- Email: [email protected]
107. l -Proline and ectoine stabilize proteins from denaturation
Abstracts / Cryobiology 63 (2011) 306–342 5 °C for 2 h. Straws were frozen in LN2 vapour and then stored at 196 °C. Samples were analyzed for motility using CASA (ISAS, Proiser; Valencia, Spain), membrane integrity (MI) with Sybr-14/PI staining, acrosome integrity (AI) with FITC-PSA, DNA fragmentation (sDF) with acridine orange and plasma membrane integrity in the tail (PMIT) by HOS test. Data were analysed by linear mixed-effects models. Cooling rate at 0.28 °C/min yielded the highest (P < 0.05) of spermatozoa with progressive motility, MI and AI to respect of the other treatments. No difference was found in HOS and DNA fragmentation. Results showed that cooling rate at 0.28 °C/min is the most adequate to cryopreserve Pelibuey ram sperm. Source of funding: Supported by CONACYT-FOMIX 107996. Conflicts of interest: None declared. doi:10.1016/j.cryobiol.2011.09.107
105. High throughput microwave processing of small biological samples for anhydrous storage. Stephanie Cellemme, Elisha Paramore, Mathew Van Vorst, Gloria D. Elliott *, Department of Mechanical Engineering and Engineering Science, 9201 University City Blvd., University of North Carolina at Charlotte, Charlotte, NC 28221-0001, USA Recently our group demonstrated that microwave processing can be used to reproducibly dehydrate samples for preservation in an anhydrous state, yielding samples that are more uniform in water content than those produced by other dry processing methods. The current work expands upon the original proof-of-concept, to enable higher throughput drying with more process control. Utilization of a commercial microwave (CEM SAM 255, Matthews, NC) enabled continuous drying at low power settings, with automatic feedback of average temperature. A new turntable was manufactured from ultra-high molecular weight polyethylene to enable drying of up to 12 samples at a time. Samples were processed, stored, and rehydrated in the same container. Samples were dried on polycarbonate Isopore track-etched membranes positioned within one side of a syringe filter holder (MilliporeTM, Billerica, MA). At the end of the drying period, the syringe filter was assembled thus enabling aseptic storage in a humidity-controlled environment or vacuum-packing for shipping and/or long-term storage. To determine sample repeatability and uniformity of drying within the microwave cavity, solutions of trehalose in media were processed for specific intervals and then weighed at the end of each processing period. Samples of 10 lL volume could be completely dehydrated by continuous processing at 20% power (120 W) for 8 min, with excellent sample-to-sample repeatability. During this drying period the average temperature measured in the 7 inch diameter IR sensing window increased by 4 °C. As the sample size was decreased to 5 lL, the samples could be completely dehydrated within 90 s at the same power level, at the expense of sample-to-sample reproducibility. Average temperature increased by 2 °C during processing. Higher resolution temperature mapping experiments are underway to determine individual sample temperature history during processing. In preliminary testing of biological sample recovery, a known number of mouse macrophage cells (J774) suspended in trehalose solution (200 mM) were deposited in 5 lL volumes onto Millipore track-etched membranes and microwaved in a CEM SAM 255 microwave at 120 W for 30 s, achieving an approximate moisture content of 6 gH2O/gdw. The samples were rehydrated by attaching a syringe and pulling DMEM through the filter holder, while providing several minutes for sample equilibration. The cells recovered within the solution were then counted with a hemocytometer, using Trypan Blue to distinguish live from dead cells. Results indicated that there was a 15% recovery of cells from the membrane. Of the cells that were recovered, 63% were viable, leading to a total viable recovery of 10%. Due to dilution effects, the statistical power of cell counts was low, but results were observed to be within range of the viability results from other processing methods, at this moisture content level. Recovery levels of other cell types are currently being investigated. The new microwave procedure was successful in producing repeatable results for multiple simultaneously processed samples. Though the recovery of cells was low, the viability was high, suggesting that it will be possible to improve the recoverability with continued refinements of the process design. Source of funding: This work was funded by a Prototype Grant from the Charlotte Research Institute at UNC Charlotte. Conflicts of interest: None declared. doi:10.1016/j.cryobiol.2011.09.108
106. Supercooling-facilitating activities in larch (Larix kaempferi) dormant buds. Keita Endoh *, Seizo Fujikawa, Keita Arakawa, Research Faculty and Graduate School of Agriculture, Hokkaido University, Japan Dormant buds in many trees adapt to subzero temperatures by a unique mechanism, so-called extraorgan freezing. In the process of extraorgan freezing in dormant buds of larch (Larix kaempferi), dehydrated water from tissue cells locally accumulates and forms large masses of ice crystals in specific areas between scale layers and beneath crown tissue during freezing, and cellular freezing behaviors are different
