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W H Tsien

Publications and source records attributed to W H Tsien.

18 recordsLinked to original sources
PubMed1993

Identification of sites on the human Fc epsilon RI alpha subunit which are involved in binding human and rat IgE.

The high affinity IgE Fc receptor (Fc epsilon RI), found on mast cells and basophils, is a tetrameric receptor complex. The extracellular portion of the Fc epsilon RI alpha subunit consists of two immunoglobulin-like domains and binds IgE in the absence of the other subunits. To localize the high affinity IgE binding site within the Fc epsilon RI alpha subunit, we generated a series of chimeric receptor constructs where one of the two immunoglobulin-like domains was either deleted or substituted with those from the human Fc gamma RIIIA alpha or the rat Fc epsilon RI alpha subunit. The chimeric receptors were monitored for their capacity to bind human and rat IgE, and their reactivity with different antireceptor antibodies. Domain I substitutions maintained high affinity human IgE binding. Domain II substitutions resulted in a total loss of both human and rat IgE binding. Single-domain alpha subunits could not bind IgE, suggesting that both extracellular domains are required for proper protein folding or IgE binding. To further localize the IgE binding sites, homolog-scanning mutagenesis was performed. At least three independent regions of domain II encompassing residues 118-129, 136-150, and 148-162 were required for IgE binding. Our results suggest that domain II of the human Fc epsilon RI alpha confers most of the important contributions to the binding of the human IgE Fc molecule, whereas domain I of the rat Fc epsilon RI alpha makes important contributions to the binding of rat IgE.

Amino Acid Sequence↗
PubMed1993

Humanized Mik beta 1, a humanized antibody to the IL-2 receptor beta-chain that acts synergistically with humanized anti-TAC.

Mik beta 1 is a mouse mAb directed at the beta-subunit of the human IL-2R (Tac) that inhibits IL-2 binding and inhibits IL-2 induction of large granular lymphocytes (LGL). Mik beta 1 alone does not inhibit IL-2-induced T-cell proliferation, but acts synergistically with anti-Tac to inhibit IL-2-induced proliferation of activated T cells. To evaluate these effects for possible therapy in humans, we constructed two humanized Mik beta 1 antibodies by combining the complementarity-determining regions of the murine antibody with human framework and constant regions. Compared with murine Mik beta 1, the two humanized Mik beta 1 antibodies, which differ in their degree of humanization, had similar affinities for IL-2R beta. The murine Mik beta 1 and one of the humanized Mik beta 1 antibodies were equivalent in competing for IL-2 binding to IL-2R beta and inhibiting IL-2 induction of LGL cytotoxicity. The activity of the second humanized antibody was significantly reduced. The three Mik beta 1 antibodies act synergistically to varying degrees with humanized anti-Tac to prevent IL-2-induced proliferation of activated T cells. This capacity to synergize paralleled their abilities to inhibit IL-2 binding. In addition, both humanized antibodies directed antibody-dependent cell-mediated cytotoxicity. We hope that humanized Mik beta 1 in combination with humanized anti-Tac will provide a new immunosuppressive therapy for the treatment of autoimmune diseases, graft-versus-host disease, and prevention of allograft rejection.

Amino Acid Sequence↗
PubMed1993

Purification and characterization of human recombinant IgE-Fc fragments that bind to the human high affinity IgE receptor.

