Thermodynamics of glutathione binding to the tyrosine 7 to phenylalanine mutant of glutathione S-transferase from Schistosoma japonicum

The binding of glutathione (GSH) to the tyrosine 7 to phenylalanine mutant of Schistosoma japonicum glutathione S-transferase (SjGST-Y7F) has been studied by isothermal titration calorimetry (ITC). At pH 6.5 and 25 °C this mutant shows a higher affinity for glutathione than wild type enzyme despite an almost complete loss of activity in the presence of 1-chloro-2,4-dinitrobenzene (CDNB) as second substrate. The enthalpy change upon binding of GSH is more negative for the mutant than for the wild type GST (SjGST). Changes in accessible solvent areas (ASA) have been calculated based on enthalpy and heat capacity changes. ASA values indicated the burial of apolar surfaces of protein and ligand upon binding. A more negative ΔC_p value has been obtained for the mutant enzyme, suggesting a more hydrophobic interaction, as may be expected from the change of a tyrosine residue to phenylalanine.     

Synthesis, protonation behavior, conformational analysis, and regioselective enzymatic acylation of the novel diamino analogue of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU)

(E)-3',5'-Diamino-5-(2-bromovinyl)-2',3',5'-trideoxyuridine (5), the diamino analogue of BVDU (1), was synthesized from BVDU. The protonation behavior of 5 has been studied by means of pH-metric measurements and NMR spectroscopy. This study allows the determination of the basicity constants and the stepwise protonation sites. Thus, the main species at physiological pH is the monoprotonated form. The conformational analysis of this nucleoside analogue was also carried out through 1H NMR spectroscopy. In addition, a convenient synthesis of N-3' and N-5' acylated derivatives was developed by regioselective enzymatic acylation. Thus, Candida antarctica lipase B (CAL-B) selectively acylated the 5'-amino group, thus furnishing nucleosides 8. On the other hand, immobilized Pseudomonas cepacia lipase (PSL-C) exhibited the opposite selectivity, conferring acylation at the 3'-amino group, thus affording derivatives 9.     

Modelling the production and transport of dissolved organic carbon in forest soils

DyDOC describes soil carbon dynamics, with a focus on dissolved organic carbon (DOC). The model treats the soil as a three-horizon profile, and simulates metabolic carbon transformations, sorption reactions and water transport. Humic substances are partitioned into three fractions, one of which is immobile, while the other two (hydrophilic and hydrophobic) can pass into solution as DOC. DyDOC requires site-specific soil characteristics, and is driven by inputs of litter and water, and air and soil temperatures. The model operates on hourly and daily time steps, and can simulate carbon cycling over both long (hundreds-to-thousands of years) and short (daily) time scales. An important feature of DyDOC is the tracking of ¹⁴C, from its entry in litter to its loss as DO¹⁴C in drainage water, enabling information about C dynamics to be obtained from both long-term radioactive decay, and the characteristic ¹⁴C pulse caused by thermonuclear weapon testing during the 1960s ("bomb carbon"). Parameterisation is performed by assuming a current steady state. Values of a range of variables, including C pools, annual DOC fluxes, and ¹⁴C signals, are combined into objective functions for least-squares minimisation. DyDOC has been applied successfully to spruce forest sites at Birkenes (Norway) and Waldstein (Germany), and most of the parameters have similar values at the two sites. The results indicate that the supply of DOC from the surface soil horizon to percolating water depends upon the continual metabolic production of easily leached humic material. In contrast, concentrations and fluxes of DOC in the deeper soil horizons are controlled by sorption processes, involving comparatively large pools of leachable organic matter. Times to reach steady state are calculated to be several hundred years in the organic layer, and hundreds-to-thousands of years in the deeper mineral layers. It is estimated that DOC supplies 89% of the mineral soil carbon at Birkenes, and 73% at Waldstein. The model, parameterised with "steady state" data, simulates short-term variations in DOC concentrations and fluxes, and in DO¹⁴C, which are in approximate agreement with observations.     

