The promise and potential of biotechnology rests, ultimately, on our understanding of biological ... more The promise and potential of biotechnology rests, ultimately, on our understanding of biological processes at a molecular level. Such understanding allows manipulation and modulation of complex biochemical, metabolic and genetic processes in microorganisms, plants, animals and humans. The vast beneficial potential in manipulating biological processes and systems derives from the diversity of biological organisms and their inherent versatility and adaptability, as reflected in their validation of Darwinian paradigms concerning evolution and natural selection. Within living systems, the obviously chaotic nature of diversity based biological environments is transformed into complex and intricate physiological structures and biochemical pathways based on spatial and temporal organization of an organism’s biochemical components. Utilizing structures and pathways, the myriad possible chemical consequences inherent in a collection of molecules in living systems reduces to organized metabolic processes. This ordered biochemical architecture in living systems is based almost entirely on molecular recognition and biospecificity. It is, therefore, through intervention of selective effectors, inhibitors, substrates, hormones and other pairs of transmitter and receptor molecules that living organisms maintain control over the course of chemical change within their systems. Examples of such control range from control of gene expression to self/non self discrimination via the immune system. Biotechnology, in essence, attempts to exploit the known factors affecting the flow of chemical information in biological systems for human use.
An entry from the Cambridge Structural Database, the world's repository for small molecule cr... more An entry from the Cambridge Structural Database, the world's repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.
Antifreeze proteins (AFPs) lower the non-equilibrium freezing point of water (in the presence of ... more Antifreeze proteins (AFPs) lower the non-equilibrium freezing point of water (in the presence of ice) below the melting point, thereby producing a difference between the freezing and melting points that has been termed thermal hysteresis. In general, the magnitude of the thermal hysteresis depends upon the specific activity and concentration of the AFP. This study describes several low-molecular-mass solutes that enhance the thermal hysteresis activity of an AFP from overwintering larvae of the beetle Dendroides canadensis. The most active of these is citrate, which increases the thermal hysteresis nearly sixfold from 1.2 degrees C in its absence to 6.8 degrees C. Solutes which increase activity approximately fourfold are succinate, malate, aspartate, glutamate and ammonium sulfate. Glycerol, sorbitol, alanine and ammonium bicarbonate increased thermal hysteresis approximately threefold. Interestingly, 0.5 mol l-1 sodium sulfate eliminated activity. Solute concentrations between 0.2...
Overwintering larvae of the beetle Dendroides canadensis produce potent antifreeze proteins to in... more Overwintering larvae of the beetle Dendroides canadensis produce potent antifreeze proteins to inhibit inoculative freezing and promote supercooling. We hypothesized that addition of Dendroides canadensis recombinant antifreeze proteins (DAFPs) in the extender will improve the quality and fertility of cryopreserved Nili-Ravi buffalo (Bubalus bubalis) sperm. The study was divided into two parts: (1) Evaluation of the effect of DAFPs on the quality of frozen-thawed buffalo bull sperm and (2) Examination of the fertilizing ability of frozen-thawed buffalo bull sperm. Semen was collected from three bulls using an artificial vagina (42 °C). Qualifying ejaculates from each bull were divided into four aliquots and diluted (at 37 °C, 50 × 10(6) sperm/mL) in tris-citric acid extender containing DAFP (at 0.1, 1.0, and 10 μg/mL), and the sperm were evaluated for important characteristics relative to a control without DAFP. D canadensis recombinant antifreeze proteins at any of the three concen...
SUMMARY Most aquatic insects do not survive subzero temperatures and, for those that do, the phys... more SUMMARY Most aquatic insects do not survive subzero temperatures and, for those that do, the physiology has not been well characterized. Nemoura arctica is a species of stonefly widely distributed throughout arctic and subarctic Alaska. We collected nymphs from the headwaters of the Chandalar River, where we recorded streambed temperatures as low as –12.7°C in midwinter. When in contact with ice, autumn-collected N. arctica cool to –1.5±0.4°C before freezing, but individuals survived temperatures as low as –15°C, making this the first described species of freeze-tolerant stonefly. N. arctica clearly survive freezing in nature, as winter-collected nymphs encased in ice demonstrated high survivorship when thawed. In the laboratory, 87% of N. arcticanymphs frozen to –15°C for 2.5 weeks survived and, within one month of thawing, 95% of the last-instar nymphs emerged. N. arctica produce both glycerol and ice-binding factors (e.g. antifreeze protein) in response to low temperature. Hemoly...
