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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/10—Protozoa; Culture media therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/18—Testing for antimicrobial activity of a material
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56905—Protozoa
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

• HAT Human African trypanosomiasis
• the present invention includes a method of preparing a cell free preparation of protozoan microsomes, wherein the cell free preparation of protozoan microsomes demonstrate the ability to translocate a plasma membrane polypeptide into the microsome.
• the present invention includes a method of endoplasmic reticulum (ER) translocation of a polypeptide using a cell free preparation of protozoan microsomes, wherein the cell free preparation of protozoan microsomes demonstrate the ability to translocate a plasma membrane polypeptide into the microsome.
• the method further includes contacting the cell free preparation of protozoan microsomes with a cell free preparation of protozoan cytosol, wherein the cytosol demonstrates the ability to facilitate the translocation of a plasma membrane polypeptide into a cell free preparation of protozoan microsomes.
• the present invention includes a method of screening for an agent that modulates the ER translocation of a polypeptide in a protozoan, the method including contacting the cell free preparation of protozoan microsomes, wherein the microsomes demonstrate the ability to translocate a plasma membrane polypeptide into the microsome with an agent and monitoring ER translocation of a polypeptide.
• the methods further includes contacting the cell free preparation of protozoan microsomes with a cell free preparation of protozoan cytosol, wherein the cytosol demonstrates the ability to facilitate the translocation of a plasma membrane polypeptide into a cell free preparation of protozoan microsomes.
• FIG. IA is a flowchart of protocol for post-translational import of VSG_ 117 5 ⁇ Depicted are the various steps and temperatures at which reactions took place.
• the translation product for VSG_1 17 500 is presented along with [ 14 C] methylated protein markers (Amersham).
• Fig. 1 B shows the import of VSG_117 500 into T. brucei microsomes (TbRM).
• VSG_1 17 g6 mRNA was translated in rabbit reticulocyte lysate for 15 minutes and then treated with cycloheximide (50 ⁇ g/ml, final concentration).
• hrucei cytosoHc Hsp70 (Van der Ploeg et al., 1985, Science; 228(4706): 1443-6) are 70% identical.
• MAL3-101 was used as a small molecule inhibitor of Hsp40-mediated Hsp70 ATPase (Fewell et al., 2004, J Biol Chem;
• Equisetin an analog of CJ-21, 058, is produced by the soil fungus Fusa ⁇ um heterosporum (Vesonder et al., 1979, J Antibiot (Tokyo); 32(7):759-61). Since CJ-21, 058 blocked protein translocation into TbRM, it was tested if equisetin had similar effects. Equisetin (25 ⁇ M) inhibited translocation of VSGJ.17 into TbRM by 95% (Fig. 2B, compare lanes 9 and 10). Hence, both CJ-21,058 and equisetin block VSG_117 translocation into TbRM.
• equisetin and CJ-21,058 inhibit import of proteins into TbRM (Fig. 2), and both compounds are trypanocidal (Fig. 3), with little effect on human HeLa cells (Fig. 4).
• TC 50 of equisetin (3.3 ⁇ M) and CJ-21,058 (7 ⁇ M) is comparable to that of suramin that is used for treatment of HAT.
• Equisetin has been considered for treatment of HIV infection (reviewed in De Clercq, 200O 5 Med Res Rev; 20(5):323-49).
• the present invention has developed, for the first time, a cell-free microsomal system for import of trypanosome proteins into the parasite ER.
• This example shows that cytosol from T. brucei is required for translocation of the full-length variant surface glycoproteins VSG_1 17 and VSG_MVAT7 into T. brucei microsomes (TbRM). Trypanosome microsome preparation. Following procedures also described in Examples 1 and 2, T.
• T. brucei rough microsomes TbRM
• the supernatant from the first centrifugation was centrifuged at 12,000-x g (20 minutes, 4°C), and the pellet saved as T. brucei rough microsomes (TbRM).
• the supernatant was centrifuged at 186,000-x g (4°C, 60 min) to obtain an 186,00Og-RM. All pellets were resuspended in rough microsome buffer (RMB) (at OD 280 — 50 per ml) and stored at -80°C until use.
• RMB rough microsome buffer
• ⁇ l ⁇ l
• T. brucei signal sequences will be studied. Specifically, this example will test whether peptide motifs that have discovered in h-regions (i.e., h-motifs) contribute to VSG translocation into the ER. The importance of specific amino acids (e.g., Leu and VaI), order of residues, and hydrophobicity of the amino acids will be examined using both in vitro and in vivo approaches.
• specific amino acids e.g., Leu and VaI

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• 2007
  • 2007-08-07 EP EP07811144A patent/EP2056870A4/de not_active Withdrawn
  • 2007-08-07 CA CA002660010A patent/CA2660010A1/en not_active Abandoned
  • 2007-08-07 WO PCT/US2007/017555 patent/WO2008021092A2/en not_active Ceased

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