A separation-based biosensor has been developed that is capable of near-real-time analysis of aspartate and glutamate with a temporal resolution of less than 2 min in anesthetized or awake, freely moving animals. The instrument consists of a microdialysis sampling system, an on-line reactor, an injection interface, and a CE-LIF system. Primary amine analytes are derivatized with NDA/CN following microdialysis sampling using an on-line reactor to produce fluorescent CBI derivatives. The reaction takes approximately 1 min. The derivatized sample then travels to a microinjection valve which alternately sends CE running buffer and reacted microdialysis sample to the CE column via an injection interface. The interface allows a controllable volume of 10-20 nL to be injected onto the CE separation capillary. Separation of aspartate and glutamate from the other amino acids present in the microdialysis sample was achieved within 70 s. Detection limits for glutamate and aspartate using laser-induced fluorescence detection were 0.1 microM. The linear dynamic range was acceptable for the determination of aspartate and glutamate in dialysate samples where the levels are between 1 and 10 microM. Full automation of the system was achieved by computer control of the valve, the interface, and the data collection system. The performance of this system was demonstrated in an anesthetized rat by monitoring ECF levels of aspartate and glutamate released in brain after stimulation with high concentrations of K+.

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. Matriptase activation and shedding with HAI-1 is induced by steroid sex hormones in human prostate cancer cells, but not in breast cancer cells. Am J Physiol Cell Physiol 291: C40 -C49, 2006. First published February 8, 2006 doi:10.1152/ajpcell.00351.2005.-Matriptase and its cognate inhibitor, hepatocyte growth factor activator inhibitor-1 (HAI-1), have been implicated in carcinoma onset and malignant progression. However, the pathological mechanisms of matriptase activation are not defined. Steroid sex hormones play crucial roles in prostate and breast cancer. Therefore, we investigated the questions of whether and how steroid sex hormones regulate matriptase activation in these cancer cells. Treatment of cells with 17␤-estradiol had no effect on activation of matriptase in hormonestarved breast cancer cells, in part due to their high constitutive level of activated matriptase. In striking contrast, very low levels of activated matriptase were detected in hormone-starved lymph node prostatic adenocarcinoma (LNCaP) cells. Robust activation of matriptase was observed as early as 6 h after exposure of these cells to 5␣-dihydrotestosterone (DHT). Activation of matriptase was closely followed by shedding of the activated matriptase with Ͼ90% of total activated matriptase present in the culture media 24 h after DHT treatment. Activated matriptase was shed in a complex with HAI-1 and may result from simultaneously proteolytic cleavages of both membrane-bound proteins. Latent matriptase and free HAI-1 were also shed into culture media. As a result of shedding, the cellular levels of matriptase and HAI-1 were significantly reduced 24 h after exposure to DHT. DHT-induced matriptase activation and shedding were significantly inhibited by the androgen antagonist bicalutamide, by the RNA transcription inhibitor actinomycin D, and by the protein synthesis inhibitor cycloheximide. These results suggest that in LNCaP cells, androgen induces matriptase activation via the androgen receptor, and requires transcription and protein synthesis.androgen; hepatocyte growth factor activator inhibitor-1 MATRIPTASE and its inhibitor, hepatocyte growth factor activator inhibitor-1 (HAI-1), represent a cognate pair, a cell surface proteolytic enzyme and its inhibitor (29). Both are broadly coexpressed by epithelial elements in most epithelium-containing tissues (18, 45). Matriptase, a type 2 transmembrane serine protease (40, 54), contains a COOH-terminal serine protease domain and several noncatalytic domains, including a sperm protein-enterokinase-agrin module, two tandem C1r/s-UegfBone morphogenic protein-1 domains, and four tandem lowdensity lipoprotein (LDL) receptor class A domains (19,30,57,59). Matriptase was shown to play an important role in epidermal barrier function, terminal epidermal differentiation, hair follicle development and thymic homeostasis in matriptase knockout mice (32). Some of these functions may result from its role in the maturation of pro-filaggrin (34), a marker for the terminal differentiation of keratinoc...

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