Enzyme activity down to -100 degrees C

The activities of two enzymes, beef liver catalase (EC 1.11.1.6) and calf intestine alkaline phosphatase (EC 3.1.3.1), have been measured down to -97 degrees C and -100 degrees C, respectively. Enzyme activity has not previously been measured at such low temperatures. For catalase, the cryosolvents used were methanol:ethylene glycol:water (70:10:20) and DMSO:ethylene glycol:water (60:20:20). For alkaline phosphatase, methanol:ethylene glycol:water (70:10:20) was used. All of the Arrhenius plots were linear over the whole of the temperature range examined. Since the lowest temperatures at which activity was measured are well below the dynamic transition observed for proteins, the results indicate that the motions which cease below the dynamic transition are not essential for enzyme activity. In all cases the use of cryosolvent led to substantial increases in Arrhenius activation energies, and this imposed practical limitations on the measurement of enzyme activity below -100 degrees C. At even lower temperatures, enzyme activity may be limited by the effect of solvent fluidity on substrate/product diffusion, but overall there is no evidence that any intrinsic enzyme property imposes a lower temperature limit for enzyme activity.     

Freeze-fracturing in ultrahigh vacuum at -196 degrees C

Conventional freeze-etching is carried out in a vacuum of approximately 10(-6) torr and at a specimen temperature of -100 degrees C. The relatively poor topographic resolution of most freeze-etch replicas, and the lack of complementarity of morphological details in double replicas have been thought to be caused by structural distortions during fracturing, and radiation damage during replication. Both phenomena can be reduced by lowering the specimen temperature. To prevent condensation of residual gases (especially H2O) on the fracture faces at lower specimen temperature, an improved vacuum is required. Therefore, an ultrahigh vacuum freeze-fracture apparatus has been developed which allows fracturing and Pt/C-shadowing of specimens at -196 degrees C while maintaining a vacuum of 10(-9) torr. It consists of a modified Balzers BA 350 ultrahigh vacuum (UHV) unit, equipped with an airlock which enables the input of nonhoar-frosted specimens directly into the evacuated bell jar. A comparison of the paracrystalline plasmalemma structure in yeast cells portrayed by the conventional technique and by UHV-freeze-fracturing at -196 degrees C shows the improved topographic resolution which has been achieved with the new technique. The improvement is explained by less structural distortions during fracturing at lower temperatures. The particles of the paracrystalline regions on the P face are more regularly arranged and exhibit a craterlike substructure which corresponds with a ringlike depression in the E face. The optical diffraction patterns of these paracrystalline regions demonstrate the improvement of the structural record by showing well-defined third- and fourth-order spots.     

An evaluation of five preservation techniques and conventional freezing temperatures of -20 degrees C and -85 degrees C for long-term preservation of Campylobacter jejuni

AIMS: This study aimed to identify a simple, inexpensive preservation technique that will allow a quick and reliable recovery of Campylobacter jejuni following long-term periods of preservation. METHODS AND RESULTS: Preservation techniques include (i) Cryobank microbial preservation system using hypertonic 'cryopreservative solution' and glass beads, (ii) Cryobank microbial preservation system using defibrinated lysed horse blood and glass beads, (iii) FBP medium, (iv) 15% glycerol/85% nutrient broth no. 2 culture, and (v) 50% glycerol/50% nutrient broth no. 2 culture. Each preservation technique was evaluated over a 1-year period at conventional freezing temperatures of -20 degrees C and -85 degrees C. Replacement of 'cryopreservative fluid' in commercially prepared vials of glass beads with lysed horse blood increased the duration of preservation of Camp. jejuni by up to 6 months. CONCLUSIONS: FBP medium proved the most successful preservation technique with 100 and 80% recovery after 1 year at -85 degrees C and -20 degrees C, respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated a simple inexpensive preservation method for long-term storage of Camp. jejuni.     

