Cyclosporine A inhibits TGF-ß2-induced myofibroblasts of primary cultured human pterygium fibroblasts.

Cyclosporine A (CsA), an immunomodulatory drug, and is increasingly used to treat moderate dry eye syndrome and ocular surface inflammation. However, any inhibitory effect on differentiation of fibroblasts to myofibroblasts remains unclear. Here, we show that the inhibitory effect of CsA on transforming growth factor-beta2 (TGF-ß2)-induced myofibroblasts in primary cultured human pterygium fibroblasts. CsA significantly decreased mRNA and protein expression of myofibroblast-related markers including α-SMA, laminin, and fibronectin. These findings were supported by the results from immunofluorescence staining. Taken together, these results indicate the therapeutic potential of CsA against pterygium progression. Further studies are necessary to elucidate the precise intracellular signal mechanism responsible for CsA-induced downregulation of myofibroblast markers in pterygium fibroblasts.

Engineered Escherichia coli with periplasmic carbonic anhydrase as a biocatalyst for CO2 sequestration.

Carbonic anhydrase is an enzyme that reversibly catalyzes the hydration of carbon dioxide (CO2). It has been suggested recently that this remarkably fast enzyme can be used for sequestration of CO2, a major greenhouse gas, making this a promising alternative for chemical CO2 mitigation. To promote the economical use of enzymes, we engineered the carbonic anhydrase from Neisseria gonorrhoeae (ngCA) in the periplasm of Escherichia coli, thereby creating a bacterial whole-cell catalyst. We then investigated the application of this system to CO2 sequestration by mineral carbonation, a process with the potential to store large quantities of CO2. ngCA was highly expressed in the periplasm of E. coli in a soluble form, and the recombinant bacterial cell displayed the distinct ability to hydrate CO2 compared with its cytoplasmic ngCA counterpart and previously reported whole-cell CA systems. The expression of ngCA in the periplasm of E. coli greatly accelerated the rate of calcium carbonate (CaCO3) formation and exerted a striking impact on the maximal amount of CaCO3 produced under conditions of relatively low pH. It was also shown that the thermal stability of the periplasmic enzyme was significantly improved. These results demonstrate that the engineered bacterial cell with periplasmic ngCA can successfully serve as an efficient biocatalyst for CO2 sequestration.

Biomineralization-based conversion of carbon dioxide to calcium carbonate using recombinant carbonic anhydrase.

Recently, as a mimic of the natural biomineralization process, the use of carbonic anhydrase (CA), which is an enzyme catalyzing fast reversible hydration of carbon dioxide to bicarbonate, has been suggested for biological conversion of CO(2) to valuable chemicals. While purified bovine CA (BCA) has been used in previous studies, its practical utilization in CO(2) conversion has been limited due to the expense of BCA preparation. In the present work, we investigated conversion of CO(2) into calcium carbonate as a target carbonate mineral by using a more economical, recombinant CA. To our knowledge, this is the first report of the usage of recombinant CA for biological CO(2) conversion. Recombinant α-type CA originating in Neisseria gonorrhoeae (NCA) was highly expressed as a soluble form in Escherichia coli. We found that purified recombinant NCA which showed comparable CO(2) hydration activity to commercial BCA significantly promoted formation of solid CaCO(3) through the acceleration of CO(2) hydration rate, which is naturally slow. In addition, the rate of calcite crystal formation was also accelerated using recombinant NCA. Moreover, non-purified crude recombinant NCA also showed relatively significant ability. Therefore, recombinant CA could be an effective, economical biocatalyst in practical CO(2) conversion system.

Coexpression of molecular chaperone enhances activity and export of organophosphorus hydrolase in Escherichia coli.

Periplasmic secretion has been used in attempts to construct an efficient whole-cell biocatalyst with greatly reduced diffusion limitations. Previously, we developed recombinant Escherichia coli that express organophosphorus hydrolase (OPH) in the periplasmic space using the twin-arginine translocation (Tat) pathway to degrade environmental toxic organophosphate compounds. This system has the advantage of secreting protein into the periplasm after folding in the cytoplasm. However, when OPH was expressed with a Tat signal sequence in E. coli, we found that the predominant OPH was an insoluble premature form in the cytoplasm, and thus, the whole-cell OPH activity was significantly lower than its cell lysate activity. In this work, we, for the first time, used a molecular chaperone coexpression strategy to enhance whole-cell OPH activity by improving the periplasmic translocation of soluble OPH. We found that the effect of GroEL-GroES (GroEL/ES) assistance on the periplasmic localization of OPH was secretory pathway dependent. We observed a significant increase in the amount of soluble mature OPH when cytoplasmic GroEL/ES was expressed; this increase in the amount of mature OPH might be due to enhanced OPH folding in the cytoplasm. Importantly, the whole-cell OPH activity of the chaperone-coexpressing cells was ∼5.5-fold greater at 12 h after induction than that of cells that did not express the chaperone as a result of significant Tat-based periplasmic translocation of OPH in the chaperone-coexpressing cells. Collectively, these data suggest that molecular chaperones significantly enhance the whole-cell activity of periplasmic OPH-secreting cells, yielding an effective whole-cell biocatalyst system with highly reduced diffusion limitations.