- Email: [email protected]
123. Brillouin and Raman scattering of lysozyme crystals in lower alcohol aqueous solutions
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Abstracts / Cryobiology 59 (2009) 370–418
it is well known that low temperature induces a cold-denaturation of proteins. According to previous thermodynamic studies of cold-denaturation of proteins [Adv. Protein Chem. 46 (1995)], it was suggested that the cold-denaturation structure of proteins is more compact than the native structure. Recently, there have been studies focusing on the structural stability of proteins in aqueous solution at 77 K using a rapid cooling method (500 K/min) [Biophys. J. 66 (1994) 249]. The structural flexibility of proteins becomes slower with decreasing temperature, and results in a glass transition [Eur. Biophys. J. 26 (1997) 327]. The glass transition temperature (Tg) of proteins has been investigated using a differential scanning calorimetry (DSC) method [Biophys. J. 90 (2006) 3732]. However, there have been few studies of protein secondary structural change in aqueous solution at 77 K. It is necessary to investigate the protein structure at 77 K for understanding the cold-denaturation mechanism and freezing preservation of biomolecules. Here, we carried out the systematic investigation of secondary structural change in proteins at 77 K. We have measured FTIR and CD spectra of various proteins (a-, a/b-, and b-proteins) at 298 and 77 K. Our results showed that the buried a-helical structure of a-proteins (bovine serum albumin (BSA) and cytochrome c), which is located in the interior of the protein, decreases on going from 298 to 77 K. Whereas, the solvated a-helix, which is fully exposed to the water, increases. This means that the a-helical structures of a-proteins essentially remain unchanged at 77 K. For a/b-proteins (lysozyme and ribonucleae A), low temperature (77 K) induced the a ? b transition. This means that a/bproteins prefer the b-sheet structure to the a-helical structure at 77 K, but this behavior depends on the b-sheet content of a/b-proteins. Moreover, the b-sheet structure of b-proteins (trypsin and a-chymotrypsin) transforms to a disordered structure on going from 298 to 77 K. On the basis of these results, we conclude that the transformation of secondary structure of proteins at 77 K depends on the contents of the secondary structures of proteins. (Conflicts of interest: None declared. Source of funding: None declared.)
doi:10.1016/j.cryobiol.2009.10.134
121. The three-dimensional structure and dynamics of trehalose transporter TRET1 in Polypedilum vandeplanki—as revealed by computer simulations. *Taku Okawa a, Takahiro Kikawada b, Takashi Okuda b, Minoru Sakurai a, a Center for Biological Resources and Informatics, Tokyo Institute of Technology, B-62 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan, b Anhydrobiosis Research Unit, National Institute of Agrobiological Sciences, Ohwashi 1-2, Tsukuba, Ibaraki 305-8634, Japan Potentially, trehalose has a useful anhydro-protectant ability for cells and biomolecules such as proteins and nucleotides. A major obstacle to application is that cellular membranes are impermeable to trehalose. Kikawada et al. isolated a novel trehalose transporter (TRET1) from an anhydrobiotic insect, Polypedilum vandeplanki [1]. They indicated that TRET1 is a trehalose-specific facilitated transporter and that the direction of transport is reversible depending on the concentration gradient of trehalose. It is expected that the use of TRET1 paves the way for the development of new desiccation–preservation technologies in the future. However, its functional mechanism remains unclear, partly because its three-dimensional (3-D) structure has not been elucidated experimentally. At present, it is most urgent and interesting to predict a 3-D structure of TRET1 by use of bioinfomatics techniques such as homology modeling and to investigate its functional mechanism at molecular level. TRET1 is a member of the major facilitator superfamily (MFS). In this study, the 3D structure (PDB code 1SUK) of GLUT1, glucose transporter from the human, was selected as a template and homology modeling was performed with MODELLER 9.4. In addition, the prediction of the TM region and secondary structures in TRET1 was done by using SOSUI and PSIPRED, respectively. Before performing homology modeling, we removed 20 residues from the N-terminus of TRET1. The validity of such a truncation was supported from results of the secondary structure analyses. Consequently, it was found that TRET1 has 12 transmembrane (TM) helices with an inward-facing conformation as a whole and involves several cavities inside the TM region. We performed MD simulation starting from the above homology modeling structure and explored the dynamics of the protein. After 26 ns simulation, we could successfully obtain the equilibrium structure of TRET1 in the lipid bilayer membrane. To examine the correlated motion of the protein, principal component analysis was applied to the trajectory data obtained. And we visualized the first principal component, which corresponds to the largest correlated motion of the protein. As a result, this vibration mode was shown to correspond to a hinge-bending motion, that is, the helices on the intracellular side come close or draw apart together. This dynamic may be essential for substrate uptake. Furthermore, we performed docking simulation combined with binding energy calculation to investigate the substrate selectivity of TRET1. Here, we selected trehalose and isotrehalose as a substrate. As a result it was found that trehalose more strongly binds to TRET1 than isotrehalose. This explains well the substrate selectivity of TRET1 reported by Kikawada et al. [1]. (Conflicts of interest: None declared. Source of funding: Grant-in-Aid for Scientific Research (No. 21370068) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) and also in part by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN).)
