Wound-Healing Effect of Antheraea pernyi Epidermal Growth Factor.

To evaluate the wound-healing effect of Antheraea pernyi epidermal growth factor (ApEGF), we performed the sequence analysis, cloning, and prokaryotic expression of cDNA from the ApEGF gene, examined the transcriptional changes, and investigated the wound-healing effect of this protein in cells and rat epidermis. Primers were designed based on available sequence information related to the ApEGF gene in a public database, and part of the ApEGF sequence was obtained. The full-length cDNA sequence of ApEGF was obtained using inverse PCR. The gene sequence fragment of ApEGF was 666 bp in length, encoding 221 amino acids, with a predicted protein mass of 24.19 kD, an isoelectric point of 5.15, and no signal peptide sequence. Sequence homology analysis revealed 86.1% sequence homology with Bombyx mori, 92.7% with Manducal sexta, 92.6% with Trichoplusia ni, and 91.8% with Helicoverpa armigera. ApEGF was truncated and then subjected to prokaryotic expression, isolation, and purification. Truncated ApEGF was used for wound-healing experiments in vitro and in vivo. The results showed that after 48 h, transforming growth factor (TGF)-ß1 had 187.32% cell growth effects, and the ApEGF group had 211.15% cell growth compared to the control group in vitro. In rat epidermis, truncated ApEGF showed a significantly better healing effect than the control. This result indicated that ApEGF, which exerted a direct wound-healing effect, could be used in wound-healing therapy.

Modeling and Re-Engineering of Azotobacter vinelandii Alginate Lyase to Enhance Its Catalytic Efficiency for Accelerating Biofilm Degradation.

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 ß-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.

Cell-based biological evaluations of 5-(3-bromo-4-phenethoxybenzylidene)thiazolidine-2,4-dione as promising wound healing agent.

Recent studies have focused on prostaglandin E2 (PGE2) because PGE2 regulates vertebrate hematopoietic stem cell induction and engraftment. PGE2 acts through EP2 and EP4 receptors to mediate regeneration and hematopoietic stem cell (HSC) development via the Wnt signaling pathway. Previously we reported that inhibitors of 15-PGDH can control the intracellular levels of PGE2. Therefore, we developed new potent 15-PGDH inhibitor, 5-(3-bromo-4-phenethoxybenzylidene)thiazolidine-2,4-dione (TD191), with an IC50 of 4 nM and tested cell-based wound healing effects. This compound significantly increased the level of PGE2 (451 pg/mL) in A549 cells, which was about 7-fold higher than that of control. HaCaT cells exposed to TD191 showed significantly improved wound healing after 48 h in scratch wound healing test, whereas treatment of TD191 to the fibroblast Hs27 cells slightly decreased cell growth compared with control. SCL is a basic helix-loop-helix transcription factor that is an essential for HSC development. By qPCR, SCL expression in HaCaT cells was 2-fold enhanced after addition of TD191, while treatment of TD191 into fibroblast Hs27 cells was not significantly changed the expression levels of the gene. This data provides in vitro evidence that TD191 may have utility for the therapeutic management of wound healing without scar formation.

Wound healing effects of new 15-hydroxyprostaglandin dehydrogenase inhibitors.

Previously, we reported that the antidiabetic drug ciglitazone and its analogs were potent inhibitors of 15-hydroxyprostaglandin dehydrogenase (15-PGDH). In continuing attempts to develop highly potent 15-PGDH inhibitors, a series of thiazolidinedione analogs were synthesized and tested. Compound 17 exhibited IC50 of 45 nM. This compound also significantly increased levels of prostaglandin E2 (PGE2) in A549 cells by approximately eight-fold that in the control. Much experimental data suggests that PGE2 plays a role in the prevention of excessive scarring. However, it has a very short half-life in blood, its oxidization to 15-ketoprostaglandins is catalyzed by 15-PGDH. Therefore, 15-PGDH inhibitors may have utility for the therapeutic management of diseases requiring elevated PGE2 levels. Scratch wounds were analyzed in confluent monolayers of HaCaT cells. Cells exposed to compound 17 showed significantly improved wound healing with respect to a control.

Control of the intracellular levels of prostaglandin E2 through inhibition of the 15-hydroxyprostaglandin dehydrogenase for wound healing.

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.

Insight into the binding of the wild type and mutated alginate lyase (AlyVI) with its substrate: a computational and experimental study.

The homology model of the wild type alginate lyase (AlyVI) marine bacterium Vibrio sp. protein, was built using the crystal structure of the Family 7 alginate lyase from Sphingomonas sp. A1. To rationalize the observed structure-affinity relationships of aliginate lyase alyVI with its (GGG) substrate, molecular docking, MD imulations and binding free energy calculations followed by site-directed mutagenesis and alyVI activity assays were carried out. Per-residue decomposition of the (GGG) binding energy revealed that the most important contributions were from polar and charged residues, such as Asn138, Arg143, Asn217, and Lys308, while van der Waals interactions were responsible for binding with the catalytic His200 and Tyr312 residues. The mutants H200A, K308A, Y312A, Y312F, and W165A were found to be inactive or almost inactive. However, the catalytic efficiency (k(cat)/K(m)) of the double mutant L224V/D226G increased by two-fold compared to the wild type enzyme. This first structural model with its substrate binding mode and the agreement with experimental results provide a suitable base for the future rational design of new mutated alyVI structures with improved catalytic activity.

Production of oligosaccharide from alginate using Pseudoalteromonas agarovorans.

A marine bacterium was isolated from seawater near the Korean south coast for efficient saccharification from alginate. Based on 16S rDNA sequence, the isolated strain was identified as Pseudoalteromonas agarovorans. Various environmental factors affecting saccharification of alginate using P. agarovorans CHO-12 have been investigated in flask cultures. The optimum concentration of sugar was obtained at 30 rpm and 29 degrees C. Among various NaCl concentrations, when NaCl concentration was increased from 10 to 30 g/l, the cell concentration sharply increased, while there is no increase at above 40 g/l. The maximum sugar concentration was obtained at 13.8 when 30 g/l of NaCl was used. Yeast extract and corn steep liquor were the best nitrogen source for efficient saccharification. Especially, the sugar concentration of 14.9 g/l was obtained after 3 days of culture using a mixture of 1.0 g/l of yeast extract and 1.5 g/l of corn steep liquor. Scale up was carried out at 50 l of reactor for 3 days using P. agarovorans CHO-12 and Stenotrophomonas maltophilia sp. When S. maltophilia was used, cell concentration was about twofold higher than that of P. agarovorans CHO-12. On the other hand, when P. agarovorans CHO-12 was used, the maximum saccharification rate was obtained, 7.5 g/l/day after 2 days of culture, which was about tenfold higher than that of S. maltophilia.

Gene expression changes at metamorphosis induced by thyroid hormone in Xenopus laevis tadpoles.

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.