The complex of Sphingomonas elodea ATCC 31461 glucose-1-phosphate uridylyltransferase with glucose-1-phosphate reveals a novel quaternary structure, unique among nucleoside diphosphate-sugar pyrophosphorylase members

Gellan gum is a widely used commercial material, available in many different forms. Its economic importance has led to studies into the biosynthesis of exopolysaccharide gellan gum, which is industrially prepared in high yields using Sphingomonas elodea ATCC 31461. Glucose-1-phosphate uridylyltransferase mediates the reversible conversion of glucose-1-phosphate and UTP into UDP-glucose and pyrophosphate, which is a key step in the biosynthetic pathway of gellan gums. Here we present the X-ray crystal structure of the glucose-1-phosphate uridylyltransferase from S. elodea. The S. elodea enzyme shares strong monomeric similarity with glucose-1-phosphate thymidylyltransferase, several structures of which are known, although the quaternary structures of the active enzymes are rather different. A detailed comparison between S. elodea glucose-1-phosphate uridylyltransferase and available thymidylyltransferases is described and shows remarkable structural similarities, despite the low sequence identities between the two divergent groups of proteins.     

Solanum melongena polyphenol oxidase biosensor for the electrochemical analysis of paracetamol

A new strategy for the construction of a polyphenol oxidase carbon paste biosensor for paracetamol detection is reported. The eggplant (Solanum melongena) was processed to collect the polyphenol oxidase as an enzyme that was incorporated in the carbon paste sensor construction. The constructed sensor displayed high sensitivity and good selection for paracetamol detection and recognition. Optimized conditions included pH 6.0 (highest activity), pH 7.0 (highest stability), pulse amplitude of 50?mV, and 15% of vegetable extract per carbon paste. The sensor displayed a linear range from 20 to 200?µM, with a detection limit of 5?µM. Application of the sensor to paracetamol determination in tablet and oral solutions have shown satisfactory results. The efficiency of the method showed very good repeatability ranging between 1.26 and 1.72% relative standard deviation for interday analysis, while recoveries for paracetamol varied between 97.5 and 99.8% for the voltammetric determination. The strategy for a simple, low cost, and efficient eggplant polyphenol oxidase sensor showcased in this work provides an opportunity for the detection of other phenolic compounds in various matrices.     

Photosynthesis in the water-stressed C4 grass Setaria sphacelata is mainly limited by stomata with both rapidly and slowly imposed water deficits

A comparison of the effects of a rapid and a slowly imposed water deficit on photosynthesis was performed in Setaria sphacelata var. splendida (Stapf) Clayton, a C₄ NADP‐ME grass. Gas exchange was measured in rapidly and slowly dehydrated adult leaves either under atmospheric CO₂ partial pressure with an infrared gas analyser or under saturating CO₂ partial pressure with a leaf disc oxygen electrode. These measurements were used to calculate stomatal and non‐stomatal limitations to photosynthesis. These were further investigated using modulated chlorophyll a fluorescence measurements and photosynthetic pigment quantification. The decrease of net photosynthesis, leaf conductance and water use efficiency was more pronounced under rapid stress than in slow stress. However, photosynthesis is always mainly limited by stomata in both types of stress, albeit the contribution of non‐stomatal limitations increases at severe water deficits in slow stress experiments. The substomatal CO₂ partial pressure significantly increased in both types of stress, suggesting an increased resistance due to an internal barrier to CO₂ diffusion. Physical alterations in the structure of the intercellular spaces due to leaf shrinkage may account for these results. The maximal photochemical efficiency of photosystem II (PSII) was remarkably resistant to stress, as the F_v/F_m ratio decreased only at severe water deficit. On the contrary, the effective photochemical efficiency of PSII (ΔF/F′_m) measured under high actinic light decreased linearly in both types of stress, although in a more pronounced way under rapid stress. A similar variation in photochemical quenching suggests that the decrease of ΔF/F′_m is mainly due to the closure of PSII reaction centres. The non‐photochemical quenching did not change significantly except under severe dehydration indicating that the energization state of thylakoids remained stable under stress. The decrease observed in photosynthetic pigments may be an adaptation to stress rather than a limiting factor to photosynthesis. Results suggests that, although intrinsic mesophyll metabolic inhibitions occur, stomatal limitation to CO₂ diffusion is the main reason for the decrease in photosynthesis.     

