Steroid specificity of the putative DHB receptor: evidence that the receptor is not 11βHSD

1998 ◽  
Vol 275 (1) ◽  
pp. E124-E131 ◽  
Author(s):  
Karen E. Sheppard ◽  
Karen Khoo ◽  
Zygmunt S. Krozowski ◽  
Kevin X. Z. Li
Keyword(s):  
Steroid Receptors ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Rat Colon ◽  
Colonic Crypt ◽  
Binding Assays ◽  
Ovary Cells ◽  
High Affinity ◽  
Steroid Binding ◽  
Crypt Cells

Recently, we identified a novel putative nuclear receptor in colonic crypt cells distinct from both mineralocorticoid receptor and glucocorticoid receptor, with high affinity for 11-dehydrocorticosterone (11-DHB) (33). In the present study, competitive nuclear binding assays demonstrated that this site has a unique steroid binding specificity that distinguishes it from other steroid receptors. Western blot analysis showed the presence of 11β-hydroxysteroid dehydrogenase-2 (11βHSD2) but not 11βHSD1 in colonic crypt cells and showed that 11βHSD2 was present in the nuclear pellet. Differences in steroid specificity between the putative DHB receptor and inhibition of 11βHSD activity indicate that binding is not to the enzyme. Furthermore, modified Chinese hamster ovary cells transfected with the 11βHSD2 gene express nuclear 11βHSD2 but not a nuclear DHB binding site. In conclusion, these data support the existence of a novel nuclear DHB receptor in rat colon that is distinct from the classic steroid receptors and from both 11βHSD1 and 11βHSD2.

New endomorphin analogs with mu-agonist and delta-antagonist properties

2004 ◽  
Vol 76 (5) ◽  
pp. 951-957 ◽  
Author(s):  
G. Tóth ◽  
A. Keresztes ◽  
Cs. Tömböly ◽  
A. Péter ◽  
F. Fülöp ◽  
...  
Keyword(s):  
Opioid Receptor ◽  
Opioid Receptors ◽  
Radioligand Binding ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Binding Assays ◽  
Brain Membrane ◽  
Analgesic Tolerance ◽  
Ovary Cells ◽  
High Affinity

Endomorphins (endomorphin-1,H-Tyr-Pro-Trp-Phe-NH 2 ,endomorphin-2, Tyr-Pro-Phe-Phe-NH2) are potent and selective µ-opioid receptor agonists. In order to improve the affinity and chemical stability of endomorphins, we have designed, synthesized, and characterized novel analogs with unnatural (2 ',6 '-dimethyltyrosine, Dmt) and/or ß-alicyclic amino acids (ACPC and ACHC). Radioligand binding assay indicated that several of the novel analogs exhibit high affinity for both µ-and d-opioid receptors in rat-or mouse-brain membrane preparations. The most promising derivatives—such as Dmt-Pro-Trp/Phe-Phe-NH2, Dmt-(1 S,2R)-ACPC-Phe-Phe-NH2, and Dmt-(1S,2R)-ACHC-Phe-Phe-NH2 )—were characterized in recombinant cell lines expressing human µ-or d-opioid receptors. Interestingly, while these novel peptides were potent opioid agonists in the functional [35S]GTPgammaS binding assays in Chinese hamster ovary cells expressing the µ-opioid receptors, some behaved as antagonist or inverse agonist in the human d-opioid receptor-expressing CHO cells. Since it has previously been demonstrated that the coadministration of d-antagonists with µ-analgesics attenuates the development of analgesic tolerance, introduction of high-affinity d-antagonist properties into the µ-agonist endomorphins is expected to lead to potent analgesics that produce limited tolerance.