Proceedings of the National Academy of Sciences, 2009
Thermal hysteresis (TH), a difference between the melting and freezing points of a solution that ... more Thermal hysteresis (TH), a difference between the melting and freezing points of a solution that is indicative of the presence of large-molecular-mass antifreezes (e.g., antifreeze proteins), has been described in animals, plants, bacteria, and fungi. Although all previously described TH-producing biomolecules are proteins, most thermal hysteresis factors (THFs) have not yet been structurally characterized, and none have been characterized from a freeze-tolerant animal. We isolated a highly active THF from the freeze-tolerant beetle, Upis ceramboides , by means of ice affinity. Amino acid chromatographic analysis, polyacrylamide gel electrophoresis, UV-Vis spectrophotometry, and NMR spectroscopy indicated that the THF contained little or no protein, yet it produced 3.7 ± 0.3 °C of TH at 5 mg/ml, comparable to that of the most active insect antifreeze proteins. Compositional and structural analyses indicated that this antifreeze contains a β-mannopyranosyl-(1→4) β-xylopyranose backbo...
Journal of Comparative Physiology B: Biochemical, Systemic, and Environmental Physiology, 2002
Antifreeze proteins depress the freezing point of water while not affecting the melting point, pr... more Antifreeze proteins depress the freezing point of water while not affecting the melting point, producing a characteristic difference in freezing and melting points termed thermal hysteresis. Larvae of the beetle Dendroides canadensis accumulate potent antifreeze proteins (DAFPs) in their hemolymph and gut, but to achieve high levels of thermal hysteresis requires enhancers, such as glycerol. DAFPs have previously been shown to inhibit the activity of bacterial and hemolymph protein ice nucleators, however, the effect was not large and therefore the effectiveness of the DAFPs in promoting supercooling of the larvae in winter was doubtful. However, this study demonstrates that DAFPs, in combination with the thermal hysteresis enhancers glycerol (1 M) or citrate (0.5 M), eliminated the activity of hemolymph protein ice nucleators and Pseudomonas syringae ice-nucleating active bacteria, and lowered the supercooling points (nucleation temperatures) of aqueous solutions containing these ice nucleators to those of water or buffer alone. This shows that the DAFPs, along with glycerol, play a critical role in promoting hemolymph supercooling in overwintering D. canadensis. Also, DAFPs in combination with enhancers may be useful in applications which require inhibition of ice nucleators.
Ice structuring proteins (ISPs) protect organisms from damage or death by freezing. They depress ... more Ice structuring proteins (ISPs) protect organisms from damage or death by freezing. They depress the non-equilibrium freezing point of water and prevent recrystallization, probably by binding to the surface of ice crystals. Many ISPs have been described and it is likely that many more exist in nature that have not yet been identified. ISPs come in many forms and thus cannot be reliably identified by their structure or consensus ice-binding motifs. Recombinant protein expression is the gold standard for proving the activity of a candidate ISP. Among existing expression systems, cell-free protein expression is the simplest and gives the fastest access to the protein of interest, but selection of the appropriate cell-free expression system is crucial for functionality. Here we describe cell-free expression methods for three ISPs that differ widely in structure and glycosylation status from three organisms: a fish (Macrozoarces americanus), an insect (Dendroides canadensis) and an alga (Chlamydomonas sp. CCMP681). We use both prokaryotic and eukaryotic expression systems for the production of ISPs. An ice recrystallization inhibition assay is used to test functionality. The techniques described here should improve the success of cell-free expression of ISPs in future applications.
SUMMARY Larvae of the freeze-avoiding beetle Cucujus clavipes puniceus (Coleoptera: Cucujidae) in... more SUMMARY Larvae of the freeze-avoiding beetle Cucujus clavipes puniceus (Coleoptera: Cucujidae) in Alaska have mean supercooling points in winter of –35 to –42°C, with the lowest supercooling point recorded for an individual of –58°C. We previously noted that some larvae did not freeze when cooled to –80°C, and we speculated that these larvae vitrified. Here we present evidence through differential scanning calorimetry that C. c. puniceus larvae transition into a glass-like state at temperatures <–58°C and can avoid freezing to at least –150°C. This novel finding adds vitrification to the list of insect overwintering strategies. While overwintering beneath the bark of fallen trees, C. c. puniceus larvae may experience low ambient temperatures of around –40°C (and lower) when microhabitat is un-insulated because of low snow cover. Decreasing temperatures in winter are correlated with loss of body water from summer high levels near 2.0 to winter lows near 0.4 mg mg–1 dry mass and co...
