Venkataraman Bringi

Metabolic inhibitors, elicitors, and precursors as tools for probing yield limitation in taxane production by Taxus chinensis cell cultures

Inhibition of biosynthetic enzymes and translation and translocation processes, elicitation, and precursor feeding were used to probe biosynthetic pathway compartmentation, substrate−product relationships, and yield limitation of the diterpenoid taxanes in cell cultures of Taxus chinensis (PRO1–95). The results suggest the following: (i) the source of isopentenyl pyrophosphate in taxane production is likely plastidic rather than cytoplasmic; (ii) baccatin III may not be a direct precusor of Taxol (Taxol is a registered trademark of Bristol‐Myers Squibb for paclitaxel); (iii) baccatin III appears to have cytoplasmic and plastidic biosynthetic components, while Taxol production is essentially plastidic; and (iv) arachidonic acid specifically stimulates Taxol production but does not have a significant effect on baccatin III yield. Semiempirical mathematical models were used to describe these results and predict potential yield‐limiting steps. Model simulations suggest that, under current operating conditions, Taxol production in Taxus chinensis (PRO1–95) cultures is limited by the ability of the cells to convert phenylalanine to phenylisoserine rather than by the branch‐point acyl transferase. This result is supported by the lack of improvement of Taxol yield by feeding phenylalanine or benzoylglycine. The methods described in this article, while specifically expanding our knowledge of taxane production in PRO1–95 cultures, could be generally useful in investigating complex aspects of secondary metabolic pathways in plant cell cultures, especially when details of the pathway and compartmentation are sparse.

Enhanced yeast immobilization by nutrient starvation

Saccharomycesuvarum NRRL Y1347 cells were immobilized in a porous support. Cell loadings of up to 600 mg dry cell/g support or 70 mg dry cell/cm3 support were obtained. Starvation in a marine environment increased the adhesion strength of immobilized cells.

Effect of ammonia fiber expansion on the available energy content of wheat straw fed to lactating cattle and buffalo in India

The seasonal lack of availability of lush green forages can force dairy farmers in developing nations to rely on crop residues such as wheat and rice straw as the major feed source. We tested whether ammonia fiber expansion (AFEX) treatment of wheat straw would increase the energy available to Murrah buffalo and Karan-Fries cattle consuming 70% of their diet as wheat straw in India. Forty lactating animals of each species were blocked by parity and days in milk and randomly assigned to 1 of 4 treatment diets (n = 10). Treatments were a nutrient-rich diet with 0 to 20% straw (positive control; PC) and 3 high-straw diets with various levels of AFEX-treatment: (1) 70% untreated straw (no AFEX), (2) 40 to 45% untreated straw with 25 to 30% AFEX-treated straw (low AFEX), and (3) 20% untreated straw with 50% AFEX-treated straw (high AFEX). The AFEX-treated straw was pelleted. Urea was added to the no and low AFEX diets so they were isonitrogenous with the high AFEX diet. Animals were individually fed the PC diet for 14 d followed by 7 d of adaptation to treatments, full treatments for 28 to 35 d, and finally PC diets for 21 d. Compared with buffalo fed the PC diet, those fed high-straw diets consumed 29% less feed dry matter, put out 16% less milk energy, and lost 0.8 kg/d more body weight; the AFEX treatment of straw did not alter intake or milk production but greatly ameliorated the body weight loss (-1.0 kg/d for no AFEX and -0.07 kg/d for high AFEX). In Karan-Fries cattle, high-straw diets decreased dry matter intake by 39% and milk energy by 24%, and the high AFEX diet increased intake by 42% and milk energy by 18%. The AFEX treatment increased digestibilities of organic matter, dry matter, neutral detergent fiber, acid detergent fiber, and crude protein by 6 to 13 percentage points in buffalo and 5 to 10 points in cattle. In conclusion, AFEX treatment increased the digestibility and energy availability of wheat straw for lactating buffalo and cattle and has commercial potential to improve milk production and feed efficiency when high-quality forages or grains are not available.

Exposure Assessment of Acetamide in Milk, Beef, and Coffee Using Xanthydrol Derivatization and Gas Chromatography/Mass Spectrometry

Acetamide has been classified as a possible human carcinogen, but uncertainties exist about its levels in foods. This report presents evidence that thermal decomposition of N-acetylated sugars and amino acids in heated gas chromatograph injectors contributes to artifactual acetamide in milk and beef. An alternative gas chromatography/mass spectrometry protocol based on derivatization of acetamide with 9-xanthydrol was optimized and shown to be free of artifactual acetamide formation. The protocol was validated using a surrogate analyte approach based on d3-acetamide and applied to analyze 23 pasteurized whole milk, 44 raw sirloin beef, and raw milk samples from 14 different cows, and yielded levels about 10-fold lower than those obtained by direct injection without derivatization. The xanthydrol derivatization procedure detected acetamide in every food sample tested at 390 ± 60 ppb in milk, 400 ± 80 ppb in beef, and 39 000 ± 9000 ppb in roasted coffee beans.