Effects of the antidepressant fluoxetine on the subcellular localization of 5-HT1A receptors and SERT

Serotonin (5-HT) 5-HT(1A) autoreceptors (5-HT(1A)autoR) and the plasmalemmal 5-HT transporter (SERT) are key elements in the regulation of central 5-HT function and its responsiveness to antidepressant drugs. Previous immuno-electron microscopic studies in rats have demonstrated an internalization of 5-HT(1A)autoR upon acute administration of the selective agonist 8-OH-DPAT or the selective serotonin reuptake inhibitor antidepressant fluoxetine. Interestingly, it was subsequently shown in cats as well as in humans that this internalization is detectable by positron emission tomography (PET) imaging with the 5-HT(1A) radioligand [(18)F]MPPF. Further immunocytochemical studies also revealed that, after chronic fluoxetine treatment, the 5-HT(1A)autoR, although present in normal density on the plasma membrane of 5-HT cell bodies and dendrites, do not internalize when challenged with 8-OH-DPAT. Resensitization requires several weeks after discontinuation of the chronic fluoxetine treatment. In contrast, the SERT internalizes in both the cell bodies and axon terminals of 5-HT neurons after chronic but not acute fluoxetine treatment. Moreover, the total amount of SERT immunoreactivity is then reduced, suggesting that SERT is not only internalized, but also degraded in the course of the treatment. Ongoing and future investigations prompted by these finding are briefly outlined by way of conclusion.     

Analysis of Na+,K+-ATPase motion and incorporation into the plasma membrane in response to G protein-coupled receptor signals in living cells

Dopamine (DA) increases Na(+),K(+)-ATPase activity in lung alveolar epithelial cells. This effect is associated with an increase in Na(+),K(+)-ATPase molecules within the plasma membrane (). Analysis of Na(+),K(+)-ATPase motion was performed in real-time in alveolar cells stably expressing Na(+),K(+)-ATPase molecules carrying a fluorescent tag (green fluorescent protein) in the alpha-subunit. The data demonstrate a distinct (random walk) pattern of basal movement of Na(+),K(+)-ATPase-containing vesicles in nontreated cells. DA increased the directional movement (by 3.5 fold) of the vesicles and an increase in their velocity (by 25%) that consequently promoted the incorporation of vesicles into the plasma membrane. The movement of Na(+),K(+)-ATPase-containing vesicles and incorporation into the plasma membrane were microtubule dependent, and disruption of this network perturbed vesicle motion toward the plasma membrane and prevented the increase in the Na(+),K(+)-ATPase activity induced by DA. Thus, recruitment of new Na(+),K(+)-ATPase molecules into the plasma membrane appears to be a major mechanism by which dopamine increases total cell Na(+),K(+)-ATPase activity.     

Immunoglobulin heavy- and light-chain repertoire in splenic marginal zone lymphoma

The considerable heterogeneity in morphology, immunophenotype, genotype, and clinical behavior of splenic marginal zone lymphoma (SMZL) hinders firm conclusions on the origin and differentiation stage of the neoplastic cells. Immunoglobulin (IG) gene usage and somatic mutation patterns were studied in a series of 43 SMZL cases. Clonal IGHV-D-J rearrangements were amplified in 42/43 cases (4 cases carried double rearrangements). Among IGHV-D-J rearrangements, IGHV3 and IGHV4 subgroup genes were used with the highest frequency. Nineteen IGHV genes were unmutated (> 98% homology to the closest germline IGHV gene), whereas 27/46 were mutated. Clonal IGKV-J and IGLV-J gene rearrangements were amplified in 36/43 cases, including 31 IGKV-J (8/31 in lambda light-chain expressing cases) and 12 IGLV-J rearrangements; 9/31 IGKV and 6/12 IGLV sequences were mutated. IGKV-J and IGLV-J rearrangements used 14 IGKV and 9 IGLV different germline genes. Significant evidence for positive selection by classical T-dependent antigen was found in only 5/27 IGHV and 6/15 IGKV+IGLV mutated genes. These results provide evidence for the diverse B-cell subpopulations residing in the SMZ, which could represent physiologic equivalents of distinct SMZL subtypes. Furthermore, they indicate that in SMZL, as in other B cell malignancies, a complementarity imprint of antigen selection might be witnessed either by IGHV, IGKV, or IGLV rearranged sequences.     

