Physiological studies of a temperature-sensitive sporulation mutant of Bacillus cereus T.

Growth of temperature-sensitive mutant Bacillus cereus T JS22-C occurred normally at the restrictive temperature (37 degrees C), but sporulation was blocked at stage 0. The production of extracellular and intracellular proteases and of alkaline phosphatase occurred at 37 degrees C, but the expression of a functional tricarboxylic acid cycle did not. At the permissive temperature (26 degrees C), the mutant sporulated at a slightly lower frequency (60%) and at a lower rate than the parent strain. The oxidation of organic acids, which accumulate in the growth medium began at T0 in cultures of the parent strain but was delayed until about T3 in cultures of the mutant. Later events in sporulation were also delayed in the mutant by about 3 h. Experiments in which the temperature of growth was shifted from 37 to 26 degrees C or from 26 to 37 degrees C at various times showed that the temperature-sensitive event began approximately 1 h after the end of exponential growth and ended when the cells reached the end of stage II (septum formation). The absence of a functional tricarboxylic acid cycle in cells of the mutant grown at 37 degrees C or shifted from 26 to 37 degrees C before T1 did not appear to be due to a lesion in one of the structural genes of the tricarboxylic acid cycle but was more likely due to the inability of the cells to derepress the synthesis of some of the enzymes of that cycle.     

Effects of preculturing conditions on lag time and specific growth rate of Enterobacter sakazakii in reconstituted powdered infant formula

Enterobacter sakazakii can be present, although in low levels, in dry powdered infant formulae, and it has been linked to cases of meningitis in neonates, especially those born prematurely. In order to prevent illness, product contamination at manufacture and during preparation, as well as growth after reconstitution, must be minimized by appropriate control measures. In this publication, several determinants of the growth of E. sakazakii in reconstituted infant formula are reported. The following key growth parameters were determined: lag time, specific growth rate, and maximum population density. Cells were harvested at different phases of growth and spiked into powdered infant formula. After reconstitution in sterile water, E. sakazakii was able to grow at temperatures between 8 and 47 degrees C. The estimated optimal growth temperature was 39.4 degrees C, whereas the optimal specific growth rate was 2.31 h(-1). The effect of temperature on the specific growth rate was described with two secondary growth models. The resulting minimum and maximum temperatures estimated with the secondary Rosso equation were 3.6 degrees C and 47.6 degrees C, respectively. The estimated lag time varied from 83.3 +/- 18.7 h at 10 degrees C to 1.73 +/- 0.43 h at 37 degrees C and could be described with the hyperbolic model and reciprocal square root relation. Cells harvested at different phases of growth did not exhibit significant differences in either specific growth rate or lag time. Strains did not have different lag times, and lag times were short given that the cells had spent several (3 to 10) days in dry powdered infant formula. The growth rates and lag times at various temperatures obtained in this study may help in calculations of the period for which reconstituted infant formula can be stored at a specific temperature without detrimental impact on health.     

Effect of operating parameters on specific production rate of a cloned-gene product and performance of recombinant fermentation process