Role of the α-subunit326GRV sequence in the surface expression of fibrinogen and vitronectin receptors

1998 ◽  
Vol 275 (5) ◽  
pp. C1239-C1246 ◽  
Author(s):  
Milagros Ferrer ◽  
Matilde S. Ayuso ◽  
Nora Butta ◽  
Roberto Parrilla ◽  
Consuelo González-Manchón
Keyword(s):  
Functional Role ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Surface Expression ◽  
Platelet Surface ◽  
Α Subunit ◽  
Vitronectin Receptor ◽  
Ovary Cells ◽  
High Affinity

The platelet GPIIb-GPIIIa heterodimer (integrin αIIbβ3) binds fibrinogen with high affinity in response to activation by agonists, leading to platelet aggregation and formation of a hemostatic plug. The326GRV motif in GPIIb is highly conserved in the α-subunit of other integrins, suggesting that it might play an important functional role. Moreover, Arg327→His substitution in GPIIb has been associated with defective platelet surface expression of GPIIb-IIIa and thrombasthenic phenotype. This work aimed at elucidating whether the absence of Arg327or its substitution by His was responsible for the impaired surface expression of GPIIb-IIIa complexes. Transfection of cDNA encoding [Ala327]GPIIb, [Gln327]GPIIb, or [Phe327]GPIIb into Chinese hamster ovary cells inherently expressing GPIIIa permitted surface exposure of GPIIb-IIIa complexes, whereas [Glu327]GPIIb did not. These observations indicate that it is not the loss of [Arg327]GPIIb but the presence of His327or a negatively charged residue like Glu at position 327 of GPIIb that prevents the surface exposure of GPIIb-IIIa heterodimers. In contrast, changing Gln344, the homologue to Arg327in the α-subunit of the vitronectin receptor, to His did not prevent the surface expression of αv-GPIIIa complexes. Thus the conformational constraint imposed by His327seems to be rather specific for the heterodimerization and/or processing of GPIIb-IIIa complexes.

scholarly journals Development of high-affinity ligands and photoaffinity labels for the d-fructose transporter GLUT5

2002 ◽  
Vol 367 (2) ◽  
pp. 533-539 ◽  
Author(s):  
Jing YANG ◽  
James DOWDEN ◽  
Arnaud TATIBOUËT ◽  
Yasumaru HATANAKA ◽  
Geoffrey D. HOLMAN
Keyword(s):  
Secondary Amine ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Amine Group ◽  
Ovary Cells ◽  
Affinity Ligands ◽  
High Affinity ◽  
Ring Form ◽  
Secondary Amine Group ◽  
Specific Uptake

The GLUT5 transporter catalyses the specific uptake of d-fructose and can accept this hexose in its furanose and pyranose ring forms. The transporter does not accept fructose epimers and has very limited tolerance of bulky groups substituted at the 2-, 3-, 4- and 5-OH positions [Tatibouët, Yang, Morin and Holman (2000) Bioorg. Med. Chem. 8, 1825—1833]. To further explore whether bulky groups can be tolerated at the primary OH positions, a d-fructose analogue with an allylamine group substitution to replace the 1-OH group was synthesized and was found to be quite well tolerated (Ki = 27.1mM). However, this analogue occurs in multiple ring forms. By contrast, 2,5-anhydro-d-mannitol is a symmetrical molecule that occurs only in a furanose ring form in which C-1 and C-6 are equivalent. We have therefore synthesized new 2,5-anhydro-d-mannitol analogues (substituted at the equivalent of the 6-OH of d-fructose) and from studies in Chinese hamster ovary cells expressing GLUT5 cells report that (i) the allylamine derivative of 2,5-anhydro-d-mannitol is well tolerated (Ki = 2.66mM); (ii) introduction of a di-nitrophenyl-substituted secondary amine group enhances affinity (Ki = 0.56mM); (iii) introduction of amide-linked biotinylated photolabel moieties is possible without loss of affinity relative to 2,5-anhydro-d-mannitol but a small secondary amine spacer between the biotinylated photolabelling moiety and the fructofuranose ring increases affinity (fructose photolabel 2; Ki = 1.16mM); (iv) introduction of a hydrophilic tartarate spacer between biotin and the diazirine photoreactive groups can be accomplished without reduction in affinity and (v) photoactivation of biotinylated fructose photolabels leads to specific biotin tagging of GLUT5. These data suggest that substitution of a secondary amine group (-NH) to replace the C-6 (or C-1) -OH of 2,5-anhydro-d-mannitol results in compounds of high affinity; the affinity is enhanced over 10-fold compared with d-fructose.

