Enhancing Protein Expression in HEK-293 Cells by Lowering Culture Temperature

Animal cells and cell lines, such as HEK-293 cells, are commonly cultured at 37°C. These cells are often used to express recombinant proteins. Having a higher expression level or a higher protein yield is generally desirable. As we demonstrate in this study, dropping culture temperature to 33°C, but not lower, 24 hours after transient transfection in HEK-293S cells will give rise to ~1.5-fold higher expression of green fluorescent protein (GFP) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. By following the time course of the GFP-expressing cells growing at 37°C and 33°C from 24 hours after transfection (including 19 hours recovery at 37°C in the normal growth medium), we found that a mild hypothermia (i.e., 33°C) reduces the growth rate of HEK-293S cells, while increasing cellular productivity of recombinant proteins. As a result, green cells remain undivided in a longer period of time. Not surprisingly, the property of a recombinant protein expressed in the cells grown at 33°C is unaffected, as shown by the use of AMPA receptors. We further demonstrate with the use of PC12 cells that this method may be especially useful when a recombinant protein is difficult to express using a chemical-based, transient transfection method.     

Effects of cycling temperatures on fiber metabolism in cultured cotton ovules

The effects of temperature on rates of cellulose synthesis, respiration, and long-term glucose uptake were investigated using cultured cotton ovules (Gossypium hirsutum L. cv Acala SJ1). Ovules were cultured either at constant 34°C or under cycling temperatures (12 h at 34°C/12 h at 15-40°C). Rates of respiration and cellulose synthesis at various temperatures were determined on day 21 during the stage of secondary wall synthesis by feeding cultured ovules with [¹⁴C]glucose. Respiration increased between 18 and approximately 34°C, then remained constant up to 40°C. In contrast, the rate of cellulose synthesis increased above 18°C, reached a plateau between about 28 and 37°C, and then decreased at 40°C. Therefore, the optimum temperature for rapid and metabolically efficient cellulose synthesis in Acala SJ1 is near 28°C. In ovules cycled to 15°C, respiration recovered to the control rate immediately upon rewarming to 34°C, but the rate of cellulose synthesis did not fully recover for several hours. These data indicate that cellulose synthesis and respiration respond differently to cool temperatures. The long-term uptake of glucose, which is the carbon source in the culture medium, increased as the low temperature in the cycle increased between 15 and 28°C. However, glucose uptake did not increase in cultures grown constantly at 34°C compared to those cycled at 34/28°C. These observations are consistent with previous observations on the responses of fiber elongation and weight gain to cycling temperatures in vitro and in the field.     

A rapid slide plaque technic for bacteriophage assay

1. A modified rapid plaque-counting procedure is described, whereby staphylococcal phage host-cell suspensions in agar are spread over constant areas on glass slides and are incubated in moist chambers. 2. Statistical analysis indicated a precision of ±8.2 for means of sets of eight slides incubated at 37°C. and ±7.6 for means of sets of eight incubated at 28°C. 3. With the staphylococcus system, a temperature of 28°C. yielded significantly higher counts than at 37°C. 4. Counts made after 4 hours may be used as an estimate of the final plaque count bearing in mind the fact that the yield at this time is significantly less than at 24 hours. 5. The data presented compared favorably with the results obtained by the plate method used by Hershey, and suggest that a greater precision may be obtained by this method—at least for the staph. K phage system.     

Heat-shock proteins are associated with hnRNA in Drosophila melanogaster tissue culture cells

Ribonucleoprotein complexes of Drosophila melanogaster Kc tissue culture cells grown at 24°C or heat-shocked at 37°C were cross-linked in vivo by u.v. irradiation. Cross-linked heterogeneous nuclear ribonucleoprotein (hnRNP) complexes were fractionated by oligo(dT)-cellulose chromatography and CsCI density centrifugation. The hnRNP complexes of both 24°C and 37°C culture cells possess buoyant densities in CsCI between = 1.38 g/cm^-3 and 1.43 g/cm^-3. The ³⁵S-labelled proteins bound to the hnRNA of 37°C culture cells correspond in mol. wt. to the so-called heat-shock proteins of 70 K, 68 K, 27 K, 26 K, 23 K and 22 K. The 70 K and 68 K proteins are also present in hnRNP complexes of 24°C culture cells. In addition, several other Drosophila hnRNPs of 140 K, 56 K, 45 K, 43 K, 38 K, 37 K and 34 K, whose synthesis is strongly repressed under heat-shock conditions, could be identified. The results demonstrate that the so-called heat-shock proteins possess a function as RNPs.     