Cryopreservation of mouse embryos at -196 degrees C by vitrification

Embryos (8-16 cell) were obtained from random bred albino mice (6-8 weeks old) that were induced to superovulate by injections of 5 I.U. PMSG and 5 I.U. hCG given 48 hr apart. Embryos were exposed to intracellular cryoprotecting medium (glycerol 10%, 1-2 propanediol 20% in PBS) for 10 min and then transferred to extracellular vitrification medium (25% glycerol, 25% 1-2 propanediol in PBS). Vitrification medium containing embryos, and diluent (1 M sucrose) were loaded in a straw and immediately plunged into liquid N2. After thawing at 20 degrees C, the contents of the straw were mixed by shaking (1 step dilution) and emptied in a petri dish. After 3 washings in culture medium the embryos were kept in CO2 incubator for further development. In 3-step dilution procedure the dilution of cryoprotectants was done in 0.5 and 0.25 M sucrose before culture. Embryos in 3-step dilution of cryoprotectants exhibited high survival as compared to 1-step dilution (20.23% vs 6.55%).     

Rapid-cooling and storage of plant nematodes at -140 degrees C

Low temperatures can assure the long-term or even indefinite preservation of important biological specimens. Nematode cryopreservation allows for the availability of large numbers of living nematodes at any one time, especially for experimental purposes. New isolates of Bursaphelenchus have recently been collected, including Bursaphelenchus eremus (Rühm) Goodey. This species was identified in north-central Italy on dying oak trees and from the bark beetle Scolytus intricatus Ratzeburg as dauer larvae. We therefore, sought to develop a cryopreservation technique for the long-term storage of all available Bursaphelenchus spp. The technique consists of a rapid-cooling protocol involving immersion in a liquid nitrogen bath before storage of the frozen samples in a mechanical freezer at -140 degrees C. The survival of nematodes subjected to this rapid-cooling protocol was higher than previously reported using slow-cooling methods and is suitable for several species of Bursaphelenchus and other phytoparasitic nematodes.     

Post-mortem changes in cytochemical localization and enzymological measurement of marker enzymes of the mitochondria, SDH and Mg-ATPase, of porcine muscle stored at 4 degrees C, -18 degrees C, or -80 degrees C

As a series of studies on postmortem changes in the fine structure of porcine muscle, activity of two mitochondrial marker enzymes, succinate dehydrogenase (SDH) and magnesium dependent adenosine triphosphatase (Mg-ATPase), was measured and localized in cardiac, red and white muscles stored at 4 degrees C, -18 degrees C or -80 degrees C. The postmortem loss of SDH activity was most remarkable in cardiac muscle. The variation of SDH activity was proportional to the amount of absolute activity. The postmortem change of Mg-ATPase was more variable than SFH, though the activity was well preserved up to 15 weeks in all three types of porcine muscle stored at -80 degrees C. The loss of Mg-ATPase was most remarkable in red muscle stored at -18 degrees C or -80 degrees C. Cytochemical localization of SDH was between the outer and the inner mitochondrial membranes while that of Mg-ATPase was on the inner surface or matrix side of the inner membrane. Those localization was not altered by the difference in temperature and the duration of storage.     

Nucleotide binding by myosin in glycerol-extracted rabbit skeletal muscle fibres at temperatures between -35 degrees C and 10 degrees C

Glycerol-extracted rabbit psoas fibres were incubated at temperatures between -35 degrees C and +10 degrees C in a low-ionic-strength relaxing solution containing 50% ethyleneglycol, 100 microM [3H]MgATP, 1 mM [14C]mannitol and less than 0.01 microM Ca2+. The fibres were then rinsed in a solution containing 1 mM ATP and the bound nucleotide eluted in trichloroacetic acid; all these operations were carried out at the cold temperature. Residual bound nucleotide was eluted with trichloroacetic acid at room temperature. The fibres were found to bind approximately 180 microM nucleotide, which is consistent with binding to the enzymatic site of myosin. The eluate, obtained in the cold, was analysed on poly(ethyleneimine)-cellulose for its ATP and ADP content. At temperatures down to -22 degrees C most of the bound nucleotide was ADP and there was little variation of this fraction with temperature. As the temperature was lowered below -22 degrees C the ATP fraction rose sharply; by -35 degrees C it predominated. These results are similar in type to those found by Biosca et al. [(1984) Biochemistry 23, 1947-1953] on isolated subfragment 1, but are displaced to a much lower temperature range. Thus in a muscle fibre only a low thermal energy is needed for myosin to hold its nucleotide in a constant balance between ATP and ADP.     