Ice-binding proteins (IBPs) assist in subzero tolerance of multiple cold-tolerant organisms: anim... more Ice-binding proteins (IBPs) assist in subzero tolerance of multiple cold-tolerant organisms: animals, plants, fungi, bacteria etc. IBPs include: (1) antifreeze proteins (AFPs) with high thermal hysteresis antifreeze activity; (2) low thermal hysteresis IBPs; and (3) ice-nucleating proteins (INPs). Several structurally different IBPs have evolved, even within related taxa. Proteins that produce thermal hysteresis inhibit freezing by a non-colligative mechanism, whereby they adsorb onto ice crystals or ice-nucleating surfaces and prevent further growth. This lowers the so-called hysteretic freezing point below the normal equilibrium freezing/melting point, producing a difference between the two, termed thermal hysteresis. True AFPs with high thermal hysteresis are found in freeze-avoiding animals (those that must prevent freezing, as they die if frozen) especially marine fish, insects and other terrestrial arthropods where they function to prevent freezing at temperatures below those ...
... John G. Duman and T. Mark Olsen. ... fulva, day-dily, bulbs; Populus deltoides, eastern cotto... more ... John G. Duman and T. Mark Olsen. ... fulva, day-dily, bulbs; Populus deltoides, eastern cottonwood; Quercus alba, white oak, acorns; and Triticum aestivum, winter wheat), thermal hysteresis activity (THA) was demonstrated in the evergreen Christmas fern (Polystichum ...
The haemolymph of larvae of Tenebrio molitor contains a factor which produces a thermal hysteresi... more The haemolymph of larvae of Tenebrio molitor contains a factor which produces a thermal hysteresis (a difference between the freezing and melting points) of approximately 0.75 °C. When larvae were acclimated to low temperatures or short photoperiod the thermal hysteresis increased more than twofold. Coincident with the increase in thermal hysteresis the supercooling points and lower lethal temperatures of the larvae were depressed. Therefore, the thermal-hysteresis-producing factor seems to function as an antifreeze. The factor may also act as an adaptation to prevent desiccation. Thermal hysteresis increased almost three-fold in larvae acclimated to low relative humidity. Also, larvae with high levels of the thermal hysteresis factor survived low relative humidities much better than did larvae with lower levels.
The promise and potential of biotechnology rests, ultimately, on our understanding of biological ... more The promise and potential of biotechnology rests, ultimately, on our understanding of biological processes at a molecular level. Such understanding allows manipulation and modulation of complex biochemical, metabolic and genetic processes in microorganisms, plants, animals and humans. The vast beneficial potential in manipulating biological processes and systems derives from the diversity of biological organisms and their inherent versatility and adaptability, as reflected in their validation of Darwinian paradigms concerning evolution and natural selection. Within living systems, the obviously chaotic nature of diversity based biological environments is transformed into complex and intricate physiological structures and biochemical pathways based on spatial and temporal organization of an organism’s biochemical components. Utilizing structures and pathways, the myriad possible chemical consequences inherent in a collection of molecules in living systems reduces to organized metabolic processes. This ordered biochemical architecture in living systems is based almost entirely on molecular recognition and biospecificity. It is, therefore, through intervention of selective effectors, inhibitors, substrates, hormones and other pairs of transmitter and receptor molecules that living organisms maintain control over the course of chemical change within their systems. Examples of such control range from control of gene expression to self/non self discrimination via the immune system. Biotechnology, in essence, attempts to exploit the known factors affecting the flow of chemical information in biological systems for human use.
An entry from the Cambridge Structural Database, the world's repository for small molecule cr... more An entry from the Cambridge Structural Database, the world's repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.
Antifreeze proteins (AFPs) lower the non-equilibrium freezing point of water (in the presence of ... more Antifreeze proteins (AFPs) lower the non-equilibrium freezing point of water (in the presence of ice) below the melting point, thereby producing a difference between the freezing and melting points that has been termed thermal hysteresis. In general, the magnitude of the thermal hysteresis depends upon the specific activity and concentration of the AFP. This study describes several low-molecular-mass solutes that enhance the thermal hysteresis activity of an AFP from overwintering larvae of the beetle Dendroides canadensis. The most active of these is citrate, which increases the thermal hysteresis nearly sixfold from 1.2 degrees C in its absence to 6.8 degrees C. Solutes which increase activity approximately fourfold are succinate, malate, aspartate, glutamate and ammonium sulfate. Glycerol, sorbitol, alanine and ammonium bicarbonate increased thermal hysteresis approximately threefold. Interestingly, 0.5 mol l-1 sodium sulfate eliminated activity. Solute concentrations between 0.2...
