Abscisic add levels of individual leaf cells

Abscisic acid (ABA) concentrations of guard cells, subsidiary cells and epidermis cells of Comrnelina communis L. and Tradescantia virginiana L. were determined in cell sap samples extracted by means of micro glass capillaries. Concentrations up to 6.7 m M were indicated by commercial immunoassay test kits. A gradient of ABA concentration was found between guard cells (2,49 ± 1.81 m M , n = 25), subsidiary cells (1.25 ± 1.46 m M , n = 21) and epidermis cells (0.86 ± 0.76 m M , n = 20; mean values ± SD).     

Predicting physiological concentrations of metabolites from their molecular structure.

Physiological concentrations of metabolites can partly be explained by their molecular structure. We hypothesize that substances containing certain chemical groups show increased or decreased concentration in cells. We consider here, as chemical groups, local atomic configurations, describing an atom, its bonds, and its direct neighbor atoms. To test our hypothesis, we fitted a linear statistical model that relates experimentally determined logarithmic concentrations to feature vectors containing count numbers of the chemical groups. In order to determine chemical groups that have a clear effect on the concentration, we use a regularized (lasso) regression. In a dataset on 41 substances of central metabolism in different organisms, we found that the physical concentrations are increased by the occurrence of amino and hydroxyl groups, while aldehydes, ketones, and phosphates show decreased concentrations. The model explains about 22% of the variance of the logarithmic mean concentrations.     

Pre-imaginal flight motor pattern in Locusta

In a wind stream, larval stages of Locusta usually show a tonic muscle activity but they can also exhibit a rhythmic motor output. With ageing such a pattern can be released sooner, the trains become longer. The basic rhythm of 10 Hz does not change. The initial co-contraction of specific muscles is substituted later in development by an antagonistic recruitment. This activity resembles the flight motor pattern of young locusts which lack phasic sensory feedback from the wing region. Azadirachtin, an insect growth regulator, has been used to produce a permanent 5th larval instar. However, the extension of the last larval stage does not lead to a further development of the motor pattern to a level comparable to mature animals.     

Abscisic acid relations of plants grown on tungsten enriched substrates

The effect of tungsten as a potential inhibitor of ABA biosynthesis in plants was tested both under controlled conditions in the greenhouse or laboratory and in the field at a site naturally enriched with tungsten. Although as predicted in all cases the activity of aldehyde oxidase was strongly inhibited by tungsten, in well watered, greenhouse-grown maize plants increased concentrations of tungsten did not reduce ABA concentration in tissues. Depending on the time of incubation, mostly an accumulation due to an increased net ABA synthesis could be observed. Under drought stress, however, tungsten may have an inhibitory effect on ABA accumulation. Field experiments with drought-stressed clover and dandelion plants from a habitat close to a former tungsten mine confirm those of the greenhouse and laboratory. The ineffectiveness of tungsten in decreasing ABA levels in well-watered plants and its minimal effects in inhibiting ABA accumulation of droughted plants indicate that it is unsuitable as an inhibitor of ABA biosynthesis.     

Crystal structures of MMP-9 complexes with five inhibitors: contribution of the flexible Arg424 side-chain to selectivity

Human matrix metalloproteinase 9 (MMP-9), also called gelatinase B, is particularly involved in inflammatory processes, bone remodelling and wound healing, but is also implicated in pathological processes such as rheumatoid arthritis, atherosclerosis, tumour growth, and metastasis. We have prepared the inactive E402Q mutant of the truncated catalytic domain of human MMP-9 and co-crystallized it with active site-directed synthetic inhibitors of different binding types. Here, we present the X-ray structures of five MMP-9 complexes with gelatinase-specific, tight binding inhibitors: a phosphinic acid (AM-409), a pyrimidine-2,4,6-trione (RO-206-0222), two carboxylate (An-1 and MJ-24), and a trifluoromethyl hydroxamic acid inhibitor (MS-560). These compounds bind by making a compromise between optimal coordination of the catalytic zinc, favourable hydrogen bond formation in the active-site cleft, and accommodation of their large hydrophobic P1' groups in the slightly flexible S1' cavity, which exhibits distinct rotational conformations of the Pro421 carbonyl group in each complex. In all these structures, the side-chain of Arg424 located at the bottom of the S1' cavity is not defined in the electron density beyond C(gamma), indicating its mobility. However, we suggest that the mobile Arg424 side-chain partially blocks the S1' cavity, which might explain the weaker binding of most inhibitors with a long P1' side-chain for MMP-9 compared with the closely related MMP-2 (gelatinase A), which exhibits a short threonine side-chain at the equivalent position. These novel structural details should facilitate the design of more selective MMP-9 inhibitors.     

Tissue factor coagulant function is enhanced by protein-disulfide isomerase independent of oxidoreductase activity

Protein-disulfide isomerase (PDI) switches tissue factor (TF) from coagulation to signaling by targeting the allosteric Cys186-Cys209 disulfide. Here, we further characterize the interaction of purified PDI with TF. We find that PDI enhances factor VIIa-dependent substrate factor X activation 5-10-fold in the presence of wild-type, oxidized soluble TF but not TF mutants that contain an unpaired Cys186 or Cys209. PDI-accelerated factor Xa generation was blocked by bacitracin but not influenced by inhibition of vicinal thiols, reduction of PDI, changes in redox gradients, or covalent thiol modification of reduced PDI by N-ethylmaleimide or methyl-methanethiosulfonate, which abolished PDI oxidoreductase but not chaperone activity. PDI had no effect on fully active TF on either negatively charged phospholipids or in activating detergent, indicating that PDI selectively acts upon cryptic TF to facilitate ternary complex formation and macromolecular substrate turnover. PDI activation was reduced upon mutation of TF residues in proximity to the macromolecular substrate binding site, consistent with a primary interaction of PDI with TF. PDI enhanced TF coagulant activity on microvesicles shed from cells, suggesting that PDI plays a role as an activating chaperone for circulating cryptic TF.     

The first agmatine/cadaverine aminopropyl transferase: biochemical and structural characterization of an enzyme involved in polyamine biosynthesis in the hyperthermophilic archaeon Pyrococcus furiosus

We report here the characterization of the first agmatine/cadaverine aminopropyl transferase (ACAPT), the enzyme responsible for polyamine biosynthesis from an archaeon. The gene PF0127 encoding ACAPT in the hyperthermophile Pyrococcus furiosus was cloned and expressed in Escherichia coli, and the recombinant protein was purified to homogeneity. P. furiosus ACAPT is a homodimer of 65 kDa. The broad substrate specificity of the enzyme toward the amine acceptors is unique, as agmatine, 1,3-diaminopropane, putrescine, cadaverine, and sym-nor-spermidine all serve as substrates. While maximal catalytic activity was observed with cadaverine, agmatine was the preferred substrate on the basis of the k(cat)/K(m) value. P. furiosus ACAPT is thermoactive and thermostable with an apparent melting temperature of 108 degrees C that increases to 112 degrees C in the presence of cadaverine. Limited proteolysis indicated that the only proteolytic cleavage site is localized in the C-terminal region and that the C-terminal peptide is not necessary for the integrity of the active site. The crystal structure of the enzyme determined to 1.8-A resolution confirmed its dimeric nature and provided insight into the proteolytic analyses as well as into mechanisms of thermal stability. Analysis of the polyamine content of P. furiosus showed that spermidine, cadaverine, and sym-nor-spermidine are the major components, with small amounts of sym-nor-spermine and N-(3-aminopropyl)cadaverine (APC). This is the first report in Archaea of an unusual polyamine APC that is proposed to play a role in stress adaptation.