A two-stage continuous system in combination with a temperature-sensitive expression system were used as model systems to maximize the productivity of a cloned gene and minimize the problem associated with the plasmid instability for a high-expression recombinant. In order to optimize the two-stage fermentation process, the effects of such operational variables as temperature and dilution rate on productivity of cloned gene were studied using the model systems and a recombinant, Escherichia coli K12 DeltaH1 Deltatrp/pPLc23trp A1. When the expression of cloned gene is induced by raising the operating temperature above 38 degrees C, a significant decrease in the colony-forming-units (CFU) of the plasmid-harboring cell was observed, and the decrease was related to the product concentration. In order to describe this phenomenon, a new kinetic parameter related to the metabolic stress (metabolic stress factor) was introduced. It is defined as the ratio of the rate of change of pheno-type from colony-forming to non-colony-forming cells to the product accumulation per unit cell mass. At a fixed temperature of 40 degrees C, the varying dilution rate D in the range of 0.35-0.90 h(-1) did not affect the metabolic stress factor significantly. At a fixed dilution rate of D = 0.35 h(-1), this factor remained practically constant up to 41 degrees C but increased rapidly beyond 41 degrees C. The effects of temperature and dilution rate in the second stage on the specific production rate were also studied while maintaining the apparent specific growth rate (mu(2) (app)) of the second stage constant at or near mu(2) (app) = 0.26 h(-1). Under a constant dilution rate, D(2) = 0.35 h(-1), the maximum specific production rate obtained was about q(p, max) = 38 units TrpA/mg cell/h at 41 degrees C. At a constant temperature, T(2) = 40 degrees C, specific production rate increased with decreasing dilution rate with in the dilution rate range of D(2) = 0.35-0.90 h(-1). Based on the results of our study, the optimal operating conditions found were dilution rate D(2) = 0.35 h(-1) and operating temperature T(2) = 41 degrees C at the apparent specific growth rate of 0.26 h(-1). Under the optimal operating conditions, about threefold increase in productivity was achieved compared to the best batch culture result. In addition, the fermentation period could be extended for more than 100 h.     

Overheating is detrimental to meiotic spindles within in vitro matured human oocytes

The present study was designed to examine the effects of overheating on meiotic spindle morphology within in vitro matured human oocytes using a polarized light microscope (Polscope). Immature human oocytes at either germinal vesicle or metaphase I stage were cultured in vitro for 24-36 h until they reached metaphase II (M-II) stage. After maturation, oocytes at M-II stage were imaged in the living state with the Polscope at 37, 38, 39 and 40 degrees C for up to 20 min. After heating, oocytes were returned to 37 degrees C and then imaged for another 20 min at 37 degrees C. The microtubules in the spindles were quantified by their maximum retardance, which represents the amount of microtubules. Spindles were intact at 37 degrees C during 40 min of examination and their maximum retardance (1.72-1.79) did not change significantly during imaging. More microtubules were formed in the spindles heated to 38 degrees C and the maximum retardance was increased from 1.77 before heating to 1.95 at 20 min after heating. By contrast, spindles started to disassemble when the temperature was increased to 39 degrees C for 10 min (maximum retardance was reduced from 1.76 to 1.65) or 40 degrees C for 1 min (maximum retardance was reduced from 1.75 to 1.5). At the end of heating (20 min), fewer microtubules were present in the spindles and the maximum retardance was reduced to 0.8 and 0.78 in the oocytes heated to 39 degrees C and 40 degrees C, respectively. Heating to 40 degrees C also induced spindles to relocate in the cytoplasm in some oocytes. After the temperature was returned to 37 degrees C, microtubules were repolymerized to form spindles, but the spindles were not reconstituted completely compared with the spindles imaged before heating. These results indicate that spindles in human eggs are sensitive to high temperature. Moreover, maintenance of an in vitro manipulation temperature of 37 degrees C is crucial for normal spindle morphology.     

Dispersed mammary epithelial cells. Receptors of lactogenic hormones in virgin, pregnant, and lactating rabbits.