References [1] Kikawada T, Saito A, Kanamori Y, Nakahara Y, Iwata K, Tanaka D, Watanabe M, Okuda T. Proc. Natl. Acad. Sci. USA 2007;104:11585–90. doi:10.1016/j.cryobiol.2009.10.135
122. Measurement of membrane hydraulic conductivity (Lp and its activation energy (ELp in endothelial cells from the bovine carotid artery using a perfusion microscope. *Gang Zhao a,b, Tetsuya Yamamoto a, Hiroshi Takamatsu a, a Department of Mechanical Engineering, Kyushu University, Fukuoka 819-0395, Japan, b Department of Modern Mechanics, University of Science and Technology of China, Hefei 230027, Anhui, PR China Cryopreservation is an important technique for preserving cells, tissues, and even organs, while at the same time cryoinjury is more or less inevitable. Developing successful cryopreservation strategies for small elastic arteries and corneas have proven to be more difficult than expectation due to detachment and loss of viability of endothelial cells. The final goal of our study is to determine the best protocol for cryopreservation of endothelial cells using a simulation model for the freezing process. To this end, we need to find cell survival as a function of biophysical events estimated by the simulation. The model takes into account of osmotic volume change of cells and intracellular ice formation. As a first step in this study, we determined the osmotic properties of bovine carotid artery endothelial cells (HH cells) that are associated with cryobiological characteristics. These include the hydraulic conductivity (Lp) and its activation energy (ELp). A perfusion chamber developed and used in our previous studies [1,2] was used to subject cells to a programmed change of the extracellular NaCl concentration due to two important features of this perfusion chamber, i.e. the simple structure and the excellent reproducibility of the programmed extracellular concentration change. The response of isolated cells that were immobilized in a transparent perfusion chamber mounted on the microscope stage was observed during concentration increase from 0.15 to 0.5 M NaCl at three different temperatures, i.e. 23, 11, and 0 °C. The volumetric change of the cells was calculated from the measurements of the projected areas, assuming a spherical shape, which has been shown to be acceptable in the determination of Lp by 3-D measurement of cell volumes [3]. The membrane water transport model [2] was used to calculate volume change of cells. The hydraulic conductivity and the osmotically inactive volume of HH cells were determined using nonlinear regression to fit a membrane transport model to measured volume changes. The value of Lp was 0.26 lm/atm/min at 23 °C. The activation of Lp was then calculated from the data at 23, 11, and 0 °C using the Arrhenius equation. (Conflicts of interest: None declared. Source of funding: Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (No. 20 08060), and the National Natural Science Foundation of China (No. 50506029).)
References [1] Takamatsu H, Komori Y, Zawlodzka S, Fujii M. Quantitative examination of a perfusion microscope for the study of osmotic response of cells. J. Biomech. Eng. 2004;126:402–9. [2] Zawlodzka S, Takamatsu H. Osmotic injury of PC-3 cells by hypertonic NaCl solutions at temperature above 0 °C. Cryobiology 2005;50:58–70. [3] Yoshimori T, Takamatsu H. 3-D measurement of osmotic dehydration of isolated and adhered PC-3 cells. Cryobiology 2009;58:52–61. doi:10.1016/j.cryobiol.2009.10.136
123. Brillouin and Raman scattering of lysozyme crystals in lower alcohol aqueous solutions. *Hitoshi Kanazawa, Eiji Hashimoto, Yuichiro Aoki, Takahiro Ishii, Seiji Kojima, Graduate School of Pure and Applied Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8573, Japan The cryopreservation of biological substances has attracted much attention in biotechnology and food science. Although crystal structures of small protein crystals have been well studied by X-ray and neutron diffraction methods, the understanding of vibrational properties of these crystals is not enough. In the present study, we examine a tetragonal hen egg white lysozyme (HEWL) crystal that is immersed in lower alcohols such as propylene glycol (PG) and glycerol aqueous solutions, well known as cryoprotectants. HEWL crystals were grown by the two liquid interface (TLI) method, which employs insoluble and dense liquids. The crystals were grown and float on the interface of two different kinds of liquids, the HEWL mother solution and a dense liquid of Fluorinate. The TLI method enables the growth of high quality crystals [1]. The Raman scattering was excited by a diode-pumped solid state laser (DPSS532), operating in a single mode at 532 nm. The scattering light was collected
Abstracts / Cryobiology 59 (2009) 370–418 into the entrance slit of the triple grating monochromator with additive dispersion (Jobin Yvon T64000). Brillouin scattering was measured by using a diode-pumped solid-state laser at 532 nm wavelength and a Sandercock type 3 + 3 pass tandem Fabry–Perot Interferometer [2]. The Raman scattering of a HEWL crystal in lower alcohol aqueous solutions was investigated to characterize the conformations of molecular structure. It is observed that a HWEL crystal can be immersed in lower alcohol aqueous solutions without destroying the crystal structure and denaturation of the protein. We can obtain two kinds of spectra, related to a HEWL crystal and solutions by Brillouin scattering. Typical relaxation behavior is observed in the gigahertz frequency range. From Brillouin spectra of a HEWL crystal, we determine the temperature dependences of elastic properties and relaxation times. It is found that the relaxation time obeys the Arrhenius law and relaxation parameters are much different from those of bulk PG aqueous solutions. (Conflicts of interest: None declared. Source of funding: None declared.)
