scholarly journals Are the IL-2 Receptors Expressed in the Murine Fetal Thymus Functional?

1990 ◽  
Vol 1 (1) ◽  
pp. 59-66 ◽  
Author(s):  
Juan Carlos Zoúñiga-Pflücker ◽  
Kendall A. Smith ◽  
Lucio Tentori ◽  
Drew M. Pardoll ◽  
Dan L. Longo ◽  
...  
Keyword(s):  
Interleukin 2 ◽  
Binding Studies ◽  
Developmentally Regulated ◽  
Fetal Thymus ◽  
High Affinity ◽  
Hybridization Data ◽  
Regulated Expression ◽  
Dependent Growth

It is well established that the majority of murine fetal thymocytes (day 15 of gestation) express receptors for interleukin 2 (IL-2), but the functional significance of these IL-2 receptors (IL-2Rs) is not clear. In situ hybridization data show a developmentally regulated expression of IL-2 and IL-2R mRNA. IL-2 binding studies were performed on fetal thymocytes and the results show the presence of both high (kD ≅ 20 pM) and low (kD ≅ 10 nM) affinity IL-2Rs. These IL-2Rs are indeed functional: intact fetal thymic lobes (but not cell suspensions) cultured in IL-2 exhibited an in vitro proliferative response at 20 pM of IL-2, corresponding with the presence of a functional high-affinity IL-2R on fetal thymocytes. The IL-2-dependent growth was primarily observed in the IL-2R + thymic subset, which contains the CD3-/CD4-/CD8- precursor thymocytes. Furthermore, in vitro blocking of IL-2 in intact fetal thymic lobes resulted in a reduction in the cell yield, which predominantly affected the expansion of the immature CD3-/CD4-/CD8-thymocytes. Our findings strongly support the concept that the IL-2/IL-2R pathway is responsible for the proliferation of precursor cells within the fetal thymus.

scholarly journals Developmentally Regulated Expression of Msx1, Msx2 and Fgfs in the Developing Mouse Cranial Base

2006 ◽  
Vol 76 (6) ◽  
pp. 990-995 ◽  
Author(s):  
Xuguang Nie
Keyword(s):  
Gene Expression ◽  
In Vitro Transcription ◽  
Cranial Base ◽  
Developmentally Regulated ◽  
Embryonic Mouse ◽  
Osteogenic Cells ◽  
Regulated Expression ◽  
Msx Genes

Abstract Objective: To examine the expression pattern of the Fgf and Msx genes in cranial base development. Materials and Methods: To detect the expression of these genes, antisense riboprobes were synthesized by in vitro transcription. Radioactive in situ hybridization was performed on parasagittal sections of embryonic mouse heads. Results: Msx2 was observed in the underlying perichondrium at restricted stages. Msx1 was not observed in cranial base development. Fgf1 was localized in osteogenic cells from the time of ossification; Fgf10 was highly expressed in the occipital-vertebral joint during E13 to E14; Fgf2, Fgf7, and Fgf18 were localized in the perichondria; Fgf12 was transitorily expressed at early chondrocranium; Fgf9 was seen in the hypertrophic chondrocytes. Conclusions: The Fgf and Msx gene expression in the cranial base was different from that of other skeletons.

Vasodilation elicited by liposomal VIP is unimpeded by anti-VIP antibody in hamster cheek pouch

1998 ◽  
Vol 275 (1) ◽  
pp. R56-R62 ◽  
Author(s):  
Hiroyuki Ikezaki ◽  
Sudhir Paul ◽  
Hayat Alkan-Önyüksel ◽  
Manisha Patel ◽  
Xiao-Pei Gao ◽  
...  
Keyword(s):  
Calcium Ionophore ◽  
Myeloma Cell ◽  
Cheek Pouch ◽  
Myeloma Cell Line ◽  
Hamster Cheek Pouch ◽  
High Affinity ◽  
Sterically Stabilized Liposomes