The number and affinity of binding sites for lactogenic hormones have been determined in dispersed mammary cells from virgin, pregnant, and lactating rabbits. Dispersed epithelial cells, prepared from mammary glands by enzyme digestion, calcium chelation, and gentle shearing, were separated from nonepithelial cells by density centrifugation. 125I-labeled ovine prolactin (oPRL) and 125I-labeled human growth hormone (/GH) were used as tracers. Association and dissociation of 125I-oPRL or 125I-hGH were time- and temperature-dependent. The rate of association followed a second order reversible reaction with a rate constant of approximately 0.5 at 4 degrees C, approximately 2.0 at 23 degrees C, and approximately 9 x 10(7) M-1 min-1 at 37 degrees C. Maximum binding was achieved after 120 h at 4 degrees C, 48 h at 23 degrees C, and 2 to 4 h at 37 degrees C. Dissociation of 125I-oPRL or hGH from cells by unlabeled oPRL was complete at 4 degrees C after 160 h, following a first order reaction (5-1 = 9.9 x 10(-5) min) and incomplete at 23 degrees C and 37 degrees C even after prolonged time. Internalization of receptor-bound 125I-oPRL was studied by quantitative electron microscope autoradiography. Grain distribution over- and volume densities of cellular organelles was analyzed as a function of time and temperature. At 37 degrees C, there was a rapid and specific translocation of lactogenic hormones to intracellular organelles. Autoradiographic grains were found associated with vesicles, Golgi elements, lysosome-like structures, and the nucleus. One class of high affinity binding sites was estimated from Scatchard plot and direct kinetic analyses at 4 degrees C. Whereas the apparent affinity constant (approximately 10(10) M-1) did not change significantly throughout pregnancy and early lactation, the number of receptors extrapolated from Scatchard plots at 4 degrees C varied in an inverse relation to serum progesterone concentration. Thus, approximately 1900 sites were detected in virgin rabbits (progesterone, approximately 200 pg/ml), and midpregnancy (progesterone, approximately 15,000 pg/ml), and approximately 1800 during early lactation (progesterone, approximately 500 pg/ml). The binding properties of lactogenic hormones to dispersed cells was compared with those to Triton X-100 solubilized microsomal membrane preparations. Good correlation between the two systems was found indicating that cell dispersion did not alter binding properties. Our results indicate that dispersed mammary cells bind lactogenic hormones in a saturable and reversible process, that the number of exposed receptors varies throughout gestation and lactation, and finally that lactogenic hormones are internalized following interaction with their membrane receptors.     

Investigating the warming and cooling rates of human cadavers by development of a gel-filled model to validate core temperature

Tissue Services (within NHS Blood and Transplant) plans to bring deceased donors to its state of the art retrieval suite at its new centre in Speke, Liverpool in air-conditioned transport at circa 20 degrees C but without dedicated active cooling. The aim of this study was to determine how quickly a refrigerated body would warm at different ambient temperatures using a gel-filled model. Two models of a human body were prepared consisting of neoprene wetsuits filled with approximately 7 or 18 l of a viscous solution, which once set has similar properties to ballistics gel. This gel consisted of 47.5% distilled water, 47.5% glycerol and 5% agar. Final "dummy" weights were 7.4 and 18.6 kg respectively, representing "virtual" weights of approximately 40 kg and 70 kg. A K-class thermocouple probe was then inserted into a "rectal" position within each model and the models were cooled to a series of different core temperatures: 5 degrees C, 10 degrees C and 15 degrees C and then were placed in an orbital incubator set at 20 degrees C or 30 degrees C ambient temperature. The rate of temperature increase, in the dummy, was measured, until the model's core temperature was close to the ambient temperature. This was done in triplicate for each size model and ambient temperature. Data indicate that increase in core temperature depends on the size of the model and the initial core temperature. For an equivalent donor weight of 70 kg and background temperature of 20 degrees C, core temperature rises from 5 degrees C to 9.2 degrees C; 10 degrees C to 13.3 degrees C and 15 degrees C to 15.5 degrees C after 2 h. The final core temperatures after 2 h are likely to retard bacterial growth, movement or contamination during transport. Cooling rate data indicated that a 70 kg donor equivalent cooled from 37 degrees C to 15 degrees C within 6 h in a cold room at 4 degrees C. This work has shown that a body can be transported without refrigeration and not cause further tissue deterioration as a result.     

