G alpha minigenes expressing C-terminal peptides serve as specific inhibitors of thrombin-mediated endothelial activation

The C termini of G protein alpha subunits are critical for binding to their cognate receptors, and peptides corresponding to the C terminus can serve as competitive inhibitors of G protein-coupled receptor-G protein interactions. This interface is quite specific as a single amino acid difference annuls the ability of a G alpha(i) peptide to bind the A(1) adenosine receptor (Gilchrist, A., Mazzoni, M., Dineen, B., Dice, A., Linden, J., Dunwiddie, T., and Hamm, H. E. (1998 ) J. Biol. Chem. 273, 14912--14919). Recently, we demonstrated that a plasmid minigene vector encoding the C-terminal sequence of G alpha(i) could specifically inhibit downstream responses to agonist stimulation of the muscarinic M(2) receptor (Gilchrist, A., Bunemann, M., Li, A., Hosey, M. M., and H. E. Hamm (1999) J. Biol. Chem. 274, 6610--6616). To selectively antagonize G protein signal transduction events and determine which G protein underlies a given thrombin-induced response, we generated minigene vectors that encode the C-terminal sequence for each family of G alpha subunits. Minigene vectors expressing G alpha C-terminal peptides (G alpha(i), G alpha(q), G alpha(12), and G alpha(13)) or the control minigene vector, which expresses the G alpha(i) peptide in random order (G(iR)), were systematically introduced into a human microvascular endothelial cell line. The C-terminal peptides serve as competitive inhibitors presumably by blocking the site on the G protein-coupled receptor that normally binds the G protein. Our results not only confirm that each G protein can control certain signaling events, they emphasize the specificity of the G protein-coupled receptor-G protein interface. In addition, the C-terminal G alpha minigenes appear to be a powerful tool for dissecting out the G protein that mediates a given physiological function following thrombin activation.     

Thrombin induces proteinase-activated receptor-1 gene expression in endothelial cells via activation of Gi-linked Ras/mitogen-activated protein kinase pathway

We addressed the mechanisms of restoration of cell surface proteinase-activated receptor-1 (PAR-1) by investigating thrombin-activated signaling pathways involved in PAR-1 re-expression in endothelial cells. Exposure of endothelial cells transfected with PAR-1 promoter-luciferase reporter construct to either thrombin or PAR-1 activating peptide increased the steady-state PAR-1 mRNA and reporter activity, respectively. Pretreatment of reporter-transfected endothelial cells with pertussis toxin or co-expression of a minigene encoding 11-amino acid sequence of COOH-terminal Galphai prevented the thrombin-induced increase in reporter activity. Pertussis toxin treatment also prevented thrombin-induced MAPK phosphorylation, indicating a role of Galphai in activating the downstream MAPK pathway. Expression of constitutively active Galphai2 mutant or Gbeta1gamma2 subunits increased reporter activity 3-4-fold in the absence of thrombin stimulation. Co-expression of dominant negative mutants of either Ras or MEK1 with the reporter construct inhibited the thrombin-induced PAR-1 expression, whereas constitutively active forms of either Ras or MEK1 activated PAR-1 expression in the absence of thrombin stimulation. Expression of dominant negative Src kinase or inhibitors of phosphoinositide 3-kinase also prevented the MAPK activation and PAR-1 expression. We conclude that thrombin-induced activation of PAR-1 mediates PAR-1 expression by signaling through Gi1/2 coupled to Src and phosphoinositide 3-kinase, and thereby activating the downstream Ras/MAPK cascade.     

Reproductive investment and parental roles in Sabine's gulls<Emphasis Type="Italic"> Xema sabini</Emphasis>

More than 90% of avian species exhibit biparental care, though parental activities are often shared unequally between the members of a pair. Among gull species (Laridae), males and females generally share parental activities, although there appear to be considerable differences between species in the relative contribution of each sex. This study examined the behaviour of male and female Sabine's gulls (Xema sabini) during the incubation period and immediately post-hatch; particularly the amount of time they each invested in breeding activities. Although considered an aberrant species in aspects of behaviour and biology, the Sabine's gull showed a high reproductive investment by both sexes, as other gull species do. Males fed females prior to egg laying and contributed equally to incubation and chick provisioning, and females contributed equally to nest defence. Overall, there was no difference between the sexes in the extent of their contributions to parental care, although there was considerable individual variation within pairs. Sabine's gulls are Arctic breeders and the extent of their contributions to parental activities could have been influenced by their extreme breeding environment and short breeding season.     

