Neutralization of NGF-TrkA receptor interaction by the novel antagonistic anti-TrkA monoclonal antibody MNAC13: a structural insight

MNAC13, a mouse monoclonal antibody, recognizes with high affinity and specificity the neurotrophin receptor TrkA and displays a neutralizing activity toward the NGF/TrkA interaction. Detailed knowledge of the molecular basis determining the specificity of this antibody is of importance because of its potential use as a modulator of the TrkA-mediated NGF activity. Here, we report a full biochemical and structural characterization of the MNAC13 antibody. Epitope mapping studies, by serial deletion mutants and by phage display, reveal a conformational epitope that is localized on the carboxy-terminal region of the first immunoglobulin-like domain (d4) of TrkA. The X-ray crystal structure of the MNAC13 Fab fragment has been determined and refined to 1.8 A resolution. The antigen-binding site is characterized by a crevice, surrounded by hydrophilic-charged residues on either side, dipping deep toward three mainly hydrophobic subsites. Remarkably an isopropanol molecule has been found to bind in one of the hydrophobic crevices. Overall, the surface topology (shape and electrostatic potential) of the combining site is consistent with the binding data on TrkA ECD serial deletions mutants. The structure of the MNAC13 Fab fragment may assist in the rational structure-based design of high affinity humanized forms of MNAC13, appropriate for therapeutic approaches in neuropathy and inflammatory pain states.     

Isolation, Characterization, and Heterologous Expression of a Carboxylesterase of Pseudomonas aeruginosa PAO1

We purified to homogeneity an intracellular esterase from the opportunistic pathogen Pseudomonas aeruginosa PAO1. The enzyme hydrolyzes p-nitrophenyl acetate and other acetylated substrates. The N-terminal amino acid sequence was analyzed and 11 residues, SEPLILDAPNA, were determined. The corresponding gene PA3859 was identified in the P. aeruginosa PAO1 genome as the only gene encoding for a protein with this N-terminus. The encoding gene was cloned in Escherichia coli, and the recombinant protein expressed and purified to homogeneity. According to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis and analytical gel filtration chromatography, the esterase was found to be a monomer of approximately 24 kDa. The experimentally determined isoelectric point was 5.2 and the optimal enzyme activity was at 55°C and at pH 9.0. The esterase preferentially hydrolyzed short-chain fatty acids. It is inhibited by phenylmethylsulfonyl fluoride (PMSF) but not by ethylendiaminotetraacetic acid (EDTA). Native enzyme preparations typically showed a Michaelis constant (K_m) and V_max of 0.43 mM and 12,500 U mg^–1, respectively, using p-nitrophenyl acetate as substrate. Homology-based database searches clearly revealed the presence of the consensus GXSXG signature motif that is present in the serine-dependent acylhydrolase protein family.     

甲功五项化学发光免疫分析的基质效应对临床测值的影响

目的:探索评价基质效应在化学发光免疫分析中对甲状腺功能五项指标的影响。方法:选取甲状腺功能五项高值血清,用10种基质牛血清、马血清、山羊血清、水解明胶、BSA、PBS、生理盐水、正常人血清、甲减人血清、甲亢人血清分别对T3、T4、FT3、FT4、TSH的高值血清进行倍比稀释,观察基质效应,另将10种基质用考马斯亮兰法检测蛋白含量,分析蛋白含量与基质效应的关系。结果:T3项目牛血清、水解明胶、BSA有明显基质效应;T4和FT3项目牛血清、水解明胶、BSA、PBS、生理盐水有明显基质效应;FT4项目牛血清、马血清、水解明胶、BSA、PBS、生理盐水有明显基质效应;TSH项目没有发现基质效应,正常人血清、甲减人血清和甲亢人血清对甲状腺功能五项无基质效应。检测结果显示蛋白含量多少与基质效应无关。结论:人血清基质是用于稀释样本,基质效应最小的液体,针对个体差异性进行的选择,稀释T3、T4、FT3、FT4高值血清选择甲减人血清,稀释TSH高值血清选择甲亢人血清,可以得到较为满意的结果。     