Characterization of Thermoanaerobacter Glucose Isomerase in Relation to Saccharidase Synthesis and Development of Single-Step Processes for Sweetener Production

Regulation of glucose isomerase synthesis was studied in Thermoanaerobacter strain B6A, which fermented a wide variety of carbohydrates including glucose, xylose, lactose, starch, and xylan. Glucogenic amylase activities and β-galactosidase were produced constitutively, whereas the synthesis of glucose isomerase was induced by either xylose or xylan. Production of these saccharidase activities was not significantly repressed by the presence of glucose or 2-deoxyglucose in the growth media. Glucose isomerase production was optimized by controlling the culture pH at 5.5 during xylose fermentation. The apparent temperature and pH optima for these cell-bound saccharidase activities were as follows: glucose isomerase, 80°C, pH 7.0 to 7.5; glucogenic amylase, 70°C, pH 5.0 to 5.5; and β-galactosidase, 60°C, pH 6.0 to 6.5 Glucose isomerase, glucogenic amylase, and β-galactosidase were produced in xylose-grown cells that were active and stable at 60 to 70°C and pH 6.0 to 6.5. Under single-step process conditions, these saccharidase activities in whole cells or cell extracts converted starch or lactose directly into fructose mixtures. A total of 96% of initial liquefied starch was converted into a 49:51 mixture of glucose and fructose, whereas 85% of initial lactose was converted into a 40:31:29 mixture of galactose, glucose, and fructose.     

INFLUENCE OF TEMPERATURE AND HYDROGEN ION CONCENTRATION UPON THE SPORE CYCLE OF BACILLUS SUBTILIS

1. At 5°C. no germination took place. 2. At 25°C. and at 37°C. germination occurs if the hydrogen ion concentration of the broth is kept between pH 5 and pH 10, but not at higher or lower pH values. 3. The completion of the spore cycle likewise requires a hydrogen ion concentration between pH 5 and pH 10. 4. The spores can germinate when the pH value is 10, although after germination the vegetative cells multiply only to a very slight extent and soon pass into spores. 5. The slight growth and multiplication of vegetative cells in broth of pH 10 suggest that the formation of endospores in this medium must be caused largely by the unfavorable reaction of the medium rather than by the accumulation of metabolic products. 6. Automatic adjustment of the medium seems to play a rôle in the completion of the spore cycle. 7. The results are not only of theoretical importance but they have a practical application to the preservation of food by canning and by other methods.     

Influence of pH and Temperature on Enumeration of Cellulose- and Hemicellulose-Degrading Thermophilic Anaerobes in Neutral and Alkaline Icelandic Hot Springs

Cellulose- and hemicellulose-degrading thermophilic anaerobes were enumerated in biomat samples of various temperatures from two different hot springs in the Hveragerǒi area of Iceland: one spring had a pH near 7, the second had a pH near 9. The most-probable-number technique was used for enumeration of bacteria in the samples, with media at many different temperatures (37 to 90°C) and two pH values (7 and 9). There were generally more xylan-degrading then cellulose-utilizing organisms in both environments. There was no growth at 80°C in the neutral spring or at 37°C in the alkaline spring. However, there were large numbers of both types of organisms in the alkaline spring at 80°C and in the neutral spring at 37°C. No cultures grew from the most-probable-number tubes inoculated with the Hveragerǒi samples and incubated at 90°C or with media at pH 9. However, xylan-degrading cultures at 70°C were enriched at pH 9 with samples from some other Icelandic hot springs.     

