- Email: [email protected]
142. Cloning and functional analysis of the trehalose transporter (TRET) gene from an insect
Abstracts / Cryobiology 55 (2007) 324–378 and the cooling and warming protocols on recovery growth of hairy root cultures of medicinal plants cryopreserved using the vitrification protocol with a new culture vessel incorporating a sieve to facilitate filling in and removal of solutions. The highest recovery growth of cryopreserved hairy roots (82.7-89.4 %) was achieved after progressive preculture for 1 day in liquid medium with 0.3 M sucrose and 5-6 hours with 0.5 M sucrose, loading for 30 min in 2 M glycerol + 0.5 M sucrose, dehydration with PVS2 (glycerol 30 % + Me2SO 15 % + EG 15 % in MS with 0.4 M sucrose) for 5-10 min, depending on the species, cooling roots in 5 ll droplets of PVS2 vitrification solution placed on aluminum foil strips by dipping these strips in liquid nitrogen, warming them by plunging the foil strips into preheated (40 C) 0.8 M sucrose solution for 30 s and further incubation in the same solution for 30 min. (Conflicts of interest: None declared. Source of funding: None declared). doi:10.1016/j.cryobiol.2007.10.142
140. Implementing cryopreservation of garlic germplasm collections using the droplet-vitrification technique. Hae-Sung Hwang, Haeng-Hoon Kim, Yoon-Geol Lee, Ho-Cheol Ko, National Institute of Agricultural Biotechnology, Suwon, Republic of Korea The droplet-vitrification protocol was applied to involucres of plants of two Korean garlic collections, Danyang and Mokpo, to establish a safety backup collection of cryopreserved germplasm. Garlic involucres of the 59 accessions harvested at Danyang showed a mean percentage survival of 83.3% and 73.5% regeneration after cryopreservation. Involucres of accessions cryopreserved at sub-optimal stages displayed lower survival and/or regeneration. Of these 59 accessions, 53 were cryopreserved and samples were stored for safety back-up/long-term conservation. In the Mokpo collection, involucres of 149 accessions were cryopreserved, displaying a mean percentage of 79.9% survival and 78.2% regeneration. Of these 149 accessions, 116 were cryopreserved and stored for the longterm. A total of 252 accessions of five clonal Allium species, including garlic, were cryopreserved using involucres, cloves or bulbils, with a mean percentage of 80.9% survival and 77.0% regeneration, from which 221 accessions were stored in liquid nitrogen for long-term conservation. The real-time quantitative PCR assay of several garlic viruses showed that virus concentration was much lower in plantlets originating from cryopreserved material, compared to plantlets originating from preculture control and dehydration control samples. These results demonstrate that largescale implementation of cryopreservation of Allium germplasm is feasible and that it results in the regeneration of virus-free or little-infected material. This will significantly facilitate the conservation and international exchange of Allium germplasm. (Conflicts of interest: None declared. Source of funding: None declared). doi:10.1016/j.cryobiol.2007.10.143
141. Evaluation of non-penetrating cryoprotective additives: effects of sugars on the attachment ability of freshly isolated rat hepatocytes. Kong Heng Lee a, Raquel Magalhaes a, Sok Siam Gouk a, Hanry Yu b, Lilia L. Kuleshova a, a Low Temperature Preservation Unit, National University Medical Institutes, National University of Singapore, Singapore, Singapore; b Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore Vitrification protocols based on high concentrations may present potential problems of toxicity to biological systems. Substitution of penetrating cryoprotectants with non-penetrating cryoprotectants in large amounts, as components of a vitrification solution, may reduce overall toxicity. Using this approach we reported successful vitrification for an hepatocyte-matrix system [Wu YN, Yu H, Chang S, Magalha˜es R & Kuleshova L (2007) Tissue Engineering 13, 649-658] and self assembled hepatocyte aggregates [Magalha˜es R, Wang XW, Gouk SS, Ten CM, Lee KH, Yu H, Kuleshova L. (2006) NanoBio-Tokyo 2006 Conference proceedings, T1-2018] in 40%ethylene glycol supplemented with 0.6 M sucrose