Combining fluorescence lifetime and polarization microscopy to discriminate phase separated domains in giant unilamellar vesicles

Using fluorescence lifetime microscopy we study the structure of lipid domains in giant unilamellar vesicles made from sphingomyelin, 1,2-dioleoyl-sn-glycero-3-phosphocholine, and cholesterol. Lifetimes and orientation of a derivative of the fluorescent probe DPH embedded in the membrane were measured for binary and ternary lipid mixtures incorporating up to 42 mol % of cholesterol. The results show that adding cholesterol always increases the lifetime of the probe studied. In addition, the analysis of the probe orientation indicates that cholesterol has little influence on the ordering of the sphingomyelin alkyl chains whereas it has a noticeable effect on the structure of the 1,2-dioleoyl-sn-glycero-3-phosphocholine chains. The measurements made on the orientation and lifetime of the probe show the structure of the membrane in its liquid ordered and liquid disordered domains.     

Arthropod Biodiversity in the Canopy of Vochysia divergens (Vochysiaceae), a Forest Dominant in the Brazilian Pantanal

The canopy of an invasive tree species in the Pantanal floodplains, Vochysia divergens Pohl (Vochysiaceae; height 12 m) was sprayed once with 5% synthetic pyrethrum during the dry, non-flooded season near Poconé, Mato Grosso. About 4200 arthropods collected (43.7 ind./m 2) represented 14 orders. The Hymenoptera dominated (21.4 ind./ 2 ; mostly ants), followed by Coleoptera, Araneae and Diptera (85% of the total catch). Of the 26 Coleoptera families (6.2 ind./ 2) the Curculionidae, Scolytidae, Elateridae and Dryopidae were most abundant (70% of the total catch), and on trophic level the herbivores dominated (42%). This is the first quantitative canopy study of a tree species in the northern Pantanal floodplains. The type of data needed on arthropods from tree canopies in future studies to aid in the management and conservation of Pantanal floodplains is discussed.     

Antioxidant activities of sulfated polysaccharides from brown and red seaweeds

The in vitro antioxidant activities of the following six sulfated polysaccharides were investigated: iota, kappa and lambda carrageenans, which are widely used in the food industry, fucoidan (homofucan) from the edible seaweed Fucus vesiculosus and fucans (heterofucans) F0.5 and F1.1 from the seaweed Padina gymnospora. With respect to the inhibition of superoxide radical formation, fucoidan had an IC₅₀ (the half maximal inhibitory concentration) of 0.058 mg·mL⁻¹, while the IC₅₀ for the kappa, iota and lambda carrageenans were 0.112, 0.332 and 0.046 mg·mL⁻¹, respectively. All of the samples had an inhibitory effect on the formation of hydroxyl radicals. The results of peroxidation tests showed that fucoidan had an IC₅₀ of 1.250 mg·mL⁻¹ and that the kappa, iota and lambda carrageenans had an IC₅₀ of 2.753 and 2.338 and 0.323 mg·mL⁻¹, respectively. Fucan fractions showed low antioxidant activity relative to fucoidan. These results clearly indicate the beneficial effect of algal polysaccharides as antioxidants.     

Defining Immune Engagement Thresholds for In Vivo Control of Virus-Driven Lymphoproliferation

Persistent infections are subject to constant surveillance by CD8⁺ cytotoxic T cells (CTL). Their control should therefore depend on MHC class I-restricted epitope presentation. Many epitopes are described for γ-herpesviruses and form a basis for prospective immunotherapies and vaccines. However the quantitative requirements of in vivo immune control for epitope presentation and recognition remain poorly defined. We used Murid Herpesvirus-4 (MuHV-4) to determine for a latently expressed viral epitope how MHC class-I binding and CTL functional avidity impact on host colonization. Tracking MuHV-4 recombinants that differed only in epitope presentation, we found little latitude for sub-optimal MHC class I binding before immune control failed. By contrast, control remained effective across a wide range of T cell functional avidities. Thus, we could define critical engagement thresholds for the in vivo immune control of virus-driven B cell proliferation.