Effect of Storage Temperature on Cultured Epidermal Cell Sheets Stored in Xenobiotic-Free Medium

Cultured epidermal cell sheets (CECS) are used in regenerative medicine in patients with burns, and have potential to treat limbal stem cell deficiency (LSCD), as demonstrated in animal models. Despite widespread use, short-term storage options for CECS are limited. Advantages of storage include: flexibility in scheduling surgery, reserve sheets for repeat operations, more opportunity for quality control, and improved transportation to allow wider distribution. Studies on storage of CECS have thus far focused on cryopreservation, whereas refrigeration is a convenient method commonly used for whole skin graft storage in burns clinics. It has been shown that preservation of viable cells using these methods is variable. This study evaluated the effect of different temperatures spanning 4°C to 37°C, on the cell viability, morphology, proliferation and metabolic status of CECS stored over a two week period in a xenobiotic–free system. Compared to non-stored control, best cell viability was obtained at 24°C (95.2±9.9%); reduced cell viability, at approximately 60%, was demonstrated at several of the temperatures (12°C, 28°C, 32°C and 37°C). Metabolic activity was significantly higher between 24°C and 37°C, where glucose, lactate, lactate/glucose ratios, and oxygen tension indicated increased activation of the glycolytic pathway under aerobic conditions. Preservation of morphology as shown by phase contrast and scanning electron micrographs was best at 12°C and 16°C. PCNA immunocytochemistry indicated that only 12°C and 20°C allowed maintenance of proliferative function at a similar level to non-stored control. In conclusion, results indicate that 12°C and 24°C merit further investigation as the prospective optimum temperature for short-term storage of cultured epidermal cell sheets.     

Effect of temperature conditioning on chilling injury of cucumber cotyledons: possible role of abscisic Acid and heat shock proteins

Endogenous abscisic acid levels and induced heat shock proteins were measured in tissue exposed for 6 hours to temperatures that reduced their subsequent chilling sensitivity. One-centimeter discs excised from fully expanded cotyledons of 11-day-old seedlings of cucumber (Cucumis sativus L., cv Poinsett 76) were exposed to 12.5 or 37°C for 6 hours followed by 4 days at 2.5 or 12.5°C. Ion leakage, a qualitative indicator of chilling injury, increased after 2 to 3 day exposure to 2.5°C, but not to 12.5°C, a nonchilling temperature. Exposure to 37°C before chilling significantly reduced the rate of ion leakage by about 60% compared to tissue exposed to 12.5°C before chilling, but slightly increased leakage compared to tissue exposed to 12.5 or 37°C and held at the nonchilling temperature of 12.5°C. There was no relationship between abscisic acid content following exposure to 12.5 or 37°C and chilling tolerance. Five heat shock proteins, with apparent molecular mass of 25, 38, 50, 70, and 80 kilodaltons, were induced by exposure to 37 or 42°C for 6 hours, and their appearance coincided with increased chilling resistance. Heat shock treatments reduced the synthesis of three proteins with apparent molecular mass of 14, 17, and 43 kilodaltons. Induction of heat shock proteins could be a possible cause of reduced chilling injury in tissue exposed to 37 or 42°C.     

Potential Role of Pyrophosphate:Fructose 6-Phosphate Phosphotransferase in Carbohydrate Metabolism of Cold Stored Tubers of Solanum tuberosum cv Bintje

To gain a better understanding of the mechanism of cold induced sweetening, sugar accumulation in potato, Solanum tuberosum cv Bintje, was compared to the maximum activity of inorganic pyrophosphate (PPi):fructose 6-phosphate 1-phosphotransferase (EC 2.7.1.90) and the concentration of two regulatory metabolites. Mature tubers accumulated reducing sugars and sucrose at an almost linear rate of 13.4 and 5.2 micromole per day per gram dry weight at 2°C and 4.5 and 1.3 micromole per day per gram dry weight, respectively, at 4°C. During storage at 8°C sugar accumulation was nil. Sugar accumulation was preceded by a lag phase of about 4 days. The accumulation of reducing sugars persisted for at least 4 weeks, whereas sucrose accumulation declined after 2 weeks of storage. The ratio of glucose:fructose changed concomitantly with sugar increase from 65:35 to equimolarity. The maximum activity of PPi:fructose 6-phosphate 1-phosphotransferase was 2.51 and 2.25 units per gram dry weight during storage at 2 and 8°C, respectively. The temperature coefficient of this enzyme from potatoes kept at 2 or 8°C was 2.12 and 2.48, respectively. The endogenous concentration of fructose 2,6-biphosphate increased from 0.15 to 1 nanomole per gram dry weight during storage at 2 and 4°C but remained the same throughout storage at 8°C. After exposure to 2°C an initial increase in the concentration of PPi was observed from 4.0 to 5.6 nanomoles per gram dry weight. Pyrophosphate concentration did not change during storage at 4°C but decreased slightly at 8°C. All observed changes became annulled after transfer of cold stored tubers to 18°C. These data strongly indicate that PPi:fructose 6-phosphate 1-phosphotransferase can be fully operational in cold stored potato tubers and the lack of increase in PPi concentration supports the functioning of this enzyme during sugar accumulation.     