Overwintering larvae of the beetle Dendroides canadensis produce potent antifreeze proteins to in... more Overwintering larvae of the beetle Dendroides canadensis produce potent antifreeze proteins to inhibit inoculative freezing and promote supercooling. We hypothesized that addition of Dendroides canadensis recombinant antifreeze proteins (DAFPs) in the extender will improve the quality and fertility of cryopreserved Nili-Ravi buffalo (Bubalus bubalis) sperm. The study was divided into two parts: (1) Evaluation of the effect of DAFPs on the quality of frozen-thawed buffalo bull sperm and (2) Examination of the fertilizing ability of frozen-thawed buffalo bull sperm. Semen was collected from three bulls using an artificial vagina (42 °C). Qualifying ejaculates from each bull were divided into four aliquots and diluted (at 37 °C, 50 × 10(6) sperm/mL) in tris-citric acid extender containing DAFP (at 0.1, 1.0, and 10 μg/mL), and the sperm were evaluated for important characteristics relative to a control without DAFP. D canadensis recombinant antifreeze proteins at any of the three concen...
SUMMARY Most aquatic insects do not survive subzero temperatures and, for those that do, the phys... more SUMMARY Most aquatic insects do not survive subzero temperatures and, for those that do, the physiology has not been well characterized. Nemoura arctica is a species of stonefly widely distributed throughout arctic and subarctic Alaska. We collected nymphs from the headwaters of the Chandalar River, where we recorded streambed temperatures as low as –12.7°C in midwinter. When in contact with ice, autumn-collected N. arctica cool to –1.5±0.4°C before freezing, but individuals survived temperatures as low as –15°C, making this the first described species of freeze-tolerant stonefly. N. arctica clearly survive freezing in nature, as winter-collected nymphs encased in ice demonstrated high survivorship when thawed. In the laboratory, 87% of N. arcticanymphs frozen to –15°C for 2.5 weeks survived and, within one month of thawing, 95% of the last-instar nymphs emerged. N. arctica produce both glycerol and ice-binding factors (e.g. antifreeze protein) in response to low temperature. Hemoly...
Proceedings of the National Academy of Sciences, 2009
Thermal hysteresis (TH), a difference between the melting and freezing points of a solution that ... more Thermal hysteresis (TH), a difference between the melting and freezing points of a solution that is indicative of the presence of large-molecular-mass antifreezes (e.g., antifreeze proteins), has been described in animals, plants, bacteria, and fungi. Although all previously described TH-producing biomolecules are proteins, most thermal hysteresis factors (THFs) have not yet been structurally characterized, and none have been characterized from a freeze-tolerant animal. We isolated a highly active THF from the freeze-tolerant beetle, Upis ceramboides , by means of ice affinity. Amino acid chromatographic analysis, polyacrylamide gel electrophoresis, UV-Vis spectrophotometry, and NMR spectroscopy indicated that the THF contained little or no protein, yet it produced 3.7 ± 0.3 °C of TH at 5 mg/ml, comparable to that of the most active insect antifreeze proteins. Compositional and structural analyses indicated that this antifreeze contains a β-mannopyranosyl-(1→4) β-xylopyranose backbo...
Journal of Comparative Physiology B: Biochemical, Systemic, and Environmental Physiology, 2002
Antifreeze proteins depress the freezing point of water while not affecting the melting point, pr... more Antifreeze proteins depress the freezing point of water while not affecting the melting point, producing a characteristic difference in freezing and melting points termed thermal hysteresis. Larvae of the beetle Dendroides canadensis accumulate potent antifreeze proteins (DAFPs) in their hemolymph and gut, but to achieve high levels of thermal hysteresis requires enhancers, such as glycerol. DAFPs have previously been shown to inhibit the activity of bacterial and hemolymph protein ice nucleators, however, the effect was not large and therefore the effectiveness of the DAFPs in promoting supercooling of the larvae in winter was doubtful. However, this study demonstrates that DAFPs, in combination with the thermal hysteresis enhancers glycerol (1 M) or citrate (0.5 M), eliminated the activity of hemolymph protein ice nucleators and Pseudomonas syringae ice-nucleating active bacteria, and lowered the supercooling points (nucleation temperatures) of aqueous solutions containing these ice nucleators to those of water or buffer alone. This shows that the DAFPs, along with glycerol, play a critical role in promoting hemolymph supercooling in overwintering D. canadensis. Also, DAFPs in combination with enhancers may be useful in applications which require inhibition of ice nucleators.
