Behavioral and sleep/wake characteristics of mice lacking norepinephrine and hypocretin.

We investigated the interaction between norepinephrine (NE) and orexin/hypocretin (Hcrt) in the control of sleep behavior and narcoleptic symptoms by creating mice that were deficient in both neurotransmitters. Mice with a targeted disruption of the dopamine beta-hydroxylase (Dbh) gene (deficient in NE and epinephrine) or the Hcrt gene were bred to generate double knockouts (DKOs), each single KO (Dbh-KO and Hcrt-KO), and control mice. The duration of wake, non-rapid eye movement (NREM) and REM sleep were monitored by electroencephalogram (EEG)/electromyogram (EMG) recording over a 24-h period, and the occurrence of behavioral arrests was monitored by video/EEG recording for 4 h. Overall, there was very little interaction between the two genes; for most parameters that were measured, the DKO mice resembled either Dbh-KO or Hcrt-KO mice. REM sleep was increased in both DKO and Hcrt-KO mice at night relative to the other groups, but DKO mice had significantly more REM sleep during the day than the other three groups. Sleep latency in response to saline or amphetamine injections was reduced in Dbh-KO and DKO mice relative to other groups. Behavioral arrests, that are frequent in Hcrt-KO mice, were not exacerbated in DKO mice.

Cultivation of roots in bioreactors.

Over the past few years, the number of reports of roots cultured in reactors has increased dramatically. This has provided much-needed information on the physical and metabolic requirements of cultured roots, and has established techniques for the manipulation of root tissue. Most of this work, however, is conducted on a relatively small scale (less than 5 l), and many are demonstrations as opposed to systematic studies. The real challenge of scale-up will be encountered when moving to the 100-1000 l scale, while maintaining dry tissue densities in excess of 10-20 (g dry weight) l-1. Achievement of this goal will require small-scale studies, focused on obtaining design and operational information, coupled with measurements of fundamental scale-up parameters in large-scale systems.

Intrinsic oxygen use kinetics of transformed plant root culture.

Root meristem oxygen uptake, root tip extension rate, and specific growth rate are assessed as a function of dissolved oxygen level for three transformed root cultures. The influence of hydrodynamic boundary layer was considered for all measurements to permit correlation of oxygen-dependent kinetics with the concentration of oxygen at the surface of the root meristem. Oxygen uptake rate is shown to be saturated at ambient conditions, and a saturation level of approximately 300 micromole O2/(cm(3) tissue.hr) was observed for all three of these morphologically diverse root types. In nearly all cases, the observation of a minimum oxygen pressure, below which respiration, extension, or root growth would not occur, could be accounted for as a boundary layer mass transfer resistance. The critical oxygen pressure below which respiration declines is below saturated ambient oxygen conditions. In contrast, critical oxygen pressures for root tip extension were much higher; extension was nearly linear for the two thicker root types (Hyoscyamus muticus, henbain; Solanum tuberosum, potato) above ambient oxygen levels. The performance of the thinnest root, Brassica juncea (Indian mustard) was consistent with reduced internal limitations for oxygen transport. Extension rates did not correlate with biomass accumulation. The fastest growing henbain culture micro = 0.44 day(-)(1)) displayed the slowest extension rate (0.16 mm/hr), and the slowest growing mustard culture (micro = 0.22 day(-)(1)) had the fastest tip extension rate (0.3 mm/hr). This apparent paradox is explained in terms of root branching patterns, where the root branching ratio is shown to be dependent upon the oxygen-limited mersitem extension rate. The implications of these observations on the performance of root culture in bioreactors is discussed.

Direct Agrobacterium tumefaciens-mediated transformation of Hyoscyamus muticus hairy roots using green fluorescent protein.

Hyoscyamus muticus hairy root segments were infected with Agrobacterium tumefaciens ASE containing the binary vector pCGN1548 with a green fluorescent protein (GFP) reporter gene under the control of the CaMV 35S promoter. The roots were incubated on callus-inducing medium to generate transformed cells. Transformants were selected on medium containing 50 and 100 mg/L kanamycin and screened by visual inspection for GFP expression. Highly fluorescent cells were incubated on phytohormone-free medium for regeneration of the hairy root phenotype. This infection technique can be applied directly to existing hairy root cultures which have been previously characterized and selected for desirable physiological traits. These studies also indicate that GFP is not toxic to H. muticus plant tissue and that H. muticus hairy roots have minimal autofluorescence which allows for clear observation of GFP.

