Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-11-07
2003-11-04
Whisenant, Ethan (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023500, C536S024310, C435S004000, C435S021000, C435S963000, C435S242000, C435S320100
Reexamination Certificate
active
06642004
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to the field of nucleotides. In particular, the invention relates to measuring the potential biological activity of a compound by measuring nucleotide levels in cells following chemical treatment, and more particularly it pertains to measuring the levels of cyclic phosphate nucleotides present in cells following chemical treatment for measuring the potential biological activity of a compound.
BACKGROUND OF THE INVENTION
The physiological responses to many biologically active compounds are mediated through “second messengers”. Nucleotides, for example cyclic adenosine 3′,5′-monophosphate or cyclic AMP (cAMP), play important roles as second messengers in signal transduction pathways after hormones or other biologically active compounds bind to cell surface receptors. Increased levels of nucleotides, resulting from receptor activation, cause activation of specific nucleotide-dependent protein kinases, which in turn cause phosphorylation of various target proteins. It is the activation of these phosphorylated target proteins that bring about the diverse physiological responses in the cells including, but not limited to, biological activity. Thus, the higher the level of the nucleotide present the more biological activity a compound may possess.
The measuring of intracellular nucleotide levels following chemical treatment of cells has been reported in the literature, for example Takeda et al., J. Biochem., Vol. 105, pp. 327-329 (1989), Berg et al., Biotechniques, Vol. 15, No. 1, pp. 56-59 (1993), Brown et al., Biochem. J. 121, pp. 561-562 (1971), and A. G. Gilman, PNAS, Vol. 67, No. 1, pp. 305-312 (1970). However, these methods tend to involve multiple reaction vessels and be time-intensive limiting the number of measurements that can be carried out in a given time period. As such, these methods preclude a single vessel, i.e. “one-pot”, high-throughput method for measuring changes in nucleotides. In addition, “one-pot” methods reported in the literature, for example Amersham LifeScience's commercially available Biotrak™ product and Kariv et al., J. Biomolecular Screening, Vol. 4, No. 1, pp. 27-32, (1999), tend to be expensive. As a result, there is a need for an inexpensive, single vessel, high-throughput method for measuring changes in the amount of a nucleotide present in a cell.
SUMMARY OF THE INVENTION
One embodiment of the present invention describes a single vessel, high-throughput method for measuring levels of nucleotides generated in a testing medium. The present invention measures changes in the amount of a nucleotide in a test medium in response to the addition of a test compound to the test medium. The present invention is particularly effective in measuring changes in a nucleotide, such as cyclic adenosine 3′,5′-monophosphate (cAMP), in a cell, particularly in cells of insects.
In another embodiment of the present invention, a single vessel, high-throughput method of identifying compounds that which increase the amount of a nucleotide generated by a testing medium by comparing test compounds to the test medium alone or to the test medium following chemical treatment with compounds that increase nucleotides is disclosed. This method can be useful in identify compounds suspected of exhibiting biological activity.
In yet another embodiment of the present invention, a single vessel, high-throughput method of identifying compounds with biological activity through the generation of cAMP in a cell is disclosed.
The present invention is less complex, more cost effective, and comparable in sensitivity to those disclosed in the art.
DEFINITIONS
The modifier “about” as utilized herein indicates that certain preferred operating ranges, such as ranges for molar ratios for reactants, material amounts, and temperature, are not fixedly determined. The meaning will often be apparent to one of ordinary skill in the art of molecular biology. For example, a recitation of a temperature range of about 120° C. to about 135° C. in reference to an experiment would be interpreted to include other like temperatures that can be expected to favor a useful completion of the experiment, such as 105° C. or 150° C. In general, unless more particular ranges are disclosed, “about” shall indicate not more than 10% of the absolute value of an end point or 10% of the range recited, whichever is less.
The term “ambient temperature” as utilized herein shall mean any suitable temperature found in a laboratory or other working quarter, and is generally not below about 15° C. nor above about 30° C.
The term “biological activity” as utilized herein shall mean the ability of a substance, such as a chemical, including but not limited to drugs (i.e. pharmaceuticals) and pesticides, to act on a cell, virus, organ or organism and which creates a change in the functioning of the cell, virus, organ or organism.
The term “testing vessel” as utilized herein shall mean any device, such as a petri-dish, a microtiter plate, a test-tube, or beaker, which may be utilized to perform an assay, a reaction, a method, an experiment, or other procedure.
The term “testing medium” as used herein shall mean any environment, such as a cell or a cellular membrane, suitable for generating a nucleotide.
The term “nucleotide binding protein” as utilized herein shall mean any protein, for example, a protein derived from a bovine adrenal gland, a protein derived from a bovine muscle, or an antibody, that selectively binds or attaches to a nucleotide.
As used herein, the term “lysing agent” shall mean any substance, such as a detergent, capable of causing cell lysis.
DETAILED DESCRIPTION OF THE INVENTION
One embodiment of the present invention involves a method of measuring levels of a nucleotide generated in a testing medium, for example, cells or cellular membranes, following chemical treatment. The method comprises:
(a) contacting a test compound with the testing medium in a testing vessel, for example, a microtiter plate, a test-tube, or beaker;
(b) maintaining the test compound in contact with the testing medium in the testing vessel for a time sufficient to allow nucleotides to be generated in the testing medium;
(c) releasing nucleotides: generated in the testing medium into the testing vessel;
(d) adding a radiolabeled nucleotide ligand and a fixed amount of a nucleotide binding protein to the testing vessel, wherein the radiolabeled nucleotide ligand competes with nucleotides generated in the testing medium to bind to the nucleotide binding protein;
(e) maintaining the testing vessel for a period of time at a temperature sufficient to allow nucleotides generated in the testing medium and the radiolabeled nucleotide ligand to bind to the nucleotide binding protein to form a nucleotide binding protein complex;
(f) separating the nucleotide binding protein complex from uncomplexed radiolabeled nucleotide ligand; and
(g) measuring the level of radioactivity of the nucleotide binding protein complex, wherein the level of radioactivity is inversely proportional to the amount of the nucleotide generated in said testing medium.
Suitable nucleotides that may be generated and measured by the present invention include, but are not limited to, cyclic phosphates. Preferable nucleotides generated and measured by the present invention are cyclic monophosphates, for example, cyclic adenosine 3′,5′-monophosphate (cAMP), dibutyryl cyclic adenosine 3′,5′-monophosphate (dbcAMP), cyclic guanosine 3′,5′-monophosphate (cGMP), cyclic inosine 3′,5′-monophosphate (cIMP), and cyclic uridine 3′,5′-monophosphate(cUMP). Particularly preferred and useful nucleotides generated and measured by the present invention are cAMP and cGMP.
Suitable cells that may be used as the test medium in the present invention include, but are not limited to, native or cloned invertebrate or vertebrate cells which are either adherent or nonadherent, for example Sf9 cells, Sf21 cells, KC cells, CHO cells, COS7 cells, and HEK293 cells.
Chaguturu Muniratham K.
Dargar Ratna
FMC Corporation
FMC Corporation
Hashemi Shar
Whisenant Ethan
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