Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-10-13
2003-12-09
Kunz, Gary (Department: 1647)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S004000
Reexamination Certificate
active
06660483
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to a human phospholipase C expressed in the brain (B-PLC), and to compositions and methods useful for diagnosing and treating physiologic pathologic conditions mediated by B-PLC. The invention finds application in the medical sciences.
BACKGROUND OF THE INVENTION
Intracellular signaling in the central nervous system (CNS) occurs predominantly by chemical signaling in which neurotransmitter molecules are released from one cell upon a challenge and are received and translated in the other into an appropriate pattern of response by specialized, cell membrane spanning polypeptides termed receptors. Some cellular responses are mediated by G-protein coupled receptors (GPCRs), members of a large family of transmembrane receptors. Upon their activation by neurotransmitters, GPCRs stimulate the GTPase activity of bound G-proteins and the latter are released and can activate several intracellular effector systems within the cell, including phospholipase C.
Phospholipase C (PLC) is an enzyme that breaks down phosphatidylinositol, a component of plasma membrane highly enriched in the CNS, to two important intracellular signaling molecules: diacylglycerol (DAG) and inositol-1,4,5-phosphate (IP3). Both DAG and IP3 can trigger numerous signaling cascades in response to extracellular stimuli to maintain normal cellular homeostasis in the case of normal neuronal activity, or, when the stimulus is associated with a neuropathologic challenge, to manage the balance between cell death and cell survival, repair and regeneration. DAGs activate an important intracellular kinase, protein kinase C, in Ca
2+
-dependent manner, while IP3 activates specific receptors on the endoplasmic reticulum to release Ca
2+
from intracellular stores, as well as IP3-dependent kinase (IP3-K). Via these pathways, the PLC-mediated signaling impacts such cellular phenomena as neurotransmitter release, receptor desensitization, transcriptional activation.
Proteins with homology to PLCs, but apparently lacking lipase activity, are also known. Like PLCs, these proteins bind inositol-1,4,5-phosphate, and are believed to play roles in regulation of activities mediated by IP
3
. See, e.g., Kanematsu et al., 1996
, Biochem J
313:319-25.
SUMMARY OF THE INVENTION
In one aspect, the invention provides an isolated, substantially pure, or recombinant B-PLC polypeptide, or immunogenic fragment thereof, for example, a polypeptide having an amino acid sequence exactly or substantially (e.g., at least about 80% or about 90%) identical to SEQ ID NO:2 or SEQ ID NO:4 or a fragment of the full-length sequence (e.g., a fragment having at least about 20 contiguous amino acid residues exactly identical to SEQ ID NO:2). In one embodiment, the invention provides a fusion protein having a sequence of a B-PLC polypeptide. In embodiment, the polypeptide has IP
3
binding activity. In embodiments, the polypeptide has PLC catalytic activity.
In another aspect, the invention provides an isolated polynucleotide that encodes, or is complementary to a sequence that encodes, a B-PLC polypeptide (e.g., having a sequence of SEQ ID NO:2 or a fragment thereof). In embodiments, the isolated polynucleotide has at least 15, at least 50, or at least 100 or more contiguous bases identical or exactly complementary to SEQ ID NO:1. In one embodiment, the invention provides an isolated polynucleotide that selectively hybridizes under stringent hybridization conditions to a polynucleotide sequence of SEQ ID NO:1. The polynucleotides of the invention may be operably linked to a promoter. The invention further provides a recombinant vector (such as an expression vector) containing a B-PLC polynucleotide, as well as cells (e.g., eukaryotic, mammalian or human cells) that contain the vector.
In another aspect, the invention provides an antibody (e.g., a monoclonal antibody), or fragment thereof, wherein the antibody or antibody fragment specifically binds to a B-PLC polypeptide.
In one aspect, the invention provides a method of detecting an B-PLC gene product in a sample by (a) contacting the sample with a probe that specifically binds the gene product, wherein the probe and the gene product form a complex, and detecting the formation of the complex; or (b) specifically amplifying the gene product in the biological sample, wherein said gene product is a polynucleotide, and detecting the amplification product; where the formation of the complex or presence of the amplification product is correlated with the presence of the B-PLC gene product in the biological sample. In this assay, the sample may be from human. The gene product may be, in various embodiments, a protein or RNA, and the probe may be an antibody or polynucleotide.
In a different aspect, the invention provides a method of treating an B-PLC mediated condition in a mammal by modulating (e.g., increasing or reducing) the activity or expression of B-PLC in a cell or tissue in the mammal.
The invention further provides methods for the diagnosis of a neurological condition in an animal, such as a human patient, by obtaining a biological sample from the animal and detecting an increased level of B-PLC in the sample compared to the level in a healthy animal, wherein said increased level is diagnostic of a neurological condition in the animal. In an embodiment the biological sample is blood. In an embodiment, the method of claim 17 wherein the increased level of B-PLC is detected using an immunoassay. The neurological condition that can be diagnosed may be hypoxic-ischemic brain insult (e.g., stroke), a neuroinflammatory disease, or other conditions.
Any method of detection can be used. In an embodiment, the increased level of B-PLC is detected using a detectably labeled polynucleotide probe comprising the sequence of SEQ ID NO:1 or 6 or a fragment thereof. In a different embodiment the the increased level of B-PLC is detected using an antibody that specifically binds the polypeptide of SEQ ID NO: 2 or 7.
In another aspect, the invention provides a method of treating a condition characterized by increased B-PLC expression in a mammal comprising modulating the activity or expression of B-PLC in a cell or tissue in the mammal. Examples of conditions susceptible to treatment include hypoxic-ischemic brain insult (e.g., stroke).
In another aspect, the invention provides a method of identifying an agent that modulates of B-PLC activity comprising contacting a cell or composition comprising a B-PLC protein encoded by a B-PLC polynucleotide and detecting a difference in protein activity in the presence of the agent compared to the absence of the agent. In embodiment the activity is IP3-binding activity.
A method of identifying an agent that modulates of B-PLC expression in a cell, comprising contacting a cell that expresses the B-PLC protein of SEQ ID NO:2 or a naturally occurring allele thereof, and detecting a change in B-PLC expression in the presence of the agent compared to the absence of the agent.
The invention also provides a method for identifying an agent for treatment or prevention of a neurological condition (e.g., hypoxic-ischemic brain insult), by determining whether a compound or treatment is a modulator of activity or expression of a B-PLC. In an embodiment, the activity is IP
3
-binding activity.
REFERENCES:
Nakashima et al.J. Cerebral Blood Flow and Metab., vol. 13, pp. 255-259, 1993.*
Singer, et al., (1997),Annu. Rev. Biochem., 66:475-509.
Genbank accession No. AB029015 (1999).
Genbank accession No. AW408450 (2000).
Genbank accession No. AA277075 (1997).
Genbank accession No. AA772973 (1998).
Genbank accession No. AW493796 (2000).
Genbank accession No. AW500653 (2000).
Genbank accession No. BE655266 (2000).
Genbank accession No. AA690679 (1997).
Genbank accession No. AL024179 (1999).
Genbank accession No. AW478640 (2000).
Genbank accession No. T78839 (1995).
Genbank accession No. AW428387 (2000).
Genbank accession No. AW466123 (2000).
Kanematsu et al., (1996),Biochem. J.1(313):319-325.
Takeuchi et al., (1999),Chem. Phys. Lipids, 98:35-47.
Yagisawa et
Chin Daniel J.
Kask Kalev
Melcher Thorsten
AGY Therapeutics, Inc.
Bozicevic Field & Francis LLP
Gucker Stephen
Kunz Gary
Sherwood Pamela J.
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