Growth of plant root cultures in liquid- and gas-dispersed reactor environments.

The growth of Agrobacterium transformed "hairy root" cultures of Hyoscyamus muticus was examined in various liquid- and gas-dispersed bioreactor configurations. Reactor runs were replicated to provide statistical comparisons of nutrient availability on culture performance. Accumulated tissue mass in submerged air-sparged reactors was 31% of gyratory shake-flask controls. Experiments demonstrate that poor performance of sparged reactors is not due to bubble shear damage, carbon dioxide stripping, settling, or flotation of roots. Impaired oxygen transfer due to channeling and stagnation of the liquid phase are the apparent causes of poor growth. Roots grown on a medium-perfused inclined plane grew at 48% of gyratory controls. This demonstrates the ability of cultures to partially compensate for poor liquid distribution through vascular transport of nutrients. A reactor configuration in which the medium is sprayed over the roots and permitted to drain down through the root tissue was able to provide growth rates which are statistically indistinguishable (95% T-test) from gyratory shake-flask controls. In this type of spray/trickle-bed configuration, it is shown that distribution of the roots becomes a key factor in controlling the rate of growth. Implications of these results regarding design and scale-up of bioreactors to produce fine chemicals from root cultures are discussed.

Interaction of methyl jasmonate, wounding and fungal elicitation during sesquiterpene induction in Hyoscyamus muticus in root cultures.

The ability of methyl jasmonate (MeJa) to induce sesquiterpene production in root cultures of Hyoscyamus muticus has been studied. Although MeJa alone could not induce sesquiterpene in unwounded culture, MeJa added in the presence of wounding displayed a dose-dependent response, saturating at 50 µM. The ability to respond to MeJa declined with an increase in time between MeJa contact and wounding; however, responsiveness could be recovered by re-wounding of tissue prior to MeJa contact, suggesting that additional signaling related to wounding is required for sesquiterpene pathway induction. The saturation level of sesquiterpene induction with fungal elicitor was four times higher than the saturation level achieved by MeJa, with clear differences in sesquiterpene composition. Fungal elicitation results in a higher level of lubimin and a lower level of solavetivone production; whereas, methyl jasmonate induces predominantly solavetivone and little or no lubimin production. This suggests that fungal elicitation induces enzymes further down the sesquiterpene pathway which are not affected by MeJa. The induction of roots in contact with subsaturated levels of elicitor can be enhanced to saturation production levels by the addition of small amounts of MeJa (5-10 µmoles/l). In these studies, MeJa was consistently found to favor the earlier metabolite (solavetivone), while fungal elicitation promoted conversion to subsequent metabolites in the pathway (lubimin). The interactive role of MeJa in signal transduction for secondary metabolic production is discussed.

The role of liquid mixing and gas-phase dispersion in a submerged, sparged root reactor.

An Agrobacterium-transformed root culture of Solanum tuberosum was grown in a 15-1 bubble column. The specific respiration rate decreased by a factor of ten as the tissue grew over a 25-day culture period. On days 5, 8, 13, and 21, respiration was shown to be independent of aeration rate over a range of 0.05-0.4 vvm (volume of air per volume of liquid min-1). Gas dispersion measured from argon tracer residence time distributions increased fourfold due to increased stagnation and channeling of gas through the bed of growing roots; however, introduction of an antifoam surfactant on day 20 greatly reduced dispersion with no accompanying change in respiration. Taken together, the gas dispersion and respiration studies suggest that the gas-liquid interface is not the dominant resistance to oxygen mass transfer. Liquid mixing time measured with a dye tracer increased from 1.45 +/- 0.45 min with no root tissue to 40.2 +/- 1.6 min with 180 g FW l-1 of roots in the column. In addition, the oxygen uptake rate of growing tips (5.2 +/- 0.2 mm) of individual root segments of S. tuberosum measured in a stirred microcell (600 microliters) increased with the oxygen tension of the medium. Based on these results, the role of liquid mixing, gas-phase dispersion, and diffusion in the tissue in the scaleup of root culture is discussed.

From state hospital to integrated human service system: managing the transition.

If a human service facility can treat a mental patient at one-fifth the cost incurred at a parallel state mental hospital, and do it better, there is compelling reason for mental health care managers to consider the benefits of matrix organization.

Plant cell suspension culture rheology.