The Fc-region of immunoglobulin E (IgE) comprising C epsilon 2, C epsilon 3, and C epsilon 4 domains is sufficient for binding to the alpha chain of the high affinity IgE-Fc receptor (Fc epsilon RI alpha). In order to identify the smallest Fc fragment capable of binding to the Fc epsilon RI alpha with high affinity, various regions of the IgE-Fc molecule were expressed in COS cells and investigated for their ability to bind Fc epsilon RI alpha. The smallest fragment that showed Fc epsilon RI alpha binding activity spans amino acids 329-547 and lacks the entire C epsilon 2 domain. Two active fragments, viz. Fc epsilon(315-547) (containing Cys328 which is responsible for interchain S-S bonding) and Fc epsilon(329-547), have been overexpressed in CHO cells and purified to homogeneity. The purified proteins bind to the Fc epsilon RI alpha with high affinity, similar to native IgE. SDS-polyacrylamide gel electrophoresis analyses indicate that Fc epsilon(315-547) is an S-S-linked dimer of apparent molecular mass of 68 kDa. Fc epsilon(329-547) appears on SDS-gel as three distinct bands at approximately 32 kDa, both under reducing and nonreducing conditions. However, size exclusion chromatography and analytical ultracentrifugation studies suggest that Fc epsilon(329-547) also remains associated as a dimer. The presence of N-linked glycosylation was detected in both proteins. The deglycosylated form of Fc epsilon(315-547) was isolated after Endo F/N-glycosidase F digestion and demonstrated to have binding activity comparable to that of the mock-digested protein. These results suggest that the presence of N-linked sugars is not necessary for Fc epsilon RI alpha binding. Both proteins blocked the release of histamine from RBL cells expressing human Fc epsilon RI alpha in a dose-dependent manner. The availability of these recombinant IgE-Fc proteins will be helpful in elucidating the key epitopes essential for the binding of IgE to its high affinity receptor.

Animals↗
PubMed1991

Localization in human interleukin 2 of the binding site to the alpha chain (p55) of the interleukin 2 receptor.

Human interleukin 2 (IL-2) analogs with defined amino acid substitutions were used to identify specific residues that interact with the 55-kDa subunit (p55) or alpha chain of the human IL-2 receptor. Analog proteins containing specific substitutions for Lys-35, Arg-38, Phe-42, or Lys-43 were inactive in competitive binding assays for p55. All of these analogs retained substantial competitive binding to the intermediate-affinity p70 subunit (beta chain) of the receptor complex. The analogs varied in ability to interact with the high-affinity p55/p70 receptor. Despite the lack of binding to p55, all analogs exhibited significant biological activity, as assayed on the murine CTLL cell line. The dissociation constants of Arg-38 and Phe-42 analogs for p70 were consistent with intermediate-affinity binding; the Kd values were not significantly affected by the presence of p55 in binding to the high-affinity IL-2 receptor complex. These results confirm the importance of the B alpha-helix in IL-2 as the locus for p55-receptor binding and support a revised model of IL-2-IL-2 receptor interaction.

Amino Acid Sequence↗
PubMed1988

Identification of specific residues of human interleukin 2 that affect binding to the 70-kDa subunit (p70) of the interleukin 2 receptor.

Analogs of interleukin 2 containing defined amino acid substitutions and deletions were assayed for bioactivity and for competitive binding to the high-affinity human interleukin 2 receptor complex and its two component subunits, a 55-kDa subunit (p55 or TAC) and a 70-kDa subunit (p70). Substitution of Asp20 or deletion of Phe124 resulted in inactive analog proteins that were unable to interact with the high-affinity p55/p70 complex or the intermediate-affinity p70 subunit of the interleukin 2 receptor. These analogs, however, retained the capacity to compete for binding to the low-affinity p55 subunit. The presence of the carboxylic acid in the side chain of Asp20 was necessary for effective binding to the p70 protein. In contrast, substitution of Trp121 and Leu17 created analogs that were inactive in the bioassay and all three binding assays. The effects of these mutations on protein conformation were assessed by circular dichroism. These results demonstrate that specific residues in the NH2 and COOH termini of interleukin 2 are crucial for its structure and activity.

Binding, Competitive↗
PubMed1988

Human interleukin-2 anti-idiotypes.

