Chemical screens against a reconstituted multiprotein complex: myricetin blocks DnaJ regulation of DnaK through an allosteric mechanism

DnaK is a molecular chaperone responsible for multiple aspects of bacterial proteostasis. The intrinsically slow ATPase activity of DnaK is stimulated by its co-chaperone, DnaJ, and these proteins often work in concert. To identify inhibitors we screened plant-derived extracts against a reconstituted mixture of DnaK and DnaJ. This approach resulted in the identification of flavonoids, including myricetin, which inhibited activity by up to 75%. Interestingly, myricetin prevented DnaJ-mediated stimulation of ATPase activity, with minimal impact on either DnaK's intrinsic turnover rate or its stimulation by another co-chaperone, GrpE. Using NMR, we found that myricetin binds DnaK at an unanticipated site between the IB and IIB subdomains and that it allosterically blocked binding of DnaK to DnaJ. Together, these results highlight a "gray box" screening approach, which might facilitate the identification of inhibitors of other protein-protein interactions.     

Accurate whole-spectrum measurements of intracellular pH and [Na+]

Fluorescent measurements of intracellular H⁺ and Na⁺ are improved by using whole spectra of the fluorescent indicators BCECF and SBFI, respectively. The extra data in whole spectra enable both an accurate calibration and a ready detection of artifacts which are not possible to identify using a more conventional data analysis that relies upon only two wavelength windows in the fluorescence spectra. The whole-spectrum technique is applicable to cell suspensions in a conventional fluorimeter (as is reported here with SBFI), as well as to attached cells using a fluorimeter combined with an inverted epifluorescence microscope. The spectral method was highly reproducible in that pairs of successive pH measurements differed, on average, by only 0.01±0.02 U. Random uncertainty from sample to sample was estimated numerically from the standard deviation of measurements on ionophore-treated cells. When full-spectrum analysis was employed, this scatter showed a two-fold improvement over results obtained using the two-wavelength ratio method. Because SBFI has a relatively narrow dynamic range, whole-spectrum analysis has been applied to improve the accuracy of sodium determinations. The calibrated system measured [Na⁺]_i with excellent linearity over the range 2–150 mM and with an accuracy of approximately 5 mM.     

A preliminary study on the stabilization of blood typing antibodies sorbed into paper

This study investigated the stability of the primary blood typing antibodies (Anti-A, Anti-B and Anti-D IgM) on paper. This knowledge is critical to manufacture a new type of paper-based blood typing device where blood group antibodies must be kept active on paper for extended periods. Two strategies were explored. The first involved mixing additives such as polyvinylpyrrolidone (PVP), dextran and glycerol, with antibodies before sorption onto paper. While all the additives tested improved the antibody stability on paper, their protection for storage at room temperature was limited; dextran provided the longest protection, followed by PVP and then glycerol. The second strategy relied on freeze-drying to stabilize the antibodies in paper. Freeze dried antibodies sorbed into paper could be stored for long periods at ambient conditions without significantly loss of their activity. The thermal stability of antibodies in paper was also improved by freeze-drying. Our work shows that the use of additives and freeze-drying are effective approaches to retain the activities of IgM blood group antibodies on paper. These approaches will be further explored for the large scale development of a new generation of clinical and home-care blood testing devices.     

Vinyl chloride suspension polymerisation and the control of polymer properties

A number of poly(vinyl alcohols), used in the suspension polymerisation of vinyl chloride, have been fractionated and characterised. The most effective had the highest molecular weight and contained the most unsaturation. The more insoluble fractions gave the best balance of product properties. Reactor sampling experiments have been used to determine the mechanism of polymerisation. In most polymerisations, the final grain size is determined by factors that control the coalescence and break-up of the monomer droplets during the first 15% conversion. It is suggested that the structure within the grains is controlled by the formation of a continuous network of PVC primaries which retards droplet contraction.