The Characterization of PG0228 in Porphyromonas gingivalis W83

Periodontitis affects 10 to 15 percent of most adult populations and can contribute to numerous systemic diseases. Porphyromonas gingivalis, a gram-negative anaerobic bacterium, is a recognized prime causative agent in periodontitis. Studies have shown a number of small non-coding RNAs (sRNAs) have been related to bacterial virulence. Many of these sRNAs require the facilitation of the bacterial Sm-like protein, Hfq, for optimum function. Hfq is a RNA chaperone involved in RNA stability, sRNA function, and polyadenylation. Mutants lacking in Hfq often show pleiotropic phenotypes, although the extent and severity of hfq null phenotypes is often species-specific. Hfq has been encoded by nearly half of eubacteria, including pathogens. Based on a standard BLAST search, hfq has not been detected in P. gingivalis. It is highly likely, however, that the bacterium possesses an Hfq homologue due to its importance as an overall RNA cofactor. The P. gingivalis hypothetical protein, PG0228, possesses the Sm-like protein motif, thus we believe it is an excellent Hfq candidate. Our goal was to characterize PG0228 so we can gain a better insight into the function of this hypothetical protein and determine if it indeed behaves like Hfq. Microarray analysis, growth studies, and a survival study were done on a Δ0228 mutant to determine the biological role of the protein encoded by PG0228. PG0228 was also tagged in vivo in order to determine if the protein binds to RNA. Our results show P. gingivalis deficient in PG0228 show significant similarities to other bacterium deficient in hfq. The Δ0228 strain showed significant sensitivity to host defense mechanisms and an overall gene regulation in 15% of the genome. In addition, the mutant is viable but produces a lower final cell density. Thus, we believe PG0228 is an excellent Hfq candidate, and suggest further studies will show PG0228 is an Hfq homologue

Congenital adrenal hyperplasia: The influence of puberty on cortisol pharmacokinetics.

In Congenital Adrenal Hyperplasia (CAH) due to 21-hydroxylase deficiency, appropriate glucocorticoid substitution does not often result in adequate suppression of the hypothalamic-pituitary-adrenal (HPA) axis. Suboptimal control is especially observed in pubertal patients, despite adherence to medical treatment. The clinical studies described in this thesis were performed to address the effect of puberty on cortisol pharmacokinetics and HPA axis activity in patients with CAH due to 21-hydroxylase deficiency. Six studies were conducted on forty patients (14 prepubertal, 20 pubertal and 6 postpubertal) with classical 21-hydroxylase deficiency. They revealed: i) An alteration in the pharmacokinetic parameters of total and free cortisol at puberty: There was an increase in cortisol clearance and volume of distribution but no change in half life. The half life of free cortisol was shorter in females compared to males. ii) An increase in cortisol clearance and a decrease in half life in association with specific alterations in the endocrine milieu at puberty, including alterations in the growth hormone (GH) ~ insulin-like growth factors and 11β-hydroxysteroid dehydrogenase activity. Also, a decrease in cortisol half life in association with elevated adrenocorticotrophic hormone (ACTH) concentrations. iii) Essentially complete bioavailability of oral hydrocortisone tablets used as substitution therapy in these patients. iv) A significant negative correlation between cortisol and 17-hydroxyprogesterone concentrations. Also, a difference in the activity of HPA axis between daytime and night-time, with the minimal activity observed between 1600h and 0400h. v) No alteration in the negative feedback effect of cortisol at the pituitary level. vi) A significant positive correlation between serum cortisol and GH concentrations. The above findings may explain the difficulties encountered in the management of CAH patients at puberty and suggest that replacement therapy should be with frequent doses of glucocorticoid substitution in all pubertal patients, particularly females. Management should also aim at providing adequate HPA axis suppression between 0400h and 1600h, as well as preventing and/or treating hyperandrogenism