We describe the generation of a monoclonal anti-idiotypic (anti-id) antibody directed against affinity purified rabbit antibodies (id) to recombinant human IL-2. This monoclonal antibody, 6A12, has the ability to inhibit the neutralization of IL-2 activity by the id, suggesting that it may bind at or near the IL-2 neutralizing site on the id. 6A12 exhibits IL-2 agonist activity on PHA-activated human T cells. The purified IgG of 6A12 is also shown to bind to a purified soluble recombinant p55 subunit of the IL-2 receptor. Furthermore, purified 6A12 shows inhibition of IL-2 activity in an IL-2 dependent mouse T cell line (CTLL) and this inhibition can be reversed by excess IL-2. These results suggest that although 6A12 may not be an exact 'internal image' of the receptor binding site of IL-2, it may bind to at least the P55 subunit of the ligand binding site on the high affinity IL-2 receptor complex.

Animals↗
PubMed1987

Structure-function analysis of human interleukin-2. Identification of amino acid residues required for biological activity.

To locate functional domains of the interleukin-2 (IL-2) protein, a cDNA clone encoding biologically active human IL-2 was mutagenized using synthetic oligonucleotides to incorporate defined amino acid substitutions and deletions in the mature protein. The IL-2 analogs were then produced in Escherichia coli and assayed for the ability to induce proliferation of IL-2-dependent cells and the ability to compete for binding to the IL-2 receptor. Our analysis of over 50 different mutations demonstrated that the integrity of at least three regions of the IL-2 molecule is required for full biological activity: the NH2 terminus (residues 1-20), the COOH terminus (residues 121-133), and 2 of the 3 cysteine residues (58 and 105). Deletion of the NH2-terminal 20 amino acids or the COOH-terminal 10 amino acids resulted in the loss of greater than 99% of bioactivity and binding. Amino acid substitutions at specific positions in these regions also resulted in proteins which retained less than 1% activity. The NH2 terminus and an adjacent internal region were recognized by neutralizing anti-IL-2 antibodies. In combination with the results from epitope competition analysis with neutralizing antibodies, these data are consistent with the IL-2 protein being folded such that the NH2 terminus, the COOH terminus, and the internal 30- to 60-region are juxtaposed to form the binding site recognized by the IL-2 receptor.

Amino Acid Sequence↗
PubMed1982

The lack of correlation between inhibition of aggregation and cAMP levels with canine platelets.

Arachidonic acid (AA) induced aggregation of canine platelets can be inhibited by various phosphodiesterase inhibitors (PDIs) with the order of potency IBMX greater than or equal to papaverine greater than Ro 20-1724 greater than theophylline. With aggregation induced by AA plus epinephrine (EPI), only IBMX and papaverine inhibited at 100 microM. None of these PDIs affected the basal cAMP levels but all potentiated the PGE1-stimulated cAMP production, with the order of potency being Ro 20-1724 greater than papaverine greater than IBMX greater than theophylline. PGE1 at 1 microM caused a sharp increase in cAMP and complete inhibition of platelet aggregation induced by AA plus EPI. However, when EPI was added before PGE1, there was no elevation of cAMP yet inhibition of aggregation still occurred. Our results indicated that inhibition of platelet aggregation does not require a measurable increase in cAMP.

1-Methyl-3-isobutylxanthine↗
PubMed1981

The elevation of mouse thymus cell cyclic adenosine monophosphate (cAMP) by lipopolysaccharide.

The ability of certain lipopolysaccharide (LPS) preparations to elevate cyclic adenosine monophosphate (cAMP) in mouse thymus cells in the presence of Ro 20-1724, 4-(3-butoxyl 4-methoxybenzyl)-2-imidazolidinone, was not related to the source of supply, bacterial strain, or method of extraction. Under the same conditions adenosine is a potent stimulator of thymus cell cAMP and is, of course, blocked by the further addition of theophylline. When theophylline was added to the LPS preparations with Ro 20-1724, the cAMP production was also blocked. These studies suggested that the observed stimulation of cAMP by LPS preparations was due to adenosine and/or its nucleotides present as contaminants.

4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone↗
PubMed1980

Effects of ascorbic acid on 3H-thymidine incorporation by isolated mouse thymocytes.

