Cellular basis for the olfactory response to nicotine.

Smokers regulate their smoking behavior on the basis of sensory stimuli independently of the pharmacological effects of nicotine (Rose J. E., et al. (1993) Pharmacol., Biochem. Behav.44 (4), 891-900). A better understanding of sensory mechanisms underlying smoking behavior may help to develop more effective smoking alternatives. Olfactory stimulation by nicotine makes up a considerable part of the flavor of tobacco smoke, yet our understanding of the cellular mechanisms responsible for olfactory detection of nicotine remains incomplete. We used biophysical methods to characterize the nicotine sensitivity and response mechanisms of neurons from olfactory epithelium. In view of substantial differences in the olfactory receptor repertoire between rodent and human (Mombaerts P. (1999) Annu. Rev. Neurosci.22, 487-509), we studied biopsied human olfactory sensory neurons (OSNs), cultured human olfactory cells (Gomez G., et al. (2000) J. Neurosci. Res.62 (5), 737-749), and rat olfactory neurons. Rat and human OSNs responded to S(-)-nicotine with a concentration dependent influx of calcium and activation of adenylate cyclase. Some rat OSNs displayed some stereoselectivity, with neurons responding to either enantiomer alone or to both. Freshly biopsied and primary cultured human olfactory neurons were less stereoselective. Nicotinic cholinergic antagonists had no effect on the responses of rat or human OSNs to nicotine. Patch clamp recording of rat OSNs revealed a nicotine-activated, calcium-sensitive nonspecific cation channel. These results indicate that nicotine activates a canonical olfactory receptor pathway rather than nicotinic cholinergic receptors on OSNs. Further, because the nicotine-sensitive mechanisms of rodents appear generally similar to those of humans, this animal model is an appropriate one for studies of nicotine sensation.

Engineered Saccharomyces cerevisiae strain BioS-1, for the detection of water-borne toxic metal contaminants.

Saccharomyces cerevisiae responds to extracellular toxic stimuli by increasing intracellular cyclic AMP levels, leading to activation of a cAMP-dependent protein kinase, protein kinase A (PKA). Activated PKA phosphorylates downstream substrates, including specific DNA-binding proteins, to turn off the expression of most or all of the yeast genes. Such cAMP-PKA-mediated inhibition of gene expression in response to toxic stimuli appears to be unique to S. cerevisiae. For instance, in mammalian cells, the cAMP-PKA signaling pathway is rather responsive to growth factors and hormones in addition to being primarily involved in the activation of gene expression. Activation of gene expression by the cAMP-PKA pathway in mammalian cells is due mainly to the presence of cAMP-response elements (CREs) located in the promoters of many mammalian genes, and the expression of PKA-responsive stimulatory transcription factor CRE-binding protein, commonly referred as CREBP, which binds to the CREs. Thus, activation of the cAMP-PKA signaling pathway results in the phosphorylation of CREBP by PKA, and phosphorylated CREBP transactivates specific gene expression by interacting with the cognate CRE. Based on these findings, we sought to engineer a yeast-based biosensor, in which the stress-sensing cAMP-PKA pathway of yeast is coupled to the mammalian CREBP-CRE-stimulated gene expression pathway, which drives the expression of a reporter protein, such as green fluorescent protein (GFP). As a primary step toward the development of this biosensor, we engineered a yeast strain, BioS-1, by genetically altering YPH 501, a wild-type strain of S. cerevisiae, to express human CREBP and human CRE promoter-driven GFP. Exposure of BioS-1 to varying concentrations of As3+, Fe2+, Pb2+, and Cd2+ elicits concentration-dependent expression of the GFP reporter that can be easily monitored by the fluorescence emitted by GFP. The results also indicate that the engineered BioS-1 yeast cells can detect 2.5 ppm of these toxic metals and report it through the expression of GFP within 3 h. The results presented herein demonstrate that this engineered yeast strain can detect metal toxicants and can validate the use of this prototypic yeast strain to develop a biosensor that can be used to detect and monitor cytotoxic water-borne toxic heavy metals.

Proliferation-specific genes activated by Galpha(12): a role for PDGFRalpha and JAK3 in Galpha(12)-mediated cell proliferation.

Galpha(12), the alpha-subunit of G protein G12, is ubiquitously expressed and it has been identified as a putative "causative oncogene" of soft-tissue sarcomas. Overexpression of wild-type or GTPase-deficient mutant of Galpha(12) (Galpha(12)Q229L or Galpha(12)QL) leads to the oncogenic transformation of NIH3T3 cells. Galpha(12)QL-tramsformed NIH3T3 cells show a distinct oncogenic phenotype defined by increased cell proliferation, anchorage-independent growth, reduced growth-factor dependency, attenuation of apoptotic signals, and neoplastic cytoskeletal changes. In this study, the genes contributing to the reduced growth-factor dependency of Galpha(12)QL-NIH3T3 cells were identified by transcription profiling of serum-starved Galpha(12)QL-transformed NIH3T3 (Galpha(12)QL-NIH3T3) cells. Results from these studies indicate that Galpha(12)QL stimulates the expression of genes that promote cell growth. The increased expressions of growth-promoting genes in Galpha(12)QL-NIH3T3 cells were validated by semiquantitative reverse transcription-polymerase chain reaction and immunoblot analyses. Further studies aimed at investigating the critical role of two of such upregulated genes, namely PDGFRalpha and JAK3, indicated that the inhibition of PDGFRalpha or JAK3 activity-attenuated Galpha(12)QL-mediated serum-independent cell proliferation. These studies point to possible novel autocrine and/or paracrine control mechanisms involving PDGFRalpha and JAK3 in Galpha(12)-mediated proliferation and oncogenesis.