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In insects, the cuticle provides protection against physical injury and water loss, rigidness for muscle attachment and mechanical support, and flexibility in inter-segmental and joint areas for mobility. As most insects undergo metamorphosis, they need to shred off old cuticle and synthesize new cuticle to fit the body shape and size throughout their life cycles. The newly formed cuticle, mainly composed of cuticular proteins, chitin, and sclerotizing reagents, needs to be hardened through the crosslinks between cuticular proteins and sclerotizing reagents. This dissertation concerns the biochemical activities of several pyridoxal 5-phosphate (PLP)-dependent decarboxylases with most of them involved in insect cuticle hardening. Herein, we first present a detailed overview of topics in reactions and enzymes involved in insect cuticle hardening. Aspartate 1-decarboxylase (ADC) is at the center of this dissertation. beta-alanine, the product of ADC-catalyzed reaction from aspartate, is the component of an important sclerotizing reagent, N-beta-alanyldopamine; the levels of beta-alanine in insects regulate the concentrations of dopamine, therefore affecting insect sclerotization and tanning (collectively referred as cuticle hardening in this dissertation). Biochemical characterization of insect ADC has revealed that this enzyme has typical mammalian cysteine sulfinic acid decarboxylase (CSADC) activity, able to generate hypotaurine and taurine. The result throws lights on research in the physiological roles of insect ADC and the pathway of insect taurine biosynthesis. Cysteine was found to be an inactivator of several PLP-dependent decarboxylases, such as ADC, glutamate decarboxylase (GAD) and CSADC. This study helps to understand symptoms associated with the abnormal cysteine concentrations in several neurodegenerative diseases. A mammalian enzyme, glutamate decarboxylase like-1 (GADL1), has been shown to have the same substrate usage as insect ADC does, potentially

Cystamine has shown significant neuroprotective properties in preclinical studies of Parkinson’s disease (PD) and Huntington’s disease (HD). Cysteamine, its FDA-approved reduced form, is scheduled to be tested for clinical efficacy in HD patients. Here, we studied the key cystamine metabolites, namely cysteamine, hypotaurine and taurine, as well as cysteine, in order to identify which one is more distinctively responsible for the neuroprotective action of cystamine. After a single administration of cystamine (10, 50 or 200 mg/kg), naïve mice were perfused with phosphate-buffered saline (PBS) at 1, 3, 12, 24 or 48 h post-injection and brain and plasma samples were analyzed by two distinct HPLC methods. Although plasma levels remained under the detection threshold, significant increases in cysteamine brain levels were detected with the 50 and 200 mg/kg doses in mice perfused 1 and 3 h following cystamine injection. To further assess cysteamine as the candidate molecule for pre-clinical and clinical trials in PD, we evaluated its capacity to cross the blood brain barrier. Using an in situ cerebral perfusion technique, we determined that the brain transport coefficient (Clup) of cysteamine (259 μM) was 0.15 ± 0.02 μL/g/s and was increased up to 0.34 ± 0.07 μL/g/s when co-perfused in the presence of cysteine. Taken together, these results strongly suggest that cysteamine is the neuroactive metabolite of cystamine and may further support its therapeutic use in neurodegenerative diseases, particularly in HD and PD.

Vesicomyid clams, vestimentiferans, and some bathymodiolin mussels from hydrothermal vents and cold seeps possess thiotrophic endosymbionts, high levels of hypotaurine and, in tissues with symbionts, thiotaurine. The latter, a product of hypotaurine and sulfide, may store and/or transport sulfide non-toxically, and the ratio to hypotaurine plus thiotauririe (Th/[H + Th]) may reflect an animal's sulfide exposure. To test this, we analyzed seep and vent animals with in situ sulfide measurements. Calyptogena kilmeri clams occur at high-sulfide seeps in Monterey Canyon, while C. (Vesicomya) pacifica clams occur at seeps with lower levels but take up and metabolize sulfide more effectively. From one seep where they co-occur, both had gill thiotaurine contents at 22-25 mmol kg(-1) wet mass, and while C. (V.) pacifica had a higher blood sulfide level, it had a lower Th/[H + Th] (0.39) than C. kilmeri (0.63). However, these same species from different seeps with lower sulfide exposures had lower ratios. Bathymodiolus thermophilus [East Pacific Rise (EPR 9 degrees 50' N)] from high-(84 mu M) and a low-(7 mu M) sulfide vents had gill ratios of 0.40 and 0.12, respectively. Trophosomes of Riftia pachyptila (EPR 9 degrees 50' N) from medium-(33 mu M) and low-(4 mu M) sulfide vents had ratios of 0.23 and 0.20, respectively (not significantly different). Ridgeia piscesae vestimentiferans (Juan de Fuca Ridge) have very different phenotypes at high- and low-sulfide sites, and their trophosomes had the greatest differences: 0.81 and 0.04 ratios from high- and low-sulfide sites, respectively. Thus Th/ [H + Th] may indicate sulfide exposure levels within species, but not in interspecies comparisons, possibly due to phylogenetic and metabolic differences. Total H + Th was constant within each species (except in R. piscesae); the sum may indicate the maximum potential sulfide load that a species faces.

