brasiliensis. The knowledge of conserved and non-conserved domains in inteins have opened up new opportunities for the study of population variability in pathogenic fungi, including their phylogenetic relationships and recognition or diagnoses of species. Furthermore, inteins in pathogenic fungi should also be considered a promising therapeutic drug target, since once the autocatalytic splicing is inhibited, the host protein, which is typically vital, will not be able to perform its normal function and the fungal cell will not survive or reproduce.

Expression of bacterial virulence factors and cytokines during in vitro macrophage infection by enteroinvasive Escherichia coli and Shigella flexneri: a comparative study

Enteroinvasive Escherichia coli (EIEC) and Shigellaspp cause bacillary dysentery in humans by invading and multiplying within epithelial cells of the colonic mucosa. Although EIEC and Shigellashare many genetic and biochemical similarities, the illness caused by Shigellais more severe. Thus, genomic and structure-function molecular studies on the biological interactions of these invasive enterobacteria with eukaryotic cells have focused on Shigella rather than EIEC. Here we comparatively studied the interactions of EIEC and of Shigella flexneriwith cultured J774 macrophage-like cells. We evaluated several phenotypes: (i) bacterial escape from macrophages after phagocytosis, (ii) macrophage death induced by EIEC and S. flexneri, (iii) macrophage cytokine expression in response to infection and (iv) expression of plasmidial (pINV) virulence genes. The results showed thatS. flexneri caused macrophage killing earlier and more intensely than EIEC. Both pathogens induced significant macrophage production of TNF, IL-1 and IL-10 after 7 h of infection. Transcription levels of the gene invasion plasmid antigen-C were lower in EIEC than in S. flexneri throughout the course of the infection; this could explain the diminished virulence of EIEC compared to S. flexneri.

Prokaryotic cells: structural organisation of the cytoskeleton and organelles

For many years, prokaryotic cells were distinguished from eukaryotic cells based on the simplicity of their cytoplasm, in which the presence of organelles and cytoskeletal structures had not been discovered. Based on current knowledge, this review describes the complex components of the prokaryotic cell cytoskeleton, including (i) tubulin homologues composed of FtsZ, BtuA, BtuB and several associated proteins, which play a fundamental role in cell division, (ii) actin-like homologues, such as MreB and Mb1, which are involved in controlling cell width and cell length, and (iii) intermediate filament homologues, including crescentin and CfpA, which localise on the concave side of a bacterium and along its inner curvature and associate with its membrane. Some prokaryotes exhibit specialised membrane-bound organelles in the cytoplasm, such as magnetosomes and acidocalcisomes, as well as protein complexes, such as carboxysomes. This review also examines recent data on the presence of nanotubes, which are structures that are well characterised in mammalian cells that allow direct contact and communication between cells.

Distinct subcellular localization of tRNA-derived fragments in the infective metacyclic forms of Trypanosoma cruzi

Small non-coding RNAs derived from transfer RNAs have been identified as a broadly conserved prokaryotic and eukaryotic response to stress. Their presence coincides with changes in developmental state associated with gene expression regulation. In the epimastigote form of Trypanosoma cruzi, tRNA fragments localize to posterior cytoplasmic granules. In the infective metacyclic form of the parasite, we found tRNA-derived fragments to be abundant and evenly distributed within the cytoplasm. The fragments were not associated with polysomes, suggesting that the tRNA-derived fragments may not be directly involved in translation control in metacyclics.

Mycobacterial laminin-binding histone-like protein mediates collagen-dependent cytoadherence

When grown in the presence of exogenous collagen I, Mycobacterium bovis BCG was shown to form clumps. Scanning electron microscopy examination of these clumps revealed the presence of collagen fibres cross-linking the bacilli. Since collagen is a major constituent of the eukaryotic extracellular matrices, we assayed BCG cytoadherence in the presence of exogenous collagen I. Collagen increased the interaction of the bacilli with A549 type II pneumocytes or U937 macrophages, suggesting that BCG is able to recruit collagen to facilitate its attachment to host cells. Using an affinity chromatography approach, we have isolated a BCG collagen-binding protein corresponding to the previously described mycobacterial laminin-binding histone-like protein (LBP/Hlp), a highly conserved protein associated with the mycobacterial cell wall. Moreover, Mycobacterium leprae LBP/Hlp, a well-characterized adhesin, was also able to bind collagen I. Finally, using recombinant fragments of M. leprae LBP/Hlp, we mapped the collagen-binding activity within the C-terminal domain of the adhesin. Since this protein was already shown to be involved in the recognition of laminin and heparan sulphate-containing proteoglycans, the present observations reinforce the adhesive activities of LBP/Hlp, which can be therefore considered as a multifaceted mycobacterial adhesin, playing an important role in both leprosy and tuberculosis pathogenesis.

