Evolution of C4 phosphoenolpyruvate carboxylase in Flaveria, a conserved serine residue in the carboxyl-terminal part of the enzyme is a major determinant for C4-specific characteristics

C4 phosphoenolpyruvate carboxylases have evolved from ancestral C3 isoforms during the evolution of angiosperms and gained distinct kinetic and regulatory properties compared with the C3 isozymes. To identify amino acid residues and/or domains responsible for these C4-specific properties the C4 phosphoenolpyruvate carboxylase of Flaveria trinervia (C4) was compared with its orthologue in the closely related C3 plant Flaveria pringlei. Reciprocal enzyme chimera were constructed and the kinetic constants, K(0.5) and k(cat), as well as the Hill coefficient, h, were determined for the substrate phosphoenolpyruvate both in the presence and absence of the activator glucose 6-phosphate. By this approach two regions were identified which determined most of the kinetic differences of the C4 and C3 ppcA phosphoenolpyruvate carboxylases with respect to the substrate PEP. In addition, the experiments suggest that the two regions do not act additively but interact with each other. The region between amino acids 296 and 437 is essential for activation by glucose 6-phosphate. The carboxyl-terminal segment between amino acids 645 and 966 contains a C4 conserved serine or a C3 invariant alanine at position 774 in the respective enzyme isoform. Site-directed mutagenesis shows that this position is a key determinant for the kinetic properties of the two isozymes.     

Polar organic phase liquid chromatography with packed capillary columns using a vancomycin chiral stationary phase

Vancomycin immobilized on silica served as the chiral stationary phase (CSP) in this investigation with polar organic solvents as the mobile phase in liquid chromatography (LC). It was shown that trace amounts of water were beneficial for improving peak shape and efficiency. To regulate the retention and selectivity an acid and/or base were added to the mobile phase where an excess of acid was shown to be preferential for enantioseparation. An unusual increase in selectivity with increasing temperature was shown for the acidic drug, thalidomide. Additionally, nonlinear van't Hoff plots were obtained for metoprolol enantiomers that showed increased retention with increasing temperature. Metoprolol also showed unusual behavior in the polar organic phase when water was added to resemble reversed-phase chromatography, with minimum retention observed at high water or high methanol concentrations. In both instances a high degree of electrostatic interaction between metoprolol and vancomycin was concluded. Metoprolol and ten of its analogs were examined on this CSP to evaluate the enantiorecognition process. A comparison in enantioselectivity for a number of acidic and basic drugs using this CSP was also carried out using the polar organic phase, reversed phase, and normal phase LC which were all compared to the results obtained in supercritical fluid chromatography (SFC). Polar organic phase LC offered a better separation of basic molecules while reversed phase LC was preferred for the resolution of acids. SFC showed the broadest enantioselectivity overall and normal phase LC indicated similar properties, as expected, to SFC but with lower column efficiency. Copyright 2000 Wiley-Liss, Inc.     

黄腐酸对干旱胁迫下黄瓜光合特性及产量和品质的影响

黄腐酸(FA)可参与植物耐旱性的调控,但关于其对干旱胁迫下黄瓜光合作用的调控机制尚不清楚。本研究以‘津优35'黄瓜为试材,采用聚乙二醇(PEG-6000)模拟干旱,通过喷施不同浓度(0、100、300、500、700、900 mg·L^-1)FA,研究其缓解黄瓜干旱胁迫的浓度效应及其对光合关键酶活性、叶绿体超微结构、叶绿素荧光参数、水分利用效率及产量和品质的影响。结果表明: 室内试验中,与对照(0 mg·L^-1)相比,不同浓度FA处理均显著提高了干旱胁迫下黄瓜幼苗的叶片相对含水量和叶面积,降低旱害指数、丙二醛含量和电解质渗漏率,随着FA浓度的增加其缓解效应呈现先升高后下降的趋势,且以700 mg·L^-1 FA的作用效果最好。FA显著增加干旱胁迫下黄瓜幼苗的叶绿素含量、Rubisco和Rubisco活化酶(RCA)活性及基因表达、净光合速率(P_n)、最大光化学效率和实际光化学效率、单位面积吸收光能、捕获光能、电子传递的量子产额和PSⅠ活性,降低K点的上升,维持叶绿体超微结构。温室控水试验表明,FA可显著增加干旱胁迫下温室黄瓜的水分利用效率,促进干物质量的积累,增加果实中Vc、可溶性糖、可溶性蛋白和游离氨基酸含量,降低单宁含量。综上,施用FA可在干旱条件下提高温室黄瓜产量,改善果实品质。     

The antimicrobial peptide LL-37 triggers release of apoptosis-inducing factor and shows direct effects on mitochondria

The human antimicrobial peptide LL-37 permeabilizes the plasma membrane of host cells, but LL-37-induced direct effects on mitochondrial membrane permeability and function has not been reported. Here, we demonstrate that LL-37 is rapidly (within 20 min) internalized by human osteoblast-like MG63 cells, and that the peptide co-localizes with MitoTracker arguing for accumulation in mitochondria. Subcellular fractionation and Western blot disclose that stimulation with LL-37 (8 μM) for 2 h triggers release of the mitochondrial protein apoptosis-inducing factor (AIF) to the cytosol, whereas LL-37 causes no release of cytochrome C oxidase subunit IV of the inner mitochondrial membrane, suggesting that LL-37 affects mitochondrial membrane permeability in a specific manner. Next, we investigated release of AIF and cytochrome C from isolated mitochondria by measuring immunoreactivity by dot blot. The media of mitochondria treated with LL-37 (8 μM) for 2 h contained 50% more AIF and three times more cytochrome C than that of control mitochondria, showing that LL-37 promotes release of both AIF and cytochrome C. Moreover, in vesicles reflecting mitochondrial membrane lipid composition, LL-37 stimulates membrane permeabilization and release of tracer molecules. We conclude that LL-37 is rapidly internalized by MG63 cells and accumulates in mitochondria, and that the peptide triggers release of pro-apoptotic AIF and directly affects mitochondrial membrane structural properties.     