Influence of saline stress on root hydraulic conductance and PIP expression in Arabidopsis

Measurements of the root hydraulic conductance (L0) of roots of Arabidopsis thaliana were carried out and the results were compared with the expression of aquaporins present in the plasma membrane of A. thaliana. L0 of plants treated with different NaCl concentrations was progressively reduced as NaCl concentration was increased compared to control plants. Also, L0 of plants treated with 60 mmol/L NaCl for different lengths of time was measured. Variations during the light period were seen, but only for the controls. A good correlation between mRNA expression and L0 was observed in both experiments. Control plants and plants treated with 60 mmol/L NaCl were incubated with Hg and then with DTT. For these plants, L0 and cell-to-cell pathway contributions to root water transport were determined. These results revealed that in control plants most water movement occurs via the cell-to-cell pathway, thus implying aquaporin involvement. But, in NaCl-stressed plants, the Hg-sensitive cell-to-cell pathway could be inhibited already by the effect of NaCl on water channels. Therefore, short periods of NaCl application to Arabidopsis plants are characterised by decreases in the L0 of roots, and are related to down-regulation of the expression of the PIP aquaporins. This finding indicates that the well known effect of salinity on L0 could involve regulation of aquaporin expression.     

Different blocking effects of HgCl2 and NaCl on aquaporins of pepper plants

In this study we have compared the short-term effects of both NaCl and HgCl₂ on aquaporins of Capsicum annuum L. plants, in order to determine whether or not they are similar. Stomatal conductance, turgor, root hydraulic conductance and water status were measured after 0.5, 2, 4 and 6 h of NaCl (60 mmol/L) or HgCl₂ (50 μmol/L) treatment. When 60 mmol/L NaCl was added to the nutrient solution, a large decrease in stomatal conductance was observed after 2 h. However, when HgCl₂ (50 μmol/L) was added, the decrease occurred after 4 h. The number of open stomata closed was always lower in plants treated with HgCl₂ than in plants treated with NaCl. The water content of the Hg²⁺-treated plants was decreased, compared with controls and NaCl-treated. The root hydraulic conductance decreased after HgCl₂ and NaCl treatment plants. Turgor of leaf epidermal cells was greatly reduced in plants treated with HgCl₂, but remained constant in the NaCl treatment, compared with control plants. The fact that the stomatal conductance was reduced more rapidly after NaCl addition, followed by the stomatal closure, and that both water content and turgor did not differ from the control suggests that in NaCl-treated plants there must be a signal moving from root to shoot. Therefore, the control of plant homeostasis through a combined regulation of root and stomatal exchanges may be dependent on aquaporin regulation.     

Role of the carboxy terminus of Escherichia coli FtsA in self-interaction and cell division

The role of the carboxy terminus of the Escherichia coli cell division protein FtsA in bacterial division has been studied by making a series of short sequential deletions spanning from residue 394 to 420. Deletions as short as 5 residues destroy the biological function of the protein. Residue W415 is essential for the localization of the protein into septal rings. Overexpression of the ftsA alleles harboring these deletions caused a coiled cell phenotype previously described for another carboxy-terminal mutation (Gayda et al., J. Bacteriol. 174:5362-5370, 1992), suggesting that an interaction of FtsA with itself might play a role in its function. The existence of such an interaction was demonstrated using the yeast two-hybrid system and a protein overlay assay. Even these short deletions are sufficient for impairing the interaction of the truncated FtsA forms with the wild-type protein in the yeast two-hybrid system. The existence of additional interactions between FtsA molecules, involving other domains, can be postulated from the interaction properties shown by the FtsA deletion mutant forms, because although unable to interact with the wild-type and with FtsADelta1, they can interact with themselves and cross-interact with each other. The secondary structures of an extensive deletion, FtsADelta27, and the wild-type protein are indistinguishable when analyzed by Fourier transform infrared spectroscopy, and moreover, FtsADelta27 retains the ability to bind ATP. These results indicate that deletion of the carboxy-terminal 27 residues does not alter substantially the structure of the protein and suggest that the loss of biological function of the carboxy-terminal deletion mutants might be related to the modification of their interacting properties.     