References [1] Ike Yuji, Hashimoto Eiji, Aoki Yuichiro, Kanazawa Hitoshi, Kojima Seiji. J. Mol. Struct. 2009;924–926:157. [2] Ike Yuji, Seshimo Yuichi, Kojima Seiji. J. Mol. Struct. 2009;924–926:127. doi:10.1016/j.cryobiol.2009.10.137
124. Salt effects on the conformational structure of LEA protein from Polypedilum vanderplanki and its model peptide. *Takao Furuki a, Tempei Shimizu a, Mitsuhiro Miyazawa b, Takahiro Kikawada b, Takashi Okuda b, Minoru Sakurai a, a Center for Biological Resources and Informatics, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8501, Japan, b National Institute of Agrobiological Sciences, Ohwashi 1-2, Tsukuba, Ibaraki 305-8634, Japan Larvae of P. vanderplanki are able to enter so-called ‘anhydrobiosis’ when they are almost completely dehydrated and fall into suspended animation, although they can revive upon rehydration. One of the most characteristic phenomena is the accumulation of a large amount of endogenous trehalose up to ca. 20 wt% of their dried bodies [1], together with late embryogenesis abundant (LEA) proteins [2]. Previously we have demonstrated that vitrification of the larvae is indispensable to their successful anhydrobiosis: this was studied by differential scanning calorimetry (DSC) and temperature-controlled Fourier transform infrared (FT-IR) spectroscopy [3] but the structural features and actual function of LEA proteins were less clear. The present study aims toward testing the ion-sequestration mechanism as one of possible functions of LEA proteins [4], investigating salt effects on the conformational structure for one of LEA proteins from P. vanderplanki (denoted as PvLEA2 in Ref. [2]) and of its model peptide. PvLEA2 is composed of 180 amino acid residues, with a molecular weight of 20.6 kDa [2]. The model peptide was designed as two repetition of the 11-mer amino acid unit forming the major segment characteristic of PvLEA2. Feature aspects of these molecular structures were studied by FT-IR spectroscopic measurements. The dried LEA protein had a content of a-helical structure at ca. 60% relative to the whole, except for the case of slow drying at RH = 98% which yielded ca 50% content of a-helical structure of the dehydrated LEA protein. The conformational structure of the dried LEA protein depended on the kinds of co-existing salts, being predominantly a-helical and b-sheet structures in the presence of NaCl and CaCl2, respectively. The dried model peptide also showed the similar trend in the observed molecular structure to PvLEA2, suggesting that the overall conformational property of the LEA protein arose from the segments composed of the 11-mer amino acid sequences. The ion-sequestration mechanism was based upon on the assumption that dried LEA proteins adopted a-helical coiled coil structure [4]. From this point of view it is controversial to apply the ion-sequestration mechanism to all kinds of ionic species. (Conflicts of interest: None declared. Source of funding: In part by Grant-in-Aid for Scientific Research (No. 21370068) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan and also in part by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN).)