Nitrogen washing from C3 and C4 cover grasses residues by rain

Crop species with the C4 photosynthetic pathway are more efficient in assimilating N than C3 plants, which results in different N amounts prone to be washed from its straw by rain water. Such differences may affect N recycling in agricultural systems where these species are grown as cover crops. In this experiment, phytomass production and N leaching from the straw of grasses with different photosynthetic pathways were studied in response to N application. Pearl millet (Pennisetum glaucum) and congo grass (Brachiaria ruziziensis) with the C4 photosynthetic pathway, and black oat (Avena Strigosa) and triticale (X Triticosecale), with the C3 photosynthetic pathway, were grown for 47 days. After determining dry matter yields and N and C contents, a 30 mm rainfall was simulated over 8 t ha-1 of dry matter of each plant residue and the leached amounts of ammonium and nitrate were determined. C4 grasses responded to higher fertilizer rates, whereas N contents in plant tissue were lower. The amount of N leached from C4 grass residues was lower, probably because the C/N ratio is higher and N is more tightly bound to organic compounds. When planning a crop rotation system it is important to take into account the difference in N release of different plant residues which may affect N nutrition of the subsequent crop.

Photosynthesis rate, chlorophyll content and initial development of physic nut without micronutrient fertilization

Few studies in Brazil have addressed the need for micronutrients of physic nut focusing on physiological responses, especially in terms of photosynthesis. The objective of this study was to evaluate the effects of omission of boron (B), copper (Cu), iron (Fe), manganese (Mn) and zinc (Zn) on Jatropha curcas L.. The experimental design was a randomized block with four replications. The treatments were complete solution (control) and solution without B, Cu, Fe, Mn, and Zn. We evaluated the chlorophyll content (SPAD units), photosynthetic rate, dry matter production and accumulation of micronutrients in plants, resulting from different treatments. The first signs of deficiency were observed for Fe and B, followed by Mn and Zn, while no symptoms were observed for Cu deficiency. The micronutrient omission reduced the dry matter yield, chlorophyll content and photosynthetic rate of the plants differently for each omitted nutrient. It was, however, the omission of Fe that most affected the development of this species in all parameters evaluated. The treatments negatively affected the chlorophyll content, evaluated in SPAD units, and the photosynthetic rate, except for the omission of B. However this result was probably due to the concentration effect, since there was a significant reduction in the dry matter production of B-deficient plants.

Chemical and biological properties of phosphorus-fertilized soil under legume and grass cover (Cerrado region, Brazil)

The use of cover crops has been suggested as an effective method to maintain and/or increase the organic matter content, while maintaining and/or enhancing the soil physical, chemical and biological properties. The fertility of Cerrado soils is low and, consequently, phosphorus levels as well. Phosphorus is required at every metabolic stage of the plant, as it plays a role in the processes of protein and energy synthesis and influences the photosynthetic process. This study evaluated the influence of cover crops and phosphorus rates on soil chemical and biological properties after two consecutive years of common bean. The study analyzed an Oxisol in Selvíria (Mato Grosso do Sul, Brazil), in a randomized block, split plot design, in a total of 24 treatments with three replications. The plot treatments consisted of cover crops (millet, pigeon pea, crotalaria, velvet bean, millet + pigeon pea, millet + crotalaria, and millet + velvet bean) and one plot was left fallow. The subplots were represented by phosphorus rates applied as monoammonium phosphate (0, 60 and 90 kg ha-1 P2O5). In August 2011, the soil chemical properties were evaluated (pH, organic matter, phosphorus, potential acidity, cation exchange capacity, and base saturation) as well as biological variables (carbon of released CO2, microbial carbon, metabolic quotient and microbial quotient). After two years of cover crops in rotation with common bean, the cover crop biomass had not altered the soil chemical properties and barely influenced the microbial activity. The biomass production of millet and crotalaria (monoculture or intercropped) was highest. The biological variables were sensitive and responded to increasing phosphorus rates with increases in microbial carbon and reduction of the metabolic quotient.

