Kinetics of growth and ribosome-inactivating protein production from Trichosanthes kirilowii plant cell cultures in a 5-L bioreactor.

Ribosome-inactivating proteins, named for their ability to inhibit protein translation in cell-free systems, are an important class of natural plant defense proteins with potential human therapeutic and agricultural applications. The kinetics of growth, nutrient consumption, and extracellular protein translation inhibitory activity are presented for Trichosanthes kirilowii plant cell suspensions in 5-L bioreactors at two agitation rates (50 and 100 rpm). The cultures had a 7-9.5 day lag phase followed by exponential growth with a doubling time of less than 2 days. Biomass concentrations reached levels of approximately 19 g (dry weight)/L. Protein translation inhibitory activity was observed in the culture broths during the exponential growth phase and reached levels of approximately 50-60 units. No detrimental effects of agitation were observed at 100 rpm. These studies demonstrate the potential for plant cell culture production of ribosome-inactivating proteins in bioreactor systems.

Peripheral limitations to exercise.

We present a computer simulation of a two-compartment model of the systemic circulation which demonstrates how this model can be used to understand the mechanism(s) for the maximal exercise cardiac output (Q). The model consists of two parallel vascular channels, the splanchnic channel (all blood draining through the hepatic veins) and the peripheral channel (all other vascular beds). The distinguishing characteristic of each channel is the product of its venous compliance and venous resistance. Model parameters for the human circulation were estimated from similar parameters obtained directly from animal experiments. "Exercise" was achieved by decreasing the compliance of both channels to 40% of their initial value and by redistributing the Q such that the fraction of Q perfusing the splanchnic channel fell from 38 to 5%, while that perfusing the peripheral channel (skeletal muscles) increased from 62 to 95%. These combined changes increased Q from 4.4 to 22.0 l X min-1 and suggest that maximal adjustments of the two-compartment model parameters lead to a prediction of a maximal Q that approaches the maximal Q usually obtained by humans during exercise.

A novel type-1 ribosome-inactivating protein isolated from the supernatant of transformed suspension cultures of Trichosanthes kirilowii.

A type-1 ribosome-inactivating protein (RIP) designated TK-35 has been purified from the supernatant of suspension cultures of Agrobacterium rhizogenes-transformed stem sections of Trichosanthes kirilowii. The protein was purified from the supernatant by PerSeptive SH/M cation exchange and Sephadex G-75 S gel permeation chromatography. The protein occurs as a monomer, with a molecular weight of 35,117, and is glycosylated. A protein translation inhibition assay indicates that TK-35 has an IC50 value of 2.45 nM and is able to release the rRNA N-glycosidase diagnostic fragment from rabbit reticulocyte lysate. TK-35 is quite thermally stable. Analysis of its N-terminal sequence and two lys-C-protease-digested polypeptides (internal) amino acid sequence indicates that this protein is not homologous to trichosanthin and other type-1 RIPs in Cucurbitaceae family.

Interparticle migration of metal cations in stream sediments as a factor in toxics transport.

Sorption of metal cations by stream sediments is an important process affecting the movement of released contaminants in the environment. The ability of cations to desorb from one sediment particle and subsequently sorb to another can greatly affect metal transport rates but rates for this process have not been reported. The objective of this study was to determine the rate at which sorbed metals can migrate from contaminated sediment particles to uncontaminated sediment particles as a function of the concentration of the contaminating solution and the duration of the contact with the contaminating solution. Samples of small sediment particles were exposed to solutions containing cobalt, after which they were rinsed and combined with larger uncontaminated sediment particles in the presence of stream water. Initial concentrations of the contaminating solution ranged from 1ng/l to 1000mg/l and exposures to the contaminating solution ranged from 6h to 14 days. The rate of the migration increased with increasing concentrations in the contaminating solution and with decreasing times of exposure to the contaminating solution. Under the conditions of these experiments, the time required for the migration to reach equilibrium was on the order of months or longer. In separate experiments, the kinetics of adsorption and desorption of cobalt were measured as a function of concentration of the contaminating solution. The time required to reach adsorption equilibrium increased with increasing concentration in the contaminating solution. Times to sorption equilibrium were on the order of months. Desorption was much slower than adsorption and, together with intraparticle diffusion, probably controls the rate of migration from contaminated to uncontaminated sediment. The results of this study show that interparticle migration of metal cations can proceed at significant rates that are strongly influenced by the length of time that the metal has been in contact with the sediment.

Slow desorption of volatile organic compounds from soil: evidence of desorption step limitations.

Transient adsorption and desorption of 1,2 dichloroethane and toluene on dry Yolo silt loam soil were studied by continuously measuring the composition of the effluent from a soil-packed chromatography column with a mass spectrometer. After obtaining complete breakthrough at approximately 30% relative saturation of one chemical in nitrogen, pure nitrogen feed was initiated and maintained for several hours. Of the material adsorbed at breakthrough, 9.7% of the 1,2 dichloroethane and 14.2% of the toluene were highly resistant to desorption and remained sorbed on the soil even after 5 h of nitrogen flow. When a second chemical with a higher adsorption affinity was introduced into the soil column (water following toluene or toluene following 1,2 dichloroethane), the majority of the first chemical was quickly desorbed and began leaving the soil column before breakthrough of the second chemical. Conversely, when a second chemical with a smaller adsorption affinity was introduced into the soil column, only a small amount of the first chemical was displaced and began leaving the soil column after breakthrough of the second chemical. The results of this study indicate that the desorption step itself may be the rate-limiting step for sorbate which remains after prolonged exposure to sorbate-free gas.