References [1] Watanabe M, Kikawada T, Minagawa N, Yukihiro F, Okuda T. J. Exp. Biol. 2002;205:2799–802. [2] Kikawada T, Nakahara Y, Kanamori Y, Iwata K, Watanabe M, McGee B, Tunnacliffe A, Okuda T. Biochem. Biophys. Res. Commun. 2006;348:56–61. [3] Sakurai M, Furuki T, Akao K, Tanaka D, Nakahara Y, Kikawada T, Watanabe M, Okuda T. Proc. Natl. Acad. Sci. USA 2008;105:5093–8. [4] Dure III L. Plant J. 1993;3:363–9. doi:10.1016/j.cryobiol.2009.10.138
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125. In situ observation of xenon hydrate formation in onion tissue by using NMR and powder X-ray diffraction measurement. *Hiroko Ando a, Satoshi Takeya b, Yoshinori Kawagoe a, Yoshio Makino a, Toru Suzuki c, Seiichi Oshita a, a Graduate School of Agriculture and life science, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan, b National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan, c Department of Food Science and Technology, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japan Gas hydrates are ice-like crystals which are composed of water and small molecules as guests. Various studies have been conducted about physical properties of the gas hydrate in water from an interesting point of view in many kinds of fields. In 1961, Pauling suggested that the anesthetic effect of xenon (Xe) may relate to the hydrates [1]. While hydrates are not formed under body conditions, Pauling suggested they may be stabilized by changed side chains of proteins and solutions in the encephalonic fluid. On the other hand, for understanding the hydrate formation in biological tissue, Hulle and Fennema (1971) attempted to observe ethylene oxide (EO) hydrate formation in the biological tissue by using an optical microscopy [2]. However, clear evidence supporting the hydrate formation in the biological tissue has not been shown until now. Recently, we proposed a new preservation method for fresh vegetable by using Xe, since the deterioration of vegetables was prevented under Xe atmosphere after harvest. The results suggested that the suppression mechanism is related to the formation process of the Xe hydrate [3]. However, these previous studies were not enough to understand the suppression mechanism because actual formation of the hydrate crystals in the biological tissue had not been clarified. For further understanding, in this study, the formation of the hydrate in onion tissue was examined by using both powder X-ray diffraction (PXRD) and NMR measurements. Onion tissue, cut into 4 4 10 mm3, was preserved under Xe pressure up to 0.8 MPa at 5 °C. A few hours later, ice-like crystals were repeatedly recognized on the tissue surface. PXRD (Ultima III, Rigaku) measurement was conducted using the same tissue. The result of PXRD showed the crystals were structure I hydrate, that is Xe hydrate. Additionally, the mass ratio of Xe hydrate to water in the onion tissue was estimated to be 10% by quantitative analysis using PXRD pattern. The solid content in the tissue, corresponding to the Xe hydrate, was also measured by using 25 MHz pulse NMR spectrometer (MU25A, JOEL) with solid echo pulse sequence. The result indicated that the 12% of water molecules in the tissue turned into the Xe hydrate after the introduction of Xe. The hydrate ratios in the onion tissue determined by both methods were consistent. From these results, it was confirmed that Xe hydrate could be formed in biological tissue such as onion. Further, it is suggested that the NMR measurement with the solid echo pulse sequence is suitable to observe the hydrate formation in intact tissue. (Conflicts of interest: None declared. Source of funding: Part support by JSPS (Nos. 19-11965 and 20380139).)
References [1] Pauling L. Science 1961;134:15. [2] Hulle GV, Fennema O. Cryobiology 1971;7:223. [3] Makino Y et al.. Agric. Eng. Int.: CIGR E J. 2006;VIII:1.
doi:10.1016/j.cryobiol.2009.10.139
126. Anomalous ice nucleation behavior in aqueous polyvinyl alcohol solutions. *Maito Koga, Shigesaburo Ogawa, Shuichi Osanai, Department of Applied Chemistry, Graduate School of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku, Yokohama 223-8522, Japan Recently, there have been many reports of the cold behavior of aqueous polyvinyl alcohol (PVA) solutions such as the ice recrystallization inhibition and the formation of PVA physical gel by freeze/thaw treatments. Based on these characteristics, PVA is expected to be used in the field of biomedical and pharmaceutical applications. The study of the ice nucleation behavior of aqueous PVA solutions, however, has remained less developed than that of the other polymer solutions. It was reported that their ice nucleation often occurred at a higher temperature than that of the control solution. In addition, PVA seemed to inhibit the heterogeneous nucleation induced by some organic nucleators, but many ice grains remained together. This is the first study to investigate the ice nucleation behavior of aqueous PVA solutions using a W/O (water in oil) emulsion with 5 lm diameter droplets. This paper describes a study of aqueous polymer solutions of five PVA samples with different molecular weights in comparison with polyethylene glycol (PEG), polyvinyl pyrrolidone (PVP) and dextran (DX). The W/O emulsion with 5 lm diameter droplets were prepared using n-heptane, sorbitan tristearate (Span65) as the emulsifier and an aqueous polymer solution by mechanical stirring and membrane emulsification. All ice nucleation temperatures (Tf)) were measured by a DSC-60 at the scan rate of 5 °C/min and determined as the onset point of the exothermic peak during the cooling process. The overlap concentration (C*) and the freezing point depression (DTm) were determined by conventional
Abstracts / Cryobiology 59 (2009) 370–418
