With NASA support this research aimed at evaluating early opportunities in Microgravity Sciences to commercialize space and to develop the biotechnology facility for the International Space Station . The main task was to evaluate the production of taxol (generic name: paclitaxel) with cell suspensions in bioreactors designed for the Space Shuttle. Unexpectedly, this work led to the early demonstration of L-arginine-dependent nitric oxide (NO) bursts in mechanically and gravitationally stressed plant cells, to NO-induced programmed cell death (apoptosis) (reviewed in ), and to a model describing how these factors contribute to increased taxol and taxane recovery from conifers.
Earlier, when intermediates of the Krebs-Henseleit or urea cycle (See Figure 1) were fed to conifers, several substituted guanidino compounds were derived from uniformly labeled 14C-L-arginine, and less so from 14C-L-citrulline [3, 4]. At that time, the substituted guanidines were considered mainly as respiratory inhibitors [5, 6]. Today, they are natural inhibitors of plant, animal and human nitric oxide synthases (NOSs). NOS substrates are L-arginine and oxygen. NOS products are L-citrulline and NO.
Since nitrate and nitrite reductases were also known sources of NO, we obtained an Arabidopsis nitrate reductase double mutant with the aim of finding out if cells could produce NO in the absence of nitrate, nitrite, and their reductases . With this mutant we reaffirmed that the source of NO was putative NOS activity. The production of NO from L-arginine was blocked by D-arginine, and by the NOS inhibitor, NG-monomethyl-L-arginine (L-NMMA) ensuring that NO was produced in the absence of any residual nitrate reductase activity. NOS-dependent NO production in cells was inhibited by other guanidino compounds but not by D-arginine. Subsequently, the discovery by others of two plant NOS genes provided evidence that plant genomes code for NOS. In our work with Taxus cell suspensions, the substituted guanidines offered protection against mechanically induced stress and cell-death or apoptosis.
Taxol is an effective anti-cancer agent that was first isolated from the bark of Taxus brevifolia . Taxol binds to microtubules thereby offering a novel mechanism of blocking cell proliferation. It became the best-selling anticancer drug in history. By 2000, commercial sales of taxol were well over $1.5 billion. New models for taxol and taxane biosynthesis emerged  so that taxol biosynthesis could be followed at the subcellular level by immunocytochemistry, and by laser confocal and scanning electron microscopy [10–13, 20]. The use of NO donors, NOS substrates, products, inhibitors (substituted guanidines), and NO traps provided new opportunities to control the citrulline-NO cycle (See Figure 1), apoptosis, and taxol production in unit gravity, simulated microgravity, and in hypergravity.
Haploid egg cells from female trees of T. brevifolia were selected as the experimental material [12, 19]. These cells are easily screened without the effects of dominance, recessive, and epistatic interactions characteristic of diploid cells. Lethal genes are directly expressed, and removed by apoptosis making cell populations genetically more uniform. Diploid cell suspensions were also established from T. cuspidata  needles on 3-year-old stock obtained in 1995 from Zelenka Nursery (Grand Haven MI), and from seeds of T. chinensis. This provided better comparisons with all other published work with using diploid cells.
Cell suspensions were maintained in darkness at 25 ± 2°C on semisolid media, in 125 ml Erlenmeyer flasks (60 rpm), 1 L nippled flasks on a clinostat (1 rpm simulating 2 × 10-4 × g, but with significant convective mixing of the gaseous and liquid environment), 100 ml high-aspect rotating vessels (HARV 12.5 cm dia.), and rotating cylindrical culture vessels (RCCV 7.5 cm dia.) both at ca.10-2 × g (Synthecon, Houston TX). The HARV and RCCV were used by NASA in early space shuttle experiments, and in a mini-payload integration center, designed as an in-flight laboratory.
In all cell assays, apoptotic cells were distinguished morphologically and by the TUNEL reaction [14, 15]. Free taxol, taxanes, and baccatin III in cells and the culture medium, or bound taxol and taxanes, released after xylanase activity, were determined with competitive inhibition enzyme-linked immunoassays kits from Hawaii Biotechnology. Cells were examined by laser confocal microscopy (Zeiss LSM 410 Invert Scan Microscope) using single or double-labeling immunocytochemical fluorescence (FITC, Cy3) and colloidal gold to reaffirm the subcellular locations of taxol, the taxane ring (baccatin III), the C-13 side chain of taxol, and taxanes in general. For samples larger than 10 g fresh biomass, these compounds were determined by HPLC using authentic standards and taxil columns (MetaChem Technologies Inc.) .