371
at a cooling rate 400 C/min. For clinical applications, large quantities of cells enclosed in large containers have to undergo vitrification-warming and this will lead to an unavoidable reduction of cooling and warming rates. One of the ways to compensate the reduction in cooling/warming rates, is to use a higher concentration of chemicals in the vitrification solution, resulting in greater dehydration of cells. Therefore, we have studied the dehydration limits of hepatocytes by exposure to various concentrations of non-penetrating chemicals, namely 1M Sucrose, 0.6 M Sucrose, 9% to 12% Ficoll, 1M Trehalose, 2M Fructose and 2M Glucose. Subsequently, we assessed the efficacy of hepatocyte vitrification in 40%EG supplemented with non-penetrating additives (selected in the first stage of this study) to compensate for lower cooling/warming rates (80 C /min). Hepatocytes were re-suspended in each of the test solutions and incubated for 10, 20 minutes, and 1 hour. Sequential 1 minute steps followed to achieve working concentrations of: 66%, 50%, 25% and 12.5% of initial concentration. Finally, cells were centrifuged at ·30 g for 3 minutes in 4 C, retrieved, re-suspended in Hepatozyme and plated in collagen-coated dishes. Attachment efficiency was assessed after 24 hours incubation at 37 C, 5% CO2 humidified incubator. It was found that for short exposure to Sucrose and Trehalose at concentrations of 1M as well as 06.MSucrose 12% Ficoll (33%w/v), marginally influenced cell attachment efficiency, which ranged from 95 to100%. Fructose and Glucose are less suitable additives as the attachment efficiency ranged between 56% to 78% for both. Results did not differ by more than 5% for 10 and 20 minutes of exposure. However, with longer exposure times to 1M Trehalose and 1M Sucrose, hepatocytes showed a significant reduction in cell attachment, especially in the 1M Trehalose group which had a reduction at 64% wrt controls. Levels of cell attachment efficiency was retained (<60%) after 1 h of exposure to 2M Fructose and 2M Glucose. For 0.6M Sucrose 9 - 12% Ficoll, levels of cell attachment, ranging from 95 to 99%, indicating that this mixture in combination with penetrating cryoprotectant might be a better option for the vitrification of hepatocytes. Using 40%EG supplemented with either 1M sucrose, 0.6M sucrose or 12% Ficoll was found to be promising for vitrification of hepatocytes under low cooling/warming rates. Further functional analysis of post-vitrified hepatocytes is underway to fully determine the most effective solution for vitrification at these conditions. (Conflicts of interest: None declared. Source of funding: BMRC of Singapore, (grant no.04/1/21/19/317) doi:10.1016/j.cryobiol.2007.10.144
Dessication 142. Cloning and functional analysis of the trehalose transporter (TRET) gene from an insect. Yasushi Kanamori, Ayako Saito, Daisuke Tanaka, Takahiro Kikawada, Masahiko Watanabe, Takashi Okuda, National Institute of Agrobiological Sciences, Ibaraki, Japan Accumulation of trehalose in the cell is a key for desiccation tolerance in many organisms. Two ways are considered to accumulate trehalose: one is synthesis inside the cell and the other is introduction from outside the cell. The facilitated sugar transporter family is a useful tool for the later and maintains isotonicity across both sides of cell membrane by introducing an exogenous gene. A facilitated trehalose transporter gene has been successfully cloned only from the sleeping chironomid, Polypedilum vanderplanki (PvTRET1). In insects, Tret1 orthologs are considered to exist on other insect genomes because trehalose is the main haemolymph sugar and is synthesized from the fat body. Therefore, we isolated six orthologs of PvTret1 from four insect species (three clones from Drosophila melanogaster and one each from Apis mellifera, Bombyx mori and Anopheles gambiae). The amino acid sequences of these are 58.5 - 80.4% identical to that of PvTRET1 and the E-values between them were very high (0.0 - e-165). To investigate the mechanism of recognition and transportation of trehalose, we performed a comparative sequence analysis, revealing that the consensus of amino acid motifs of the mammalian class 1 SLC2A family of glucose transporters (GLUTs) were found in the amino acid sequences of insect TRETs. The N-glycosylation site, which is involved in maintaining the structure of transporters with the high affinity for glucose in