it is well known that low temperature induces a cold-denaturation of proteins. According to previous thermodynamic studies of cold-denaturation of proteins [Adv. Protein Chem. 46 (1995)], it was suggested that the cold-denaturation structure of proteins is more compact than the native structure. Recently, there have been studies focusing on the structural stability of proteins in aqueous solution at 77 K using a rapid cooling method (500 K/min) [Biophys. J. 66 (1994) 249]. The structural flexibility of proteins becomes slower with decreasing temperature, and results in a glass transition [Eur. Biophys. J. 26 (1997) 327]. The glass transition temperature (Tg) of proteins has been investigated using a differential scanning calorimetry (DSC) method [Biophys. J. 90 (2006) 3732]. However, there have been few studies of protein secondary structural change in aqueous solution at 77 K. It is necessary to investigate the protein structure at 77 K for understanding the cold-denaturation mechanism and freezing preservation of biomolecules. Here, we carried out the systematic investigation of secondary structural change in proteins at 77 K. We have measured FTIR and CD spectra of various proteins (a-, a/b-, and b-proteins) at 298 and 77 K. Our results showed that the buried a-helical structure of a-proteins (bovine serum albumin (BSA) and cytochrome c), which is located in the interior of the protein, decreases on going from 298 to 77 K. Whereas, the solvated a-helix, which is fully exposed to the water, increases. This means that the a-helical structures of a-proteins essentially remain unchanged at 77 K. For a/b-proteins (lysozyme and ribonucleae A), low temperature (77 K) induced the a ? b transition. This means that a/bproteins prefer the b-sheet structure to the a-helical structure at 77 K, but this behavior depends on the b-sheet content of a/b-proteins. Moreover, the b-sheet structure of b-proteins (trypsin and a-chymotrypsin) transforms to a disordered structure on going from 298 to 77 K. On the basis of these results, we conclude that the transformation of secondary structure of proteins at 77 K depends on the contents of the secondary structures of proteins. (Conflicts of interest: None declared. Source of funding: None declared.)
doi:10.1016/j.cryobiol.2009.10.134
121. The three-dimensional structure and dynamics of trehalose transporter TRET1 in Polypedilum vandeplanki—as revealed by computer simulations. *Taku Okawa a, Takahiro Kikawada b, Takashi Okuda b, Minoru Sakurai a, a Center for Biological Resources and Informatics, Tokyo Institute of Technology, B-62 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan, b Anhydrobiosis Research Unit, National Institute of Agrobiological Sciences, Ohwashi 1-2, Tsukuba, Ibaraki 305-8634, Japan Potentially, trehalose has a useful anhydro-protectant ability for cells and biomolecules such as proteins and nucleotides. A major obstacle to application is that cellular membranes are impermeable to trehalose. Kikawada et al. isolated a novel trehalose transporter (TRET1) from an anhydrobiotic insect, Polypedilum vandeplanki [1]. They indicated that TRET1 is a trehalose-specific facilitated transporter and that the direction of transport is reversible depending on the concentration gradient of trehalose. It is expected that the use of TRET1 paves the way for the development of new desiccation–preservation technologies in the future. However, its functional mechanism remains unclear, partly because its three-dimensional (3-D) structure has not been elucidated experimentally. At present, it is most urgent and interesting to predict a 3-D structure of TRET1 by use of bioinfomatics techniques such as homology modeling and to investigate its functional mechanism at molecular level. TRET1 is a member of the major facilitator superfamily (MFS). In this study, the 3D structure (PDB code 1SUK) of GLUT1, glucose transporter from the human, was selected as a template and homology modeling was performed with MODELLER 9.4. In addition, the prediction of the TM region and secondary structures in TRET1 was done by using SOSUI and PSIPRED, respectively. Before performing homology modeling, we removed 20 residues from the N-terminus of TRET1. The validity of such a truncation was supported from results of the secondary structure analyses. Consequently, it was found that TRET1 has 12 transmembrane (TM) helices with an inward-facing conformation as a whole and involves several cavities inside the TM region. We performed MD simulation starting from the above homology modeling structure and explored the dynamics of the protein. After 26 ns simulation, we could successfully obtain the equilibrium structure of TRET1 in the lipid bilayer membrane. To examine the correlated motion of the protein, principal component analysis was applied to the trajectory data obtained. And we visualized the first principal component, which corresponds to the largest correlated motion of the protein. As a result, this vibration mode was shown to correspond to a hinge-bending motion, that is, the helices on the intracellular side come close or draw apart together. This dynamic may be essential for substrate uptake. Furthermore, we performed docking simulation combined with binding energy calculation to investigate the substrate selectivity of TRET1. Here, we selected trehalose and isotrehalose as a substrate. As a result it was found that trehalose more strongly binds to TRET1 than isotrehalose. This explains well the substrate selectivity of TRET1 reported by Kikawada et al. [1]. (Conflicts of interest: None declared. Source of funding: Grant-in-Aid for Scientific Research (No. 21370068) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) and also in part by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN).)
