UT Chem/Biochem Dept

The 1999-2000 Beckman Scholars: Cindy V. Ly, MD/PhD

Faculty Mentor: Professor Jason B. Shear Length of term: Fall 99, Spring 00, Summer 00; Summer 99 was spent in a neuroscience internship at UCSF. Honors & Awards: University Honors (Spring 99, Fall 99, Spring 00); Anonymous No. 1 Endowed Presidential Scholarship (Fall 99 – Spring 00); Deans Scholars Program (Fall 99 – Spring 00); Barrow Scholar (2001); 2006 National Research Service Award (NRSA), National Institute Neurological Disease and Stroke (NINDS) Publications: Neely, G.G. et.al., Cell, 143(4), 628-638, 2010. Giagtzoglou, N., Ly, C.V., Bellen, H.J. From Cell-cell Junctions, 295-319, 2010.Yao, C-K. et al., Cell,138(5), 947-60, 2009. Giagtzoglou, N., Ly, C.V., Bellen, H.J. in Cold Spring Harbor perspectives in Biology, 1(4) a003079, 2009. Verstreken, P.et al. Neuron 63(2): 203-215, 2009. Ly, C.V. et al,, Cell Biol, 181(1): 157-170, 2008. Ly, C., Verstreken, P. (2006) Mitochondria at the synapse. Neuroscientist 12(4): 291-299.; Verstreken, P., Ly, C., Venken, K.J.T., Koh, T.W., Zhou, Y., Bellen, H.J. (2005) Synaptic mitochondria are critical for mobilization of reserve pool vesicles at Drosophila neuromuscular junctions. Neuron, 47: 365-378.; Hayman L.A., Shukla V., Ly C., Taber K.H. (2003) Clinical and imaging anatomy of the scalp. Journal of Computer Assisted Tomography, 27: 454-459.; Shukla, V., Hayman, L.A., Ly, C., Fuller, G., Taber, K.H. (2002) Adult cranial dura I: intrinsic vessels. Journal Computer Assisted Tomography, 26: 1069-1074.; Wei J, Okerberg E, Dunlap J, Ly C, Shear JB (2000) Determination of Biological Toxins Using Capillary Electrokinetic Chromatography with Multiphoton-Excited Fluorescence. Analytical Chemistry, 72: 1360.; Debad, J.D., Ly, C. (2004) Methods, Compositions, and Kits for Biomarker Extraction. Patent #: WO2004059280.; Poster. 47th Drosophila Research Conf. (2006); Poster. 46th Drosophila Research Conf. (2005). Where is she now? Graduated with Bachelor of Science in Biochemistry with High Honors, May 2000. Cindy worked for IGEN International, a medical diagnostics biotech company based in Maryland for a year, completed the MD/PhD program at Baylor U in 2009, and is currently a Neurology resident at Barnes Jewish Hospital, St Louis. How can I contact her? cindy.ly at bcm.tmc.edu

Beckman research project in the Shear group:

Sensitive and selective strategy for probing D-amino acids in biological microenvironments

Contrary to prevailing dogma regarding dependence of biological systems on L-amino acids, trace levels of the D-enantiomers of some amino acids have been detected in neurological tissue. The objective of my research was to develop a sensitive and selective method that would allow analysis of D-amino acid neuromodulators at cellular and sub-cellular levels, enabling us to explore the potential role of these molecules in neuronal activity.

An enzymatic approach was established in which D-amino acid oxidase catalyzes the production of H₂O₂ from D-alanine; peroxidase then mediates the oxidation of tyramine, in the presence of H₂O₂, to produce a fluorescent dimer. Thus, D-alanine levels correspond stoichiometrically to the amount of dimerized tyramine produced. Detection was achieved using capillary-zone electrophoresis coupled to multi-photon excited fluorescence (CZE-MPE). CZE-MPE is an analytical system well-suited to this application because CZE accommodates volume-limited samples in the nanoliter to picoliter range and permits separation of these samples into their component parts; MPE fluorescence provides sensitive and simultaneous detection of multiple chromophores in a biological matrix using a single laser source.

Analysis of reactions performed off-column gave concentration and mass detection limits of 1.4 +/- 0.6 microM and 90 +/- 40 amole, respectively. However, on-column methods in which a portion of the capillary is used as a microreactor are more suited to analysis of biological microenvironments because dilution of the analyte is limited and only minimal reagent is needed. On-column approaches yielded detection limits of 13 +/- 5 microM and 400 +/- 100 amole. No significant response is observed when assay reagents are incubated with a mixture of other neuronally relevant amino acids, including D-aspartate, glycine, D-glutamate, D-serine, and L-alanine; therefore, this assay is selective for D-alanine. Although D-amino acid oxidase is reported to have activity with several other D-amino acids, evidence suggests that these alternative substrates are not biologically prevalent.

Although close to publication, several experimental parameters must be adjusted before drafting a manuscript for a journal such as Analytical Chemistry. Firstly, our sensitivity can be improved significantly by using optical filters that maximize collection of emission and minimize collection of background (e.g., laser scatter). Furthermore, reducing the capillary diameter from 6 micrometers, presently used, to 1 micrometer or smaller would lead to substantial gains in mass detection limits. In addition, a multi-component analysis will be performed to demonstrate the ability of the technique to simultaneously analyze various neuroactive compounds.

In estimating the sensitivity that will be required to analyze possible D-alanine content at a subcellular level, we can begin by considering the total alanine content of a cell: neurons from the land snail Helix aspersa, for example, have been reported to contain alanine at picomole quantities and millimolar concentrations.¹ It is not currently known what fraction of this total alanine may exist as the D-enantiomer; the established approach may be adequate for analysis at this cellular scale. However, by making the suggested system modifications, we expect improvements that would make our detection strategy conducive to subcellular determination of D-amino acids for analysis of secretory and storage vesicles within a single-cell.

1. Oates, Mary D., Cooper, Bruce R., Jorgenson, James W., Analytical Chemistry, 1990, 62, 1573–1577.