372
Abstracts / Cryobiology 55 (2007) 324–378
GLUT1, was predicted on the loop domain located between putative transmembrane domain 1 and 2, like GLUT1. Phylogenic analysis with the nucleotide sequences of 44 sugar transporter-like genes in D. melanogaster considered that the insect Tret genes had diverged at a common ancestor species. The functional expression assay using the Xenopus oocyte showed that translation products of all orthologs could transport trehalose except for one ortholog from D. melanogaster even though all of the proteins were localized in the plasma membrane. (Conflicts of interest: None declared. Source of funding: None declared). doi:10.1016/j.cryobiol.2007.10.145
143. Cellular states of dried Polypedirum Vanderplanki as studied by temperature-controlled Fourier transform infrared spectroscopy. Takao Furuki a, Ken-ichi Akao b, Masahiko Watanabe c, Takahiro Kikawada c, Minoru Sakurai a, Takashi Okuda c, a Tokyo Institute of Technology, Yokohama, Japan; b JASCO Corporation, Hachioji, Japan; c National Institute of Agrobiological Science, Tsukuba, Japan Larvae of Polypedirum vanderplanki enter the so-called anhydrobiosis where organisms are almost completely dehydrated and the metabolic activity is undetectable with the ability to revive on rehydration. One of the most characteristic phenomena is the accumulation of large amounts of intra-cellular trehalose, up to ca. 20 wt % of their dried bodies. We have already demonstrated that vitrification of trehalose is indispensable to successful anhydrobiosis as studied by the differential scanning calorimeter (DSC). In the present study cellular states of dried larvae of Polypedirum vanderplanki were investigated with Fourier transform infrared (FT-IR) spectroscopy under temperature-controlled conditions. Two different types of samples were prepared. One was a slowly-dried larva (Slow sample) which accumulated much trehalose in the dried body and successfully entered anhydrobiosis. The other was a quickly-dried one (Quick sample) which included little or no trehalose and failed to enter anhydrobiosis. In order to elucidate the thermotropic states of the cellular membrane, the following two regions of FT-IR spectra were focused upon: (1) Anti-symmetric vibrational band of the phosphate group constituting polar head groups of lipid membranes (Peak P), and (2) CH vibrational bands, most of which originates in acyl chains of phospholipids (Peak C). The peak P for the Slow sample was found to be lower in the wave number than the Quick one, suggesting that for the Slow sample the polar head group of the cell membrane formed hydrogen bonds with trehalose. Furthermore it is of particular interest that the wave number of the peak P for the Slow sample remained constant even at relatively high temperatures where the given sample fell into a rubbery state, which were estimated to be over 70 from DSC measurements. This finding indicates that trehalose in the dried body underwent a transition from the glassy to the rubber state, keeping the hydrogen bonds with cellular membranes. For the Slow and Quick samples, the lower limit temperature where the cell membrane remained entirely in the liquid crystalline phase was found to be around 20 and 45 respectively, as determined from the temperature dependence of wave numbers for the peak C. That is, for the Slow sample at room temperature the cellular membranes keep a high fluidity, close to the ordinary physiological state, even with little or no body water. For the Quick sample, in contrast, the cell membranes are largely in the gel phase, i.e. hardened, where the acyl chains of the phospholipids are tightly packed. It should be noted that the cell membrane in liquid crystalline phase is necessary, but not sufficient as a prerequisite for successful revival on rehydration. For the Slow sample the cell membrane stays in the liquid crystalline phase at high temperatures as trehalose is devitrified. However, on rehydration in the corresponding high temperature region, the recovery rate of the Slow sample is down to only 10 % or lower. Consequently, the vitrification of trehalose is indispensable for successful anhydrobiosis of larvae of Polypedirum vanderplanki. (Conflicts of interest: None declared. Source of funding: None declared). doi:10.1016/j.cryobiol.2007.10.146