Zebularine: a unique molecule for an epigenetically based strategy in cancer chemotherapy. The magic of its chemistry and biology

1-(beta-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one (zebularine) is structurally 4-deamino cytidine. The increased electrophilic character of this simple aglycon endows the molecule with unique chemical and biological properties, making zebularine a versatile starting material for the synthesis of complex nucleosides and an effective inhibitor of cytidine deaminase and DNA cytosine methyltransferase. Zebularine is a stable, antitumor agent that preferentially targets cancer cells and shows activity both in vitro and in experimental animals, even after oral administration.     

Relevance of dopamine signals anchoring dynamin-2 to the plasma membrane during Na+,K+-ATPase endocytosis

Clathrin-dependent endocytosis of Na(+),K(+)-ATPase in response to dopamine regulates its catalytic activity in intact cells. Because fission of clathrin-coated pits requires dynamin, we examined the mechanisms by which dopamine receptor signals promote dynamin-2 recruitment and assembly at the site of Na(+),K(+)-ATPase endocytosis. Western blotting revealed that dopamine increased the association of dynamin-2 with the plasma membrane and with phosphatidylinositol 3-kinase. Dopamine inhibited Na(+),K(+)-ATPase activity in OK cells and in those overexpressing wild type dynamin-2 but not in cells expressing a dominant-negative mutant. Dephosphorylation of dynamin is important for its assembly. Dopamine increased protein phosphatase 2A activity and dephosphorylated dynamin-2. In cells expressing a dominant-negative mutant of protein phosphatase 2A, dopamine failed to dephosphorylate dynamin-2 and to reduce Na(+),K(+)-ATPase activity. Dynamin-2 is phosphorylated at Ser(848), and expression of the S848A mutant significantly blocked the inhibitory effect of dopamine. These results demonstrate a distinct signaling network originating from the dopamine receptor that regulates the state of dynamin-2 phosphorylation and that promotes its location (by interaction with phosphatidylinositol 3-kinase) at the site of Na(+),K(+)-ATPase endocytosis.     

Utility of a pre-optimized kit for random amplified polymorphic DNA in typing Candida albicans

The utility of a pre-optimized kit for random amplified polymorphic DNA (RAPD) was assessed in typing diverse strains of Candida albicans from epidemiologically unrelated inpatients (interpatient analysis) and in detecting clonal variations that maybe present within individual patient isolates (intrapatient analysis). Stool samples from inpatients were cultured on Inhibitory Mold agar. Nine individual colonies from all patients with > or =9 colonies of C. albicans (n = 18) were selected, frozen, and karyotyped using CHEF genomic DNA plug kits and CHEF-DRIII. Each of the selected colonies was then analyzed by RAPD, utilizing the selected kit, with 6 primers. Interpatient analysis revealed 9 karyotypes and 17 RAPD composites. RAPD discrimination was significantly better (p < 0.001). Intrapatient analysis revealed 34 (21%) and 33 (20.4%) variants among 162 colonies tested by RAPD and karyotyping, respectively. The results were discordant in 25 variants, all with differences of 1-3 bands. These results illustrate that this pre-optimized kit for RAPD provides excellent discrimination of genetically unrelated strains. Its performance in delineating subtle clonal differences was comparable with karyotyping; both methods failed to detect all minor genetic variations. The ease of use and quick turnaround time of this kit offer a practical and reliable method for typing diverse strains of C. albicans, but may be inadequate for assessing microevolution.     

Molecular characteristics of diverse populations are consistent with the hypothesis of a recent invasion of Drosophila melanogaster by mobile P elements

Approximately 100 strains derived from natural populations of Drosophila melanogaster were tested for the presence or absence of P- element sequences by using two molecular probes derived from internal regions of a full-sized P element. Strains that had been collected from several continents at varying times during the past 60 years were examined. The oldest available strains, representing most major geographical regions of the world, exhibited no detectable hybridization to the P-element probes. In contrast, all recently collected natural populations that were tested carried P-element sequences. The earliest appearance of P elements occurred in collections made during the 1950s and early 1960s in the Americas and during the late 1960s on other continents. The youngest strains that were completely devoid of P elements originated in populations sampled during the mid-1960s in America, but as late as 1974 in populations from the USSR. There are differences in the patterns of hybridization to the two P-element probes between populations from different geographical regions. These differences are consistent with the varying P-M phenotypic properties of these populations. Taken together with the results of phenotypic tests reported in earlier studies, the available evidence is consistent with the hypothesis of a worldwide P-element invasion of D. melanogaster during the past 30 years and suggests that the putative invasion of the Americas possibly preceded by approximately a decade that in Europe, Africa, and the rest of the world.      