scholarly journals Expression, purification and characterization of a mouse-human chimeric antibody and chimeric Fab' fragment

1992 ◽  
Vol 281 (2) ◽  
pp. 317-323 ◽  
Author(s):  
D J King ◽  
J R Adair ◽  
S Angal ◽  
D C Low ◽  
K A Proudfoot ◽  
...  
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Antibody Fragment ◽  
Chimeric Antibody ◽  
Attractive Alternative ◽  
Binding Assays ◽  
Fab Fragment ◽  
Colorectal Tumour ◽  
Ovary Cells

B72.3 is a mouse monoclonal antibody against a tumour-associated antigen, TAG72, which recognizes breast, ovarian and colorectal tumour tissue. A mouse-human chimeric version of B72.3 has been expressed in Chinese-hamster ovary cells. This molecule has the binding specificity of B72.3 and constant regions from human IgG4. The chimeric B72.3 assembles to intact IgG and recognizes TAG72 as well as B72.3 in competitive binding assays. A proportion of the chimeric B72.3 (approx. 10%) does not form inter-heavy-chain disulphide bonds but still assembles into the IgG tetramer. This appears to be a general property of human IgG4 molecules. Co-expression of the chimeric light chain with a chimeric Fd' gene resulted in the expression of functional Fab'. Very little F(ab')2 is produced, although the Fab' can be oxidized to the dimeric F(ab')2 in vitro. The production of Fab' and F(ab')2 by this method is an attractive alternative to proteolytic digestion of IgG. The ability to produce these molecules in large quantities will allow the production and testing of a range of anti-tumour antibody and antibody fragment conjugates.

scholarly journals Rap1 binding to the talin 1 F0 domain makes a minimal contribution to murine platelet GPIIb-IIIa activation

2018 ◽  
Vol 2 (18) ◽  
pp. 2358-2368 ◽  
Author(s):  
Frederic Lagarrigue ◽  
Alexandre R. Gingras ◽  
David S. Paul ◽  
Andrew J. Valadez ◽  
Monica N. Cuevas ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Platelet Glycoprotein ◽  
Low Doses ◽  
Wild Type ◽  
Ovary Cells ◽  
High Affinity ◽  
Fibrinogen Binding ◽  
Platelet Functions

Abstract Activation of platelet glycoprotein IIb-IIIa (GPIIb-IIIa; integrin αIIbβ3) leads to high-affinity fibrinogen binding and platelet aggregation during hemostasis. Whereas GTP-bound Rap1 GTPase promotes talin 1 binding to the β3 cytoplasmic domain to activate platelet GPIIb-IIIa, the Rap1 effector that regulates talin association with β3 in platelets is unknown. Rap1 binding to the talin 1 F0 subdomain was proposed to forge the talin 1–Rap1 link in platelets. Here, we report a talin 1 point mutant (R35E) that significantly reduces Rap1 affinity without a significant effect on its structure or expression. Talin 1 head domain (THD) (R35E) was of similar potency to wild-type THD in activating αIIbβ3 in Chinese hamster ovary cells. Coexpression with activated Rap1b increased activation, and coexpression with Rap1GAP1 reduced activation caused by transfection of wild-type THD or THD(R35E). Furthermore, platelets from Tln1R35E/R35E mice showed similar GPIIb-IIIa activation to those from wild-type littermates in response to multiple agonists. Tln1R35E/R35E platelets exhibited slightly reduced platelet aggregation in response to low doses of agonists; however, there was not a significant hemostatic defect, as judged by tail bleeding times. Thus, the Rap1–talin 1 F0 interaction has little effect on platelet GPIIb-IIIa activation and hemostasis and cannot account for the dramatic effects of loss of Rap1 activity on these platelet functions.

scholarly journals Vitronectin binds to the gonococcal adhesin OpaA through a glycosaminoglycan molecular bridge