The 15-min incorporation of 3H-thymidine (Tdr) and 3H-uridine (Ur) into nucleic acids of freshly isolated mouse thymocytes decreased steadily with time of preincubation at 37 degrees C. Sodium ascorbate at 5 mM prevented the decline of 3H-Tdr incorporation by preventing the decrease in its uptake. No such effect was noted on the incorporation of 3H-Ur, suggesting that ascorbate might be more specific for cells in or near the S phase of the cell cycle. The ene-diol group on the ascorbate molecule was required for this function, as ascorbyl-2-sulfate was ineffective and dehydroascorbate (DHA) reduced 3H-Tdr incorporation even further. Ascorbate was also inhibitory at a lower concentration (0.1 mM) or lower cell density. Thiols such as dithiothreitol or reduced glutathione seemed to act like 0.1 mM rather than 5 mM ascorbate. The inhibition by 0.1 mM ascorbate was presented by 0.1% bovine serum albumin (BSA), catalase or anaerobiosis. BSA had its own protective effects on the cells, since at 0.1% it increased the uptake of both 3H-Tdr and 3H-Ur. The combined effects of 5 mM ascorbate and 0.1% BSA on 3H-Tdr uptake were additive, but some synergism was noted at the lower BSA concentrations. These results suggest that with low concentrations of ascorbate (0.1 mM) oxidative reactions occur in vitro, resulting in the accumulation of the toxic hydroxyl radical (. OH). High concentrations apparently override this inhibition by a mechanism possibly involving an increase in critical cellular thiol groups.

Animals↗
PubMed1980

The regulation of nucleotide metabolism of immune cells: papaverine induced nucleotide breakdown.

During a period of prelabeling of mouse thymus cells with any nucleoside at 4 degrees C, nucleoside phosphates accumulated, but no nucleic acid synthesis occurred. Elevating the temperature to 37 degrees C then led to incorporation into the respective nucleic acid reaching a maximum in 5--15 min. Papaverine inhibited this incorporation (IC50:50 muM) and caused an efflux of label into the medium as a nonphosphorylated product. The responses of the different nucleotide phosphate pools showed more dependency on the base then the sugar moeity. The effect of papaverine could not be altered or mimicked by deprivation of oxygen, glucose, or calcium. Mouse spleen cells responded like thymocytes to papaverine, but rat GH3 pituitary cell DNA syntesis was only transiently inhibited with no concomitant efflux of 3H into the medium. As expected, thymus cellular adenosine triphosphate (ATP), determined by the luciferin-luciferase reaction, decreased in the presence of papaverine; suprisingly, extracellular ATP fell as well. The results suggest that decreases in cellular ATP of mouse thymus cells leads to reductions of all nucleoside phosphates and the efflux of the resultant nucleosides. Papaverine may effect a decrease in the ATP levels by activating a phosphohydrolase rather than, or in addition to, the previously suggested inhibition of mitochondrial electron transport.

Animals↗
PubMed1975

Alterations in the hydrolytic activity, inhibitor sensitivity and molecular size of the rat erythrocyte cyclic AMP phosphodiesterase by calcium and hypertonic sodium chloride.

Hypertonic NaCl irreversibly reduced the sensitivity of the rat erythrocyte cyclic AMP phosphodiesterase to inhibition by Ro 20-1724 but not by papavarine. This effect could be prevented by 1 mM EDTA and mimicked by by CaCl2. The reduction in inhibitor sensitivity was associated with an increase in hydrolytic activity and a four-fold reduction in molecular weight as estimated by Sephadex G-200 chromatography. The response to CaCl2, like that to NaCl, was time and concentration dependent and proceeded more slowly in 2 M NaCl. This concentration of NaCl contained sufficient Ca++ as an impurity to account for its effects on inhibitor sensitivity and molecular size. However, the initial and reversible increase in inhibitor sensitivity and hydrolytic activity obtained in the presence of EDTA, probably resulted from the high salt concentration, per se.

3',5'-Cyclic-AMP Phosphodiesterases↗