144. Molecular dynamics simulation on the interaction between trehalose and benzene in aqueous medium. Atsutoshi Okabe a, Takao Furuki a, Kazuyuki Oku b, Shigeharu Fukuda b, Minoru Sakurai a, a Tokyo Institute of Technology, Yokohama, Japan; b Hayashibara Biochemical laboratories, Inc, Okayama, Japan Trehalose is a nonreducing disaccharide consisting of two D-glucose units linked by a,a-1,1-linkage. This sugar acts as a protectant against various stresses, such as desiccation, heat, freezing and osmotic shock. In addition to the protection function against these water stresses, trehalose effectively depresses the oxidation of unsaturated fatty acid (UFA) in aqueous solution, where trehalose forms an intermolecular 1:1 complex with a cis-type double bond of UFA[1]. According to our recent NMR study, trehalose also directly binds to benzene in aqueous medium. Interestingly, such intriguing complexation with the unsaturated bonds was not observed for other disaccharides such as maltose, neotrehalose and sucrose. Our quantum chemical calculations indicated that the OH. . .p and CH. . .O types of hydrogen bonds are responsible for the stabilization of the above trehalose/unsaturated bond complexes[K. Oku, H. Watanabe, M. Kubota, S. Fukuda, M. Kurimoto, Y. Tsujisaka, M. Komori, Y. Inoue, and M. Sakurai, J. Am. Chem. Soc., 125, 12739-12748 (2003): K. Oku, M. Kurose, M. Kubota, S. Fukuda, M. Kurimoto, Y. Tujisaka, A. Okabe, and M. Sakurai, J. Phys. Chem. B, 109, 3032-3040 (2005)]. It is of great interest to elucidate what is the main driving force of such complex formation processes. To address this problem, we performed explicitsolvent molecular dynamics (MD) simulations for trehalose/benzene systems. Using the umbrella sampling method and the weighted histogram analysis, we obtained the potential of mean force (PMF) for the complex formation process. PMF was plotted against the distance (R) between the center of mass of trehalose and that of benzene. The resulting free energy profile indicated that the two molecules form a stable intermolecular com˚ . The structure of the complex was examined in plex at a value of R 5A detail by visualizing the MD trajectory data, which indicated that the center of mass of benzene was always located around the C1(C1 0 ) and C2(C2 0 ) methyne groups and the exocyclic methylene groups of trehalose. This was in good agreement with the result predicted from the 1H-1H NOESY spectrum. On the other hand, the MD simulation of the aqueous solution of trehalose indicated that the solvation shell around the C1(C1 0 ) and C2(C2 0 ) atoms and the exocyclic methylene groups were less hydrated than that around the other portion of the glucose ring. In other words, a hydration pocket is formed near this region. In conclusion, benzene binds to trehalose in water in a fashion that dehydration penalty, accompanied by association of the two molecules, is minimized. efficacy. (Conflicts of interest: None declared. Source of funding: None declared). doi:10.1016/j.cryobiol.2007.10.147
145. Malondialdehydes as indicators of oxidative damage in dried mammalian cells. Tamir Kanias a,b, Jason P. Acker a,b, a University of Alberta, Edmonton, AB, Canada; b Canadian Blood Services, Edmonton, AB, Canada Stabilization of mammalian cells in the dried state is one of the most actively investigated areas of cell preservation research today. The drying process often results in deleterious injuries including membrane rupture, loss of cell functionality, and cell death. We hypothesize that desiccation stress is accompanied by the formation of reactive oxygen species (ROS) that contribute to cell destruction. Very little is known about the extent of oxidative damage that occurs during desiccation of mammalian cells. The evaluation of oxidative damage in dry biomaterial is challenging since ROS are extremely unstable in the dry state. The objective of this study is to develop a method to determine the extent of oxidative damage in dry mammalian cells. Red blood cells (RBCs) are extremely susceptible to free radical attack and were, therefore, chosen as a cell model. Membrane lipid peroxidation is an indicator for oxidative damage. The extent of the damage can be measured by malondialdehydes (MDAs) that are the decomposition products of oxidized polyunsaturated fatty acids. The reaction of MDAs with thiobarbituric acid (TBA) forms a thiobarbituric acid reactive