Synthesis and antiviral evaluation of benzimidazoles, quinoxalines and indoles from dehydroabietic acid

Several heterocycles, such as benzimidazoles, quinoxalines and indoles incorporated into a hydrophenanthrene and naphthalene skeleton, were synthesised from two useful ortho-bromonitro precursors derived from dehydroabietic acid: methyl 12-bromo-13-nitro-deisopropyldehydroabietate and methyl 12-bromo-13,14-dinitro-deisopropyldehydroabietate. The new heterocycles were evaluated for their activity in vitro against several RNA and DNA viruses.     

Tetrahydrobiopterin, a critical factor in the production and role of nitric oxide in mast cells

Mast cells (MC) are biologically potent, ubiquitously distributed immune cells with fundamental roles in host integrity and disease. MC diversity and function is regulated by exogenous nitric oxide; however, the production and function of endogenously produced NO in MC is enigmatic. We used rat peritoneal MC (PMC) as an in vivo model to examine intracellular NO production. Live cell confocal analysis of PMC using the NO-sensitive probe diaminofluorescein showed distinct patterns of intracellular NO formation with either antigen (Ag)/IgE (short term) or interferon-gamma (IFN-gamma) (long term). Ag/IgE-induced NO production is preceded by increased intracellular Ca2+, implying constitutive nitric-oxide synthase (NOS) activity. NO formation inhibits MC degranulation. NOS has obligate requirements for tetrahydrobiopterin (BH4), a product of GTP-cyclohydrolase I (CHI), IFN-gamma-stimulated PMC increased CHI mRNA, protein, and enzymatic activity, while decreasing CHI feedback regulatory protein mRNA, causing sustained NO production. Treatment with the CHI inhibitor, 2,4-diamino-6-hydroxypyrimidine, inhibited NO in both IFN-gamma and Ag/IgE systems, increasing MC degranulation. Reconstitution with the exogenous BH4 substrate, sepiapterin, restored NO formation and inhibited exocytosis. Thus, Ag/IgE and IFN-gamma induced intracellular NO plays a key role in MC mediator release, and alterations in NOS activity via BH4 availability may be critical to the heterogeneous responsiveness of MC.     

Accelerating the rate of disassembly of karyopherin.cargo complexes

Transport of macromolecules across the nuclear pore complex (NPC) occurs in seconds and involves assembly of a karyopherin.cargo complex and docking to the NPC, translocation of the complex across the NPC via interaction with nucleoporins (Nups), and dissociation of the complex in the nucleoplasm. To identify rate-limiting steps in the Kap95p.Kap60p-mediated nuclear import pathway of Saccharomyces cerevisiae, we reconstituted key intermediate complexes and measured their rates of dissociation and affinities of interaction. We found that a nuclear localization signal-containing protein (NLS-cargo) dissociates slowly from Kap60p monomers and Kap60p.Kap95p heterodimers with half-lives (t(12)) of 7 and 73 min, respectively; that Kap60p and Kap60p.NLS-cargo complexes dissociate slowly from Kap95p (t(12) = 36 and 73 min, respectively); and that Kap95p.Kap60p.NLS-cargo complexes and Kap95p.Kap60p heterodimers dissociate rapidly from the nucleoporin Nup1p (t(12) < or = 21 s) and other Nups. A search for factors that accelerate disassembly of the long-lived intermediates revealed that Nup1p and Nup2p accelerate 16- and 19-fold the rate of dissociation of NLS-cargo from Kap60p.Kap95p heterodimers; that Gsp1p-GTP accelerates > or = 447-fold the rate of dissociation of Kap60p.NLS-cargo from Kap95p; and that Nup2p and the Cse1p.Gsp1p-GTP complex independently accelerate > or = 22- and > or = 39-fold the rate of dissociation of NLS-cargo from Kap60p. We suggest that Nup1p, Nup2p, Cse1p, and Gsp1p accelerate disassembly of Kap95p.Kap60p.NLS-cargo complexes by triggering allosteric mechanisms within Kaps that cause rapid release of binding partners. In that way, Nup1p, Nup2p, Cse1p, and Gsp1p may function as karyopherin release factors (or KaRFs) in the nuclear basket structure of the S. cerevisiae NPC.     

