Desensitization of the δ-Opioid Receptor Correlates with Its Phosphorylation in SK-N-BE Cells: Involvement of a G Protein-Coupled Receptor Kinase

Abstract: Phosphorylation of G protein-coupled receptors is considered an important step during their desensitization. In SK-N-BE cells, recently presented as a pertinent model for the studies of the human δ-opioid receptor, pretreatment with the opioid agonist etorphine increased time-dependently the rate of phosphorylation of a 51-kDa membrane protein. Immunological characterization of this protein with an antibody, raised against the amino-terminal region of the cloned human δ-opioid receptor, revealed that it corresponded to the δ-opioid receptor. During prolonged treatment with etorphine, phosphorylation increased as early as 15 min to reach a maximum within 1 h. Phosphorylation and desensitization of adenylyl cyclase inhibition paralleled closely and okadaic acid inhibited the resensitization, a result strongly suggesting that phosphorylation of the δ-opioid receptor plays a prominent role in its rapid desensitization. The increase in phosphorylation of the δ-opioid receptor, as well as its desensitization, was not affected by H7, an inhibitor of protein kinase A and protein kinase C, but was drastically reduced by heparin or Zn²⁺, known to act as G protein-coupled receptor kinase (GRK) inhibitors. These results are the first to show, on endogenously expressed human δ-opioid receptor, that a close link exists between receptor phosphorylation and agonist-promoted desensitization and that desensitization involves a GRK.     

Alkyl-dihydroxyacetone phosphate synthase and dihydroxyacetone phosphate acyltransferase form a protein complex in peroxisomes.

Dihydroxyacetone phosphate (GrnP) acyltransferase and alkyl-GrnP synthase are the key enzymes involved in the biosynthesis of ether phospholipids. Both enzymes are located on the inside of the peroxisomal membrane. Here we report evidence for a direct interaction between these enzymes obtained by the use of chemical cross-linking. After cross-linking and immunoblot analysis alkyl-GrnP synthase could be detected in a 210-kDa complex which was located entirely on the lumenal side of the peroxisomal membrane. Two-dimensional SDS/PAGE demonstrated that GrnP-acyltransferase is also cross-linked in a 210-kDa complex. Co-immunoprecipitation confirmed that the two enzymes interact, in a heterotrimeric complex. Furthermore, alkyl-GrnP synthase can form a homotrimeric complex in the absence of GrnP-acyltransferase as was demonstrated by immunoblot analysis after cross-linking experiments with either GrnP-acyltransferase deficient human fibroblast homogenates or recombinant (His)6-tagged alkyl-GrnP synthase. We conclude that alkyl-GrnP synthase interacts selectively with GrnP-acyltransferase in a heterotrimeric complex and in the absence of GrnP-acyltransferase can also form a homotrimeric complex.     

Rates of mutation to growth factor autonomy and tumorigenicity differ in hematopoietic stem and precursor cells expressing the multilineage colony-stimulating factor gene.

At least two separate but interdependent events are required to attain autonomous growth as a consequence of ectopic expression of the multilineage colony-stimulating factor gene in hematopoietic progenitor cells. The rate at which the second event occurs is more than 3 orders of magnitude higher in precursor cell lines (FDC-P1 or FDC-P2) than in stem cell lines (FDC-Pmix). Autonomous, but not density-dependent, growth is tightly coupled to tumorigenicity in precursor cells; however, neither growth-factor-independent nor autonomously growing stem cell lines are tumorigenic.     

The rho gene product expressed in E. coli is a substrate of botulinum ADP-ribosyltransferase C3

The ras-related rho A protein expressed in E. coli, was ADP-ribosylated by botulinum ADP-ribosyltransferase C3. C3 also modified the valine-14 mutant rho protein but not the products of H-ras, R-ras, ral, ypt, and rap 1 genes. A ras-rho chimaera consisting of 60 amino acids from the amino terminus of ras fused to 133 amino acids from the carboxy terminus of rho was not modified by C3. Antibodies raised against the porcine brain cytosolic substrate of C3 cross reacted with the rho, valine-14 rho and ras-rho proteins, but not with the gene products of H-ras, R-ras, ral or rap 1. Polyclonal anti-H-ras antibodies cross reacted with H-ras but not with ral, rho, or the C3 substrate purified from porcine brain.     

