Constitutive secretion of β-trace protein by cultivated porcine choroid plexus epithelial cells: Elucidation of its complete amino acid and cDNA sequences

Primary porcine choroid plexus epithelial cells cultivated in chemically defined medium maintain their epithelial characteristics and form confluent monolayers. They produce a fluid the composition of which resembles cerebrospinal fluid. The present study demonstrates constitutive secretion of large amounts of β-trace protein. This intrathecally synthesized protein is a prominent polypeptide constituent of natural cerebrospinal fluid. According to the identity of amino acid sequences it has previously been tentatively identified as a prostaglandin-D synthase and as a member of the lipocalin protein family. β-Trace was purified from cell culture supernatants and was subjected to tryptic digestion and amino acid sequencing of the resulting peptides. The complete primary structure of the protein was obtained by additional isolation of the cDNA from cultured epithelial cells. The porcine 163-amino acid polypeptide showed 69% identity with the human β-trace and contained two N-glycosylation sites occupied by complex-type oligosaccharides as is the case for the human protein. The amino acid sequences around the N-glycosylation sites of mammalian β-trace proteins (porcine, human, murine, and rat) were highly conserved. The nucleotide sequence was found to be less conserved; the porcine cDNA had a strikingly high GC-content (67%). The constitutive secretion of β-trace protein from the in vitro cultivated porcine choroid plexus epithelial cells demonstrates that the cells have retained their major in vivo physiological properties: secretion of cerebrospinal fluid proteins. Therefore, this in vitro culture system may be used as a versatile tool for studying the regulation of the formation of cerebrospinal fluid. © 1996 Wiley-Liss, Inc.     

Occlusion of the human serotonin transporter is mediated by serotonin-induced conformational changes in the bundle domain

The human serotonin transporter (hSERT) terminates neurotransmission by removing serotonin (5HT) from the synaptic cleft, an essential process for proper functioning of serotonergic neurons. Structures of the hSERT have revealed its molecular architecture in four conformations, including the outward-open and occluded states, and show the transporter’s engagement with co-transported ions and the binding mode of inhibitors. In this study, we investigated the molecular mechanism by which the hSERT occludes and sequesters the substrate 5HT. This first step of substrate uptake into cells is a structural change consisting of the transition from the outward-open to the occluded state. Inhibitors such as the antidepressants citalopram, fluoxetine, and sertraline inhibit this step of the transport cycle. Using molecular dynamics simulations, we reached a fully occluded state, in which the transporter-bound 5HT becomes fully shielded from both sides of the membrane by two closed hydrophobic gates. Analysis of 5HT-triggered occlusion showed that bound 5HT serves as an essential trigger for transporter occlusion. Moreover, simulations revealed a complex sequence of steps and showed that movements of bundle domain helices are only partially correlated. 5HT-triggered occlusion is initially dominated by movements of transmembrane helix 1b, while in the final step, only transmembrane helix 6a moves and relaxes an intermediate change in its secondary structure.     

A cell cycle study of human mammary epithelial cells.

The growth regulation of human mammary epithelial cells (HMEC) cultured in a growth factor/hormone-enriched (e.g. EGF, insulin) medium with bovine pituitary extract as the only undefined supplement was studied. The doubling times of the cultures, in which the cells appear in colonies, was 55-72 h, and a considerable intercolonial heterogenecity in proliferative activity could be demonstrated. However, every colony, irrespective of the size of the growth fraction, comprised a sub-population of rapidly growing cells which had a mean generation time of approximately 22 h. When insulin was removed from the culture medium, HMEC proliferation was inhibited. This growth inhibition was shown to be a result of a cell cycle-specific block.     

Aromatic acetylenes and dehydrofalcarinone derivatives within the Artemisia dracunculus group

Root polyacetylenes from 7 taxa belonging to the Artemisia dracunculus group have been analysed and compared. Aromatic acetylenes, together with dehydrofalcarinone derivatives, are widespread and appear to be characteristic of the group. Distinct accumulation tendencies towards capillen or isocoumarin formation underline the taxonomic separation of A. dracunculiformis, A. glauca and A. pamirica from A. dracunculus s.str. Two different types of isocoumarins may additionally contribute to an infraspecific grouping within the latter species. Considering the biosynthetic pathway of aromatic acetylenes, the capillen-containing A. dracunculiformis, A. glauca and A. pamirica occupy a more primitive position, whereas the strains of A. dracunculus s.str. appear progressively more advanced by the accumulation of different isocoumarins.     

Receptor clustering is involved in Reelin signaling

The Reelin signaling cascade plays a crucial role in the correct positioning of neurons during embryonic brain development. Reelin binding to apolipoprotein E receptor 2 (ApoER2) and very-low-density-lipoprotein receptor (VLDLR) leads to phosphorylation of disabled 1 (Dab1), an adaptor protein which associates with the intracellular domains of both receptors. Coreceptors for Reelin have been postulated to be necessary for Dab1 phosphorylation. We show that bivalent agents specifically binding to ApoER2 or VLDLR are sufficient to mimic the Reelin signal. These agents induce Dab1 phosphorylation, activate members of the Src family of nonreceptor tyrosine kinases, modulate protein kinase B/Akt phosphorylation, and increase long-term potentiation in hippocampal slices. Induced dimerization of Dab1 in HEK293 cells leads to its phosphorylation even in the absence of Reelin receptors. The mechanism for and the sites of these phosphorylations are identical to those effected by Reelin in primary neurons. These results suggest that binding of Reelin, which exists as a homodimer in vivo, to ApoER2 and VLDLR induces clustering of ApoER2 and VLDLR. As a consequence, Dab1 becomes dimerized or oligomerized on the cytosolic side of the plasma membrane, constituting the active substrate for the kinase; this process seems to be sufficient to transmit the signal and does not appear to require any coreceptor.     

Ion channels and amino acid transporters support the growth and invasion of primary brain tumors

The malignant growth of glial support cells causes gliomas, highly invasive, primary brain tumors that are largely resistant to therapy. Individual tumor cells spread by active cell migration, invading diffusely into the normal brain. This process is facilitated by Cl⁻ channels that endow glioma cells with an enhanced ability to quickly adjust their shape and cell volume to fit the narrow and tortuous extracellular brain spaces. Once satellite tumors enlarge, their growth is limited by the spatial constraints imposed by the bony cavity of the skull and spinal column. Glioma cells circumvent this limitation by active destruction of peritumoral neural tissue through the release of glutamate, inducing peritumoral seizures and ultimately excitotoxic neuronal cell death. Hence, primary brain tumors support their unusual biology by taking advantage of ion channels and transporters that are designed to support ion homeostatic functions in normal brain.