Thubunaea dactyluris Sensu Fabio and Rolas, a synonym of Physalopteroides venancioi (Spirurida, Physalopteridae)

Specimens of Thubunaea dactyluris Karve, 1938 sensu Fabio and Rolas, 1974 from the lizard Ameiva ameiva of Brazil and types of Physalopteroides venancioi (Lent, Freitas, and Proen?a, 1946) from the toad Bufo paracnemis of Paraguay were re-examined. The male caudal papillae and the tooth arrangement demonstrated that these specimens were conspecific and are all assigned to P. venancioi. The occurrence of Physalopteroides dactyluris (Karve, 1938), a nematode parasite of lizards of India and Turkmenistan, is invalidated for Brazil.     

Localization of Cell Division Protein FtsQ by Immunofluorescence Microscopy in Dividing and Nondividing Cells of Escherichia coli

The localization of cell division protein FtsQ in Escherichia coli wild-type cells was studied by immunofluorescence microscopy with specific monoclonal antibodies. FtsQ could be localized to the division site in constricting cells. FtsQ could also localize to the division site in ftsQ1(Ts) cells grown at the permissive temperature. A hybrid protein in which the cytoplasmic domain and the transmembrane domain were derived from the γ form of penicillin-binding protein 1B and the periplasmic domain was derived from FtsQ was also able to localize to the division site. This result indicates that the periplasmic domain of FtsQ determines the localization of FtsQ, as has also been concluded by others for the periplasmic domain of FtsN. Noncentral FtsQ foci were found in the area of the cell where the nucleoid resides and were therefore assumed to represent sites where the FtsQ protein is synthesized and simultaneously inserted into the cytoplasmic membrane.     

Relationships between phenolics-conjugated polyamines and sensitivity of sugarcane to smut (Ustilago scitaminea)

Infection of sugarcane buds (var. Barbados 42231) with teliospores of Ustilago scitaminea changes the pattern of polyamine conjugation in several organs of 2-month-old plants. Stalks of infected plants contain SH-spermidine that does not occur in the healthy organ. Similar results have been obtained for SH-spermine in the first expanded leaf and in the stem. The amount of SH-cadaverine in the first expanded leaf, roots and stem of infected plants is always higher than that found for healthy plants. Some phenolics are also associated with different polyamine fractions. So, the amount of p-hydroxybenzoic acid in both SH and PH fractions of polyamines extracted from the root increases after infection. Syringic acid is the main phenol associated with the PH fraction in the first expanded leaf of infected plants, whereas this phenol is mainly associated with both SH and PH fractions isolated from the stem and the whip. Infection enhances conjugation of p-courmaric acid to PH polyamines, whereas caffeic acid appears in the SH fraction in leaf, root and stem. However, ferulic acid seems to be the main hydroxycinnamic acid derivative in the whip. Chlorogenic acid is associated with the SH fraction from the stem of healthy plants although this changes to free phenolics after infection.Key words:Saccharum officinarum, Ustilago sciaminea, phenolics, polyamines.      

Light‐dependent changes of tomato glutamine synthetase in response to Pseudomonas syringae infection or phosphinothricin treatment

Glutamine synthetase (GS, EC 6.3.1.2), a key enzyme in nitrogen assimilation, was investigated in tomato ( Lycopersicon esculentum Mill. cv. Hellfrucht Frühstamm) leaves infected with Pseudomonas syringae pv. tomato (Pst) or treated with the herbicide phosphinothricin (PPT), an irreversible inhibitor of GS. GS activity decreased markedly when Pst infection occurred in illuminated leaves, but only a slight decrease in relation to control leaves was observed under non‐photosynthetic conditions. In leaves treated with PPT, a rapid inhibition of GS activity was observed under all experimental conditions. When bacterial infection or herbicide treatment was carried out in the light, cytosolic GS (GS1) appeared as the predominant GS polypeptide; however, under non‐photosynthetic conditions GS2 remained the most abundant molecular GS species as occurs in non‐stressed plants. These results suggest a close correlation between the photosynthetic process and changes in the relative proportions of GS polypeptides during infection or herbicide treatment. Ammonium has been described as an inducer of GS genes, but as ammonium accumulated during all treatments, other light‐dependent factors could be involved in GS regulation of stressed leaves.