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Novel Carboxylesterase Nucleic Acid Molecules, Proteins and Uses Thereof

Inactive Publication Date: 2009-07-02
SILVER GARY M +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0023]Another embodiment of the present invention is a method to identify a compound capable of inhibiting flea carboxylesterase activity, the method comprising: (a) contacting an isolated flea carboxylesterase with a putative inhibitory compound under conditions in which, in the absence of the compound, the protein has carboxylesterase activity; and (b) determining if the putative inhibitory compound inhibits the activity. Also included in the present invention is a test kit to identify a compound capable of inhibiting flea carboxylesterase activity, the test kit comprising an isolated flea carboxylesterase protein having esterase activity and a means for determining the extent of inhibition of the activity in the presence of a putative inhibitory compound.
[0024]Yet another embodiment of the present invention is a therapeutic composition that is capable of reducing hematophagous ectoparasite infestation. Such a therapeutic composition includes at least one of the following prot

Problems solved by technology

In particular, the bites of hematophagous arthropods are a problem for animals maintained as pets because the infestation becomes a source of annoyance not only for the pet but also for the pet owner who may find his or her home generally contaminated with insects.
As such, hematophagous arthropods are a problem not only when they are on an animal but also when they are in the general environment of the animal.
Bites from hematophagous arthropods are a particular problem because they not only can lead to disease transmission but also can cause a hypersensitive response in animals which is manifested as disease.
A hypersensitive response in animals typically results in localized tissue inflammation and damage, causing substantial discomfort to the animal.
While some of these products are efficacious, most, at best, offer protection of a very limited duration.
Furthermore, many of the methods are often not successful in reducing arthropod populations.
Reduction of hematophagous arthropod infestation on the pet has been unsuccessful for one or more of the following reasons: (1) failure of owner compliance (frequent administration is required); (2) behavioral or physiological intolerance of the pet to the pesticide product or means of administration; and (3) the emergence of hematophagous arthropod populations resistant to the prescribed dose of pesticide.

Method used

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  • Novel Carboxylesterase Nucleic Acid Molecules, Proteins and Uses Thereof
  • Novel Carboxylesterase Nucleic Acid Molecules, Proteins and Uses Thereof
  • Novel Carboxylesterase Nucleic Acid Molecules, Proteins and Uses Thereof

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0178]This example describes labeling of proteases and esterases with radiolabeled diisopropylfluorophosphate.

[0179]Tissue samples were isolated from unfed or bovine blood-fed 1st instar Ctenocephalides felis flea larvae; bovine blood-fed or cat blood-fed 3rd instar Ctenocephalides felis flea larvae; bovine blood-fed or cat blood-fed Ctenocephalides felis prepupal flea larvae; bovine blood-fed or cat blood-fed adult Ctenocephalides felis flea midgut tissue, and whole unfed, bovine blood-fed or cat blood-fed adult Ctenocephalides felis fleas. The 1st instar, 3rd instar, prepupal and adult midgut tissues were then homogenized by freeze-fracture and sonicated in a Tris buffer comprising 50 mM Tris, pH 8.0 and 100 mM CaCl2. The whole adult flea sample was then homogenized by freeze-fracture and ground with a microtube mortar and pestle. The extracts were centrifuged at about 14,000×g for 20 minutes (min.) and the soluble material recovered. The soluble material was then diluted to a fin...

example 2

[0181]This example describes the identification of general CE activity in flea tissue extracts.

[0182]Tissue samples and soluble extracts were prepared as described above in Example 1, except not labelled, from unfed (UF) and bovine blood-fed 1st instar flea larvae, bovine blood-fed 3rd instar flea larvae, bovine blood-fed prepupal flea larvae, unfed whole adult fleas, cat blood-fed adult (ACF) whole fleas, cat blood-fed adult fleas that have had their heads and midguts removed (referred to herein as fed adult partial fleas), unfed adult flea midguts and cat blood-fed adult flea midguts. About 5 tissue equivalents of each tissue were assayed for general CE activity using the following method. Tissue samples of about 5 μl were added to separate wells of flat-bottomed microtiter plate (available from Becton Dickinson, Lincoln Park, N.J.). A control well was prepared by adding about 5 μl of Tris buffer to an empty well of the plate. About 95 μl of 25 mM Tris-HCl (pH 8.0) was then added ...

example 3

[0185]This example describes the determination of general CE activity using isoelectric focusing (JEF)-PAGE and non-reducing SDS-PAGE.

[0186]A. Non-Reducing SDS-PAGE.

[0187]Soluble extracts from unfed and bovine blood-fed 1st instar flea larvae, bovine blood-fed 3rd instar flea larvae, bovine blood-fed prepupal flea larvae, bovine blood-fed adult (ABF) whole fleas and cat blood-fed adult whole fleas were prepared using the method described in Example 1. Each soluble extract sample was combined with SDS sample buffer (available from Novex) and proteins in the samples were resolved by gel electrophoresis using 14% Tris-glycine SDS electrophoresis gels (available from Novex). The gels were run at room temperature for about 1 hour at 200 volts. After electrophoresis, the gels were soaked for about for 30 minutes in 50 mM Tris, pH 8.0, containing 2.5% Triton X-100 to renature the proteins. The gels were then soaked in 50 mM Tris, pH 8.0, for about 5 minutes and then stained for about 5 min...

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Abstract

The present invention relates to arthropod esterase proteins; to arthropod esterase nucleic acid molecules, including those that encode such esterase proteins; to antibodies raised against such esterase proteins; and to other compounds that inhibit arthropod esterase activity. The present invention also includes methods to obtain such proteins, nucleic acid molecules, antibodies, and inhibitory compounds. Also included in the present invention are therapeutic compositions comprising such proteins, nucleic acid molecules, antibodies and / or inhibitory compounds as well as the use of such therapeutic compositions to protect animals from hematophagous arthropod infestation.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a Continuation of co-pending U.S. patent application Ser. No. 10 / 678,521, filed Oct. 2, 2003; which is a Divisional of U.S. patent application Ser. No. 09 / 403,942, filed May 2, 2000, and issued as U.S. Pat. No. 6,664,090; which is a 371 filing of International Patent Application No. PCT / US97 / 20598, filed Nov. 10, 1997, which is a continuation-in-part of U.S. Ser. No. 08 / 747,221, filed Nov. 12, 1996, which issued as U.S. Pat. No. 6,063,610 on May 16, 2000; all of which are entitled “NOVEL CARBOXYLESTERASE NUCLEIC ACID MOLECULES, PROTEINS AND USES THEREOF” and are incorporated herein by reference.FIELD OF THE INVENTION[0002]The present invention relates to arthropod esterase nucleic acid molecules, proteins encoded by such nucleic acid molecules, antibodies raised against such proteins, and inhibitors of such proteins. The present invention also includes therapeutic compositions comprising such nucleic acid molecules, pr...

Claims

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Application Information

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IPC IPC(8): C12Q1/44C12N15/12C12N5/10C12N9/18A61K38/00G01N33/53A61K38/46A61K39/395A61K45/00A61K48/00A61P33/12C07K16/40C12N1/15C12N1/19C12N1/21C12N7/00C12N15/09
CPCC12N9/18A61K38/00A61P33/12
Inventor SILVER, GARY M.WISNEWSKI, NANCYBRANDT, KEVIN S.
Owner SILVER GARY M
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