Quantification of Bacterial Histidine Kinase Autophosphorylation Using a Nitrocellulose Binding Assay.

We demonstrate a useful method for quantifying autophosphorylation of purified bacterial histidine kinases. Histidine kinases are known for their involvement in two-component signal transduction, a ubiquitous system through which bacteria sense and respond to environmental stimuli. Two-component signaling features autophosphorylation of a histidine kinase, followed by phosphotransfer to the receiver domain of a response regulator protein, which ultimately leads to an output response.

Optimized assay for the quantification of histidine kinase autophosphorylation.

Although two-component signaling systems, comprising a sensory histidine kinase and a response regulator, are a primary means by which bacteria detect and respond to environmental stimuli, they are poorly characterized. Here we report optimized conditions for detecting histidine phosphorylation using a facile medium-throughput filter paper-binding assay. Employing this assay we report the kinetic parameters of previously uncharacterized histidine kinases from Vibrio haveyi, Vibrio parahaemolytius, Shewanella oneidensis, and Legionella pneumophila.

Modification of a bi-functional diguanylate cyclase-phosphodiesterase to efficiently produce cyclic diguanylate monophosphate.

Cyclic-diGMP is a bacterial messenger that regulates many physiological processes, including many attributed to pathogenicity. Bacteria synthesize cyclic-diGMP from GTP using diguanylate cyclases; its hydrolysis is catalyzed by phosphodiesterases. Here we report the over-expression and purification of a bi-functional diguanylate cyclase-phosphodiesterase from S4.

Cancer wars: natural products strike back.

Natural products have historically been a mainstay source of anticancer drugs, but in the 90's they fell out of favor in pharmaceutical companies with the emergence of targeted therapies, which rely on antibodies or small synthetic molecules identified by high throughput screening. Although targeted therapies greatly improved the treatment of a few cancers, the benefit has remained disappointing for many solid tumors, which revitalized the interest in natural products. With the approval of rapamycin in 2007, 12 novel natural product derivatives have been brought to market.

Differential inhibition of adenylyl cyclase isoforms and soluble guanylyl cyclase by purine and pyrimidine nucleotides.

Mammals express nine membranous adenylyl cyclase isoforms (ACs 1-9), a structurally related soluble guanylyl cyclase (sGC) and a soluble AC (sAC). Moreover, Bacillus anthracis and Bacillus pertussis produce the AC toxins, edema factor (EF), and adenylyl cyclase toxin (ACT), respectively. 2'(3')-O-(N-methylanthraniloyl)-guanosine 5'-[gamma-thio]triphosphate is a potent competitive inhibitor of AC in S49 lymphoma cell membranes.

Synthesis of 2',5'-dideoxy-2-fluoroadenosine and 2',5'-dideoxy-2,5'-difluoroadenosine: potent P-site inhibitors of adenylyl cyclase.

Glycosylation of 2-fluoroadenine with the appropriate protected thioglycoside derivatives, followed by deprotection and anomer separation, produced the alpha- and beta-anomers of 2',5'-dideoxy-2-fluoroadenosine (1), 2',5'-dideoxy-2,5'-difluoroadenosine (2), and 2'-deoxy-2-fluoroadenosine (3). These were examined as P-site inhibitors of adenylyl cyclase. The presence of fluorine on the purine ring increased potency of inhibition, and the most potent compound, beta-2',5'-dideoxy-2-fluoroadenosine (1b), was 3 times more potent than beta-2',5'-dideoxyadenosine.

Pro-nucleotide inhibitors of adenylyl cyclases in intact cells.

9-substituted adenine derivatives with protected phosphoryl groups were synthesized and tested as inhibitors of adenylyl cyclase in isolated enzyme and intact cell systems. Protected 3'-phosphoryl derivatives of 2',5'-dideoxyadenosine (2',5'-dd-Ado) and beta-l-2',5'-dd-Ado, protected 5'-phosphoryl derivatives of beta-l-2',3'-dd-Ado, and protected phosphoryl derivatives of two 9-(2-phosphonomethoxy-acyl)-adenines were synthesized. Protection was afforded by two cyclosaligenyl- or three S-acyl-2-thioethyl-substituents.

Vision examinations for all children entering public school--the new Kentucky law.

Background: Kentucky was the first state in the United States to pass a law requiring an eye examination by an optometrist or ophthalmologist for each child entering public school, public preschool, or Head Start program for the first time. The law became effective on July 15, 2000.

Method: Forty-three of 334 Kentucky Optometric Association members were surveyed by the Kentucky Optometric Association.