Effect of light irradiance on the production of sulfolipids from Anabaena 7120 in a fed-batch photobioreactor.

Sulfolipids have recently emerged as promising antiHIV and antitumor therapeutics. These lipids have been found in association with the photosynthetic apparatus in most photoautotrophic organisms. To date there have been no quantitative studies on the effect of environmental factors on the production of sulfolipid. In this study, we present results on the effect of light irradiance on the production of sulfolipids using the cyanobacterium Anabaena 7120. The cyanobacteria are grown in a 2 L fed-batch photobioreactor at various external-light intensities. Total lipids are extracted using the Folsch procedure and sulfolipids are quantified using thin-layer chromatography and scanning densitometry. We have achieved a maximum of 14 mg sulfolipid/g dry weight of cell. Our results indicate that there are two stages in the specific rate of production of sulfolipids, one in the declining exponential-growth phase of the cells and the other in the light-limited stage of growth.

A theoretical description of arterial pressure-flow relationships with verification in the isolated hindlimb of the dog.

We developed and tested a new two-compartment serial model of the arterial vasculature which unifies the capacitance (downstream arterial compliance) and waterfall (constant downstream pressure load) theories of blood flow through the arteries. In this model, blood drains from an upstream compliance through a resistance into a downstream compliance which empties into the veins through a downstream resistance which terminates in a constant pressure load. Using transient arterial pressure data obtained from an isolated canine hindlimb preparation, we tested this model, using a stop-flow technique. Numerical parameter estimation techniques were used to estimate the physiologic parameters of the model. The downstream compliance was found to be more than ten times larger than the upstream compliance and the constant pressure load was significantly above venous pressures but decreased in response to vasodilation. Our results support the applicability of both the capacitance and waterfall theories.

Bioreactor studies of growth and nutrient utilization in alfalfa suspension cultures.

Alfalfa (Medicago sativa L.) cells were grown in 500 ml, aerated and stirred batch bioreactors using Schenk and Hildebrant medium. For cultures in which the pH was allowed to vary, we observed two fairly distinct growth phases. Evidence is presented which indicates that the two-phase growth is most likely a result of the two nitrogen sources in the medium. The ammonium present in the medium is directly utilized during the first growth phase and ammonium resulting from intracellular nitrate reduction is utilized during the second phase. During the first growth phase, sucrose is completely hydrolyzed to glucose and fructose with some glucose and fructose consumption. In the second growth phase glucose is consumed preferentially over fructose. Attempts at maintaining the pH at 5.5 using 1N NaOH as the base titrant resulted in very little cell growth compared with cultures for which the pH was allowed to vary.

Documentation of a venous resistance in the baboon.

In the steady state, blood flow out of the heart must be the same as blood flow into the heart. Thus, flow in the cardiovascular system should depend not only on cardiac determinants of flow--pericardial pressure, afterload, heart rate, and contractility--but also on systemic venous determinants of flow as well. We studied the venous determinants of flow in nine baboons. The mean systemic pressure, the upstream driving pressure for venous return, was 10 mm Hg higher than the right atrial pressure. Flow generated by this upstream pressure was modulated by a venous resistance. This resistance acted as if it were interposed between the upstream capacitance vessels and the right atrium. These determinants of venous return in the baboon would be inadequately described by classical concepts of cardiovascular physiology. We suspect that classical concepts would be inadequate in man as well.

A simplified procedure for the purification of trichosanthin (a type 1 ribosome inactivating protein) from Trichosanthes kirilowii root tubers.

A one step rapid and simple purification procedure for trichosanthin, a type 1 ribosome inactivating protein, from root tubers of Trichosanthes kirilowii has been developed using cation-exchange perfusion chromatography. The identity of the protein has been confirmed by its size, immunoreactivity, and sequence information. Yields of 0.16% of electrophoretically pure trichosanthin from dried root tuber have been achieved with a single 10-min chromatographic step giving the ability to purify gram quantities of trichosanthin in 1 day.

Ribosome-inactivating proteins (RIPs) of wild Oregon cucumber (Marah oreganus).

Two type 1 RIPs, designated as MOR-I and MOR-II, have been isolated from Marah oreganus (manroot) seed extract. They are similar but not identical to trichosanthin, a type 1 RIP in the same family. MOR-I and MOR-II are monomeric proteins with molecular weights of 27989.0 and 27632.8 respectively and have pI values greater than 8.8. MOR-I and MOR-II inhibit cell-free protein synthesis with IC50s of 0.063 and 0.071 nM, respectively, and are relatively stable with respect to temperature and pH variations. They share a conserved N-terminal amino acid sequence (D-SF-LS) and cross-react with goat anti-trichosanthin polyclonal serum.