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N-formyl peptide receptor complex with a G-protein kinase signal pathway modification agent

A G protein and complex technology, applied in hormone receptors, peptide/protein components, animal/human proteins, etc., can solve unexplained and unanswered questions

Inactive Publication Date: 2005-06-29
莫维克借贷有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0022] Clearly, many questions remain unanswered regarding the function and mechanism of action of FPR
Moreover, the finding that FPR is expressed in nonleukocytes such as brain and dendritic cells suggests that FPR may have novel functions in other cell types that remain to be elucidated

Method used

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  • N-formyl peptide receptor complex with a G-protein kinase signal pathway modification agent
  • N-formyl peptide receptor complex with a G-protein kinase signal pathway modification agent
  • N-formyl peptide receptor complex with a G-protein kinase signal pathway modification agent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1: Binding of labeled HK-X to peripheral blood nucleated cells

[0066] Kd and Bmax (saturation binding) of HK-X ("f-Met-Leu-Phe-Phe") on peripheral blood nucleated cells were determined. 2 × 10 of the various fractions (monocytes, lymphocytes, granulocytes) prepared by density centrifugation in 100 μl of the solution were pre-washed in 1% BSA-PBS solution containing 0.1% sodium azide. 5 cells. According to Table 1, add 100 μl of FITC-labeled HK-X (dissolved in 1% BSA-PBS solution) at the following molar concentration to each group of test tubes:

[0067] pipe number

FITC-labeled HK-X molar concentration

1

2

3

4

5

6

7

8

9

9.06×10 -12

2.71×10 -11

5.43×10 -11

7.25×10 -11

9.06×10 -11

2.71×10 -10

5.43×10 -10

7.25×10 -10

9.06×10 -10

[0068]The tubes were mixed and vortexed for 30 seconds. The tubes were then placed at 4-8°C for 30 minutes. Then add 100 μl Cal-Lyse and incubate for 5 minutes,...

Embodiment 2

[0070] Example 2: Binding of HK-X to activated receptors

[0071] At 24 or 120 hours of culture, peripheral blood lymphocytes were stimulated with the mitogen Concanavalin A (ConA). These cells were then either exposed or not exposed to 100 nM FITC-labeled HK-X. Cells were stained with DAPI to determine the cell cycle. Cells were then analyzed by flow cytometry.

[0072] Figures 2A-2C The relationship between ConA-activated lymphocytes and the appearance of binding sites for FITC-labeled HK-X is shown. These four quadrants display the following characteristics:

[0073] Upper left quadrant represents DNA content above 1n with background levels (using figure 1 Cells with increased FITC HK-X binding levels above as determined by the distribution curve of

[0074] The upper right quadrant represents cells with DNA content exceeding 1n and FITC-ligand binding above background;

[0075] The lower right quadrant contains cells with 1 n DNA content but bound FITC-ligand above...

Embodiment 3

[0082] Example 3: Identification and Characterization of HK-X Receptors

[0083] Isolation and characterization of receptors that bind HK-X and other N-formyl peptides from murine peritoneal mast cells, human polymorphonuclear and mononuclear cell populations. Moreover, G protein activation of FCεR receptors acts as an increase in PKC, PI3 and Ca 2+ As a downstream consequence of mobilization, FCε receptors become downstream effector receptors of interfering G proteins.

[0084] Cells that bind HK-X include mast cells, basophils, and eosinophils, as evidenced by changes in bioreactivity upon exposure to HK-X, and binding to fluorescently or radioactively labeled HK-X cell. A receptor for HK-X has not been identified. In contrast, neutrophils express formyl peptide receptors on their surface. However, blood lymphocytes and monocytes appear to bind HK-X less than neutrophils.

[0085] Detailed Materials and Methods

[0086] 1. Isolation of Cells - Rat peritoneal mast cells...

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Abstract

The present invention describes methods of inhibiting the pro-inflammatory response of human peripheral blood mononuclear cells or polymorphonuclear cells, or fixed tissue cells. The cells are contacted with a pro-inflammatory agent to stimulate a pro-inflammatory response. The cells are then contacted with G protein kinase signaling pathway modifiers, thereby inhibiting G protein mediated inflammatory response signaling pathways. The present invention describes receptor complexes wherein G protein kinase signaling pathway modifiers bind to cell surface receptors on human peripheral blood mononuclear cells or polymorphonuclear cells stimulated with proinflammatory agents.

Description

field of invention [0001] The present invention relates to N-formyl peptide receptors found on the surface of peripheral blood cells, in particular complexes of such receptors with agents that alter or disrupt G protein signaling pathways, particularly certain N-formyl peptides, and Methods involving alteration of signal transduction by co-stimulation of pro-inflammatory agents. Background of the invention [0002] Humans have evolved mechanisms to defend against bacterial infection by using bacterially produced N-formylmethionyl peptides as chemoattractants for phagocytes, especially neutrophils and monocytes. Among the N-formyl peptides, f-Met-Leu-Phe (FMLP) was identified as having the strongest ability to recruit phagocytes and stimulate the release of lysosomal enzymes from neutrophils (Showell et al., J.Exp.Med .143:1154-1169, 1976). It was subsequently shown that synthetic tetrapeptides, especially f-Met-Ile-Phe-Leu and f-Met-Leu-Phe-Ile, can also elicit neutrophil ...

Claims

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Application Information

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IPC IPC(8): G01N33/50A61K38/00A61K38/05A61K38/07C07K5/083C07K5/103C07K14/705C07K14/72C12N5/07C12N5/078C12Q1/02G01N33/15
CPCA61K38/05A61K38/07C07K5/081C07K5/1013C07K14/723A61P37/06A61K38/16
Inventor J·A·克拉格特C·帕尔默
Owner 莫维克借贷有限责任公司
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