Changes in bleb formation following hyperthermia treatment of Chinese hamster ovary cells

Chinese hamster ovary cells in suspension cultures were heated for various times at 41.5, 43.5, and 45.5 degrees C, and quantitative determinations of microblebbing and macroblebbing of the cell membrane were performed for cells maintained at 4, 25, and 37 degrees C after hyperthermia. The percentage of cells with blebs following heating at 45.5 degrees C was dependent upon the duration of heating with increases from 40% for 5 min to 90% for 30 min. Cells exposed to lower temperatures exhibited less blebbing which was not quantifiable. The changes in bleb formation following 45.5 degrees C were dependent upon the posthyperthermia temperature: a slight decrease of macroblebbing at 25 degrees C, a decrease to 50% by 2 h at 37 degrees C, and a sharp decrease of macroblebbing to less than 10% by 1 h at 4 degrees C. Microblebbing increased slightly at 37 degrees C. When cells were transferred rapidly from the 4 degrees C posthyperthermia incubation to 37 degrees C, the bleb formation percentages returned rapidly to the higher levels which existed before posthyperthermia incubation at the lower temperatures. Gamma irradiation of 20 and 50 Gy produced only a small increase in microblebbing at longer periods (5 to 6 h) but no increase in macroblebbing. The survival of cells heated for 20 min at 45.5 degrees C was decreased 40% for suspension cells maintained at 4 degrees C for 2 to 3 h before incubation at 37 degrees C for colony formation compared to cells immediately incubated at 37 degrees C after heating. The survival of cells maintained at 25 degrees C after heating was not altered in comparison.     

Levels of major proteins of Escherichia coli during growth at different temperatures.

The adaptation of Escherichia coli B/r to temperature was studied by measuring the levels of 133 proteins (comprising 70% of the cell's protein mass) during balanced growth in rich medium at seven temperatures from 13.5 to 46 degrees C. The growth rate of this strain in either rich or minimal medium varies as a simple function of temperature with an Arrhenius constant of approximately 13,500 cal (ca. 56,500 J) per mol from 23 to 37 degrees C, the so-called normal range; above and below this range the growth rate decreases sharply. Analysis of the detailed results indicates that (i) metabolic coordination within the normal (Arrhenius) range is largely achieved by modulation of enzyme activity rather than amount; (ii) the restricted growth that occurs outside this range is accompanied by marked changes in the levels of most of these proteins; (iii) a few proteins are thermometer-like in varying simply with temperature over the whole temperature range irrespective of the influence of temperature on cell growth; and (iv) the temperature response of half of the proteins can be predicted from current information on their metabolic role or from their variation in level in different media at 37 degrees C.     

Spatio-temporal dynamics of expansion growth in roots: automatic quantification of diurnal course and temperature response by digital image sequence processing

A newly developed technique based on image sequence analysis allows automatic and precise quantification of the dynamics of the growth velocity of the root tip, the distribution of expansion growth rates along the entire growth zone and the oscillation frequencies of the root tip during growth without the need of artificial landmarks. These three major parameters characterizing expansion growth of primary roots can be analysed over several days with high spatial (20 microm) and temporal resolution (several minutes) as the camera follows the growing root by an image-controlled root tracking device. In combination with a rhizotron set up for hydroponic plant cultivation the impact of rapid changes of environmental factors can be assessed. First applications of this new system proved the absence of diurnal variation of root growth in Zea mays under constant temperature conditions. The distribution profile of relative elemental growth rate (REGR) showed two maxima under constant and varying growth conditions. Lateral oscillatory movements of growing root tips were present even under constant environmental conditions. Dynamic changes in velocity- and REGR-distribution within 1 h could be quantified after a step change in temperature from 21 degrees C to 26 degrees C. Most prominent growth responses were found in the zone of maximal root elongation.     

Development, survivorship, and reproduction of Helicoverpa armigera (Lepidoptera: Noctuidae) under constant and alternating temperatures