Site-directed perturbation of protein kinase C- integrin interaction blocks carcinoma cell chemotaxis

Polarized cell movement is an essential requisite for cancer metastasis; thus, interference with the tumor cell motility machinery would significantly modify its metastatic behavior. Protein kinase C alpha (PKC alpha) has been implicated in the promotion of a migratory cell phenotype. We report that the phorbol ester-induced cell polarization and directional motility in breast carcinoma cells is determined by a 12-amino-acid motif (amino acids 313 to 325) within the PKC alpha V3 hinge domain. This motif is also required for a direct association between PKC alpha and beta 1 integrin. Efficient binding of beta 1 integrin to PKC alpha requires the presence of both NPXY motifs (Cyto-2 and Cyto-3) in the integrin distal cytoplasmic domains. A cell-permeant inhibitor based on the PKC-binding sequence of beta 1 integrin was shown to block both PKC alpha-driven and epidermal growth factor (EGF)-induced chemotaxis. When introduced as a minigene by retroviral transduction into human breast carcinoma cells, this inhibitor caused a striking reduction in chemotaxis towards an EGF gradient. Taken together, these findings identify a direct link between PKC alpha and beta 1 integrin that is critical for directed tumor cell migration. Importantly, our findings outline a new concept as to how carcinoma cell chemotaxis is enhanced and provide a conceptual basis for interfering with tumor cell dissemination.     

Improved detection of small deletions in complex pools of DNA

About 40% of the genes in the nematode Caenorhabditis elegans have homologs in humans. Based on the history of this model system, it is clear that the application of genetic methods to the study of this set of genes would provide important clues to their function in humans. To facilitate such genetic studies, we are engaged in a project to derive deletion alleles in every gene in this set. Our standard methods make use of nested PCR to hunt for animals in mutagenized populations that carry deletions at a given locus. The deletion bearing animals exist initially in mixed populations where the majority of the animals are wild type at the target. Therefore, the production of the PCR fragment representing the deletion allele competes with the production of the wild type fragment. The size of the deletion fragment relative to wild type determines whether it can compete to a level where it can be detected above the background. Using our standard conditions, we have found that when the deletion is <600 bp, the deletion fragment does not compete effectively with the production of the wild type fragment in PCR. Therefore, although our standard methods work well to detect mutants with deletions >600 bp, they do not work well to detect mutants with smaller deletions. Here we report a new strategy to detect small deletion alleles in complex DNA pools. Our new strategy is a modification of our standard PCR based screens. In the first round of the nested PCR, we include a third PCR primer between the two external primers. The presence of this third primer leads to the production of three fragments from wild type DNA. We configure the system so that two of these three fragments cannot serve as a template in the second round of the nested PCR. The addition of this third primer, therefore, handicaps the amplification from wild type template. On the other hand, the amplification of mutant fragments where the binding site for the third primer is deleted is unabated. Overall, we see at least a 500-fold increase in the sensitivity for small deletion fragments using our new method. Using this new method, we report the recovery of new deletion alleles within 12 C.elegans genes.     

Expression of leptin and its receptor in the murine ovary: possible role in the regulation of oocyte maturation

Leptin is a product of the ob gene that is produced primarily by adipose tissue. Leptin and its receptors are found within the ovary, but it is unclear what function this hormone has in the ovary. Using immunohistochemistry, we determined that leptin is found in most cell types in the murine ovary, with the highest staining levels observed in the oocyte. Leptin receptor was also expressed in all of the main ovarian cell types, with the thecal cell layer exhibiting the highest staining levels. Leptin administration did not affect spontaneous or induced maturation of either isolated denuded oocytes or cumulus-oocyte complexes, but it did significantly increase the rate of meiotic resumption in preovulatory follicle-enclosed oocytes (P < 0.01). Measurements of cAMP within oocytes cultured with leptin showed that this enhanced ability to resume meiosis does not occur via activation of phosphodiesterase 3B and subsequent cAMP reduction. These results provide evidence that leptin affects oocyte maturation when the oocyte is cultured within its normal follicular environment. It is suggested that leptin may induce the production of another factor, possibly from thecal cells, that directly or indirectly acts on the oocyte to initiate germinal vesicle breakdown in this species.