140. Implementing cryopreservation of garlic germplasm collections using the droplet-vitrification technique. Hae-Sung Hwang, Haeng-Hoon Kim, Yoon-Geol Lee, Ho-Cheol Ko, National Institute of Agricultural Biotechnology, Suwon, Republic of Korea The droplet-vitrification protocol was applied to involucres of plants of two Korean garlic collections, Danyang and Mokpo, to establish a safety backup collection of cryopreserved germplasm. Garlic involucres of the 59 accessions harvested at Danyang showed a mean percentage survival of 83.3% and 73.5% regeneration after cryopreservation. Involucres of accessions cryopreserved at sub-optimal stages displayed lower survival and/or regeneration. Of these 59 accessions, 53 were cryopreserved and samples were stored for safety back-up/long-term conservation. In the Mokpo collection, involucres of 149 accessions were cryopreserved, displaying a mean percentage of 79.9% survival and 78.2% regeneration. Of these 149 accessions, 116 were cryopreserved and stored for the longterm. A total of 252 accessions of five clonal Allium species, including garlic, were cryopreserved using involucres, cloves or bulbils, with a mean percentage of 80.9% survival and 77.0% regeneration, from which 221 accessions were stored in liquid nitrogen for long-term conservation. The real-time quantitative PCR assay of several garlic viruses showed that virus concentration was much lower in plantlets originating from cryopreserved material, compared to plantlets originating from preculture control and dehydration control samples. These results demonstrate that largescale implementation of cryopreservation of Allium germplasm is feasible and that it results in the regeneration of virus-free or little-infected material. This will significantly facilitate the conservation and international exchange of Allium germplasm. (Conflicts of interest: None declared. Source of funding: None declared). doi:10.1016/j.cryobiol.2007.10.143
141. Evaluation of non-penetrating cryoprotective additives: effects of sugars on the attachment ability of freshly isolated rat hepatocytes. Kong Heng Lee a, Raquel Magalhaes a, Sok Siam Gouk a, Hanry Yu b, Lilia L. Kuleshova a, a Low Temperature Preservation Unit, National University Medical Institutes, National University of Singapore, Singapore, Singapore; b Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore Vitrification protocols based on high concentrations may present potential problems of toxicity to biological systems. Substitution of penetrating cryoprotectants with non-penetrating cryoprotectants in large amounts, as components of a vitrification solution, may reduce overall toxicity. Using this approach we reported successful vitrification for an hepatocyte-matrix system [Wu YN, Yu H, Chang S, Magalha˜es R & Kuleshova L (2007) Tissue Engineering 13, 649-658] and self assembled hepatocyte aggregates [Magalha˜es R, Wang XW, Gouk SS, Ten CM, Lee KH, Yu H, Kuleshova L. (2006) NanoBio-Tokyo 2006 Conference proceedings, T1-2018] in 40%ethylene glycol supplemented with 0.6 M sucrose
371