Laboratory studies were conducted to assess the effect of temperature on the survival, development, fecundity, and longevity of Helicoverpa armigera (Hübner) at 11 constant temperatures ranging from 12.5 to 40 degrees C, as well as at five alternating temperature regimes (25-10, 30-15, 32.5-17.5, 35-20, and 35-27.5 degrees C) and under a photoperiod of 16:8 (L:D) h. H. armigera reared at constant temperatures did not develop from egg to adult (emergence) outside the temperature range of 17.5-32.5 degrees C. The alternating conditions expanded this range from 10 to 35 degrees C. The lowest developmental thresholds of the immature stages were estimated by a linear model and ranged from 10.17 (pupal stage) to 11.95 degrees C (egg stage) at constant temperature regimes and from 1.1 to 5.5 degrees C, respectively at alternating temperatures. The values of developmental thresholds estimated using the nonlinear (Lactin-2) model were lower than those estimated by the linear model for constant and alternating temperature regimes except for larval and pupal stages at constant temperatures. Mean adult longevity fluctuated from 34.4 d at 15 degrees C to 7.6 d at 35 degrees C. Females reared under all alternating temperature regimes laid more eggs than females reared at any, except the 25 degrees C, constant temperature treatment. The intrinsic rate of increase was highest at 27.5 degrees C, at both the constant and the corresponding alternating temperature regimes (0.147 and 0.139, respectively). Extreme temperatures had a negative effect on life table parameters.     

Expression of a temperature-sensitive esterase in a novel chaperone-based Escherichia coli strain

A new principle for expression of heat-sensitive recombinant proteins in Escherichia coli at temperatures close to 4 degrees C was experimentally evaluated. This principle was based on simultaneous expression of the target protein with chaperones (Cpn60 and Cpn10) from a psychrophilic bacterium, Oleispira antarctica RB8(T), that allow E. coli to grow at high rates at 4 degrees C (maximum growth rate, 0.28 h(-1)). The expression of a temperature-sensitive esterase in this host at 4 to 10 degrees C yielded enzyme specific activity that was 180-fold higher than the activity purified from the non-chaperonin-producing E. coli strain grown at 37 degrees C (32,380 versus 190 micromol min(-1) g(-1)). We present evidence that the increased specific activity was not due to the low growth temperature per se but was due to the fact that low temperature was beneficial to folding, with or without chaperones. This is the first report of successful use of a chaperone-based E. coli strain to express heat-labile recombinant proteins at temperatures below the theoretical minimum growth temperature of a common E. coli strain (7.5 degrees C).     

Collagen fibrils in vitro grow from pointed tips in the C- to N-terminal direction.

Growth of collagen fibrils was examined in a system in which collagen monomers are generated by specific enzymic cleavage of type IpCcollagen with procollagen C-proteinase. Fibrils formed at 37 degrees C had highly tapered and symmetrical pointed tips. The pattern of cross-striations in the pointed tips indicated that all the molecules were oriented so that the N-termini were directed towards the tip. At 29 degrees C and 32 degrees C, the fibrils formed were thicker. One end of fibrils formed at 29 degrees C was blunt, and the other was pointed. Growth of the fibrils was exclusively from pointed tips. Occasionally a spear-like projection appeared at a blunted end. The spear-like projection then became a new pointed tip for growth in the opposite direction. The results suggested a model for fibril growth with at least three distinct binding sites for monomers. In the model, the pointed tip is the site with the highest affinity for the binding of monomers and most probably defines the critical concentration for fibril assembly. The main shaft of the fibril is a site with very low affinity for binding. The blunted end defines a low-affinity binding site where monomers can bind in opposite orientation to produce growth from a new pointed end.     

Effect of temperature on hybridoma cell cycle and MAb production

The kinetics of growth and antibody formation of an anti-interleukin-2 producing hybridoma line were studied in suspension culture at temperatures ranging from 34 degrees C to 39 degrees C. Flow cytometry was used to determine the effect of temperature on the cell cycle. Maximum cell density and monoclonal antibody yield were observed at 37 degrees C. The specific monoclonal antibody production rate was approximately constant throughout each batch experiment. Lower temperatures caused cells to stay longer in the G(1)-phase of the cell cycle, but temperature had only a marginal effect on the specific antibody production rate. Arresting of cells in the G(1)-phase by means of temperature was, therefore, not suited for enhanced monoclonal antibody production. Rather, antibody production for this hybridoma was directly linked to viable cell concentration. (c) 1992 John Wiley & Sons, Inc.     

Kinetic modeling of Sendai virus fusion with PC-12 cells. Effect of pH and temperature on fusion and viral inactivation.

