Archaeal alcohol dehydrogenase active at increased temperatures and in the presence of organic solvents

The adhA gene of the extreme thermoacidophilic Archaeon Picrophilus torridus was identified by the means of genome analysis and was subsequently cloned in Escherichia coli. PTO 0846, encoding AdhA, consists of 954 bp corresponding to 317 aa. Sequence comparison revealed that the novel biocatalyst has a low sequence identity (<26%) to previously characterized enzymes. The recombinant alcohol dehydrogenase was purified using hydroxyapatite, and alcohol oxidative activity of the purified AdhA was measured over a wide pH and temperature range with maximal activity at 83°C and pH 7.8. Detailed analysis suggests that the active AdhA is a multimer, consisting of 12 identical subunits, with a molecular mass of 35 kDa each. AdhA represents the first dodecameric alcohol dehydrogenase characterized until to date. AdhA is able to oxidize primary and secondary alcohols with ethanol and 1-phenylalcohol as preferred substrates and NAD⁺ as preferred cofactor. In addition, isopropanol, which has been used successfully as cosubstrate in cofactor regeneration, is oxidized as well by AdhA. Besides being thermostable (t _1/2 = 42 min at 70°C), AdhA is also active in the presence of increased concentrations of urea (up to 5 M) and in the presence of organic solvents [up to 50% (v/v)] commonly used for organic synthesis.     

Excited state properties of dinaphthyl ditelluride

Di-1-naphthyl ditelluride (Te₂naphthyl₂) is characterized by two low-energy excited states. The corresponding electronic transitions nTe → σ¹ Te–Te and nTe → π¹ naphthyl CT give rise to absorptions at λ_max = 403 and 311 nm, respectively. In solution nTe → σ¹ excitation leads to the cleavage of the Te–Te bond. In contrast to Te₂naphthyl₂ in the dissolved state the solid compound shows a luminescence (λ_max = 576 nm) which originates from nTe → π¹ naphthyl CT triplet.     

Calorimetric properties of water and triacylglycerols in fern spores relating to storage at cryogenic temperatures

Storing spores is a promising method to conserve genetic diversity of ferns ex situ. Inappropriate water contents or damaging effects of triacylglycerol (TAG) crystallization may cause initial damage and deterioration with time in spores placed at -15 degrees C or liquid nitrogen temperatures. We used differential scanning calorimetry (DSC) to monitor enthalpy and temperature of water and TAG phase transitions within spores of five fern species: Pteris vittata, Thelypteris palustris, Dryopteris filix-mas, Polystichum aculeatum, Polystichum setiferum. The analyses suggested that these fern spores contained between 26% and 39% TAG, and were comprised of mostly oleic (P. vittata) or linoleic acid (other species) depending on species. The water contents at which water melting events were first observable ranged from 0.06 (P. vittata) to 0.12 (P. setiferum)gH(2)Og(-1)dry weight, and were highly correlated with water affinity parameters. In spores containing more than 0.09 (P. vittata) to 0.25 (P. setiferum)gH(2)Og(-1)dry weight, some water partitioned into a near pure water fraction that melted at about 0 degrees C. These sharp peaks near 0 degrees C were associated with lethal freezing treatments. The enthalpy of water melting transitions was similar in fern spores, pollen and seeds; however, the unfrozen water content was much lower in fern spores compared to other forms of germplasm. Though there is a narrow range of water contents appropriate for low temperature storage of fern spores, water content can be precisely manipulated to avoid both desiccation and freezing damage.     