Seed germination of Pilosocereus arrabidae (Cactaceae) from a semiarid region of south‐east Brazil

We evaluated the effect of temperature regimes (six constant and four alternating temperatures), light qualities (five red : far red ratios) and water potentials (ΨW; seven NaCl and polyethylene glycol 6000 [PEG] solutions) on the percentage and germination rate, as well as the post‐seminal development morphology, that allow Pilosocereus arrabidae seeds to germinate in a hot semiarid climate on the south‐eastern Brazilian coast. The results showed that seeds germinated similarly between constant and alternating temperatures, with an optimal germination at 25/20°C and 20°C. Pilosocereus arrabidae seeds were photoblastic positive and the final germination percentage was inhibited at low red : far red ratios. Maximum germination was obtained in distilled water (0 MPa) and decreases of Ψ_W in the solutions reduced the germination, which was lower in NaCl than in iso‐osmotic PEG solutions. Germination inhibition appears to be osmotic because the recovery response was high when non‐germinated seeds from both iso‐osmotic solutions were transferred to water. Seeds of P. arrabidae are small and germination is phaneroepigeal. Despite the slow growth typically seen in seedlings and adults of Cactaceae, germination in this species depends on the ability of the seeds to appropriately sense and react to environmental cues that correlate with times and places under low‐risk growth conditions.     

Study of the mode of action of a polygalacturonase from the phytopathogen Burkholderia cepacia

We have recently isolated and heterologously expressed BcPeh28A, an endopolygalacturonase from the phytopathogenic Gram-negative bacterium Burkholderia cepacia. Endopolygalacturonases belong to glycoside hydrolase family 28 and are responsible for the hydrolysis of the non-esterified regions of pectins. The mode of action of BcPeh28A on different substrates has been investigated and its enzymatic mechanism elucidated. The hydrolysis of polygalacturonate indicates that BcPeh28A is a non-processive enzyme that releases oligomers with chain lengths ranging from two to eight. By inspection of product progression curves, a kinetic model has been generated and extensively tested. It has been used to derive the kinetic parameters that describe the time course of the formation of six predominant products. Moreover, an investigation of the enzymatic activity on shorter substrates that differ in their overall length and methylation patterns sheds light on the architecture of the BcPeh28A active site. Specifically the tolerance of individual sites towards methylated saccharide units was rationalized on the basis of the hydrolysis of hexagalacturonides with different methylation patterns.     

Development and characterization of microsatellite loci from Olea europaea

This study reports 19 simple sequence repeat loci developed from a genomic library of the olive tree (Olea europaea L.), of which 12 revealed to be polymorphic and informative, ranging from two to 14 alleles.     

People recognise when they are really anonymous in an economic game

Mounting evidence that cues of being watched can enhance cooperative behaviour questions the existence of ‘anonymous', one-shot, non-kin directed cooperation and the validity of using ‘anonymous' economic games to empirically measure such behaviour in humans. Here we investigate how sensitive people are to such cuing effects. We test whether people playing an ultimatum game can use explicit information about experimental anonymity to override any effects of cuing in a public context, when faced with both simultaneously. The aims of our study were to investigate whether, (1) individuals respond to experimentally imposed anonymity within a public context and (2) the presence of known others affects cooperative behaviour over and above merely the presence of others. We find that proposer offers did not vary with changes in context (i.e., there was no “eyes effect”) but did vary with the degree of actual anonymity and the specific presence of known others. Hence, we infer that people recognise when their decisions are anonymous or not and proposers respond to reputation concerns when they are not anonymous. Responder behaviour did not vary with changes in context, degree of actual anonymity or the specific presence of known others. Hence, responders do not respond to reputation concerns and use one uniform strategy, perhaps as long as the payoff structure remains constant. This latter finding may hint at selection in favour of strategies that uniformly ensure near-equal splits of resources in some environments, and thus manifest as strong fairness norms in a population.