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P J Weathers

Publications and source records attributed to P J Weathers.

At least 19 recordsLinked to original sources
PubMed2007

Sugars proportionately affect artemisinin production.

Little is known about the effect of sugars in controlling secondary metabolism. In this study, sugars alone or in combination with their analogs were used to investigate their role in the production of the antimalarial drug, artemisinin, in Artemisia annua L. seedlings. Compared to sucrose, a 200% increase in artemisinin by glucose was observed. Different ratios of fructose to glucose yielded artemisinin levels directly proportional to increases in relative glucose concentration. When the glucose analog, 3-O-methylglucose, was added with glucose, artemisinin production was dramatically decreased, but hexokinase activity was significantly increased compared to glucose alone. In contrast, neither mannose nor mannitol had any significant effect on artemisinin yield. In comparison with 30 g/l sucrose, artemisinin levels were significantly reduced by 80% in the presence of 27 g/l sucrose + 3 g/l palatinose, which cannot be transported into cells through the sucrose transporter. Together these results suggest that both monosaccharide and disaccharide sugars are likely acting not only as carbon sources but also as signals to affect the downstream production of artemisinin, and that the mechanism of these effects appears to be complex.

Artemisia annua↗
PubMed2004

Alteration of biomass and artemisinin production in Artemisia annua hairy roots by media sterilization method and sugars.

Transformed root cultures of Artemisia annua grown in autoclaved medium show large variations in biomass and artemisinin production regardless of the culture conditions or clonal type. However, using filter-sterilized sugars singly or in combination while holding the carbon level in the medium constant resulted in an unexpected variability in biomass production and artemisinin yield. Autoclaving results in variable hydrolysis of sucrose in the culture medium. Subsequent experiments using combinations of filter-sterilized sugars at a constant total carbon level in the medium showed a stimulation of artemisinin production by glucose. Growth in sucrose was equivalent to growth in fructose and significantly better than in glucose. These results suggest that sugars may be affecting terpenoid metabolism not only as carbon sources, but also as signal molecules.

Artemisia annua↗
PubMed2003

Tetraploid Artemisia annua hairy roots produce more artemisinin than diploids.

Hairy root cultures of diploid Artemisia annua L. (clone YUT16) grow rapidly and produce the antimalarial sesquiterpene artemisinin. Little is known about how polyploidy affects the growth of transformed hairy roots and the production of secondary metabolites. Using colchicine, we produced four stable tetraploid clones of A. annua L. from the YUT16 hairy root clone. Analysis showed major differences in growth and artemisinin production compared to the diploid clone. Tetraploid clones produced up to six times more artemisinin than the diploid parent. This study provides an initial step in increasing our understanding of the role of polyploidy in secondary metabolite production, especially in hairy roots.

Artemisia annua↗
PubMed2001

One-step acclimatization of plantlets using a mist reactor.

A mist reactor was used to grow and acclimatize carnation plants in vitro without using ex vitro acclimatization techniques. The acclimatization protocol in the reactor consisted of altering the mist-on period during the course of the culture period and a stepwise reduction in the relative humidity surrounding the plants from 98% to 70% relative humidity (RH) during the final week of in vitro growth. After transfer and further growth in a greenhouse for 5 weeks, survival was 91% for plants grown in reactors, 81% from vented boxes, and 50% from unvented boxes. Ex vitro survival directly correlated with increased in vitro rooting and decreased hyperhydration. In vitro rooting also correlated with high-quality plants, but did not significantly correlate with low hyperhydration, as normal plants often lacked roots. After 5 weeks in the greenhouse, the quantity of mid- and high-quality plants obtained from reactors and ventilated boxes was similar. Conditions in the mist reactor can be manipulated to produce plants that are readily acclimatized and are equal or better in quality and yield than plants produced using conventional methods.

Acclimatization↗
PubMed2000

The growth of single roots of Artemisia annua in nutrient mist reactors.

To better characterize the development and growth of hairy roots in a mist-fed root bed, a single root aerosol reactor was developed. Growth kinetics studies were conducted on hairy roots of Artemisia annua as a function of the mist cycle, carrier gas, and nutrient compositions. Sustained rapid growth was only observed when conditioned medium was fed to the roots. The presence of 1% CO(2) in the carrier gas did not enhance the growth kinetics but it did prevent necrosis of the tissue at the highest mist cycle.

