Analysis of binding residues in monoclonal antibody with high affinity for the head domain of the rat P2X4 receptor.

P2X4 receptor is known to be involved in neuropathic pain. In order to detect the expression of P2X4 receptor on microglia at the time of onset of neuropathic pain, one approach consists on the preparation of the monoclonal antibodies with both selective binding and high affinity. We have recently established a monoclonal antibody (named 12-10H) which had high affinity to rat P2X4 receptor expressed in 1321N1 cells.

PCBP1 and PCBP2 both bind heavily oxidized RNA but cause opposing outcomes, suppressing or increasing apoptosis under oxidative conditions.

PCBP1, a member of the poly(C)-binding protein (PCBP) family, has the capability of binding heavily oxidized RNA and therefore participates in the cellular response to oxidative conditions, helping to induce apoptosis. There are four other members of this family, PCBP2, PCBP3, PCBP4, and hnRNPK, but it is not known whether they play similar roles. To learn more, we first tested their affinity for an RNA strand carrying two 8-oxoguanine (8-oxoG) residues at sites located in close proximity to each other, representative of a heavily oxidized strand or RNA with one 8-oxoG or none.

Evidence for detection of rat P2X4 receptor expressed on cells by generating monoclonal antibodies recognizing the native structure.

P2X purinergic receptors are ATP-driven ionic channels expressed as trimers and showing various functions. A subtype, the P2X4 receptor present on microglial cells is highly involved in neuropathic pain. In this study, in order to prepare antibodies recognizing the native structure of rat P2X4 (rP2X4) receptor, we immunized mice with rP2X4's head domain (rHD, Gln111-Val167), which possesses an intact structure stabilized by S-S bond formation (Igawa and Abe et al.

Specific binding of PCBP1 to heavily oxidized RNA to induce cell death.

In aerobically growing cells, the guanine base of RNA is oxidized to 8-oxo-7,8-dihydroguanine (8-oxoG), which induces alteration in their gene expression. We previously demonstrated that the human AUF1 protein binds to 8-oxoG in RNA to induce the selective degradation of oxidized messenger RNA. We herein report that the poly(C)-binding protein PCBP1 binds to more severely oxidized RNA to activate apoptosis-related reactions.

Solution structure of the rat P2X4 receptor head domain involved in inhibitory metal binding.

The P2X receptor is an ATP-gated cation channel expressed on the plasma membrane. The head domain (Gln111-Val167 in the rat P2X4 receptor) regulates ATP-induced cation influx. In this study, we prepared a head domain with three disulfide bonds, such as the intact rat P2X4 receptor contains.

Preparation and characterization of a monoclonal antibody against the refolded and functional extracellular domain of rat P2X4 receptor.

The P2X4 purinergic receptor is a key molecule in neuropathic pain, particularly in the allodynia after peripheral nerve injury. We therefore sought to establish an anti-P2X4 receptor monoclonal antibody that would be useful for detection and characterization of the P2X4 receptor. We first prepared the refolded extracellular domain of the rat P2X4 receptor expressed in Escherichia coli.