Validation of a musculo-skeletal model of the mandible and its application to mandibular distraction osteogenesis

Mandibular distraction osteogenesis will lead to a change in muscle coordination and load transfer to the temporomandibular joints (TMJ). The objective of this work is to present and validate a rigid-body musculo-skeletal model of the mandible based on inverse dynamics for calculation of the muscle activations, muscle forces and TMJ reaction forces for different types of clenching tasks and dynamic tasks. This approach is validated on a symmetric mandible model and an application will be presented where the TMJ reaction forces during unilateral clenching are estimated for a virtual distraction patient with a shortened left ramus. The mandible model consists of 2 rigid segments and has 4 degrees-of-freedom. The model was equipped with 24 hill-type musculotendon actuators. During the validation experiment one subject was asked to do several tasks while measuring EMG activity, bite force and kinematics. The bite force and kinematics were used as input for the simulations of the same tasks after which the estimated muscle activities were compared with the measured muscle activities. This resulted in an average correlation coefficient of 0.580 and an average of the Mean Absolute Error of 0.109. The virtual distraction model showed a large difference in the TMJ reaction forces between left and right compared with the symmetric model for the same loading case. The present work is a step in the direction of building patient-specific mandible models, which can assess the mechanical effects on the TMJ before mandibular distraction osteogenesis surgery.     

Evaluation of long-term mating disruption of Ephestia kuehniella and Plodia interpunctella (Lepidoptera: Pyralidae) in indoor storage facilities by pheromone traps and monitoring of relative aerial concentrations of pheromone

The potential for pheromone-based mating disruption (MD) of Ephestia kuehniella (Walker) and Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae) was investigated in two flour mills and a pet food distributor. Plastic sachets emitting 2-3 mg per d (Z,E)-9,12-tetradecadienyl acetate, the major pheromone component of both moth species, were used as MD dispensers, which were applied in grid systems resulting in one dispenser per 100 m(3) of air volume. Pheromone traps with sticky inserts were used to monitor moth population fluctuations. To monitor pheromone levels in the air before, during, and after the treatment, electroantennographic (EAG) measurements were performed using a portable device. All localities showed decreased trap catches after application of MD. In two localities with low initial population densities, trap catches were reduced immediately after application of MD and remained very low, even several months after the MD treatment was terminated. In contrast, in a locality with a higher initial population density the reduction in trap catches was slower, and trap catches increased again soon after the termination of the MD treatment. Electrophysiological data showed not only increased aerial levels of pheromone during the treatment period but also levels that were higher than during pretreatment, even 12 mo after removal of MD dispensers. The localities had good ventilation, and the memory effect observed indicates that the pheromone adhered to surfaces that subsequently functioned as secondary dispensers. Customer complaints registered by one of the mills were 49% less in 2004, after 2 yr of MD compared with 2002, the year before the treatments began.     

Determination of aldehydes in human breath by on-fibre derivatization, solid-phase microextraction and GC-MS

A GC-MS method for the simultaneous determination of hexanal, heptanal, octanal, nonanal and decanal in exhaled breath was established and validated. The aldehydes were derivatized on PDMS/DVB fibres using O-2,2,4,5,6-(pentafluorobenzyl) hydroxylamine hydrochloride (PFBHA) as the headspace derivatization reagent. The resultant oximes were quantified by GC-MS in selected ion monitoring (SIM) mode. The method provides detection limits of 0.01-0.03 nM for the aldehydes, with a linear response in the concentration range 0.002-20 nM. Within-day precision values for the five aldehydes at 0.02-0.04 nM and 0.2-0.4 nM were in the ranges: 3-9% and 3-8%, respectively; the corresponding between-day precision values were 11-22% and 10-24%. Exhaled breath samples could be stored at -20 degrees C for 48 h.     

Spatial mapping of affinity changes for the integrin LFA‐1 during cell migration using clusters identified based on local density

Localization microscopy methods like Stochastic Optical Reconstruction Microscopy (STORM) are very well suited for exploring clustering of proteins, as the data inherently provide a list of molecular coordinates. Here we use state‐of‐art cluster analysis algorithms (DBSCAN) to explore the clustering behaviour of different affinity forms of the integrin LFA‐1. It has been suggested that LFA‐1 may form clusters, in order to increase the avidity to ICAM‐1. However, this hypothesis still seems to be controversial. In this study, we found, variations in clustering behaviour among the different affinity forms of LFA‐1 in migrating T‐cells. We found that panLFA‐1 is located in clusters throughout the polarised cell on ICAM‐1, with an increased density of molecules and clusters in the mid area and rear of the cell, whereas the intermediate and high affinity form of LFA‐1 showed an increased number in the mid area of a migrating cell and the high affinity form of LFA‐1 in the front and rear. Together, these data suggest that, in addition to LFA‐1 conformation, protein clustering might play a role in controlling cell‐substrate adhesion on ICAM‐1.By applying the cluster analysis algorithm DBSCAN to localization microscopy data, integrin clusters could be identified and different cluster parameters could be quantified.