Artemisia↗
PubMed2000

Changes in Hechtian strands in cold-hardened cells measured by optical microsurgery.

Optical microsurgical techniques were employed to investigate the mechanical properties of Hechtian strands in tobacco (Nicotiana tabacum) and Ginkgo biloba callus cells. Using optical tweezers, a 1. 5-microm diameter microsphere coated with concanavalin A was inserted though an ablated hole in the cell wall of a plasmolyzed cell and attached to a Hechtian strand. By displacing the adhered microsphere from equilibrium using the optical trapping force, the tensions of individual strands were determined. Measurements were made using both normal and cold-hardened cells, and in both cases, tensions were on the order of 10(-12) N. Significant differences were found in the binding strengths of cold-hardened and normal cultured cells. An increased number density of strands in cold-hardened G. biloba compared with normal cultured cells was also observed. Although no Hechtian strands were detected in any Arabidopsis callus cells, strands were present in leaf epidermal cells. Finally, the movement of attached microspheres was monitored along the outside of a strand while cycling the osmotic pressure.

Cold Temperature↗
PubMed1996

Cryopreservation of transformed (hairy) roots of Artemisia annua.

The antimalarial drug artemisinin has been found in transformed (hairy) roots of Artemisia annua. A protocol was developed to preserve A. annua hairy roots in liquid nitrogen. Root tips were excised from 7-day-old cultures and held on solid White's medium for 24 h prior to cryoprotection. They were then treated with a cryoprotecting mixture containing 8% (v/v) dimethyl sulfoxide (Me2SO) and 20% (w/v) sucrose at 25 degrees C for 1 h followed by cooling at 0.09 degrees C/min to 4 degrees C then cooling to -35 degrees C at 0.72 degrees C/min. Vials containing the root tips were then plunged into liquid nitrogen. After thawing in a water bath to 37 degrees C, root tips were held in the cryoprotecting mixture for 10 min before it was diluted to 25% of its original concentration. Root tips were washed once with fresh liquid White's medium and held for 1 h prior to culturing on White's medium with 0.2% Gelrite and 3% (w/v) sucrose. Regrowth of root tips averaged 65%. Independent variables in this study included 1) Me2SO concentration; 2) the type and concentration of cosolutes; 3) cooling rate(s); 4) the temperature at which the sample is transferred to liquid nitrogen; 5) the age of the culture from which root tips are taken; 6) the recovery period between root tip excision and immersion in cryoprotectant; and 7) the amount of Gelrite used in the postthaw plating medium.

Antimalarials↗
PubMed1992

Culture of amebocytes in a nutrient mist bioreactor.

We report the first use of nutrient mist bioreactor (NMB) technology to culture animal cells. The nutrient mist approximated the amebocyte stem tissue's natural environment, which is a thin layer of fluid in the gill leaflets of the horseshoe crab Limulus polyphemus. NMB culture was tried in an attempt to increase production of amebocytes, which are the source of the Limulus Amebocyte Lysate (LAL), the basis for a sensitive and commercially valuable endotoxin assay. Amebocyte growth in the nutrient mist bioreactor is comparable to growth in liquid medium. However, the current design of the bioreactor presents problems for primary cultures such as ours where a pyrogen-free environment is necessary and fungal decontamination is difficult.

Animals↗
PubMed1992

Aeroponics for the culture of organisms, tissues and cells.

Characteristics of aeroponics are discussed. Contrast is made, where appropriate, with hydroponics and aero-hydroponics as applies to research and commercial applications of nutrient mist technology. Topics include whole plants, plant tissue cultures, cell and microbial cultures, and animal tissue cultures with regard to operational considerations (moisture, temperature, minerals, gaseous atmosphere) and design of apparati.

Journal Article↗
PubMed1984

N(2)O evolution by green algae.

Evidence is presented here that axenic cultures of Chlorella, Scenedesmus, Coelastrum, and Chlorococcum spp. evolve N(2)O when grown on NO(2), showing that the Chlorophyceae are a source of N(2)O in aquatic systems.