We have studied the fusion activity of Sendai virus, a lipid-enveloped paramyxovirus, towards a line of adherent cells designated PC-12. Fusion was monitored by the dequenching of octadecyl-rhodamine, a fluorescent non-exchangeable probe. The results were analysed with a mass action kinetic model which could explain and predict the kinetics of virus-cell fusion. When the temperature was lowered from 37 degrees C to 25 degrees C, a sharp inhibition of the fusion process was observed, probably reflecting a constraint in the movement of viral glycoproteins at low temperatures. The rate constants of adhesion and fusion were reduced 3.5-fold and 7-fold, respectively, as the temperature was lowered from 37 degrees C to 25 degrees C. The fusion process seemed essentially pH-independent, unlike the case of liposomes and erythrocyte ghosts. Preincubation of the virus in the absence of target cell membranes at neutral and alkaline pH (37 degrees C, 30 min) did not affect the fusion process. However, a similar preincubation of the virus at pH = 5.0 resulted in marked, though slow, inhibition in fusion with the fusion rate constant being reduced 8-fold. Viral preincubation for 5 min in the same acidic conditions yielded a mild inhibition of fusogenic activity, while preincubation in the cold (4 degrees C, 30 min) did not alter viral fusion activity. These acid-induced inhibitory effects could not be fully reversed by further viral preincubation at pH = 7.4 (37 degrees C, 30 min). Changes in internal pH as well as endocytic activity of PC-12 cells had small effect on the fusion process, thus indicating that Sendai virus fuses primarily with the plasma membranes.     

Formation of glycosidases in batch and continuous culture of Bacteroides fragilis.

Nine strains of bacteroides fragilis were cultivated in stirred fermentors and tested for their ability to produce glycosidases. B. fragilis subsp. vulgatus B70 was used for optimizing the production of glycosidases. The highest bacterial yield was obtained in proteose peptone-yeast extract medium. The optimum pH for maximal bacterial yield was 7.0, and the optimum temperature for growth was 37 degrees C. The formation of glycosidases was optimal between pH 6.5 and 7.5, and the optimum temperature for synthesis of glycosidases was between 33 and 37 degrees C. Culture under controlled conditions in fermentors gave more reproducible production of glycosidases than static cultures in bottles. The strain was also grown in continuous culture at a dilution rate of 0.1 liter/h at pH 7.0 and 37 degrees C with a yield of 2.0 mg of dry weight per ml in the complex medium. The formation of glycosidases remained constant during the entire continuous process.     

Voltage-sensitive nitrendipine binding in an isolated cardiac sarcolemma preparation

Nitrendipine binding has been evaluated in a highly enriched sarcolemma preparation isolated from canine ventricle. The binding was found to be specific, saturable, rapid, and reversible. The dissociation constant (Kd) determined by equilibrium binding studies at 20 degrees C was 0.0880 nM. The Kd increased to 0.670 nM at 37 degrees C. The maximal binding capacity of this preparation ranged from 437 to 1775 fmol/mg protein and was not significantly affected by changes in temperature between 20 and 37 degrees C. The Kd, determined kinetically from the ratio of the dissociation and association rate constants (k-1/k1), was 0.112 and 0.285 nM at 20 and 37 degrees C, respectively. In order to test the hypothesis that nitrendipine binding changes with membrane potential potassium, Nernst potentials were developed, in the presence of valinomycin, by the establishment of potassium gradients across the vesicular membrane. Evaluation of the rates of dissociation of [3H]nitrendipine from the sarcolemma preparation identified a component of binding that was rapidly lost when the transmembrane potential was polarized to inside-negative values. The magnitude of the loss of nitrendipine binding was 25-27% at the most negative potentials examined. Evaluation of the rate of association of nitrendipine revealed that the component of binding that was rapidly lost upon hyperpolarization of the membrane returned over a time course similar to the rate of dissipation of the membrane potential, suggesting that the effects of potential on nitrendipine binding are reversible. These findings are consistent with the hypothesis that nitrendipine binding affinity changes with membrane potential.     