at a cooling rate 400 C/min. For clinical applications, large quantities of cells enclosed in large containers have to undergo vitrification-warming and this will lead to an unavoidable reduction of cooling and warming rates. One of the ways to compensate the reduction in cooling/warming rates, is to use a higher concentration of chemicals in the vitrification solution, resulting in greater dehydration of cells. Therefore, we have studied the dehydration limits of hepatocytes by exposure to various concentrations of non-penetrating chemicals, namely 1M Sucrose, 0.6 M Sucrose, 9% to 12% Ficoll, 1M Trehalose, 2M Fructose and 2M Glucose. Subsequently, we assessed the efficacy of hepatocyte vitrification in 40%EG supplemented with non-penetrating additives (selected in the first stage of this study) to compensate for lower cooling/warming rates (80 C /min). Hepatocytes were re-suspended in each of the test solutions and incubated for 10, 20 minutes, and 1 hour. Sequential 1 minute steps followed to achieve working concentrations of: 66%, 50%, 25% and 12.5% of initial concentration. Finally, cells were centrifuged at ·30 g for 3 minutes in 4 C, retrieved, re-suspended in Hepatozyme and plated in collagen-coated dishes. Attachment efficiency was assessed after 24 hours incubation at 37 C, 5% CO2 humidified incubator. It was found that for short exposure to Sucrose and Trehalose at concentrations of 1M as well as 06.MSucrose 12% Ficoll (33%w/v), marginally influenced cell attachment efficiency, which ranged from 95 to100%. Fructose and Glucose are less suitable additives as the attachment efficiency ranged between 56% to 78% for both. Results did not differ by more than 5% for 10 and 20 minutes of exposure. However, with longer exposure times to 1M Trehalose and 1M Sucrose, hepatocytes showed a significant reduction in cell attachment, especially in the 1M Trehalose group which had a reduction at 64% wrt controls. Levels of cell attachment efficiency was retained (<60%) after 1 h of exposure to 2M Fructose and 2M Glucose. For 0.6M Sucrose 9 - 12% Ficoll, levels of cell attachment, ranging from 95 to 99%, indicating that this mixture in combination with penetrating cryoprotectant might be a better option for the vitrification of hepatocytes. Using 40%EG supplemented with either 1M sucrose, 0.6M sucrose or 12% Ficoll was found to be promising for vitrification of hepatocytes under low cooling/warming rates. Further functional analysis of post-vitrified hepatocytes is underway to fully determine the most effective solution for vitrification at these conditions. (Conflicts of interest: None declared. Source of funding: BMRC of Singapore, (grant no.04/1/21/19/317) doi:10.1016/j.cryobiol.2007.10.144
Dessication 142. Cloning and functional analysis of the trehalose transporter (TRET) gene from an insect. Yasushi Kanamori, Ayako Saito, Daisuke Tanaka, Takahiro Kikawada, Masahiko Watanabe, Takashi Okuda, National Institute of Agrobiological Sciences, Ibaraki, Japan Accumulation of trehalose in the cell is a key for desiccation tolerance in many organisms. Two ways are considered to accumulate trehalose: one is synthesis inside the cell and the other is introduction from outside the cell. The facilitated sugar transporter family is a useful tool for the later and maintains isotonicity across both sides of cell membrane by introducing an exogenous gene. A facilitated trehalose transporter gene has been successfully cloned only from the sleeping chironomid, Polypedilum vanderplanki (PvTRET1). In insects, Tret1 orthologs are considered to exist on other insect genomes because trehalose is the main haemolymph sugar and is synthesized from the fat body. Therefore, we isolated six orthologs of PvTret1 from four insect species (three clones from Drosophila melanogaster and one each from Apis mellifera, Bombyx mori and Anopheles gambiae). The amino acid sequences of these are 58.5 - 80.4% identical to that of PvTRET1 and the E-values between them were very high (0.0 - e-165). To investigate the mechanism of recognition and transportation of trehalose, we performed a comparative sequence analysis, revealing that the consensus of amino acid motifs of the mammalian class 1 SLC2A family of glucose transporters (GLUTs) were found in the amino acid sequences of insect TRETs. The N-glycosylation site, which is involved in maintaining the structure of transporters with the high affinity for glucose in
372
Abstracts / Cryobiology 55 (2007) 324–378
