Lipase of Pseudomonas cepacia for biotechnological purposes: purification,crystallization and characterization

Commercial lipase (triacylglycerol lipase, EC 3.1.1.3) of Pseudomonas cepacia (Amano) has been purified to homogeneity by a single chromatography on phenyl Sepharose. The eluted lipase crystallized spontaneously at 4°C in the eluent, containing 58–69% 2-propanol. The yield of the lipase was 87–100% and the specific activity during the hydrolysis of triolein 5800 U/mg protein. This protein has a molecular weight of 34.1 kDa as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Its purity was determined by SDS-Page and capillary zone electrophoresis to be ≥ 99%. Immobilization on Sepharose increased its stability in organic solvents. This lipase of P. cepacia differs from that of other Pseudomonas strains in respect of substrate specificity and during crystallization. It exhibits a high stability in organic solvents and supercritical carbon dioxide.     

Distinguishing Family from Friends

Kinship and friendship are key human relationships. Increasingly, data suggest that people are not less altruistic toward friends than close kin. Some accounts suggest that psychologically we do not distinguish between them; countering this is evidence that kinship provides a unique explanatory factor. Using the Implicit Association Test, we examined how people implicitly think about close friends versus close kin in three contexts. In Experiment 1, we examined generic attitudinal dispositions toward friends and family. In Experiment 2, attitude similarity as a marker of family and friends was examined, and in Experiments 3 and 4, strength of in-group membership for family and friends was examined. Findings show that differences exist in implicit cognitive associations toward family and friends. There is some evidence that people hold more positive general dispositions toward friends, associate attitude similarity more with friends, consider family as more representative of the in-group than friends, but see friends as more in-group than distant kin.     

Novel sulfenamides and sulfonamides based on pyridazinone and pyridazine scaffolds as CB1 receptor ligand antagonists

A series of sulfenamide and sulfonamide derivatives was synthesized and evaluated for the affinity at CB₁ and CB₂ receptors. The N-bornyl-S-(5,6-di-p-tolylpyridazin-3-yl)-sulfenamide, compound 11, displayed good affinity and high selectivity for CB₁ receptors (K_i values of 44.6?nM for CB₁ receptors and >40?μM for CB₂ receptors, respectively). The N-isopinocampheyl-sulfenamide 12 and its sulfonamide analogue 22 showed similar selectivity for CB₁ receptors with K_i values of 75.5 and 73.2?nM, respectively. These novel compounds behave as antagonists/inverse agonists at CB₁ receptor in the [³⁵S]-GTPγS binding assays, and none showed adequate predictive blood–brain barrier permeation, exhibiting low estimated LD₅₀. However, testing compound 12 in a supraspinal analgesic test (hot-plate) revealed that it was as effective as the classic CB₁ receptor antagonist rimonabant, in reversing the analgesic effect of a cannabinoid agonist.     

Raf-1 Cysteine-Rich Domain Increases the Affinity of K-Ras/Raf at the Membrane,Promoting MAPK Signaling

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Trackways of the American Crocodile (Crocodylus acutus) in Northwestern Costa Rica: Implications for Crocodylian Ichnology

We documented trackways of free-living Crocodylus acutus on beaches at the mouths of Tamarindo and Ventanas estuaries, Costa Rica. Our crocodiles had estimated total lengths of 1–3 meters or more. Manus prints have five digits, with digits I–III bearing claw marks. Pes prints have four digits, with claw marks on digits I–III. The pes is plantigrade. Claws generally dig into the substrate. Apart from claw marks, digit I and the heel of the pes are usually the most deeply impressed parts of footprints. Trackways are wide-gauge. Pes prints are usually positioned just behind ipsilateral manus prints of the same set and may overlap them. Manus and pes prints angle slightly outward with respect to the crocodile's direction of movement. Claw-bearing digits of both the manus and pes may create curved, concave-toward-the-midline drag marks as the autopodium is protracted. The tail mark varies in depth and clarity, and in shape from nearly linear to markedly sinuous. Sometimes the tail mark hugs the trackway midline, but sometimes it is closer to, or even cuts across, prints of one side. American crocodile footprints and trackways are similar to those observed in other extant crocodylian species, indicating substantial trackway conservatism across the group.     

