Role of acetyl-phosphate in activation of the Rrp2-RpoN-RpoS pathway in Borrelia burgdorferi.

Borrelia burgdorferi, the Lyme disease spirochete, dramatically alters its transcriptome and proteome as it cycles between the arthropod vector and mammalian host. During this enzootic cycle, a novel regulatory network, the Rrp2-RpoN-RpoS pathway (also known as the σ(54)-σ(S) sigma factor cascade), plays a central role in modulating the differential expression of more than 10% of all B. burgdorferi genes, including the major virulence genes ospA and ospC. However, the mechanism(s) by which the upstream activator and response regulator Rrp2 is activated remains unclear. Here, we show that none of the histidine kinases present in the B. burgdorferi genome are required for the activation of Rrp2. Instead, we present biochemical and genetic evidence that supports the hypothesis that activation of the Rrp2-RpoN-RpoS pathway occurs via the small, high-energy, phosphoryl-donor acetyl phosphate (acetyl∼P), the intermediate of the Ack-Pta (acetate kinase-phosphate acetyltransferase) pathway that converts acetate to acetyl-CoA. Supplementation of the growth medium with acetate induced activation of the Rrp2-RpoN-RpoS pathway in a dose-dependent manner. Conversely, the overexpression of Pta virtually abolished acetate-induced activation of this pathway, suggesting that acetate works through acetyl∼P. Overexpression of Pta also greatly inhibited temperature and cell density-induced activation of RpoS and OspC, suggesting that these environmental cues affect the Rrp2-RpoN-RpoS pathway by influencing acetyl∼P. Finally, overexpression of Pta partially reduced infectivity of B. burgdorferi in mice. Taken together, these findings suggest that acetyl∼P is one of the key activating molecule for the activation of the Rrp2-RpoN-RpoS pathway and support the emerging concept that acetyl∼P can serve as a global signal in bacterial pathogenesis.

Pharmacological characterization of nipecotic acid ethyl ester: A novel cholinergic muscarinic agonist

The aim of this dissertation was to examine the hypothesis that (R)-nipecotic acid ethyl ester ((R)-NAEE) is a cholinergic agonist that is selective for a particular subclass (M$\sb1$ or M$\sb2$) of muscarinic receptors.^ Ligand binding studies indicated that like cholinergic agonists (R)-NAEE selectively interacts with rat heart (M$\sb2$) and brain (M$\sb1$) muscarinic binding sites. Physiological studies revealed that unlike cholinergic agonists (R)-NAEE stimulated only those responses coupled to M$\sb2$ muscarinic receptors (acid secretion, negative inotropic response, smooth muscle contraction). Moreover, in rat brain (R)-NAEE differentiated between M$\sb2$ receptors negatively coupled to adenylate cyclase activity and M$\sb1$ receptors mediating PI turnover, being a weak competitive antagonist at these latter sites. In isolated rat gastric mucosal cells (R)-NAEE also differentiated between two M$\sb2$ coupled responses where it potentiated acid secretion but could not stimulate PI turnover. Atropine, a selective antimuscarinic agent, competitively antagonized all agonist effects of (R)-NAEE.^ Unlike (R)-NAEE, the muscarinic agonist arecoline, which is structurally similar to (R)-NAEE, stimulates both M$\sb1$ and M$\sb2$ receptors. Structure activity studies revealed that saturation of the piperidine ring and the length of the ester side chain of (R)-NAEE are the most important determinants for both M$\sb2$ efficacy and selectivity.^ The results of this dissertation establish that (R)-NAEE is a cholinergic muscarinic receptor agonist that displays greater efficacy at M$\sb2$ than at M$\sb1$ receptors, being a weak antagonist at the M$\sb1$ site. With such selectivity, (R)-NAEE may be regarded as a prototype for a unique class of cholinergic muscarinic M$\sb2$ receptor agonists. Because of these unique properties, (R)-NAEE should be useful in the further characterization of muscarinic receptors, and could lead to the development of a new class of therapeutic agents. ^

Liposome -mediated delivery of all-{\it trans\/}-retinoic acid

Because of its antiproliferative and differentiation-inducing properties, all-trans-retinoic acid (ATRA) has been used as a chemopreventive and therapeutic agent, for treatment various cancers including squamous cell carcinomas (SCCs). Long-term treatment with ATRA is associated with toxic effects in patients leading to acute or chronic hypervitaminosis syndrome. Moreover, prolonged treatment with oral ATRA leads to acquired resistance to the differentiation-inducing effects of the drug. This resistance is attributed to the induction of cytochrome P-450-dependent catabolic enzymes that lead to accelerated ATRA metabolism and decline in circulating levels. Most of these problems could be circumvented by incorporating ATRA in liposomes (L-ATRA) which results in sustained drug release, decrease in drug-associated toxicity, and protection of the drug from metabolism in the host. Liposomes also function as a solubilization matrix enabling lipophilic drugs like ATRA to be aerosolized and delivered directly to target areas in the aerodigestive tract and lungs. Of the 14 formulations tested, the positively-charged liposome, DPPC:SA (9:1, w/w) was found to be most effective in interacting with SCC cell lines. This, L-ATRA formulation was stable in the presence of serum proteins and buffered the toxic effects of the drug against several normal and malignant cell lines. The positive charge attributed by the presence of SA was critical for increased uptake and retention of L-ATRA by SCC cell lines and tumor spheroids. L-ATRA was highly effective in mediating differentiation in normal and transformed epithelial cells. Moreover, liposomal incorporation significantly reduced the rate of ATRA metabolism by cells and isolated liver microsomes. In vivo studies revealed that aerosol delivery is an effective way of administering L-ATRA, in terms of its safety and retention by lung tissue. The drug so delivered, is biologically active and had no toxic effects in mice. From these results, we conclude that liposome-incorporation is an excellent way of delivering ATRA to target tissues. The results obtained may have important clinical implications in treating patients with SCCs of the aerodigestive tract. ^