GLUT1, was predicted on the loop domain located between putative transmembrane domain 1 and 2, like GLUT1. Phylogenic analysis with the nucleotide sequences of 44 sugar transporter-like genes in D. melanogaster considered that the insect Tret genes had diverged at a common ancestor species. The functional expression assay using the Xenopus oocyte showed that translation products of all orthologs could transport trehalose except for one ortholog from D. melanogaster even though all of the proteins were localized in the plasma membrane. (Conflicts of interest: None declared. Source of funding: None declared). doi:10.1016/j.cryobiol.2007.10.145
143. Cellular states of dried Polypedirum Vanderplanki as studied by temperature-controlled Fourier transform infrared spectroscopy. Takao Furuki a, Ken-ichi Akao b, Masahiko Watanabe c, Takahiro Kikawada c, Minoru Sakurai a, Takashi Okuda c, a Tokyo Institute of Technology, Yokohama, Japan; b JASCO Corporation, Hachioji, Japan; c National Institute of Agrobiological Science, Tsukuba, Japan Larvae of Polypedirum vanderplanki enter the so-called anhydrobiosis where organisms are almost completely dehydrated and the metabolic activity is undetectable with the ability to revive on rehydration. One of the most characteristic phenomena is the accumulation of large amounts of intra-cellular trehalose, up to ca. 20 wt % of their dried bodies. We have already demonstrated that vitrification of trehalose is indispensable to successful anhydrobiosis as studied by the differential scanning calorimeter (DSC). In the present study cellular states of dried larvae of Polypedirum vanderplanki were investigated with Fourier transform infrared (FT-IR) spectroscopy under temperature-controlled conditions. Two different types of samples were prepared. One was a slowly-dried larva (Slow sample) which accumulated much trehalose in the dried body and successfully entered anhydrobiosis. The other was a quickly-dried one (Quick sample) which included little or no trehalose and failed to enter anhydrobiosis. In order to elucidate the thermotropic states of the cellular membrane, the following two regions of FT-IR spectra were focused upon: (1) Anti-symmetric vibrational band of the phosphate group constituting polar head groups of lipid membranes (Peak P), and (2) CH vibrational bands, most of which originates in acyl chains of phospholipids (Peak C). The peak P for the Slow sample was found to be lower in the wave number than the Quick one, suggesting that for the Slow sample the polar head group of the cell membrane formed hydrogen bonds with trehalose. Furthermore it is of particular interest that the wave number of the peak P for the Slow sample remained constant even at relatively high temperatures where the given sample fell into a rubbery state, which were estimated to be over 70 from DSC measurements. This finding indicates that trehalose in the dried body underwent a transition from the glassy to the rubber state, keeping the hydrogen bonds with cellular membranes. For the Slow and Quick samples, the lower limit temperature where the cell membrane remained entirely in the liquid crystalline phase was found to be around 20 and 45 respectively, as determined from the temperature dependence of wave numbers for the peak C. That is, for the Slow sample at room temperature the cellular membranes keep a high fluidity, close to the ordinary physiological state, even with little or no body water. For the Quick sample, in contrast, the cell membranes are largely in the gel phase, i.e. hardened, where the acyl chains of the phospholipids are tightly packed. It should be noted that the cell membrane in liquid crystalline phase is necessary, but not sufficient as a prerequisite for successful revival on rehydration. For the Slow sample the cell membrane stays in the liquid crystalline phase at high temperatures as trehalose is devitrified. However, on rehydration in the corresponding high temperature region, the recovery rate of the Slow sample is down to only 10 % or lower. Consequently, the vitrification of trehalose is indispensable for successful anhydrobiosis of larvae of Polypedirum vanderplanki. (Conflicts of interest: None declared. Source of funding: None declared). doi:10.1016/j.cryobiol.2007.10.146
