Synergistic stimulation of gamma-glutamyl transpeptidase and alkaline phosphatase activities by retinoic acid and astroglial factors in immortalized rat brain microvessel endothelial cells

The immortalized rat brain microvessel endothelial cell line RBE4 was used to investigate the in vitro regulation of two blood-brain barrier specific enzymes, gamma-glutamyl transpeptidase (GTP) and alkaline phosphatase (ALP). The effects of bFGF, astroglial factors, and retinoic acid (a cell differentiation agent) on GTP and ALP activities were separately or simultaneously studied in order to define optimal culture conditions for induction of these two specific enzymes of the blood-brain barrier. In the present study, a phenotypically distinct subpopulation of endothelial cells has been shown to develop from confluent cobblestone monolayers of RBE4 immortalized cerebral endothelial cells. These distinct cells were present within multicellular aggregates and specifically exhibited GTP and ALP activities. Addition of bFGF, astroglial factors, or retinoic acid induced the formation of these three-dimensional structures and in consequence an increase in GTP and ALP activities. For retinoic acid and astroglial factors, this increase could also be explained by the stimulation of either GTP or ALP expression in the phenotypically distinct positive cells associated with aggregates. Simultaneous treatment with retinoic acid and astroglial factors had a synergistic effect on GTP and ALP expression and thus may allow these distinct cells to evolve toward a more differentiated state. Since such results were also obtained with physiological concentrations of retinoic acid, we suggest that addition of this agent might contribute to greater differentiation of cells in in vitro blood-brain barrier models where endothelial cells are cocultured with astrocytes. © 1996 Wiley-Liss, Inc.     

“Rule of Six”: How Does the Sendai Virus RNA Polymerase Keep Count?

The "rule of six" stipulates that the Paramyxovirus RNA polymerase efficiently replicates only viral genomes counting 6n + 0 nucleotides. Because the nucleocapsid proteins (N) interact with 6 nucleotides, an exact nucleotide-N match at the RNA 3'-OH end (3'-OH congruence) may be required for recognition of an active replication promoter. Alternatively, assuming that the six positions for the interaction of N with the nucleotides are not equivalent, the nucleotide position relative to N may be critical (N phase context). The replication abilities of various minireplicons, designed so that the 3'-OH congruence could be discriminated from the N phase context, were studied. The results strongly suggest that the application of the rule of six depends on the recognition of nucleotides positioned in the proper N phase context.     

Characterization of transferrin receptor in an immortalized cell line of rat brain endothelial cells, RBE4

The content and distribution of transferrin receptors in an immortalized cell line, RBE4, derived from rat cerebral capillary endothelial cells was investigated using the monoclonal antibody MRC OX-26 (OX-26 mAb) specific for the rat transferrin receptor. An ELISA assay was developed with which the OX-26 mAb can be determined quantiatively. The detection limit of the assay was 10 pg or 0.07 fmol of murine antibody. With this technique accurate measurement of native antibody is now possible without the need for isotope labeling (iodination). Immunostaining of confluent monolayers of RBE4 cells using an antibody directed against the tight junction associated protein ZO-1 was indicative for structural intactness of RBE4 cell monolayers. OX-26 immunostaining demonstrated localization of the transferrin receptor at the plasma membrane and/or in the cytosol. Binding studies showed saturation of OX-26 mAb binding. The antibody binding analysis gave a dissociation constant (KD) of 17.1 +/- 1.2 nmol/l. The total amount of transferrin receptors present per cell was 70,800 +/- 17,000. Our results indicate that receptor binding of OX-26 mAb can be studied using an in vitro cell culture model of rat brain mircrovessel endothelium in conjunction with an ELISA technique for detection of native antibody. This approach will be used to investigate mechanisms of transendothelial transport of OX-26 in vitro.     

Conformational change in the human glucocorticoid receptor induced by ligand binding is altered by mutation of isoleucine 747 by a threonine

Limited proteolysis experiments were performed to study conformation changes induced by ligand binding on in vitro produced wild-type and I747T mutant glucocorticoid receptors. Dexamethasone-induced conformational changes were characterized by two resistant proteolysis fragments of 30 and 27 kDa. Although dexamethasone binding affinity was only slightly altered by the I747T substitution (Roux, S., Térouanne, B., Balaguer, P., Loffreda-Jausons, N., Pons, M., Chambon, P., Gronemeyer, H., and Nicolas, J.-C. (1996) Mol. Endocrinol. 10, 1214-1226), higher dexamethasone concentrations were required to obtain the same proteolysis pattern. This difference was less marked when proteolysis experiments were conducted at 0 degrees C, indicating that a step of the conformational change after ligand binding was affected by the mutation. In contrast, RU486 binding to the wild-type receptor induced a different conformational change that was not affected by the mutation. Analysis of proteolysis fragments obtained in the presence of dexamethasone or RU486 indicated that the RU486-induced conformational change affected the C-terminal part of the ligand binding domain differently. These data suggest that the ligand-induced conformational change occurs via a multistep process. In the first step, characterized by compaction of the ligand binding domain, the mutation has no effect. The second step, which stabilizes the activated conformation and does not occur at 4 degrees C, seems to be a key element in the activation process that can be altered by the mutation. This step could involve modification of the helix H12 position, explaining why the conformation induced by RU486 is not affected by the mutation.     