The purpose of this study was to determine whether a monoclonal anti-vasoactive intestinal peptide (VIP) antibody, which binds VIP with high affinity and specificity and catalyzes cleavage of the peptide in vitro, attenuates VIP vasorelaxation in vivo and, if so, whether insertion of VIP on the surface of sterically stabilized liposomes (SSL), which protects the peptide from trypsin- and plasma-catalyzed cleavage in vitro, curtails this response. Using intravital microscopy, we found that suffusion of monoclonal anti-VIP antibody (clone c23.5, IgG2ak), but not of nonimmune antibody (myeloma cell line UPC10, IgG2ak) or empty SSL, significantly attenuates VIP-induced vasodilation in the in situ hamster cheek pouch ( P < 0.05). By contrast, anti-VIP antibody has no significant effects on vasodilation elicited by isoproterenol, nitroglycerin, and calcium ionophore A-23187, agonists that activate intracellular effector systems in blood vessels that mediate, in part, VIP vasoreactivity. Suffusion of VIP on SSL, but not of empty SSL, restores the vasorelaxant effects of VIP in the presence of anti-VIP antibody. Collectively, these data suggest that VIP catalysis by high affinity and specific VIP autoantibodies displaying protease-like activity constitutes a novel mechanism whereby VIP vasoreactivity is regulated in vivo.

scholarly journals Small-Molecule Inhibition of Choline Catabolism in Pseudomonas aeruginosa and Other Aerobic Choline-Catabolizing Bacteria

2011 ◽  
Vol 77 (13) ◽  
pp. 4383-4389 ◽  
Author(s):  
Liam F. Fitzsimmons ◽  
Stevenson Flemer ◽  
A. Sandy Wurthmann ◽  
P. Bruce Deker ◽  
Indra Neil Sarkar ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Burkholderia Cepacia ◽  
Sinorhizobium Meliloti ◽  
Growth Inhibitor ◽  
Bioinformatic Analysis ◽  
Content Type ◽  
Genes Encoding ◽  
Dependent Growth

ABSTRACTCholine is abundant in association with eukaryotes and plays roles in osmoprotection, thermoprotection, and membrane biosynthesis in many bacteria. Aerobic catabolism of choline is widespread among soil proteobacteria, particularly those associated with eukaryotes. Catabolism of choline as a carbon, nitrogen, and/or energy source may play important roles in association with eukaryotes, including pathogenesis, symbioses, and nutrient cycling. We sought to generate choline analogues to study bacterial choline catabolismin vitroandin situ. Here we report the characterization of a choline analogue, propargylcholine, which inhibits choline catabolism at the level of Dgc enzyme-catalyzed dimethylglycine demethylation inPseudomonas aeruginosa. We used genetic analyses and13C nuclear magnetic resonance to demonstrate that propargylcholine is catabolized to its inhibitory form, propargylmethylglycine. Chemically synthesized propargylmethylglycine was also an inhibitor of growth on choline. Bioinformatic analysis suggests that there are genes encoding DgcA homologues in a variety of proteobacteria. We examined the broader utility of propargylcholine and propargylmethylglycine by assessing growth of other members of the proteobacteria that are known to grow on choline and possess putative DgcA homologues. Propargylcholine showed utility as a growth inhibitor inP. aeruginosabut did not inhibit growth in other proteobacteria tested. In contrast, propargylmethylglycine was able to inhibit choline-dependent growth in all tested proteobacteria, includingPseudomonas mendocina,Pseudomonas fluorescens,Pseudomonas putida,Burkholderia cepacia,Burkholderia ambifaria, andSinorhizobium meliloti. We predict that chemical inhibitors of choline catabolism will be useful for studying this pathway in clinical and environmental isolates and could be a useful tool to study proteobacterial choline catabolismin situ.

scholarly journals The Developmentally Regulated Expression of Twisted Gastrulation Reveals a Role for Bone Morphogenetic Proteins in the Control of T Cell Development

2002 ◽  
Vol 196 (2) ◽  
pp. 163-171 ◽  
Author(s):  
Daniel Graf ◽  
Suran Nethisinghe ◽  
Donald B. Palmer ◽  
Amanda G. Fisher ◽  
Matthias Merkenschlager
Keyword(s):  
T Cell ◽  
Bone Morphogenetic Proteins ◽  
Cell Receptor ◽  
Mammalian Development ◽  
Double Positive ◽  
Developmentally Regulated ◽  
Twisted Gastrulation ◽  
Thymocyte Proliferation ◽  
Regulated Expression

