We have designed a modular protein tagging system that allows different functionalities to be linked onto a single genetic fusion, either in solution, in living cells, or in chemically fixed cells. The protein tag (HaloTag) is a modified haloalkane dehalogenase designed to covalently bind to synthetic ligands (HaloTag ligands). The synthetic ligands comprise a chloroalkane linker attached to a variety of useful molecules, such as fluorescent dyes, affinity handles, or solid surfaces. Covalent bond formation between the protein tag and the chloroalkane linker is highly specific, occurs rapidly under physiological conditions, and is essentially irreversible. We demonstrate the utility of this system for cellular imaging and protein immobilization by analyzing multiple molecular processes associated with NF-kappaB-mediated cellular physiology, including imaging of subcellular protein translocation and capture of protein--protein and protein--DNA complexes.

Results suggest that for cats that have been vaccinated, detection of FHV-1-, FCV-, and FPV-specific antibodies is predictive of whether cats are susceptible to disease, regardless of vaccine type or vaccination interval. Because most client-owned cats had detectable serum antibodies suggestive of resistance to infection, use of arbitrary booster vaccination intervals is likely to lead to unnecessary vaccination of some cats.

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Production of coenzyme F 420 and its biosynthetic precursor FO was examined with a variety of aerobic actinomycetes to identify an improved source for these materials. Based on fermentation costs, safety, and ease of growth, Mycobacterium smegmatis was the best source for F 420 -5,6. M. smegmatis produced 1 to 3 mol of intracellular F 420 per liter of culture, which was more than the 0.85 to 1.0 mol of F 420 -2 per liter usually obtained with Methanobacterium thermoautotrophicum and ϳ10-fold higher than what was previously reported for the best aerobic actinomycetes. An improved chromatography system using rapidly flowing quaternary aminoethyl ion-exchange material and Florisil was used to more quickly and easily purify F 420 than with previous methods.

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These results suggest that the Clinitek strips lack sufficient concordance with results obtained by ELISA to be a reliable screening for test microalbuminuria in the dog. A reliable semiquantitative point-of-care test for canine urine albumin concentrations below those detected by standard urine dipsticks is still needed.

Clostridial neurotoxins are the most powerful toxins known. There are no available antidotes to neutralize neurotoxins after they have been internalized by neuronal cells. Enzymatic domains of clostridial neurotoxins are zinc-endopeptidases specific for protein components of the neuroexocytosis apparatus. Thus, attempts were made to find such antidotes among molecules possessing chelating properties. Subsequently, it was proposed that the process of interaction between clostridial neurotoxins and their substrates might be more complex than viewed previously and may include several separate regions of interaction. Phage display technology is free from bias toward any particular model. This technology in combination with recombinantly produced light chains of botulinum neurotoxins serotypes A, B, and C was used to identify potential inhibitors of clostridial neurotoxins. Identified sequences did not show substantial similarity with substrate proteins of clostridial neurotoxins. Nevertheless, three peptides chosen for further analysis were able to inhibit enzymatic activity of all clostridial neurotoxins tested. This work demonstrates that at least one of these peptides could not be cleaved by clostridial neurotoxin. Attempts to delete amino acid residues from this peptide resulted in dramatic loss of its inhibitory activity. Finally, this work presents a novel approach to searching for inhibitors of clostridial neurotoxins.

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