GLUT4 and Transferrin Receptor Are Differentially Sorted Along the Endocytic Pathway in CHO Cells

The trafficking of GLUT4, a facilitative glucose transporter, is examined in transfected CHO cells. In previous work, we expressed GLUT4 in neuroendocrine cells and fibroblasts and found that it was targeted to a population of small vesicles slightly larger than synaptic vesicles (Herman, G.A, F. Bonzelius, A.M. Cieutat, and R.B. Kelly. 1994. Proc. Natl. Acad. Sci. USA. 91: 12750–12754.). In this study, we demonstrate that at 37°C, GLUT4-containing small vesicles (GSVs) are detected after cell surface radiolabeling of GLUT4 whereas uptake of radioiodinated human transferrin does not show appreciable accumulation within these small vesicles. Immunofluorescence microscopy experiments show that at 37°C, cell surface–labeled GLUT4 as well as transferrin is internalized into peripheral and perinuclear structures. At 15°C, endocytosis of GLUT4 continues to occur at a slowed rate, but whereas fluorescently labeled GLUT4 is seen to accumulate within large peripheral endosomes, no perinuclear structures are labeled, and no radiolabeled GSVs are detectable. Shifting cells to 37°C after accumulating labeled GLUT4 at 15°C results in the reappearance of GLUT4 in perinuclear structures and GSV reformation. Cytosol acidification or treatment with hypertonic media containing sucrose prevents the exit of GLUT4 from peripheral endosomes as well as GSV formation, suggesting that coat proteins may be involved in the endocytic trafficking of GLUT4. In contrast, at 15°C, transferrin continues to traffic to perinuclear structures and overall labels structures similar in distribution to those observed at 37°C. Furthermore, treatment with hypertonic media has no apparent effect on transferrin trafficking from peripheral endosomes. Double-labeling experiments after the internalization of both transferrin and surface-labeled GLUT4 show that GLUT4 accumulates within peripheral compartments that exclude the transferrin receptor (TfR) at both 15° and 37°C. Thus, GLUT4 is sorted differently from the transferrin receptor as evidenced by the targeting of each protein to distinct early endosomal compartments and by the formation of GSVs. These results suggest that the sorting of GLUT4 from TfR may occur primarily at the level of the plasma membrane into distinct endosomes and that the organization of the endocytic system in CHO cells more closely resembles that of neuroendocrine cells than previously appreciated.     

Heat Stress Responses in Cultured Plant Cells : Heat Tolerance Induced by Heat Shock versus Elevated Growing Temperature

Using cultured pear (Pyrus communis cv Bartlett) cells, heat tolerance induced by heat shock was compared to that developed during growth at high temperature. After growth at 22°C, cells exposed to 38°C for 20 minutes (heat shock) showed maximum increased tolerance within 6 hours. Cells grown at 30°C developed maximum heat tolerance after 5 to 6 days; this maximum was well below that induced by heat shock. Heat shock-induced tolerance was fully retained at 22°C for 2 days and was only partly lost after 4 days. However, pear cells acclimated at 30°C lost all acquired heat tolerance 1 to 2 days after transfer to 22°C. In addition, cells which had been heat-acclimated by growth at 30°C showed an additional increase in heat tolerance in response to 39°C heat shock. The most striking difference between heat shock and high growth temperature effects on heat tolerance was revealed when tolerance was determined using viability tests based on different cell functions. Growth at 30°C produced a general hardening, i.e. increased heat tolerance was observed with all three viability tests. In contrast, significantly increased tolerance of heat-shocked cells was observed only with the culture regrowth test. The two types of treatment evoke different mechanisms of heat acclimation.     