The evolutionarily conserved, secreted protein Twisted gastrulation (Tsg) modulates morphogenetic effects of decapentaplegic (dpp) and its orthologs, the bone morphogenetic proteins 2 and 4 (BMP2/4), in early Drosophila and vertebrate embryos. We have uncovered a role for Tsg at a much later stage of mammalian development, during T cell differentiation in the thymus. BMP4 is expressed by thymic stroma and inhibits the proliferation of CD4−CD8− double-negative (DN) thymocytes and their differentiation to the CD4+CD8+ double-positive (DP) stage in vitro. Tsg is expressed by thymocytes and up-regulated after T cell receptor signaling at two developmental checkpoints, the transition from the DN to the DP and from the DP to the CD4+ or CD8+ single-positive stage. Tsg can synergize with the BMP inhibitor chordin to block the BMP4-mediated inhibition of thymocyte proliferation and differentiation. These data suggest that the developmentally regulated expression of Tsg may allow thymocytes to temporarily withdraw from inhibitory BMP signals.

scholarly journals Analysis of pre- and postsynaptic factors of the serotonin system in rabbit retina.

1985 ◽  
Vol 100 (1) ◽  
pp. 64-73 ◽  
Author(s):  
C K Mitchell ◽  
D A Redburn
Keyword(s):  
Serotonin Receptor ◽  
Amacrine Cells ◽  
Uptake System ◽  
Rabbit Retina ◽  
Binding Studies ◽  
High Affinity ◽  
Serotonin System ◽  
Significant Difference

[3H]Serotonin is accumulated by a specific set of amacrine cells in the rabbit retina. These cells also accumulate the neurotoxin, 5,7-dihydroxytryptamine, and show signs of necrosis within 4 h of in vivo exposure to the drug. Biochemical analysis of [3H]serotonin uptake reveal a sodium- and temperature-dependent, high affinity uptake system with a Km of 0.94 microM and Vmax of 1.08 pmol/mg protein/min. [3H]Tryptophan is also accumulated in rabbit retinal homogenates by a high affinity process. Accumulated [3H]serotonin is released in response to potassium-induced depolarization of intact, isolated retinas. In vitro binding studies of rabbit retinal homogenate membranes demonstrate specific sets of binding sites with characteristics of the postsynaptic serotonin receptor. These data strongly suggest that rabbit retina contains virtually all of the molecular components required for a functional serotonergic neurotransmitter system. The only significant difference between the serotonin system in rabbit retina and that in the well-established serotonin transmitter systems in nonmammalin retinas and in brains of most species is the relatively low concentration of endogenous serotonin in rabbit retinas, as demonstrated by high-performance liquid chromatography, histofluorescence, or immunocytochemistry.

The thyroid hormone analogue SKF L-94901: nuclear occupancy and serum binding studies

1989 ◽  
Vol 76 (5) ◽  
pp. 495-501 ◽  
Author(s):  
John W. Barlow ◽  
Lorna E. Raggatt ◽  
Chen-Fee Lim ◽  
Sharon L. Munro ◽  
Duncan J. Topliss ◽  
...  
Keyword(s):  
Thyroid Hormone ◽  
Serum Proteins ◽  
Binding Studies ◽  
Hormone Analogue ◽  
Isolated Nuclei ◽  
Whole Cells ◽  
High Affinity ◽  
Nuclear Extracts

