Novel non-ß-lactam inhibitor of ß-lactamase TEM-171 based on acylated phenoxyaniline.

The microbial resistance to antibiotics is a genuine global threat. Consequently, a search of new inhibitors remains of acute importance due to the increasing spread of multidrug resistance. Here we present a new type of non-ß-lactam ß-lactamase inhibitor PA-34 based on natural phenoxyaniline, identified using computer-assisted screening of scaffolds related to those of known low-affinity inhibitors. The compound displays reversible competitive inhibition of bacterial ß-lactamase TEM-171, with a Ki of 88 µM. Using enzyme kinetics, infra-red spectroscopy, fluorescence quenching and computer docking, we propose that the inhibitor binds at the entrance to the enzyme active site. This is a novel inhibition mechanism compared to binding covalently to the catalytic serine in the active site or non-covalently to the allosteric site. The residues involved in binding the inhibitor are conserved among molecular class A ß-lactamases. The identified compound and its proposed binding mode may have a potential for a regulation of the catalytic activity of a wide range of class A ß-lactamases. We also hypothesise that the presented route for finding non-ß-lactam compounds may be an effective and durable approach for combating bacterial antibiotic resistance.

Tomography of a Cryo-immobilized Yeast Cell Using Ptychographic Coherent X-Ray Diffractive Imaging.

The structural investigation of noncrystalline, soft biological matter using x-rays is of rapidly increasing interest. Large-scale x-ray sources, such as synchrotrons and x-ray free electron lasers, are becoming ever brighter and make the study of such weakly scattering materials more feasible. Variants of coherent diffractive imaging (CDI) are particularly attractive, as the absence of an objective lens between sample and detector ensures that no x-ray photons scattered by a sample are lost in a limited-efficiency imaging system. Furthermore, the reconstructed complex image contains quantitative density information, most directly accessible through its phase, which is proportional to the projected electron density of the sample. If applied in three dimensions, CDI can thus recover the sample's electron density distribution. As the extension to three dimensions is accompanied by a considerable dose applied to the sample, cryogenic cooling is necessary to optimize the structural preservation of a unique sample in the beam. This, however, imposes considerable technical challenges on the experimental realization. Here, we show a route toward the solution of these challenges using ptychographic CDI (PCDI), a scanning variant of coherent imaging. We present an experimental demonstration of the combination of three-dimensional structure determination through PCDI with a cryogenically cooled biological sample--a budding yeast cell (Saccharomyces cerevisiae)--using hard (7.9 keV) synchrotron x-rays. This proof-of-principle demonstration in particular illustrates the potential of PCDI for highly sensitive, quantitative three-dimensional density determination of cryogenically cooled, hydrated, and unstained biological matter and paves the way to future studies of unique, nonreproducible biological cells at higher resolution.

Pseudomonas chlororaphis Strain Sm3, Bacterial Antagonist of Pratylenchus penetrans.

The interaction of Pseudomonas chlororaphis strain Sm3 and the root-lesion nematode Pratylenchus penetrans was investigated in three separate greenhouse experiments with soils from southern British Columbia, Canada. The bacteria were applied to the roots of strawberry plants and planted in unpasteurized field soils, with natural or supplemented infestation of P. penetrans. Nematode suppression in roots was evident after 6 or 10 weeks in all experiments. Root or shoot growth were increased after 10 weeks in two experiments. Population dynamics of P. chlororaphis Sm3 in the rhizosphere was followed using an antibiotic-resistant mutant of P. chlororaphis Sm3. There was no apparent correlation between bacterial density in the rhizosphere and P. penetrans suppression in strawberry roots and rhizosphere soil, although the soil with the highest nematode reduction also had the largest P. chlororaphis Sm3 population in the rhizosphere.

Separation of the protein-tyrosine kinase and phosphatidylinositol kinase activities of the human placental insulin receptor.

On immunoprecipitation using a specific antiphosphotyrosine antibody, phosphatidylinositol kinase (EC 2.7.1.67) activity was separated from the protein-tyrosine kinase (EC 2.7.1.112) activity of the wheat germ agglutinin (WGA) -purified insulin receptor from human placenta. This clearly indicates that protein-tyrosine kinase and phosphatidylinositol kinase activity do not reside on the same polypeptide chain as previously has been suggested. Quantitatively, the fraction of phosphatidylinositol kinase that was bound to WGA sepharose and eluted together with the insulin receptor amounted to 2% of the Triton X-100 soluble phosphatidylinositol kinase. The apparent Km values of the bound and unbound phosphatidylinositol kinase with respect to PI and ATP were very similar (0.4 and 0.3 mmol/l and 8 and 7 mumol/l, respectively) suggesting that the WGA-bound phosphatidylinositol kinase is not a different enzyme, but rather represents a small portion of the bulk Triton X-100-soluble phosphatidylinositol kinase that is bound to the lectin tightly associated with the insulin receptor. The synthetic polymer (Glu80Tyr20)n, a model substrate of the insulin receptor tyrosine kinase, at 0.5 mmol/l, inhibited phosphatidylinositol kinase of WGA-purified insulin receptor by 70-90%. This inhibition was not overcome by increasing the concentrations of ATP or PI as one would expect if a functional interrelationship of the protein-tyrosine kinase and the phosphatidylinositol kinase would exist.