Yoshiko Aoki

CLONING AND FUNCTIONAL EXPRESSION OF A CDNA ENCODING A MOUSE TYPE 2 NEUROPEPTIDE Y RECEPTOR

A cDNA clone homologous with the human neuropeptide Y (NPY)-Y2 receptor has been isolated from a mouse brain cDNA library. Analysis of the predicted amino-acid sequence indicates that the polypeptide encoded by this cDNA is 94% homologous to the human NPY-Y2 receptor. In Chinese hamster ovary (CHO) cells expressing the mouse NPY-Y2 receptor, an increase in intracellular Ca2+ and inhibition of forskolin-induced cAMP accumulation were observed due to stimulation with NPY, NPY-(13-36) and peptide YY, but not with pancreatic polypeptide or [Leu31, Pro34]NPY. The fact that the NPY-induced increase in intracellular Ca2+ and inhibition of forskolin-induced cAMP accumulation were eliminated by pretreatment with pertussis toxin suggests that the NPY-Y2 receptor couples to PTX-sensitive G-protein(s), probably Gi/Go, in CHO cells.

Induction of IL-6 production by bone marrow stromal cells on the adhesion of IL-6-dependent hematopoietic cells

Cellular interactions between hematopoietic cells and stromal cells play crucial roles in the proliferation and differentiation of the hematopoietic cells. Interleukin‐6 (IL‐6)‐dependent 7TD1 cells markedly proliferated without IL‐6 when they were co‐cultured with hematopoietic‐supportive bone marrow stromal cells, HESS‐5 cells and HESS‐1 CL.3 cells, which can support long‐term hematopoiesis in vitro with but not without direct cell contact, cell contact being prevented with a microporous membrane. The production of IL‐6 and the amount of IL‐6 mRNA in hematopoietic‐supportive stromal cells but not 7TD1 cells significantly increased only when the stromal cells were co‐cultured in direct contact with 7TD1 cells. Furthermore, the amount of IL‐6 mRNA increased according to the number of 7TD1 cells co‐cultured. These inductions were not observed on co‐culture with a murine myeloid cell line, M1 cells, or on the addition of the co‐culture supernatant. These results suggest that 7TD1 cells transmit the signal to stromal cells that enhances IL‐6 production by stromal cells via direct cell contact. A certain specific molecule for transduction of the signals may exist on the surface membrane of stromal cells and hematopoietic cells.

A radioreceptor binding assay for platelet-activating factor (PAF) using membranes from CHO cells expressing human PAF receptor

A simple and reproducible radioreceptor assay (RRA) has been developed using membranes from CHO cells which can stably express human platelet-activating factor (PAF) receptor. The CHO cells expressing the PAF receptor, termed CHO.1F8, showed a significant intracellular Ca2+ response to PAF, and the same binding properties to [3H]WEB 2086, a PAF antagonist, as reported (Kd, 13.6 +/- 1.9 nM; Bmax, 2.5 +/- 0.4 pmol/mg protein (n = 6)). A competitive binding assay was done using the CHO.1F8 cell membranes and [3H]WEB 2086. The minimum detectable dose of PAF was 0.3 nM (approximately 30 pg per well) and the assay was highly specific for PAF. This method makes it possible to handle large numbers of samples rapidly and simultaneously, since the receptor membrane is prepared in advance and the binding assay can be completed within 3 h. Using this method, we have determined the production and cell association of PAF in human neutrophils.

Comparison of phosphonate transition state analogs for inducing catalytic antibodies and evaluation of key structural factors by an ab initio study

The relation between the structure of haptens and the esterolytic activities of antibodies was investigated. We synthesized two phenylalanine analogs, the negatively charged phosphonate derivative 1 and the neutral phosphonamidate derivative 2. Seventeen out of 41 monoclonal antibodies generated against the hapten 1 hydrolyzed the relevant phenylalanine ester R-12. On the contrary, none of 27 monoclonal antibodies generated against the hapten 2 had catalytic activity. An ab initio study of the structural and electronic properties of the modeled haptens showed that the value of the negative electrostatic potential around the phosphonyl oxygen was an important factor affecting the induction of esterolytic antibodies.

A Simple, Rapid and Specific Radioreceptor Assay for Platelet-Activating Factor

Platelet-activating factor (PAF) is a mediator with physiological and inflammatory activities. Numerous actions of PAF are mediated through specific membrane receptors.1 Accurate and rapid determination of PAF in biological samples is essential for a comprehensive understanding of its role and mode of action. To date, various biological,2 massspectrometrical3 and immunological4 assays for PAF have been developed, but all have problems such as interference by endogenous PAF inhibitors, lack of specificity, data standardization or reproduction, tedious procedures for derivatization of PAF, or contamination with other membrane phospholipids which interfer with the determination5. Ra-dioreceptor binding assays for PAF were developed to measure PAF produced by cells or homogenates of tissues.6,7 However, these methods also have drawbacks in being tedious procedures involving the preparation of rabbit platelet membranes or unreliable due to significant deviations in binding capacity depending on the membrane batches. We have now overcome these difficulties by using membranes from CHO cells carrying a cloned human PAF receptor and [3H]WEB 2086, a PAF antagonist. We describe in this paper the characteristics of this novel assay system and its application in quantifying PAF in biological samples.