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Cyathin A(3), produced by the fungus Cyathus helenae, is a member of the cyathane family of diterpene natural products. While many of the cyathanes display antibacterial/antimicrobial activity or have cytotoxic activity against human cancer cell lines, their most exciting therapeutic potential is derived from their ability to induce nerve growth factor (NGF) release from glial cells, making the cyathanes attractive lead molecules for the development of neuroprotective therapeutics to prevent/treat Alzheimer's disease. To investigate if cyathin A(3) has NGF-inducing activity, we set out to obtain it using published C. helenae bench-scale fungal fermentations. However, to overcome nonproducing fermentations, we developed an alternative, bacteria-induced static batch fermentation approach to the production of cyathin A(3), as described in this report. HPLC, UV absorption spectra, and mass spectrometry identify cyathin A(3) in fungal fermentations induced by the timely addition of Escherichia coli K12 or Bacillus megabacterium. Pre-filtration of the bacterial culture abolishes cyathin A(3) induction, suggesting that bacteria-associated media changes or physical interaction between the fungus and bacteria underlie the induction mechanism. Through alteration of incubation conditions, including agitation, the timing of induction, and media composition, we optimized the fermentation to yield nearly 1 mg cyathin A(3)/ml media, a sixfold increase over previously described yields. Additionally, by comparison of fermentation profiles, we reveal that cyathin A(3) biosynthesis is regulated by carbon catabolite repression. We have used an enzyme-linked immunosorbent assay to illustrate that cyathin A(3) induces NGF release from cultured glial cells, and therefore cyathin A(3) warrants further examination in the development of neuroprotective therapeutics.

Raising organic loading rate, and the behavior of dissolved CODcr (D-CODcr), VFA and specific methanogen activity, were investigated through a laboratory experiment for the start-up of a sludge recycling center. Moreover, application for MPN-PCR methods using a gene as a direct technique to measure the quantity of methanogen was attempted. It was recognized that specific methanogen activity depends on the quantity of methanogen, and that gas production does not reflect the condition of methane fermentation. The methane fermentation condition was checked through the specific methanogen activity and analysis of D-CODcr. The target organic loading rate was reached in the short period of about 30 days, and rapid startup was successfully attained for a full-scale anaerobic digester. [ABSTRACT FROM AUTHOR]