Thyroid hormone (TH) controlled gene expression profiles have been studied in the tail, hind limb and brain tissues during TH-induced and spontaneous Xenopus laevis metamorphosis. Amplified cRNA probes mixed with a universal standard were hybridized to a set of 21,807-sense strand 60-mer oligonucleotides on each slide representing the entries in X. laevis UniGene Build 48. Most of the up-regulated genes in hind limb and brain are the same. This reflects in part the fact that the initial response to TH induction in both tissues is cell proliferation. A large number of up-regulated genes in the limb and brain programs encode common components of the cell cycle, DNA and RNA metabolism, transcription and translation. Notch is one of the few genes that is differentially expressed exclusively in the brain in the first 48 h of TH induction studied in these experiments. The TH-induced gene expression changes in the tail are different from the limb and brain programs. Distinct muscle and fibroblast programs were identified in the tail. Dying muscle fibers in tail (marked by active caspase-3) up-regulate a group of genes that include proteolytic enzymes. At the climax of metamorphosis, tail muscle down-regulates more than half of the genes that encode the glycolytic enzymes in the cytoplasm and the tricarboxylic acid pathway and all five complexes of the electron transport system in mitochondria. These changes in gene expression precede the activation of caspase-3. Some of these same energy metabolism-related genes are up-regulated in the limb and brain programs by TH. A prominent feature of the tail fibroblasts is the down-regulation of several collagen and other extra cellular matrix genes and the up-regulation of hydrolytic enzymes that are responsible for dissolving the notochord and resorbing the tail.

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Excessive scar formation is an aberrant form of wound healing and is an indication of an exaggerated function of fibroblasts and excess accumulation of extracellular matrix during wound healing. Much experimental data suggests that prostaglandin E₂ (PGE₂) plays a role in the prevention of excessive scarring. However, it has a very short half-live in blood, its oxidization to 15-ketoprostaglandins is catalyzed by 15-hydroxyprostaglandin dehydrogenase (15-PGDH). Previously, we reported that 15-PGDH inhibitors significantly increased PGE₂ levels in A549 cells. In our continuing attempts to develop highly potent 15-PGDH inhibitors, we newly synthesized various thiazolidine-2,4-dione derivatives. Compound 27, 28, 29, and 30 demonstrated IC₅₀ values of 0.048, 0.020, 0.038 and 0.048 μM, respectively. They also increased levels of PGE₂ in A549 cells. Especially, compound 28 significantly increased level of PGE₂ at 260 pg/mL, which was approximately fivefold higher than that of control. Scratch wounds were analyzed in confluent monolayers of HaCaT cells. Cells exposed to compound 28 showed significantly improved wound healing with respect to control.

Alginate is known to prevent elimination of Pseudomonas aeruginosa biofilms. Alginate lyase (AlgL) might therefore facilitate treatment of Pseudomonas aeruginosa-infected cystic fibrosis patients. However, the catalytic activity of wild-type AlgL is not sufficiently high. Therefore, molecular modeling and site-directed mutagenesis of AlgL might assist in enzyme engineering for therapeutic development. AlgL, isolated from Azotobacter vinelandii, catalyzes depolymerization of alginate via a β-elimination reaction. AlgL was modeled based on the crystal structure template of Sphingomonas AlgL species A1-III. Based on this computational analysis, AlgL was subjected to site-directed mutagenesis to improve its catalytic activity. The kcat/Km of the K194E mutant showed a nearly 5-fold increase against the acetylated alginate substrate, as compared to the wild-type. Double and triple mutants (K194E/K245D, K245D/K319A, K194E/K245D/E312D, and K194E/K245D/K319A) were also prepared. The most potent mutant was observed to be K194E/K245D/K319A, which has a 10-fold improved kcat value (against acetylated alginate) compared to the wild-type enzyme. The antibiofilm effect of both AlgL forms was identified in combination with piperacillin/tazobactam (PT) and the disruption effect was significantly higher in mutant AlgL combined with PT than wild-type AlgL. However, for both the wild-type and K194E/K245D/K319A mutant, the use of the AlgL enzyme alone did not show significant antibiofilm effect.

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