The studies described in this proposal are designed to respond to the RFP for the development of antibodies to receptors and signal transduction macromolecules important in the expression of the effects of ethanol on brain function and behavior. We plan to generate new antibodies to various NMDA receptor subunits and to specific domains of these proteins. The antibodies we plan to raise are targeted to regions of the proteins that represent key regulatory sites for the function of the proteins. Numerous studies have confirmed that a prevalent site of ethanol action in the central nervous system is at the glutamate receptors, in particular the NMDA receptors. Although there is remarkable agreement on the interference by ethanol with normal glutamatergic neurotransmission in brain as a result of the inhibition of NMDA receptor-ion channels, there are still several issues that need to be examined with the use of new molecular probes. The antibodies we plan to raise should help many investigators interested in probing some of these issues.
The specific aims for this project are:
Specific Aim #1. The subcloning and expression of antigenic proteins or the synthesis of peptides based on the structure of the NMDAR1/NMDAR2 receptor proteins and of the glutamate, CPP, and glycine binding protein subunits of a receptor-like complex for the purpose of raising polyclonal and monoclonal antibodies.
Specific Aim #2. The characterization of the immune reactivity of the antibodies raised by means of immunochemical assays, Western blots, immunoextraction procedures, and immunocytochemical tracing of the expression of the proteins in brain.
Specific Aim #3. The characterization of the activity of select antibodies as inhibitors of NMDA-induced Ca2+ influx into neurons and as probes of the altered expression specific subunit isoforms in the brains of animals exposed to chronic ethanol treatment or in primary neuronal cultures treated in vitro with ethanol.
Specific Aim #4. The full documentation of the properties of each antibody, either polyclonal or monoclonal, generated and the production, storage and distribution to other scientists of these antibodies.

Agency
National Institute of Health (NIH)
Institute
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
Type
Research Project (R01)
Project #
5R01AA011419-04
Application #
2894185
Study Section
Special Emphasis Panel (SRCA)
Project Start
1996-09-30
Project End
2002-08-31
Budget Start
1999-09-01
Budget End
2002-08-31
Support Year
4
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of Kansas Lawrence
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
072933393
City
Lawrence
State
KS
Country
United States
Zip Code
66045
Badawi, Yomna; Pal, Ranu; Hui, Dongwei et al. (2015) Ischemic tolerance in an in vivo model of glutamate preconditioning. J Neurosci Res 93:623-32
Wang, Xinkun; Patel, Nilam D; Hui, Dongwei et al. (2014) Gene expression patterns in the hippocampus during the development and aging of Glud1 (Glutamate Dehydrogenase 1) transgenic and wild type mice. BMC Neurosci 15:37
Michaelis, E K; Wang, X; Pal, R et al. (2011) Neuronal Glud1 (glutamate dehydrogenase 1) over-expressing mice: increased glutamate formation and synaptic release, loss of synaptic activity, and adaptive changes in genomic expression. Neurochem Int 59:473-81
Hascup, Kevin N; Bao, Xiaodong; Hascup, Erin R et al. (2011) Differential levels of glutamate dehydrogenase 1 (GLUD1) in Balb/c and C57BL/6 mice and the effects of overexpression of the Glud1 gene on glutamate release in striatum. ASN Neuro 3:
Wang, Xinkun; Bao, Xiaodong; Pal, Ranu et al. (2010) Transcriptomic responses in mouse brain exposed to chronic excess of the neurotransmitter glutamate. BMC Genomics 11:360
Wang, Xinkun; Michaelis, Elias K (2010) Selective neuronal vulnerability to oxidative stress in the brain. Front Aging Neurosci 2:12
Hui, Dongwei; Kumar, Keshava N; Mach, Julie R et al. (2009) A rat brain bicistronic gene with an internal ribosome entry site codes for a phencyclidine-binding protein with cytotoxic activity. J Biol Chem 284:2245-57
Bao, Xiaodong; Pal, Ranu; Hascup, Kevin N et al. (2009) Transgenic expression of Glud1 (glutamate dehydrogenase 1) in neurons: in vivo model of enhanced glutamate release, altered synaptic plasticity, and selective neuronal vulnerability. J Neurosci 29:13929-44
Pal, Ranu; Agbas, Abdulbaki; Bao, Xiaodong et al. (2003) Selective dendrite-targeting of mRNAs of NR1 splice variants without exon 5: identification of a cis-acting sequence and isolation of sequence-binding proteins. Brain Res 994:1-18
Hui, D; Bao, X; Michaelis, E K (2001) High-probability amplification of genomic DNA starting from cDNA. Biotechniques 31:268-70, 272

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