probe 150 nM, containing 6 FAM as ARQ197 clinical 5 reporter dye. Primers and probes for p38 and p38 were used as recommended by the manufacturer. PCR reaction param eters were as follows incubation at 50 C for 2 min, incu bation at 95 C for 10 min and thereafter 40 cycles of denaturation at 95 C for 15 sec and annealing and exten sion at 60 C for 1 min. Each sample was determined in duplicate. A standard curve method was used to determine the rela tive iNOS and GAPDH mRNA levels as described in Applied Biosystems User Bulletin 2. In short, a standard curve for each gene was created using mRNA isolated from LPS stimulated J774. 2 macrophages. Isolated RNA was reverse transcribed as described. Dilution series were made from obtained cDNA ranging from 10 ng to 1 pg and were subjected to real time PCR as described.

Obtained threshold cycle values were plotted against dilu tion factor to create a standard curve. Relative mRNA lev els in test samples were then calculated from the standard curve. Statistics Results are expressed as mean standard error of mean. When indicated, statistical significance was cal culated by analysis of variances supported by Bonferroni multiple comparisons test. Background C. roseus produces terpenoid indole alkaloids as a part of its secondary metabolism. TIAs provide protection against microbial infection, herbivores and abiotic envi ronmental stresses such as UV irradiation. Some of the TIAs are of pharmaceutical importance such as the antitumor dimeric alkaloids, vincristine and vinblastine, and the anti hypertensive monomeric alkaloids, ajmali cine and serpentine.

The anti tumor dimeric alkaloids, which accumulate in the leaves of C. roseus, are composed of catharanthine and vindoline monomers and are exclu sively found in C. roseus plants. In plants, the dimeric alka loids and the monomer catharanthine accumulate in low amounts whereas the monomer vindoline accumulates at a relatively higher level. C. roseus cell cultures have been investigated as alternative means of production of terpenoid indole alkaloids, but they failed to produce vin doline. Therefore, it has been considered desirable to produce the dimers by coupling catharanthine obtained from cell cultures with vindoline obtained from the culti vated plants. The production of catharanthine by C. roseus cell cultures has been one of the most extensively explored areas of plant cell culture and is still limited due to the low yield.

Elicitations are considered to be an important strategy towards improved in vitro production of secondary metab olites. In cell cultures, biotic and Entinostat abiotic elicitors have effectively stimulated the production of plant secondary metabolites. Fungal elicitors have been widely tested for elicitation of catharanthine production in various C. roseus cells.