The effect of ajmalicine spiking and resin addition timing on the production of indole alkaloids from Catharanthus roseus cell cultures

The potential for the feedback inhibition of indole alkaloid synthesis was investigated by spiking suspension cultures of Catharanthus roseus with 0, 9, or 18 mg/L ajmalicine on day 0. The production of ajmalicine, catharanthine, and serpentine were inhibited in a dose-dependent manner. The inhibition was transient as the exogenous ajmalicine was ultimately either metabolized in the medium or within the cell. The addition of neutral resin has previously been shown to enhance ajmalicine production. To minimize product inhibition and product metabolism, Amberlite XAD-7 resin was added to immobilized cultures of C. roseus starting on either day 0, 5, or 15, and fresh resin was exchanged for spent resin every 5 days. The addition of resin did not decrease the viability of the culture. Growth was reduced only in cultures with resin added on day 0. Alkaloid production was enhanced to different extents by the timing of resin addition, suggesting that feedback inhibition or product metabolism was present throughout the culture period. Ajmalicine recovery was nearly 100% when the resin was added initially either on day 0 or day 5. Ajmalicine recovery was reduced to 55% when the resin was added later in the culture period starting on day 15, presumably because of resin saturation or the inaccessibility of alkaloids trapped in the vacuole. Delaying the addition of XAD-7 resin until 5 days after the start of the culture resulted in the highest improvement in ajmalicine production, i.e approximately 70% and also resulted in the complete recovery of ajmalicine from the cell.     

Origin of two isoprenoid units in a lavandulyl moiety of sophoraflavanone G from Sophora flavescens cultured cells

Cell suspension cultures of Sophora flavescens produced large amounts of sophoraflavanone G, an 8-lavandulylated flavanone and lupalbigenin, a 6,3'-di-dimethylallylated isoflavone, by the simultaneous addition of cork tissues and methyl jasmonate. The labeling pattern of the isoprene units resulting after administration of [1-13C] glucose into the cell cultures in the presence of the above additives revealed that two isoprene units in the lavandulyl group of sophoraflavanone G and two dimethylallyl groups of lupalbigenin were biosynthesized via the 1-deoxy-D-xylulose-5-phosphate pathway.     

Quantitative detection of probiotic Bifidobacterium strains in bacterial mixtures by using real-time PCR

Strain-specific rRNA-targeted primers were designed for the quantitative detection of Bifidobacterium infantis Y1, B. breve Y8 and B. longum Y10 used in a pharmaceutical probiotic product (VSL-3). PCR and real-time PCR techniques with the selected primers were employed for the direct enumeration of the bifidobacteria in the probiotic preparation and for studying their kinetic characteristics in batch cultures. These analysis revealed that B. infantis Y1 was the predominant strain in the probiotic product and that its growth rate was the highest. Since B. infantis Y1, B. breve Y8 and B. longum Y10 are co-cultured during the industrial production of VSL-3, the kinetic characteristics of these strains can explain their different concentrations in the probiotic preparation. A validation of the PCR quantification method was performed by identifying a representative number of isolates from the bacterial mixtures with automated ribotyping. The methodology described represents a useful tool for the specific quantitative detection of bacterial strains and species in complex mixtures such as pharmaceutical preparations, dairy starter cultures, faecal samples and biopsies.     

Effect of nitrogen and sucrose on the primary and secondary metabolism of transformed root cultures of Hyoscyamus muticus

Abatract The effect of carbon and nitrogen sources on two well-established hairy root clones, LBA1S and C58A, of Hyoscyamus muticus strain Cairo, were investigated. Both clones exhibited completely different patterns with regards to their growth rate, hyoscyamine accumulation, and fatty acid contents. Clone C58A grew faster and yielded more biomass (17.4 g l^-1, in 21 days), but produced less hyoscyamine. The maximum hyoscyamine content (120 mg l^-1) in clone LBA1S was reached in 28 days. Neither of the clones could use lactose or fructose as the sole carbon source, nor ammonium as the sole nitrogen source. The growth in the medium containing glucose was significantly reduced compared to that containing sucrose. Clone LBA1S was sensitive to the changes in sucrose concentration and an increase in ammonium in the culture medium, whereas C58A tolerated these changes better but was more sensitive to the increase in total nitrogen. Lipid synthesis was active in the exponential growth phase, and the total fatty acid content varied from 5 to 34 mg g^-1 of dry root material. The major fatty acids were linoleic, palmitic and linolenic. There were considerable differences in the total amount of lipids and in their relative ratios when different nutrients were applied.Abbreviations DW dry weight - FA fatty acids - FFA free fatty acids - FW fresh weight     

Glycine cleavage and generation of one-carbon units in Euglena gracilis

Euglena gracilis Klebs (strain Z) was maintained in division synchronized autotrophic culture, receiving either air (low CO₂) or 5% CO₂ in     

Callus cultures and bark from Norway spruce clones show similar cellular features and relative resistance to fungal pathogens