Electron transfer in photosystem II at cryogenic temperatures

The photochemistry in photosystem II of spinach has been characterized by electron paramagnetic resonance (EPR) spectroscopy in the temperature range of 77-235 K, and the yields of the photooxidized species have been determined by integration of their EPR signals. In samples treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), a single stable charge separation occurred throughout the temperature range studied as reflected by the constant yield of the Fe(II)-QA-EPR signal. Three distinct electron donation pathways were observed, however. Below 100 K, one molecule of cytochrome b559 was photooxidized per reaction center. Between 100 and 200 K, cytochrome b559 and the S1 state competed for electron donation to P680+. Photooxidation of the S1 state occurred via two intermediates: the g = 4.1 EPR signal species first reported by Casey and Sauer [Casey, J. L., & Sauer, K. (1984) Biochim. Biophys. Acta 767, 21-28] was photooxidized between 100 and 160 K, and upon being warmed to 200 K in the dark, this EPR signal yielded the multiline EPR signal associated with the S2-state. Only the S1 state donated electrons to P680+ at 200 K or above, giving rise to the light-induced S2-state multiline EPR signal. These results demonstrate that the maximum S2-state multiline EPR signal accounts for 100% of the reaction center concentration. In samples where electron donation from cytochrome b559 was prevented by chemical oxidation, illumination at 77 K produced a radical, probably a chlorophyll cation, which accounted for 95% of the reaction center concentration. This electron donor competed with the S1 state for electron donation to P680+ below 100 K.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanical testing of spider silk at cryogenic temperatures

A method and results for mechanical testing of spider silk in extreme environments is presented. In particular, silk from the spider Steatoda triangulosa is harvested, and samples are subjected to cryogenic temperatures by means of liquid nitrogen submersion. Samples are destructively tested while immersed in liquid nitrogen, and the stress-strain characteristics are compared to those of silk at room temperature. The strength, elasticity, and toughness of the cryogenically submersed silk are determined. It is found that on average, silk is 64% stronger while immersed in liquid nitrogen (i.e., at -196°C). The testing method could also be used for testing of silk in chemically hostile environments.     

Enhancement of transesterification-catalyzing capability of bio-imprinted tannase in organic solvents by cryogenic protection and immobilization

Improvement of transesterification-catalyzing capability of bio-imprinted tannase is a crucial question of whether to be efficiently utilized in organic media. As for biotransformation of tannic acid to propyl gallate, bio-imprinting technique can dramatically enhance the transesterification-catalyzing capability of tannase. In this work, both cryogenic protection and immobilization were utilized to further improve its apparent catalytic capability in organic media. The results show that Triton-X-100, mannose, and magnesium ion all have a positive effect on cryogenic protection of the tannase. Particularly, combinational application of the three cryoprotectants increases its catalytic performance by 2.7-fold factor. Also, immobilization further elevates its catalytic capability by 2.1 folds. Noteworthily, the coupling application of immobilization and cryo-protection can cause the conversion rate of substrate of the bio-imprinted tannase to increase to a promising 70%. Consequently, it will be helpful to fully utilize tannase in organic phase.     

Extended stability of restriction enzymes at ambient temperatures

The stability of restriction enzymes as supplied by manufacturers without any modification has been examined. No reduction in activity was observed for three enzymes (HindIII, EcoRI and Tsp509I) held at ambient temperature or 4 degrees C for the period of study (12 months), while activity was observed for up to 12 weeks after storage at 37 degrees C, which was considerably better than following desiccation with trehalose, a recognized preservation technique. A larger trial of 23 different restriction enzymes held at room temperature for one week showed that all enzymes retained significant activity. As a practical demonstration of the usefulness of this finding, enzymes were posted to Africa by conventional mail (cost $1 US) and shown to retain activity upon arrival after three weeks in transit (compared to a cost of $1000 US by cold-chain transportation). Supplying enzymes to third-world markets should now be possible by removing the necessity for cold-chain transport. After arrival, enzymes can simply be stored in a standard domestic refrigerator.     

Cytotoxicity of commonly used solvents at elevated temperatures

At 43 degrees C (but not at 41 degrees C), organic solvents used to dissolve water-insoluble chemotherapeutic agents become themselves lethal to cells. This finding is not unique to Chinese hamster cells (HA-1); mouse mammary sarcoma cells (EMT-6) behave similarly. The solvent concentrations involved are in the range of those needed to make drug solutions. Hence experiments measuring drug-cell interactions at elevated temperatures must include controls which independently measure solvent effects.     