1998 ◽  
Vol 334 (1) ◽  
pp. 133-139 ◽  
Author(s):  
Thomas D. DUENSING ◽  
Jos P. M. van PUTTEN
Keyword(s):  
Binding Sites ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Heparin Binding ◽  
Dextran Sulphate ◽  
Binding Assays ◽  
Sulphated Polysaccharides ◽  
Ovary Cells ◽  
Internalization Process ◽  
Micro Organisms

Several bacterial pathogens including Neisseria gonorrhoeae bind the human serum glycoprotein vitronectin. We aimed at defining the gonococcal receptor for vitronectin. Ligand blots demonstrated that vitronectin bound specifically to the heparin-binding outer-membrane protein OpaA, but that coating OpaA with the sulphated polysaccharide heparin was required for the interaction to occur. Bound vitronectin could be dissociated from OpaA–heparin–vitronectin complexes by the addition of excess heparin, indicating that sulphated polysaccharides provided the main linkage between the two proteins. Binding assays with intact micro-organisms substantiated the requirement of sulphated polysaccharides such as heparin and dextran sulphate for the efficient binding of vitronectin to OpaA+ gonococci. This was underscored by the increased binding of vitronectin to gonococci that had been preincubated with saturating concentrations of dextran sulphate, as opposed to the inhibition of vitronectin binding observed when bacteria were incubated simultaneously with vitronectin and saturating concentrations of dextran sulphate. Binding assays with dextran sulphates of various sizes indicated that vitronectin binding correlated with the size of the polysaccharide rather than with the amount of OpaA produced by the bacteria. The inability of zero-length cross-linking agents to couple vitronectin to OpaA provided further evidence that sulphated polysaccharides formed the linkage between vitronectin and OpaA. Infection experiments demonstrated that proteoglycan-deficient Chinese hamster ovary cells efficiently internalized dextran sulphate/vitronectin-coated gonococci, suggesting that soluble sulphated polysaccharides could substitute for cell surface glycosaminoglycans in the internalization process. On the basis of our results, we propose a novel mechanism of vitronectin binding in which sulphated polysaccharides act as molecular bridges, linking the glycosaminoglycan-binding sites of vitronectin and gonococcal OpaA.

Recombinant Human Soluble Thrombomodulin Delivers Bounded Thrombin to Antithrombin III: Thrombomodulin Associates with Free Thrombin and Is Recycled to Activate Protein C

1993 ◽  
Vol 70 (03) ◽  
pp. 418-422 ◽  
Author(s):  
Masaharu Aritomi ◽  
Naoko Watanabe ◽  
Rika Ohishi ◽  
Komakazu Gomi ◽  
Takao Kiyota ◽  
...  
Keyword(s):  
Protein C ◽  
Antithrombin Iii ◽  
Transmembrane Domain ◽  
Chinese Hamster Ovary Cells ◽  
Time Lag ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
Soluble Thrombomodulin ◽  
Simulation Based ◽  
Recombinant Human Soluble Thrombomodulin

SummaryRecombinant human soluble thrombomodulin (rhs-TM), having no transmembrane domain or chondroitin sulfate, was expressed in Chinese hamster ovary cells. Interactions between rhs-TM, thrombin (Th), protein C (PC) and antithrombin III (ATIII) were studied. Equilibrium between rhs-TM and Th had no detectable time lag in clotting inhibition (K d = 26 nM) or PC activation (K d = 22 nM), while ATIII inhibited Th at a bimolecular rate constant = 5,200 M-1s-1 (K d <0.2 nM). A mixture of ATIII, Th and rhs-TM showed that ATIII reacted with Th slower than rhs-TM, whose presence did not affect the reaction between ATIII and Th. In a mixture of rhs-TM, ATIII and PC, the repeated addition of Th caused the repeated activation of PC; which was consistent with the Simulation based on the assumption that rhs-TM is recycled as a Th cofactor. From these results, we concluded that upon inhibition of the rhs-TM-Th complex by ATIII, rhs-TM is released to recombine with free Th and begins to activate PC, while the Th-ATIII complex does not affect rhs-TM-Th equilibrium.

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