Mammalian sphingosine kinase type 2 isoforms, cloning, expression and methods of use thereof
AssigneesSankyo Company, Limited, Georgetown University
InventorsSarah Spiegel, Takafumi Kohama
Life of this patent
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Abstract
Nucleic acids encoding mouse and human sphingosine kinase type 2 isoforms, methods for detecting agents or drugs which inhibit or promote sphingosine activity and therapeutic agents containing peptides or antibodies to peptides encoded by such nucleic acids.
Claims68
An isolated and purified DNA which encodes a mammalian sphingosine kinase type 2 isoform.
An isolated and purified DNA selected from the group consisting of
- a) a DNA having a nucleotide sequence consisting essentially of the nucleotide sequence of SEQ ID NO:
- 11 and
- b) a DNA encoding protein consisting essentially of the amino acid sequence of SEQ ID NO:
- 12.
An isolated and purified DNA selected from the group consisting of
- a) a DNA having a nucleotide sequence consisting essentially of the nucleotide sequence of SEQ ID NO:
- 13 and
- b) a DNA encoding protein consisting essentially of the amino acid sequence of SEQ ID NO:
- 14.
An isolated protein comprising an amino acid sequence of murine SPHK2 consisting essentially of SEQ ID NO:
- 12.
An isolated and purified DNA which encodes a protein of a sphingosine kinase type 2 isoform, said DNA comprising a sequence selected from the group consisting of the sequence of Genbank Accession No. bankit325787 and the sequence of Genbank Accession No. bankit325752.
A method for screening agents or drugs which reduce or eliminate sphingosine kinase type 2 activity, the method comprising detecting a decrease in sphingosine kinase type 2 enzyme activity in the presence of said agent or drug.
A method for detecting the presence of a sphingosine kinase type 2 isoform in a sample comprising
- (i) contacting a sample with antibodies which recognize sphingosine kinase type 2;
- and
- (ii) detecting the presence or absence of a complex formed between sphingosine kinase type 2 and antibodies specific therefor.
A method for detecting sphingosine kinase type 2 in a sample comprising subjecting the sample to a polymerase chain reaction, a northern hybridization assay or an in situ hybridization assay and detecting for the presence of sphingosine kinase type 2.
A diagnostic kit for detecting sphingosine kinase type 2 RNA/cDNA in a sample comprising primers or nucleotides specific for sphingosine kinase type 2 RNA or cDNA suitable for hybridization to sphingosine kinase type 2 RNA or cDNA and/or amplification of sphingosine kinase type 2 RNA or cDNA and suitable ancillary reagents.
A method for detecting an agent or a drug which inhibits or promotes sphingosine kinase type 2 activity comprising:
- (a) introducing a recombinant DNA construct comprising a vector and a DNA encoding a mammalian sphingosine kinase type 2 isoform in to a cell such that sphingosine kinase type 2 isoform is produced in said cell;
- (b) adding at least one drug or agent to said cell;
- and
- (c) detecting whether or not said drug or agent inhibits or promotes sphingosine kinase type 2 activity by measuring sphingosine kinase-dependent phosphorylation of lipids in said cells and comparing the resultant measurement to a control which did not receive such drug or agent, wherein a decrease in the amount of sphingosine kinase-dependent phosphorylation of lipids as compared to the control indicates an inhibitory drug or agent, or an increase in the amount of sphingosine kinase-dependent phosphorylation of lipids in said cell as compared to the control indicates a stimulatory drug or agent.
Dependencies are inferred from the claim text.