Vesicomyid clams, vestimentiferans, and some bathymodiolin mussels from hydrothermal vents and cold seeps possess thiotrophic endosymbionts, high levels of hypotaurine and, in tissues with symbionts, thiotaurine. The latter, a product of hypotaurine and sulfide, may store and/or transport sulfide non-toxically, and the ratio to hypotaurine plus thiotauririe (Th/[H + Th]) may reflect an animal's sulfide exposure. To test this, we analyzed seep and vent animals with in situ sulfide measurements. Calyptogena kilmeri clams occur at high-sulfide seeps in Monterey Canyon, while C. (Vesicomya) pacifica clams occur at seeps with lower levels but take up and metabolize sulfide more effectively. From one seep where they co-occur, both had gill thiotaurine contents at 22-25 mmol kg(-1) wet mass, and while C. (V.) pacifica had a higher blood sulfide level, it had a lower Th/[H + Th] (0.39) than C. kilmeri (0.63). However, these same species from different seeps with lower sulfide exposures had lower ratios. Bathymodiolus thermophilus [East Pacific Rise (EPR 9 degrees 50' N)] from high-(84 mu M) and a low-(7 mu M) sulfide vents had gill ratios of 0.40 and 0.12, respectively. Trophosomes of Riftia pachyptila (EPR 9 degrees 50' N) from medium-(33 mu M) and low-(4 mu M) sulfide vents had ratios of 0.23 and 0.20, respectively (not significantly different). Ridgeia piscesae vestimentiferans (Juan de Fuca Ridge) have very different phenotypes at high- and low-sulfide sites, and their trophosomes had the greatest differences: 0.81 and 0.04 ratios from high- and low-sulfide sites, respectively. Thus Th/ [H + Th] may indicate sulfide exposure levels within species, but not in interspecies comparisons, possibly due to phylogenetic and metabolic differences. Total H + Th was constant within each species (except in R. piscesae); the sum may indicate the maximum potential sulfide load that a species faces.

Vesicomyid clams, vestimentiferans, and some bathymodiolin mussels from hydrothermal vents and cold seeps possess thiotrophic endosymbionts, high levels of hypotaurine and, in tissues with symbionts, thiotaurine. The latter, a product of hypotaurine and sulfide, may store and/or transport sulfide non-toxically, and the ratio to hypotaurine plus thiotauririe (Th/[H + Th]) may reflect an animal's sulfide exposure. To test this, we analyzed seep and vent animals with in situ sulfide measurements. Calyptogena kilmeri clams occur at high-sulfide seeps in Monterey Canyon, while C. (Vesicomya) pacifica clams occur at seeps with lower levels but take up and metabolize sulfide more effectively. From one seep where they co-occur, both had gill thiotaurine contents at 22-25 mmol kg(-1) wet mass, and while C. (V.) pacifica had a higher blood sulfide level, it had a lower Th/[H + Th] (0.39) than C. kilmeri (0.63). However, these same species from different seeps with lower sulfide exposures had lower ratios. Bathymodiolus thermophilus [East Pacific Rise (EPR 9 degrees 50' N)] from high-(84 mu M) and a low-(7 mu M) sulfide vents had gill ratios of 0.40 and 0.12, respectively. Trophosomes of Riftia pachyptila (EPR 9 degrees 50' N) from medium-(33 mu M) and low-(4 mu M) sulfide vents had ratios of 0.23 and 0.20, respectively (not significantly different). Ridgeia piscesae vestimentiferans (Juan de Fuca Ridge) have very different phenotypes at high- and low-sulfide sites, and their trophosomes had the greatest differences: 0.81 and 0.04 ratios from high- and low-sulfide sites, respectively. Thus Th/ [H + Th] may indicate sulfide exposure levels within species, but not in interspecies comparisons, possibly due to phylogenetic and metabolic differences. Total H + Th was constant within each species (except in R. piscesae); the sum may indicate the maximum potential sulfide load that a species faces.