Esterase Autodisplay: Enzyme Engineering and Whole-Cell Activity Determination in Microplates with pH Sensors

Among the GDSL family of serine esterases/lipases is a group of bacterial enzymes that posses C-terminal extensions involved in outer membrane anchoring or translocation. ApeE from Salmonella enterica serovar Typhimurium, a member of this group, has been expressed in Escherichia coli and was resistant to protease digestion when the protease was added to whole cells, indicating a periplasmic localization. The five consensus blocks conserved within all GDSL esterases were identified in ApeE by multiple sequence alignment and separated from the C-terminal extension. The DNA sequence spanning the four invariant residues Ser, Gly, Asn, and His, and hence representing the catalytic domains of ApeE, was amplified by PCR and fused in frame to the transport domains of the autodisplay system. The resulting artificial esterase, called EsjA, was overexpressed in the cell envelope of E. coli and was shown to be active by the use of α-naphthyl acetate (α-NA) as a substrate in an in-gel activity stain after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Surface exposure of EsjA was indicated by its accessibility to protease added to whole cells. The esterase activity of whole cells displaying EsjA was determined by a pH agar assay and by the use of microplates with integrated pH-dependent optical sensors. α-NA, α-naphthyl butyrate, and α-naphthyl caproate were used as substrates, and it turned out that the substrate preferences of artificial EsjA were altered in comparison to original ApeE. Our results indicate that autodisplay of esterase in combination with pH sensor microplates can provide a new platform technology for the screening of tailor-made hydrolase activities.     

Telemetered epidural pressure.

An implantable transducer capable of telemetering epidural brain pressure during long periods of time is described. The transducer is constructed from conventionally available materials, and routine workshop techniques are used. The coplanarity principle has been applied while the described mounting clamp allows discrete positioning of the transducer relative to the brain. Basically, the transducer consists of a coil-capacitor circuit in which alterations of epidural pressure induce changes in capacity and, thus, the transducer's resonance frequency. An external electromagnetically coupled impedance-measuring device converts the resonance frequency to a pressure analogous voltage. In vitro tests showed that the transducer had good accuracy and reliability for a period of more than 1 month. An in vivo experiment with a cat showed that the measured epidural pressure was linearly related to ventricular fluid pressure.     

Synthesis and evaluation of radiolabeled antagonists for imaging of beta-adrenoceptors in the brain with PET.

Five potent, lipophilic beta-adrenoceptor antagonists (carvedilol, pindolol, toliprolol and fluorinated analogs of bupranolol and penbutolol) were labeled with either carbon-11 or fluorine-18 and evaluated for cerebral beta-adrenoceptor imaging in experimental animals. The standard radioligand for autoradiography of beta-adrenoceptors, [125I]-iodocyanopindolol, was also included in this survey. All compounds showed either very low uptake in rat brain or a regional distribution that was not related to beta-adrenoceptors, whereas some ligands did display specific binding in heart and lungs. Apparently, the criteria of a high affinity and a moderately high lipophilicity were insufficient to predict the suitability of beta-adrenergic antagonists for visualization of beta-adrenoceptors in the central nervous system.     

Gibberellin and phytochrome control senescence in alstroemeria leaves independently

In alstroemeria ( Alstroemeria hybrida ), leaf senescence is effectively retarded by the application of gibberellins and by low fluences of red light. In this study we examined the possible interaction of gibberellins and red light in the regulation of senescence. Determination of endogenous gibberellins revealed that leaf senescence is accompanied by significant changes in the concentrations of non‐ 13‐hydroxylated gibberellins, the onset of senescence coinciding with a dramatic drop in GA₄, whereas concentrations of 13‐hydroxylated gibberellins are far less influenced. However, no direct effect of red light on a specific GA‐metabolic step could be determined. When exogenously applied, non‐13‐hydroxylated GAs were more active than the 13‐hydroxylated GAs. It appeared that the effect of red light is additive to that of active GAs. We hypothesise that GA₄ and phytochrome control senescence in alstroemeria mainly through separate mechanisms and have independent effects and that the observed differences in gibberellin concentrations are a consequence of delayed leaf senescence rather than a cause for it.     

Growth and reproduction in culture of ten Phytophthora fragariae races

Cultural characteristics were employed to develop a basis for characterizing each of the ten recognized races ofPhytophthora fragariae Hickman in the United States. Investigations were made into the relationships of temperature to growth and colony morphology, the effects of various media and ß-sitosterol on growth and oospore production, and relative zoosporangium production capabilities of the ten races. On the basis of this information, the ten races were divided into two groups according to their colony morphologies. Further characterization was possible on the basis of differential oospore and zoosporangium production in different media, and, to a lesser extent, with contrasting increments in mycelial mass and linear extension.