Journal Article↗
PubMed1980

Cyanophycin granule polypeptide formation and degradation in the cyanobacterium Aphanocapsa 6308.

The effect of a number of conditions on the amount of cyanophycin granule polypeptide [multi-L-arginyl poly(L-aspartic acid)] formed in the unicellular cyanobacterium Aphanocapsa 6308 was determined. Light, CO2, sulfur, and phosphorus starvation as well as the addition of arginine to culture media increased the amount of cyanophycin granule polypeptide in cells when compared with that in cells grown under conditions optimal for growth. Nitrogen limitation and reduction of growth temperature to 30 degrees C decreased the amount of cyanophycin granule polypeptide on a dry-weight basis. Shift-up and shift-down experiments suggest cyanophycin granule polypeptide may be a reserve nitrogen polymer in Aphanocapsa 6308.

Arginine↗
PubMed1980

Structure and composition of cyanophycin granules in the cyanobacterium Aphanocapsa 6308.

Cyanophycin granules in the unicellular cyanobacterium Aphanocapsa 6308 were examined with the electron microscope in both thin section and by freeze-fracture techniques. Purified granules were examined with the electron microscope, by arginine determinations, by chromatography, and by elemental analysis. They are similar in ultrastructure and composition to those isolated from the nitrogen-fixing cyanobacterium Anabaena cylindrica, consisting of equal molar quantities of L-arginine and L-aspartic acid.

Arginine↗
PubMed1978

Arginine catabolism in Aphanocapsa 6308.

The catabolic products of arginine metabolism were observed in Aphanocapsa 6308, a unicellular cyanobacterium, by thin layer chromatography of growth media, by limiting growth conditions, and by enzymatic analysis. Of the organic, nitrogenous compounds examined, only arginine supported growth in CO2-free media. The excretion of ornithine at a concentration level greater than citrulline suggested the existence in Aphanocapsa 6308 of the arginine dihydrolase pathway which produced ornithine, CO2,NH4,+ adenosine 5'-triphosphate. Its existence was confirmed by enzymatic analysis. Although cells could not grow on urea as a sole carbon source a very active urease and subsequently an arginase were also demonstrated, indicating that Aphanocapsa can metabolize arginine via the arginase pathway. The level of enzymes for both pathways indicates a lack of genetic control. It is suggested that the arginase pathway provides only nitrogen for the cells wheras the arginine dihydrolase pathway provides not only nitrogen, but also CO2 and adenosine 5'-triphosphate.

Adenosine Triphosphate↗
PubMed1978

Variations in short term products of inorganic carbon fixation in exponential and stationary phase cultures of Aphanocapsa 6308.

Aphanocapsa 6308 metabolizes both NaHCO3 and Na2CO3. The short term incorporation (5-s) metabolic pattern and the patterns of incorporation of bicarbonate for exponential versus stationary phase cultures differ, however. Cells were equilibrated for 10 min in air and distilled water prior to injection of either NaH14CO3 at pH 8.0, or Na214CO3 at pH 11.0. Hot ethanol extracts were analyzed via paper chromatography and autoradiography for products of CO2 fixation. At 5 s, malate (51.5%) predominates slightly as a primary bicarbonate fixation product over 3-phosphoglycerate (40.3%); 3-phosphoglycerate is the primary product of carbonate fixation. At 60 s, the carbonate and bicarbonate labelling patterns are similar. Cells in stationary phase fix in 5 s a greater proportion of bicarbonate into malate (36% vs. 14% for 3-phosphoglycerate) than do cells in exponential growth. Likewise, 60 s incorporations show a large amount of bicarbonate fixed into aspartate (30.9%) in stationary phase cells over that of exponential phase (11.6%). These data suggest an operative C4 pathway for purposes not related to carbohydrate synthesis but rather as compensation for the incomplete tricarboxylic acid cycle in cyanobacteria. The enhancement of both aspartate fixation and CO2 fixation into citrulline in stationary phase correlates with an increase in cyanophycin granule production which requires both aspartate and arginine.

Aspartic Acid↗