References [1] Kikawada T, Saito A, Kanamori Y, Nakahara Y, Iwata K, Tanaka D, Watanabe M, Okuda T. Proc. Natl. Acad. Sci. USA 2007;104:11585–90. doi:10.1016/j.cryobiol.2009.10.135
122. Measurement of membrane hydraulic conductivity (Lp and its activation energy (ELp in endothelial cells from the bovine carotid artery using a perfusion microscope. *Gang Zhao a,b, Tetsuya Yamamoto a, Hiroshi Takamatsu a, a Department of Mechanical Engineering, Kyushu University, Fukuoka 819-0395, Japan, b Department of Modern Mechanics, University of Science and Technology of China, Hefei 230027, Anhui, PR China Cryopreservation is an important technique for preserving cells, tissues, and even organs, while at the same time cryoinjury is more or less inevitable. Developing successful cryopreservation strategies for small elastic arteries and corneas have proven to be more difficult than expectation due to detachment and loss of viability of endothelial cells. The final goal of our study is to determine the best protocol for cryopreservation of endothelial cells using a simulation model for the freezing process. To this end, we need to find cell survival as a function of biophysical events estimated by the simulation. The model takes into account of osmotic volume change of cells and intracellular ice formation. As a first step in this study, we determined the osmotic properties of bovine carotid artery endothelial cells (HH cells) that are associated with cryobiological characteristics. These include the hydraulic conductivity (Lp) and its activation energy (ELp). A perfusion chamber developed and used in our previous studies [1,2] was used to subject cells to a programmed change of the extracellular NaCl concentration due to two important features of this perfusion chamber, i.e. the simple structure and the excellent reproducibility of the programmed extracellular concentration change. The response of isolated cells that were immobilized in a transparent perfusion chamber mounted on the microscope stage was observed during concentration increase from 0.15 to 0.5 M NaCl at three different temperatures, i.e. 23, 11, and 0 °C. The volumetric change of the cells was calculated from the measurements of the projected areas, assuming a spherical shape, which has been shown to be acceptable in the determination of Lp by 3-D measurement of cell volumes [3]. The membrane water transport model [2] was used to calculate volume change of cells. The hydraulic conductivity and the osmotically inactive volume of HH cells were determined using nonlinear regression to fit a membrane transport model to measured volume changes. The value of Lp was 0.26 lm/atm/min at 23 °C. The activation of Lp was then calculated from the data at 23, 11, and 0 °C using the Arrhenius equation. (Conflicts of interest: None declared. Source of funding: Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (No. 20 08060), and the National Natural Science Foundation of China (No. 50506029).)
References [1] Takamatsu H, Komori Y, Zawlodzka S, Fujii M. Quantitative examination of a perfusion microscope for the study of osmotic response of cells. J. Biomech. Eng. 2004;126:402–9. [2] Zawlodzka S, Takamatsu H. Osmotic injury of PC-3 cells by hypertonic NaCl solutions at temperature above 0 °C. Cryobiology 2005;50:58–70. [3] Yoshimori T, Takamatsu H. 3-D measurement of osmotic dehydration of isolated and adhered PC-3 cells. Cryobiology 2009;58:52–61. doi:10.1016/j.cryobiol.2009.10.136
123. Brillouin and Raman scattering of lysozyme crystals in lower alcohol aqueous solutions. *Hitoshi Kanazawa, Eiji Hashimoto, Yuichiro Aoki, Takahiro Ishii, Seiji Kojima, Graduate School of Pure and Applied Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8573, Japan The cryopreservation of biological substances has attracted much attention in biotechnology and food science. Although crystal structures of small protein crystals have been well studied by X-ray and neutron diffraction methods, the understanding of vibrational properties of these crystals is not enough. In the present study, we examine a tetragonal hen egg white lysozyme (HEWL) crystal that is immersed in lower alcohols such as propylene glycol (PG) and glycerol aqueous solutions, well known as cryoprotectants. HEWL crystals were grown by the two liquid interface (TLI) method, which employs insoluble and dense liquids. The crystals were grown and float on the interface of two different kinds of liquids, the HEWL mother solution and a dense liquid of Fluorinate. The TLI method enables the growth of high quality crystals [1]. The Raman scattering was excited by a diode-pumped solid state laser (DPSS532), operating in a single mode at 532 nm. The scattering light was collected
Abstracts / Cryobiology 59 (2009) 370–418 into the entrance slit of the triple grating monochromator with additive dispersion (Jobin Yvon T64000). Brillouin scattering was measured by using a diode-pumped solid-state laser at 532 nm wavelength and a Sandercock type 3 + 3 pass tandem Fabry–Perot Interferometer [2]. The Raman scattering of a HEWL crystal in lower alcohol aqueous solutions was investigated to characterize the conformations of molecular structure. It is observed that a HWEL crystal can be immersed in lower alcohol aqueous solutions without destroying the crystal structure and denaturation of the protein. We can obtain two kinds of spectra, related to a HEWL crystal and solutions by Brillouin scattering. Typical relaxation behavior is observed in the gigahertz frequency range. From Brillouin spectra of a HEWL crystal, we determine the temperature dependences of elastic properties and relaxation times. It is found that the relaxation time obeys the Arrhenius law and relaxation parameters are much different from those of bulk PG aqueous solutions. (Conflicts of interest: None declared. Source of funding: None declared.)