1. We studied a brominated thyroid hormone analogue, SKF L-94901, which has the potential to lower serum cholesterol without adverse cardiovascular effects. This compound is about 50% as active as tri-iodothyronine (T3) in liver nuclear receptor binding in vivo but only 1% as active in vitro and has nearly 200 times more enzyme-inducing activity in liver than in heart. Our aim was to examine the interaction of SKF L-94901 with [125I]T3 binding to the intact nuclei in whole cells, isolated nuclei and nuclear extracts of human HeLa cells and to investigate the binding of this compound to human serum. 2. Relative to thyroxine (T4), the affinity of this compound for T4-binding globulin was 0.0035%, for transthyretin 1.66% and for albumin 1.26%. Low affinity for serum proteins, with a relatively high circulating free fraction, could explain why SKF L-94901 is more potent in vivo than in vitro. 3. Human HeLa cell nuclei, isolated after whole-cell incubations, bound [125I]T3 with high affinity (Kd = 78 ± 8 pmol/l, mean ± sem), which was displaceable by T3 analogues in the order Triac {[4-(4-hydroxy-3-iodophenoxy)-3,5-di-iodophenyl]acetic acid} > T3 > T4 ≫ reverse T3. Similar high-affinity (Kd = 58 ± 6 pmol/l, mean ± sem) and identical specificity was observed in high-salt (0.4 mol/l KCl) nuclear extracts. In nuclei of whole cells incubated with [125I]T3 and SKF L-94901, the analogue was 0.8% as potent as T3, whereas in experiments with nuclear extract, the analogue was 7.7% as potent as T3. Results from incubation of T3 with isolated nuclei were virtually identical to those obtained with nuclear extracts. 4. These results suggest an extranuclear component may be involved in restricting access of SKF L-94901 to the nucleus. Whether such mechanisms account for observed differences in its effects on different tissues with reduced influence of SKF L-94901 on cardiac tissue remains to be established. 5. We conclude that SKF L-94901 is weakly bound in serum and shows less potent competition for T3 nuclear binding after incubation of whole cells than after incubation with nuclear extracts or isolated nuclei. This compound may allow further analysis of intracellular mechanisms of thyroid hormone transport and action.

scholarly journals Identification of Domains Required for Developmentally Regulated SNARE Function in Saccharomyces cerevisiae

Genetics ◽  
2000 ◽  
Vol 155 (4) ◽  
pp. 1643-1655 ◽  
Author(s):  
Aaron M Neiman ◽  
Luba Katz ◽  
Patrick J Brennwald
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Snare Complex ◽  
Binding Studies ◽  
Developmentally Regulated ◽  
Vegetative Cells ◽  
Additional Layer ◽  
Prospore Membrane ◽  
N Terminus ◽  
Developmental Conditions

Abstract Saccharomyces cerevisiae cells contain two homologues of the mammalian t-SNARE protein SNAP-25, encoded by the SEC9 and SPO20 genes. Although both gene products participate in post-Golgi vesicle fusion events, they cannot substitute for one another; Sec9p is active primarily in vegetative cells while Spo20p functions only during sporulation. We have investigated the basis for the developmental stage-specific differences in the function of these two proteins. Localization of the other plasma membrane SNARE subunits, Ssop and Sncp, in sporulating cells suggests that these proteins act in conjunction with Spo20p in the formation of the prospore membrane. In vitro binding studies demonstrate that, like Sec9p, Spo20p binds specifically to the t-SNARE Sso1p and, once bound to Sso1p, can complex with the v-SNARE Snc2p. Therefore, Sec9p and Spo20p interact with the same binding partners, but developmental conditions appear to favor the assembly of complexes with Spo20p in sporulating cells. Analysis of chimeric Sec9p/Spo20p molecules indicates that regions in both the SNAP-25 domain and the unique N terminus of Spo20p are required for activity during sporulation. Additionally, the N terminus of Spo20p is inhibitory in vegetative cells. Deletion studies indicate that activation and inhibition are separable functions of the Spo20p N terminus. Our results reveal an additional layer of regulation of the SNARE complex, which is necessary only in sporulating cells.

Uptake of labeled palmitate by the intact liver: role of intracellular binding sites.

1978 ◽  
Vol 234 (6) ◽  
pp. E542
Author(s):  
C A Goresky ◽  
D S Daly ◽  
S Mishkin ◽  
I M Arias
Keyword(s):  
Fatty Acids ◽  
Free Fatty Acids ◽  
Binding Sites ◽  
Hepatic Uptake ◽  
Binding Studies ◽  
High Affinity ◽  
Multiple Indicator Dilution ◽  
Intracellular Binding ◽  
Z Protein