Formation of glycosidases in batch and continuous culture of Bacteroides fragilis.

Nine strains of bacteroides fragilis were cultivated in stirred fermentors and tested for their ability to produce glycosidases. B. fragilis subsp. vulgatus B70 was used for optimizing the production of glycosidases. The highest bacterial yield was obtained in proteose peptone-yeast extract medium. The optimum pH for maximal bacterial yield was 7.0, and the optimum temperature for growth was 37 degrees C. The formation of glycosidases was optimal between pH 6.5 and 7.5, and the optimum temperature for synthesis of glycosidases was between 33 and 37 degrees C. Culture under controlled conditions in fermentors gave more reproducible production of glycosidases than static cultures in bottles. The strain was also grown in continuous culture at a dilution rate of 0.1 liter/h at pH 7.0 and 37 degrees C with a yield of 2.0 mg of dry weight per ml in the complex medium. The formation of glycosidases remained constant during the entire continuous process.     

Maximizing interferon-gamma production by Chinese hamster ovary cells through temperature shift optimization: experimental and modeling

The Chinese hamster ovary (CHO) cell line producing interferon-gamma (IFN-gamma) exhibits a 2-fold increase in specific productivity when grown at 32 degrees C compared to 37 degrees C. Low temperature also causes growth arrest, meaning that the cell density is significantly lower at 32 degrees C, nutrients are consumed at a slower rate and the batch culture can be run for a longer period of time prior to the onset of cell death. At the end of the batch, product concentration is doubled at the low temperature. However, the batch time is nearly doubled as well, and this causes volumetric productivity to only marginally improve by using low temperature. One approach to alleviate the problem of slow growth at low temperature is to utilize a biphasic process, wherein cells are cultured at 37 degrees C for a period of time in order to obtain reasonably high cell density and then the temperature is shifted to 32 degrees C to achieve high specific productivity. Using this approach, it is hypothesized that IFN-gamma volumetric productivity would be maximized. We developed and validated a model for predicting the optimal point in time at which to shift the culture temperature from 37 degrees C to 32 degrees C. It was found that by shifting the temperature after 3 days of growth, the IFN-gamma volumetric productivity is increased by 40% compared to growth and production at 32 degrees C and by 90% compared to 37 degrees C, without any decrease in total production relative to culturing at 32 degrees C alone. The modeling framework presented here is applicable for optimizing controlled proliferation processes in general.     

Effects of temperature acclimation on Neurospora phospholipids. Fatty acid desaturation appears to be a key element in modifying phospholipid fluid properties

Experiments were conducted on the effect of growth temperature on phospholipids of Neurospora. Strains grown at high (37 degrees C) and low (15 degrees C) temperatures show large differences in the proportions of phospholipid fatty acid alpha-linolenate (18 : 3) which can vary by 10-fold over this temperature range. Changes in the phospholipid base composition are less dramatic; the most significant is an increase in phosphatidylethanolamines at low temperatures accompanied by a concomitant decrease in phosphatidylcholine. It appears that phospholipid fatty acid desaturation is closely regulated with respect to growth temperature. Over the 37 to 15 degrees C growth temperature range there appear to be at least two desaturase systems in Neurospora which are under different controls. Production of 18 : 1 and 18 : 2 species appears to occur at high levels over the entire temperature range, whereas the production of 18 : 3 seems to be inversely related to growth temperature. Shifting 37 degrees C-acclimated cultures to 15 degrees C produces a growth lag period of approximately 3 h, during which the level of 18 : 3 increases markedly. Differential scanning calorimetry of phospholipids from 37 degrees C cells shows a phase transition at -22 degrees C while lipids from 15 degrees C cultures exhibit a phase transition with reduced enthalpy at about -41 degrees C. The data are consistent with the idea that phospholipid composition in Neurospora is under strict control and suggest that membrane fluidity is regulated with respect to growth temperature through changes in membrane lipid composition.