Ice structuring proteins (ISPs) protect organisms from damage or death by freezing. They depress ... more Ice structuring proteins (ISPs) protect organisms from damage or death by freezing. They depress the non-equilibrium freezing point of water and prevent recrystallization, probably by binding to the surface of ice crystals. Many ISPs have been described and it is likely that many more exist in nature that have not yet been identified. ISPs come in many forms and thus cannot be reliably identified by their structure or consensus ice-binding motifs. Recombinant protein expression is the gold standard for proving the activity of a candidate ISP. Among existing expression systems, cell-free protein expression is the simplest and gives the fastest access to the protein of interest, but selection of the appropriate cell-free expression system is crucial for functionality. Here we describe cell-free expression methods for three ISPs that differ widely in structure and glycosylation status from three organisms: a fish (Macrozoarces americanus), an insect (Dendroides canadensis) and an alga (Chlamydomonas sp. CCMP681). We use both prokaryotic and eukaryotic expression systems for the production of ISPs. An ice recrystallization inhibition assay is used to test functionality. The techniques described here should improve the success of cell-free expression of ISPs in future applications.
SUMMARY Larvae of the freeze-avoiding beetle Cucujus clavipes puniceus (Coleoptera: Cucujidae) in... more SUMMARY Larvae of the freeze-avoiding beetle Cucujus clavipes puniceus (Coleoptera: Cucujidae) in Alaska have mean supercooling points in winter of –35 to –42°C, with the lowest supercooling point recorded for an individual of –58°C. We previously noted that some larvae did not freeze when cooled to –80°C, and we speculated that these larvae vitrified. Here we present evidence through differential scanning calorimetry that C. c. puniceus larvae transition into a glass-like state at temperatures <–58°C and can avoid freezing to at least –150°C. This novel finding adds vitrification to the list of insect overwintering strategies. While overwintering beneath the bark of fallen trees, C. c. puniceus larvae may experience low ambient temperatures of around –40°C (and lower) when microhabitat is un-insulated because of low snow cover. Decreasing temperatures in winter are correlated with loss of body water from summer high levels near 2.0 to winter lows near 0.4 mg mg–1 dry mass and co...
Ice-binding proteins (IBPs) assist in subzero tolerance of multiple cold-tolerant organisms: anim... more Ice-binding proteins (IBPs) assist in subzero tolerance of multiple cold-tolerant organisms: animals, plants, fungi, bacteria etc. IBPs include: (1) antifreeze proteins (AFPs) with high thermal hysteresis antifreeze activity; (2) low thermal hysteresis IBPs; and (3) ice-nucleating proteins (INPs). Several structurally different IBPs have evolved, even within related taxa. Proteins that produce thermal hysteresis inhibit freezing by a non-colligative mechanism, whereby they adsorb onto ice crystals or ice-nucleating surfaces and prevent further growth. This lowers the so-called hysteretic freezing point below the normal equilibrium freezing/melting point, producing a difference between the two, termed thermal hysteresis. True AFPs with high thermal hysteresis are found in freeze-avoiding animals (those that must prevent freezing, as they die if frozen) especially marine fish, insects and other terrestrial arthropods where they function to prevent freezing at temperatures below those ...
... John G. Duman and T. Mark Olsen. ... fulva, day-dily, bulbs; Populus deltoides, eastern cotto... more ... John G. Duman and T. Mark Olsen. ... fulva, day-dily, bulbs; Populus deltoides, eastern cottonwood; Quercus alba, white oak, acorns; and Triticum aestivum, winter wheat), thermal hysteresis activity (THA) was demonstrated in the evergreen Christmas fern (Polystichum ...
The haemolymph of larvae of Tenebrio molitor contains a factor which produces a thermal hysteresi... more The haemolymph of larvae of Tenebrio molitor contains a factor which produces a thermal hysteresis (a difference between the freezing and melting points) of approximately 0.75 °C. When larvae were acclimated to low temperatures or short photoperiod the thermal hysteresis increased more than twofold. Coincident with the increase in thermal hysteresis the supercooling points and lower lethal temperatures of the larvae were depressed. Therefore, the thermal-hysteresis-producing factor seems to function as an antifreeze. The factor may also act as an adaptation to prevent desiccation. Thermal hysteresis increased almost three-fold in larvae acclimated to low relative humidity. Also, larvae with high levels of the thermal hysteresis factor survived low relative humidities much better than did larvae with lower levels.
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