Stereoselective inhibition of serotonin re-uptake and phosphodiesterase by dual inhibitors as potential agents for depression

Multi-target compounds where more than one functional activity is incorporated into the same molecule may have advantages in treating disease states. Selective serotonin re-uptake inhibitors (SSRIs)^a (i.e., (R)- and (S)-norfluoxetine) were chemically linked to a PDE4 inhibitor via a five carbon bridge. The new dual PDE4 inhibitor/SSRIs (i.e., (R)-8 and (S)-8) showed moderately potent but highly selective serotonin re-uptake inhibition (IC₅₀ values of 173 and 42 nM, respectively) in vitro. The dual PDE4 inhibitor/SSRIs (R)-8 and (S)-8 also inhibited PDE4D2 (i.e., K_i values of 106 and 253 nM, respectively). Due to the synergistic functional activity, PDE4 inhibitor/SSRIs may be effective in treating diseases such as depression.     

Dissection of CENP-C–directed Centromere and Kinetochore Assembly

Eukaryotic cells ensure accurate chromosome segregation in mitosis by assembling a microtubule-binding site on each chromosome called the kinetochore that attaches to the mitotic spindle. The kinetochore is assembled specifically during mitosis on a specialized region of each chromosome called the centromere, which is constitutively bound by >15 centromere-specific proteins. These proteins, including centromere proteins A and C (CENP-A and -C), are essential for kinetochore assembly and proper chromosome segregation. How the centromere is assembled and how the centromere promotes mitotic kinetochore formation are poorly understood. We have used Xenopus egg extracts as an in vitro system to study the role of CENP-C in centromere and kinetochore assembly. We show that, unlike the histone variant CENP-A, CENP-C is not maintained at centromeres through spermatogenesis but is assembled at the sperm centromere from the egg cytoplasm. Immunodepletion of CENP-C from metaphase egg extract prevents kinetochore formation on sperm chromatin, and depleted extracts can be complemented with in vitro–translated CENP-C. Using this complementation assay, we have identified CENP-C mutants that localized to centromeres but failed to support kinetochore assembly. We find that the amino terminus of CENP-C promotes kinetochore assembly by ensuring proper targeting of the Mis12/MIND complex and CENP-K.     

Filamin A Regulates Caveolae Internalization and Trafficking in Endothelial Cells

Transcytosis via caveolae is critical for maintaining vascular homeostasis by regulating the tissue delivery of macromolecules, hormones, and lipids. In the present study, we test the hypothesis that interactions between F-actin cross-linking protein filamin A and caveolin-1 facilitate the internalization and trafficking of caveolae. Small interfering RNA-mediated knockdown of filamin A, but not filamin B, reduced the uptake and transcytosis of albumin by ∼35 and 60%, respectively, without altering the actin cytoskeletal structure or cell–cell adherens junctions. Mobility of both intracellular caveolin-1–green fluorescent protein (GFP)-labeled vesicles measured by fluorescence recovery after photobleaching and membrane-associated vesicles measured by total internal reflection-fluorescence microscopy was decreased in cells with reduced filamin A expression. In addition, in melanoma cells that lack filamin A (M2 cells), the majority of caveolin-1-GFP was localized on the plasma membrane, whereas in cells in which filamin A expression was reconstituted (A7 cells and M2 cells transfected with filamin A-RFP), caveolin-1-GFP was concentrated in intracellular vesicles. Filamin A association with caveolin-1 in endothelial cells was confirmed by cofractionation of these proteins in density gradients, as well as by coimmunoprecipitation. Moreover, this interaction was enhanced by Src activation, associated with increased caveolin-1 phosphorylation, and blocked by Src inhibition. Taken together, these data suggest that filamin A association with caveolin-1 promotes caveolae-mediated transport by regulating vesicle internalization, clustering, and trafficking.     

The anaphase promoting complex targeting subunit Ama1 links meiotic exit to cytokinesis during sporulation in Saccharomyces cerevisiae

Ascospore formation in yeast is accomplished through a cell division in which daughter nuclei are engulfed by newly formed plasma membranes, termed prospore membranes. Closure of the prospore membrane must be coordinated with the end of meiosis II to ensure proper cell division. AMA1 encodes a meiosis-specific activator of the anaphase promoting complex (APC). The activity of APC^Ama1 is inhibited before meiosis II, but the substrates specifically targeted for degradation by Ama1 at the end of meiosis are unknown. We show here that ama1Δ mutants are defective in prospore membrane closure. Ssp1, a protein found at the leading edge of the prospore membrane, is stabilized in ama1Δ mutants. Inactivation of a conditional form of Ssp1 can partially rescue the sporulation defect of the ama1Δ mutant, indicating that an essential function of Ama1 is to lead to the removal of Ssp1. Depletion of Cdc15 causes a defect in meiotic exit. We find that prospore membrane closure is also defective in Cdc15 and that this defect can be overcome by expression of a form of Ama1 in which multiple consensus cyclin-dependent kinase phosphorylation sites have been mutated. These results demonstrate that APC^Ama1 functions to coordinate the exit from meiosis II with cytokinesis.     

Patterns of distribution and abundance of bonefish larvae <Emphasis Type="Italic">Albula</Emphasis> spp. (Albulidae) in the western Caribbean and adjacent areas

Overall patterns of distribution and abundance of Albula spp. leptocephali larvae offshore in the western Caribbean Sea (CAS) and Gulf of Mexico (GOM), and in coastal waters of the Mexican Caribbean (MXC) were analyzed from: (a) cruise data available from the Academy of Natural Sciences of Philadelphia (CAS, GOM) and (b) coastal plankton surveys (1998–2002 and January 2004) (MXC). We found striking inshore-offshore differences in the larval catch and size structure. Offshore cruises yielded 57 leptocephali, mostly determined as early stage I (18.0 ± 8.2 mm SL, mean ± SD). In contrast, coastal samples yielded 2,466 larvae 51.4 ± 3.6 mm SL, mostly late stage I; of these, 2,345 (95%) were caught over 4 nights in January 2004. The relationship between the larval length (mm, SL) and the distance to the coastline (km) was best represented by the regression model with a distinct variance for each locality. To ascertain whether the coastal inflow of leptocephali follows a regular seasonal pattern or depends on episodic events will require further monitoring; available evidence suggests that the southern coast of the MXC offers favorable conditions for the recruitment of Albula spp. larvae.