The results of rheological measurements on 10 different plant cell suspension cultures are presented. Nicotiana tabacum (tobacco) suspension cultures grown in serial batch subculture display high viscosity and power law rheology. This "undesirable" rheology is shown to be a result of elongated cell morphology. The rheology of Papaver somniferum (poppy) cell suspensions is quite different; poppy suspensions behave as Newtonian fluids and have relatively low viscosity (less than 15 cP) at fresh cell densities up to 250 g/L. This flow behavior can be attributed to a lack of elongation in batch-grown poppy cells. A simple correlation for the viscosity as a function of cell density is developed for poppy suspensions up to 300 g fresh weight (FW)/L. It is shown that tobacco cells do not elongate when grown in semicontinuous culture (daily media replacement). These semicontinuously cultured cells have rheological behavior that is indistinguishable from that of poppy, further confirming the dependence of rheology on plant cell morphology. The rheology of a wide variety of other plant suspensions at 200 g FW/L is presented. Most cell suspensions, including soybean, cotton, bindweed, and potato, display low viscosities similar to poppy suspensions. Only carrot and atriplex exhibit slight pseudoplastic behavior which corresponded to a slight degree of cellular elongation for these cultures. This demonstrates that complex rheology associated with elongated cell morphology is much less common than low-viscosity Newtonian behavior. High viscosity in plant cell culture is therefore not an intrinsic characteristic of plant cells but, instead, is a result of the ability to grow cultures to extremely high cell densities due to low biological oxygen demand.

Inhibitory role of root hairs on transport within root culture bioreactors.

An experimental system was developed to produce root cultures of Hyoscyamus muticus with and without the profuse root hairs. Growth in the presence of 7.6 microM pyrene butyric acid (PBA) and 2.2 mM phosphate virtually eliminated root hairs, whereas growth rate, general morphology and nutrient yields remained unchanged in well-mixed flask culture. These root cultures were used to demonstrate decreased flow resistance in a tubular reactor as a result of root hair removal. To assess the impact on bioreactor performance, hairy and hairless root cultures were grown in a highly characterized 15-L bubble column bioreactor. In the absence of root hairs, the mixing was greatly enhanced; mixing times became shorter for the hairless culture at roughly 100 g (fresh weight)/L. By the end of the 3-week culture period, the mixing time of the hairy culture was 29 times longer than that of the hairless culture. The growth rate of the hairless culture in the bioreactor was as much as 2.4 times greater than growth of the hairy culture under the same conditions. The improved reactor performance was reflected in greater biomass accumulation and respiratory activity. These results show that the root hairs-which facilitate nutrient uptake in a static soil environment-are detrimental to growth in a liquid environment as an effect of their stagnating fluid flow and limiting oxygen availability.

Modeling linear and variable growth in phosphate limited suspension cultures of Opium poppy.

In examining the growth kinetics of cell suspensions of opium poppy (Papaver somniferum), the increase in biomass with time was observed to be linear over the entire batch growth period of up to 20 days. Although batch growth profiles were reproducible utilizing the same inoculum, growth rates varied tremendously when experiments were inoculated with cells from different flasks. Both of these phenomena are difficult to explain with conventional batch growth models. In a series of a experiments, phosphate was determined to be the growth-rate-limiting substrate. By expressing growth rate in terms of the intracellular reserves of phosphorus, a growth model which expresses kinetics in terms of the intracellular phosphorus contents of the cells is shown to predict both linear growth character and inoculum dependent variability in growth. The stationary phase phosphate content of seven plant suspension cultures of different plant species was found to be comparable to phosphorus levels of phosphate-starved poppy cells, which suggests that phosphate limitation may be common for plant tissue culture. The applicability of this model to other biological systems which display similar batch growth patterns when subjected to inorganic nutrient deprivation is discussed.

Enhanced recovery of solavetivone from Agrobacterium transformed root cultures of Hyoscyamus muticus using integrated product extraction.

The integrated recovery of solavetivone from fungus elicited "hairy root" cultures of Hyoscyamus muticus is examined using volatile organic solvents and solid-phase adsorbents in an external loop extraction configuration. Hexane and pentane are shown to be toxic when added directly to the culture; however, growth of roots is not inhibited when cultures are exposed to media saturated with these hydrocarbons. Solid-phase neutral adsorbents, XAD-7 and XAD-16, display higher capacity and better solavetivone partitioning capability than the hydrocarbons; however, their selectivity for the sesquiterpene solavetivone is poor in comparison with hexane. In both cases, the integration of product recovery through extraction resulted in a doubling of product formation by alleviating feedback repression. Implications of these results to the recovery of secondary metabolites from plant root cultures are discussed. .