335
among tissues in the bud. Cells of the crown tissue and scale, which are adjacent to extracellular ice crystals, adapt by extracellular freezing. On the other hand, primordial tissue cells in dormant buds adapt by deep supercooling with incomplete dehydration. These cells maintain an intracellular water liquid state below 30 °C by sufficient slow cooling in mid-winter. Thus, it is possible that supercooling-facilitating substances exist in primordial tissue cells for protection from lethal intracellular freezing at subzero temperatures. In the present study, supercooling-facilitating activity was examined to clarify the molecular mechanism of freezing adaptation of dormant bud cells. First, supercooling-facilitating activity was examined in the extract of dormant buds of larch. In crude methanol extract from buds, supercooling-facilitating activities in the presence of Erwinia ananans (2 mg/ml) as an ice nucleator were detected by measuring freezing temperatures using thermocouples when the samples were cooled at a cooling rate of 0.2 °C/min. Freezing temperatures of Milli-Q water and the crude extract at 200 mmol/kg in the presence of E. ananas was 4.7 and 6.6 °C, respectively, and supercooling-facilitating activity was estimated to be 1.9 °C. This suggested the existence of supercooling-facilitating substances in the crude extract of larch dormant buds. Then the crude extracts were further separated into aqueous and EtOAc fractions by extraction with EtOAc for partial purification of the supercooling-facilitating substances. Freezing temperatures of aqueous and EtOAc fractions at 200 mmol/kg were 5.2 and 10.9 °C, respectively. Therefore, supercooling-facilitating activities of aqueous and EtOAc fractions were estimated to be 0.5 and 6.2 °C, respectively. This suggested that supercooling-facilitating substances in dormant buds are mostly distributed in the EtOAc fraction. When silver iodide (1 mM) was used as an ice nucleator, instead of E. ananas, for measuring freezing temperatures using thermocouples, higher supercooling-facilitating activities were detected in both fractions. The activities of aqueous and EtOAc fractions at 200 mmol/kg were 15.8 and 17.4 °C, respectively. However, when freezing temperatures of crude extract, aqueous and EtOAc fractions were analyzed by measuring freezing temperatures using thermocouples without an ice nucleator, freezing temperatures of crude extract, aqueous and EtOAc fractions at 200 mmol/kg were almost same with that of Milli-Q water as a control. This means that none of the three fractions showed supercooling-facilitating activity in pure water. These results suggested that supercooling-facilitating substances exist in dormant buds of larch that adapt to subzero temperatures by extraorgan freezing. Conflict of interest: None declared. Source of funding: This work is supported by the Japan Society for Promotion of Science since 2010 (22 3319). doi:10.1016/j.cryobiol.2011.09.109
107. L-Proline and ectoine stabilize proteins from denaturation. H. Sun a, H. Wolfes b, B. Glasmacher * a, N. Hofmann a, a Institute for Multiphase Processes/Leibniz Universitaet Hannover, Hannover, Germany, b Institute for Biophysical Chemistry, Hannover Medical School, Hannover, Germany Compatible solutes such as ectoine, hydroxyectoine and L-proline have been reported to be able to stabilize lipids and proteins. Recently it was found that L-proline and ectoine could be applied in cryopreservation of human stem cells as cryoprotective agents (CPA). Thus, the conventional CPA dimethyl sulfoxide which is cell toxic could be replaced. The working mechanism of compatible solutes was explained as ‘‘preferential exclusion”. In this study we have studied effects of L-proline and ectoine on the denaturation of the model protein bovine RNase A with differential scanning calorimeter (DSC). Nano DSC from TA Instruments was used in this study. Bovine RNase A was used as model protein, since it is a relatively well studied protein. Protein was dissolved in sodium phosphate buffer with pH 6 and dialysed overnight. Lproline and ectoine were dissolved in bi-distillate water. Protein solution was mixed with different concentrations of L-proline and ectoine in the range from 10 mM to 4 M, end concentration of RNase A was kept at 2 mg/ml. Samples were examined with DSC in a temperature range from 20 °C to 100 °C. Protein denaturation onset temperature, melting temperature, calorimetric enthalpy, change of the specific heat capacity and the fraction of the denatured protein were determined by analysing DSC data curves. DSC results showed that L-proline and ectoine elevate melting temperature with a positive relationship with their concentration. In the presence of 2 M L-proline, protein melting temperature was 1.5 K higher than that without L-proline. Melting enthalpy of bovine RNase A was also increased, small concentrations of L-proline (10–100 mM) could lead to the maximum increasement. Ectoine, however, requires much higher concentrations (500 mM). In the presence of 2 M L-proline and 4 M ectoine, protein denaturation was highly postponed. These results are in agreement with literature data. Compatible solutes L-proline and ectoine are able to stabilize bovine RNase A from denaturation by elevating the melting temperature and the melting enthalpy. L-Proline needs relatively lower concentrations compared to ectoine. This work is supported by funding from Deutsche Forschungsgemeinschaft (DFG, German research foundation) for the Cluster of Excellent REBIRTH (From Regenerative Biology to Reconstructive Therapy). Conflicts of interest: None declared. doi:10.1016/j.cryobiol.2011.09.110
