Franklin F. Fannin

Production of loline alkaloids by the grass endophyte, Neotyphodium uncinatum, in defined media.

Lolines (saturated 1-aminopyrrolizidines with an oxygen bridge) are insecticidal alkaloids produced in symbioses of certain Epichloë (anamorph-Neotyphodium) species (fungal endophytes) with grasses, particularly of the genera Lolium and Festuca. Prior to the present study, it was unknown whether lolines were of plant or fungal origin. Neotyphodium uncinatum, the common endophyte of meadow fescue (Lolium pratense=Festuca pratensis) produced loline, N-acetylnorloline, and N-formylloline when grown in the defined minimal media at pH 5.0-7.5, with both organic and inorganic nitrogen sources and sugars as carbon sources. In contrast, lolines were not detected in complex medium cultures. GC-MS and 13C NMR spectroscopic analyses confirmed the identity of the alkaloids isolated from the defined medium cultures. Lolines accumulated to ca. 700 mg/l (4 mM) in cultures with 16.7 mM sucrose and 15-30 mM asparagine, ornithine or urea. Kinetics of loline production and fungal growth were assessed in defined medium with 16.7 mM sucrose and 30 mM ornithine. The alkaloid production rate peaked after the onset of stationary phase, as is common for secondary metabolism in other microbes.

Evidence for concurrent spread of tobacco mosaic virus from infected epidermal cells to neighboring epidermal and mesophyll cells

Abstract The upper surfaces of tobacco ( Nicotiana tabacum L.) leaves were mechanically inoculated with tobacco mosaic virus (TMV) and, at various times thereafter, protoplasts from upper epidermal and pallisade mesophyll cells were isolated. The percentage of each type of protoplast infected with TMV was then determined by fluorescent antibody analysis. The results provided an estimation of the numbers of infections initially established and of the direction and rate of spread of virus to neighboring leaf cells. From 0.4 to 2% of the epidermal protoplasts, but none of the mesophyll protoplasts, isolated during the first 5–6 h after inoculation of leaves were infected. With leaves sampled during the next 1–2 h, the percentage of infected epidermal protoplasts increased; at the same time, the percentage of infected mesophyll protoplasts increased from zero to values approximating the initial percentage of infected epidermal protoplasts. This suggests that only epidermal cells were initially infected and that virus later spread from these cells to both epidermal and mesophyll cells in the inoculated leaves.

The kinetic advantage for transport into hamster intestine of glucose generated from phlorizin by brush border β-glucosidase

Abstract Phlorizin, labeled with tritium only in the glucose moiety, was used as substrate for the β-glucosidase present in brush border membranes from hamster intestine in order to study, simultaneously, the kinetics of hydrolysis and the fate of the [3H]glucose liberated by the enzyme. The [3H]glucose seems to experience the same hydrolase related transport into the intestinal villi as the hexoses liberated from the common disaccharides by their respective hydrolases. The released [3H]glucose accumulation rate is only partially inhibited by unlabelled glucose added to the medium as either the free sugar or as the precursors sucrose, lactose or glucose 1-phosphate, and then only when these sugars are present at very high levels. Furthermore, glucose oxidase, added to the medium as a glucose scavenger, has no effect on the uptake rate of the phlorizin hydrolase-liberated sugar. These and other findings are presented as evidence that, under conditions where the Na+-dependent glucose carrier is more than 97% inhibited by phlorizin, the glucose derived from the inhibitor, like the hexoses from disaccharides, has a kinetic advantage for transfer into the intestinal tissue.

A hydrolase-related transport system is not required to explain the intestinal uptake of glucose liberated from phlorizin

The fate of [3H]glucose released from a wide range of [3H]phlorizin concentrations by phlorizin hydrolase has been studied under conditions where the Na+-dependent glucose transport system in hamster intestine is profoundly inhibited by the glucoside. At 0.2-2.0 mM phlorizin, the [3H]glucose uptake was a constant 11-12% of that generated by the enzyme and at the highest level, it was reduced to that of passive diffusion. Glucose liberated from 0.2 mM [3H]phlorizin is accumulated at a rate nearly equal to that found for 0.2 mM [14C]glucose when this free sugar uptake is measured in a medium containing 0.2 mM unlabeled phlorizin. Furthermore, without sodium, the accumulation rates of hydrolase-derived or exogenous glucose are both reduced to the rate of [14C]mannitol. Our results indicate that the glucose released from phlorizin enters the tissue via the small fraction of the Na+-dependent glucose carriers which escape phlorizin blockade together with a mannitol-like passive diffusion. It enjoys a kinetic advantage for tissue entry over free glucose in the medum by virtue of the position of the site where it is formed, i.e inside the unstirred water layer and near normal entry portals. No special hydrolase-related transport system, like the one proposed for disaccharides, needs to be considered to account for our findings.

Tall fescue seed extraction and partial purification of ergot alkaloids

Many substances in the tall fescue/endophyte association (Schedonorus arundinaceus/Epichloë coenophiala) have biological activity. Of these compounds only the ergot alkaloids are known to have significant mammalian toxicity and the predominant ergot alkaloids are ergovaline and ergovalinine. Because synthetically produced ergovaline is difficult to obtain, we developed a seed extraction and partial purification protocol for ergovaline/ergovalinine that provided a biologically active product. Tall fescue seed was ground and packed into several different sized columns for liquid extraction. Smaller particle size and increased extraction time increased efficiency of extraction. Our largest column was a 114 × 52 × 61 cm (W × L × D) stainless steel tub. Approximately 150 kg of seed could be extracted in this tub. The extraction was done with 80% ethanol. When the solvent front migrated to bottom of the column, flow was stopped and seed was allowed to steep for at least 48 h. Light was excluded from the solvent from the beginning of this step to the end of the purification process. Following elution, ethanol was removed from the eluate by evaporation at room temperature and the resulting syrup was freeze-dried. About 80% recovery of alkaloids was achieved with 18-fold increase in concentration of ergovaline. Initial purification of the dried product was accomplished by extracting with hexane/water (6:1, v/v). The aqueous fraction was extracted with chloroform, the aqueous layer discarded, after which the chloroform was removed with a resulting 20-fold increase of ergovaline. About 65% of the ergovaline was recovered from the chloroform residue for an overall recovery of 50%. The resultant partially purified ergovaline had biological activities in in vivo and in vitro bovine bioassays that approximate that of synthetic ergovaline.

Production of extracellular fibers by tobacco leaf epidermal protoplasts

Protoplasts prepared from the upper epidermis of tobacco (Nicotiana tabacum L.) leaves produce tubular structures which are highly fluorescent in the presence of Calcofluor white. The presence of Ca2+ in the incubation medium appears to be necessary for the production of these fibers. A commercial cellulase preparation destroyed these structures and inhibited their production.