Mapping of Ras-related GTP-binding proteins by GTP overlay following two-dimensional gel electrophoresis

For identification of Rab, Rac, Rho, Ral, Rap, and Arf proteins on two-dimensional polyacrylamide gels, we have expressed full-length cDNAs of members of these protein families with the T7 RNA polymerase-recombinant vaccinia virus expression system. Membrane preparations from cells expressing the cDNAs were subjected to high-resolution two-dimensional polyacrylamide gel electrophoresis followed by [alpha-32P]GTP ligand blotting. We have mapped 28 small GTP-binding proteins relative to their isoelectric points and according to their molecular weights and by immunoblotting with specific antibodies. Rab and Rho proteins could be specifically identified by extraction of streptolysin O-permeabilized Madin-Darby canine kidney (MDCK) cells with Rab- and Rho-GDP dissociation inhibitor. We applied the reference mapping to analyze the GTP-binding patterns of synaptosome fractions from rat brain. The purified synaptosomes exhibited specific enrichment of Rab3a, Rab5a, Ral, and several other GTPases. This approach and the map we have produced should provide a useful aid for the analysis of the expression and localization of members of all families of small GTP-binding proteins in various cell types and subcellular fractions.     

Evidence for plasmid-associated crystal toxin production in Bacillus thuringiensis subsp. israelensis

Three crystalliferous (Cry+) strains of Bacillus thuringiensis subsp. israelensis (serotype 14) that produce parasporal protein crystals toxic to dipteran larvae and several acrystalliferous (Cry-) mutants, either induced or spontaneously derived from a single Cry+ parent, were examined for the presence of covalently closed circular (CCC) DNA in attempts to correlate toxin production with the presence of a specific plasmid. The plasmid profiles of both Cry+ and Cry- variants were analyzed by both a cleared lysate- and a modified Eckhardt lysate-electrophoresis technique. All of the Cry- mutants derived from the Cry+ parental strain had lost a 4.0- to 4.4-megadalton (Mdal) plasmid. Bioassay data confirmed loss of toxin production by the Cry- variants. All three Cry+ strains, including the parent of the Cry- strains, contained CCC plasmids DNAs of the following approximate molecular weights: 4.0 to 4.4, 5.2 to 6.0, and 11.4 to 13.0 Mdal. One Cry+ strain contained an additional CCC plasmid of 6.7 to 7.2 Mdal. The plasmid patterns for several Cry- derivatives differed in other respects from the pattern for their parent strain. The various Cry+ and Cry- strains could be distinguished either by phenotypical differences in antibiotic sensitivity, crystal production, and toxicity, or by differences in their plasmid profiles.     

Isolation of a spotted fever group rickettsia from the Pacific Coast tick, Ixodes pacificus, in Oregon

A rickettsia of the spotted fever group was isolated on three occasions from Ixodes pacificus in western Oregon. These isolations, and additional evidence furnished by complement fixation tests on guinea pigs inoculated with other Oregon ticks of this species, indicate that the association of this rickettsia with the Pacific Coast tick may be widespread. This is the first isolation of a spotted fever group rickettsia from I. pacificus. Because the Oregon isolates are mildly virulent for guinea pigs they resemble the Western U and Rickettsia montana strains of rickettsiae. However, preliminary evidence from cross-immunofluorescence tests of mouse antisera suggests the Tillamook and Grants Pass strains are antigenically different from all known spotted fever group agents.     

LINEARIZATION OF EMPIRICAL RHEOLOGICAL DATA FOR USE IN COMPOSITION CONTROL OF MULTICOMPONENT FOODSTUFFS

Abstract An empirical measure of viscosity, which is often far from being a linear function of composition, was used together with refractive index to build up a function which bears a linear relationship to the composition of tomato paste-water-sucrose mixtures. The new function can be used directly for rapid composition control by linear vector-vector transformation.     

An investigation of liquid carryover and sample residual for a high-throughput flow cytometer sample delivery system

The phenomenon of intersample contamination in air-segmented continuous-flow assays has been studied for many years, and new uses are being found for these sampling techniques every day. One application that has been developed recently employs a flow cytometer to conduct high-throughput screening assays of biological compounds. We have explored the sources of intersample contamination in the system and shown how methods developed previously can be applied to describe these phenomena. Using a simple model, we were able to accurately measure liquid film thickness in the sample tubing and demonstrate the effects of intersample contamination in a flow cytometer assay. Also, measures have been taken to reduce the level of intersample contamination in cytometric screening assays, helping to make the system a more viable tool for drug screening applications.     

Application of iterative suboptimal strategies for improving adaptation transients in adaptive systems

Some solutions which involve iterative optimization techniques are given to improve adaptation transients in adaptive systems. Two suboptimal controllers are developed and applied to an equivalent discrete-time system valid for describing the behaviour of a recent adaptive control scheme when one of the (time-varying) parameters entering the adaptation algorithm varies within a closed domain admissible from a convergence point of view. The resulting suboptimal control strategies are translated into corrections of this parameter in order to recompute the adaptation algorithm over a finite horizon. Some numerical simulations illustrate the usefulness of the proposed scheme.