Storage Temperature Alters the Expression of Differentiation-Related Genes in Cultured Oral Keratinocytes

Purpose

Storage of cultured human oral keratinocytes (HOK) allows for transportation of cultured transplants to eye clinics worldwide. In a previous study, one-week storage of cultured HOK was found to be superior with regard to viability and morphology at 12°C compared to 4°C and 37°C. To understand more of how storage temperature affects cell phenotype, gene expression of HOK before and after storage at 4°C, 12°C, and 37°C was assessed.

Materials and Methods

Cultured HOK were stored in HEPES- and sodium bicarbonate-buffered Minimum Essential Medium at 4°C, 12°C, and 37°C for one week. Total RNA was isolated and the gene expression profile was determined using DNA microarrays and analyzed with Partek Genomics Suite software and Ingenuity Pathway Analysis. Differentially expressed genes (fold change > 1.5 and P < 0.05) were identified by one-way ANOVA. Key genes were validated using qPCR.

Results

Gene expression of cultures stored at 4°C and 12°C clustered close to the unstored control cultures. Cultures stored at 37°C displayed substantial change in gene expression compared to the other groups. In comparison with 12°C, 2,981 genes were differentially expressed at 37°C. In contrast, only 67 genes were differentially expressed between the unstored control and the cells stored at 12°C. The 12°C and 37°C culture groups differed most significantly with regard to the expression of differentiation markers. The Hedgehog signaling pathway was significantly downregulated at 37°C compared to 12°C.

Conclusion

HOK cultures stored at 37°C showed considerably larger changes in gene expression compared to unstored cells than cultured HOK stored at 4°C and 12°C. The changes observed at 37°C consisted of differentiation of the cells towards a squamous epithelium-specific phenotype. Storing cultured ocular surface transplants at 37°C is therefore not recommended. This is particularly interesting as 37°C is the standard incubation temperature used for cell culture.     

Efflux of Red Cell Water into Buffered Hypertonic Solutions

Buffered NaCl solutions hypertonic to rabbit serum were prepared and freezing point depressions of each determined after dilution with measured amounts of water. Freezing point depression of these dilutions was a linear function of the amount of water added. One ml. of rabbit red cells was added to each 4 ml. of the hypertonic solutions and after incubation at 38°C. for 30 minutes the mixture was centrifuged and a freezing point depression determined on the supernatant fluid. The amount of water added to the hypertonic solutions by the red cells was calcuated from this freezing point depression. For each decrease in the freezing point of -0.093°C. of the surrounding solution red cells gave up approximately 5 ml. of water per 100 ml. of red cells in the range of -0.560 to -0.930°C. Beyond -0.930°C. the amount of water given up by 100 ml. of red cells fits best a parabolic equation. The maximum of this equation occurred at a freezing point of the hypertonic solution of -2.001°C. at which time the maximum amount of water leaving the red cells would be 39.9 ml. per 100 ml. of red cells. The data suggest that only about 43 per cent of the red cell water is available for exchange into solutions of increasing tonicity.     

Relevance of amadori and maillard products to seed deterioration

The possible role of Amadori and Maillard reactions in the deterioration of dry seeds was investigated using model systems and whole soybean seeds, Glycine max cv Hodgson. In model systems of glucose plus an enzyme (lysozyme), the production of Amadori products was accelerated by higher temperature and relative humidity. The reaction between glucose and lysozyme at 50°C, 75% relative humidity, leads to a progressive decline in enzymatic activity. During accelerated aging of soybean seeds (40°C, 100% relative humidity), a sequence is observed in which the Amadori products increase with time and then decline under conditions in which the Maillard products increase in the axes. Loss of germinability occurs at the time when the Maillard products increase in the soybean axes. These results are suggestive of a role for nonenzymic glycation in soybean seed deterioration during accelerated aging.     