Seed limitation,not soil legacy effects,prevents native understorey from establishing in oak woodlands in Scotland after removal of Rhododendron ponticum

Following removal of the invasive species Rhododendron ponticum, the native understorey plant community typically fails to reestablish itself. Potential explanations for this failure include (1) lack of an appropriate native seed source; (2) inability of seed to penetrate a dense bryophyte layer; and (3) persistence of chemical “legacy effects” in the soil. We established an experiment to test these competing hypotheses in an Atlantic oak woodland where R. ponticum had been removed. The following experimental treatments were applied singly and in combination: (1) addition of a native seed mix to test for seed limitation; (2) removal of the established ground vegetation at the start of the experiment (which principally consisted of bryophytes) to test for the impact of a barrier layer; (3) addition of activated carbon to test for chemical legacy effects in the soil; and (4) fertilization as an additional measure to promote the establishment of native vascular plants. Application of the native seed mix was revealed to be an effective way to increase the cover of native vascular plants and was particularly effective when applied after the removal of the bryophyte layer. The application of activated carbon and/or fertilizer, however, had no effect on the cover of native vegetation. We conclude that reports of R. ponticum exerting chemical legacy effects long after its removal may have been overstated and that seed limitation and inability to successfully establish in a dense bryophyte layer provided the strongest barriers to natural recolonization by the native plant community following R. ponticum removal.     

Physiological and proteomic analyses of Fe(III)‐reducing co‐cultures of Desulfotomaculum reducens MI‐1 and Geobacter sulfurreducens PCA

We established Fe(III)‐reducing co‐cultures of two species of metal‐reducing bacteria, the Gram‐positive Desulfotomaculum reducens MI‐1 and the Gram‐negative Geobacter sulfurreducens PCA. Co‐cultures were given pyruvate, a substrate that D. reducens can ferment and use as electron donor for Fe(III) reduction. G. sulfurreducens relied upon products of pyruvate oxidation by D. reducens (acetate, hydrogen) for use as electron donor in the co‐culture. Co‐cultures reduced Fe(III) to Fe(II) robustly, and Fe(II) was consistently detected earlier in co‐cultures than pure cultures. Notably, faster cell growth, and correspondingly faster pyruvate oxidation, was observed by D. reducens in co‐cultures. Global comparative proteomic analysis was performed to observe differential protein abundance during co‐culture vs. pure culture growth. Proteins previously associated with Fe(III) reduction in G. sulfurreducens, namely c‐type cytochromes and type IV pili proteins, were significantly increased in abundance in co‐cultures relative to pure cultures. D. reducens ribosomal proteins were significantly increased in co‐cultures, likely a reflection of faster growth rates observed for D. reducens cells while in co‐culture. Furthermore, we developed multiple reaction monitoring (MRM) assays to quantitate specific biomarker peptides. The assays were validated in pure and co‐cultures, and protein abundance ratios from targeted MRM and global proteomic analysis correlate significantly.     

Local and global anatomy of antibody‐protein antigen recognition

Deciphering antibody‐protein antigen recognition is of fundamental and practical significance. We constructed an antibody structural dataset, partitioned it into human and murine subgroups, and compared it with nonantibody protein‐protein complexes. We investigated the physicochemical properties of regions on and away from the antibody‐antigen interfaces, including net charge, overall antibody charge distributions, and their potential role in antigen interaction. We observed that amino acid preference in antibody‐protein antigen recognition is entropy driven, with residues having low side‐chain entropy appearing to compensate for the high backbone entropy in interaction with protein antigens. Antibodies prefer charged and polar antigen residues and bridging water molecules. They also prefer positive net charge, presumably to promote interaction with negatively charged protein antigens, which are common in proteomes. Antibody‐antigen interfaces have large percentages of Tyr, Ser, and Asp, but little Lys. Electrostatic and hydrophobic interactions in the Ag binding sites might be coupled with Fab domains through organized charge and residue distributions away from the binding interfaces. Here we describe some features of antibody‐antigen interfaces and of Fab domains as compared with nonantibody protein‐protein interactions. The distributions of interface residues in human and murine antibodies do not differ significantly. Overall, our results provide not only a local but also a global anatomy of antibody structures.