Proline-directed phosphorylation and isomerization in mitotic regulation and in Alzheimer's Disease

The reversible phosphorylation of proteins on serine/threonine residues preceding proline (Ser/Thr-Pro) is a major regulatory mechanism for the control of a series of cell cycle events. Although phosphorylation is thought to regulate protein function by inducing conformational changes, little is known about most of these conformational changes and their significance. Recent studies indicate that the conformation and function of a subset of these phosphorylated proteins are controlled by the prolyl isomerase Pin1 through isomerization of specific phosphorylated Ser/Thr-Pro bonds. Furthermore, compelling evidence supports the idea that proline-directed phosphorylation and subsequent isomerization play a critical role not only in cell cycle control, but also in the development of Alzheimer's disease, where postmitotic neurons display various cell cycle markers, especially mitotic events, prior to degeneration.     

Biphasic intracellular expression of Staphylococcus aureus virulence factors and evidence for Agr-mediated diffusion sensing

Staphylococcus aureus invades a variety of mammalian cells and escapes from the endosome to multiply in the cytoplasm. We had previously hypothesized that the molecular events leading to escape of S. aureus from the endosome involved the Agr virulence factor regulatory system. In this report we demonstrate that temporal changes in intracellular activation of the Agr regulon correlates with expression of membrane active toxins. Also, the initial expression of Agr by even small numbers of staphylococci resulted in the permeabilization of the endosomal membrane and the eventual escape of bacteria into the cytoplasm by 3 h post invasion. After Agr downregulation, a second peak of expression coincided with increased permeability of the host cell membrane. In contrast to the parental strain, an Agr-mutant was unable to escape into the cytoplasm and was observed in intact endosomes as late as 5 h post invasion. These data provide evidence that staphylococcal virulence factor production during invasion of host cells is mediated by an Agr-dependent process that is most accurately described in the context of diffusion sensing.     

Bone-targeted 2,6,9-trisubstituted purines: novel inhibitors of Src tyrosine kinase for the treatment of bone diseases

Novel bone-targeted 2,6,9-trisubstituted purine template-based inhibitors of Src tyrosine kinase are described. Drug design studies of known purine compounds revealed that both positions-2 and -6 were suitable for incorporating bone-seeking moieties. A variety of bone-targeting groups with different affinity to hydroxyapatite were utilized in the study. Compound 3d was determined to be a potent Src inhibitor and was quite selective against a panel of other protein kinases.     

Clastogenic effect for cigarette smoking but not areca quid chewing as measured by micronuclei in exfoliated buccal mucosal cells

The objective of this study was to use the micronuclei from exfoliated buccal mucosal cells to investigate the clastogenic effects of areca quid chewing and cigarette smoking, as well as the interaction between the two. The study population was selected from residents of seven villages recruited for a community-cohort study. A total of 141 subjects were recruited based on the regular consumption of cigarettes and betel quid. Salient personal characteristics were collected from interview using a specially designed questionnaire. Micronuclei were scored on Feulgen/fast green-stained smear preparations of exfoliated cells obtained by scraping the surface of the buccal mucosa. There was no significant interaction between the chewing of betel nut and cigarette smoking. Heavy smoking was positively associated with MN frequency, with areca quid chewing negatively associated. A significant positive trend was demonstrated for the relationship between MN frequency and either daily cigarette consumption or cumulative smoking pack-years. By contrast, negative trends were demonstrated for the analogous relationships with areca quid chewing. These results indicate that heavy smoking, but not areca quid chewing, increases MN formation. These findings suggest that the carcinogenesis of the oral cancers induced by areca quid chewing in Taiwan may be through a pathway other than genotoxicity.     

Conversion of a trans-spliced C. elegans gene into a conventional gene by introduction of a splice donor site.

In Caenorhabditis elegans, pre-mRNAs that are trans-spliced are distinguished by the presence of an 'outron', intron-like RNA at the 5' end followed by a splice acceptor. We report that trans-splicing of the rol-6 gene can be completely suppressed simply by introducing a donor site into its 173 nt outron, at a site 50 nt upstream of the trans-splice site, thereby converting rol-6 into a conventional gene with a spliced intron near its 5' end. When the consensus donor site was inserted at sites further upstream it was less effective in replacing transplicing with cis-splicing. Surprisingly, the length of the intron was not the important variable, since lengthening of the 50 nt intron to 250 nt did not restore trans-splicing. Apparently the context into which the splice site was introduced determined the efficiency of its use. These results support the conclusion that the sole signal for trans-splicing is the presence of an outron. Clearly, cis- and trans-splice acceptor sites are interchangeable, allowing the possibility of competition between the two types of splicing.