Studies of fluid exchanges between rat liver slices and simple media

1. The exchange of fluid between slices of rat liver and solutions of monosaccharides, disaccharides, and sodium chloride has been studied in relation to the temperature of incubation. The point of apparent isotonicity (P.A.I.) of the tissue was defined as the concentration of the solution in which the slices neither gained nor lost weight after immersion for a period of 10 minutes. 2. In solutions of glucose, the P.A.I. of the slices was significantly lower at 4°C. than at 20°C. but similar at 20°C. and 37°C. Upon immersion for 15 to 60 minutes in 0.66 molar glucose the slices always swelled more at 37°C. than at 20°C. In solutions of sucrose change in the temperature of incubation was without effect on the hydration of the tissues. 3. In solutions of sodium chloride, the P.A.I. and the content of water, chloride, and sodium plus potassium were lower at 37°C. than at 20°C. 4. These findings emphasize the role of translocation of solute in providing an osmotic gradient for the movement of water between the tissue slices and the media.     

Temperature-Regulated Formation of Mycelial Mat-Like Biofilms by Legionella pneumophila

Fifty strains representing 38 species of the genus Legionella were examined for biofilm formation on glass, polystyrene, and polypropylene surfaces in static cultures at 25°C, 37°C, and 42°C. Strains of Legionella pneumophila, the most common causative agent of Legionnaires' disease, were found to have the highest ability to form biofilms among the test strains. The quantity, rate of formation, and adherence stability of L. pneumophila biofilms showed considerable dependence on both temperature and surface material. Glass and polystyrene surfaces gave between two- to sevenfold-higher yields of biofilms at 37°C or 42°C than at 25°C; conversely, polypropylene surface had between 2 to 16 times higher yields at 25°C than at 37°C or 42°C. On glass surfaces, the biofilms were formed faster but attached less stably at 37°C or 42°C than at 25°C. Both scanning electron microscopy and confocal laser scanning microscopy revealed that biofilms formed at 37°C or 42°C were mycelial mat like and were composed of filamentous cells, while at 25°C, cells were rod shaped. Planktonic cells outside of biofilms or in shaken liquid cultures were rod shaped. Notably, the filamentous cells were found to be multinucleate and lacking septa, but a recA null mutant of L. pneumophila was unaffected in its temperature-regulated filamentation within biofilms. Our data also showed that filamentous cells were able to rapidly give rise to a large number of short rods in a fresh liquid culture at 37°C. The possibility of this biofilm to represent a novel strategy by L. pneumophila to compete for proliferation among the environmental microbiota is discussed.     

RNA METABOLISM IN HELA CELLS AT REDUCED TEMPERATURE : I. Modified Processing of 45S RNA

Incubation of HeLa cells at suboptimal temperature has been used to study the synthesis of 45S ribosomal RNA precursor and the individual steps of the subsequent processing to 28S RNA. Below 20°C no detectable 45S RNA is formed. The processing of 45S RNA to 32S RNA ceases around 15°C, and the processing of 32S RNA to 28S RNA is inhibited near 25°C. Prolonged incubation at reduced temperature results in further modification of the processing, resulting in the apparent accumulation of 41S RNA. The products of these reactions at reduced temperature appear normal in that the ribosomal RNA made at 27°C can be isolated from functional polyribosomes in the cytoplasm after a short incubation at 37°C.     

Effects of Abiotic Factors on Acetylene Reduction by Cyanobacteria Epiphytic on Moss at a Subantarctic Island

Acetylene reduction (AR) rates by cyanobacteria epiphytic on a moss at Marion Island (46°54′ S, 37°45′ E) increased from −5°C to a maximum at 25 to 27°C. Q₁₀ values between 0 and 25°C were between 2.3 and 2.9, depending on photosynthetic photon flux density. AR rates declined sharply at temperatures above the optimum and were lower at 35°C than at 0°C. Photosynthetic photon flux density at low levels markedly influenced AR, and half of the maximum rate occurred at 84 μmol m⁻² s⁻¹, saturation occurring at ca. 1,000 μmol m⁻² s⁻¹. Higher photosynthetic photon flux density levels decreased AR rates. AR increased up to the highest sample moisture content investigated (3,405%), and the pH optimum was between 5.9 and 6.2. The addition of P, Co, and Mo, individually or together, depressed AR.