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Tryparedoxin, expression plasmid, process of preparation, method of use, test kit and pharmaceutical composition

Inactive Publication Date: 2005-06-30
FLOHE LEOPOLD +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0005] The uniqueness of this cascade of oxidoreductases offers the possibility to inhibit the parasitic metabolism without causing adverse effects in the host organism.

Problems solved by technology

1996). Vaccination strategies have so far failed and most of the chemotherapeutic drugs currently used for treatment are unsatisfactory in terms of both efficacy and toxicity (Risse
However, their ability to cope with such oxidative stress appears to be surprisingly weak.
However, a pertinent enzymatic entity could never be purified (Henderson et al., 1987; Penketh et al., 1987) and doubts about its existence were raised (Penketh and Klein, 1986).

Method used

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  • Tryparedoxin, expression plasmid, process of preparation, method of use, test kit and pharmaceutical composition
  • Tryparedoxin, expression plasmid, process of preparation, method of use, test kit and pharmaceutical composition
  • Tryparedoxin, expression plasmid, process of preparation, method of use, test kit and pharmaceutical composition

Examples

Experimental program
Comparison scheme
Effect test

example 2

Determination of Tryparedoxin Activity

[0043] In essence, the activity of tryparedoxin activity is measured by coupling the catalytic reduction of hydroperoxide mediated by tryparedoxin peroxidase to NADPH consumption by means of trypanothione and trypanothione reductase. For example, an assay sample may contain 0.1 mM NADPH in 50 mM Hepes pH 7.6, 1 mM EDTA, 50 M H2O2 or t-butyl hydroperoxide (t-bOOH), 45 M T(SH)2, 16.5 μg / ml tryparedoxin peroxidase and 0.34 U trypanothione reductase and an unknown amount of tryparedoxin. Unless otherwise stated, the reaction is started with the addition of the hydroperoxide. Dihydro-trypanothione is obtained by chemical reduction of TS2 (Bachem, Switzerland) as described (Fairlamb et al., 1986). t-BOOH may be replaced by other hydroperoxides, such as H2O2, linoleic acid hydroperoxide or phosphatidylcholine hydroperoxide.

[0044]FIG. 6 demonstrates that trypanothione reductase, T(SH)2, tryparedoxin and tryparedoxin peroxidase are indispensable for th...

example 3

Characterisation of Tryparedoxin I by Partial Proteins Sequencing

[0045] Since the N-terminus of tryparedoxin I was blocked, the protein was digested with either bovine trypsin or endoproteinase Glu-C from Staphylococcus aureus (both sequencing grade, Promega) according to Stone and Williams (1993). The peptides were separated by HPLC (Applied Biosystems 172A) on an Aquapore OD-300 RP-18 column. Automated Edman degradation was performed with an Applied Biosystems, Inc. sequencer with an on-line C-18 reverse phase HPLC. Database searches were performed with the BLAST and FASTA programs. Peptides were aligned with the Bestfit program, Genetics Computer Group (GCG), Madison, Wis., USA.

[0046] Seven fragments could be sequenced and could be aligned to a thioredoxin-like protein of C. elegans (FIG. 7).

example 4

Use of Sequenced Fragments of Tryparedoxin I to Elucidate the Encoding DNA

[0047] Cells culture and DNA extraction: C. fasciculata (HS6) was grown as described by Shim and Fairlamb (1988). The cells were harvested by centrifugation for 15 min at 7000 rpm, washed twice with saline solution (0.9% NaCl) and resuspended in 5 ml buffer (50 mM TrisHCl, 100 mM EDTA, 15 mM NaCl, 0.5% SDS, 100 μg ml−1 Proteinase K, pH 8.0). Resuspended cells were preincubated at 50° C. for 40 min. The genomic DNA was extracted twice with equivalent volumes of phenol (incubation: 60° C. for 45 min; centrifugation: 20 min, 4500 rpm) followed by phenol:chloroform:isoamyl alcohol (25:24:1) and chloroform:isoamyl alcohol extraction (24:1). Genomic DNA was precipitated with sodium acetate and ethanol.

[0048] Primers, hybridization probes and sequence analysis: Based on the peptide sequences of tryparedoxin I (Nogoceke et al., 1997) degenerate oligodeoxyribonucleotides were synthesized. Polymerase chain reaction (P...

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Abstract

The present invention provides novel enzymes, tryparedoxins, their isolation from Crithidia fasciculata, a method for the production thereof in genetically transformed bacteria, and their use as molecular targets for the discovery of trypanocidal drugs

Description

[0001] This is a continuation of International Application No. PCT / EP97 / 06983 filed Dec. 12, 1997, the entire disclosure of which is incorporated herein by reference.INTRODUCTION [0002] Flagellated protozoan parasites of the family Trypanosomatidae are among the most prevalent human pathogens in tropical and subtropical areas. These organisms have complex life cycles and some of them are the causative agents of debilitating or life-threatening diseases, such as American Chagas' disease (Trypanosoma cruzi), African sleeping sickness (T. brucei ganibienise and T. b. rhodesienise), oriental sore (Leishmania tropica), kala azar (L. donovani) and mucocutaneous leishmaniasis (L. brasiliensis). Others infect hosts as diverse as plants (Phytomonas species), insects (Crithidia and Leptomonas species) and livestock (T. congolenise, T. b. brucei, T. evansi). Many of the human pathogens are also endemic in wildlife. Worldwide, more than 30 million people are estimated to suffer from trypanosoma...

Claims

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Application Information

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IPC IPC(8): C12N15/09A61K38/00C07K14/44C12N1/15C12N1/19C12N1/21C12N5/10C12N9/00C12N9/02C12N9/08C12Q1/28
CPCA61K38/00C12N9/0004C07K14/44C12N15/52
Inventor FLOHE, LEOPOLDNOGOCEKE, EVERSONKALISZ, HENRYKMONTEMARTINI, MARISA
Owner FLOHE LEOPOLD
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