References [1] Ike Yuji, Hashimoto Eiji, Aoki Yuichiro, Kanazawa Hitoshi, Kojima Seiji. J. Mol. Struct. 2009;924–926:157. [2] Ike Yuji, Seshimo Yuichi, Kojima Seiji. J. Mol. Struct. 2009;924–926:127. doi:10.1016/j.cryobiol.2009.10.137
124. Salt effects on the conformational structure of LEA protein from Polypedilum vanderplanki and its model peptide. *Takao Furuki a, Tempei Shimizu a, Mitsuhiro Miyazawa b, Takahiro Kikawada b, Takashi Okuda b, Minoru Sakurai a, a Center for Biological Resources and Informatics, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8501, Japan, b National Institute of Agrobiological Sciences, Ohwashi 1-2, Tsukuba, Ibaraki 305-8634, Japan Larvae of P. vanderplanki are able to enter so-called ‘anhydrobiosis’ when they are almost completely dehydrated and fall into suspended animation, although they can revive upon rehydration. One of the most characteristic phenomena is the accumulation of a large amount of endogenous trehalose up to ca. 20 wt% of their dried bodies [1], together with late embryogenesis abundant (LEA) proteins [2]. Previously we have demonstrated that vitrification of the larvae is indispensable to their successful anhydrobiosis: this was studied by differential scanning calorimetry (DSC) and temperature-controlled Fourier transform infrared (FT-IR) spectroscopy [3] but the structural features and actual function of LEA proteins were less clear. The present study aims toward testing the ion-sequestration mechanism as one of possible functions of LEA proteins [4], investigating salt effects on the conformational structure for one of LEA proteins from P. vanderplanki (denoted as PvLEA2 in Ref. [2]) and of its model peptide. PvLEA2 is composed of 180 amino acid residues, with a molecular weight of 20.6 kDa [2]. The model peptide was designed as two repetition of the 11-mer amino acid unit forming the major segment characteristic of PvLEA2. Feature aspects of these molecular structures were studied by FT-IR spectroscopic measurements. The dried LEA protein had a content of a-helical structure at ca. 60% relative to the whole, except for the case of slow drying at RH = 98% which yielded ca 50% content of a-helical structure of the dehydrated LEA protein. The conformational structure of the dried LEA protein depended on the kinds of co-existing salts, being predominantly a-helical and b-sheet structures in the presence of NaCl and CaCl2, respectively. The dried model peptide also showed the similar trend in the observed molecular structure to PvLEA2, suggesting that the overall conformational property of the LEA protein arose from the segments composed of the 11-mer amino acid sequences. The ion-sequestration mechanism was based upon on the assumption that dried LEA proteins adopted a-helical coiled coil structure [4]. From this point of view it is controversial to apply the ion-sequestration mechanism to all kinds of ionic species. (Conflicts of interest: None declared. Source of funding: In part by Grant-in-Aid for Scientific Research (No. 21370068) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan and also in part by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN).)