Expression of the GM-CSF gene after retroviral transfer in hematopoietic stem cell lines induces synchronous granulocyte-macrophage differentiation

Multipotent murine stem cell lines (FDC-Pmix) depend on IL-3 for self-renewal and proliferation and can be induced to differentiate into multiple hematopoietic lineages. Single FDC-Pmix cells infected with retroviral vectors expressing GM-CSF are induced to differentiate into granulocytes and macrophages. This results in a complete loss of clonogenic cells if IL-3 is not exogenously supplied; however, multipotent variants can be selected that do not terminally differentiate if cells are kept in the presence of IL-3. Unidirectional and synchronous granulocyte and macrophage differentiation accompanied with loss of self-renewal capacity is induced when IL-3 is removed. Our data indicate that activation of the GM-CSF receptor induces differentiation of stem cells by an instructive mechanism that can be blocked by the activated IL-3 receptor. A model of how receptors can induce proliferation and cell-specific differentiation by two separate pathways is discussed.     

Seasonal dynamics of the zoobenthic communities in the mesohaline zone of the Loire estuary (France)

The application of correspondence factorial analysis to four types of sediment in the upper reaches of the Loire estuary enabled the significance of environmental factors in the spatial distribution, abundance and seasonal dynamics of the biotic communities to be determined. In coarse sand, the communities are stable but species are few in number and population densities are low. In muddy sediments (from muddy sand to fluid mud) population densities are high but undergo seasonal fluctuations due to changes in the hydrological regime (especially the dissolved oxygen regime). This is true for the composition of the biotic communities. The oligohaline phase, in which the fluvial element is predominant, is characterized by abundance maxima due to freshwater species. The mesohaline phase, in which the marine element is predominant, exerts a restrictive action on population densities; certain species are supplanted by others, their development being governed by the quality of the environment.     

Preparation from chitin of (1-4)-2-amino-2-deoxy-beta-D-glucopyranuronan and its 2-sulfoamino analog having blood-anticoagulant properties

Chitosan, prepared by total N-deacetylation of chitin, underwent complete and specific carboxylation at C-6 when oxidized, as the perchlorate salt 2, with chromium trioxide in acetic acid. The resultant (1→4)-2-amino-2-deoxy-β-D-glucopyranuronan, obtained as its perchlorate (3), was N-sulfated with chlorosulfonic acid in pyridine to afford a (1→4)-2-deoxy-2-sulfoamino-β-D-glucopyranuronan, isolated as its amorphous sodium salt 4; the latter displayed moderate blood-anticoagulant activity. The products 3 and 4 showed marked in vitro growth inhibition of leukemia L-1210 cells.     

Effects of wounds on the terpene content of twigs of maritime pine (Pinus pinaster Ait.)

Summary The effect of wounds on the volatile terpene composition of the living bark of Pinus pinaster Ait. (maritime pine) twigs was investigated with respect to the processes of mono- and sesquiterpene hydrocarbon biosynthesis. The large increase in the amounts of - and -pinene is a characteristic feature after a mechanical injury, whereas the quantities of the other terpenes are only slightly increased. This is due to the reactivation of the resin duct secretory cells of primary origin located in cortical tissues. The effect of wounding is observed over a long period and the terpene profiles are very different at the end of the experiments as compared with the initial profiles of the same tissues. The traumatic essential oil (obtained after mechanical traumatism) resembles an oleoresin extracted from tissues of secondary origin. Statistical analysis underlines the effects of the between-tree variations and of the dates of application of the wound.