105. High throughput microwave processing of small biological samples for anhydrous storage. Stephanie Cellemme, Elisha Paramore, Mathew Van Vorst, Gloria D. Elliott *, Department of Mechanical Engineering and Engineering Science, 9201 University City Blvd., University of North Carolina at Charlotte, Charlotte, NC 28221-0001, USA Recently our group demonstrated that microwave processing can be used to reproducibly dehydrate samples for preservation in an anhydrous state, yielding samples that are more uniform in water content than those produced by other dry processing methods. The current work expands upon the original proof-of-concept, to enable higher throughput drying with more process control. Utilization of a commercial microwave (CEM SAM 255, Matthews, NC) enabled continuous drying at low power settings, with automatic feedback of average temperature. A new turntable was manufactured from ultra-high molecular weight polyethylene to enable drying of up to 12 samples at a time. Samples were processed, stored, and rehydrated in the same container. Samples were dried on polycarbonate Isopore track-etched membranes positioned within one side of a syringe filter holder (MilliporeTM, Billerica, MA). At the end of the drying period, the syringe filter was assembled thus enabling aseptic storage in a humidity-controlled environment or vacuum-packing for shipping and/or long-term storage. To determine sample repeatability and uniformity of drying within the microwave cavity, solutions of trehalose in media were processed for specific intervals and then weighed at the end of each processing period. Samples of 10 lL volume could be completely dehydrated by continuous processing at 20% power (120 W) for 8 min, with excellent sample-to-sample repeatability. During this drying period the average temperature measured in the 7 inch diameter IR sensing window increased by 4 °C. As the sample size was decreased to 5 lL, the samples could be completely dehydrated within 90 s at the same power level, at the expense of sample-to-sample reproducibility. Average temperature increased by 2 °C during processing. Higher resolution temperature mapping experiments are underway to determine individual sample temperature history during processing. In preliminary testing of biological sample recovery, a known number of mouse macrophage cells (J774) suspended in trehalose solution (200 mM) were deposited in 5 lL volumes onto Millipore track-etched membranes and microwaved in a CEM SAM 255 microwave at 120 W for 30 s, achieving an approximate moisture content of 6 gH2O/gdw. The samples were rehydrated by attaching a syringe and pulling DMEM through the filter holder, while providing several minutes for sample equilibration. The cells recovered within the solution were then counted with a hemocytometer, using Trypan Blue to distinguish live from dead cells. Results indicated that there was a 15% recovery of cells from the membrane. Of the cells that were recovered, 63% were viable, leading to a total viable recovery of 10%. Due to dilution effects, the statistical power of cell counts was low, but results were observed to be within range of the viability results from other processing methods, at this moisture content level. Recovery levels of other cell types are currently being investigated. The new microwave procedure was successful in producing repeatable results for multiple simultaneously processed samples. Though the recovery of cells was low, the viability was high, suggesting that it will be possible to improve the recoverability with continued refinements of the process design. Source of funding: This work was funded by a Prototype Grant from the Charlotte Research Institute at UNC Charlotte. Conflicts of interest: None declared. doi:10.1016/j.cryobiol.2011.09.108
106. Supercooling-facilitating activities in larch (Larix kaempferi) dormant buds. Keita Endoh *, Seizo Fujikawa, Keita Arakawa, Research Faculty and Graduate School of Agriculture, Hokkaido University, Japan Dormant buds in many trees adapt to subzero temperatures by a unique mechanism, so-called extraorgan freezing. In the process of extraorgan freezing in dormant buds of larch (Larix kaempferi), dehydrated water from tissue cells locally accumulates and forms large masses of ice crystals in specific areas between scale layers and beneath crown tissue during freezing, and cellular freezing behaviors are different
335