References [1] Watanabe M, Kikawada T, Minagawa N, Yukihiro F, Okuda T. J. Exp. Biol. 2002;205:2799–802. [2] Kikawada T, Nakahara Y, Kanamori Y, Iwata K, Watanabe M, McGee B, Tunnacliffe A, Okuda T. Biochem. Biophys. Res. Commun. 2006;348:56–61. [3] Sakurai M, Furuki T, Akao K, Tanaka D, Nakahara Y, Kikawada T, Watanabe M, Okuda T. Proc. Natl. Acad. Sci. USA 2008;105:5093–8. [4] Dure III L. Plant J. 1993;3:363–9. doi:10.1016/j.cryobiol.2009.10.138
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125. In situ observation of xenon hydrate formation in onion tissue by using NMR and powder X-ray diffraction measurement. *Hiroko Ando a, Satoshi Takeya b, Yoshinori Kawagoe a, Yoshio Makino a, Toru Suzuki c, Seiichi Oshita a, a Graduate School of Agriculture and life science, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan, b National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan, c Department of Food Science and Technology, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japan Gas hydrates are ice-like crystals which are composed of water and small molecules as guests. Various studies have been conducted about physical properties of the gas hydrate in water from an interesting point of view in many kinds of fields. In 1961, Pauling suggested that the anesthetic effect of xenon (Xe) may relate to the hydrates [1]. While hydrates are not formed under body conditions, Pauling suggested they may be stabilized by changed side chains of proteins and solutions in the encephalonic fluid. On the other hand, for understanding the hydrate formation in biological tissue, Hulle and Fennema (1971) attempted to observe ethylene oxide (EO) hydrate formation in the biological tissue by using an optical microscopy [2]. However, clear evidence supporting the hydrate formation in the biological tissue has not been shown until now. Recently, we proposed a new preservation method for fresh vegetable by using Xe, since the deterioration of vegetables was prevented under Xe atmosphere after harvest. The results suggested that the suppression mechanism is related to the formation process of the Xe hydrate [3]. However, these previous studies were not enough to understand the suppression mechanism because actual formation of the hydrate crystals in the biological tissue had not been clarified. For further understanding, in this study, the formation of the hydrate in onion tissue was examined by using both powder X-ray diffraction (PXRD) and NMR measurements. Onion tissue, cut into 4 4 10 mm3, was preserved under Xe pressure up to 0.8 MPa at 5 °C. A few hours later, ice-like crystals were repeatedly recognized on the tissue surface. PXRD (Ultima III, Rigaku) measurement was conducted using the same tissue. The result of PXRD showed the crystals were structure I hydrate, that is Xe hydrate. Additionally, the mass ratio of Xe hydrate to water in the onion tissue was estimated to be 10% by quantitative analysis using PXRD pattern. The solid content in the tissue, corresponding to the Xe hydrate, was also measured by using 25 MHz pulse NMR spectrometer (MU25A, JOEL) with solid echo pulse sequence. The result indicated that the 12% of water molecules in the tissue turned into the Xe hydrate after the introduction of Xe. The hydrate ratios in the onion tissue determined by both methods were consistent. From these results, it was confirmed that Xe hydrate could be formed in biological tissue such as onion. Further, it is suggested that the NMR measurement with the solid echo pulse sequence is suitable to observe the hydrate formation in intact tissue. (Conflicts of interest: None declared. Source of funding: Part support by JSPS (Nos. 19-11965 and 20380139).)
References [1] Pauling L. Science 1961;134:15. [2] Hulle GV, Fennema O. Cryobiology 1971;7:223. [3] Makino Y et al.. Agric. Eng. Int.: CIGR E J. 2006;VIII:1.
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126. Anomalous ice nucleation behavior in aqueous polyvinyl alcohol solutions. *Maito Koga, Shigesaburo Ogawa, Shuichi Osanai, Department of Applied Chemistry, Graduate School of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku, Yokohama 223-8522, Japan Recently, there have been many reports of the cold behavior of aqueous polyvinyl alcohol (PVA) solutions such as the ice recrystallization inhibition and the formation of PVA physical gel by freeze/thaw treatments. Based on these characteristics, PVA is expected to be used in the field of biomedical and pharmaceutical applications. The study of the ice nucleation behavior of aqueous PVA solutions, however, has remained less developed than that of the other polymer solutions. It was reported that their ice nucleation often occurred at a higher temperature than that of the control solution. In addition, PVA seemed to inhibit the heterogeneous nucleation induced by some organic nucleators, but many ice grains remained together. This is the first study to investigate the ice nucleation behavior of aqueous PVA solutions using a W/O (water in oil) emulsion with 5 lm diameter droplets. This paper describes a study of aqueous polymer solutions of five PVA samples with different molecular weights in comparison with polyethylene glycol (PEG), polyvinyl pyrrolidone (PVP) and dextran (DX). The W/O emulsion with 5 lm diameter droplets were prepared using n-heptane, sorbitan tristearate (Span65) as the emulsifier and an aqueous polymer solution by mechanical stirring and membrane emulsification. All ice nucleation temperatures (Tf)) were measured by a DSC-60 at the scan rate of 5 °C/min and determined as the onset point of the exothermic peak during the cooling process. The overlap concentration (C*) and the freezing point depression (DTm) were determined by conventional