EFFECTS OF SILICON ON ALLEVIATING ARSENIC TOXICITY IN MAIZE PLANTS

Arsenic is a metalloid highly toxic to plants and animals, causing reduced plant growth and various health problems for humans and animals. Silicon, however, has excelled in alleviating stress caused by toxic elements in plants. The aim of this study was to investigate the effects of Si in alleviating As stress in maize plants grown in a nutrient solution and evaluate the potential of the spectral emission parameters and the red fluorescence (Fr) and far-red fluorescence (FFr) ratio obtained in analysis of chlorophyll fluorescence in determination of this interaction. An experiment was carried out in a nutrient solution containing a toxic rate of As (68 μmol L-1) and six increasing rates of Si (0, 0.25, 0.5, 1.0, 1.5, and 2.0 mmol L-1). Dry matter production and concentrations of As, Si, and photosynthetic pigments were then evaluated. Chlorophyll fluorescence was also measured throughout plant growth. Si has positive effects in alleviating As stress in maize plants, evidenced by the increase in photosynthetic pigments. Silicon application resulted in higher As levels in plant tissue; therefore, using Si for soil phytoremediation may be a promising choice. Chlorophyll fluorescence analysis proved to be a sensitive tool, and it can be successfully used in the study of the ameliorating effects of Si in plant protection, with the Fr/FFr ratio as the variable recommended for identification of temporal changes in plants.

Physiological responses of two tropical weeds to shade: II. Leaf gas exchange and nitrogen content

Ipomoea asarifolia (Desr.) Roem. & Schultz (Convolvulaceae) and Stachytarpheta cayennensis (Rich) Vahl. (Verbenaceae), two weeds found in pastures and crop areas in the Brazilian Amazonia, Brazil, were grown in controlled environment cabinets under high (800-1000 µmol m-² s-¹) and low (200-350 µmol m-² s-¹) light regimes during a 40-day period. The objective was to determine the effect of shade on photosynthetic features and leaf nitrogen content of I. asarifolia and S. cayennensis. High-irradiance grown I. asarifolia leaves had significantly higher dark respiration and light saturated rates of photosynthesis than low-irradiance leaves. No significant differences for these traits, between treatments, were observed in S. cayennensis. Low-irradiance leaves of both species displayed higher CO2 assimilation rates under low irradiance. High-irradiance grown leaves of both species had less nitrogen per unit of weight. Low-irradiance S. cayennensis had more nitrogen per unit of leaf area than high-irradiance plants; however, I. asarifolia showed no consistent pattern for this variable through time. For S. cayennensis, leaf nitrogen content and CO2 assimilation were inversely correlated to the amount of biomass allocated to developing reproductive structures. These results are discussed in relation to their ecological and weed management implications.

Molecular biology of baculovirus and its use in biological control in Brazil

Baculoviruses are insect viruses found mainly in Lepidoptera. The family Baculoviridae is taxonomically divided in two genera, Nucleopolyhedrovirus and Granulovirus, which differ by occlusion body morphology. NPVs (Nucleopolyhedroviruses) have polyhedrical inclusion bodies (PIBs) containing multiple viral particles, while GVs (Granuloviruses) appear to be generally single particles occluded in oval shaped occlusion bodies. During the life cycle, two different viral progenies are produced: BV (Budded Virus) and PDV (Polyhedra Derived Virus), which are essential for the infectious process and virus propagation in host cells. Baculoviruses are being used for pest control and they are especially safe due to their specificity and invertebrate-restricted host range. Baculoviruses have been used as vectors for high level protein expression ofheterologous genes from prokaryotic and eukaryotic organisms. Also, recombinant DNA techniques have allowed the production of genetically modified viral insecticides. This study is a review on the taxonomy, structure, replication and molecular biology of baculoviruses, as well as their use as bioinsecticides in Brazil.