among tissues in the bud. Cells of the crown tissue and scale, which are adjacent to extracellular ice crystals, adapt by extracellular freezing. On the other hand, primordial tissue cells in dormant buds adapt by deep supercooling with incomplete dehydration. These cells maintain an intracellular water liquid state below 30 °C by sufficient slow cooling in mid-winter. Thus, it is possible that supercooling-facilitating substances exist in primordial tissue cells for protection from lethal intracellular freezing at subzero temperatures. In the present study, supercooling-facilitating activity was examined to clarify the molecular mechanism of freezing adaptation of dormant bud cells. First, supercooling-facilitating activity was examined in the extract of dormant buds of larch. In crude methanol extract from buds, supercooling-facilitating activities in the presence of Erwinia ananans (2 mg/ml) as an ice nucleator were detected by measuring freezing temperatures using thermocouples when the samples were cooled at a cooling rate of 0.2 °C/min. Freezing temperatures of Milli-Q water and the crude extract at 200 mmol/kg in the presence of E. ananas was 4.7 and 6.6 °C, respectively, and supercooling-facilitating activity was estimated to be 1.9 °C. This suggested the existence of supercooling-facilitating substances in the crude extract of larch dormant buds. Then the crude extracts were further separated into aqueous and EtOAc fractions by extraction with EtOAc for partial purification of the supercooling-facilitating substances. Freezing temperatures of aqueous and EtOAc fractions at 200 mmol/kg were 5.2 and 10.9 °C, respectively. Therefore, supercooling-facilitating activities of aqueous and EtOAc fractions were estimated to be 0.5 and 6.2 °C, respectively. This suggested that supercooling-facilitating substances in dormant buds are mostly distributed in the EtOAc fraction. When silver iodide (1 mM) was used as an ice nucleator, instead of E. ananas, for measuring freezing temperatures using thermocouples, higher supercooling-facilitating activities were detected in both fractions. The activities of aqueous and EtOAc fractions at 200 mmol/kg were 15.8 and 17.4 °C, respectively. However, when freezing temperatures of crude extract, aqueous and EtOAc fractions were analyzed by measuring freezing temperatures using thermocouples without an ice nucleator, freezing temperatures of crude extract, aqueous and EtOAc fractions at 200 mmol/kg were almost same with that of Milli-Q water as a control. This means that none of the three fractions showed supercooling-facilitating activity in pure water. These results suggested that supercooling-facilitating substances exist in dormant buds of larch that adapt to subzero temperatures by extraorgan freezing. Conflict of interest: None declared. Source of funding: This work is supported by the Japan Society for Promotion of Science since 2010 (22 3319). doi:10.1016/j.cryobiol.2011.09.109
107. L-Proline and ectoine stabilize proteins from denaturation. H. Sun a, H. Wolfes b, B. Glasmacher * a, N. Hofmann a, a Institute for Multiphase Processes/Leibniz Universitaet Hannover, Hannover, Germany, b Institute for Biophysical Chemistry, Hannover Medical School, Hannover, Germany Compatible solutes such as ectoine, hydroxyectoine and L-proline have been reported to be able to stabilize lipids and proteins. Recently it was found that L-proline and ectoine could be applied in cryopreservation of human stem cells as cryoprotective agents (CPA). Thus, the conventional CPA dimethyl sulfoxide which is cell toxic could be replaced. The working mechanism of compatible solutes was explained as ‘‘preferential exclusion”. In this study we have studied effects of L-proline and ectoine on the denaturation of the model protein bovine RNase A with differential scanning calorimeter (DSC). Nano DSC from TA Instruments was used in this study. Bovine RNase A was used as model protein, since it is a relatively well studied protein. Protein was dissolved in sodium phosphate buffer with pH 6 and dialysed overnight. Lproline and ectoine were dissolved in bi-distillate water. Protein solution was mixed with different concentrations of L-proline and ectoine in the range from 10 mM to 4 M, end concentration of RNase A was kept at 2 mg/ml. Samples were examined with DSC in a temperature range from 20 °C to 100 °C. Protein denaturation onset temperature, melting temperature, calorimetric enthalpy, change of the specific heat capacity and the fraction of the denatured protein were determined by analysing DSC data curves. DSC results showed that L-proline and ectoine elevate melting temperature with a positive relationship with their concentration. In the presence of 2 M L-proline, protein melting temperature was 1.5 K higher than that without L-proline. Melting enthalpy of bovine RNase A was also increased, small concentrations of L-proline (10–100 mM) could lead to the maximum increasement. Ectoine, however, requires much higher concentrations (500 mM). In the presence of 2 M L-proline and 4 M ectoine, protein denaturation was highly postponed. These results are in agreement with literature data. Compatible solutes L-proline and ectoine are able to stabilize bovine RNase A from denaturation by elevating the melting temperature and the melting enthalpy. L-Proline needs relatively lower concentrations compared to ectoine. This work is supported by funding from Deutsche Forschungsgemeinschaft (DFG, German research foundation) for the Cluster of Excellent REBIRTH (From Regenerative Biology to Reconstructive Therapy). Conflicts of interest: None declared. doi:10.1016/j.cryobiol.2011.09.110