144. Molecular dynamics simulation on the interaction between trehalose and benzene in aqueous medium. Atsutoshi Okabe a, Takao Furuki a, Kazuyuki Oku b, Shigeharu Fukuda b, Minoru Sakurai a, a Tokyo Institute of Technology, Yokohama, Japan; b Hayashibara Biochemical laboratories, Inc, Okayama, Japan Trehalose is a nonreducing disaccharide consisting of two D-glucose units linked by a,a-1,1-linkage. This sugar acts as a protectant against various stresses, such as desiccation, heat, freezing and osmotic shock. In addition to the protection function against these water stresses, trehalose effectively depresses the oxidation of unsaturated fatty acid (UFA) in aqueous solution, where trehalose forms an intermolecular 1:1 complex with a cis-type double bond of UFA[1]. According to our recent NMR study, trehalose also directly binds to benzene in aqueous medium. Interestingly, such intriguing complexation with the unsaturated bonds was not observed for other disaccharides such as maltose, neotrehalose and sucrose. Our quantum chemical calculations indicated that the OH. . .p and CH. . .O types of hydrogen bonds are responsible for the stabilization of the above trehalose/unsaturated bond complexes[K. Oku, H. Watanabe, M. Kubota, S. Fukuda, M. Kurimoto, Y. Tsujisaka, M. Komori, Y. Inoue, and M. Sakurai, J. Am. Chem. Soc., 125, 12739-12748 (2003): K. Oku, M. Kurose, M. Kubota, S. Fukuda, M. Kurimoto, Y. Tujisaka, A. Okabe, and M. Sakurai, J. Phys. Chem. B, 109, 3032-3040 (2005)]. It is of great interest to elucidate what is the main driving force of such complex formation processes. To address this problem, we performed explicitsolvent molecular dynamics (MD) simulations for trehalose/benzene systems. Using the umbrella sampling method and the weighted histogram analysis, we obtained the potential of mean force (PMF) for the complex formation process. PMF was plotted against the distance (R) between the center of mass of trehalose and that of benzene. The resulting free energy profile indicated that the two molecules form a stable intermolecular com˚ . The structure of the complex was examined in plex at a value of R 5A detail by visualizing the MD trajectory data, which indicated that the center of mass of benzene was always located around the C1(C1 0 ) and C2(C2 0 ) methyne groups and the exocyclic methylene groups of trehalose. This was in good agreement with the result predicted from the 1H-1H NOESY spectrum. On the other hand, the MD simulation of the aqueous solution of trehalose indicated that the solvation shell around the C1(C1 0 ) and C2(C2 0 ) atoms and the exocyclic methylene groups were less hydrated than that around the other portion of the glucose ring. In other words, a hydration pocket is formed near this region. In conclusion, benzene binds to trehalose in water in a fashion that dehydration penalty, accompanied by association of the two molecules, is minimized. efficacy. (Conflicts of interest: None declared. Source of funding: None declared). doi:10.1016/j.cryobiol.2007.10.147
145. Malondialdehydes as indicators of oxidative damage in dried mammalian cells. Tamir Kanias a,b, Jason P. Acker a,b, a University of Alberta, Edmonton, AB, Canada; b Canadian Blood Services, Edmonton, AB, Canada Stabilization of mammalian cells in the dried state is one of the most actively investigated areas of cell preservation research today. The drying process often results in deleterious injuries including membrane rupture, loss of cell functionality, and cell death. We hypothesize that desiccation stress is accompanied by the formation of reactive oxygen species (ROS) that contribute to cell destruction. Very little is known about the extent of oxidative damage that occurs during desiccation of mammalian cells. The evaluation of oxidative damage in dry biomaterial is challenging since ROS are extremely unstable in the dry state. The objective of this study is to develop a method to determine the extent of oxidative damage in dry mammalian cells. Red blood cells (RBCs) are extremely susceptible to free radical attack and were, therefore, chosen as a cell model. Membrane lipid peroxidation is an indicator for oxidative damage. The extent of the damage can be measured by malondialdehydes (MDAs) that are the decomposition products of oxidized polyunsaturated fatty acids. The reaction of MDAs with thiobarbituric acid (TBA) forms a thiobarbituric acid reactive