Floral development and the formation of unisexual spikelets in the Andropogoneae (Poaceae)

We investigated spikelet development in four distantly related species of the grass tribe Andropogoneae to determine whether spikelet development and the formation of unisexual florets are uniform throughout the tribe. We studied development in Bothriochloa bladhii, Coelorachis aurita, Heteropogon contortus, and Hyparrhenia hirta, and compared these with Panicum, a member of the sister tribe Paniceae. Many aspects of spikelet development in the species we have studied correlate with what is already known for Tripsacum and maize (both Andropogoneae), despite variation in how unisexual florets are distributed on the plant. The formation of unisexual spikelets is also uniform. All florets initiate both pistil and stamen primordia. In florets destined to be male, cell death occurs in the subepidermal layers of the gynoecium after the formation of a gynoecial ridge. In florets destined to be female, there is no apparent cell death in the stamens, but growth ceases after anther formation. The similarity in spikelet development and the formation of unisexual florets point to a common genetic mechanism for sex determination throughout the Andropogoneae and possibly the entire Panicoideae. Use of a cell death pathway to cause gynoecial abortion may be the basis of one morphological character that defines the subfamily.     

Mapping 245 SSR markers on the <Emphasis Type="Italic">Vitis vinifera</Emphasis> genome: a tool for grape genetics

The aim of the present work was to develop a microsatellite marker-based map of the Vitis vinifera genome (n=19), useful for genetic studies in this perennial heterozygous species, as SSR markers are highly transferable co-dominant markers. A total of 346 primer pairs were tested on the two parents (Syrah and Grenache) of a full sib population of 96 individuals (S × G population), successfully amplifying 310 markers. Of these, 88.4% markers were heterozygous for at least one of the two parents. A total of 292 primer pairs were then tested on Riesling, the parent of the RS₁ population derived from selfing (96 individuals), successfully amplifying 299 markers among which 207 (62.9%) were heterozygous. Only 6.7% of the markers were homozygous in all three genotypes, stressing the interest of such markers in grape genetics. Four maps were constructed based on the segregation of 245 SSR markers in the two populations. The Syrah map was constructed from the segregations of 177 markers that could be ordered into 19 linkage groups (total length 1,172.2 cM). The Grenache map was constructed with the segregations of 178 markers that could be ordered into 18 linkage groups (total length 1,360.6 cM). The consensus S × G map was constructed with the segregations of 220 markers that were ordered into 19 linkage groups (total length 1,406.1 cM). One hundred and eleven markers were scored on the RS₁ population, among them 27 that were not mapped using the S × G map. Out of these 111 markers, 110 allowed to us to construct a map of a total length of 1,191.7 cM. Using these four maps, the genome length of V. vinifera was estimated to be around 2,200 cM. The present work allowed us to map 123 new SSR markers on the V. vinifera genome that had not been ordered in a previous SSR-based map (Riaz et al. 2004), representing an average of 6.5 new markers per linkage group. Any new SSR marker mapped is of great potential usefulness for many applications such as the transfer of well-scattered markers to other maps for QTL detection, the use of markers in specific regions for the fine mapping of genes/QTL, or for the choice of markers for MAS.     

6-aryl-2,4-dioxo-5-hexenoic acids, novel integrase inhibitors active against HIV-1 multiplication in cell-based assays

A series of 6-aryl-2,4-dioxo-5-hexenoic acids, were synthesized and tested against HIV-1 in cell-based assays and against recombinant HIV-1 integrase (rIN) in enzyme assays. Compound 8a showed potent antiretroviral activity (EC(50)=1.5 microM) and significant inhibition against rIN (strand transfer: IC(50)=7.9 microM; 3'-processing: IC(50)=7.0 microM). A preliminary molecular modeling study was carried out to compare the spatial conformation of 8a with those of L-731988 (4) and 5CITEP (7) in the IN core.     

Tuberous sclerosis complex gene products,Tuberin and Hamartin,control mTOR signaling by acting as a GTPase-activating protein complex toward Rheb

BACKGROUND: Tuberous Sclerosis Complex (TSC) is a genetic disorder that occurs through the loss of heterozygosity of either TSC1 or TSC2, which encode Hamartin or Tuberin, respectively. Tuberin and Hamartin form a tumor suppressor heterodimer that inhibits the mammalian target of rapamycin (mTOR) nutrient signaling input, but how this occurs is unclear. RESULTS: We show that the small G protein Rheb (Ras homolog enriched in brain) is a molecular target of TSC1/TSC2 that regulates mTOR signaling. Overexpression of Rheb activates 40S ribosomal protein S6 kinase 1 (S6K1) but not p90 ribosomal S6 kinase 1 (RSK1) or Akt. Furthermore, Rheb induces phosphorylation of eukaryotic initiation factor 4E binding protein 1 (4E-BP1) and causes 4E-BP1 to dissociate from eIF4E. This dissociation is completely sensitive to rapamycin (an mTOR inhibitor) but not wortmannin (a phosphoinositide 3-kinase [PI3K] inhibitor). Rheb also activates S6K1 during amino acid insufficiency via a rapamycin-sensitive mechanism, suggesting that Rheb participates in nutrient signaling through mTOR. Moreover, Rheb does not activate a S6K1 mutant that is unresponsive to mTOR-mediated signals, confirming that Rheb functions upstream of mTOR. Overexpression of the Tuberin-Hamartin heterodimer inhibits Rheb-mediated S6K1 activation, suggesting that Tuberin functions as a Rheb GTPase activating protein (GAP). Supporting this notion, TSC patient-derived Tuberin GAP domain mutants were unable to inactivate Rheb in vivo. Moreover, in vitro studies reveal that Tuberin, when associated with Hamartin, acts as a Rheb GTPase-activating protein. Finally, we show that membrane localization of Rheb is important for its biological activity because a farnesylation-defective mutant of Rheb stimulated S6K1 activation less efficiently. CONCLUSIONS: We show that Rheb acts as a novel mediator of the nutrient signaling input to mTOR and is the molecular target of TSC1 and TSC2 within mammalian cells.