In field experiments, clones of Norway spruce [Picea abies (L.) Karst.] showed different degrees of resistance against pathogenic fungi inoculated into the bark that correlate with differences in polyphenolic parenchyma (PP) cells of the bark. Cells of spruce callus cultures, particularly towards the callus surface, resemble PP cells and this study looks at changes in callus cells during infection and the relative resistance of cultures from clones of low (weak) or high (strong) resistance to fungal infection. Callus cultures, initiated from trees with different resistance, were co-inoculated with Ceratocystis polonica (Siem.) C. Moreau and Heterobasidion annosum (Fr.) Bref. Callus cells from strong clones resemble PP cells of bark tissue from strong clones, having more polyphenolic bodies, while callus cells from weak clones are more similar to PP cells from those clones, which have less extensive phenolic bodies. Callus cultures from trees with weak resistance were more quickly overgrown by both species of pathogenic fungi than cultures from trees with strong resistance. Callus cells of infected cultures showed changes similar to activated PP cells of bark, including enhanced accumulation of polyphenolics. Phenolic bodies were more numerous and more extensive (larger and denser) in callus cells of strong versus weak clones under all conditions. Thus, callus cells may perform similar functions in defense as PP cells in the bark. Callus from trees of varying resistance seem to reflect the relative resistance of the trees from which they are derived, and this study indicates that some mechanisms of resistance can be studied using callus from trees of different resistance.     

Scopolamine and hyoscyamine production by hairy root cultures of Brugmansia candida: influence of calcium chloride, hemicellulase and theophylline

CaCl₂ (50 mM) and hemicellulase (0.5 U mg^–1) increased the intracellular accumulation (60–250%), release (60–200%) and production (45–200%) of hyoscyamine and scopolamine in hairy roots of Brugmansia candida. Theophylline (0.25 mM), alone or in combination with hemicellulase, was ineffective in increasing hyoscyamine and scopolamine production.     

Cryopreservation of Digitalis lanata Ehrh. cell cultures: Preculture and freeze tolerance

Cryopreservation experiments were performed with Digitalis lanata cell cultures. The main stress was laid on the behaviour of the cells during the preculture period and the capacity of various preculture additives to induce freeze tolerance. The following compounds were used as preculture additives: trehalose, mannitol, sucrose, melibiose, proline, and sorbitol. They are listed in the order of their respective efficiency. Using trehalose, high post-thaw viability rates were achieved and the cells resumed growth after a short lag period. Melibiose was used as a preculture additive for the first time. Its suitability was in the range of that of sucrose. Proline and sorbitol were not able to induce freeze tolerance in Digitalis cells. Cell viability showed a considerable decrease at the beginning of the preculture period. This reduction was found to be transient in the presence of trehalose, mannitol, sucrose, and melibiose. The damaging effects of proline and sorbitol were too severe to be compensated for by the cells. The PAL activity increased markedly in the presence of proline, whereas the trehalose-treated and the control cells behaved nearly identical to one another.     

Enrichment and characterization of clonogenic epithelial cells from adult rat liver and initiation of epithelial cell strains

Summary A highly efficient method is described for obtaining prolifertive epithelial cells from adult rat livers for the reproducible establishment of liver epithelial cell strains. When cells were isolated from livers of 10-to 15-wk-old male Fischer 344 rats by a collagenase-perfusion method, collected by centrifugation at 50×g for 5 min, and cultured in Williams' medium E containing fetal bovine serum and dexamethasone, colonies of epithelial cells different in size and morphology from hepatocytes were obtained. Sequential perfusion with collagenase and dispase yielded numerous epithelial cell colonies. When isolated cells were fractionated by differential centrifugation, the great majority of hepatocytes were sedimented at 50 ×g for 1 min, whereas many non-hepatocytic cells remiined in the supernatant and could be sedimented by a second centrifugation at 50×g for 5 min. Culture of the two fractions revealed that almost all the epithelial cell colonies were derived from cells in the non-hepatocytic cell fraction. The epithelial cells were cytochemically negative for γ-glutamyl transpeptidase activity, whereas an increase in the activity was detected in hepatocytes with duration in culture. Ultrastructural characteristics of hepatocytes were not found in the cells of newly established cell strains. These results suggest that adult rat liver epithelial cells propagable in culture were derived from a cell type other than the hepatocyte.     

The role of in vitro cultivation on symbiotic trait and function variation in a single species of arbuscular mycorrhizal fungus

In vitro propagation of AM fungi using transformed root cultures (TRC) is commonly used to obtain pure AM fungal propagules for use in research and industry. Early observations indicate that such an artificial environment can alter traits and function of AM fungi over time. We hypothesized that increased in vitro cultivation may promote ruderal strategies in fungi by enhancing propagule production and reducing mutualistic quality. To examine the effect of in vitro cultivation on the trait and function of AM fungi, we inoculated plants with 11 Rhizoglomus irregulare isolates which fell along a cultivation gradient spanning 80 generations. We harvested plants at 10, 20 and 30 d post inoculation to observe differences in fungal and plant traits post infection. In vitro cultivation led to increased spore production but reduced plant shoot phosphorus. Our results indicate that in vitro propagation may indirectly select for traits that affect symbiotic quality.