Treatment of suspension-cultured Glycine max cv Harosoy 63 cells with soluble chitosan (20-500 micrograms per milliliter) increased membrane permeability as shown by leakage of electrolytes, protein, and UV absorbing material. Severe damage to the cell membrane by chitosan (100 and 500 ug/ml) was also indicated by reduced staining with fluorescein diacetate and the leakage of fluorescein from preloaded cells. Other basic polymers (poly-L-lysine, histone, DEAE-dextran, protamine sulfate, and glycol chitosan) also increased permeability, whereas the basic monomers L-lysine and D-glucosamine, and acidic or neutral polymers were not active. Chitosan-induced leakage was inhibited by divalent cations, the order of effectiveness being Ba2 > Ca2+ > Sr2 > Mg2+. Na polygalacturonate and Na poly-L-aspartate also reduced polycation-induced leakage, probably by formation of polycation-polyanion complexes. A chitosan-polygalacturonate complex precipitated on mixing solutions of the two polymers containing approximately equal numbers of galacturonate and glucosamine residues, but not with either polymer in excess. A similar concentrationdependent precipitation of chitosan by Na poly-L-aspartate was found. Leakage from Phaseolus vulgaris cv Grandessa cells was also induced by chitosan, and was inhibited by Ca2+ and Na polygalacturonate.Chitosan (,8-1,4- MATERIALS AND METHODSCell Cultures. The cell suspension culture of Glycine max cv Harosoy 63 was a gift from J. Ebel, Freiburg University, Germany and that of Phaseolus vulgaris cv Grandessa (seeds from Bruno Nebelung, Munster, Germany) was initiated by inoculation of callus derived from sterile hypocotyl explants of 6-d-old seedlings. Suspension cultures were grown at 26°C in the dark on a 1.5-cm radius rotary shaker at 120 rpm in Erlenmeyer flasks containing B5 medium (9) which was modified by using 50 jLM FeSO4-EDTA as the source of iron (8), and subcultured at 6-and 12-d intervals, respectively.Chemicals. Chitosan from crab shells (Sigma) was dissolved in 90 ml 0.1 N acetic acid/g chitosan by stirring overnight, centrifuged at 27,000g for 20 min to remove insoluble material, then precipitated by neutralization to pH 8.0 with 5 N NaOH. The precipitate was washed extensively with distilled H20 by centrifugation and freeze-dried. The glucosamine content of this purified preparation was estimated to be 100%o by the method of Ride and Drysdale (23). Aqueous solutions of purified chitosan and glycol chitosan (Sigma) were prepared for use by dissolving 100 mg in 18 ml 0.1 N acetic acid and dialyzing four times against 2 liters of distilled H20. Poly-L-lysine hydrobromide (mol wt, >70,000), DEAE-dextran (approximate mol wt, 500,000), histone (calf thymus, type II), Na polygalacturonate (grade II), Na poly-L-aspartate (mol wt, 20,000-50,000), glucosamine hydrochloride, L-lysine monohydrochloride, L-aspartic acid, FDA, and BSA (fraction V) were from Sigma. Protamine sulfate and galacturonic acid were from Merck (Darmstadt, Germany). Solutions of aspartic acid and galacturonic...

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In the obese state profound metabolic disturbances exist and it is not known how this disrupted metabolism in obese subjects (body mass index greater than 30) may change their ability to respond to the superimposed, injury-induced stress. Understanding the mechanisms that modify the metabolic parameters in traumatized obese patients is essential in their nutritional assessment and further treatment. We have investigated in 7 obese and 10 nonobese multiple trauma patients, on a whole-body level, the energy metabolism, protein kinetics, and lipolysis in the early catabolic "flow phase" of severe injury when they were receiving maintenance fluids without calories or nitrogen. Traumatized obese patients mobilized relatively more protein and less fat compared with nonobese subjects. A relative block both in lipolysis and fat oxidation is experienced by injured obese patients that results in a shift to preferential use of proteins and carbohydrates. Reduced endogenous protein synthetic efficiency observed in obese patients implies increased protein recycling. Thus obese patients could not effectively use their most abundant fat fuel sources and have to depend on other fuel sources. The nutritional management of obese trauma victims should therefore be tailored towards provision of enough glucose calories to spare protein.

Treatment with chitosan of suspension-cultured Glycine max cells labeled with 45Ca2" caused a rapid release of calcium, which was complete much earlier than the chitosan-induced leakage of intracellular electrolytes and probably reflects calcium loss primarily from the cell wall and/ or plasma membrane. A linear correlation was found between calcium release from chitosan-treated whole cells or isolated cell walls and the amount of chitosan bound. Other polycations (poly-L-lysine, histone, DEAE-dextran, and protamine sulfate), low molecular weight polyamines (spermine, spermidine, and putrescine) and polyanions (polygalacturonate and poly-L-aspartate, which act as chelating agents) also released calcium from whole cells and isolated cell walls; however, only the polycations increased membrane permeability. Poly-L-lysines of differing molecular weight showed a similar ability to release calcium, but their effect on membrane permeability increased with increasing molecular weight. The results suggest that the effect of polycations on permeability is not the direct result of calcium displacement from the cell surface but is probably due to cross-linking of surface components. The order of effectiveness of inorganic cations in displacing calcium from whole cells and isolated cell walls was Ca2l, Ba2 , Sr2+ > Mg2'> K+, Na+.Chitosan (8-1,4-linked glucosamine) has been implicated as an important regulatory component in host-fungal interactions (10) based on histochemical and immunochemical evidence of its production at the host-pathogen interface during infection (7-9), and its many physiological effects on plant and fungal cells (1,7,12,14,19,20 branes (5).In this report, we compare the abilities ofdifferent polycations, polyamines, and other molecules to release Ca2" from Glycine max cells with their effects on membrane permeability, and discuss the results in relation to the mechanism by which basic polymers increase permeability. In addition, the possible significance of Ca2" displacement by chitosan during fungal infection is considered. MATERIALS AND METHODS Cell Culture. Cell suspension cultures of Glycine max cvHarosoy 63 were grown as described previously (20) for 6 d in B5 medium (6), modified by using 50 ,M FeSO4-EDTA as the source of iron (3). 45Ca Labeling of Cells. Cells were washed three times in an incubation medium consisting of 1 mm NaH2PO4 and 2% sucrose (w/v), adjusted to pH 5.5 with 0.1 N NaOH, by centrifugation at 1 5OOg for 5 min. After settling in a measuring cylinder for 20 min, the cells were resuspended in incubation medium to give a 4:1 ratio of total volume to settled volume. [45Ca]C12 (100 mM) in incubation medium was added to give a final concentration of 2 mm containing 0.4 gCi/ml, and the suspension was shaken in an Erlenmeyer flask on a 1.5-cm rotary shaker at 120 rpm for 30 min at 26°C. The cells were then washed and resuspended in incubation medium as above.Uniform labeling of cells was achieved by growing cultures for 6 d in culture medium containing 0.2 ,Ci 45Ca/ml (the medi...