Temperature dependence of the microwave properties of aqueous solutions of ethylene glycol between +15 degrees C and -70 degrees C.

The values of the dielectric constant and of the loss tangent for samples of 0 m (distilled H₂O), 1 m, 2 m, 3 m, 4 m, 5 m, 10 m, and 100% ethylene glycol were determined over the temperature range of +15 °C to ?70 °C. An operating frequency of 1.40 GHz to 1.55 GHz was used, allowing the results to be applied directly to both 0.915 GHz and 2.450 GHz studies. Strong temperature and concentration dependencies were found; low concentration solutions tended to behave similarly to water, while higher concentration solutions were more independent. Peak values and discontinuities occurred at different temperatures, depending on concentration. Five molar ethylene glycol was indicated as a near optimum concentration based on toxicity, cryoprotection, and microwave power absorption considerations. A study of blends of cryoprotectants is suggested.     

Thermophilic (55-65 degrees C) and extreme thermophilic (70-80 degrees C) sulfate reduction in methanol and formate-fed UASB reactors

The feasibility of thermophilic (55-65 degrees C) and extreme thermophilic (70-80 degrees C) sulfate-reducing processes was investigated in three lab-scale upflow anaerobic sludge bed (UASB) reactors fed with either methanol or formate as the sole substrates and inoculated with mesophilic granular sludge previously not exposed to high temperatures. Full methanol and formate degradation at temperatures up to, respectively, 70 and 75 degrees C, were achieved when operating UASB reactors fed with sulfate rich (COD/SO4(2-)=0.5) synthetic wastewater. Methane-producing archaea (MPA) outcompeted sulfate-reducing bacteria (SRB) in the formate-fed UASB reactor at all temperatures tested (65-75 degrees C). In contrast, SRB outcompeted MPA in methanol-fed UASB reactors at temperatures equal to or exceeding 65 degrees C, whereas strong competition between SRB and MPA was observed in these reactors at 55 degrees C. A short-term (5 days) temperature increase from 55 to 65 degrees C was an effective strategy to suppress methanogenesis in methanol-fed sulfidogenic UASB reactors operated at 55 degrees C. Methanol was found to be a suitable electron donor for sulfate-reducing processes at a maximal temperature of 70 degrees C, with sulfide as the sole mineralization product of methanol degradation at that temperature.     

Magnesium chelation inactivates beta-galactosidase in -20 degrees C storage.

Mammalian cell lysate containing beta-galactosidase (beta Gal) derived from the transient expression of the bacterial lacZ gene driven by the human beta-actin promoter loses activity progressively over time in storage at -20 degrees C in the presence of EDTA. The simultaneous presence of NaCl with EDTA exacerbates such an inactivation, although NaCl by itself does not. However, EGTA, a chelating agent that preferentially binds Ca2+ over Mg2+, does not inactivate beta Gal. Addition of equal or higher molar concentration of Mg2+ (as MgCl2) or Ca2+ (as CaCl2), both effectively chelated by EDTA, to an EDTA-containing lysate prevents this cold-related inactivation, but does not reactivate the enzyme. Therefore, the chelation of Mg2+ by EDTA at -20 degrees C inactivates beta Gal. Storage of cell lysate at -70 degrees C completely prevents the EDTA-induced inactivation of beta Gal. It is recommended that when beta Gal activity is used as the reporter for gene expression 1) EDTA should not be used to prepare cell lysate and 2) the cell lysate should be stored in a -70 degrees C freezer to preserve full activity.     

Preservation at -195 degrees C of trypsinized organs for primary cultures

Trypsinized cells from rabbit kidney can be kept at -195, 8 degrees C indefinitely. When necessary, propagation in monolayer cultures is readily obtained as primary cultures, provided that some conditions be respected: 1) only glass culture flask must be used. Falcon plastic vessels do not allow cell attachment in this type of primary culture 2) the lag period is particularly long: it taken about 20 days before the beginning of cell multiplication 3) fetal calf serum is toxic for the cells in the 2nd and 3rd change of the culture medium, during the lag period.