The multiple-indicator dilution technique was utilized to examine the hepatic uptake of albumin-bound labeled palmitate from the portal vein blood of the pentobarbital-anesthetized dog, in a fasted state and after infusion of a variety of compounds that were expected to bind to Z protein, the cellular cytosolic protein binding free fatty acids, and their acyl-CoA derivatives. Analysis of the data indicates that after infusion of alpha-bromopalmitate, 16-bromo-9-hexadecenoate, and sulfobromophthalein sodium (which also bind to albumin), the palmitate label influx, efflux, and metabolic sequestration (removal of label from the pool of free fatty acids able to leave the cell) all increase and that, after infusion of flavaspidic acid, label efflux and metabolic sequestration increase. In vitro competitive binding studies carried out on the cellular cytosol indicat that the basis for the increase in efflux and metabolic sequestration is displacement of labeled palmitate from high affinity sites on the intracellular Z protein (which are presumably in equilibrium with and may be taken to be representative of other intracellular binding sites). These studies also suggest that increased uptake is due to similar displacement from high affinity sites on serum albumin.

HIV TAT Basic Peptide Is Not a High-Affinity Ligand for VEGF Receptor 2

2003 ◽  
Vol 384 (10-11) ◽  
pp. 1435-1441 ◽  
Author(s):  
A. Rubio Demirovic ◽  
J. Canadi ◽  
W. Weiglhofer ◽  
P. Scheidegger ◽  
R. Jaussi ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Peptide Sequence ◽  
Vegf Receptor ◽  
Binding Studies ◽  
Basic Peptide ◽  
Signalling Molecules ◽  
High Affinity ◽  
Affinity Ligand ◽  
Domain 3

AbstractThe 'transactivator of transcription' (TAT) protein of human immunodeficiency virus transforms cells in culture and promotes the development of tumors, socalled Kaposi's sarcoma, in AIDS patients. TAT induces growth and differentiation of blood vessels and has been suggested to directly activate VEGF receptor 2 expressed on endothelial cells through a peptide sequence located between amino acids 46 and 64, the so-called basic domain. This peptide mimics many aspects of TAT function when added to endothelial cells, even when expressed in the context of recombinant chimeric proteins. To define the exact sites of interaction between this peptide and VEGF receptor 2 we performed binding studies with recombinant proteins derived from the extracellular ligand binding domain of VEGF receptor 2. These in vitro binding studies showed that the TAT peptide binds with only low specificity to Iglike domain 3 of the receptor, while VEGF interacts with receptor-derived proteins encompassing at least extracellular domains 1 through 3. The original concept that the angiogenic properties of TAT basic peptide result from specific, high-affinity interaction with VEGF receptor 2 must therefore be revised. Apparently this peptide interacts with cells in multiple ways: by directly activating acidic cell surface-exposed receptors, by releasing extracellular matrix-bound growth factors such as bFGF and VEGF which then bind to their cognate receptors, and by activating intracellular signalling molecules with which basic peptide interacts upon translocation into cells.

The promoter of the CD19 gene is a target for the B-cell-specific transcription factor BSAP

1992 ◽  
Vol 12 (6) ◽  
pp. 2662-2672
Author(s):  
Z Kozmik ◽  
S Wang ◽  
P Dörfler ◽  
B Adams ◽  
M Busslinger
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
B Cell ◽  
Lymphoid Cells ◽  
Binding Studies ◽  
Specific Transcription Factor ◽  
High Affinity ◽  
B Lymphoid

The CD19 protein is expressed on the surface of all B-lymphoid cells with the exception of terminally differentiated plasma cells and has been implicated as a signal-transducing receptor in the control of proliferation and differentiation. Here we demonstrate complete correlation between the expression pattern of the CD19 gene and the B-cell-specific transcription factor BSAP in a large panel of B-lymphoid cell lines. The human CD19 gene has been cloned, and several BSAP-binding sites have been mapped by in vitro protein-DNA binding studies. In particular, a high-affinity BSAP-binding site instead of a TATA sequence is located in the -30 promoter region upstream of a cluster of heterogeneous transcription start sites. Moreover, this site is occupied by BSAP in vivo in a CD19-expressing B-cell line but not in plasma or HeLa cells. This high-affinity site has been conserved in the promoters of both human and mouse CD19 genes and was furthermore shown to confer B-cell specificity to a beta-globin reporter gene in transient transfection experiments. In addition, BSAP was found to be the only abundant DNA-binding activity of B-cell nuclear extracts that interacts with the CD19 promoter. Together, this evidence strongly implicates BSAP in the regulation of the CD19 gene.

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