Growth Yields and Maintenance Coefficients of Unadapted and NaCl-Adapted Tobacco Cells Grown in Semicontinuous Culture.

Comparison of carbon utilization between unadapted and NaCl (428 millimolar) adapted tobacco (Nicotiana tabacum L.) cells under substrate limited growth conditions was facilitated using semicontinuous culture. Growth yields (Y(g)) and maintenance coefficients (m) of unadapted and NaCl adapted cells were similar, indicating that the efficiency of carbon utilization for growth was not altered as a result of salt adaptation and that no additional metabolic costs were associated with growth of adapted cells in the presence of a high concentration (428 millimolar) of NaCl. The Y(g) (0.588 grams organic dry weight gain per gram sugar uptake) and m values (0.117 grams sugar uptake per gram organic dry weight per day) were comparable in spite of substantial physiological and biochemical differences that exist between unadapted and NaCl adapted cells. Apparently, a metabolic homeostasis governs biomass production of cells before and after adaptation to salinity.

Estimation of growth yield and maintenance coefficient of plant cell suspensions.

Methodology is presented for the determination of growth yield (Y(g)) and maintenance coefficient (m) for carbon utilization of plant cells grown in suspension culture. Estimation of Y(g) and m requires measurements of specific growth rate (micro) and specific rate of substrate uptake (q) at different growth limiting substrate concentrations. Batch culture of tobacco cells did not permit evaluation of Y(g) and m because micro is constant and maximal during most of the growth cycle. In batch culture, the period of declining specific growth rate is extremely brief because of the rapid transition from logarithmic growth to stationary phase. This occurs because the K(m) for growth is relatively small compared to the initial sucrose concentration. Thus, when the substrate level reaches the K(m), the large mass of cells rapidly depletes the remaining substrate. In contrast, semicontinuous culture facilitates the determination of Y(g) and m because various steady-state growth rates can be achieved. Mathematical expressions were developed to determine the effective values of micro and q over the semicontinuous replacement interval. The validity of this approach was verified by conducting simulations using experimentally determined parameters.

11-hydroperoxyeicosatetraenoic acid is the major dioxygenation product of lipoxygenase isolated from hairy root cultures of Solanum tuberosum.

The profile of primary dioxygenation products of arachidonic acid catalyzed by lipoxygenase isolated from hairy root cultures of Solanum tuberosum treated with a fungal elicitor was compared to that obtained for the enzyme from potato tubers. 11-Hydroperoxyeicosatetraenoic acid (11-HPETE) was the most abundant dioxygenation product formed followed by 8- and 5-HPETEs in the decreasing order of abundance. In contrast, 5-HPETE is the predominant oxidation product of lipoxygenase from potato tubers. Differences in the defense requirements of storage tuber as compared to roots may be the basis of the differences in regio-specificity demonstrated in this work.

Approximation of continuous growth of Cephalotaxus harringtonia plant cell cultures using fed-batch operation.

In Cephalotaxus harringtonia plant cell cultures, periods of batch growth that are limited by hexose uptake are too short to make an accurate estimate of the Monod saturation constant. Continuous cultures are infeasible on a laboratory scale, and semicontinuous cultures require too frequent sampling. Fed-batch operation, consisting of intermittent removal from a culture that is fed continuously, was investigated as a possible solution to these problems. For a constant feed rate, computer simulations showed that a steady state can be achieved which is useful for studying growth at different specific growth rates. In terms of the dilution rate it was confirmed that the operation is essentially equivalent to continuous culture when the samples represent a small fraction of the total culture volume. Experiments with glucose or fructose as the carbon source were carried out in shake flasks fed by a multichannel syringe pump. Results indicate that Monod kinetics based on medium glucose levels cannot adequately describe growth under these conditions. Monod's expression for specific growth rate using internal glucose concentration gives an improved correlation.

Gonadal dysgenesis with mosaicism and a nonfluorescent Y chromosome: report of two cases with correlation of clinical, pathologic, and cytogenetic findings.

Two phenotypic girls with nonfluorescent Y chromosome mosaicism and histologic streak gonads were presented. H-Y antigen (a Y chromosome--determined antigen) was negative in both patients. Electron microscopic findings of "streak gonads" were presented for the first time. The authors recommend bilateral gonadectomy in patients with nonfluorescent Y chromosomes and correlation between the histologic findings and H-Y antigen status.