Rapid measurement of protein kinase and phosphatase activities by slot-filtration.

A slot-filtration method has been developed for the detection and quantitation of protein kinase and phosphatase activities. In this technique, after kinase-dependent phosphorylation or phosphatase-dependent dephosphorylation of different substrates, samples are transferred under vacuum onto nitrocellulose using a slot-blotting apparatus. Non-incorporated or released radioactivity is then removed by filtration and washing under vacuum. Quantitation is performed by scintillation or Cerenkov counting of the excised membrane slots. Application of the method to the assay of four different protein kinases (protein kinase N, cyclic AMP-dependent protein kinase and calcium/calmodulin-dependent protein kinases type I and type III) and one phosphatase is presented. A number of protein substrates with varying molecular masses and isoelectric points were found suitable for the slot-filtration technique. The method is applicable to impure as well as purified kinase and phosphatase preparations, can be used over a wide range of concentrations of substrates, has a very low background of nonspecific ATP binding and provides highly reproducible data. The slot-filtration method can also be adapted for use with ion-exchange paper, particularly for assays using peptides as substrates. The technique, with either nitrocellulose or ion-exchange paper, can be used to rapidly process large numbers of samples and can be simultaneously applied to direct comparison of different kinases, phosphatases and/or substrates in the same experiment.     

Synergistic Inhibition of Microbial Sulfide Production by Combinations of the Metabolic Inhibitor Nitrite and Biocides

MICs of six broad-spectrum biocides and two specific metabolic inhibitors and fractional inhibitory concentration indexes (FICIs) for controlling a sulfide-producing consortium were determined. Nitrite was synergistic (FICI < 1) with all but one biocide due to its specific inhibition of dissimilatory sulfite reductase. Hence, combining nitrite with biocides allows more efficient and cost-effective control of sulfate-reducing bacteria.     

Development of HIV fusion inhibitors.

In the past 25 years, the worldwide AIDS epidemic has grown such that roughly 38 million people were estimated to be living with the disease worldwide at the end of 2003. The introduction of antiretroviral-based therapies, beginning in 1987, has enabled many to live with HIV as a chronic, rather than terminal, disease. However, the emergence and spread of drug-resistant strains highlights the continued need for new therapies with novel modes of action. In 2003, the FDA and EMEA approved enfuvirtide (Fuzeon), a 36 amino acid peptide derived from the natural gp41 HR2 sequence, as the first HIV fusion inhibitor. T-1249, a 39 amino acid fusion inhibitor, is active against viruses that develop resistance to enfuvirtide. The development of FIs and the processes to manufacture enfuvirtide and T-1249 on an unprecedented scale for peptide therapeutics are presented. Synthetic routes based on a combination of solid phase peptide synthesis and solution phase fragment condensation as well as the analytical controls necessary to insure a robust process are discussed.