Alleviation of the alelrgenic potential of airborne and contact allergens by thioredoxin

A thioredoxin and hypoallergenic technology, applied in the fields of oxidoreductase, plant protein processing, peptide/protein composition, etc., can solve the problems that have not been studied in depth, allergic reactions, and the ability to reduce are not understood

Inactive Publication Date: 2002-05-22
RGT UNIV OF CALIFORNIA
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The intracellular disulfide bonds of the 2S large subunit show homology to the soybean Bowman-Birk arrestin (Kreis et al., 1989), but the ability of the 2S protein to undergo reduction under physiological conditions is unknown
A second subgroup such as fascitoxins (e.g. fascin1 and fascin2) are cholinesterase inhibitory proteins, which have not been well studied
However, these treatments, especially in children, carry the risk of potentially fatal allergic reactions

Method used

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  • Alleviation of the alelrgenic potential of airborne and contact allergens by thioredoxin
  • Alleviation of the alelrgenic potential of airborne and contact allergens by thioredoxin
  • Alleviation of the alelrgenic potential of airborne and contact allergens by thioredoxin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0144] Thioredoxin-associated reduction of alpha-amylase inhibitor protein

[0145] enzyme activation test

[0146] The ability to displace a specific thioredoxin in chloroplast enzyme activation is a test for the ability of a given protein's thiol group to undergo a reversible redox change. Although not a physiological state in the case of extraplastid proteins, this assay has proven useful in several studies. A related example is the purine thionin, which activates chloroplast FBPase when reduced by thioredoxin h (Wada, K. et al. (1981) FEBS Lett. 124:237-240 and Johnson, T.C. et al. (1987) Plant Physiol .85: 446-451). FBPase (whose physiological activator protein is thioredoxin f) is not affected by thioredoxin h. In this example, cysteine-rich proteins were tested for their ability to activate FBPase and NADP-MDH as previously described. The α-amylase inhibitors of durum wheat (DSG-1 and DSG-2) were found to be effective for enzyme activation; however they differ from ...

Embodiment 2

[0157] DTNB reduction test

[0158]A second test of thiol redox capacity was the ability to catalyze the reduction of the sulfhydryl reagent 2',5'-dithiobis(2-nitrobenzoic acid) (DTNB), as measured by an increase in absorbance at 412 nm. Here, the analyzed protein was reduced by NADPH via NTR and thioredoxin. The DTNB assay was confirmed to be effective for both durum (DSG-1 and 2) and bread wheat (CM-1 ) alpha-amylase inhibitory proteins. When reduced by the NADP / thioredoxin system (in this case using NTR and thioredoxin from E. coli), DSG-1 or DSG-2 significantly enhanced the reduction of DTNB (NADPH → NTR → thioredoxin protein → DSG → DTNB). DTNB reduction assays were performed with 10 µg thioredoxin and 10 µg NTR and 20 µg DSG-1 or DSG-2. CM-1 was also potent in the DTNB reduction assay and, as activated with NADPH-MDH (Table I), a higher activity than the DSG protein was detectable. The conditions for the CM-1 assay were the same as for the DSG / DTNB assay, except that...

Embodiment 3

[0162] Determination of protein reduction

[0163] The availability of monobromoperphrine (mBBr) and its suitability for use in plant systems provides a new technique for measuring the sulfhydryl groups of plant proteins (Crawford, N.A. et al. (1989) Arch. Biochem. Biophys. 271: 223- 239). When coupled with SDS-polyacrylamide gel electrophoresis, mBBr can even be used to quantify changes in the sulfhydryl state of redox-active proteins in complex mixtures. The technique was therefore applied to this inhibitory protein to demonstrate its ability to be reduced by thioredoxin. Here, the tested proteins were reduced with thioredoxin, which itself had been previously reduced with DTT or NADPH and NTR. The mBBr derivative of the reduced protein was then prepared, separated from other components by SDS-polyacrylamide gel electrophoresis, and its reduced state was detected by fluorescence. In the experiments described below, E. coli thioredoxin was found to be effective in reducing...

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Abstract

Thioredoxin, a small dithiol protein, is a specific reductant for allergenic proteins and particularly allergenic proteins present in pollen and animal and plant sources. All targeted proteins contain disulfide (S-S) bonds that are reduced to the sulfhydryl (SH) level by thioredoxin. The proteins are allergenically active and less digestible in the oxidized (S-S) state. When reduced (SH state), they lose their allergenicity and / or become more digestible. Thioredoxin achieved this reduction when activated (reduced) either by NADPH via NADP-thioredoxin reductase (physiological conditions) or by lipoic acid chemical reductant. Skin tests carried out with sensitized dogs showed that treatment of the pollens with reduced thioredoxin prior to injection eliminated or decreased the allergenicity of the pollen. Studies showed increased digestion of the pollen proteins by pepsin following reduction by thioredoxin. Pollen proteins that have been reduced by thioredoxin are effective and safe immunotherapeutic agents for decreasing or eliminating an animal's allergic reaction that would otherwise occur upon exposure to the non-reduced pollen protein.

Description

[0001] cross reference [0002] This application is a continuation-in-part of Serial No. 08 / 953,703, filed October 17, 1997, which is a continuation-in-part of Serial No. 08 / 326,976, filed October 21, 1994, which was filed on August 11, 1998. issued U.S. Patent No. 5,792,506 on 12 April 1994, which is a continuation-in-part of Serial No. 08 / 211,673, filed April 12, 1994, which in turn is a continuation-in-part of Serial No. 07 / 935,002, filed August 25, 1992 Application (abandoned), which in turn is a successor-in-part of Serial No. 07 / 776,109 filed October 12, 1991 (abandoned). field of invention [0003] The present invention relates to the use of thiol redox proteins to reduce intramolecular disulfide bonds of seed proteins (eg, cereal proteins), enzyme inhibitor proteins (eg, venom, pollen proteins), and certain other proteins. More particularly, the present invention relates to the reduction of gliadin, glutenin, albumin and globulin using thioredoxin and glutaredoxin to ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/26A21D2/26A21D2/36A21D8/04A21D13/06A23C9/12A23C9/152A23J1/12A23J1/14A23J3/08A23J3/18A23K1/16A23L1/305A23L5/20A23L7/109A23L11/30A23L13/40A61K35/12A61K35/20A61K38/00A61K38/44A61K39/35A61K39/36A61P37/00A61P37/08C07K1/113C07K14/415C07K14/435C07K14/46C07K14/76C07K14/81C12N9/02C12N9/20C12P21/02C12Q1/34
CPCC12Y108/01009C07K14/415A61K35/20C07K14/46A23J3/18C12N9/0036C07K1/1133A23L1/2115A61K38/44C07K14/76C12Q1/34C07K14/43504A61K39/35A21D2/26A23C9/1209A23L1/305A23L1/0155A21D2/266C07K14/811A23L1/3149A23C9/152A23K1/1631A21D2/265C12N9/20A21D13/064C12Q1/26A21D8/04A61K2035/122A23C9/1213C12P21/02A21D2/36A23J1/12A23J1/14A21D2/264C12Y108/01008A23J3/08Y10S436/904A23L1/16A23K20/147A23L5/27A23L7/109A23L11/34A23L13/48A23L33/17A61P37/00A61P37/08Y10T436/182
Inventor B·B·布坎南G·德瓦尔R·M·洛萨诺J·H·翁B·C·伊O·L·弗里克
Owner RGT UNIV OF CALIFORNIA
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