Pharmacological Evidence for the Involvement of Calciuml Calmodulin in Serotonin 5-HT3 Receptor-Mediated Cation Permeability in NG 108-15 Cells

Abstract: In NG 108–15 clonal cells, extracellular application of micromolar concentrations of serotonin [5-hydroxy-tryptamine (5-HT)] and substance P induces the opening of a cation permeability monitored by the influx of [¹⁴C]-guanidinium. The serotoninergic component of this cation permeability Is linked to 5-HT₃ receptor activation, whereas the substance P component probably involves an “N-terminal-dependent substance P receptor.” In this study, [¹⁴C]guanidinium influx triggered by 1 μM 5-HT plus 10 μM substance P was shown to be insensitive to tetrodotoxin, verapamil, diltiazem, nimodipine, and ω-conotoxin, as expected from a process independent of voltage-sensitive sodium and calcium channels. In contrast, [¹⁴C]guanidinium influx was inhibited by millimolar concentrations of extracellular calcium and by the chelation of intracellular calcium by bis-O-aminophenoxyethanetetraacetic acid. The inhibition by extracellular calcium apparently involved a competition between the divalent cation and [¹⁴C]guanidinium for the same channel. When NG 108–15 cells were exposed to X537A, an ionophore that specifically induces release of calcium from intracellular stores, [¹⁴C]guanidinium uptake was markedly increased even in the absence of 5-HT and/or substance P. Conversely, [¹⁴C]guanidinium influx due to the latter substances could be reversibly and dose-dependently blocked by various drugs that possess calmodulin-antagonizing properties. These results strongly suggest that the cation permeability opened by 5-HT and substance P in NG 108–15 cells involves a calcium/calmodulin-dependent process. However, as the phosphodiesterase inhibitor isobutylmethylxanthine, the nitric oxide synthase inhibitor A/monomethylarginine, the protein kinase C inhibitor staurosporine, and the protein kinase C activator 12-O-tetradeca-noylphorbol 13-acetate did not alter [¹⁴C]guanidinium influx in NG 108–15 cells exposed to 5-HT and substance P., this process probably does not involve the calcium-dependent nitric oxide pathway and protein kinase C activation.     

Mitochondrial control-region sequences in two shorebird species, the turnstone and the dunlin, and their utility in population genetic studies

We determined the mitochondrial control-region sequences of five turnstones (Arenaria interpres) and three dunlins (Calidris alpina). Comparisons revealed that the central part (part II) is conserved relative to much more variable parts at the beginning (part I) and the end (part III). This pattern of sequence conservation is also found in the control regions of other vertebrates. The average sequence divergence between turnstone and dunlin was 21.8% for part I, 7.5% for part II, and 29.5% for part III. Within-species sequence divergence over the entire control region was much lower, at 0.9% for turnstones and 2.0% for dunlins. In both shorebird species, part III contains a repetitive sequence composed only of A and C nucleotides, which has not been found in the control regions of other birds. A survey of the part I sequences of 25 turnstones and 25 dunlins sampled around the world revealed that these species have very different population genetic structures. Dunlins are not only much more differentiated in their sequences but also have a strongly subdivided population genetic structure. Pleistocene vicariant events combined with strong natal philopatry and high mutation rates of the sequences are likely responsible for this population genetic subdivision. Conversely, part I sequences of turnstones are weakly differentiated and are geographically unstructured. We argue that this is not the result of global gene flow but that, instead turnstones have recently expanded from a refugial population that was bottlenecked.      

Ensuring performance and provider profit through data replication in cloud systems

Cloud computing is a relatively recent computing paradigm that is often the answer for dealing with large amounts of data. Tenants expect the cloud providers to keep supplying an agreed upon quality of service, while cloud providers aim to increase profits as it is a key ingredient of any economic enterprise. In this paper, we propose a data replication strategy for cloud systems that satisfies the response time objective for executing queries while simultaneously enables the provider to return a profit from each execution. The proposed strategy estimates the response time of the queries and performs data replication in a way that the execution of any particular query is still estimated to be profitable for the provider. We show with simulations that how the proposed strategy fulfills these two criteria.