Broad Long-Term Objectives: Develop and apply tools for the determination of biologically active peptides at physiologically relevant concentrations and volumes of sample.
Specific Aims : Technical Advance: Establish a novel detection method for the liquid chromatographic determination of peptides that is peptide selective-only peptides give peaks, and sensitive on a concentration basic-picomolar. Such peptides as oxytocin, vasopressin, neuropeptide Y, substance P, delta sleep-inducing peptide, enkephalins, and others will be measurable without immunoassay at physiological levels. Biological Problem: Through the application of this new and powerful method, we will work to understand the mechanisms of excitotoxicity-the events in which highly stimulated nerve cells die. Methods: Technical: We will develop a novel detection technique with the selectivity of fluorescence. Biological: Models of ischemia: in vitro: oxygen starvation of rat hippocampus slices. in vivo: clamping of carotid arteries of adult rats; hypoxia/ishemia in neonatal rats. Sampling: in vitro, perfusion; in vivo, microdialysis, also tissue extraction. Health Relatedness: What the proposed procedure offers over traditional immunoassays is the ability to see the whole peptide picture, including what is happening to peptide precursors, and how active peptides are metabolized. This is because the analysis does not rely upon specific biological binding reactions. In studies of virtually any regulatory system, such information will be useful in understanding pathology. Our specific application is to the role of peptides on the glutamatergic system in the rat brain, and in particular trying to understand the phenomena that lead to nerve cell death in such incidents as stroke, head trauma, epilepsy, deep hypoglycemia, and several syndromes that include dementias such as AIDS-related dementia. Current knowledge allows us to say that it is possible in principle to minimize cell death, and the related practical consequences, resulting from exitotoxicity, but it does not allow us to say how. A more detailed understanding of the mechanisms leading to cell death, including the roles of peptides, will allow evaluation of drug strategies against nerve damage.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM044842-07
Application #
2701538
Study Section
Metallobiochemistry Study Section (BMT)
Project Start
1991-05-01
Project End
1999-04-30
Budget Start
1998-05-01
Budget End
1999-04-30
Support Year
7
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of Pittsburgh
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
053785812
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Ou, Yangguang; Wilson, Rachael E; Weber, Stephen G (2018) Methods of Measuring Enzyme Activity Ex Vivo and In Vivo. Annu Rev Anal Chem (Palo Alto Calif) 11:509-533
Tecuatl, Carolina; Herrrera-López, Gabriel; Martín-Ávila, Alejandro et al. (2018) TrkB-mediated activation of the phosphatidylinositol-3-kinase/Akt cascade reduces the damage inflicted by oxygen-glucose deprivation in area CA3 of the rat hippocampus. Eur J Neurosci 47:1096-1109
Ou, Yangguang; Weber, Stephen G (2018) Higher Aminopeptidase Activity Determined by Electroosmotic Push-Pull Perfusion Contributes to Selective Vulnerability of the Hippocampal CA1 Region to Oxygen Glucose Deprivation. ACS Chem Neurosci 9:535-544
Yin, Bocheng; Barrionuevo, Germán; Weber, Stephen G (2018) Mitochondrial GSH Systems in CA1 Pyramidal Cells and Astrocytes React Differently during Oxygen-Glucose Deprivation and Reperfusion. ACS Chem Neurosci 9:738-748
Wilson, Rachael E; Jaquins-Gerstl, Andrea; Weber, Stephen G (2018) On-Column Dimethylation with Capillary Liquid Chromatography-Tandem Mass Spectrometry for Online Determination of Neuropeptides in Rat Brain Microdialysate. Anal Chem 90:4561-4568
Patil, Jaspal; Matte, Ashok; Mallard, Carina et al. (2018) Spirulina diet to lactating mothers protects the antioxidant system and reduces inflammation in post-natal brain after systemic inflammation. Nutr Neurosci 21:59-69
Ou, Yangguang; Weber, Stephen G (2017) Numerical Modeling of Electroosmotic Push-Pull Perfusion and Assessment of Its Application to Quantitative Determination of Enzymatic Activity in the Extracellular Space of Mammalian Tissue. Anal Chem 89:5864-5873
Wilson, Rachael E; Groskreutz, Stephen R; Weber, Stephen G (2016) Improving the Sensitivity, Resolution, and Peak Capacity of Gradient Elution in Capillary Liquid Chromatography with Large-Volume Injections by Using Temperature-Assisted On-Column Solute Focusing. Anal Chem 88:5112-21
Patil, Jaspal; Matte, Ashok; Nissbrandt, Hans et al. (2016) Sustained Effects of Neonatal Systemic Lipopolysaccharide on IL-1? and Nrf2 in Adult Rat Substantia Nigra Are Partly Normalized by a Spirulina-Enriched Diet. Neuroimmunomodulation 23:250-259
D'Angelo, Barbara; Ek, C Joakim; Sun, Yanyan et al. (2016) GSK3? inhibition protects the immature brain from hypoxic-ischaemic insult via reduced STAT3 signalling. Neuropharmacology 101:13-23

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