A quantitative analysis of the thermal properties of porcine liver with glycerol at subzero and cryogenic temperatures

There is a lack of information on the effect of cryoprotective agents (CPAs) on the thermal properties of biomaterials at cryobiologically relevant temperatures (i.e. <233.15 K, −40 °C). Thermal properties that are of most interest include: thermal conductivity, density, specific heat, and latent heat resulting from phase change in tissue systems. Availability of such information would be beneficial for accurate mathematical modeling of cryobiological applications. Recently, we reported these thermal properties in phosphate buffered saline (PBS) with varying concentrations of glycerol, a widely used cryoprotective agent. In this study we extend these results by assessing the effects of glycerol on the thermal properties of porcine liver at subzero temperatures. Differential scanning calorimeter (DSC) was used to measure the specific heat and the latent heat release of porcine liver immersed in PBS and varying concentrations of glycerol. The specific heat data obtained from the DSC experiments were also used to predict the bulk thermal conductivity. This was done using a transient heat transfer model with a thermistor probe technique. Results show that the introduction of glycerol significantly alters thermal properties from known values for H₂O and non-treated liver. Therefore, inaccuracies in thermal predictions can be expected due to the application of measured vs. predicted thermal properties such as from weight averaging. This supports the need for these and other measurements of biomaterial thermal properties, with and without CPA addition, in the cryogenic regime.     

Spectral and catalytic properties of cytochrome oxidase in organic solvents

Isolated bovine heart cytochrome oxidase has been extracted into n-hexane, probably in reverse micelles, by the use of asolectin and calcium. The diluted extracts are composed of particles with the hydrodynamic radius of 42 nm. Spectral characteristics of the extracted oxidase are similar to those in aqueous solutions. At the high molar ratio of water to phospholipid (W0 = 8) in an organic solvent both cytochrome a and a3 are reducible and oxygen uptake is observed. However, at low W0 (W0 = 1.8) the rate of cytochrome a reduction is decreased and reduction of cytochrome a3 is inhibited.     

Oxygen consumption of brown noddy (Anous stolidus) embryos in a quasiequilibrium state at lowered ambient temperatures

1. The oxygen consumption (MO2) of the semi-precocial Brown Noddy embryos at different stages of development was measured at 36 degrees C and again after 5-hr exposure to lowered ambient temperatures (30 and 32 degrees C). 2. The MO2 measured in a quasiequilibrium state was equal to the value predicted by a temperature coefficient of 2. 3. In contrast to precocial chickens, the semi-precocial Noddy had no apparent metabolic response to cooling before hatching.     

Macromolecular cryocrystallography--methods for cooling and mounting protein crystals at cryogenic temperatures

Cryocrystallography is routinely used in macromolecular crystallography laboratories. The main advantage of X-ray diffraction data collection near 100K is that crystals display much less radiation damage than seen at room temperature. Techniques and tools are described to facilitate cryoprotecting and flash-cooling crystals for data collection.     

Comparability of tympanic and oral mercury thermometers at high ambient temperatures

ABSTRACT: BACKGROUND: Body temperature can be measured in seconds with tympanic thermometers as opposed to minutes with mercury ones. The aim of this study was to compare tympanic and oral mercury thermometer measurements under high ambient field temperatures. RESULTS: Tympanic temperature (measured thrice by 3 operators) was compared to oral temperature measured once with a mercury-in-glass thermometer in 201 patients (aged [greater than or equal to]5 years), on the Thai-Myanmar border. Ambient temperature was measured with an electronic thermo-hygrometer. Participants had a mean [min-max] age of 27 [5-60] years and 42% (84) were febrile by oral thermometer. The mean difference in the mercury and tympanic temperature measurement for all observers/devices was 0.09 (95%CI 0.07-0.12)degreesC and intra-class correlation for repeat tympanic measurements was high ([greater than or equal to]0.97) for each observer. Deviations in tympanic temperatures were not related to ambient temperature. CONCLUSION: Clinically significant differences were not observed between oral and tympanic temperature measurements at high ambient temperatures in a rural tropical setting.