Physiological and anatomical characteristics of leaves of two clones of guarana

The objective of this work was to analyze gas exchange, photosynthetic characteristics, photochemical efficiency of photosystem II and anatomical characteristics of young plant leaves of two guarana (Paullinia cupana) clones (BRS-CG372RC and BRS-CG611RL) growing under open field. The variables of gas exchange and fluorescence of chlorophyll a were evaluated in mature leaves. The values of photosynthesis and transpiration found for BRS-CG372RC were 27% greater and 80% lesser than values found for BRS-CG611RL, respectively. The values of stomatal conductance found for the clones BRS-CG372RC and BRS-CG611RL were in the order of 224 and 614 mmol mm-2 s-1, respectively. The values of photorespiration, rate of carboxylation and rate electron transport were greater in BRS-CG372RC. The clone BRS-CG372RC exhibited stomatal density 26% greater than BRS-CG611RL. However, the area of ostiolar opening was 42% greater in BRS-CG611RL. The values of the water use efficiency in BRS-CG372RC were 134% greater than in BRS-CG611RL. High stomatal density and low stomatal conductance can be important characteristics in the selection of the clones with a good ability to assimilate carbon and optimize the use of water.

Microclimatic characterization and productivity of coffee plants grown under shade of pigeon pea in Southern Brazil

Recent studies on coffee (Coffea arabica L.) cultivation in agroforestry systems in Southern Brazil have shown the potential of partial shading to improve management of this crop. The objective of this work was to evaluate microclimatic conditions and their effects on coffee production of plants shaded with pigeon pea (Cajanus cajan) in comparison to unshaded ones, from May 2001 to August 2002 in Londrina, State of Paraná, Brazil. The appraised microclimatic characteristics were: global radiation, photosynthetic and radiation balance; air, leaf and soil temperatures; and soil humidity. Shading caused significant reduction in incident global solar radiation, photosynthetically active radiation and net radiation, and attenuated maximum leaf, air and soil temperatures, during the day. Shade also reduced the rate of cooling of night air and leaf temperatures, especially during nights with radiative frost. Soil moisture at 0-10 cm depth was higher under shade. The shaded coffee plants produced larger cherries due to slower maturation, resulting in larger bean size. Nevertheless, plants under shade emitted less plagiotropic branches, with smaller number of nodes per branch, and fewer nodes with fruits, resulting in a large reduction in coffee production. These results show the need to find an optimal tree density and management that do not compromise coffee production and protect against extreme temperatures.

Physiological analysis of leaf senescence of two rice cultivars with different yield potential

The objective of this work was to evaluate the physiological changes that occur in different leaves during the early and late grain-filling stages of two rice genotypes (Oryza sativa subsp. indica , BRS Pelota cultivar, and O. sativa subsp. japonica , BRS Firmeza cultivar), which present differences in grain yield potential. The plants were cultivated in greenhouse. Pigment content, chlorophyll fluorescence, electron transport and oxygen evolution rate were determined in the grain-filling stage, from the first to the forth leaf (top to bottom). Pigment content, photochemical efficiency of photosystem II and electron transport decreased significantly according to the position of leaves in 'BRS Pelota'. The BRS Firmeza cultivar shows higher pigment content and higher activity of the photosynthetic apparatus in comparison to 'BRS Pelota' during the grain-filling stage.

Intercepted solar radiation by maize crops subjected to different tillage systems and water availability levels

The objective of this work was to evaluate changes in the photosynthetic photon flux density (PPFD) interception efficiency and PPFD extinction coefficient for maize crop subjected to different soil tillage systems and water availability levels. Crops were subjected to no-tillage and conventional tillage systems combined with full irrigation and non-irrigation treatments. Continuous measurements of transmitted PPFD on the soil surface and incoming PPFD over the canopy were taken throughout the crop cycle. Leaf area index and soil water potential were also measured during the whole period. Considering a mean value over the maize cycle, intercepted PPFD was higher in the conventional tillage than in the no-tillage system. During the initial stages of plants, intercepted PPFD in the conventional tillage was double the PPFD interception in the no-tillage treatment. However, those differences were reduced up to the maximum leaf area index, close to tasseling stage. The lowest interception of PPFD occurred in the conventional tillage during the reproductive period, as leaf senescence progressed. Over the entire crop cycle, the interception of PPFD by the non-irrigated plants was about 20% lower than by the irrigated plants. The no-tillage system reduced the extinction coefficient for PPFD, which may have allowed a higher penetration of solar radiation into the canopy