Cyclic AMP-dependent protein kinase (PKA) is anchored at specific subcellular sites through the interaction of the regulatory subunit (R) with protein kinase A-anchoring proteins (AKAPs) via an amphipathic helix binding motif. Synthetic peptides containing this amphipathic helix domain competitively disrupt PKA binding to AKAPs and cause a loss of PKA modulation of cellular responses. In this report we use S-Ht31, a cell-permeant anchoring inhibitor peptide, to study the role of PKA anchoring in sperm. Our analysis of three species of mammalian sperm detected three isoforms of PKA (RIIalpha, RIIbeta, and RIbeta) and one 110-kDa AKAP. The addition of S-Ht31 to bovine caudal epididymal sperm inhibits motility in a time- and concentration-dependent manner. A control peptide, S-Ht31-P, identical to S-Ht31 except for a proline for isoleucine substitution to prevent amphipathic helix formation, had no effect on motility. The inhibition of motility by S-Ht31 is reversible but only if calcium is present in the suspension buffer, suggesting a role for PKA anchoring in regulating cellular calcium homeostasis. Surprisingly, inhibition of PKA catalytic activity had little effect on basal motility or motility stimulated by agents previously thought to work via PKA activation. These data suggest that the interaction of the regulatory subunit of PKA with sperm AKAPs, independent of PKA catalytic activity, is a key regulator of sperm motility and that disruption of this interaction using cell-permeable anchoring inhibitor peptides may form the basis of a sperm-targeted contraceptive.

Objective. Blau syndrome and its sporadic counterpart, early-onset sarcoidosis, share an identical phenotype featuring the classic triad of arthritis, dermatitis, and uveitis and are associated with mutations of CARD15 in 50-90% of cases. We chose the term "pediatric granulomatous arthritis" to refer to both. An international registry was established in the spring of 2005 to define the phenotype spectrum and establish the mutation frequency and variants.Methods. Histologically confirmed granuloma and arthritis were required for inclusion. Probands and relatives were genotyped for CARD15. Deidentified clinical information was collected.Results. One year after the inception of the registry, 61 individuals from 22 pedigrees had been entered. Seven pedigrees with 19 individuals (8 affected, 11 unaffected) had clinical disease that was atypical, and none of the individuals in those pedigrees showed mutations. There were 9 classic simplex pediatric granulomatous arthritis pedigrees including 19 individuals (9 affected, 10 unaffected) and 6 classic multiplex pedigrees with 22 individuals (17 affected, 5 unaffected). Cutaneous presentation was the most common. Arthritis was polyarticular in 96% of patients. Isolated eye disease was never the presenting symptom, but significant/severe visual impairment was observed in 41% of patients. Eye disease was bilateral in 21 of 22 patients and was complicated by glaucoma in 6 of 22 patients and by cataracts in 50% of patients. Skin biopsy was the best diagnostic approach (because of accuracy and low invasiveness).Conclusion. In this series, the first combining familial and sporadic pedigrees and, to our knowledge, the largest, we further defined the phenotype and showed that all affected classic (and no nonclassic) pedigrees carry a mutation and that there is no asymptomatic carriage. If these data are confirmed, mutation analysis rather than tissue sampling may prove to be the most efficient diagnostic procedure.

We have studied a leukemic stem-cell line,

SummaryEpidemiological studies support that chronic periodontal infections are associated with an increased risk of cardiovascular disease. Previously, we reported that the periodontal pathogen Porphyromonas gingivalis accelerated atherosclerotic plaque formation in hyperlipidemic apoE -/-mice, while an isogenic fimbria-deficient (FimA-) mutant did not. In this study, we utilized 41 kDa (major) and 67 kDa (minor) fimbria mutants to demonstrate that major fimbria are required for efficient P. gingivalis invasion of human aortic endothelial cells (HAEC). Enzymelinked immunosorbent assay (ELISA) revealed that only invasive P. gingivalis strains induced HAEC production of pro-inflammatory molecules interleukin (IL)-1 β β β β , IL-8, monocyte chemoattractant protein (MCP)-1, intracellular adhesion molecule (ICAM)-1, vascular cellular adhesion molecule (VCAM)-1 and Eselectin. The purified native forms of major and minor fimbria induced chemokine and adhesion molecule expression similar to invasive P. gingivalis , but failed to elicit IL-1 β β β β production. In addition, the major and minor fimbria-mediated production of MCP-1 and IL-8 was inhibited in a dose-dependent manner by P. gingivalis lipopolysaccharide (LPS). Both P. gingivalis LPS and heat-killed organisms failed to stimulate HAEC. Treatment of endothelial cells with cytochalasin D abolished the observed pro-inflammatory MCP-1 and IL-8 response to invasive P. gingivalis and both purified fimbria, but did not affect P. gingivalis induction of IL-1 β β β β . These results suggest that major and minor fimbria elicit chemokine production in HAEC through actin cytoskeletal rearrangements; however, induction of IL-1 β β β β appears to occur via a separate mechanism. Collectively, these data support that invasive P. gingivalis and fimbria stimulate endothelial cell activation, a necessary initial event in the development of atherogenesis.